U.S. patent application number 14/749599 was filed with the patent office on 2016-02-18 for compositions and methods of nucleic acid-targeting nucleic acids.
The applicant listed for this patent is Caribou Biosciences, Inc.. Invention is credited to James M. Berger, Matthew Merrill Carter, Paul Donohoue, Jennifer A. Doudna, Rachel E. Haurwitz, Andrew Paul May.
Application Number | 20160046949 14/749599 |
Document ID | / |
Family ID | 51580795 |
Filed Date | 2016-02-18 |
United States Patent
Application |
20160046949 |
Kind Code |
A1 |
May; Andrew Paul ; et
al. |
February 18, 2016 |
COMPOSITIONS AND METHODS OF NUCLEIC ACID-TARGETING NUCLEIC
ACIDS
Abstract
This disclosure provides for compositions and methods for the
use of nucleic acid-targeting nucleic acids and complexes
thereof.
Inventors: |
May; Andrew Paul; (San
Francisco, CA) ; Haurwitz; Rachel E.; (Kensington,
CA) ; Doudna; Jennifer A.; (Berkeley, CA) ;
Berger; James M.; (Baltimore, MD) ; Carter; Matthew
Merrill; (North Granby, CT) ; Donohoue; Paul;
(Berkeley, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Caribou Biosciences, Inc. |
Berkeley |
CA |
US |
|
|
Family ID: |
51580795 |
Appl. No.: |
14/749599 |
Filed: |
June 24, 2015 |
Related U.S. Patent Documents
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Application
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14206319 |
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14749599 |
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61818386 |
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Current U.S.
Class: |
536/23.2 |
Current CPC
Class: |
A61K 47/549 20170801;
C12N 15/102 20130101; A61K 38/465 20130101; C12N 2310/20 20170501;
A61P 35/00 20180101; C12Q 1/6818 20130101; C12N 2310/531 20130101;
C12N 15/11 20130101; C12N 15/907 20130101; C12N 15/85 20130101;
C12Q 1/68 20130101; C12N 15/90 20130101; A61P 11/00 20180101; A61P
27/02 20180101; C12Q 1/6869 20130101; C12Q 1/6874 20130101; A61P
13/12 20180101; A61P 9/00 20180101; A61P 29/00 20180101; C12N 9/22
20130101; C12N 15/52 20130101; C12N 2310/533 20130101; A61P 31/04
20180101; C12N 2800/80 20130101; A61K 47/6455 20170801; C12Q 1/686
20130101; A61P 9/10 20180101; C12N 15/902 20130101; C12Q 1/6806
20130101; C12N 15/113 20130101; A61P 19/02 20180101; A61P 25/00
20180101; C12Q 1/6818 20130101; C12Q 2525/203 20130101 |
International
Class: |
C12N 15/52 20060101
C12N015/52; C12N 9/22 20060101 C12N009/22 |
Claims
1-26. (canceled)
27. An engineered nucleic acid targeting nucleic acid, comprising:
an engineered CRISPR RNA (crRNA), an engineered trans-activating
CRISPR RNA (tracrRNA), or a combination thereof, wherein (i) the
engineered nucleic acid targeting nucleic acid can associate with a
Cas9 protein to form a nucleic acid targeting nucleic acid-Cas9
complex capable of binding a target DNA, and (ii) the engineered
crRNA, the engineered tracrRNA, or the combination thereof is
altered relative to a corresponding wild-type crRNA, tracrRNA, or
both; and the engineered nucleic acid targeting nucleic acid
comprises at least one of the following: a duplex variant, a spacer
extension, a mid-tracr modification, or a tracr extension.
28. The engineered nucleic acid targeting nucleic acid of claim 27,
wherein the duplex variant comprises at least one of the following
modifications: a modification of a first stem, a modification of a
bulge, a modification of a first duplex, or an affinity tag.
29. The engineered nucleic acid targeting nucleic acid of claim 28,
wherein the modification of the first stem comprises a Csy4 binding
sequence to which a Csy4 protein binds, a Cas5 binding sequence to
which a Cas5 protein binds, or a Cas6 binding sequence to which a
Cas6 protein binds.
30. The engineered nucleic acid targeting nucleic acid of claim 27,
wherein the spacer extension comprises at least one of the
following: a spacer extension less than 10 nucleotides in length, a
5' cap, a riboswitch sequence, a sequence that forms a hairpin, a
sequence that targets the nucleic acid targeting nucleic acid to a
subcellular location, a sequence that provides for tracking, a
sequence that provides a protein binding site for a protein, a
primer binding site, or an affinity tag.
31. The engineered nucleic acid targeting nucleic acid of claim 30,
wherein the sequence that provides the protein binding site for the
protein comprises a Csy4 binding sequence to which a Csy4 protein
binds, a Cas5 binding sequence to which a Cas5 protein binds, or a
Cas6 binding sequence to which a Cas6 protein binds.
32. The engineered nucleic acid targeting nucleic acid of claim 27,
wherein the mid-tracr modification comprises at least one of the
following modifications: a modification of a P domain, a
modification of a second stem, a modification of a third stem, an
added nucleic acid binding protein binding site, or an affinity
tag.
33. The engineered nucleic acid targeting nucleic acid of claim 32,
wherein the added nucleic acid binding protein binding site
comprises a Csy4 binding sequence to which a Csy4 protein binds, a
Cas5 binding sequence to which a Cas5 protein binds, or a Cas6
binding sequence to which a Cas6 protein binds.
34. The engineered nucleic acid targeting nucleic acid of claim 27,
wherein the tracr extension comprises at least one of the
following: a 3' hybridizing extension, an added nucleic acid
binding protein binding site, or an affinity tag.
35. The engineered nucleic acid targeting nucleic acid of claim 34,
wherein the added nucleic acid binding protein binding site
comprises a Csy4 binding sequence to which a Csy4 protein binds, a
Cas5 binding sequence to which a Cas5 protein binds, or a Cas6
binding sequence to which a Cas6 protein binds.
36. The engineered nucleic acid targeting nucleic acid of claim 34,
wherein a donor polynucleotide is hybridized to the 3' hybridizing
extension.
37. The engineered nucleic acid targeting nucleic acid of claim 27,
wherein the tracr extension comprises at least one of the
following: a tracr extension having a length of from 1 to 400
nucleotides, a 3' polyadenylated tail, a riboswitch sequence, a
sequence that forms a hairpin, a sequence that targets the nucleic
acid targeting nucleic acid to a subcellular location, a sequence
that provides for tracking, or a primer binding site.
38. The engineered nucleic acid targeting nucleic acid of claim 29,
wherein the Csy4 protein binds to the Csy4 binding sequence with a
Kd .about.50 pM.
39. The engineered nucleic acid targeting nucleic acid of claim 27,
wherein the engineered nucleic acid targeting nucleic acid is a
double guide nucleic acid structure.
40. The engineered nucleic acid targeting nucleic acid of claim 27,
wherein the engineered nucleic acid targeting nucleic acid is a
single guide nucleic acid structure.
41. The engineered nucleic acid targeting nucleic acid of claim 27,
wherein the engineered nucleic acid targeting nucleic acid
comprises DNA.
42. The engineered nucleic acid targeting nucleic acid of claim 27,
wherein the engineered nucleic acid targeting nucleic acid
comprises RNA.
43. The engineered nucleic acid targeting nucleic acid of claim 41,
wherein the engineered nucleic acid targeting nucleic acid
comprises RNA.
Description
CROSS-REFERENCE
[0001] This application is a continuation application of U.S.
patent application Ser. No. 14/206,319, filed on Mar. 12, 2014,
which claims the benefit of U.S. Provisional Application No.
61/818,386, filed May 1, 2013 [Attorney Docket No. 44287-710.101],
U.S. Provisional Application No. 61/902,723, filed Nov. 11, 2013
[Attorney Docket No. 44287-710.103], U.S. Provisional Application
No. 61/818,382, filed May 1, 2013 [Attorney Docket No.
44287-712.101], U.S. Provisional Application No. 61/859,661, filed
Jul. 29, 2013 [Attorney Docket No. 44287-712.102], U.S. Provisional
Application No. 61/858,767, filed Jul. 26, 2013 [Attorney Docket
No. 44287-713.102], U.S. Provisional Application No. 61/822,002,
filed May 10, 2013 [Attorney Docket No. 44287-717.101], U.S.
Provisional Application No. 61/832,690, filed Jun. 7, 2013
[Attorney Docket No. 44287-719.101], U.S. Provisional Application
No. 61/906,211, filed Nov. 19, 2013 [Attorney Docket No.
44287-719.102], U.S. Provisional Application No. 61/900,311, filed
Nov. 5, 2013 [Attorney Docket No. 44287-719.103], U.S. Provisional
Application No. 61/845,714, filed Jul. 12, 2013 [Attorney Docket
No. 44287-721.101], U.S. Provisional Application No. 61/883,804,
filed Sep. 27, 2013 [Attorney Docket No. 44287-721.102], U.S.
Provisional Application No. 61/781,598, filed Mar. 14, 2013
[Attorney Docket No. 44287-722.101], U.S. Provisional Application
No. 61/899,712, filed Nov. 4, 2013 [Attorney Docket No.
44287-727.101], U.S. Provisional Application No. 61/865,743, filed
Aug. 14, 2013 [Attorney Docket No. 44287-733.101], U.S. Provisional
Application No. 61/907,777, filed Nov. 22, 2013 [Attorney Docket
No. 44287-734.101], U.S. Provisional Application No. 61/903,232,
filed Nov. 12, 2013 [Attorney Docket No. 44287-751.101], U.S.
Provisional Application No. 61/906,335, filed Nov. 19, 2013
[Attorney Docket No. 44287-752.101], U.S. Provisional Application
No. 61/907,216, filed Nov. 21, 2013 [Attorney Docket No.
44287-753.101], the entire contents of which are incorporated
herein by reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Mar. 10, 2014, is named 44287-722-201_SeqList_.txt and is
7,828,921 bytes in size.
BACKGROUND OF THE INVENTION
[0003] Genome engineering can refer to altering the genome by
deleting, inserting, mutating, or substituting specific nucleic
acid sequences. The altering can be gene or location specific.
Genome engineering can use nucleases to cut a nucleic acid thereby
generating a site for the alteration. Engineering of non-genomic
nucleic acid is also contemplated. A protein containing a nuclease
domain can bind and cleave a target nucleic acid by forming a
complex with a nucleic acid-targeting nucleic acid. In one example,
the cleavage can introduce double stranded breaks in the target
nucleic acid. A nucleic acid can be repaired e.g. by endogenous
non-homologous end joining (NHEJ) machinery. In a further example,
a piece of nucleic acid can be inserted. NModifications of nucleic
acid-targeting nucleic acids and site-directed polypeptides can
introduce new functions to be used for genome engineering.
SUMMARY OF THE INVENTION
[0004] In one aspect, the disclosure provides for an engineered
nucleic acid-targeting nucleic acid comprising: a mutation in a
P-domain of the nucleic acid-targeting nucleic acid. In some
embodiments, the P-domain starts downstream of a last paired
nucleotide of a duplex between a CRISPR repeat and a tracrRNA
sequence of the nucleic acid-targeting nucleic acid. In some
embodiments, the engineered nucleic acid-targeting nucleic acid
further comprises a linker sequence. In some embodiments, the
linker sequence links the CRISPR repeat and the tracrRNA sequence.
In some embodiments, the engineered nucleic acid-targeting nucleic
acid is an isolated engineered nucleic acid-targeting nucleic acid.
In some embodiments, the engineered nucleic acid-targeting nucleic
acid is a recombinant engineered nucleic acid-targeting nucleic
acid. In some embodiments, the engineered nucleic acid-targeting
nucleic acid is adapted to hybridize to a target nucleic acid. In
some embodiments, the P-domain comprises 2 adjacent nucleotides. In
some embodiments, the P-domain comprises 3 adjacent nucleotides. In
some embodiments, the P-domain comprises 4 adjacent nucleotides. In
some embodiments, the P-domain comprises 5 adjacent nucleotides. In
some embodiments, the P-domain comprises 6 or more adjacent
nucleotides. In some embodiments, the P-domain starts 1 nucleotide
downstream of the last paired nucleotide of the duplex. In some
embodiments, the P-domain starts 2 nucleotides downstream of the
last paired nucleotide of the duplex. In some embodiments, the
P-domain starts 3 nucleotides downstream of the last paired
nucleotide of the duplex. In some embodiments, the P-domain starts
4 nucleotides downstream of the last paired nucleotide of the
duplex. In some embodiments, the P-domain starts 5 nucleotides
downstream of the last paired nucleotide of the duplex. In some
embodiments, the P-domain starts 6 or more nucleotides downstream
of the last paired nucleotide of the duplex. In some embodiments,
the mutation comprises one or more mutations. In some embodiments,
the one or more mutations are adjacent to each other. In some
embodiments, the one or more mutations are separated from each
other. In some embodiments, the mutation is adapted to allow the
engineered nucleic acid-targeting nucleic acid to hybridize to a
different protospacer adjacent motif. In some embodiments, the
different protospacer adjacent motif comprises at least 4
nucleotides. In some embodiments, the different protospacer
adjacent motif comprises at least 5 nucleotides. In some
embodiments, the different protospacer adjacent motif comprises at
least 6 nucleotides. In some embodiments, the different protospacer
adjacent motif comprises at least 7 or more nucleotides. In some
embodiments, the different protospacer adjacent motif comprises two
non-adjacent regions. In some embodiments, the different
protospacer adjacent motif comprises three non-adjacent regions. In
some embodiments, the mutation is adapted to allow the engineered
nucleic acid-targeting nucleic acid to bind to a target nucleic
acid with a lower dissociation constant than an un-engineered
nucleic acid-targeting nucleic acid. In some embodiments, the
mutation is adapted to allow the engineered nucleic acid-targeting
nucleic acid to bind to a target nucleic acid with greater
specificity than an un-engineered nucleic acid-targeting nucleic
acid. In some embodiments, the mutation is adapted to reduce
binding of the engineered nucleic acid-targeting nucleic acid to a
non-specific sequence in a target nucleic acid than an
un-engineered nucleic acid-targeting nucleic acid. In some
embodiments, the engineered nucleic acid-targeting nucleic acid
further comprises two hairpins, wherein one of the two hairpins
comprises a duplex between a polynucleotide comprising at least 50%
identity to a CRISPR RNA over 6 contiguous nucleotides, and a
polynucleotide comprising at least 50% identity to a tracrRNA over
6 contiguous nucleotides; and, wherein one of the two hairpins is
3' of the first hairpin, wherein the second hairpin comprises an
engineered P-domain. In some embodiments, the second hairpin is
adapted to de-duplex when the nucleic acid is in contact with a
target nucleic acid. In some embodiments, the P-domain is adapted
to: hybridize with a first polynucleotide, wherein the first
polynucleotide comprises a region of the engineered nucleic
acid-targeting nucleic acid, hybridize to a second polynucleotide,
wherein the second polynucleotide comprises a target nucleic acid,
and hybridize specifically to the first or second polynucleotide.
In some embodiments, the first polynucleotide comprises at least
50% identity to a tracrRNA over 6 contiguous nucleotides. In some
embodiments, the first polynucleotide is located downstream of a
duplex between a polynucleotide comprising at least 50% identity to
a CRISPR repeat over 6 contiguous nucleotides, and a polynucleotide
comprising at least 50% identity to a tracrRNA sequence over 6
contiguous nucleotides. In some embodiments, the second
polynucleotide comprises a protospacer adjcent motif. In some
embodiments, the engineered nucleic acid-targeting nucleic acid is
adapted to bind to a site-directed polypeptide. In some
embodiments, the mutation comprises an insertion of one or more
nucleotides into the P-domain. In some embodiments, the mutation
comprises deletion one or more nucleotides from the P-domain. In
some embodiments, the mutation comprises mutation of one or more
nucleotides. In some embodiments, the mutation is configured to
allow the nucleic acid-targeting nucleic acid to hybridize to a
different protospacer adjacent motif. In some embodiments, the
different protospacer adjacent motif comprises a protospacer
adjacent motif selected from the group consisting of: 5'-NGGNG-3',
5'-NNAAAAW-3,5'-NNNNGATT-3,5'-GNNNCNNA-3 and 5'-NNNACA-3', or any
combination thereof. In some embodiments, the mutation is
configured to allow the engineered nucleic acid-targeting nucleic
acid to bind with a lower dissociation constant than an
un-engineered nucleic acid-targeting nucleic acid. In some
embodiments, the mutation is configured to allow the engineered
nucleic acid-targeting nucleic acid to bind with greater
specificity than an un-engineered nucleic acid-targeting nucleic
acid. In some embodiments, the mutation is configured to reduce
binding of the engineered nucleic acid-targeting nucleic acid to a
non-specific sequence in a target nucleic acid than an
un-engineered nucleic acid-targeting nucleic acid.
[0005] In one aspect, the disclosure provides for a method for
modifying a target nucleic acid comprising contacting a target
nucleic acid with an engineered nucleic acid-targeting nucleic acid
comprising: a mutation in a P-domain of the nucleic acid-targeting
nucleic acid, and modifying the target nucleic acid. In some
embodiments, the method further comprises inserting a donor
polynucleotide into the target nucleic acid. In some embodiments,
the modifying comprises cleaving the target nucleic acid. In some
embodiments, the modifying comprises modifying transcription of the
target nucleic acid.
[0006] In one aspect the disclosure provides for a vector
comprising a polynucleotide sequence encoding an engineered nucleic
acid-targeting nucleic acid comprising: a mutation in a P-domain of
the nucleic acid-targeting nucleic acid.
[0007] In one aspect the disclosure provides for a kit comprising:
an engineered nucleic acid-targeting nucleic acid comprising: a
mutation in a P-domain of the nucleic acid-targeting nucleic acid;
and a buffer. In some embodiments, the kit further comprises a
site-directed polypeptide. In some embodiments, the kit further
comprises a donor polynucleotide. In some embodiments, the kit
further comprises instructions for use.
[0008] In one aspect, the disclosure provides for an engineered
nucleic acid-targeting nucleic acid comprising: a mutation in a
bulge region of a nucleic acid-targeting nucleic acid. In some
embodiments, the bulge is located within a duplex between a CRISPR
repeat and a tracrRNA sequence of the nucleic acid-targeting
nucleic acid. In some embodiments, the engineered nucleic
acid-targeting nucleic acid further comprises a linker sequence. In
some embodiments, the linker sequence links the CRISPR repeat and
the tracrRNA sequence. In some embodiments, the engineered nucleic
acid-targeting nucleic acid is an isolated engineered nucleic
acid-targeting nucleic acid. In some embodiments, the engineered
nucleic acid-targeting nucleic acid is a recombinant engineered
nucleic acid-targeting nucleic acid. In some embodiments, the bulge
comprises at least 1 unpaired nucleotide on the CRISPR repeat, and
1 unpaired nucleotide on a the tracrRNA sequence. In some
embodiments, the bulge comprises at least 1 unpaired nucleotide on
the CRISPR repeat, and at least 2 unpaired nucleotides on a the
tracrRNA sequence. In some embodiments, the bulge comprises at
least 1 unpaired nucleotide on the CRISPR repeat, and at least 3
unpaired nucleotides on a the tracrRNA sequence. In some
embodiments, the bulge comprises at least 1 unpaired nucleotide on
the CRISPR repeat, and at least 4 unpaired nucleotides on a the
tracrRNA sequence. In some embodiments, the bulge comprises at
least one 1 unpaired nucleotide on the CRISPR repeat, and at least
5 unpaired nucleotides on a the tracrRNA sequence. In some
embodiments, the bulge comprises at least 2 unpaired nucleotide on
the CRISPR repeat, and 1 unpaired nucleotide on a the tracrRNA
sequence. In some embodiments, the bulge comprises at least 3
unpaired nucleotide on the CRISPR repeat, and at least 2 unpaired
nucleotides on a the tracrRNA sequence. In some embodiments, the
bulge comprises at least 4 unpaired nucleotide on the CRISPR
repeat, and at least 3 unpaired nucleotides on a the tracrRNA
sequence. In some embodiments, the bulge comprises at least 5
unpaired nucleotide on the CRISPR repeat, and at least 4 unpaired
nucleotides on the tracrRNA sequence. In some embodiments, the
bulge comprises at least one nucleotide on the CRISPR repeat
adapted to form a wobble pair with at least one nucleotide on the
tracrRNA sequence. In some embodiments, the mutation comprises one
or more mutations. In some embodiments, the one or more mutations
are adjacent to each other. In some embodiments, the one or more
mutations are separated from each other. In some embodiments, the
mutation is adapted to allow the engineered nucleic acid-targeting
nucleic acid to bind to a different site-directed polypeptide. In
some embodiments, the different site-directed polypeptide is a
homologue of Cas9. In some embodiments, the different site-directed
polypeptide is a mutated version of Cas9. In some embodiments, the
different site-directed polypeptide comprises 10% amino acid
sequence identity to Cas9 in a nuclease domain selected from the
group consisting of: a RuvC nuclease domain, and a HNH nuclease
domain, or any combination thereof. In some embodiments, the
mutation is adapted to allow the engineered nucleic acid-targeting
nucleic acid to hybridize to a different protospacer adjacent
motif. In some embodiments, the mutation is adapted to allow the
engineered nucleic acid-targeting nucleic acid to bind to a
site-directed polypeptide with a lower dissociation constant than
an un-engineered nucleic acid-targeting nucleic acid. In some
embodiments, the mutation is adapted to allow the engineered
nucleic acid-targeting nucleic acid to bind to a site-directed
polypeptide with greater specificity than an un-engineered nucleic
acid-targeting nucleic acid. In some embodiments, the mutation is
adapted to allow the engineered nucleic acid-targeting nucleic acid
to direct a site-directed polypeptide to cleave a target nucleic
acid with greater specificity than an un-engineered nucleic
acid-targeting nucleic acid. In some embodiments, the mutation is
adapted to reduce binding of the engineered nucleic acid-targeting
nucleic acid to a non-specific sequence in a target nucleic acid
than an un-engineered nucleic acid-targeting nucleic acid. In some
embodiments, the engineered nucleic acid-targeting nucleic acid is
adapted to hybridize to a target nucleic acid. In some embodiments,
the mutation comprises insertion one or more nucleotides into the
bulge. In some embodiments, the mutation comprises deletion of one
or more nucleotides from the bulge. In some embodiments, the
mutation comprises mutation of one or more nucleotides. In some
embodiments, the mutation is configured to allow the engineered
nucleic acid-targeting nucleic acid to hybridize to a different
protospacer adjacent motif compared to an un-engineered nucleic
acid-targeting nucleic acid. In some embodiments, the mutation is
configured to allow the engineered nucleic acid-targeting nucleic
acid to bind to a site-directed polypeptide with a lower
dissociation constant than an un-engineered nucleic acid-targeting
nucleic acid. In some embodiments, the mutation is configured to
allow the engineered nucleic acid-targeting nucleic acid to bind to
a site-directed polypeptide with greater specificity than an
un-engineered nucleic acid-targeting nucleic acid. In some
embodiments, the mutation is configured to reduce binding of the
engineered nucleic acid-targeting nucleic acid to a non-specific
sequence in a target nucleic acid than an un-engineered nucleic
acid-targeting nucleic acid.
[0009] In one aspect the disclosure provides for a method for
modifying a target nucleic acid comprising: contacting the target
nucleic acid with an engineered nucleic acid-targeting nucleic acid
comprising: a mutation in a bulge region of a nucleic
acid-targeting nucleic acid; and modifying the target nucleic acid.
In some embodiments, the method further comprises inserting a donor
polynucleotide into the target nucleic acid. In some embodiments,
the modifying comprises cleaving the target nucleic acid. In some
embodiments, the modifying comprises modifying transcription of the
target nucleic acid.
[0010] In one aspect the disclosure provides for a vector
comprising a polynucleotide sequence encoding an engineered nucleic
acid-targeting nucleic acid comprising: a mutation in a bulge
region of a nucleic acid-targeting nucleic acid; and modifying the
target nucleic acid.
[0011] In one aspect the disclosure provides for a kit comprising:
an engineered nucleic acid-targeting nucleic acid comprising: a
mutation in a bulge region of a nucleic acid-targeting nucleic
acid; and modifying the target nucleic acid; and a buffer. In some
embodiments, the kit further comprises a site-directed polypeptide.
In some embodiments, the kit further comprises a donor
polynucleotide. In some embodiments, the kit further comprises
instructions for use.
[0012] In one aspect the disclosure provides for a method for
producing a donor polynucleotide-tagged cell comprising: cleaving a
target nucleic acid in a cell using a complex comprising a
site-directed polypeptide and a nucleic acid-targeting nucleic
acid, inserting a donor polynucleotide into a cleaved target
nucleic acid, propagating the cell carrying the donor
polynucleotide, and determining an origin of the
donor-polynucleotide tagged cell. In some embodiments, the method
is performed in vivo. In some embodiments, the method is performed
in vitro. In some embodiments, the method is performed in situ. In
some embodiments, the propagating produces a population of cells.
In some embodiments, the propagating produces a cell line. In some
embodiments, the method further comprises determining a nucleic
acid sequence of a nucleic acid in the cell. In some embodiments,
the nucleic acid sequence determines an origin of the cell. In some
embodiments, the determining comprises determining a genotype of
the cell. In some embodiments, the propagating comprises
differentiating the cell. In some embodiments, the propagating
comprises de-differentiating the cell. In some embodiments, the
propagating comprises differentiating the cell and then
dedifferentiating the cell. In some embodiments, the propagating
comprises passaging the cell. In some embodiments, the propagating
comprises inducing the cell to divide. In some embodiments, the
propagating comprises inducing the cell to enter the cell cycle. In
some embodiments, the propagating comprises the cell forming a
metastasis. In some embodiments, the propagating comprises
differentiating a pluripotent cell into a differentiated cell. In
some embodiments, the cell is a differentiated cell. In some
embodiments, the cell is a de-differentiated cell. In some
embodiments, the cell is a stem cell. In some embodiments, the cell
is a pluripotent stem cell. In some embodiments, the cell is a
eukaryotic cell line. In some embodiments, the cell is a primary
cell line. In some embodiments, the cell is a patient-derived cell
line. In some embodiments, the method further comprises
transplanting the cell into an organism. In some embodiments, the
organism is a human. In some embodiments, the organism is a mammal.
In some embodiments, the organism is selected from the group
consisting of: a human, a dog, a rat, a mouse, a chicken, a fish, a
cat, a plant, and a primate. In some embodiments, the method
further comprises selecting the cell. In some embodiments, the
donor polynucleotide is inserted into a target nucleic acid that is
expressed in one cell state. In some embodiments, the donor
polynucleotide is inserted into a target nucleic acid that is
expressed in a plurality of cell types. In some embodiments, the
donor polynucleotide is inserted into a target nucleic acid that is
expressed in a pluripotent state. In some embodiments, the donor
polynucleotide is inserted into a target nucleic acid that is
expressed in a differentiated state.
[0013] In one aspect the disclosure provides for a method for
making a clonally expanded cell line comprising: introducing into a
cell a complex comprising: a site-directed polypeptide and a
nucleic acid-targeting nucleic acid, contacting the complex to a
target nucleic acid, cleaving the target nucleic acid, wherein the
cleaving is performed by the complex, thereby producing a cleaved
target nucleic acid, inserting a donor polynucleotide into the
cleaved target nucleic acid, propagating the cell, wherein the
propagating produces the clonally expanded cell line. In some
embodiments, the cell is selected from the group consisting of:
HeLa cell, Chinese Hamster Ovary cell, 293-T cell, a
pheochromocytoma, a neuroblastomas fibroblast, a rhabdomyosarcoma,
a dorsal root ganglion cell, a NSO cell, CV-1 (ATCC CCL 70), COS-I
(ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3
(ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C 1271
(ATCC CRL 1616), BS-C-I. (ATCC CCL 26), MRC-5 (ATCC CCL 171),
L-cells, HEK-293 (ATCC CRL1 573) and PC 12 (ATCC CRL-1721), HEK293T
(ATCC CRL-11268), RBL (ATCC CRL-1378), SH-SY5Y (ATCC CRL-2266),
MDCK (ATCC CCL-34), SJ-RH30 (ATCC 2061), HepG2 (ATCC HB-8065),
ND7/23 (ECACC 92090903), CHO (ECACC 85050302), Vera (ATCC CCL 81),
Caco-2 (ATCC HTB 37), K562 (ATCC CCL 243), Jurkat (ATCC TIB-152),
Per.Co, Huvec (ATCC Human Primary PCS 100-010, Mouse CRL 2514, CRL
2515, CRL 2516), HuH-7D12 (ECACC 01042712), 293 (ATCC CRL 10852),
A549 (ATCC CCL 185), IMR-90 (ATCC CCL 186), MCF-7 (ATC HTB-22), U-2
OS (ATCC HTB-96), and T84 (ATCC CCL 248), or any combination
thereof. In some embodiments, the cell is stem cell. In some
embodiments, the cell is a differentiated cell. In some
embodiments, the cell is a pluripotent cell.
[0014] In one aspect the disclosure provides for a method for
multiplex cell type analysis comprising: cleaving at least one
target nucleic acid in two or more cells using a complex comprising
a site-directed polypeptide and a nucleic acid-targeting nucleic
acid, to create two cleaved target nucleic acids, inserting a
different a donor polynucleotide into each of the cleaved target
nucleic acids, and analyzing the two or more cells. In some
embodiments, the analyzing comprises simultaneously analyzing the
two or more cells. In some embodiments, the analyzing comprises
determining a sequence of the target nucleic acid. In some
embodiments, the analyzing comprises comparing the two or more
cells. In some embodiments, the analyzing comprises determining a
genotype of the two or more cells. In some embodiments, the cell is
a differentiated cell. In some embodiments, the cell is a
de-differentiated cell. In some embodiments, the cell is a stem
cell. In some embodiments, the cell is a pluripotent stem cell. In
some embodiments, the cell is a eukaryotic cell line. In some
embodiments, the cell is a primary cell line. In some embodiments,
the cell is a patient-derived cell line. In some embodiments, a
plurality of donor polynucleotides are inserted into a plurality of
cleaved target nucleic acids in the cell.
[0015] In one aspect, the disclosure provides for a composition
comprising: an engineered nucleic acid-targeting nucleic acid
comprising a 3' hybridizing extension, and a donor polynucleotide,
wherein the donor polynucleotide is hybridized to the 3'
hybridizing extension. In some embodiments, the 3' hybridizing
extension is adapted to hybridize to at least 5 nucleotides from
the 3' of the donor polynucleotide. In some embodiments, the 3'
hybridizing extension is adapted to hybridize to at least 5
nucleotides from the 5' of the donor polynucleotide. In some
embodiments, the 3' hybridizing extension is adapted to hybridize
to at least 5 adjacent nucleotides in the donor polynucleotide. In
some embodiments, the 3' hybridizing extension is adapted to
hybridize to all of the donor polynucleotide. In some embodiments,
the 3' hybridizing extension comprises a reverse transcription
template. In some embodiments, the reverse transcription template
is adapted to be reverse transcribed by a reverse transcriptase. In
some embodiments, the composition further comprises a reverse
transcribed DNA polynucleotide. In some embodiments, the reverse
transcribed DNA polynucleotide is adapted to hybridize to the
reverse transcription template. In some embodiments, the donor
polynucleotide is DNA. In some embodiments, the 3' hybridizing
extension is RNA. In some embodiments, the engineered nucleic
acid-targeting nucleic acid is an isolated engineered nucleic
acid-targeting nucleic acid. In some embodiments, the engineered
nucleic acid-targeting nucleic acid is a recombinant engineered
nucleic acid-targeting nucleic acid.
[0016] In one aspect the disclosure provides for a method for
introducing a donor polynucleotide into a target nucleic acid
comprising: contacting the target nucleic acid with a composition
comprising: an engineered nucleic acid-targeting nucleic acid
comprising a 3' hybridizing extension, and a donor polynucleotide,
wherein the donor polynucleotide is hybridized to the 3'
hybridizing extension. In some embodiments, the method further
comprises cleaving the target nucleic acid to produce a cleaved
target nucleic acid. In some embodiments, the cleaving is performed
by a site-directed polypeptide. In some embodiments, the method
further comprises inserting the donor polynucleotide into the
cleaved target nucleic acid.
[0017] In one aspect, the disclosure provides for a composition
comprising: an effector protein, and a nucleic acid, wherein the
nucleic acid comprises at least 50% sequence identity to a crRNA
over 6 contiguous nucleotides, at least 50% sequence identity to a
tracrRNA over 6 contiguous nucleotides; and a non-native sequence,
wherein the nucleic acid is adapted to bind to the effector
protein. In some embodiments, the composition further comprises a
polypeptide comprising at least 10% amino acid sequence identity to
a nuclease domain of Cas9, wherein the nucleic acid binds to the
polypeptide. In some embodiments, the polypeptide comprises at
least 60% amino acid sequence identity in a nuclease domain to a
nuclease domain of Cas9. In some embodiments, the polypeptide is
Cas9. In some embodiments, the nucleic acid further comprises a
linker sequence, wherein the linker sequence links the sequence
comprising at least 50% sequence identity to a crRNA over 6
contiguous nucleotides and the sequence comprising at least 50%
sequence identity to a tracrRNA over 6 contiguous nucleotides. In
some embodiments, the non-native sequence is located at a position
of the nucleic acid selected from the group consisting of: a 5' end
and a 3' end, or any combination thereof. In some embodiments, the
nucleic acid comprises two nucleic acid molecules. In some
embodiments, the nucleic acid comprises a single continuous nucleic
acid molecule. In some embodiments, the non-native sequence
comprises a CRISPR RNA-binding protein binding sequence. In some
embodiments, the non-native sequence comprises a binding sequence
selected from the group consisting of: a Cas5 RNA-binding sequence,
a Cas6 RNA-binding sequence, and a Csy4 RNA-binding sequence, or
any combination thereof. In some embodiments, the effector protein
comprises a CRISPR RNA-binding protein. In some embodiments, the
effector protein comprises at least 15% amino acid sequence
identity to a protein selected from the group consisting of: Cas5,
Cas6, and Csy4, or any combination thereof. In some embodiments, an
RNA-binding domain of the effector protein comprises at least 15%
amino acid sequence identity to an RNA-binding domain of a protein
selected from the group consisting of: Cas5, Cas6, and Csy4, or any
combination thereof. In some embodiments, the effector protein is
selected from the group consisting of: Cas5, Cas6, and Csy4, or any
combination thereof. In some embodiments, the effector protein
further comprises one or more non-native sequences. In some
embodiments, the non-native sequence confers an enzymatic activity
to the effector protein. In some embodiments, the enzymatic
activity is selected from the group consisting of:
methyltransferase activity, demethylase activity, acetylation
activity, deacetylation activity, ubiquitination activity,
deubiquitination activity, deamination activity, dismutase
activity, alkylation activity, depurination activity, oxidation
activity, pyrimidine dimer forming activity, transposase activity,
recombinase activity, polymerase activity, ligase activity,
helicase activity, photolyase activity or glycosylase activity,
acetyltransferase activity, deacetylase activity, kinase activity,
phosphatase activity, ubiquitin ligase activity, deubiquitinating
activity, adenylation activity, deadenylation activity, SUMOylating
activity, deSUMOylating activity, ribosylation activity,
deribosylation activity, myristoylation activity, remodelling
activity, protease activity, oxidoreductase activity, transferase
activity, hydrolase activity, lyase activity, isomerase activity,
synthase activity, synthetase activity, and demyristoylation
activity, or any combination thereof. In some embodiments, the
nucleic acid is RNA. In some embodiments, the effector protein
comprises a fusion protein comprising an RNA-binding protein and a
DNA-binding protein. In some embodiments, the composition further
comprises a donor polynucleotide. In some embodiments, the donor
polynucleotide is bound directly to the DNA binding protein, and
wherein the RNA binding protein is bound to the nucleic
acid-targeting nucleic acid. In some embodiments, the 5' end of the
donor polynucleotide is bound to the DNA-binding protein. In some
embodiments, the 3' end of the donor polynucleotide is bound to the
DNA-binding protein. In some embodiments, at least 5 nucleotides of
the donor polynucleotide bind to the DNA-binding protein. In some
embodiments, the nucleic acid is an isolated nucleic acid. In some
embodiments, the nucleic acid is a recombinant nucleic acid.
[0018] In one aspect, the disclosure provides for a method for
introducing a donor polynucleotide into a target nucleic acid
comprising: contacting a target nucleic acid with a complex
comprising a site-directed polypeptide and a composition
comprising: an effector protein, and a nucleic acid, wherein the
nucleic acid comprises at least 50% sequence identity to a crRNA
over 6 contiguous nucleotides, at least 50% sequence identity to a
tracrRNA over 6 contiguous nucleotides; and a non-native sequence,
wherein the nucleic acid is adapted to bind to the effector
protein. In some embodiments, the method further comprises cleaving
the target nucleic acid. In some embodiments, the cleaving is
performed by the site-directed polypeptide. In some embodiments,
the method further comprises inserting the donor polynucleotide
into the target nucleic acid.
[0019] In one aspect the disclosure provides for a method for
modulating a target nucleic acid comprising: contacting a target
nucleic acid with one or more complexes, each complex comprising a
site-directed polypeptide and a composition comprising: an effector
protein, and a nucleic acid, wherein the nucleic acid comprises at
least 50% sequence identity to a crRNA over 6 contiguous
nucleotides, at least 50% sequence identity to a tracrRNA over 6
contiguous nucleotides; and a non-native sequence, wherein the
nucleic acid is adapted to bind to the effector protein, and
modulating the target nucleic acid. In some embodiments, the
site-directed polypeptide comprises at least 10% amino acid
sequence identity to a nuclease domain of Cas9. In some
embodiments, the modulating is performed by the effector protein.
In some embodiments, the modulating comprises an activity selected
from the group consisting of: methyltransferase activity,
demethylase activity, acetylation activity, deacetylation activity,
ubiquitination activity, deubiquitination activity, deamination
activity, dismutase activity, alkylation activity, depurination
activity, oxidation activity, pyrimidine dimer forming activity,
transposase activity, recombinase activity, polymerase activity,
ligase activity, helicase activity, photolyase activity or
glycosylase activity, acetyltransferase activity, deacetylase
activity, kinase activity, phosphatase activity, ubiquitin ligase
activity, deubiquitinating activity, adenylation activity,
deadenylation activity, SUMOylating activity, deSUMOylating
activity, ribosylation activity, deribosylation activity,
myristoylation activity, remodelling activity, protease activity,
oxidoreductase activity, transferase activity, hydrolase activity,
lyase activity, isomerase activity, synthase activity, synthetase
activity, and demyristoylation activity, or any combination
thereof. In some embodiments, the effector protein comprises one or
more effector proteins.
[0020] In one aspect the disclosure provides for a method for
detecting if two complexes are in proximity to one another
comprising: contacting a first target nucleic acid with a first
complex, wherein the first complex comprises a first site-directed
polypeptide, a first modified nucleic acid-targeting nucleic acid,
and a first effector protein, wherein the effector protein is
adapted to bind to the modified nucleic acid-targeting nucleic
acid, and wherein the first effector protein comprises a non-native
sequence that comprises a first portion of a split system, and
contacting a second target nucleic acid with a second complex,
wherein the second complex comprises a second site-directed
polypeptide, a second modified nucleic acid-targeting nucleic acid,
and a second effector protein, wherein the effector protein is
adapted to bind to the modified nucleic acid-targeting nucleic
acid, and wherein the second effector protein comprises a
non-native sequence that comprises a second portion of a split
system. In some embodiments, the first target nucleic acid and the
second target nucleic acid are on the same polynucleotide polymer.
In some embodiments, the split system comprises two or more protein
fragments that individually are not active, but, when formed into a
complex, result in an active protein complex. In some embodiments,
the method further comprises detecting an interaction between the
first portion and the second portion. In some embodiments, the
detecting indicates the first and second complex are in proximity
to one another. In some embodiments, the site-directed polypeptide
is adapted to be unable to cleave the target nucleic acid. In some
embodiments, the detecting comprises determining the occurrence of
a genetic mobility event. In some embodiments, the genetic mobility
event comprises a translocation. In some embodiments, prior to the
genetic mobility event the two portions of the split system do not
interact. In some embodiments, after the genetic mobility event the
two portions of the split system do interact. In some embodiments,
the genetic mobility event is a translocation between a BCR and an
Abl gene. In some embodiments, the interaction activates the split
system. In some embodiments, the interaction indicates the target
nucleic acids bound by the complexes are close together. In some
embodiments, the split system is selected from the group consisting
of: split GFP system, a split ubiquitin system, a split
transcription factor system, and a split affinity tag system, or
any combination thereof. In some embodiments, the split system
comprises a split GFP system. In some embodiments, the detecting
indicates a genotype. In some embodiments, the method further
comprises: determining a course of treatment for a disease based on
the genotype. In some embodiments, the method further comprises
treating the disease. In some embodiments, the treating comprises
administering a drug. In some embodiments, the treating comprises
administering a complex comprising a nucleic acid-targeting nucleic
acid and a site-directed polypeptide, wherein the complex can
modify a genetic element involved in the disease. In some
embodiments, the modifying is selected from the group consisting
of: adding a nucleic acid sequence to the genetic element,
substituting a nucleic acid sequence in the genetic element, and
deleting a nucleic acid sequence from the genetic element, or any
combination thereof. In some embodiments, the method further
comprises: communicating the genotype from a caregiver to a
patient. In some embodiments, the communicating comprises
communicating from a storage memory system to a remote computer. In
some embodiments, the detecting diagnoses a disease. In some
embodiments, the method further comprises: communicating the
diagnosis from a caregiver to a patient. In some embodiments, the
detecting indicates the presence of a single nucleotide
polymorphism (SNP). In some embodiments, the method further
comprises: communicating the occurrence of a genetic mobility event
from a caregiver to a patient. In some embodiments, the
communicating comprises communicating from a storage memory system
to a remote computer. In some embodiments, the site-directed
polypeptide comprises at least 20% amino acid sequence identity to
Cas9. In some embodiments, the site-directed polypeptide comprises
at least 60% amino acid sequence identity to Cas9. In some
embodiments, the site-directed polypeptide comprises at least 60%
amino acid sequence identity in a nuclease domain to a nuclease
domain of Cas9. In some embodiments, the site-directed polypeptide
is Cas9. In some embodiments, the modified nucleic acid-targeting
nucleic acid comprises a non-native sequence. In some embodiments,
the non-native sequence is located at a position of the modified
nucleic acid-targeting nucleic acid selected from the group
consisting of: a 5' end, and a 3' end, or any combination thereof.
In some embodiments, the modified nucleic acid-targeting nucleic
acid comprises two nucleic acid molecules. In some embodiments, the
nucleic acid comprises a single continuous nucleic acid molecule
comprising a first portion comprising at least 50% identity to a
CRISPR repeat over 6 contiguous nucleotides and a second portion
comprising at least 50% identity to a tracrRNA sequence over 6
contiguous nucleotides. In some embodiments, the first portion and
the second portion are linked by a linker. In some embodiments, the
non-native sequence comprises a CRISPR RNA-binding protein binding
sequence. In some embodiments, the non-native sequence comprises a
binding sequence selected from the group consisting of: a Cas5
RNA-binding sequence, a Cas6 RNA-binding sequence, and a Csy4
RNA-binding sequence, or any combination thereof. In some
embodiments, the modified nucleic acid-targeting nucleic acid is
adapted to bind to an effector protein. In some embodiments, the
effector protein is a CRISPR RNA-binding protein. In some
embodiments, the effector protein comprises at least 15% amino acid
sequence identity to a protein selected from the group consisting
of: Cas5, Cas6, and Csy4, or any combination thereof. In some
embodiments, a RNA-binding domain of the effector protein comprises
at least 15% amino acid sequence identity to an RNA-binding domain
of a protein selected from the group consisting of: Cas5, Cas6, and
Csy4, or any combination thereof. In some embodiments, the effector
protein is selected from the group consisting of: Cas5, Cas6, and
Csy4, or any combination thereof. In some embodiments, the nucleic
acid-targeting nucleic acid is RNA. In some embodiments, the target
nucleic acid is DNA. In some embodiments, the interaction comprises
forming an affinity tag. In some embodiments, the detecting
comprises capturing the affinity tag. In some embodiments, the
method further comprises sequencing nucleic acid bound to the first
and second complexes. In some embodiments, the method further
comprises fragmenting the nucleic acid prior to the capturing. In
some embodiments, the interaction forms an activated system. In
some embodiments, the method further comprises altering
transcription of a first target nucleic acid or a second target
nucleic acid, wherein the altering is performed by the activated
system. In some embodiments, the second target nucleic acid is
unattached to the first target nucleic acid. In some embodiments,
the altering transcription of the second target nucleic acid is
performed in trans. In some embodiments, the altering transcription
of the first target nucleic acid is performed in cis. In some
embodiments, the first or second target nucleic acid is selected
from the group consisting of: an endogenous nucleic acid, and an
exogenous nucleic acid, or any combination thereof. In some
embodiments, the altering comprises increasing transcription of the
first or second target nucleic acids. In some embodiments, the
first or second target nucleic acid comprises a polynucleotide
encoding one or more genes that cause cell death. In some
embodiments, the first or second target nucleic acid comprises a
polynucleotide encoding a cell-lysis inducing peptide. In some
embodiments, the first or second target nucleic acid comprises a
polynucleotide encoding an immune-cell recruiting antigen. In some
embodiments, the first or second target nucleic acid comprises a
polynucleotide encoding one or more genes involved in apoptosis. In
some embodiments, the one or more genes involved in apoptosis
comprises caspases. In some embodiments, the one or more genes
involved in apoptosis comprises cytokines. In some embodiments, the
one or more genes involved in apoptosis are selected from the group
consisting of: tumor necrosis factor (TNF), TNF receptor 1 (TNFR1),
TNF receptor 2 (TNFR2), Fas receptor, FasL, caspase-8, caspase-10,
caspase-3, caspase-9, caspase-3, caspase-6, caspase-7, Bcl-2, and
apoptosis inducing factor (AlF), or any combination thereof. In
some embodiments, the first or second target nucleic acid comprises
a polynucleotide encoding one or more nucleic acid-targeting
nucleic acids. In some embodiments, the one or more nucleic
acid-targeting nucleic acids target a plurality of target nucleic
acids. In some embodiments, the detecting comprises generating
genetic data. In some embodiments, the method further comprises
communicating the genetic data from a storage memory system to a
remote computer. In some embodiments, the genetic data indicates a
genotype. In some embodiments, the genetic data indicates the
occurrence of a genetic mobility event. In some embodiments, the
genetic data indicates a spatial location of genes.
[0021] In one aspect, the disclosure provides for a kit comprising:
a site-directed polypeptide, a modified nucleic acid-targeting
nucleic acid, wherein the modified nucleic acid-targeting nucleic
acid comprises a non-native sequence, an effector protein that is
adapted to bind to the non-native sequence, and a buffer. In some
embodiments, the kit further comprises instructions for use.
[0022] In one aspect the disclosure provides for a vector
comprising a polynucleotide sequence encoding a modified nucleic
acid-targeting nucleic acid, wherein the modified nucleic
acid-targeting nucleic acid comprises a non-native sequence. In
some embodiments, the polynucleotide sequence is operably linked to
a promoter. In some embodiments, the promoter is an inducible
promoter.
[0023] In one aspect the disclosure provides for a vector
comprising: a polynucleotide sequence encoding: a modified nucleic
acid-targeting nucleic acid, wherein the modified nucleic
acid-targeting nucleic acid comprises a sequence configured to bind
to an effector protein, and a site-directed polypeptide. In some
embodiments, the polynucleotide sequence is operably linked to a
promoter. In some embodiments, the promoter is an inducible
promoter.
[0024] In one aspect, the disclosure provides for a vector
comprising: a polynucleotide sequence encoding: a modified nucleic
acid-targeting nucleic acid, wherein the modified nucleic
acid-targeting nucleic acid comprises a non-native sequence, a
site-directed polypeptide, and an effector protein. In some
embodiments, the polynucleotide sequence is operably linked to a
promoter. In some embodiments, the promoter is an inducible
promoter.
[0025] In one aspect the disclosure provides for a genetically
modified cell comprising a composition comprising: an effector
protein, and a nucleic acid, wherein the nucleic acid comprises at
least 50% sequence identity to a crRNA over 6 contiguous
nucleotides, at least 50% sequence identity to a tracrRNA over 6
contiguous nucleotides; and a non-native sequence, wherein the
nucleic acid is adapted to bind to the effector protein.
[0026] In one aspect the disclosure provides for a genetically
modified cell comprising a vector comprising a polynucleotide
sequence encoding a modified nucleic acid-targeting nucleic acid,
wherein the modified nucleic acid-targeting nucleic acid comprises
a non-native sequence.
[0027] In one aspect the disclosure provides for a genetically
modified cell comprising a vector comprising: a polynucleotide
sequence encoding: a modified nucleic acid-targeting nucleic acid,
wherein the modified nucleic acid-targeting nucleic acid comprises
a sequence configured to bind to an effector protein, and a
site-directed polypeptide.
[0028] In one aspect the disclosure provides for a genetically
modified cell comprising a vector comprising: a polynucleotide
sequence encoding: a modified nucleic acid-targeting nucleic acid,
wherein the modified nucleic acid-targeting nucleic acid comprises
a non-native sequence, a site-directed polypeptide, and an effector
protein.
[0029] In one aspect the disclosure provides for a kit comprising:
a vector comprising a polynucleotide sequence encoding a modified
nucleic acid-targeting nucleic acid, wherein the modified nucleic
acid-targeting nucleic acid comprises a non-native sequence, and a
buffer. In some embodiments, the kit further comprises instructions
for use.
[0030] In one aspect the disclosure provides for a kit comprising:
a vector comprising: a polynucleotide sequence encoding: a modified
nucleic acid-targeting nucleic acid, wherein the modified nucleic
acid-targeting nucleic acid comprises a sequence configured to bind
to an effector protein, and a site-directed polypeptide, and a
buffer. In some embodiments, the kit further comprises instructions
for use.
[0031] In one aspect the disclosure provides for a kit comprising:
a vector comprising: a polynucleotide sequence encoding: a modified
nucleic acid-targeting nucleic acid, wherein the modified nucleic
acid-targeting nucleic acid comprises a non-native sequence, a
site-directed polypeptide, and an effector protein, and a buffer.
In some embodiments, the kit further comprises instructions for
use.
[0032] In one aspect, the disclosure provides for a composition
comprising: a multiplexed genetic targeting agent, wherein the
multiplexed genetic targeting agent comprises one or more nucleic
acid modules, wherein the nucleic acid module comprises a
non-native sequence, and wherein the nucleic acid module is
configured to bind to a polypeptide comprising at least 10% amino
acid sequence identity to a nuclease domain of Cas9 and wherein the
nucleic acid module is configured to hybridize to a target nucleic
acid. In some embodiments, the nucleic acid module comprises a
first sequence comprising at least 50% sequence identity to a crRNA
over 6 contiguous nucleotides, and a second sequence comprising at
least 50% sequence identity to a tracrRNA over 6 contiguous
nucleotides. In some embodiments, the composition further comprises
a linker sequence that links the first and second sequences. In
some embodiments, the one or more nucleic acid modules hybridize to
one or more target nucleic acids. In some embodiments, the one or
more nucleic acid modules differ by at least one nucleotide in a
spacer region of the one or more nucleic acid modules. In some
embodiments, the one or more nucleic acid modules is RNA. In some
embodiments, the multiplexed genetic targeting agent is RNA. In
some embodiments, the non-native sequence comprises a ribozyme. In
some embodiments, the non-native sequence comprises an
endoribonuclease binding sequence. In some embodiments, the
endoribonuclease binding sequence is located at a 5' end of the
nucleic acid module. In some embodiments, the endoribonuclease
binding sequence is located at a 3' end of the nucleic acid module.
In some embodiments, the endoribonuclease binding sequence is
adapted to be bound by a CRISPR endoribonuclease. In some
embodiments, the endoribonuclease binding sequence is adapted to be
bound by an endoribonuclease comprising a RAMP domain. In some
embodiments, the endoribonuclease binding sequence is adapted to be
bound by an endoribonuclease selected from the group consisting of:
a Cas5 superfamily member endoribonuclease, and a Cas6 superfamily
member endoribonuclease, or any combination thereof. In some
embodiments, the endoribonuclease binding sequence is adapted to be
bound by an endoribonuclease comprising at least 15% amino acid
sequence identity to a protein selected from the group consisting
of: Csy4, Cas5, and Cas6. In some embodiments, the endoribonuclease
binding sequence is adapted to be bound by an endoribonuclease
comprising at least 15% amino acid sequence identity to a nuclease
domain of a protein selected from the group consisting of: Csy4,
Cas5, and Cas6. In some embodiments, the endoribonuclease binding
sequence comprises a hairpin. In some embodiments, the hairpin
comprises at least 4 consecutive nucleotides in a stem loop
structure. In some embodiments, the endoribonuclease binding
sequence comprises at least 60% identity to a sequence selected
from the group consisting of:
TABLE-US-00001 5'-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3';
5'-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3';
5'-GUUGCAAACCUCGUUAGCCUCGUAGAGGAUUGAAAC-3';
5'-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3';
5'-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3';
5'-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3';
5'-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3';
5'-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3'.
5'-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3';
5'-GUUGCAAACCUCGUUAGCCUCGUAGAGGAUUGAAAC-3';
5'-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3';
5'-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3';
5'-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3';
5'-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3';
5'-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3',
5'-GUCGCCCCCCACGCGGGGGCGUGGAUUGAAAC-3';
5'-CCAGCCGCCUUCGGGCGGCUGUGUGUUGAAAC-3';
5'-GUCGCACUCUACAUGAGUGCGUGGAUUGAAAU-3';
5'-UGUCGCACCUUAUAUAGGUGCGUGGAUUGAAAU-3'; and
5'-GUCGCGCCCCGCAUGGGGCGCGUGGAUUGAAA-3',
or any combination thereof. In some embodiments, the one or more
nucleic acid modules are adapted to be bound by different
endoribonucleases. In some embodiments, the multiplexed genetic
target agent is an isolated multiplexed genetic targeting agent. In
some embodiments, the multiplexed genetic target agent is a
recombinant multiplexed genetic target agent.
[0033] In one aspect the disclosure provides for a vector
comprising a polynucleotide sequence encoding a multiplexed genetic
targeting agent, wherein the multiplexed genetic targeting agent
comprises one or more nucleic acid modules, wherein the nucleic
acid module comprises a non-native sequence, and wherein the
nucleic acid module is configured to bind to a polypeptide
comprising at least 10% amino acid sequence identity to a nuclease
domain of Cas9 and wherein the nucleic acid module is configured to
hybridize to a target nucleic acid. In some embodiments, the
polynucleotide sequence is operably linked to a promoter. In some
embodiments, the promoter is an inducible promoter.
[0034] In one aspect, the disclosure provides for a genetically
modified cell comprising a multiplexed genetic targeting agent,
wherein the multiplexed genetic targeting agent comprises one or
more nucleic acid modules, wherein the nucleic acid module
comprises a non-native sequence, and wherein the nucleic acid
module is configured to bind to a polypeptide comprising at least
10% amino acid sequence identity to a nuclease domain of Cas9 and
wherein the nucleic acid module is configured to hybridize to a
target nucleic acid.
[0035] In one aspect the disclosure provides for a genetically
modified cell comprising a vector comprising a polynucleotide
sequence encoding a multiplexed genetic targeting agent, wherein
the multiplexed genetic targeting agent comprises one or more
nucleic acid modules, wherein the nucleic acid module comprises a
non-native sequence, and wherein the nucleic acid module is
configured to bind to a polypeptide comprising at least 10% amino
acid sequence identity to a nuclease domain of Cas9 and wherein the
nucleic acid module is configured to hybridize to a target nucleic
acid.
[0036] In one aspect the disclosure provides for a kit comprising a
multiplexed genetic targeting agent, wherein the multiplexed
genetic targeting agent comprises one or more nucleic acid modules,
wherein the nucleic acid module comprises a non-native sequence,
and wherein the nucleic acid module is configured to bind to a
polypeptide comprising at least 10% amino acid sequence identity to
a nuclease domain of Cas9 and wherein the nucleic acid module is
configured to hybridize to a target nucleic acid, and a buffer. In
some embodiments, the kit further comprises instructions for
use.
[0037] In one aspect the disclosure provides for a kit comprising:
a vector comprising a polynucleotide sequence encoding a
multiplexed genetic targeting agent, wherein the multiplexed
genetic targeting agent comprises one or more nucleic acid modules,
wherein the nucleic acid module comprises a non-native sequence,
and wherein the nucleic acid module is configured to bind to a
polypeptide comprising at least 10% amino acid sequence identity to
a nuclease domain of Cas9 and wherein the nucleic acid module is
configured to hybridize to a target nucleic acid, and a buffer. In
some embodiments, the kit further comprises instructions for
use.
[0038] In one aspect the disclosure provides for a method for
generating a nucleic acid, wherein the nucleic acid binds to a
polypeptide comprising at least 10% amino acid sequence identity to
a nuclease domain of Cas9 and hybridizes to a target nucleic acid
comprising: introducing the a multiplexed genetic targeting agent,
wherein the multiplexed genetic targeting agent comprises one or
more nucleic acid modules, wherein the nucleic acid module
comprises a non-native sequence, and wherein the nucleic acid
module is configured to bind to a polypeptide comprising at least
10% amino acid sequence identity to a nuclease domain of Cas9 and
wherein the nucleic acid module is configured to hybridize to a
target nucleic acidinto a host cell, processing the multiplexed
genetic targeting agent into the one or more nucleic acid modules,
and contacting the processed one or more nucleic acid modules to
one or more target nucleic acids in the cell. In some embodiments,
the method further comprises cleaving the target nucleic acid. In
some embodiments, the method further comprises modifying the target
nucleic acid. In some embodiments, the modifying comprises altering
transcription of the target nucleic acid. In some embodiments, the
modifying comprises inserting a donor polynucleotide into the
target nucleic acid.
[0039] In one aspect the disclosure provides for a modified
site-directed polypeptide comprising: a first nuclease domain, a
second nuclease domain, and an inserted nuclease domain. In some
embodiments, the site-directed polypeptide comprises at least 15%
identity to a nuclease domain of Cas9. In some embodiments, the
first nuclease domain comprises a nuclease domain selected from the
group consisting of: a HNH domain, and a RuvC domain, or any
combination thereof. In some embodiments, the second nuclease
domain comprises a nuclease domain selected from the group
consisting of: a HNH domain, and a RuvC domain, or any combination
thereof. In some embodiments, the inserted nuclease domain
comprises a HNH domain. In some embodiments, the inserted nuclease
domain comprises a RuvC domain. In some embodiments, the inserted
nuclease domain is N-terminal to the first nuclease domain. In some
embodiments, the inserted nuclease domain is N-terminal to the
second nuclease domain. In some embodiments, the inserted nuclease
domain is C-terminal to the first nuclease domain. In some
embodiments, the inserted nuclease domain is C-terminal to the
second nuclease domain. In some embodiments, the inserted nuclease
domain is in tandem to the first nuclease domain. In some
embodiments, the inserted nuclease domain is in tandem to the
second nuclease domain. In some embodiments, the inserted nuclease
domain is adapted to cleave a target nucleic acid at a site
different than the first or second nuclease domains. In some
embodiments, the inserted nuclease domain is adapted to cleave an
RNA in a DNA-RNA hybrid. In some embodiments, the inserted nuclease
domain is adapted to cleave a DNA in a DNA-RNA hybrid. In some
embodiments, the inserted nuclease domain is adapted to increase
specificity of binding of the modified site-directed polypeptide to
a target nucleic acid. In some embodiments, the inserted nuclease
domain is adapted to increase strength of binding of the modified
site-directed polypeptide to a target nucleic acid.
[0040] In one aspect the disclosure provides for a vector
comprising a polynucleotide sequence encoding a modified
site-directed polypeptide comprising: a first nuclease domain, a
second nuclease domain, and an inserted nuclease domain.
[0041] In one aspect the disclosure provides for a kit comprising:
a modified site-directed polypeptide comprising: a first nuclease
domain, a second nuclease domain, and an inserted nuclease domain,
and a buffer. In some embodiments, the kit further comprises
instructions for use.
[0042] In one aspect the disclosure provides for a composition
comprising: a modified site-directed polypeptide, wherein the
polypeptide is modified such that it is adapted to target a second
protospacer adjacent motif compared to a wild-type site-directed
polypeptide. In some embodiments, the site-directed polypeptide is
modified by a modification selected from the group consisting of:
an amino acid addition, an amino acid substitution, an amino acid
replacement, and an amino acid deletion, or any combination
thereof. In some embodiments, the modified site-directed
polypeptide comprises a non-native sequence. In some embodiments,
the modified site-directed polypeptide is adapted to target the
second protospacer adjacent motif with greater specificity than the
wild-type site-directed polypeptide. In some embodiments, the
modified site-directed polypeptide is adapted to target the second
protospacer adjacent motif with a lower dissociation constant
compared to the wild-type site-directed polypeptide. In some
embodiments, the modified site-directed polypeptide is adapted to
target the second protospacer adjacent motif with a higher
dissociation constant compared to the wild-type site-directed
polypeptide. In some embodiments, the second protospacer adjacent
motif comprises a protospacer adjacent motif selected from the
group consisting of: 5'-NGGNG-3',
5'-NNAAAAW-3,5'-NNNNGATT-3,5'-GNNNCNNA-3', and 5'-NNNACA-3', or any
combination thereof.
[0043] In one aspect the disclosure provides for a vector
comprising a polynucleotide sequence encoding a modified
site-directed polypeptide, wherein the polypeptide is modified such
that it is adapted to target a second protospacer adjacent motif
compared to a wild-type site-directed polypeptide.
[0044] In one aspect the disclosure provides for a kit comprising:
a modified site-directed polypeptide, wherein the polypeptide is
modified such that it is adapted to target a second protospacer
adjacent motif compared to a wild-type site-directed polypeptide,
and a buffer. In some embodiments, the kit further comprises
instructions for use.
[0045] In one aspect the disclosure provides for a composition
comprising: a modified site-directed polypeptide, wherein the
polypeptide is modified such that it is adapted to target a second
nucleic acid-targeting nucleic acid compared to a wild-type
site-directed polypeptide. In some embodiments, the site-directed
polypeptide is modified by a modification selected from the group
consisting of: an amino acid addition, an amino acid substitution,
an amino acid replacement, and an amino acid deletion, or any
combination thereof. In some embodiments, the modified
site-directed polypeptide comprises a non-native sequence. In some
embodiments, the modified site-directed polypeptide is adapted to
target the second nucleic acid-targeting nucleic acid with greater
specificity than the wild-type site-directed polypeptide. In some
embodiments, the modified site-directed polypeptide is adapted to
target the second nucleic acid-targeting nucleic acid with a lower
dissociation constant compared to the wild-type site-directed
polypeptide. In some embodiments, the modified site-directed
polypeptide is adapted to target the second nucleic acid-targeting
nucleic acid with a higher dissociation constant compared to the
wild-type site-directed polypeptide. In some embodiments, the
site-directed polypeptide targets a tracrRNA portion of the second
nucleic acid target nucleic acid.
[0046] In one aspect the disclosure provides for a vector
comprising a polynucleotide sequence encoding a modified
site-directed polypeptide, wherein the polypeptide is modified such
that it is adapted to target a second nucleic acid-targeting
nucleic acid compared to a wild-type site-directed polypeptide.
[0047] In one aspect the disclosure provides for a kit comprising:
a modified site-directed polypeptide, wherein the polypeptide is
modified such that it is adapted to target a second nucleic
acid-targeting nucleic acid compared to a wild-type site-directed
polypeptide, and a buffer. In some embodiments, the kit further
comprises instructions for use.
[0048] In one aspect the disclosure provides for a composition
comprising: a modified site-directed polypeptide comprising a
modification in a bridge helix as compared to SEQ ID: 8. In some
embodiments, the composition is configured to cleave a target
nucleic acid.
[0049] In one aspect the disclosure provides for a composition
comprising: a modified site-directed polypeptide comprising a
modification in a highly basic patch as compared to SEQ ID: 8. In
some embodiments, the composition is configured to cleave a target
nucleic acid.
[0050] In one aspect the disclosure provides for a composition
comprising: a modified site-directed polypeptide comprising a
modification in a polymerase-like domain as compared to SEQ ID: 8.
In some embodiments, the composition is configured to cleave a
target nucleic acid.
[0051] In one aspect the disclosure provides for a composition
comprising: a modified site-directed polypeptide comprising a
modification in a bridge helix, highly basic patch, nuclease
domain, and polymerase domain as compared to SEQ ID: 8, or any
combination thereof.
[0052] In one aspect the disclosure provides for a vector
comprising a polynucleotide sequence encoding a modified
site-directed polypeptide comprising a modification in a bridge
helix, highly basic patch, nuclease domain, and polymerase domain
as compared to SEQ ID: 8, or any combination thereof.
[0053] In one aspect the disclosure provides for a kit comprising:
a modified site-directed polypeptide comprising a modification in a
bridge helix, highly basic patch, nuclease domain, and polymerase
domain as compared to SEQ ID: 8, or any combination thereof, and a
buffer. In some embodiments, the kit further comprises instructions
for use. In some embodiments, the kit further comprises a nucleic
acid-targeting nucleic acid.
[0054] In one aspect the disclosure provides for a genetically
modified cell a modified site-directed polypeptide comprising a
modification in a bridge helix, highly basic patch, nuclease
domain, and polymerase domain as compared to SEQ ID: 8, or any
combination thereof.
[0055] In one aspect the disclosure provides for a method for
genome engineering comprising: contacting a target nucleic acid
with a complex, wherein the complex comprises a modified
site-directed polypeptide comprising a modification in a bridge
helix, highly basic patch, nuclease domain, and polymerase domain
as compared to SEQ ID: 8, or any combination thereoand a nucleic
acid-targeting nucleic acid, and modifying the target nucleic acid.
In some embodiments, the contacting comprises contacting the
complex to a protospacer adjacent motif in the target nucleic acid.
In some embodiments, the contacting comprises contacting the
complex to a longer target nucleic acid sequence compared to an
unmodified site-directed polypeptide. In some embodiments, the
modifying comprises cleaving the target nucleic acid. In some
embodiments, the target nucleic acid comprises RNA. In some
embodiments, the target nucleic acid comprises DNA. In some
embodiments, the modifying comprises cleaving the RNA strand of a
hybridized RNA and DNA. In some embodiments, the modifying
comprises cleaving the DNA strand of a hybridized RNA and DNA. In
some embodiments, the modifying comprises inserting into the target
nucleic acid a donor polynucleotide, a portion of a donor
polynucleotide, a copy of a donor polynucleotide, or a portion of a
copy of a donor polynucleotide, or any combination thereof. In some
embodiments, the modifying comprises modifying transcriptional
activity of the target nucleic acid. In some embodiments, the
modifying comprises a deleting of one or more nucleotides of the
target nucleic acid.
[0056] In one aspect the disclosure provides for a composition
comprising: a modified site-directed polypeptide comprising a
modified nuclease domain as compared to SEQ ID: 8. In some
embodiments, the composition is configured to cleave a target
nucleic acid. In some embodiments, the modified nuclease domain
comprises a RuvC domain nuclease domain. In some embodiments, the
modified nuclease domain comprises an HNH nuclease domain. In some
embodiments, the modified nuclease domain comprises duplication of
an HNH nuclease domain. In some embodiments, the modified nuclease
domain is adapted to increase specificity of the amino acid
sequence for a target nucleic acid compared to an unmodified
site-directed polypeptide. In some embodiments, the modified
nuclease domain is adapted to increase specificity of the amino
acid sequence for a nucleic acid-targeting nucleic acid compared to
an unmodified site-directed polypeptide. In some embodiments, the
modified nuclease domain comprises a modification selected from the
group consisting of: an amino acid addition, an amino acid
substitution, an amino acid replacement, and an amino acid
deletion, or any combination thereof. In some embodiments, the
modified nuclease domain comprises an inserted non-native sequence.
In some embodiments, the non-native sequence confers an enzymatic
activity to the modified site-directed polypeptide. In some
embodiments, the enzymatic activity is selected from the group
consisting of: nuclease activity, methylase activity, acetylase
activity, demethylase activity, deamination activity, dismutase
activity, alkylation activity, depurination activity, oxidation
activity, pyrimidine dimer forming activity, integrase activity,
transposase activity, recombinase activity, polymerase activity,
ligase activity, helicase activity, photolyase activity or
glycosylase activity, acetyltransferase activity, deacetylase
activity, kinase activity, phosphatase activity, ubiquitin ligase
activity, deubiquitinating activity, adenylation activity,
deadenylation activity, SUMOylating activity, deSUMOylating
activity, ribosylation activity, deribosylation activity,
myristoylation activity, remodelling activity, protease activity,
oxidoreductase activity, transferase activity, hydrolase activity,
lyase activity, isomerase activity, synthase activity, synthetase
activity, and demyristoylation activity, or any combination
thereof. In some embodiments, the enzymatic activity is adapted to
modulate transcription of a target nucleic acid. In some
embodiments, the modified nuclease domain is adapted to allow
binding of the amino acid sequence to a protospacer adjacent motif
sequence that is different from a protospacer adjacent motif
sequence to which an unmodified site-directed polypeptide is
adapted to bind. In some embodiments, the modified nuclease domain
is adapted to allow binding of the amino acid sequence to a nucleic
acid-targeting nucleic acid that is different from a nucleic
acid-targeting nucleic acid to which an unmodified site-directed
polypeptide is adapted to bind. In some embodiments, the modified
site-directed polypeptide is adapted to bind to a longer target
nucleic acid sequence than an unmodified site-directed polypeptide.
In some embodiments, the modified site-directed polypeptide is
adapted to cleave double-stranded DNA. In some embodiments, the
modified site-directed polypeptide is adapted to cleave the RNA
strand of a hybridized RNA and DNA. In some embodiments, the
modified site-directed polypeptide is adapted to cleave the DNA
strand of a hybridized RNA and DNA. In some embodiments, the
composition further comprises a modified nucleic acid-targeting
nucleic acid, wherein the modification of the site-directed
polypeptide is adapted to enable the site-directed polypeptide to
bind to the modified nucleic acid-targeting nucleic acid. In some
embodiments, the modified nucleic acid-targeting nucleic acid and
the modified site-directed polypeptide comprise compensatory
mutations.
[0057] In one aspect the disclosure provides for a method for
enriching a target nucleic acid for sequencing comprising:
contacting a target nucleic acid with a complex comprising a
nucleic acid-targeting nucleic acid and a site-directed
polypeptide, enriching the target nucleic acid using the complex,
and determining a sequence of the target nucleic acid. In some
embodiments, the method does not comprise an amplification step. In
some embodiments, the method further comprises analyzing the
sequence of the target nucleic acid. In some embodiments, the
method further comprises fragmenting the target nucleic acid prior
to the enriching. In some embodiments, the nucleic acid-targeting
nucleic acid comprises RNA. In some embodiments, the method the
nucleic acid-targeting nucleic acid comprises two RNA molecules. In
some embodiments, the method a portion of each of the two RNA
molecules hybridize together. In some embodiments, the method one
of the two RNA molecules comprises a CRISPR repeat sequence. In
some embodiments, the CRISPR repeat sequence is homologous to a
crRNA over 6 contiguous nucleotides. In some embodiments, the
CRISPR repeat sequence comprises at least 60% identity to a crRNA
over 6 contiguous nucleotides. In some embodiments, the one of the
two RNA molecules comprises a tracrRNA sequence. In some
embodiments, the tracRNA sequence is homologous to a tracrRNA over
6 contiguous nucleotides. In some embodiments, the tracRNA sequence
comprises at least 60% identity to a tracrRNA over 6 contiguous
nucleotides. In some embodiments, the nucleic acid-targeting
nucleic acid is a double guide nucleic acid. In some embodiments,
the nucleic acid-targeting nucleic acid comprises one continuous
RNA molecule wherein the continuous RNA molecule further comprises
two domains and a linker. In some embodiments, a portion of each of
the two domains of the continuous RNA molecule hybridize together.
In some embodiments, the continuous RNA molecule comprises a CRISPR
repeat sequence. In some embodiments, the CRISPR repeat sequence is
homologous to a crRNA over 6 contiguous nucleotides. In some
embodiments, the CRISPR repeat sequence comprises at least 60%
identity to a crRNA over 6 contiguous nucleotides. In some
embodiments, the continuous RNA molecule comprises a tracrRNA
sequence. In some embodiments, the tracRNA sequence is homologous
to a tracrRNA over 6 contiguous nucleotides. In some embodiments,
the tracRNA sequence comprises at least 60% identity to a tracrRNA
over 6 contiguous nucleotides. In some embodiments, the nucleic
acid-targeting nucleic acid is a single guide nucleic acid. In some
embodiments, the contacting comprises hybridizing a portion of the
nucleic acid-targeting nucleic acid with a portion of the target
nucleic acid. In some embodiments, the nucleic acid-targeting
nucleic acid hybridizes with the target nucleic acid over a region
comprising 6-20 nucleotides. In some embodiments, the site-directed
polypeptide comprises Cas9. In some embodiments, the site-directed
polypeptide comprises at least 20% homology to a nuclease domain of
Cas9. In some embodiments, the site-directed polypeptide comprises
at least 60% homology to Cas9. In some embodiments, the
site-directed polypeptide comprises an engineered nuclease domain
wherein the nuclease domain comprises reduced nuclease activity
compared to a site-directed polypeptide that comprises an
unengineered nuclease domain. In some embodiments, the
site-directed polypeptide introduces a single-strand break in the
target nucleic acid. In some embodiments, the engineered nuclease
domain comprises mutation of a conserved aspartic acid. In some
embodiments, the engineered nuclease domain comprises a D10A
mutation. In some embodiments, the engineered nuclease domain
comprises mutation of a conserved histidine. In some embodiments,
the engineered nuclease domain comprises a H840A mutation. In some
embodiments, the site-directed polypeptide comprises an affinity
tag. In some embodiments, the affinity tag is located at the
N-terminus of the site-directed polypeptide, the C-terminus of the
site-directed polypeptide, a surface-accessible region, or any
combination thereof. In some embodiments, the affinity tag is
selected from a group comprising: biotin, FLAG, His6.times.,
His9.times., and a fluorescent protein, or any combination thereof.
In some embodiments, the nucleic acid-targeting nucleic acid
comprises a nucleic acid affinity tag. In some embodiments, the
nucleic acid affinity tag is located at the 5' end of the nucleic
acid-targeting nucleic acid, the 3' end of the nucleic
acid-targeting nucleic acid, a surface-accessible region, or any
combination thereof. In some embodiments, the nucleic acid affinity
tag is selected from the group comprising a small molecule,
fluorescent label, a radioactive label, or any combination thereof.
In some embodiments, the nucleic acid affinity tag comprises a
sequence that is configured to bind to Csy4, Cas5, Cas6, or any
combination thereof. In some embodiments, the nucleic acid affinity
tag comprises 50% identity to 5'-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3'.
In some embodiments, the method further comprises diagnosing a
disease and making a patient-specific treatment decision, or any
combination thereof. In some embodiments, the determining comprises
determining a genotype. In some embodiments, the method further
comprises communicating the sequence from a storage memory system
to a remote computer. In some embodiments, the enriching comprises
contacting an affinity tag of the complex with a capture agent. In
some embodiments, the capture agent comprises an antibody. In some
embodiments, the capture agent comprises a solid support. In some
embodiments, the capture agent is selected from the group
comprising: Csy4, Cas5, and Cas6. In some embodiments, the the
capture agent comprises reduced enzymatic activity in the absence
of imidazole. In some embodiments, the capture agent comprises an
activatable enzymatic domain, wherein the activatable enzymatic
domain is activated by coming in contact with imidazole. In some
embodiments, the capture agent is a Cas6 family member. In some
embodiments, the capture agent comprises an affinity tag. In some
embodiments, the capture agent comprises a conditionally
enzymatically inactive endoribonuclease comprising a mutation in a
nuclease domain. In some embodiments, the mutation of a conserved
histidine. In some embodiments, the mutation comprises a H29A
mutation. In some embodiments, the target nucleic acid is bound to
the complex. In some embodiments, the target nucleic acid is an
excised nucleic acid that is not bound to the complex. In some
embodiments, a plurality of complexes are contacted to a plurality
of target nucleic acids. In some embodiments, the plurality of
target nucleic acids differ by at least one nucleotide. In some
embodiments, the plurality of complexes comprise a plurality of
nucleic acid-targeting nucleic acids that differ by at least one
nucleotide.
[0058] In one aspect the disclosure provides for a method for
excising a nucleic acid comprising: contacting a target nucleic
acid with two or more complexes, wherein each complex comprises a
site-directed polypeptide and a nucleic acid-targeting nucleic
acid, and cleaving the target nucleic acid, wherein the cleaving
produces an excised target nucleic acid. In some embodiments, the
cleaving is performed by a nuclease domain of the site-directed
polypeptide. In some embodiments, the method does not comprise
amplification. In some embodiments, the method further comprises
enriching the excised target nucleic acid. In some embodiments, the
method further comprises sequencing the excised target nucleic
acid. In some embodiments, the nucleic acid-targeting nucleic acid
is RNA. In some embodiments, the nucleic acid-targeting nucleic
acid comprises two RNA molecules. In some embodiments, a portion of
each of the two RNA molecules hybridize together. In some
embodiments, one of the two RNA molecules comprises a CRISPR repeat
sequence. In some embodiments, the CRISPR repeat sequence comprises
a sequence that is homologous to a crRNA over 6 contiguous
nucleotides. In some embodiments, the CRISPR repeat sequence
comprises a sequence that has at least 60% identity to a crRNA over
6 contiguous nucleotides. In some embodiments, one of the two RNA
molecules comprises a tracrRNA sequence. In some embodiments, the
tracRNA sequence is homologous to a crRNA over 6 contiguous
nucleotides. In some embodiments, the tracRNA sequence comprises at
least 60% identity to a crRNA over 6 contiguous nucleotides. In
some embodiments, the nucleic acid-targeting nucleic acid is a
double guide nucleic acid. In some embodiments, the nucleic
acid-targeting nucleic acid comprises one continuous RNA molecule
wherein the continuous RNA molecule further comprises two domains
and a linker. In some embodiments, a portion of each of the two
domains of the continuous RNA molecule hybridize together. In some
embodiments, the continuous RNA molecule comprises a CRISPR repeat
sequence. In some embodiments, the CRISPR repeat sequence is
homologous to a crRNA over 6 contiguous nucleotides. In some
embodiments, the CRISPR repeat sequence comprises at least 60%
identity to a crRNA over 6 contiguous nucleotides. In some
embodiments, the continuous RNA molecule comprises a tracrRNA
sequence. In some embodiments, the tracRNA sequence is homologous
to a crRNA over 6 contiguous nucleotides. In some embodiments, the
tracRNA sequence comprises at least 60% identity to a crRNA over 6
contiguous nucleotides. In some embodiments, the nucleic
acid-targeting nucleic acid is a single guide nucleic acid. In some
embodiments, the nucleic acid-targeting nucleic acid hybridizes
with a target nucleic acid. In some embodiments, the nucleic
acid-targeting nucleic acid hybridizes with a target nucleic acid
over a region, wherein the region comprises at least 6 nucleotides
and at most 20 nucleotides. In some embodiments, the site-directed
polypeptide is Cas9. In some embodiments, the site-directed
polypeptide comprises a polypeptide comprising at least 20%
homology to a nuclease domain of Cas9. In some embodiments, the
site-directed polypeptide comprises a polypeptide comprising at
least 60% homology to Cas9. In some embodiments, the site-directed
polypeptide comprises an affinity tag. In some embodiments, the
affinity tag is located at the N-terminus of the site-directed
polypeptide, the C-terminus of the site-directed polypeptide, a
surface-accessible region, or any combination thereof. In some
embodiments, the affinity tag is selected from a group comprising:
biotin, FLAG, His6.times., His9.times., and a fluorescent protein,
or any combination thereof. In some embodiments, the nucleic
acid-targeting nucleic acid comprises a nucleic acid affinity tag.
In some embodiments, the nucleic acid affinity tag is located at
the 5' end of the nucleic acid-targeting nucleic acid, the 3' end
of the nucleic acid-targeting nucleic acid, a surface-accessible
region, or any combination thereof. In some embodiments, the
nucleic acid affinity tag is selected from the group comprising a
small molecule, fluorescent label, a radioactive label, or any
combination thereof. In some embodiments, the nucleic acid affinity
tag is a sequence that can bind to Csy4, Cas5, Cas6, or any
combination thereof. In some embodiments, the nucleic acid affinity
tag comprises 50% identity to GUUCACUGCCGUAUAGGCAGCUAAGAAA. In some
embodiments, the target nucleic acid is an excised nucleic acid
that is not bound to the two or more complexes. In some
embodiments, the two or more complexes are contacted to a plurality
of target nucleic acids. In some embodiments, the plurality of
target nucleic acids differ by at least one nucleotide. In some
embodiments, the two or more complexes comprise nucleic
acid-targeting nucleic acids that differ by at least one
nucleotide.
[0059] In one aspect the disclosure provides for a method for
generating a library of target nucleic acids comprising: contacting
a plurality of target nucleic acids with a complex comprising a
site-directed polypeptide and a nucleic acid-targeting nucleic
acid, cleaving the plurality of target nucleic acids, and purifying
the plurality of target nucleic acids to create the library of
target nucleic acids. In some embodiments, the method further
comprises screening the library of target nucleic acids.
[0060] In one aspect the disclosure provides for a composition
comprising: a first complex comprising: a first site-directed
polypeptide and a first nucleic acid-targeting nucleic acid, a
second complex comprising: a second site-directed polypeptide and a
second nucleic acid-targeting nucleic acid, wherein, the first and
second nucleic acid-targeting nucleic acids are different. In some
embodiments, the composition further comprises a target nucleic
acid, which is bound by the first or the second complex. In some
embodiments, the first site-directed polypeptide and the second
site-directed polypeptide are the same. In some embodiments, the
first site-directed polypeptide and the second site-directed
polypeptide are different.
[0061] In one aspect the disclosure provides for a vector
comprising a polynucleotide sequence encoding: two or more nucleic
acid-targeting nucleic acids that differ by at least one
nucleotide, and a site-directed polypeptide.
[0062] In one aspect the disclosure provides a genetically modified
host cell comprising: a vector comprising a polynucleotide sequence
encoding: two or more nucleic acid-targeting nucleic acids that
differ by at least one nucleotide, and a site-directed
polypeptide.
[0063] In one aspect the disclosure provides a kit comprising: a
vector comprising a polynucleotide sequence encoding: two or more
nucleic acid-targeting nucleic acids that differ by at least one
nucleotide, a site-directed polypeptide, and a suitable buffer. In
some embodiments, the kit further comprises: a capture agent, a
solid support, sequencing adaptors, and a positive control, or any
combination thereof. In some embodiments, the kit further comprises
instructions for use.
[0064] In one aspect the disclosure provides for a kit comprising:
a site-directed polypeptide comprising reduced enzymatic activity
compared to a wild-type site-directed polypeptide, a nucleic
acid-targeting nucleic acid, and a capture agent. In some
embodiments, the kit further comprises: instructions for use. In
some embodiments, the kit further comprises a buffer selected from
the group comprising: a wash buffer, a stabilization buffer, a
reconstituting buffer, or a diluting buffer.
[0065] In one aspect the disclosure provides for a method for
cleaving a target nucleic acid using two or more nickases
comprising: contacting a target nucleic acid with a first complex
and a second complex, wherein the first complex comprises a first
nickase and a first nucleic acid-targeting nucleic acid, and
wherein the second complex comprises a second nickase and a second
nucleic acid-targeting nucleic acid, wherein the target nucleic
acid comprises a first protospacer adjacent motif on a first strand
and a second protospacer adjacent motif on a second strand, wherein
the first nucleic acid-targeting nucleic acids is adapted to
hybridize to the first protospacer adjacent motif, and wherein the
second nucleic acid-targeting nucleic acid is adapted to hybridize
to the second protospacer adjacent motif, and nicking the first and
second strands of the target nucleic acid, wherein the nicking
generates a cleaved target nucleic acid. In some embodiments, the
first and second nickase are the same. In some embodiments, the
first and second nickase are different. In some embodiments, the
first and second nucleic acid-targeting nucleic acid are different.
In some embodiments, there are less than 125 nucleotides between
the first protospacer adjacent motif and the second protospacer
adjacent motif. In some embodiments, the first and second
protospacer adjacent motifs comprise of the sequence NGG, where N
is any nucleotide. In some embodiments, the first or second nickase
comprises at least one substantially inactive nuclease domain. In
some embodiments, the first or second nickase comprises a mutation
of a conserved aspartic acid. In some embodiments, the mutation is
a D10A mutation. In some embodiments, the first or second nickase
comprises a mutation of a conserved histidine. In some embodiments,
the mutation is a H840A mutation. In some embodiments, there are
less than 15 nucleotides between the first and second protospacer
adjacent motifs. In some embodiments, there are less than 10
nucleotides between the first and second protospacer adjacent
motifs. In some embodiments, there are less than 5 nucleotides
between the first and second protospacer adjacent motifs. In some
embodiments, the first and second protospacer adjacent motifs are
adjacent to one another. In some embodiments, the nicking comprises
the first nickase nicking the first strand and the second nickase
nicking the second strand. In some embodiments, the nicking
generates a sticky end cut. In some embodiments, the nicking
generates a blunt end cut. In some embodiments, the method further
comprises inserting a donor polynucleotide into the cleaved target
nucleic acid.
[0066] In one aspect the disclosure provides for a composition
comprising: a plurality of nucleic acid molecules, wherein each
nucleic acid molecule comprises a nucleic acid-binding protein
binding site, wherein at least one of the plurality of nucleic acid
molecules encodes for a nucleic acid-targeting nucleic acid and one
of the plurality of nucleic acid molecules encodes for a
site-directed polypeptide, and a fusion polypeptide, wherein the
fusion polypeptide comprises a plurality of the nucleic
acid-binding proteins, wherein the plurality of nucleic
acid-binding proteins are adapted to bind to their cognate nucleic
acid-binding protein binding site. In some embodiments, one or more
of the plurality of nucleic acid-binding proteins comprise a
non-native sequence. In some embodiments, the non-native sequence
is located at a position selected from the group consisting of: the
N-terminus, the C-terminus, a surface accessible region, or any
combination thereof. In some embodiments, the non-native sequence
encodes for a nuclear localization signal. In some embodiments, the
plurality of nucleic acid-binding proteins are separated by a
linker. In some embodiments, some of the plurality of nucleic
acid-binding proteins are the same nucleic acid-binding protein. In
some embodiments, all of the plurality of nucleic acid-binding
proteins are the same nucleic acid-binding protein. In some
embodiments, the plurality of nucleic acid-binding proteins are
different nucleic acid-binding proteins. In some embodiments, the
plurality of nucleic acid-binding proteins comprise RNA-binding
proteins. In some embodiments, the RNA-binding proteins are
selected from the group consisting of: a Type I Clustered Regularly
Interspaced Short Palindromic Repeat system endoribonuclease, a
Type II Clustered Regularly Interspaced Short Palindromic Repeat
system endoribonuclease, or a Type III Clustered Regularly
Interspaced Short Palindromic Repeat system endoribonuclease, or
any combination thereof. In some embodiments, the RNA-binding
proteins are selected from the group consisting of: Cas5, Cas6, and
Csy4, or any combination thereof. In some embodiments, the
plurality of nucleic acid-binding proteins comprise DNA-binding
proteins. In some embodiments, the nucleic acid-binding protein
binding site is configured to bind a nucleic acid-binding protein
selected from the group consisting of: Type I, Type II, and Type
III Clustered Regularly Interspaced Short Palindromic Repeat system
nucleic acid-binding protein, or any combination thereof. In some
embodiments, the nucleic acid-binding protein binding site is
configured to bind a nucleic acid-binding protein selected from the
group consisting of: Cas6, Cas5, and Csy4, or any combination
thereof. In some embodiments, some of the plurality of nucleic acid
molecules comprise the same nucleic acid-binding protein binding
site. In some embodiments, the plurality of nucleic acid molecules
comprise the same nucleic acid-binding protein binding site. In
some embodiments, the none of the plurality of nucleic acid
molecules comprise the same nucleic acid-binding protein binding
site. In some embodiments, the site-directed polypeptide comprises
at least 20% sequence identity to a nuclease domain of Cas9. In
some embodiments, the site-directed polypeptide is Cas9. In some
embodiments, at least one of the nucleic acid molecules encodes for
a Clustered Regularly Interspaced Short Palindromic Repeat
endoribonuclease. In some embodiments, the Clustered Regularly
Interspaced Short Palindromic Repeat endoribonuclease comprises at
least 20% sequence similarity to Csy4. In some embodiments, the
Clustered Regularly Interspaced Short Palindromic Repeat
endoribonuclease comprises at least 60% sequence similarity to
Csy4. In some embodiments, the Clustered Regularly Interspaced
Short Palindromic Repeat endoribonuclease is Csy4. In some
embodiments, the plurality of nucleic acid-binding proteins
comprise reduced enzymatic activity. In some embodiments, the
plurality of nucleic acid-binding proteins are adapted to bind to
the nucleic acid-binding protein binding site but cannot cleave the
nucleic acid-binding protein binding site. In some embodiments, the
nucleic acid-targeting nucleic acid comprises two RNA molecules. In
some embodiments, a portion of each of the two RNA molecules
hybridize together. In some embodiments, a first molecule of the
two RNA molecules comprises a sequence comprising at least 60%
identity to a Clustered Regularly Interspaced Short Palindromic
Repeat RNA sequence over 8 contiguous nucleotides, and wherein a
second molecule of the two RNA molecules comprises a sequence
comprising at least 60% identity to a trans-activating-Clustered
Regularly Interspaced Short Palindromic Repeat RNA sequence over 6
contiguous nucleotides. In some embodiments, the nucleic
acid-targeting nucleic acid comprises one continuous RNA molecule
wherein the continuous RNA molecule further comprises two domains
and a linker. In some embodiments, a portion of the two domains of
the continuous RNA molecule hybridize together. In some
embodiments, a first portion of the continuous RNA molecule
comprises a sequence comprising at least 60% identity to a
Clustered Regularly Interspaced Short Palindromic Repeat RNA
sequence over 8 contiguous nucleotides, and wherein a second
portion of the continuous RNA molecule comprises a sequence
comprising at least 60% identity to a trans-activating-Clustered
Regularly Interspaced Short Palindromic Repeat RNA sequence over 6
contiguous nucleotides. In some embodiments, the nucleic acid
targeting nucleic acid is adapted to hybridize with a target
nucleic acid over 6-20 nucleotides. In some embodiments, the
composition is configured to be delivered to a cell. In some
embodiments, the composition is configured to deliver equal amounts
of the plurality of nucleic acid molecules to a cell. In some
embodiments, the composition further comprises a donor
polynucleotide molecule, wherein the donor polynucleotide molecule
comprises a nucleic acid-binding protein binding site, wherein the
binding site is bound by a nucleic acid-binding protein of the
fusion polypeptide.
[0067] In one aspect the disclosure provides for a method for
delivery of nucleic acids to a subcellular location in a cell
comprising: introducing into a cell a composition comprising: a
plurality of nucleic acid molecules, wherein each nucleic acid
molecule comprises a nucleic acid-binding protein binding site,
wherein at least one of the plurality of nucleic acid molecules
encodes for a nucleic acid-targeting nucleic acid and one of the
plurality of nucleic acid molecules encodes for a site-directed
polypeptide, and a fusion polypeptide, wherein the fusion
polypeptide comprises a plurality of the nucleic acid-binding
proteins, wherein the plurality of nucleic acid-binding proteins
are adapted to bind to their cognate nucleic acid-binding protein
binding sitestoichiometrically delivering the composition to the
subcellular location, forming a unit comprising a site-directed
polypeptide translated from the nucleic acid molecule encoding for
a site-directed polypeptide and the nucleic acid-targeting nucleic
acid, and cleaving a target nucleic acid, wherein the site-directed
polypeptide of the unit cleaves the target nucleic acid. In some
embodiments, the plurality of nucleic acid-binding proteins bind to
their cognate nucleic acid-binding protein binding site. In some
embodiments, an endoribonuclease cleaves one of the one or more
nucleic acid-binding protein binding sites. In some embodiments, an
endoribonuclease cleaves the nucleic acid-binding protein binding
sites of the nucleic acid encoding the nucleic acid-targeting
nucleic acid, thereby liberating the nucleic acid-targeting nucleic
acid. In some embodiments, the subcellular location is selected
from the group consisting of: the nuclease, the ER, the golgi, the
mitochondria, the cell wall, the lysosome, and the nucleus. In some
embodiments, the subcellular location is the nucleus.
[0068] In one aspect the disclosure provides for a vector
comprising: a polynucleotide sequence encoding a composition
comprising: a plurality of nucleic acid molecules, wherein each
nucleic acid molecule comprises a nucleic acid-binding protein
binding site, wherein at least one of the plurality of nucleic acid
molecules encodes for a nucleic acid-targeting nucleic acid and one
of the plurality of nucleic acid molecules encodes for a
site-directed polypeptide; and a fusion polypeptide, wherein the
fusion polypeptide comprises a plurality of the nucleic
acid-binding proteins, wherein the plurality of nucleic
acid-binding proteins are adapted to bind to their cognate nucleic
acid-binding protein binding site stoichiometrically delivering the
composition to the subcellular location. In some embodiments, the
vector further comprises a polynucleotide encoding a promoter. In
some embodiments, the promoter is operably linked to the
polynucleotide. In some embodiments, the promoter is an inducible
promoter.
[0069] In one aspect the disclosure provides for a genetically
modified organism comprising a vector comprising: a polynucleotide
sequence encoding for a plurality of nucleic acid molecules,
wherein each nucleic acid molecule comprises a nucleic acid-binding
protein binding site, wherein at least one of the plurality of
nucleic acid molecules encodes for a nucleic acid-targeting nucleic
acid and one of the plurality of nucleic acid molecules encodes for
a site-directed polypeptide, and a fusion polypeptide, wherein the
fusion polypeptide comprises a plurality of the nucleic
acid-binding proteins, wherein the plurality of nucleic
acid-binding proteins are adapted to bind to their cognate nucleic
acid-binding protein binding site stoichiometrically delivering the
composition to the subcellular location.
[0070] In one aspect the disclosure provides for a genetically
modified organism comprising: a composition comprising: a plurality
of nucleic acid molecules, wherein each nucleic acid molecule
comprises a nucleic acid-binding protein binding site, wherein at
least one of the plurality of nucleic acid molecules encodes for a
nucleic acid-targeting nucleic acid and one of the plurality of
nucleic acid molecules encodes for a site-directed polypeptide; and
a fusion polypeptide, wherein the fusion polypeptide comprises a
plurality of the nucleic acid-binding proteins, wherein the
plurality of nucleic acid-binding proteins are adapted to bind to
their cognate nucleic acid-binding protein binding site.
[0071] In one aspect the disclosure provides for a kit comprising:
a composition comprising: a plurality of nucleic acid molecules,
wherein each nucleic acid molecule comprises a nucleic acid-binding
protein binding site, wherein at least one of the plurality of
nucleic acid molecules encodes for a nucleic acid-targeting nucleic
acid and one of the plurality of nucleic acid molecules encodes for
a site-directed polypeptide, and a fusion polypeptide, wherein the
fusion polypeptide comprises a plurality of the nucleic
acid-binding proteins, wherein the plurality of nucleic
acid-binding proteins are adapted to bind to their cognate nucleic
acid-binding protein binding site, and a buffer.
[0072] In one aspect the disclosure provides for a kit comprising:
a vector comprising: a polynucleotide sequence encoding for a
plurality of nucleic acid molecules, wherein each nucleic acid
molecule comprises a nucleic acid-binding protein binding site,
wherein at least one of the plurality of nucleic acid molecules
encodes for a nucleic acid-targeting nucleic acid and one of the
plurality of nucleic acid molecules encodes for a site-directed
polypeptide; and a fusion polypeptide, wherein the fusion
polypeptide comprises a plurality of the nucleic acid-binding
proteins, wherein the plurality of nucleic acid-binding proteins
are adapted to bind to their cognate nucleic acid-binding protein
binding site stoichiometrically delivering the composition to the
subcellular location, and a buffer. In some embodiments, the kit
further comprises instructions for use. In some embodiments, the
buffer is selected from the group comprising: a dilution buffer, a
reconstitution buffer, and a stabilization buffer, or any
combination thereof.
[0073] In one aspect the disclosure provides for a donor
polynucleotide comprising: a genetic element of interest, and a
reporter element, wherein the reporter element comprises a
polynucleotide sequence encoding a site-directed polypeptide, and
one or more a nucleic acids, wherein the one or more nucleic acids
comprises a sequence comprising at least 50% sequence identity to a
crRNA over 6 contiguous nucleotides and a sequence comprising at
least 50% sequence identity to a tracrRNA over 6 contiguous
nucleotides. In some embodiments, the genetic element of interest
comprises a gene. In some embodiments, the genetic element of
interest comprises a non-coding nucleic acid selected from the
group consisting of: a microRNA, a siRNA, and a long non-coding
RNA, or any combination thereof. In some embodiments, the genetic
element of interest comprises a non-coding gene. In some
embodiments, the genetic element of interest comprises a non-coding
nucleic acid selected from the group consisting of: a microRNA, a
siRNA, and a long non-coding RNA, or any combination thereof. In
some embodiments, the reporter element comprises a gene selected
from the group consisting of: a gene encoding a fluorescent
protein, a gene encoding a chemiluminescent protein, and an
antibiotic resistance gene, or any combination thereof. In some
embodiments, the reporter element comprises a gene encoding a
fluorescent protein. In some embodiments, the fluorescent protein
comprises green fluorescent protein. In some embodiments, the
reporter element is operably linked to a promoter. In some
embodiments, the promoter comprises an inducible promoter. In some
embodiments, the promoter comprises a tissue-specific promoter. In
some embodiments, the site-directed polypeptide comprises at least
15% amino acid sequence identity to a nuclease domain of Cas9. In
some embodiments, the site-directed polypeptide comprises at least
95% amino acid sequence identity over 10 amino acids to Cas9. In
some embodiments, the nuclease domain is selected from the group
consisting of: an HNH domain, an FINE-like domain, a RuvC domain,
and a RuvC-like domain, or any combination thereof.
[0074] In one aspect the disclosure provides for an expression
vector comprising a polynucleotide sequence encoding for a genetic
element of interest; and a reporter element, wherein the reporter
element comprises a polynucleotide sequence encoding a
site-directed polypeptide, and one or more a nucleic acids, wherein
the one or more nucleic acids comprises a sequence comprising at
least 50% sequence identity to a crRNA over 6 contiguous
nucleotides and a sequence comprising at least 50% sequence
identity to a tracrRNA over 6 contiguous nucleotides.
[0075] In one aspect the disclosure provides for a genetically
modified cell comprising a donor polynucleotide comprising: a
genetic element of interest; and a reporter element, wherein the
reporter element comprises a polynucleotide sequence encoding a
site-directed polypeptide, and one or more a nucleic acids, wherein
the one or more nucleic acids comprises a sequence comprising at
least 50% sequence identity to a crRNA over 6 contiguous
nucleotides and a sequence comprising at least 50% sequence
identity to a tracrRNA over 6 contiguous nucleotides.
[0076] In one aspect the disclosure provides for a kit comprising:
a donor polynucleotide comprising: a genetic element of interest;
and a reporter element, wherein the reporter element comprises a
polynucleotide sequence encoding a site-directed polypeptide, and
one or more a nucleic acids, wherein the one or more nucleic acids
comprises a sequence comprising at least 50% sequence identity to a
crRNA over 6 contiguous nucleotides and a sequence comprising at
least 50% sequence identity to a tracrRNA over 6 contiguous
nucleotides; and a buffer. In some embodiments, the kit further
comprises: a polypeptide comprising at least 10% amino acid
sequence identity to Cas9; and a nucleic acid, wherein the nucleic
acid binds to the polypeptide and hybridizes to a target nucleic
acid. In some embodiments, the kit further comprises instructions
for use. In some embodiments, the kit further comprises a
polynucleotide encoding a polypeptide, wherein the polypeptide
comprises at last 15% amino acid sequence identity to Cas9. In some
embodiments, the kit further comprises a polynucleotide encoding a
nucleic acid, wherein the nucleic acid comprises a sequence
comprising at least 50% sequence identity to a crRNA over 6
contiguous nucleotides and a sequence comprising at least 50%
sequence identity to a tracrRNA over 6 contiguous nucleotides.
[0077] In one aspect the disclosure provides for a method for
selecting a cell using a reporter element and excising the reporter
element from the cell comprising: contacting a target nucleic acid
with a complex comprising a site-directed polypeptide and a nucleic
acid-targeting nucleic acid; cleaving the target nucleic acid with
the site-directed polypeptide, to generate a cleaved target nucleic
acid; inserting the donor polynucleotide comprising a genetic
element of interest; and a reporter element, wherein the reporter
element comprises a polynucleotide sequence encoding a
site-directed polypeptide, and one or more a nucleic acids, wherein
the one or more nucleic acids comprises a sequence comprising at
least 50% sequence identity to a crRNA over 6 contiguous
nucleotides and a sequence comprising at least 50% sequence
identity to a tracrRNA over 6 contiguous nucleotides into the
cleaved target nucleic acid; and selecting the cell based on the
donor polynucleotide to generate a selected cell. In some
embodiments, selecting comprises selecting the cell from a subject
being treated for a disease. In some embodiments, selecting
comprises selecting the cell from a subject being diagnosed for a
disease. In some embodiments, after the selecting, the cell
comprises the donor polynucleotide. In some embodiments, the method
further comprises excising all, some or none of the reporter
element, thereby generating a second selected cell. In some
embodiments, excising comprises contacting the 5' end of the
reporter element with a complex comprising a site-directed
polypeptide and a nucleic acid-targeting nucleic acid, wherein the
complex cleaves the 5' end. In some embodiments, excising comprises
contacting the 3' end of the reporter element with a complex
comprising a site-directed polypeptide and a nucleic acid-targeting
nucleic acid, wherein the complex cleaves the 3'end. In some
embodiments, excising comprises contacting the 5' and 3' end of the
reporter element with one or more complexes comprising a
site-directed polypeptide and a nucleic acid-targeting nucleic
acid, wherein the complex cleaves the 5' and 3'end. In some
embodiments, the method further comprises screening the second
selected cell. In some embodiments, screening comprises observing
an absence of all or some of the reporter element.
[0078] In one aspect the disclosure provides for a composition
comprising: a nucleic acid comprising: a spacer, wherein the spacer
is between 12-30 nucleotides, inclusive, and wherein the spacer is
adapted to hybridize to a sequence that is 5' to a PAM; a first
duplex, wherein the first duplex is 3' to the spacer; a bulge,
wherein the bulge comprises at least 3 unpaired nucleotides on a
first strand of the first duplex and at least 1 unpaired nucleotide
on a second strand of the first duplex; a linker, wherein the
linker links the first strand and the second strand of the duplex
and is at least 3 nucleotides in length; a P-domain; and a second
duplex, wherein the second duplex is 3' of the P-domain and is
adapted to bind to a site directed polypeptide. In some
embodiments, the sequence that is 5' to a PAM is at least 18
nucleotides in length. In some embodiments, the sequence that is 5'
to a PAM is adjacent to the PAM. In some embodiments, the PAM
comprises 5'-NGG-3'. In some embodiments, the first duplex is
adjacent to the spacer. In some embodiments, the P-domain starts
from 1-5 nucleotides downstream of the duplex, comprises at least 4
nucleotides, and is adapted to hybridize to sequence selected from
the group consisting of: a 5'-NGG-3' protospacer adjacent motif
sequence, a sequence comprising at least 50% identity to amino
acids 1096-1225 of Cas9 from S. pyogenes, or any combination
thereof. In some embodiments, the site-directed polypeptide
comprises at least 15% identity to a nuclease domain of Cas9 from
S. pyogenes. In some embodiments, the nucleic acid is RNA. In some
embodiments, the nucleic acid is an A-form RNA. In some
embodiments, the first duplex is at least 6 nucleotides in length.
In some embodiments, the 3 unpaired nucleotides of the bulge
comprise 5'-AAG-3'. In some embodiments, adjacent to the 3 unpaired
nucleotides is a nucleotide that forms a wobble pair with a
nucleotide on the second strand of the first duplex. In some
embodiments, the polypeptide binds to a region of the nucleic acid
selected from the group consisting of: the first duplex, the second
duplex, and the P-domain, or any combination thereof.
[0079] In one aspect the disclosure provides for a method of
modifying a target nucleic acid comprising: contacting a target
nucleic acid with a composition comprising: a nucleic acid
comprising: a spacer, wherein the spacer is between 12-30
nucleotides, inclusive, and wherein the spacer is adapted to
hybridize to a sequence that is 5' to a PAM; a first duplex,
wherein the first duplex is 3' to the spacer; a bulge, wherein the
bulge comprises at least 3 unpaired nucleotides on a first strand
of the first duplex and at least 1 unpaired nucleotide on a second
strand of the first duplex; a linker, wherein the linker links the
first strand and the second strand of the duplex and is at least 3
nucleotides in length; a P-domain; and a second duplex, wherein the
second duplex is 3' of the P-domain and is adapted to bind to a
site directed polypeptide; and modifying the target nucleic acid.
In some embodiments, the method further comprises contacting with a
site-directed polypeptide. In some embodiments, the contacting
comprises contacting the spacer to the target nucleic acid. In some
embodiments, the modifying comprises cleaving the target nucleic
acid to produce a cleaved target nucleic acid. In some embodiments,
the cleaving is performed by the site-directed polypeptide. In some
embodiments, the method further comprises inserting a donor
polynucleotide into the cleaved target nucleic acid. In some
embodiments, the modifying comprises modifying transcription of the
target nucleic acid.
[0080] In one aspect the disclosure provides for a vector
comprising a polynucleotide sequence encoding a nucleic acid
comprising: a spacer, wherein the spacer is between 12-30
nucleotides, inclusive, and wherein the spacer is adapted to
hybridize to a sequence that is 5' to a PAM; a first duplex,
wherein the first duplex is 3' to the spacer; a bulge, wherein the
bulge comprises at least 3 unpaired nucleotides on a first strand
of the first duplex and at least 1 unpaired nucleotide on a second
strand of the first duplex; a linker, wherein the linker links the
first strand and the second strand of the duplex and is at least 3
nucleotides in length; a P-domain; and a second duplex, wherein the
second duplex is 3' of the P-domain and is adapted to bind to a
site directed polypeptide.
[0081] In one aspect the disclosure provides for a kit comprising:
a composition comprising: a nucleic acid comprising: a spacer,
wherein the spacer is between 12-30 nucleotides, inclusive, and
wherein the spacer is adapted to hybridize to a sequence that is 5'
to a PAM; a first duplex, wherein the first duplex is 3' to the
spacer; a bulge, wherein the bulge comprises at least 3 unpaired
nucleotides on a first strand of the first duplex and at least 1
unpaired nucleotide on a second strand of the first duplex; a
linker, wherein the linker links the first strand and the second
strand of the duplex and is at least 3 nucleotides in length; a
P-domain; and a second duplex, wherein the second duplex is 3' of
the P-domain and is adapted to bind to a site directed polypeptide;
and a buffer. In some embodiments, the kit further comprises a
site-directed polypeptide. In some embodiments, the kit further
comprises a donor polynucleotide. In some embodiments, the kit
further comprises instructions for use.
[0082] In one aspect, the disclosure provides for a method of
creating a synthetically designed nucleic acid-targeting nucleic
acid comprising: designing a composition comprising: a nucleic acid
comprising: a spacer, wherein the spacer is between 12-30
nucleotides, inclusive, and wherein the spacer is adapted to
hybridize to a sequence that is 5' to a PAM; a first duplex,
wherein the first duplex is 3' to the spacer; a bulge, wherein the
bulge comprises at least 3 unpaired nucleotides on a first strand
of the first duplex and at least 1 unpaired nucleotide on a second
strand of the first duplex; a linker, wherein the linker links the
first strand and the second strand of the duplex and is at least 3
nucleotides in length; a P-domain; and a second duplex, wherein the
second duplex is 3' of the P-domain and is adapted to bind to a
site directed polypeptide.
[0083] In one aspect, the disclosure provides for a pharmaceutical
composition comprising an engineered nucleic acid-targeting nucleic
acid selected from the group consisting of: an engineered nucleic
acid-targeting nucleic acid comprising: a mutation in a P-domain of
said nucleic acid-targeting nucleic acid; an engineered nucleic
acid-targeting nucleic acid comprising: a mutation in a bulge
region of a nucleic acid-targeting nucleic acid
[0084] In one aspect the disclosure provides for a pharmaceutical
composition comprising a composition selected from the group
consisting of: A composition comprising: an engineered nucleic
acid-targeting nucleic acid comprising a 3' hybridizing extension,
and a donor polynucleotide, wherein said donor polynucleotide is
hybridized to said 3' hybridizing extension; a composition
comprising: an effector protein, and a nucleic acid, wherein said
nucleic acid comprises: at least 50% sequence identity to a crRNA
over 6 contiguous nucleotides, at least 50% sequence identity to a
tracrRNA over 6 contiguous nucleotides, and a non-native sequence,
wherein said nucleic acid is adapted to bind to said effector
protein; a composition comprising: a multiplexed genetic targeting
agent, wherein said multiplexed genetic targeting agent comprises
one or more nucleic acid modules, wherein said nucleic acid module
comprises a non-native sequence, and wherein said nucleic acid
module is configured to bind to a polypeptide comprising at least
10% amino acid sequence identity to a nuclease domain of Cas9 and
wherein said nucleic acid module is configured to hybridize to a
target nucleic acid; a composition comprising: a modified
site-directed polypeptide, wherein said polypeptide is modified
such that it is adapted to target a second protospacer adjacent
motif compared to a wild-type site-directed polypeptide; a
composition comprising: a modified site-directed polypeptide,
wherein said polypeptide is modified such that it is adapted to
target a second nucleic acid-targeting nucleic acid compared to a
wild-type site-directed polypeptide; a composition comprising: a
modified site-directed polypeptide comprising a modification in a
bridge helix as compared to SEQ ID: 8; a composition comprising: a
modified site-directed polypeptide comprising a modification in a
highly basic patch as compared to SEQ ID: 8; a composition
comprising: a modified site-directed polypeptide comprising a
modification in a polymerase-like domain as compared to SEQ ID: 8;
a composition comprising: a modified site-directed polypeptide
comprising a modification in a bridge helix, highly basic patch,
nuclease domain, and polymerase domain as compared to SEQ ID: 8, or
any combination thereof; a composition comprising: a modified
site-directed polypeptide comprising a modified nuclease domain as
compared to SEQ ID: 8; a composition comprising: a first complex
comprising: a first site-directed polypeptide and a first nucleic
acid-targeting nucleic acid, a second complex comprising: a second
site-directed polypeptide and a second nucleic acid-targeting
nucleic acid, wherein, said first and second nucleic acid-targeting
nucleic acids are different; a composition comprising: a plurality
of nucleic acid molecules, wherein each nucleic acid molecule
comprises a nucleic acid-binding protein binding site, wherein at
least one of said plurality of nucleic acid molecules encodes for a
nucleic acid-targeting nucleic acid and one of said plurality of
nucleic acid molecules encodes for a site-directed polypeptide, and
a fusion polypeptide, wherein said fusion polypeptide comprises a
plurality of said nucleic acid-binding proteins, wherein said
plurality of nucleic acid-binding proteins are adapted to bind to
their cognate nucleic acid-binding protein binding site; and a
composition comprising: a nucleic acid comprising: a spacer,
wherein said spacer is between 12-30 nucleotides, inclusive, and
wherein said spacer is adapted to hybridize to a sequence that is
5' to a PAM, a first duplex, wherein said first duplex is 3' to
said spacer, a bulge, wherein said bulge comprises at least 3
unpaired nucleotides on a first strand of said first duplex and at
least 1 unpaired nucleotide on a second strand of said first
duplex, a linker, wherein said linker links said first strand and
said second strand of said duplex and is at least 3 nucleotides in
length, a P-domain, and a second duplex, wherein said second duplex
is 3' of said P-domain and is adapted to bind to a site directed
polypeptide; or any combination thereof.
[0085] In one aspect, the disclosure provides for a pharmaceutical
composition comprising a modified site-directed polypeptide
comprising: a first nuclease domain, a second nuclease domain, and
an inserted nuclease domain.
[0086] In one aspect the disclosure provides for a pharmaceutical
composition comprising a donor polynucleotide comprising: a genetic
element of interest, and a reporter element, wherein said reporter
element comprises a polynucleotide sequence encoding a
site-directed polypeptide, and one or more a nucleic acids, wherein
said one or more nucleic acids comprises a sequence comprising at
least 50% sequence identity to a crRNA over 6 contiguous
nucleotides and a sequence comprising at least 50% sequence
identity to a tracrRNA over 6 contiguous nucleotides.
[0087] In one aspect the disclosure provides for a pharmaceutical
composition a vector selected from the group consisting of: a
vector comprising a polynucleotide sequence encoding An engineered
nucleic acid-targeting nucleic acid comprising: a mutation in a
P-domain of said nucleic acid-targeting nucleic acid; a vector
comprising a polynucleotide sequence encoding an engineered nucleic
acid-targeting nucleic acid comprising: a mutation in a bulge
region of a nucleic acid-targeting nucleic acid; and modifying the
target nucleic acid; a vector comprising a polynucleotide sequence
encoding a modified nucleic acid-targeting nucleic acid, wherein
the modified nucleic acid-targeting nucleic acid comprises a
non-native sequence; a vector comprising: a polynucleotide sequence
encoding: a modified nucleic acid-targeting nucleic acid, wherein
the modified nucleic acid-targeting nucleic acid comprises a
sequence configured to bind to an effector protein, and a
site-directed polypeptide; a vector comprising: a polynucleotide
sequence encoding: a modified nucleic acid-targeting nucleic acid,
wherein the modified nucleic acid-targeting nucleic acid comprises
a non-native sequence, a site-directed polypeptide, and an effector
protein; a vector comprising a polynucleotide sequence encoding a
multiplexed genetic targeting agent, wherein the multiplexed
genetic targeting agent comprises one or more nucleic acid modules,
wherein the nucleic acid module comprises a non-native sequence,
and wherein the nucleic acid module is configured to bind to a
polypeptide comprising at least 10% amino acid sequence identity to
a nuclease domain of Cas9 and wherein the nucleic acid module is
configured to hybridize to a target nucleic acid; a vector
comprising a polynucleotide sequence encoding a modified
site-directed polypeptide comprising a modification in a bridge
helix, highly basic patch, nuclease domain, and polymerase domain
as compared to SEQ ID: 8, or any combination thereof; a vector
comprising a polynucleotide sequence encoding: two or more nucleic
acid-targeting nucleic acids that differ by at least one
nucleotide; and a site-directed polypeptide; a vector comprising: a
polynucleotide sequence encoding a composition comprising: a
plurality of nucleic acid molecules, wherein each nucleic acid
molecule comprises a nucleic acid-binding protein binding site,
wherein at least one of the plurality of nucleic acid molecules
encodes for a nucleic acid-targeting nucleic acid and one of the
plurality of nucleic acid molecules encodes for a site-directed
polypeptide; and a fusion polypeptide, wherein the fusion
polypeptide comprises a plurality of the nucleic acid-binding
proteins, wherein the plurality of nucleic acid-binding proteins
are adapted to bind to their cognate nucleic acid-binding protein
binding sitestoichiometrically delivering the composition to the
subcellular location; an expression vector comprising a
polynucleotide sequence encoding for a genetic element of interest;
and a reporter element, wherein the reporter element comprises a
polynucleotide sequence encoding a site-directed polypeptide, and
one or more a nucleic acids, wherein the one or more nucleic acids
comprises a sequence comprising at least 50% sequence identity to a
crRNA over 6 contiguous nucleotides and a sequence comprising at
least 50% sequence identity to a tracrRNA over 6 contiguous
nucleotides; and a vector comprising a polynucleotide sequence
encoding a nucleic acid comprising: a spacer, wherein the spacer is
between 12-30 nucleotides, inclusive, and wherein the spacer is
adapted to hybridize to a sequence that is 5' to a PAM; a first
duplex, wherein the first duplex is 3' to the spacer; a bulge,
wherein the bulge comprises at least 3 unpaired nucleotides on a
first strand of the first duplex and at least 1 unpaired nucleotide
on a second strand of the first duplex; a linker, wherein the
linker links the first strand and the second strand of the duplex
and is at least 3 nucleotides in length; a P-domain; and a second
duplex, wherein the second duplex is 3' of the P-domain and is
adapted to bind to a site directed polypeptide; or any combination
thereof.
[0088] In one aspect the disclosure provides for a method of
treating a disease comprising administering to a subject: an
engineered nucleic acid-targeting comprising: a mutation in a
P-domain of said nucleic acid-targeting nucleic acid; an engineered
nucleic acid-targeting nucleic acid comprising: a mutation in a
bulge region of a nucleic acid-targeting nucleic acid; a
composition comprising: an engineered nucleic acid-targeting
nucleic acid comprising a 3' hybridizing extension, and a donor
polynucleotide, wherein said donor polynucleotide is hybridized to
said 3' hybridizing extension; a composition comprising: an
effector protein, and a nucleic acid, wherein said nucleic acid
comprises: at least 50% sequence identity to a crRNA over 6
contiguous nucleotides, at least 50% sequence identity to a
tracrRNA over 6 contiguous nucleotides, and a non-native sequence,
wherein said nucleic acid is adapted to bind to said effector
protein; a composition comprising: a multiplexed genetic targeting
agent, wherein said multiplexed genetic targeting agent comprises
one or more nucleic acid modules, wherein said nucleic acid module
comprises a non-native sequence, and wherein said nucleic acid
module is configured to bind to a polypeptide comprising at least
10% amino acid sequence identity to a nuclease domain of Cas9 and
wherein said nucleic acid module is configured to hybridize to a
target nucleic acid; a composition comprising: a modified
site-directed polypeptide, wherein said polypeptide is modified
such that it is adapted to target a second protospacer adjacent
motif compared to a wild-type site-directed polypeptide; a
composition comprising: a modified site-directed polypeptide,
wherein said polypeptide is modified such that it is adapted to
target a second nucleic acid-targeting nucleic acid compared to a
wild-type site-directed polypeptide; a composition comprising: a
modified site-directed polypeptide comprising a modification in a
bridge helix as compared to SEQ ID: 8; a composition comprising: a
modified site-directed polypeptide comprising a modification in a
highly basic patch as compared to SEQ ID: 8; a composition
comprising: a modified site-directed polypeptide comprising a
modification in a polymerase-like domain as compared to SEQ ID: 8;
a composition comprising: a modified site-directed polypeptide
comprising a modification in a bridge helix, highly basic patch,
nuclease domain, and polymerase domain as compared to SEQ ID: 8, or
any combination thereof; a composition comprising: a modified
site-directed polypeptide comprising a modified nuclease domain as
compared to SEQ ID: 8; a composition comprising: a first complex
comprising: a first site-directed polypeptide and a first nucleic
acid-targeting nucleic acid, a second complex comprising: a second
site-directed polypeptide and a second nucleic acid-targeting
nucleic acid, wherein, said first and second nucleic acid-targeting
nucleic acids are different; a composition comprising: a plurality
of nucleic acid molecules, wherein each nucleic acid molecule
comprises a nucleic acid-binding protein binding site, wherein at
least one of said plurality of nucleic acid molecules encodes for a
nucleic acid-targeting nucleic acid and one of said plurality of
nucleic acid molecules encodes for a site-directed polypeptide, and
a fusion polypeptide, wherein said fusion polypeptide comprises a
plurality of said nucleic acid-binding proteins, wherein said
plurality of nucleic acid-binding proteins are adapted to bind to
their cognate nucleic acid-binding protein binding site; a
composition comprising: a nucleic acid comprising: a spacer,
wherein said spacer is between 12-30 nucleotides, inclusive, and
wherein said spacer is adapted to hybridize to a sequence that is
5' to a PAM, a first duplex, wherein said first duplex is 3' to
said spacer, a bulge, wherein said bulge comprises at least 3
unpaired nucleotides on a first strand of said first duplex and at
least 1 unpaired nucleotide on a second strand of said first
duplex, a linker, wherein said linker links said first strand and
said second strand of said duplex and is at least 3 nucleotides in
length, a P-domain, and a second duplex, wherein said second duplex
is 3' of said P-domain and is adapted to bind to a site directed
polypeptide; a modified site-directed polypeptide comprising: a
first nuclease domain, a second nuclease domain, and an inserted
nuclease domain; a donor polynucleotide comprising: a genetic
element of interest, and a reporter element, wherein said reporter
element comprises a polynucleotide sequence encoding a
site-directed polypeptide, and one or more a nucleic acids, wherein
said one or more nucleic acids comprises a sequence comprising at
least 50% sequence identity to a crRNA over 6 contiguous
nucleotides and a sequence comprising at least 50% sequence
identity to a tracrRNA over 6 contiguous nucleotides; a vector
comprising a polynucleotide sequence encoding An engineered nucleic
acid-targeting nucleic acid comprising: a mutation in a P-domain of
said nucleic acid-targeting nucleic acid; a vector comprising a
polynucleotide sequence encoding an engineered nucleic
acid-targeting nucleic acid comprising: a mutation in a bulge
region of a nucleic acid-targeting nucleic acid; and modifying the
target nucleic acid; a vector comprising a polynucleotide sequence
encoding a modified nucleic acid-targeting nucleic acid, wherein
the modified nucleic acid-targeting nucleic acid comprises a
non-native sequence; a vector comprising: a polynucleotide sequence
encoding: a modified nucleic acid-targeting nucleic acid, wherein
the modified nucleic acid-targeting nucleic acid comprises a
sequence configured to bind to an effector protein, and a
site-directed polypeptide; a vector comprising: a polynucleotide
sequence encoding: a modified nucleic acid-targeting nucleic acid,
wherein the modified nucleic acid-targeting nucleic acid comprises
a non-native sequence, a site-directed polypeptide, and an effector
protein; a vector comprising a polynucleotide sequence encoding a
multiplexed genetic targeting agent, wherein the multiplexed
genetic targeting agent comprises one or more nucleic acid modules,
wherein the nucleic acid module comprises a non-native sequence,
and wherein the nucleic acid module is configured to bind to a
polypeptide comprising at least 10% amino acid sequence identity to
a nuclease domain of Cas9 and wherein the nucleic acid module is
configured to hybridize to a target nucleic acid; a vector
comprising a polynucleotide sequence encoding a modified
site-directed polypeptide comprising a modification in a bridge
helix, highly basic patch, nuclease domain, and polymerase domain
as compared to SEQ ID: 8, or any combination thereof; a vector
comprising a polynucleotide sequence encoding: two or more nucleic
acid-targeting nucleic acids that differ by at least one
nucleotide; and a site-directed polypeptide; a vector comprising: a
polynucleotide sequence encoding a composition comprising: a
plurality of nucleic acid molecules, wherein each nucleic acid
molecule comprises a nucleic acid-binding protein binding site,
wherein at least one of the plurality of nucleic acid molecules
encodes for a nucleic acid-targeting nucleic acid and one of the
plurality of nucleic acid molecules encodes for a site-directed
polypeptide; and a fusion polypeptide, wherein the fusion
polypeptide comprises a plurality of the nucleic acid-binding
proteins, wherein the plurality of nucleic acid-binding proteins
are adapted to bind to their cognate nucleic acid-binding protein
binding sitestoichiometrically delivering the composition to the
subcellular location; an expression vector comprising a
polynucleotide sequence encoding for a genetic element of interest;
and a reporter element, wherein the reporter element comprises a
polynucleotide sequence encoding a site-directed polypeptide, and
one or more a nucleic acids, wherein the one or more nucleic acids
comprises a sequence comprising at least 50% sequence identity to a
crRNA over 6 contiguous nucleotides and a sequence comprising at
least 50% sequence identity to a tracrRNA over 6 contiguous
nucleotides; and a vector comprising a polynucleotide sequence
encoding a nucleic acid comprising: a spacer, wherein the spacer is
between 12-30 nucleotides, inclusive, and wherein the spacer is
adapted to hybridize to a sequence that is 5' to a PAM; a first
duplex, wherein the first duplex is 3' to the spacer; a bulge,
wherein the bulge comprises at least 3 unpaired nucleotides on a
first strand of the first duplex and at least 1 unpaired nucleotide
on a second strand of the first duplex; a linker, wherein the
linker links the first strand and the second strand of the duplex
and is at least 3 nucleotides in length; a P-domain; and a second
duplex, wherein the second duplex is 3' of the P-domain and is
adapted to bind to a site directed polypeptide; or any combination
thereof. In some embodiments, the administering comprises
administering comprises administering by viral delivery. In some
embodiments, the administering comprises administering comprises
administering by electroporation. In some embodiments, the
administering comprises administering comprises administering by
nanoparticle delivery. In some embodiments, the administering
comprises administering comprises administering by liposome
delivery. In some embodiments, the administering comprises
administering by a method selected from the group consisting of:
intravenously, subcutaneously, intramuscularly, orally, rectally,
by aerosol, parenterally, ophthalmicly, pulmonarily, transdermally,
vaginally, optically, nasally, and by topical administration, or
any combination thereof. In some embodiments, the methods of the
disclosure are performed in a cell selected from the group
consisting of: plant cell, microbe cell, and fungi cell, or any
combination thereof.
Incorporation by Reference
[0089] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0090] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0091] FIG. 1A depicts an exemplary embodiment of a single guide
nucleic acid-targeting nucleic acid of the disclosure.
[0092] FIG. 1B depicts an exemplary embodiment of a single guide
nucleic acid-targeting nucleic acid of the disclosure.
[0093] FIG. 2 depicts an exemplary embodiment of a double guide
nucleic acid-targeting nucleic acid of the disclosure.
[0094] FIG. 3 depicts an exemplary embodiment of a sequence
enrichment method of the disclosure utilizing target nucleic acid
cleavage.
[0095] FIG. 4 depicts an exemplary embodiment of a sequence
enrichment method of the disclosure utilizing target nucleic acid
enrichment.
[0096] FIG. 5 depicts an exemplary embodiment of a method of the
disclosure for determining off-target binding sites of a
site-directed polypeptide utilizing purification of the
site-directed polypeptide.
[0097] FIG. 6 depicts an exemplary embodiment of a method of the
disclosure for determining off-target binding sites of a
site-directed polypeptide utilizing purification of the nucleic
acid-targeting nucleic acid.
[0098] FIG. 7 illustrates an exemplary embodiment for an
array-based sequencing method using a site-directed polypeptide of
the disclosure.
[0099] FIG. 8 illustrates an exemplary embodiment for an
array-based sequencing method using a site-directed polypeptide of
the disclosure, wherein cleaved products are sequenced.
[0100] FIG. 9 illustrates an exemplary embodiment for a
next-generation sequencing-based method using a site-directed
polypeptide of the disclosure.
[0101] FIG. 10 depicts an exemplary tagged single guide nucleic
acid-targeting nucleic acid.
[0102] FIG. 11 depicts an exemplary tagged double guide nucleic
acid-targeting nucleic acid.
[0103] FIG. 12 illustrates an exemplary embodiment of a method of
using tagged nucleic acid-targeting nucleic acid with a split
system (e.g., split fluorescent system).
[0104] FIG. 13 depicts some exemplary data on the effect of a 5'
tagged nucleic acid-targeting nucleic acid on target nucleic acid
cleavage.
[0105] FIG. 14 illustrates an exemplary 5' tagged nucleic
acid-targeting nucleic acid comprising a tag linker sequence
between the nucleic acid-targeting nucleic acid and the tag.
[0106] FIG. 15 depicts an exemplary embodiment of a method of
multiplexed target nucleic acid cleavage.
[0107] FIG. 16 depicts an exemplary embodiment of a method of
stoichiometric delivery of RNA nucleic acids.
[0108] FIG. 17 depicts an exemplary embodiment of a method of
stoichiometric delivery of nucleic acids.
[0109] FIG. 18 depicts an exemplary embodiment of seamless
insertion of a reporter element into a target nucleic acid using a
site-directed polypeptide of the disclosure.
[0110] FIG. 19 depicts an exemplary embodiment for removing a
reporter element from a target nucleic acid.
[0111] FIG. 20 depicts complementary portions of the nucleic acid
sequences of a pre-CRISPR nucleic acid and tracr nucleic acid
sequences from Streptococcus pyogenes SF370.
[0112] FIG. 21 depicts an exemplary secondary structure of a
synthetic single guide nucleic acid-targeting nucleic acid.
[0113] FIGS. 22A and B shows exemplary single-guide nucleic
acid-targeting nucleic acid backbone variants. Nucleotides in the
boxes correspond to nucleotides that have been altered relative to
CRISPR sequences labeled as FL-tracr-crRNA sequence.
[0114] FIG. 23A-C shows exemplary data from an in vitro cleavage
assay. The results demonstrate that more than one synthetic nucleic
acid-targeting nucleic acid backbone sequences can support cleavage
by a site-directed polypeptide (e.g., Cas9).
[0115] FIG. 24 shows exemplary synthetic single-guide nucleic
acid-targeting nucleic acid sequences containing variants in the
complementary region/duplex. Nucleotides in the boxes correspond to
nucleotides that have been altered relative to the CRISPR sequences
labeled as FL-tracr-crRNA sequence.
[0116] FIG. 25 shows exemplary variants to the single guide nucleic
acid-targeting nucleic acid structure within the region 3' to the
complementary region/duplex. Nucleotides in the boxes correspond to
nucleotides that have been altered relative to the naturally
occurring S. pyogenes SF370 CRISPR nucleic acid and tracr nucleic
acid sequence pairing.
[0117] FIG. 26A-B shows exemplary variants to the single guide
nucleic acid-targeting nucleic acid structure within the region 3'
to the complementary region/duplex. Nucleotides in the boxes
correspond to nucleotides that have been altered relative to the
naturally occurring S. pyogenes SF370 CRISPR nucleic acid and tracr
nucleic acid sequence pairing.
[0118] FIG. 27A-B shows exemplary variant nucleic acid-targeting
nucleic acid structures comprising additional hairpin sequences
derived from the CRISPR repeat in Pseudomonas aeruginosa (PA14).
The sequences in the boxes can bind to the ribonuclease Csy4 from
PA14.
[0119] FIG. 28 shows exemplary data from an in vitro cleavage assay
demonstrating that multiple synthetic nucleic acid-targeting
nucleic acid backbone sequences support Cas9 cleavage. The top and
bottom gel images represent two independent repeats of the
assay.
[0120] FIG. 29 shows exemplary data from an in vitro cleavage assay
demonstrating that multiple synthetic nucleic acid-targeting
nucleic acid backbone sequences support Cas9 cleavage. The top and
bottom gel images represent two independent repeats of the
assay.
[0121] FIG. 30 depicts exemplary methods of the disclosure of
bringing a donor polynucleotide to a modification site in a target
nucleic acid.
[0122] FIG. 31 depicts a system for storing and sharing electronic
information.
[0123] FIG. 32 depicts an exemplary embodiment of two nickases
generating a blunt end cut in a target nucleic acid. The
site-directed modifying polypeptides complexed with nucleic
acid-targeting nucleic acids are not shown.
[0124] FIG. 33 depicts an exemplary embodiment of staggard cutting
of a target nucleic acid using two nickases and generating sticky
ends. The site-directed modifying polypeptides complexed with
nucleic acid-targeting nucleic acids are not shown.
[0125] FIG. 34 depicts an exemplary embodiment of staggard cutting
of a target nucleic acid using two nickases and generating
medium-sized sticky ends. The site-directed modifying polypeptides
complexed with nucleic acid-targeting nucleic acids are not
shown.
[0126] FIG. 35 illustrates a sequence alignment of Cas9
orthologues. Amino acids with a "X" below them may be considered to
be similar. Amino acids with a "Y" below them can be considered to
be highly conserved or identical in all sequences. Amino acids
residues without an "X" or a "Y" may not be conserved.
[0127] FIG. 36 shows the functionality of nucleic acid-targeting
nucleic acid variants on target nucleic acid cleavage. The variants
tested in FIG. 36 correspond to the variants depicted in FIG. 22,
FIG. 24, and FIG. 25.
[0128] FIG. 37A-D shows in vitro cleavage assays using variant
nucleic acid-targeting nucleic acids.
[0129] FIG. 38 depicts exemplary amino acid sequences of Csy4 from
wild-type P. aeruginosa.
[0130] FIG. 39 depicts exemplary amino acid sequences of an
enzymatically inactive endoribonuclease (e.g., Csy4).
[0131] FIG. 40 depicts exemplary amino acid sequences of Csy4 from
P. aeruginosa.
[0132] FIG. 41A-J depicts exemplary Cas6 amino acid sequences.
[0133] FIG. 42A-C depicts exemplary Cas6 amino acid sequences.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0134] As used herein, "affinity tag" can refer to either a peptide
affinity tag or a nucleic acid affinity tag. Affinity tag generally
refer to a protein or nucleic acid sequence that can be bound to a
molecule (e.g., bound by a small molecule, protein, covalent bond).
An affinity tag can be a non-native sequence. A peptide affinity
tag can comprise a peptide. A peptide affinity tag can be one that
is able to be part of a split system (e.g., two inactive peptide
fragments can combine together in trans to form an active affinity
tag). A nucleic acid affinity tag can comprise a nucleic acid. A
nucleic acid affinity tag can be a sequence that can selectively
bind to a known nucleic acid sequence (e.g. through hybridization).
A nucleic acid affinity tag can be a sequence that can selectively
bind to a protein. An affinity tag can be fused to a native
protein. An affinity tag can be fused to a nucleotide sequence.
Sometimes, one, two, or a plurality of affinity tags can be fused
to a native protein or nucleotide sequence. An affinity tag can be
introduced into a nucleic acid-targeting nucleic acid using methods
of in vitro or in vivo transcription. Nucleic acid affinity tags
can include, for example, a chemical tag, an RNA-binding protein
binding sequence, a DNA-binding protein binding sequence, a
sequence hybridizable to an affinity-tagged polynucleotide, a
synthetic RNA aptamer, or a synthetic DNA aptamer. Examples of
chemical nucleic acid affinity tags can include, but are not
limited to, ribo-nucleotriphosphates containing biotin, fluorescent
dyes, and digoxeginin. Examples of protein-binding nucleic acid
affinity tags can include, but are not limited to, the MS2 binding
sequence, the U1A binding sequence, stem-loop binding protein
sequences, the boxB sequence, the eIF4A sequence, or any sequence
recognized by an RNA binding protein. Examples of nucleic acid
affinity-tagged oligonucleotides can include, but are not limited
to, biotinylated oligonucleotides, 2,4-dinitrophenyl
oligonucleotides, fluorescein oligonucleotides, and primary
amine-conjugated oligonucleotides.
[0135] A nucleic acid affinity tag can be an RNA aptamer. Aptamers
can include, aptamers that bind to theophylline, streptavidin,
dextran B512, adenosine, guanosine, guanine/xanthine, 7-methyl-GTP,
amino acid aptamers such as aptamers that bind to arginine,
citrulline, valine, tryptophan, cyanocobalamine,
N-methylmesoporphyrin IX, flavin, NAD, and antibiotic aptamers such
as aptamers that bind to tobramycin, neomycin, lividomycin,
kanamycin, streptomycin, viomycin, and chloramphenicol.
[0136] A nucleic acid affinity tag can comprise an RNA sequence
that can be bound by a site-directed polypeptide. The site-directed
polypeptide can be conditionally enzymatically inactive. The RNA
sequence can comprise a sequence that can be bound by a member of
Type I, Type II, and/or Type III CRISPR systems. The RNA sequence
can be bound by a RAMP family member protein. The RNA sequence can
be bound by a Cas6 family member protein (e.g., Csy4, Cas6). The
RNA sequence can be bound by a Cas5 family member protein (e.g.,
Cas5). For example, Csy4 can bind to a specific RNA hairpin
sequence with high affinity (Kd .about.50 pM) and can cleave RNA at
a site 3' to the hairpin. The Cas5 or Cas6 family member protein
can bind an RNA sequence that comprises at least about or at most
about 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%
sequence identity and/or sequence similarity to the following
nucleotide sequences:
TABLE-US-00002 5'-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3';
5'-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3'
5'-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3';
5'-GUUGCAAACCUCGUUAGCCUCGUAGAGGAUUGAAAC-3';
5'-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3';
5'-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3';
5'-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3';
5'-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3';
5'-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3'.
5'-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3';
5'-GUUGCAAACCUCGUUAGCCUCGUAGAGGAUUGAAAC-3';
5'-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3';
5'-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3';
5'-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3';
5'-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3';
5'-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3',
5'-GUCGCCCCCCACGCGGGGGCGUGGAUUGAAAC-3';
5'-CCAGCCGCCUUCGGGCGGCUGUGUGUUGAAAC-3';
5'-GUCGCACUCUACAUGAGUGCGUGGAUUGAAAU-3';
5'-UGUCGCACCUUAUAUAGGUGCGUGGAUUGAAAU-3'; and
5'-GUCGCGCCCCGCAUGGGGCGCGUGGAUUGAAA-3',
[0137] A nucleic acid affinity tag can comprise a DNA sequence that
can be bound by a site-directed polypeptide. The site-directed
polypeptide can be conditionally enzymatically inactive. The DNA
sequence can comprise a sequence that can be bound by a member of
the Type I, Type II and/or Type III CRISPR system. The DNA sequence
can be bound by an Argonaut protein. The DNA sequence can be bound
by a protein containing a zinc finger domain, a TALE domain, or any
other DNA-binding domain.
[0138] A nucleic acid affinity tag can comprise a ribozyme
sequence. Suitable ribozymes can include peptidyl transferase 23S
rRNA, RnaseP, Group I introns, Group II introns, GIR1 branching
ribozyme, Leadzyme, hairpin ribozymes, hammerhead ribozymes, HDV
ribozymes, CPEB3 ribozymes, VS ribozymes, glmS ribozyme, CoTC
ribozyme, and synthetic ribozymes.
[0139] Peptide affinity tags can comprise tags that can be used for
tracking or purification (e.g., a fluorescent protein, green
fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, a his
tag, (e.g., a 6.times.His tag), a hemagglutinin (HA) tag, a FLAG
tag, a Myc tag, a GST tag, a MBP tag, and chitin binding protein
tag, a calmodulin tag, a V5 tag, a streptavidin binding tag, and
the like).
[0140] Both nucleic acid and peptide affinity tags can comprise
small molecule tags such as biotin, or digitoxin, fluorescent label
tags, such as for example, fluoroscein, rhodamin, Alexa fluor dyes,
Cyanine3 dye, Cyanine5 dye.
[0141] Nucleic acid affinity tags can be located 5' to a nucleic
acid (e.g., a nucleic acid-targeting nucleic acid). Nucleic acid
affinity tags can be located 3' to a nucleic acid. Nucleic acid
affinity tags can be located 5' and 3' to a nucleic acid. Nucleic
acid affinity tags can be located within a nucleic acid. Peptide
affinity tags can be located N-terminal to a polypeptide sequence.
Peptide affinity tags can be located C-terminal to a polypeptide
sequence. Peptide affinity tags can be located N-terminal and
C-terminal to a polypeptide sequence. A plurality of affinity tags
can be fused to a nucleic acid and/or a polypeptide sequence.
[0142] As used herein, "capture agent" can generally refer to an
agent that can purify a polypeptide and/or a nucleic acid. A
capture agent can be a biologically active molecule or material
(e.g. any biological substance found in nature or synthetic, and
includes but is not limited to cells, viruses, subcellular
particles, proteins, including more specifically antibodies,
immunoglobulins, antigens, lipoproteins, glycoproteins, peptides,
polypeptides, protein complexes, (strept)avidin-biotin complexes,
ligands, receptors, or small molecules, aptamers, nucleic acids,
DNA, RNA, peptidic nucleic acids, oligosaccharides,
polysaccharides, lipopolysccharides, cellular metabllites, haptens,
pharmacologically active substances, alkaloids, steroids, vitamins,
amino acids, and sugures). In some embodiments, the capture agent
can comprise an affinity tag. In some embodiments, a capture agent
can preferentially bind to a target polypeptide or nucleic acid of
interest. Capture agents can be free floating in a mixture. Capture
agents can be bound to a particle (e.g. a bead, a microbead, a
nanoparticle). Capture agents can be bound to a solid or semisolid
surface. In some instances, capture agents are irreversibly bound
to a target. In other instances, capture agents are reversibly
bound to a target (e.g. if a target can be eluted, or by use of a
chemical such as imidizole).
[0143] As used herein, "Cas5" can generally refer to can refer to a
polypeptide with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 100% sequence identity and/or sequence similarity to
a wild type exemplary Cas5 polypeptide (e.g., Cas5 from D.
vulgaris, and/or any sequences depicted in FIG. 42). Cas5 can
generally refer to can refer to a polypeptide with at most about
5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequence
identity and/or sequence similarity to a wild type exemplary Cas5
polypeptide (e.g., a Cas5 from D. vulgaris). Cas5 can refer to the
wildtype or a modified form of the Cas5 protein that can comprise
an amino acid change such as a deletion, insertion, substitution,
variant, mutation, fusion, chimera, or any combination thereof.
[0144] As used herein, "Cas6" can generally refer to can refer to a
polypeptide with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 100% sequence identity and/or sequence similarity to
a wild type exemplary Cas6 polypeptide (e.g., a Cas6 from T.
thermophilus, and/or sequences depicted in FIG. 41). Cas6 can
generally refer to can refer to a polypeptide with at most about
5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequence
identity and/or sequence similarity to a wild type exemplary Cas6
polypeptide (e.g., from T. thermophilus). Cas6 can refer to the
wildtype or a modified form of the Cas6 protein that can comprise
an amino acid change such as a deletion, insertion, substitution,
variant, mutation, fusion, chimera, or any combination thereof.
[0145] As used herein, "Cas9" can generally refer to a polypeptide
with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 100% sequence identity and/or sequence similarity to a wild
type exemplary Cas9 polypeptide (e.g., Cas9 from S. pyogenes (SEQ
ID NO: 8, SEQ ID NO: 1-256, SEQ ID NO: 795-1346). Cas9 can refer to
can refer to a polypeptide with at most about 5%, 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 100% sequence identity and/or
sequence similarity to a wild type exemplary Cas9 polypeptide
(e.g., from S. pyogenes). Cas9 can refer to the wildtype or a
modified form of the Cas9 protein that can comprise an amino acid
change such as a deletion, insertion, substitution, variant,
mutation, fusion, chimera, or any combination thereof.
[0146] As used herein, a "cell" can generally refer to a biological
cell. A cell can be the basic structural, functional and/or
biological unit of a living organism. A cell can originate from any
organism having one or more cells. Some non-limiting examples
include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an
archaeal cell, a cell of a single-cell eukaryotic organism, a
protozoa cell, a cell from a plant (e.g. cells from plant crops,
fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds,
tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton,
cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns,
clubmosses, hornworts, liverworts, mosses), an algal cell, (e.g.,
Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis
gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, and
the like), seaweeds (e.g. kelp) a fungal cell (e.g., a yeast cell,
a cell from a mushroom), an animal cell, a cell from an
invertebrate animal (e.g. fruit fly, cnidarian, echinoderm,
nematode, etc.), a cell from a vertebrate animal (e.g., fish,
amphibian, reptile, bird, mammal), a cell from a mammal (e.g., a
pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human
primate, a human, etc.), and etcetera. Sometimes a cell is not
orginating from a natural organism (e.g. a cell can be a
synthetically made, sometimes termed an artificial cell).
[0147] A cell can be in vitro. A cell can be in vivo. A cell can be
an isolated cell. A cell can be a cell inside of an organism. A
cell can be an organism. A cell can be a cell in a cell culture. A
cell can be one of a collection of cells. A cell can be a
prokaryotic cell or derived from a prokaryotic cell. A cell can be
a bacterial cell or can be derived from a bacterial cell. A cell
can be an archaeal cell or derived from an archaeal cell. A cell
can be a eukaryotic cell or derived from a eukaryotic cell. A cell
can be a plant cell or derived from a plant cell. A cell can be an
animal cell or derived from an animal cell. A cell can be an
invertebrate cell or derived from an invertebrate cell. A cell can
be a vertebrate cell or derived from a vertebrate cell. A cell can
be a mammalian cell or derived from a mammalian cell. A cell can be
a rodent cell or derived from a rodent cell. A cell can be a human
cell or derived from a human cell. A cell can be a microbe cell or
derived from a microbe cell. A cell can be a fungi cell or derived
from a fungi cell.
[0148] A cell can be a stem cell or progenitor cell. Cells can
include stem cells (e.g., adult stem cells, embryonic stem cells,
iPS cells) and progenitor cells (e.g., cardiac progenitor cells,
neural progenitor cells, etc.). Cells can include mammalian stem
cells and progenitor cells, including rodent stem cells, rodent
progenitor cells, human stem cells, human progenitor cells, etc.
Clonal cells can comprise the progeny of a cell. A cell can
comprise a target nucleic acid. A cell can be in a living organism.
A cell can be a genetically modified cell. A cell can be a host
cell.
[0149] A cell can be a totipotent stem cell, however, in some
embodiments of this disclosure, the term "cell" may be used but may
not refer to a totipotent stem cell. A cell can be a plant cell,
but in some embodiments of this disclosure, the term "cell" may be
used but may not refer to a plant cell. A cell can be a pluripotent
cell. For example, a cell can be a pluripotent hematopoietic cell
that can differentiate into other cells in the hematopoietic cell
lineage but may not be able to differentiate into any other
non-hematopoetic cell. A cell may be able to develop into a whole
organism. A cell may or may not be able to develop into a whole
organism. A cell may be a whole organism.
[0150] A cell can be a primary cell. For example, cultures of
primary cells can be passaged 0 times, 1 time, 2 times, 4 times, 5
times, 10 times, 15 times or more. Cells can be unicellular
organisms. Cells can be grown in culture.
[0151] A cell can be a diseased cell. A diseased cell can have
altered metabolic, gene expression, and/or morphologic features. A
diseased cell can be a cancer cell, a a diabetic cell, and a
apoptotic cell. A diseased cell can be a cell from a diseased
subject. Exemplary diseases can include blood disorders, cancers,
metabolic disorders, eye disorders, organ disorders,
musculoskeletal disorders, cardiac disease, and the like.
[0152] If the cells are primary cells, they may be harvested from
an individual by any method. For example, leukocytes may be
harvested by apheresis, leukocytapheresis, density gradient
separation, etc. Cells from tissues such as skin, muscle, bone
marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. can
be harvested by biopsy. An appropriate solution may be used for
dispersion or suspension of the harvested cells. Such solution can
generally be a balanced salt solution, (e.g. normal saline,
phosphate-buffered saline (PBS), Hank's balanced salt solution,
etc.), conveniently supplemented with fetal calf serum or other
naturally occurring factors, in conjunction with an acceptable
buffer at low concentration. Buffers can include HEPES, phosphate
buffers, lactate buffers, etc. Cells may be used immediately, or
they may be stored (e.g., by freezing). Frozen cells can be thawed
and can be capable of being reused. Cells can be frozen in a DMSO,
serum, medium buffer (e.g., 10% DMSO, 50% serum, 40% buffered
medium), and/or some other such common solution used to preserve
cells at freezing temperatures.
[0153] As used herein, "conditionally enzymatically inactive
site-directed polypeptide" can generally refer to a polypeptide
that can bind to a nucleic acid sequence in a polynucleotide in a
sequence-specific manner, but may not cleave a target
polynucleotide except under one or more conditions that render the
enzymatic domain active. A conditionally enzymatically inactive
site-directed polypeptide can comprise an enzymatically inactive
domain that can be conditionally activated. A conditionally
enzymatically inactive site-directed polypeptide can be
conditionally activated in the presence of imidazole. A
conditionally enzymatically inactive site-directed polypeptide can
comprise a mutated active site that fails to bind its cognate
ligand, resulting in an enzymatically inactive site-directed
polypeptide. The mutated active site can be designed to bind to
ligand analogues, such that a ligand analogue can bind to the
mutated active site and reactive the site-directed polypeptide. For
example, ATP binding proteins can comprise a mutated active site
that can inhibit activity of the protein, yet are designed to
specifically bind to ATP analogues. Binding of an ATP analogue, but
not ATP, can reactivate the protein. The conditionally
enzymatically inactive site-directed polypeptide can comprise one
or more non-native sequences (e.g., a fusion, an affinity tag).
[0154] As used herein, "crRNA" can generally refer to a nucleic
acid with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 100% sequence identity and/or sequence similarity to a
wild type exemplary crRNA (e.g., a crRNA from S. pyogenes (e.g.,
SEQ ID NO: 569, SEQ ID NO: 563-679). crRNA can generally refer to a
nucleic acid with at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 100% sequence identity and/or sequence similarity to
a wild type exemplary crRNA (e.g., a crRNA from S. pyogenes). crRNA
can refer to a modified form of a crRNA that can comprise an
nucleotide change such as a deletion, insertion, or substitution,
variant, mutation, or chimera. A crRNA can be a nucleic acid having
at least about 60% identical to a wild type exemplary crRNA (e.g.,
a crRNA from S. pyogenes) sequence over a stretch of at least 6
contiguous nucleotides. For example, a crRNA sequence can be at
least about 60% identical, at least about 65% identical, at least
about 70% identical, at least about 75% identical, at least about
80% identical, at least about 85% identical, at least about 90%
identical, at least about 95% identical, at least about 98%
identical, at least about 99% identical, or 100% identical, to a
wild type exemplary crRNA sequence (e.g., a crRNA from S. pyogenes)
over a stretch of at least 6 contiguous nucleotides.
[0155] As used herein, "CRISPR repeat" or "CRISPR repeat sequence"
can refer to a minimum CRISPR repeat sequence.
[0156] As used herein, "Csy4" can generally refer to a polypeptide
with at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
100% sequence identity and/or sequence similarity to a wild type
exemplary Csy4 polypeptide (e.g., Csy4 from P. aeruginosa, see FIG.
40). Csy4 can generally refer to can refer to a polypeptide with at
least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%
sequence identity and/or sequence similarity to a wild type
exemplary Csy4 polypeptide (e.g., Csy4 from P. aeruginosa). Csy4
can refer to the wildtype or a modified form of the Csy4 protein
that can comprise an amino acid change such as a deletion,
insertion, substitution, variant, mutation, fusion, chimera, or any
combination thereof.
[0157] As used herein, "endoribonuclease" can generally refer to a
polypeptide that can cleave RNA. In some embodiments, an
endoribonuclease can be a site-directed polypeptide. An
endoribonuclease may be a member of a CRISPR system (e.g., Type I,
Type II, Type III). Endoribonuclease can refer to a Repeat
Associated Mysterious Protein (RAMP) superfamily of proteins (e.g.,
Cas6, Cas6, Cas5 families). Endoribonucleases can also include
RNase A, RNase H, RNase I, RNase III family members (e.g., Drosha,
Dicer, RNase N), RNase L, RNase P, RNase PhyM, RNase T1, RNase T2,
RNase U2, RNase V1, RNase V. An endoribonuclease can refer to a
conditionally enzymatically inactive endoribonuclease. An
endoribonuclease can refer to a catalytically inactive
endoribonuclease.
[0158] As used herein, "donor polynucleotide" can refer to a
nucleic acid that can be integrated into a site during genome
engineering or target nucleic acid engineering.
[0159] As used herein, "fixative" or "cross-linker" can generally
refer to an agent that can fix or cross-link cells. Fixed or
cross-linking cells can stabilize protein-nucleic acid complexes in
the cell. Suitable fixatives and cross-linkers can include,
formaldehyde, glutaraldehyde, ethanol-based fixatives,
methanol-based fixatives, acetone, acetic acid, osmium tetraoxide,
potassium dichromate, chromic acid, potassium permanganate,
mercurials, picrates, formalin, paraformaldehyde, amine-reactive
NHS-ester crosslinkers such as bis[sulfosuccinimidyl]suberate
(BS3), 3,3''-dithiobis[sulfosuccinimidylpropionate] (DTSSP),
ethylene glycol bis[sulfosuccinimidylsuccinate (sulfo-EGS),
disuccinimidyl glutarate (DSG), dithiobis[succinimidyl propionate]
(DSP), disuccinimidyl suberate (DSS), ethylene glycol
bis[succinimidylsuccinate] (EGS), NHS-ester/diazirine crosslinkers
such as NHS-diazirine, NHS-LC-diazirine, NHS-SS-diazirine,
sulfo-NHS-diazirine, sulfo-NHS-LC-diazirine, and
sulfo-NHS-SS-diazirine.
[0160] As used herein, "fusion" can refer to a protein and/or
nucleic acid comprising one or more non-native sequences (e.g.,
moieties). A fusion can comprise one or more of the same non-native
sequences. A fusion can comprise one or more of different
non-native sequences. A fusion can be a chimera. A fusion can
comprise a nucleic acid affinity tag. A fusion can comprise a
barcode. A fusion can comprise a peptide affinity tag. A fusion can
provide for subcellular localization of the site-directed
polypeptide (e.g., a nuclear localization signal (NLS) for
targeting to the nucleus, a mitochondrial localization signal for
targeting to the mitochondria, a chloroplast localization signal
for targeting to a chloroplast, an endoplasmic reticulum (ER)
retention signal, and the like). A fusion can provide a non-native
sequence (e.g., affinity tag) that can be used to track or purify.
A fusion can be a small molecule such as biotin or a dye such as
alexa fluor dyes, Cyanine3 dye, Cyanine5 dye. The fusion can
provide for increased or decreased stability.
[0161] In some embodiments, a fusion can comprise a detectable
label, including a moiety that can provide a detectable signal.
Suitable detectable labels and/or moieties that can provide a
detectable signal can include, but are not limited to, an enzyme, a
radioisotope, a member of a specific binding pair; a fluorophore; a
fluorescent protein; a quantum dot; and the like.
[0162] A fusion can comprise a member of a FRET pair. FRET pairs
(donor/acceptor) suitable for use can include, but are not limited
to, EDANS/fluorescein, IAEDANS/fluorescein,
fluorescein/tetramethylrhodamine, fluorescein/Cy 5, IEDANS/DABCYL,
fluorescein/QSY-7, fluorescein/LC Red 640, fluorescein/Cy 5.5 and
fluorescein/LC Red 705.
[0163] A fluorophore/quantum dot donor/acceptor pair can be used as
a fusion. Suitable fluorophores ("fluorescent label") can include
any molecule that may be detected via its inherent fluorescent
properties, which can include fluorescence detectable upon
excitation. Suitable fluorescent labels can include, but are not
limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin,
erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green,
stilbene, Lucifer Yellow, Cascade Blue.TM., Texas Red, IAEDANS,
EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705 and Oregon
green.
[0164] A fusion can comprise an enzyme. Suitable enzymes can
include, but are not limited to, horse radish peroxidase,
luciferase, beta-galactosidase, and the like.
[0165] A fusion can comprise a fluorescent protein. Suitable
fluorescent proteins can include, but are not limited to, a green
fluorescent protein (GFP), (e.g., a GFP from Aequoria victoria,
fluorescent proteins from Anguilla japonica, or a mutant or
derivative thereof), a red fluorescent protein, a yellow
fluorescent protein, any of a variety of fluorescent and colored
proteins.
[0166] A fusion can comprise a nanoparticle. Suitable nanoparticles
can include fluorescent or luminescent nanoparticles, and magnetic
nanoparticles. Any optical or magnetic property or characteristic
of the nanoparticle(s) can be detected.
[0167] A fusion can comprise quantum dots (QDs). QDs can be
rendered water soluble by applying coating layers comprising a
variety of different materials. For example, QDs can be solubilized
using amphiphilic polymers. Exemplary polymers that have been
employed can include octylamine-modified low molecular weight
polyacrylic acid, polyethylene-glycol (PEG)-derivatized
phospholipids, polyanhydrides, block copolymers, etc. QDs can be
conjugated to a polypeptide via any of a number of different
functional groups or linking agents that can be directly or
indirectly linked to a coating layer. QDs with a wide variety of
absorption and emission spectra are commercially available, e.g.,
from Quantum Dot Corp. (Hayward Calif.; now owned by Invitrogen) or
from Evident Technologies (Troy, N.Y.). For example, QDs having
peak emission wavelengths of approximately 525, 535, 545, 565, 585,
605, 655, 705, and 800 nm are available. Thus the QDs can have a
range of different colors across the visible portion of the
spectrum and in some cases even beyond.
[0168] Suitable radioisotopes can include, but are not limited to
.sup.14C, .sup.3H, .sup.32P, .sup.33P, .sup.35S, and .sup.125I.
[0169] As used herein, "genetically modified cell" can generally
refer to a cell that has been genetically modified. Some
non-limiting examples of genetic modifications can include:
insertions, deletions, inversions, translocations, gene fusions, or
changing one or more nucleotides. A genetically modified cell can
comprise a target nucleic acid with an introduced double strand
break (e.g., DNA break). A genetically modified cell can comprise
an exogenously introduced nucleic acid (e.g., a vector). A
genetically modified cell can comprise an exogenously introduced
polypeptide of the disclosure and/or nucleic acid of the
disclosure. A genetically modified cell can comprise a donor
polynucleotide. A genetically modified cell can comprise an
exogenous nucleic acid integrated into the genome of the
genetically modified cell. A gentetically modified cell can
comprise a deletion of DNA. A genetically modified cell can also
refer to a cell with modified mitochondrial or chloroplast DNA.
[0170] As used herein, "genome engineering" can refer to a process
of modifying a target nucleic acid. Genome engineering can refer to
the integration of non-native nucleic acid into native nucleic
acid. Genome engineering can refer to the targeting of a
site-directed polypeptide and a nucleic acid-targeting nucleic acid
to a target nucleic acid, without an integration or a deletion of
the target nucleic acid. Genome engineering can refer to the
cleavage of a target nucleic acid, and the rejoining of the target
nucleic acid without an integration of an exogenous sequence in the
target nucleic acid, or a deletion in the target nucleic acid. The
native nucleic acid can comprise a gene. The non-native nucleic
acid can comprise a donor polynucleotide. In the methods of the
disclosure, site-directed polypeptides (e.g., Cas9) can introduce
double-stranded breaks in nucleic acid, (e.g. genomic DNA). The
double-stranded break can stimulate a cell's endogenous DNA-repair
pathways (e.g. homologous recombination (HR) and/or non-homologous
end joining (NHEJ), or A-NHEJ (alternative non-homologous
end-joining)). Mutations, deletions, alterations, and integrations
of foreign, exogenous, and/or alternative nucleic acid can be
introduced into the site of the double-stranded DNA break.
[0171] As used herein, the term "isolated" can refer to a nucleic
acid or polypeptide that, by the hand of a human, exists apart from
its native environment and is therefore not a product of nature.
Isolated can mean substantially pure. An isolated nucleic acid or
polypeptide can exist in a purified form and/or can exist in a
non-native environment such as, for example, in a transgenic
cell.
[0172] As used herein, "non-native" can refer to a nucleic acid or
polypeptide sequence that is not found in a native nucleic acid or
protein. Non-native can refer to affinity tags. Non-native can
refer to fusions. Non-native can refer to a naturally occurring
nucleic acid or polypeptide sequence that comprises mutations,
insertions and/or deletions. A non-native sequence may exhibit
and/or encode for an activity (e.g., enzymatic activity,
methyltransferase activity, acetyltransferase activity, kinase
activity, ubiquitinating activity, etc.) that can also be exhibited
by the nucleic acid and/or polypeptide sequence to which the
non-native sequence is fused. A non-native nucleic acid or
polypeptide sequence may be linked to a naturally-occurring nucleic
acid or polypeptide sequence (or a variant thereof) by genetic
engineering to generate a chimeric nucleic acid and/or polypeptide
sequence encoding a chimeric nucleic acid and/or polypeptide. A
non-native sequence can refer to a 3' hybridizing extension
sequence.
[0173] As used herein, a "nucleic acid" can generally refer to a
polynucleotide sequence, or fragment thereof. A nucleic acid can
comprise nucleotides. A nucleic acid can be exogenous or endogenous
to a cell. A nucleic acid can exist in a cell-free environment. A
nucleic acid can be a gene or fragment thereof. A nucleic acid can
be DNA. A nucleic acid can be RNA. A nucleic acid can comprise one
or more analogs (e.g. altered backgone, sugar, or nucleobase). Some
non-limiting examples of analogs include: 5-bromouracil, peptide
nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids,
glycol nucleic acids, threose nucleic acids, dideoxynucleotides,
cordycepin, 7-deaza-GTP, florophores (e.g. rhodamine or flurescein
linked to the sugar), thiol containing nucleotides, biotin linked
nucleotides, fluorescent base analogs, CpG islands,
methyl-7-guanosine, methylated nucleotides, inosine, thiouridine,
pseudourdine, dihydrouridine, queuosine, and wyosine.
[0174] As used herein, a "nucleic acid sample" can generally refer
to a sample from a biological entity. A nucleic acid sample can
comprise nucleic acid. The nucleic acid from the nucleic acid
sample can be purified and/or enriched. The nucleic acid sample may
show the nature of the whole. Nucleic acid samples can come from
various sources. Nucleic acid samples can come from one or more
individuals. One or more nucleic acid samples can come from the
same individual. One non limiting example would be if one sample
came from an individual's blood and a second sample came from an
individual's tumor biopsy. Examples of nucleic acid samples can
include but are not limited to, blood, serum, plasma, nasal swab or
nasopharyngeal wash, saliva, urine, gastric fluid, spinal fluid,
tears, stool, mucus, sweat, earwax, oil, glandular secretion,
cerebral spinal fluid, tissue, semen, vaginal fluid, interstitial
fluids, including interstitial fluids derived from tumor tissue,
ocular fluids, spinal fluid, throat swab, cheeck swab, breath,
hair, finger nails, skin, biopsy, placental fluid, amniotic fluid,
cord blood, emphatic fluids, cavity fluids, sputum, pus,
micropiota, meconium, breast milk, buccal samples, nasopharyngeal
wash, other excretions, or any combination thereof. Nucleic acid
samples can originate from tissues. Examples of tissue samples may
include but are not limited to, connective tissue, muscle tissue,
nervous tissue, epithelial tissue, cartilage, cancerous or tumor
sample, bone marrow, or bone. The nucleic acid sample may be
provided from a human or animal. The nucleic acid sample may be
provided from a mammal, vertebrate, such as murines, simians,
humans, farm animals, sport animals, or pets. The nucleic acid
sample may be collected from a living or dead subject. The nucleic
acid sample may be collected fresh from a subject or may have
undergone some form of pre-processing, storage, or transport.
[0175] A nucleic acid sample can comprise a target nucleic acid. A
nucleic acid sample can originate from cell lysate. The cell lysate
can originate from a cell.
[0176] As used herein, "nucleic acid-targeting nucleic acid" can
refer to a nucleic acid that can hybridize to another nucleic acid.
A nucleic acid-targeting nucleic acid can be RNA. A nucleic
acid-targeting nucleic acid can be DNA. The nucleic acid-targeting
nucleic acid can be programmed to bind to a sequence of nucleic
acid site-specifically. The nucleic acid to be targeted, or the
target nucleic acid, can comprise nucleotides. The nucleic
acid-targeting nucleic acid can comprise nucleotides. A portion of
the target nucleic acid can be complementary to a portion of the
nucleic acid-targeting nucleic acid. A nucleic acid-targeting
nucleic acid can comprise a polynucleotide chain and can be called
a "single guide nucleic acid" (i.e. a "single guide nucleic
acid-targeting nucleic acid"). A nucleic acid-targeting nucleic
acid can comprise two polynucleotide chains and can be called a
"double guide nucleic acid" (i.e. a "double guide nucleic
acid-targeting nucleic acid"). If not otherwise specified, the term
"nucleic acid-targeting nucleic acid" can be inclusive, referring
to both single guide nucleic acids and double guide nucleic
acids.
[0177] A nucleic acid-targeting nucleic acid can comprise a segment
that can be referred to as a "nucleic acid-targeting segment" or a
"nucleic acid-targeting sequence," A nucleic acid-targeting nucleic
acid can comprise a segment that can be referred to as a "protein
binding segment" or "protein binding sequence."
[0178] A nucleic acid-targeting nucleic acid can comprise one or
more modifications (e.g., a base modification, a backbone
modification), to provide the nucleic acid with a new or enhanced
feature (e.g., improved stability). A nucleic acid-targeting
nucleic acid can comprise a nucleic acid affinity tag. A nucleoside
can be a base-sugar combination. The base portion of the nucleoside
can be a heterocyclic base. The two most common classes of such
heterocyclic bases are the purines and the pyrimidines. Nucleotides
can be nucleosides that further include a phosphate group
covalently linked to the sugar portion of the nucleoside. For those
nucleosides that include a pentofuranosyl sugar, the phosphate
group can be linked to the 2', the 3', or the 5' hydroxyl moiety of
the sugar. In forming nucleic acid-targeting nucleic acids, the
phosphate groups can covalently link adjacent nucleosides to one
another to form a linear polymeric compound. In turn, the
respective ends of this linear polymeric compound can be further
joined to form a circular compound; however, linear compounds are
generally suitable. In addition, linear compounds may have internal
nucleotide base complementarity and may therefore fold in a manner
as to produce a fully or partially double-stranded compound. Within
nucleic acid-targeting nucleic acids, the phosphate groups can
commonly be referred to as forming the internucleoside backbone of
the nucleic acid-targeting nucleic acid. The linkage or backbone of
the nucleic acid-targeting nucleic acid can be a 3' to 5'
phosphodiester linkage.
[0179] A nucleic acid-targeting nucleic acid can comprise a
modified backbone and/or modified internucleoside linkages.
Modified backbones can include those that retain a phosphorus atom
in the backbone and those that do not have a phosphorus atom in the
backbone.
[0180] Suitable modified nucleic acid-targeting nucleic acid
backbones containing a phosphorus atom therein can include, for
example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,
methyl and other alkyl phosphonates such as 3'-alkylene
phosphonates, 5'-alkylene phosphonates, chiral phosphonates,
phosphinates, phosphoramidates including 3'-amino phosphoramidate
and aminoalkylphosphoramidates, phosphorodiamidates,
thionophosphoramidates, thionoalkylphosphonates,
thionoalkylphosphotriesters, selenophosphates, and boranophosphates
having normal 3'-5' linkages, 2'-5' linked analogs, and those
having inverted polarity wherein one or more internucleotide
linkages is a 3' to 3', a 5' to 5' or a 2' to 2' linkage. Suitable
nucleic acid-targeting nucleic acids having inverted polarity can
comprise a single 3' to 3' linkage at the 3'-most internucleotide
linkage (i.e. a single inverted nucleoside residue in which the
nucleobase is missing or has a hydroxyl group in place thereof).
Various salts (e.g., potassium chloride or sodium chloride), mixed
salts, and free acid forms can also be included.
[0181] A nucleic acid-targeting nucleic acid can comprise one or
more phosphorothioate and/or heteroatom internucleoside linkages,
in particular --CH.sub.2--NH--O--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- (i.e. a methylene
(methylimino) or MMI backbone),
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2-- (wherein the native
phosphodiester internucleotide linkage is represented as
--O--P(.dbd.O)(OH)--O--CH.sub.2--).
[0182] A nucleic acid-targeting nucleic acid can comprise a
morpholino backbone structure. For example, a nucleic acid can
comprise a 6-membered morpholino ring in place of a ribose ring. In
some of these embodiments, a phosphorodiamidate or other
non-phosphodiester internucleoside linkage can replace a
phosphodiester linkage.
[0183] A nucleic acid-targeting nucleic acid can comprise
polynucleotide backbones that are formed by short chain alkyl or
cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or
cycloalkyl internucleoside linkages, or one or more short chain
heteroatomic or heterocyclic internucleoside linkages. These can
include those having morpholino linkages (formed in part from the
sugar portion of a nucleoside); siloxane backbones; sulfide,
sulfoxide and sulfone backbones; formacetyl and thioformacetyl
backbones; methylene formacetyl and thioformacetyl backbones;
riboacetyl backbones; alkene containing backbones; sulfamate
backbones; methyleneimino and methylenehydrazino backbones;
sulfonate and sulfonamide backbones; amide backbones; and others
having mixed N, O, S and CH.sub.2 component parts.
[0184] A nucleic acid-targeting nucleic acid can comprise a nucleic
acid mimetic. The term "mimetic" can be intended to include
polynucleotides wherein only the furanose ring or both the furanose
ring and the internucleotide linkage are replaced with non-furanose
groups, replacement of only the furanose ring can also be referred
as being a sugar surrogate. The heterocyclic base moiety or a
modified heterocyclic base moiety can be maintained for
hybridization with an appropriate target nucleic acid. One such
nucleic acid can be a peptide nucleic acid (PNA). In a PNA, the
sugar-backbone of a polynucleotide can be replaced with an amide
containing backbone, in particular an aminoethylglycine backbone.
The nucleotides can be retained and are bound directly or
indirectly to aza nitrogen atoms of the amide portion of the
backbone. The backbone in PNA compounds can comprise two or more
linked aminoethylglycine units which gives PNA an amide containing
backbone. The heterocyclic base moieties can be bound directly or
indirectly to aza nitrogen atoms of the amide portion of the
backbone.
[0185] A nucleic acid-targeting nucleic acid can comprise linked
morpholino units (i.e. morpholino nucleic acid) having heterocyclic
bases attached to the morpholino ring Linking groups can link the
morpholino monomeric units in a morpholino nucleic acid. Non-ionic
morpholino-based oligomeric compounds can have less undesired
interactions with cellular proteins. Morpholino-based
polynucleotides can be nonionic mimics of nucleic acid-targeting
nucleic acids. A variety of compounds within the morpholino class
can be joined using different linking groups. A further class of
polynucleotide mimetic can be referred to as cyclohexenyl nucleic
acids (CeNA). The furanose ring normally present in a nucleic acid
molecule can be replaced with a cyclohexenyl ring. CeNA DMT
protected phosphoramidite monomers can be prepared and used for
oligomeric compound synthesis using phosphoramidite chemistry. The
incorporation of CeNA monomers into a nucleic acid chain can
increase the stability of a DNA/RNA hybrid. CeNA oligoadenylates
can form complexes with nucleic acid complements with similar
stability to the native complexes. A further modification can
include Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group
is linked to the 4' carbon atom of the sugar ring thereby forming a
2'-C,4'-C-oxymethylene linkage thereby forming a bicyclic sugar
moiety. The linkage can be a methylene (--CH2-), group bridging the
2' oxygen atom and the 4' carbon atom wherein n is 1 or 2. LNA and
LNA analogs can display very high duplex thermal stabilities with
complementary nucleic acid (Tm=+3 to +10.degree. C.), stability
towards 3'-exonucleolytic degradation and good solubility
properties.
[0186] A nucleic acid-targeting nucleic acid can comprise one or
more substituted sugar moieties. Suitable polynucleotides can
comprise a sugar substituent group selected from: OH; F; O-, S-, or
N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or
O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be
substituted or unsubstituted C.sub.1 to C.sub.10 alkyl or C.sub.2
to C.sub.10 alkenyl and alkynyl. Particularly suitable are
O((CH2)nO)mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3,
O(CH.sub.2).sub.nNH2, O(CH.sub.2).sub.1CH.sub.3,
O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON((CH.sub.2).sub.nCH.sub.3).sub.2, where n and m
are from 1 to about 10. A sugar substituent group can be selected
from: C.sub.1 to C.sub.10 lower alkyl, substituted lower alkyl,
alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,
SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3,
SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an nucleic acid-targeting nucleic
acid, or a group for improving the pharmacodynamic properties of an
nucleic acid-targeting nucleic acid, and other substituents having
similar properties. A suitable modification can include
2'-methoxyethoxy (2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE i.e., an alkoxyalkoxy group). A
further suitable modification can include
2'-dimethylaminooxyethoxy, (i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE),
and 2'-dimethylaminoethoxyethoxy (also known as
2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e.,
2'-O--CH2-O--CH.sub.2--N(CH.sub.3).sub.2.
[0187] Other suitable sugar substituent groups can include methoxy
(--O--CH.sub.3), aminopropoxy CH.sub.2CH.sub.2CH.sub.2NH.sub.2),
allyl (--CH.sub.2--CH.dbd.CH.sub.2), --O-allyl
CH.sub.2--CH.dbd.CH.sub.2) and fluoro (F). 2'-sugar substituent
groups may be in the arabino (up) position or ribo (down) position.
A suitable 2'-arabino modification is 2'-F. Similar modifications
may also be made at other positions on the oligomeric compound,
particularly the 3' position of the sugar on the 3' terminal
nucleoside or in 2'-5' linked nucleotides and the 5' position of 5'
terminal nucleotide. Oligomeric compounds may also have sugar
mimetics such as cyclobutyl moieties in place of the pentofuranosyl
sugar.
[0188] A nucleic acid-targeting nucleic acid may also include
nucleobase (often referred to simply as "base") modifications or
substitutions. As used herein, "unmodified" or "natural"
nucleobases can include the purine bases, (e.g. adenine (A) and
guanine (G)), and the pyrimidine bases, (e.g. thymine (T), cytosine
(C) and uracil (U)). Modified nucleobases can include other
synthetic and natural nucleobases such as 5-methylcytosine
(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,
2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and
guanine, 2-propyl and other alkyl derivatives of adenine and
guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,
5-halouracil and cytosine, 5-propynyl (--C.dbd.C--CH3) uracil and
cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo
uracil, cytosine and thymine, 5-uracil (pseudouracil),
4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and
other 8-substituted adenines and guanines, 5-halo particularly
5-bromo, 5-trifluoromethyl and other 5-substituted uracils and
cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,
2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and
7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Modified
nucleobases can include tricyclic pyrimidines such as phenoxazine
cytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one),
phenothiazine cytidine
(1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as a
substituted phenoxazine cytidine (e.g.
9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one),
carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole
cytidine (Hpyrido(3',2':4,5)pyrrolo(2,3-d)pyrimidin-2-one).
[0189] Heterocyclic base moieties can include those in which the
purine or pyrimidine base is replaced with other heterocycles, for
example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and
2-pyridone. Nucleobases can be useful for increasing the binding
affinity of a polynucleotide compound. These can include
5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6
substituted purines, including 2-aminopropyladenine,
5-propynyluracil and 5-propynylcytosine. 5-methylcytosine
substitutions can increase nucleic acid duplex stability by
0.6-1.2.degree. C. and can be suitable base substitutions (e.g.,
when combined with 2'-O-methoxyethyl sugar modifications).
[0190] A modification of a nucleic acid-targeting nucleic acid can
comprise chemically linking to the nucleic acid-targeting nucleic
acid one or more moieties or conjugates that can enhance the
activity, cellular distribution or cellular uptake of the nucleic
acid-targeting nucleic acid. These moieties or conjugates can
include conjugate groups covalently bound to functional groups such
as primary or secondary hydroxyl groups. Conjugate groups can
include, but are not limited to, intercalators, reporter molecules,
polyamines, polyamides, polyethylene glycols, polyethers, groups
that enhance the pharmacodynamic properties of oligomers, and
groups that can enhance the pharmacokinetic properties of
oligomers. Conjugate groups can include, but are not limited to,
cholesterols, lipids, phospholipids, biotin, phenazine, folate,
phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,
coumarins, and dyes. Groups that enhance the pharmacodynamic
properties include groups that improve uptake, enhance resistance
to degradation, and/or strengthen sequence-specific hybridization
with the target nucleic acid. Groups that can enhance the
pharmacokinetic properties include groups that improve uptake,
distribution, metabolism or excretion of a nucleic acid. Conjugate
moieties can include but are not limited to lipid moieties such as
a cholesterol moiety, cholic acid a thioether, (e.g.,
hexyl-S-tritylthiol), a thiocholesterol, an aliphatic chain (e.g.,
dodecandiol or undecyl residues), a phospholipid (e.g.,
di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate), a polyamine or a
polyethylene glycol chain, or adamantane acetic acid, a palmityl
moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol
moiety.
[0191] A modification may include a "Protein Transduction Domain"
or PTD (i.e. a cell penetrating peptide (CPP)). The PTD can refer
to a polypeptide, polynucleotide, carbohydrate, or organic or
inorganic compound that facilitates traversing a lipid bilayer,
micelle, cell membrane, organelle membrane, or vesicle membrane. A
PTD can be attached to another molecule, which can range from a
small polar molecule to a large macromolecule and/or a
nanoparticle, and can facilitate the molecule traversing a
membrane, for example going from extracellular space to
intracellular space, or cytosol to within an organelle. A PTD can
be covalently linked to the amino terminus of a polypeptide. A PTD
can be covalently linked to the carboxyl terminus of a polypeptide.
A PTD can be covalently linked to a nucleic acid. Exemplary PTDs
can include, but are not limited to, a minimal peptide protein
transduction domain; a polyarginine sequence comprising a number of
arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6,
7, 8, 9, 10, or 10-50 arginines), a VP22 domain, a Drosophila
Antennapedia protein transduction domain, a truncated human
calcitonin peptide, polylysine, and transportan, arginine
homopolymer of from 3 arginine residues to 50 arginine residues.
The PTD can be an activatable CPP (ACPP). ACPPs can comprise a
polycationic CPP (e.g., Arg9 or "R9") connected via a cleavable
linker to a matching polyanion (e.g., G1u9 or "E9"), which can
reduce the net charge to nearly zero and thereby inhibits adhesion
and uptake into cells. Upon cleavage of the linker, the polyanion
can be released, locally unmasking the polyarginine and its
inherent adhesiveness, thus "activating" the ACPP to traverse the
membrane.
[0192] "Nucleotide" can generally refer to a base-sugar-phosphate
combination. A nucleotide can comprise a synthetic nucleotide. A
nucleotide can comprise a synthetic nucleotide analog. Nucleotides
can be monomeric units of a nucleic acid sequence (e.g.
deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term
nucleotide can include ribonucleoside triphosphates adenosine
triphosphate (ATP), uridine triphosphate (UTP), cytosine
triphosphate (CTP), guanosine triphosphate (GTP) and
deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP,
dGTP, dTTP, or derivatives thereof. Such derivatives can include,
for example, [aS]dATP, 7-deaza-dGTP and 7-deaza-dATP, and
nucleotide derivatives that confer nuclease resistance on the
nucleic acid molecule containing them. The term nucleotide as used
herein can refer to dideoxyribonucleoside triphosphates (ddNTPs)
and their derivatives. Illustrative examples of
dideoxyribonucleoside triphosphates can include, but are not
limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. A nucleotide may
be unlabeled or detectably labeled by well-known techniques.
Labeling can also be carried out with quantum dots. Detectable
labels can include, for example, radioactive isotopes, fluorescent
labels, chemiluminescent labels, bioluminescent labels and enzyme
labels. Fluorescent labels of nucleotides may include but are not
limited fluorescein, 5-carboxyfluorescein (FAM),
2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine,
6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine
(TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'dimethylaminophenylazo)
benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red,
Cyanine and 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid
(EDANS). Specific examples of fluorescently labeled nucleotides can
include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP,
[JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP,
[ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and
[dROX]ddTTP available from Perkin Elmer, Foster City, Calif.
FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink
Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and
FluoroLink Cy5-dUTP available from Amersham, Arlington Heights,
Ill.; Fluorescein-15-dATP, Fluorescein-12-dUTP,
Tetramethyl-rodamine-6-dUTP, IR770-9-dATP, Fluorescein-12-ddUTP,
Fluorescein-12-UTP, and Fluorescein-15-2'-dATP available from
Boehringer Mannheim, Indianapolis, Ind.; and Chromosome Labeled
Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP,
BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade
Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP,
fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine
Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP,
tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and
Texas Red-12-dUTP available from Molecular Probes, Eugene, Oreg.
Nucleotides can also be labeled or marked by chemical modification.
A chemically-modified single nucleotide can be biotin-dNTP. Some
non-limiting examples of biotinylated dNTPs can include,
biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP
(e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g.
biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).
[0193] As used herein, "P-domain" can refer to a region in a
nucleic acid-targeting nucleic acid. The P-domain can interact with
a protospacer adjacent motif (PAM), site-directed polypeptide,
and/or nucleic acid-targeting nucleic acid. A P-domain can interact
directly or indirectly with a protospacer adjacent motif (PAM),
site-directed polypeptide, and/or nucleic acid-targeting nucleic
acid nucleic acid. As used herein, the terms "PAM interacting
region" "anti-repeat adjacent region" and "P-domain" can be used
interchangeably.
[0194] As used here, "purified" can refer to a molecule (e.g.,
site-directed polypeptide, nucleic acid-targeting nucleic acid)
that comprises at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or
100% of the composition. For example, a sample that comprises 10%
of a site-directed polypeptide, but after a purification step
comprises 60% of the site-directed polypeptide, then the sample can
be said to be purified. A purified sample can refer to an enriched
sample, or a sample that has undergone methods to remove particles
other than the particle of interest.
[0195] As used herein, "reactivation agent" can generally refer to
any agent that can convert an enzymatically inactive polypeptide
into an enzymatically active polypeptide. Imidazole can be a
reactivation agent. A ligand analogue can be a reactivation
agent.
[0196] As used herein, "recombinant" can refer to sequence that
originates from a source foreign to the particular host (e.g.,
cell) or, if from the same source, is modified from its original
form. A recombinant nucleic acid in a cell can include a nucleic
acid that is endogenous to the particular cell but has been
modified through, for example, the use of site-directed
mutagenesis. The term can include non-naturally occurring multiple
copies of a naturally occurring DNA sequence. Thus, the term can
refer to a nucleic acid that is foreign or heterologous to the
cell, or homologous to the cell but in a position or form within
the cell in which the nucleic acid is not ordinarily found.
Similarly, when used in the context of a polypeptide or amino acid
sequence, an exogenous polypeptide or amino acid sequence can be a
polypeptide or amino acid sequence that originates from a source
foreign to the particular cell or, if from the same source, is
modified from its original form.
[0197] As used herein, "site-directed polypeptides" can generally
refer to nucleases, site-directed nucleases, endoribonucleases,
conditionally enzymatically inactive endoribonucleases, Argonauts,
and nucleic acid-binding proteins. A site-directed polypeptide or
protein can include nucleases such as homing endonucleases such as
PI-TliII, H-DreI, I-DmoI and I-CreI, I-SceI, LAGLIDADG family
nucleases, meganucleases, GIY-YIG family nucleases, His-Cys box
family nucleases, Vsr-like nucleases, endoribonucleases,
exoribonucleases, endonucleases, and exonucleases. A site-directed
polypeptide can refer to a Cas gene member of the Type I, Type II,
Type III, and/or Type U CRISPR/Cas systems. A site-directed
polypeptide can refer to a member of the Repeat Associated
Mysterious Protein (RAMP) superfamily (e.g., Cas5, Cas6
subfamilies). A site-directed polypeptide can refer to an Argonaute
protein.
[0198] A site-directed polypeptide can be a type of protein. A
site-directed polypeptide can refer to an nuclease. A site-directed
polypeptide can refer to an endoribonuclease. A site-directed
polypeptide can refer to any modified (e.g., shortened, mutated,
lengthened) polypeptide sequence or homologue of the site-directed
polypeptide. A site-directed polypeptide can be codon optimized. A
site-directed polypeptide can be a codon-optimized homologue of a
site-directed polypeptide. A site-directed polypeptide can be
enzymatically inactive, partially active, constitutively active,
fully active, inducible active and/or more active, (e.g. more than
the wild type homologue of the protein or polypeptide.). A
site-directed polypeptide can be Cas9. A site-directed polypeptide
can be Csy4. A site-directed polypeptide can be Cas5 or a Cas5
family member. A site-directed polypeptide can be Cas6 or a Cas6
family member.
[0199] In some instances, the site-directed polypeptide (e.g.,
variant, mutated, enzymatically inactive and/or conditionally
enzymatically inactive site-directed polypeptide) can target
nucleic acid. The site-directed polypeptide (e.g., variant,
mutated, enzymatically inactive and/or conditionally enzymatically
inactive endoribonuclease) can target RNA. Endoribonucleases that
can target RNA can include members of other CRISPR subfamilies such
as Cas6 and Cas5.
[0200] As used herein, the term "specific" can refer to interaction
of two molecules where one of the molecules through, for example
chemical or physical means, specifically binds to the second
molecule. Exemplary specific binding interactions can refer to
antigen-antibody binding, avidin-biotin binding, carbohydrates and
lectins, complementary nucleic acid sequences (e.g., hybridizing),
complementary peptide sequences including those formed by
recombinant methods, effector and receptor molecules, enzyme
cofactors and enzymes, enzyme inhibitors and enzymes, and the like.
"Non-specific" can refer to an interaction between two molecules
that is not specific.
[0201] As used herein, "solid support" can generally refer to any
insoluble, or partially soluble material. A solid support can refer
to a test strip, a multi-well dish, and the like. The solid support
can comprise a variety of substances (e.g., glass, polystyrene,
polyvinyl chloride, polypropylene, polyethylene, polycarbonate,
dextran, nylon, amylose, natural and modified celluloses,
polyacrylamides, agaroses, and magnetite) and can be provided in a
variety of forms, including agarose beads, polystyrene beads, latex
beads, magnetic beads, colloid metal particles, glass and/or
silicon chips and surfaces, nitrocellulose strips, nylon membranes,
sheets, wells of reaction trays (e.g., multi-well plates), plastic
tubes, etc. A solid support can be solid, semisolid, a bead, or a
surface. The support can mobile in a solution or can be immobile. A
solid support can be used to capture a polypeptide. A solid support
can comprise a capture agent.
[0202] As used herein, "target nucleic acid" can generally refer to
a nucleic acid to be used in the methods of the disclosure. A
target nucleic acid can refer to a chromosomal sequence or an
extrachromosomal sequence, (e.g. an episomal sequence, a minicircle
sequence, a mitochondrial sequence, a chloroplast sequence, etc.).
A target nucleic acid can be DNA. A target nucleic acid can be RNA.
A target nucleic acid can herein be used interchangeably with
"polynucleotide", "nucleotide sequence", and/or "target
polynucleotide". A target nucleic acid can be a nucleic acid
sequence that may not be related to any other sequence in a nucleic
acid sample by a single nucleotide substitution. A target nucleic
acid can be a nucleic acid sequence that may not be related to any
other sequence in a nucleic acid sample by a 2, 3, 4, 5, 6, 7, 8,
9, or 10 nucleotide substitutions. In some embodiments, the
substitution cannot occur within 5, 10, 15, 20, 25, 30, or 35
nucleotides of the 5' end of a target nucleic acid. In some
embodiments, the substitution cannot occur within 5, 10, 15, 20,
25, 30, 35 nucleotides of the 3' end of a target nucleic acid.
[0203] As used herein, "tracrRNA" can generally refer to a nucleic
acid with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 100% sequence identity and/or sequence similarity to a
wild type exemplary tracrRNA sequence (e.g., a tracrRNA from S.
pyogenes (SEQ ID 433), SEQ IDs 431-562). tracrRNA can refer to a
nucleic acid with at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 100% sequence identity and/or sequence similarity to
a wild type exemplary tracrRNA sequence (e.g., a tracrRNA from S.
pyogenes). tracrRNA can refer to a modified form of a tracrRNA that
can comprise an nucleotide change such as a deletion, insertion, or
substitution, variant, mutation, or chimera. A tracrRNA can refer
to a nucleic acid that can be at least about 60% identical to a
wild type exemplary tracrRNA (e.g., a tracrRNA from S. pyogenes)
sequence over a stretch of at least 6 contiguous nucleotides. For
example, a tracrRNA sequence can be at least about 60% identical,
at least about 65% identical, at least about 70% identical, at
least about 75% identical, at least about 80% identical, at least
about 85% identical, at least about 90% identical, at least about
95% identical, at least about 98% identical, at least about 99%
identical, or 100% identical, to a wild type exemplary tracrRNA
(e.g., a tracrRNA from S. pyogenes) sequence over a stretch of at
least 6 contiguous nucleotides. A tracrRNA can refer to a
mid-tracrRNA. A tracrRNA can refer to a minimum tracrRNA
sequence.
[0204] CRISPR Systems
[0205] A CRISPR (Clustered Regularly Interspaced Short Palindromic
Repeats) can be a genomic locus found in the genomes of many
prokaryotes (e.g., bacteria and archaea). CRISPR loci can provide
resistance to foreign invaders (e.g, virus, phage) in prokaryotes.
In this way, the CRISPR system can be thought to function as a type
of immune system to help defend prokaryotes against foreign
invaders. There can be three stages of CRISPR locus function:
integration of new sequences into the locus, biogenesis of CRISPR
RNA (crRNA), and silencing of foreign invader nucleic acid. There
can be four types of CRISPR systems (e.g., Type I, Type II, Type
III, TypeU).
[0206] A CRISPR locus can include a number of short repeating
sequences referred to as "repeats." Repeats can form hairpin
structures and/or repeats can be unstructured single-stranded
sequences. The repeats can occur in clusters. Repeats sequences can
frequently diverge between species. Repeats can be regularly
interspaced with unique intervening sequences referred to as
"spacers," resulting in a repeat-spacer-repeat locus architecture.
Spacers can be identical to or have high homology with known
foreign invader sequences. A spacer-repeat unit can encode a
crisprRNA (crRNA). A crRNA can refer to the mature form of the
spacer-repeat unit. A crRNA can comprise a "seed" sequence that can
be involved in targeting a target nucleic acid (e.g., possibly as a
surveillance mechansim against foreign nucleic acid). A seed
sequence can be located at the 5' or 3' end of the crRNA.
[0207] A CRISPR locus can comprise polynucleotide sequences
encoding for Crispr Associated Genes (Cas) genes. Cas genes can be
involved in the biogenesis and/or the interference stages of crRNA
function. Cas genes can display extreme sequence (e.g., primary
sequence) divergence between species and homologues. For example,
Cas1 homologues can comprise less than 10% primary sequence
identity between homologues. Some Cas genes can comprise homologous
secondary and/or tertiary structures. For example, despite extreme
sequence divergence, many members of the Cas6 family of CRISPR
proteins comprise a N-terminal ferredoxin-like fold. Cas genes can
be named according to the organism from which they are derived. For
example, Cas genes in Staphylococcus epidermidis can be referred to
as Csm-type, Cas genes in Streptococcus thermophilus can be
referred to as Csn-type, and Cas genes in Pyrococcus furiosus can
be referred to as Cmr-type.
[0208] Integration
[0209] The integration stage of CRISPR system can refer to the
ability of the CRISPR locus to integrate new spacers into the crRNA
array upon being infected by a foreign invader. Acquisition of the
foreign invader spacers can help confer immunity to subsequent
attacks by the same foreign invader. Integration can occur at the
leader end of the CRISPR locus. Cas proteins (e.g., Cas1 and Cas2)
can be involved in integration of new spacer sequences. Integration
can proceed similarly for some types of CRISPR systems (e.g., Type
I-III).
[0210] Biogenesis
[0211] Mature crRNAs can be processed from a longer polycistronic
CRISPR locus transcript (i.e., pre-crRNA array). A pre-crRNA array
can comprise a plurality of crRNAs. The repeats in the pre-crRNA
array can be recognized by Cas genes. Cas genes can bind to the
repeats and cleave the repeats. This action can liberate the
plurality of crRNAs. crRNAs can be subjected to further events to
produce the mature crRNA form such as trimming (e.g., with an
exonuclese). A crRNA may comprise all, some, or none of the CRISPR
repeat sequence.
[0212] Interference
[0213] Interference can refer to the stage in the CRISPR system
that is functionally responsible for combating infection by a
foreign invader. CRISPR interference can follow a similar mechanism
to RNA interference (RNAi (e.g., wherein a target RNA is targeted
(e.g., hybridzed) by a short interfering RNA (siRNA)), which can
result in target RNA degradation and/or destabilization. CRISPR
systems can perform interference of a target nucleic acid by
coupling crRNAs and Cas genes, thereby forming CRISPR
ribonucleoproteins (crRNPs). crRNA of the crRNP can guide the crRNP
to foreign invader nucleic acid, (e.g., by recognizing the foreign
invader nucleic acid through hybridization). Hybridized target
foreign invader nucleic acid-crRNA units can be subjected to
cleavage by Cas proteins. Target nucleic acid interference may
require a spacer adjacent motif (PAM) in a target nucleic acid.
[0214] Types of CRISPR Systems
[0215] There can be four types of CRISPR systems: Type I, Type II,
Type III, and Type U. More than one CRISPR type system can be found
in an organism. CRISPR systems can be complementary to each other,
and/or can lend functional units in trans to facilitate CRISPR
locus processing.
[0216] Type I CRISPR Systems
[0217] crRNA biogenesis in Type I CRISPR systems can comprise
endoribonuclease cleavage of repeats in the pre-crRNA array, which
can result in a plurality of crRNAs. crRNAs of Type I systems may
not be subjected to crRNA trimming. A crRNA can be processed from a
pre-crRNA array by a multi-protein complex called Cascade
(originating from CRISPR-associated complex for antiviral defense).
Cascade can comprise protein subunits (e.g, CasA-CasE). Some of the
subunits can be members of the Repeat Associated Mysterious Protein
(RAMP) superfamily (e.g., Cas5 and Casio families). The
Cascade-crRNA complex (i.e., interference complex) can recognize
target nucleic acid through hybridization of the crRNA with the
target nucleic acid. The Cascade interference complex can recruit
the Cas3 helicase/nuclease which can act in trans to facilitate
cleavage of target nucleic acid. The Cas3 nuclease can cleave
target nucleic acid (e.g., with its HD nuclease domain). Target
nucleic acid in a Type I CRISPR system can comprise a PAM. Target
nucleic acid in a Type I CRISPR system can be DNA.
[0218] Type I systems can be further subdivided by their species of
origin. Type I systems can comprise: Types IA (Aeropyrum pernix or
CASS5); IB (Thermotoga neapolitana-Haloarcula marismortui or
CASS7); IC (Desulfovibrio vulgaris or CASS1); ID; IE (Escherichia
coli or CASS2); and IF (Yersinia pestis or CASS3) subfamilies.
[0219] Type II CRISPR Systems
[0220] crRNA biogenesis in a Type II CRISPR system can comprise a
trans-activating CRISPR RNA (tracrRNA). A tracrRNA can be modified
by endogenous RNaseIII. The tracrRNA of the complex can hybridize
to a crRNA repeat in the pre-crRNA array. Endogenous RnaseIII can
be recruited to cleave the pre-crRNA. Cleaved crRNAs can be
subjected to exoribonuclease trimming to produce the mature crRNA
form (e.g., 5' trimming). The tracrRNA can remain hybridized to the
crRNA. The tracrRNA and the crRNA can associate with a
site-directed polypeptide (e.g., Cas9). The crRNA of the
crRNA-tracrRNA-Cas9 complex can guide the complex to a target
nucleic acid to which the crRNA can hybridize. Hybridization of the
crRNA to the target nucleic acid can activate Cas9 for target
nucleic acid cleavage. Target nucleic acid in a Type II CRISPR
system can comprise a PAM. In some embodiments, a PAM is essential
to facilitate binding of a site-directed polypeptide (e.g., Cas9)
to a target nucleic acid. Type II systems can be further subdivided
into II-A (Nmeni or CASS4) and II-B (Nmeni or CASS4).
[0221] Type III CRISPR Systems
[0222] crRNA biogenesis in Type III CRISPR systems can comprise a
step of endoribonuclease cleavage of repeats in the pre-crRNA
array, which can result in a plurality of crRNAs. Repeats in the
Type III CRISPR system can be unstructured single-stranded regions.
Repeats can be recognized and cleaved by a member of the RAMP
superfamily of endoribonucleases (e.g., Cas6). crRNAs of Type III
(e.g., Type III-B) systems may be subjected to crRNA trimming
(e.g., 3' trimming). Type III systems can comprise a
polymerase-like protein (e.g., Cas10). Cas10 can comprise a domain
homologous to a palm domain.
[0223] Type III systems can process pre-crRNA with a complex
comprising a plurality of RAMP superfamily member proteins and one
or more CRISPR polymerase-like proteins. Type III systems can be
divided into III-A and III-B. An interference complex of the Type
III-A system (i.e., Csm complex) can target plasmid nucleic acid.
Cleavage of the plasmid nucleic acid can occur with the HD nuclease
domain of a polymerase-like protein in the complex. An interference
complex of the Type III-B system (i.e., Cmr complex) can target
RNA.
[0224] Type U CRISPR Systems
[0225] Type U CRISPR systems may not comprise the signature genes
of either of the Type I-III CRISPR systems (e.g., Cas3, Cas9, Cas6,
Cas1, Cas2). Examples of Type U CRISPR Cas genes can include, but
are not limited to, Csf1, Csf2, Csf3, Csf4. Type U Cas genes may be
very distant homologues of Type I-III Cas genes. For example, Csf3
may be highly diverged but functionally similar to Cas5 family
members. A Type U system may function complementarily in trans with
a Type I-III system. In some instances, Type U systems may not be
associated with processing CRISPR arrays. Type U systems may
represent an alternative foreign invader defense system.
[0226] RAMP Superfamily
[0227] Repeat Associated Mysterious Proteins (RAMP proteins) can be
characterized by a protein fold comprising a
.beta..alpha..beta..beta..alpha..beta.
[beta-alpha-beta-beta-alpha-beta] motif of .beta.-strands (.beta.)
and .alpha.-helices (.alpha.). A RAMP protein can comprise an RNA
recognition motif (RRM) (which can comprise a ferredoxin or
ferredoxin-like fold). RAMP proteins can comprise an N-terminal
RRM. The C-terminal domain of RAMP proteins can vary, but can also
comprise an RRM. RAMP family members can recognize structured
and/or unstructured nucleic acid. RAMP family members can recognize
single-stranded and/or double-stranded nucleic acid. RAMP proteins
can be involved in the biogenesis and/or the interference stage of
CRISPR Type I and Type III systems. RAMP superfamily members can
comprise members of the Cas7, Cas6, and Cas5 families. RAMP
superfamily members can be endoribonucleases.
[0228] RRM domains in the RAMP superfamily can be extremely
divergent. RRM domains can comprise at least about 5%, at least
about 10%, at least about 15%, at least about 20%, at least about
25%, at least about 30%, at least about 35%, at least about 40%, at
least about 45%, at least about 50%, at least about 55%, at least
about 60%, at least about 65%, at least about 70%, at least about
75%, at least about 80%, at least about 85%, at least about 90%, at
least about 95%, or 100% sequence or structural homology to a wild
type exemplary RRM domain (e.g., an RRM domain from Cas7). RRM
domains can comprise at most about 5%, at most about 10%, at most
about 15%, at most about 20%, at most about 25%, at most about 30%,
at most about 35%, at most about 40%, at most about 45%, at most
about 50%, at most about 55%, at most about 60%, at most about 65%,
at most about 70%, at most about 75%, at most about 80%, at most
about 85%, at most about 90%, at most about 95%, or 100% sequence
or structural homology to a wild type exemplary RRM domain (e.g.,
an RRM domain from Cas7).
[0229] Cas7 Family
[0230] Cas7 family members can be a subclass of RAMP family
proteins. Cas7 family proteins can be categorized in Type I CRISPR
systems. Cas7 family members may not comprise a glycine rich loop
that is familiar to some RAMP family members. Cas7 family members
can comprise one RRM domain. Cas7 family members can include, but
are not limited to, Cas7 (COG1857), Cas7 (COG3649), Cas7 (CT1975),
Csy3, Csm3, Cmr6, Csm5, Cmr4, Cmr1, Csf2, and Csc2.
[0231] Cas6 Family
[0232] The Cas6 family can be a RAMP subfamily. Cas6 family members
can comprise two RNA recognition motif (RRM)-like domains. A Cas6
family member (e.g., Cas6f) can comprise a N-terminal RRM domain
and a distinct C-terminal domain that may show weak sequence
similarity or structural homology to an RRM domain. Cas6 family
members can comprise a catalytic histidine that may be involved in
endoribonuclease activity. A comparable motif can be found in Cas5
and Cas7 RAMP families. Cas6 family members can include, but are
not limited to, Cas6, Cas6e, Cas6f (e.g., Csy4).
[0233] Cas5 Family
[0234] The Cas5 family can be a RAMP subfamily. The Cas5 family can
be divided into two subgroups: one subgroup that can comprise two
RRM domains, and one subgroup that can comprise one RRM domain.
Cas5 family members can include, but are not limited to, Csm4,
Csx10, Cmr3, Cas5, Cas5(BH0337), Csy2, Csc1, Csf3.
[0235] Cas Genes
[0236] Exemplary CRISPR Cas genes can include Cas1, Cas2, Cas3'
(Cas3-prime), Cas3'' (Cas3-double prime), Cas4, Cas5, Cas6, Cas6e
(formerly referred to as CasE, Cse3), Cas6f (i.e., Csy4), Cas7,
Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Csy1, Csy2,
Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5,
Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14,
Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4.
Table 1 provides an exemplary categorization of CRISPR Cas genes by
CRISPR system type.
[0237] The CRISPR-Cas gene naming system has undergone extensive
rewriting since the Cas genes were discovered. For the purposes of
this application, Cas gene names used herein are based on the
naming system outlined in Makarova et al. Evolution and
classification of the CRISPR-Cas systems. Nature Reviews
Microbiology. 2011 June; 9(6): 467-477.
Doi:10.1038/nrmicro2577.
TABLE-US-00003 TABLE 1 Exemplary classification of CRISPR Cas genes
by CRISPR Type System type or subtype Gene Name Type I cas1, cas2,
cas3' Type II cas1, cas2, cas9 Type III cas1, cas2, cas10 Subtype
I-A cas3'', cas4, cas5, cas6, cas7, cas8a1, cas8a2, csa5 Subtype
I-B cas3'', cas4, cas5, cas6, cas7, cas8b Subtype I-C cas4, cas5,
cas7, cas8c Subtype I-D cas4, cas6, cas10d, csc1, csc2 Subtype I-E
cas5, cas6e, cas7, cse1, cse2 Subtype I-F cas6f, csy1, csy2, csy3
Subtype II-A csn2 Subtype II-B cas4 Subtype III-A cas6, csm2, csm3,
csm4, csm5, csm6 Subtype III-B cas6, cmr1, cmr3, cmr4, cmr5, cmr6
Subtype I-U csb1, csb2, csb3, csx17, csx14, csx10 Subtype III-U
csx16, csaX, csx3, csx1 Unknown csx15 Type U csf1, csf2, csf3,
csf4
[0238] Site-Directed Polypeptides
[0239] A site-directed polypeptide can be a polypeptide that can
bind to a target nucleic acid. A site-directed polypeptide can be a
nuclease.
[0240] A site-directed polypeptide can comprise a nucleic
acid-binding domain. The nucleic acid-binding domain can comprise a
region that contacts a nucleic acid. A nucleic acid-binding domain
can comprise a nucleic acid. A nucleic acid-binding domain can
comprise a proteinaceous material. A nucleic acid-binding domain
can comprise nucleic acid and a proteinaceous material. A nucleic
acid-binding domain can comprise RNA. There can be a single nucleic
acid-binding domain. Examples of nucleic acid-binding domains can
include, but are not limited to, a helix-turn-helix domain, a zinc
finger domain, a leucine zipper (bZIP) domain, a winged helix
domain, a winged helix turn helix domain, a helix-loop-helix
domain, a HMG-box domain, a Wor3 domain, an immunoglobulin domain,
a B3 domain, a TALE domain, a RNA-recognition motif domain, a
double-stranded RNA-binding motif domain, a double-stranded nucleic
acid binding domain, a single-stranded nucleic acid binding
domains, a KH domain, a PUF domain, a RGG box domain, a DEAD/DEAH
box domain, a PAZ domain, a Piwi domain, and a cold-shock
domain.
[0241] A nucleic acid-binding domain can be a domain of an
argonaute protein. An argonaute protein can be a eukaryotic
argonaute or a prokaryotic argonaute. An argonaute protein can bind
RNA, DNA, or both RNA and DNA. An argonaute protein can cleaved
RNA, or DNA, or both RNA and DNA. In some instances, an argonaute
protein binds a DNA and cleaves a target DNA.
[0242] In some instances, two or more nucleic acid-binding domains
can be linked together. Linking a plurality of nucleic acid-binding
domains together can provide increased polynucleotide targeting
specificity. Two or more nucleic acid-binding domains can be linked
via one or more linkers. The linker can be a flexible linker.
Linkers can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40 or more
amino acids in length. Linkers can comprise at least 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, or 100% glycine content. Linkers can comprise at
most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% glycine content. Linkers
can comprise at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% serine
content. Linkers can comprise at most 5%, 10%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or
100% serine content.
[0243] Nucleic acid-binding domains can bind to nucleic acid
sequences. Nucleic acid binding domains can bind to nucleic acids
through hybridization. Nucleic acid-binding domains can be
engineered (e.g. engineered to hybridize to a sequence in a
genome). A nucleic acid-binding domain can be engineered by
molecular cloning techniques (e.g., directed evolution,
site-specific mutation, and rational mutagenesis).
[0244] A site-directed polypeptide can comprise a nucleic
acid-cleaving domain. The nucleic acid-cleaving domain can be a
nucleic acid-cleaving domain from any nucleic acid-cleaving
protein. The nucleic acid-cleaving domain can originate from a
nuclease. Suitable nucleic acid-cleaving domains include the
nucleic acid-cleaving domain of endonucleases (e.g., AP
endonuclease, RecBCD enonuclease, T7 endonuclease, T4 endonuclease
IV, Bal 31 endonuclease, Endonucleasel (endo I), Micrococcal
nuclease, Endonuclease II (endo VI, exo III)), exonucleases,
restriction nucleases, endoribonucleases, exoribonucleases, RNases
(e.g., RNAse I, II, or III). In some instances, the nucleic
acid-cleaving domain can originate from the FokI endonuclease. A
site-directed polypeptide can comprise a plurality of nucleic
acid-cleaving domains. Nucleic acid-cleaving domains can be linked
together. Two or more nucleic acid-cleaving domains can be linked
via a linker. In some embodiments, the linker can be a flexible
linker. Linkers can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40 or
more amino acids in length. In some embodiments, a site-directed
polypeptide can comprise the plurality of nucleic acid-cleaving
domains.
[0245] A site-directed polypeptide (e.g., Cas9, argonaute) can
comprise two or more nuclease domains. Cas9 can comprise a HNH or
FINE-like nuclease domain and/or a RuvC or RuvC-like nuclease
domain. HNH or FINE-like domains can comprise a McrA-like fold. HNH
or FINE-like domains can comprise two antiparallel .beta.-strands
and an .alpha.-helix. HNH or FINE-like domains can comprise a metal
binding site (e.g., divalent cation binding site). HNH or HNH-like
domains can cleave one strand of a target nucleic acid (e.g.,
complementary strand of the crRNA targeted strand). Proteins that
comprise an HNH or HNH-like domain can include endonucleases,
clicins, restriction endonucleases, transposases, and DNA packaging
factors.
[0246] RuvC or RuvC-like domains can comprise an RNaseH or
RNaseH-like fold. RuvC/RNaseH domains can be involved in a diverse
set of nucleic acid-based functions including acting on both RNA
and DNA. The RNaseH domain can comprise 5 .beta.-strands surrounded
by a plurality of .alpha.-helices. RuvC/RNaseH or RuvC/RNaseH-like
domains can comprise a metal binding site (e.g., divalent cation
binding site). RuvC/RNaseH or RuvC/RNaseH-like domains can cleave
one strand of a target nucleic acid (e.g., non-complementary strand
of the crRNA targeted strand). Proteins that comprise a RuvC,
RuvC-like, or RNaseH-like domain can include RNaseH, RuvC, DNA
transposases, retroviral integrases, and Argonaut proteins).
[0247] The site-directed polypeptide can be an endoribonuclease.
The site-directed polypeptide can be an enzymatically inactive
site-directed polypeptide. The site-directed polypeptide can be a
conditionally enzymatically inactive site-directed polypeptide.
Site-directed polypeptides can introduce double-stranded breaks or
single-stranded breaks in nucleic acid, (e.g. genomic DNA). The
double-stranded break can stimulate a cell's endogenous DNA-repair
pathways (e.g. homologous recombination and non-homologous end
joining (NHEJ) or alternative non-homologues end joining (A-NHEJ)).
NHEJ can repair cleaved target nucleic acid without the need for a
homologous template. This can result in deletions of the target
nucleic acid. Homologous recombination (HR) can occur with a
homologous template. The homologous template can comprise sequences
that are homologous to sequences flanking the target nucleic acid
cleavage site. After a target nucleic acid is cleaved by a
site-directed polypeptide the site of cleavage can be destroyed
(e.g., the site may not be accessible for another round of cleavage
with the original nucleica acid-targeting nucleic acid and
site-directed polypeptide).
[0248] In some cases, homologous recombination can insert an
exogenous polynucleotide sequence into the target nucleic acid
cleavage site. An exogenous polynucleotide sequence can be called a
donor polynucleotide. In some instances of the methods of the
disclosure the donor polynucleotide, a portion of the donor
polynucleotide, a copy of the donor polynucleotide, or a portion of
a copy of the donor polynucleotide can be inserted into the target
nucleic acid cleavage site. A donor polynucleotide can be an
exogenous polynucleotide sequence. A donor polynucleotide can be a
sequence that does not naturally occur at the target nucleic acid
cleavage site. A vector can comprise a donor polynucleotide. The
modifications of the target DNA due to NHEJ and/or HR can lead to,
for example, mutations, deletions, alterations, integrations, gene
correction, gene replacement, gene tagging, transgene insertion,
nucleotide deletion, gene disruption, and/or gene mutation. The
process of integrating non-native nucleic acid into genomic DNA can
be referred to as genome engineering.
[0249] In some cases, the site-directed polypeptide can comprise an
amino acid sequence having at most 10%, at most 15%, at most 20%,
at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at
most 75%, at most 80%, at most 85%, at most 90%, at most 95%, at
most 99%, or 100%, amino acid sequence identity to a wild type
exemplary site-directed polypeptide (e.g, Cas9 from S. pyogenes,
SEQ ID NO: 8).
[0250] In some cases, the site-directed polypeptide can comprise an
amino acid sequence having at least 10%, at least 15%, 20%, at
least 30%, at least 40%, at least 50%, at least 60%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, at least 99%, or 100%, amino acid sequence identity to a wild
type exemplary site-directed polypeptide (e.g., Cas9 from S.
pyogenes, SEQ ID NO: 8).
[0251] In some cases, the site-directed polypeptide can comprise an
amino acid sequence having at most 10%, at most 15%, at most 20%,
at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at
most 75%, at most 80%, at most 85%, at most 90%, at most 95%, at
most 99%, or 100%, amino acid sequence identity to the nuclease
domain of a wild type exemplary site-directed polypeptide (e.g,
Cas9 from S. pyogenes, SEQ ID NO: 8).
[0252] A site-directed polypeptide can comprise at least 70, 75,
80, 85, 90, 95, 97, 99, or 100% identity to wild-type site-directed
polypeptide (e.g., Cas9 from S. pyogenes, SEQ ID NO: 8) over 10
contiguous amino acids. A site-directed polypeptide can comprise at
most 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to wild-type
site-directed polypeptide (e.g., Cas9 from S. pyogenes, SEQ ID NO:
8) over 10 contiguous amino acids. A site-directed polypeptide can
comprise at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity
to a wild-type site-directed polypeptide (e.g., Cas9 from S.
pyogenes, SEQ ID NO: 8) over 10 contiguous amino acids in a HNH
nuclease domain of the site-directed polypeptide. A site-directed
polypeptide can comprise at most 70, 75, 80, 85, 90, 95, 97, 99, or
100% identity to a wild-type site-directed polypeptide (e.g., Cas9
from S. pyogenes, SEQ ID NO: 8) over 10 contiguous amino acids in a
HNH nuclease domain of the site-directed polypeptide. A
site-directed polypeptide can comprise at least 70, 75, 80, 85, 90,
95, 97, 99, or 100% identity to a wild-type site-directed
polypeptide (e.g., Cas9 from S. pyogenes, SEQ ID NO: 8) over 10
contiguous amino acids in a RuvC nuclease domain of the
site-directed polypeptide. A site-directed polypeptide can comprise
at most 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a
wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes,
SEQ ID NO: 8) over 10 contiguous amino acids in a RuvC nuclease
domain of the site-directed polypeptide.
[0253] In some cases, the site-directed polypeptide can comprise an
amino acid sequence having at least 10%, at least 15%, at least
20%, at least 30%, at least 40%, at least 50%, at least 60%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 99%, or 100%, amino acid sequence identity
to the nuclease domain of a wild type exemplary site-directed
polypeptide (e.g., Cas9 from S. pyogenes).
[0254] The site-directed polypeptide can comprise a modified form
of a wild type exemplary site-directed polypeptide. The modified
form of the wild type exemplary site-directed polypeptide can
comprise an amino acid change (e.g., deletion, insertion, or
substitution) that reduces the nucleic acid-cleaving activity of
the site-directed polypeptide. For example, the modified form of
the wild type exemplary site-directed polypeptide can have less
than less than 90%, less than 80%, less than 70%, less than 60%,
less than 50%, less than 40%, less than 30%, less than 20%, less
than 10%, less than 5%, or less than 1% of the nucleic
acid-cleaving activity of the wild-type exemplary site-directed
polypeptide (e.g., Cas9 from S. pyogenes). The modified form of the
site-directed polypeptide can have no substantial nucleic
acid-cleaving activity. When a site-directed polypeptide is a
modified form that has no substantial nucleic acid-cleaving
activity, it can be referred to as "enzymatically inactive."
[0255] The modified form of the wild type exemplary site-directed
polypeptide can have more than 90%, more than 80%, more than 70%,
more than 60%, more than 50%, more than 40%, more than 30%, more
than 20%, more than 10%, more than 5%, or more than 1% of the
nucleic acid-cleaving activity of the wild-type exemplary
site-directed polypeptide (e.g., Cas9 from S. pyogenes).
[0256] The modified form of the site-directed polypeptide can
comprise a mutation. The modified form of the site-directed
polypeptide can comprise a mutation such that it can induce a
single stranded break (SSB) on a target nucleic acid (e.g., by
cutting only one of the sugar-phosphate backbones of the target
nucleic acid). The mutation can result in less than 90%, less than
80%, less than 70%, less than 60%, less than 50%, less than 40%,
less than 30%, less than 20%, less than 10%, less than 5%, or less
than 1% of the nucleic acid-cleaving activity in one or more of the
plurality of nucleic acid-cleaving domains of the wild-type site
directed polypeptide (e.g., Cas9 from S. pyogenes). The mutation
can result in one or more of the plurality of nucleic acid-cleaving
domains retaining the ability to cleave the complementary strand of
the target nucleic acid but reducing its ability to cleave the
non-complementary strand of the target nucleic acid. The mutation
can result in one or more of the plurality of nucleic acid-cleaving
domains retaining the ability to cleave the non-complementary
strand of the target nucleic acid but reducing its ability to
cleave the complementary strand of the target nucleic acid. For
example, residues in the wild type exemplary S. pyogenes Cas9
polypeptide such as Asp10, His840, Asn854 and Asn856 can be mutated
to inactivate one or more of the plurality of nucleic acid-cleaving
domains (e.g., nuclease domains). The residues to be mutated can
correspond to residues Asp10, His840, Asn854 and Asn856 in the wild
type exemplary S. pyogenes Cas9 polypeptide (e.g., as determined by
sequence and/or structural alignment). Non-limiting examples of
mutations can include D10A, H840A, N854A or N856A. One skilled in
the art will recognize that mutations other than alanine
substitutions are suitable.
[0257] A D10A mutation can be combined with one or more of H840A,
N854A, or N856A mutations to produce a site-directed polypeptide
substantially lacking DNA cleavage activity. A H840A mutation can
be combined with one or more of D10A, N854A, or N856A mutations to
produce a site-directed polypeptide substantially lacking DNA
cleavage activity. A N854A mutation can be combined with one or
more of H840A, D10A, or N856A mutations to produce a site-directed
polypeptide substantially lacking DNA cleavage activity. A N856A
mutation can be combined with one or more of H840A, N854A, or D10A
mutations to produce a site-directed polypeptide substantially
lacking DNA cleavage activity. Site-directed polypeptides that
comprise one substantially inactive nuclease domain can be referred
to as nickases.
[0258] Mutations of the disclosure can be produced by site-directed
mutation. Mutations can include substitutions, additions, and
deletions, or any combination thereof. In some instances, the
mutation converts the mutated amino acid to alanine. In some
instances, the mutation converts the mutated amino acid to another
amino acid (e.g., glycine, serine, threonine, cysteine, valine,
leucine, isoleucine, methionine, proline, phenylalanine, tyrosine,
tryptophan, aspartic acid, glutamic acid, asparagines, glutamine,
histidine, lysine, or arginine). The mutation can convert the
mutated amino acid to a non-natural amino acid (e.g.,
selenomethionine). The mutation can convert the mutated amino acid
to amino acid mimics (e.g., phosphomimics). The mutation can be a
conservative mutation. For example, the mutation can convert the
mutated amino acid to amino acids that resemble the size, shape,
charge, polarity, conformation, and/or rotamers of the mutated
amino acids (e.g., cysteine/serine mutation, lysine/asparagine
mutation, histidine/phenylalanine mutation).
[0259] In some instances, the site-directed polypeptide (e.g.,
variant, mutated, enzymatically inactive and/or conditionally
enzymatically inactive site-directed polypeptide) can target
nucleic acid. The site-directed polypeptide (e.g., variant,
mutated, enzymatically inactive and/or conditionally enzymatically
inactive endoribonuclease) can target RNA. Site-directed
polypeptides that can target RNA can include members of other
CRISPR subfamilies such as Cas6 and Cas5.
[0260] The site-directed polypeptide can comprise one or more
non-native sequences (e.g., a fusion).
[0261] A site-directed polypeptide can comprise an amino acid
sequence comprising at least 15% amino acid identity to a Cas9 from
a bacterium (e.g., S. pyogenes), a nucleic acid binding domain, and
two nucleic acid cleaving domains (i.e., an HNH domain and a RuvC
domain).
[0262] A site-directed polypeptide can comprise an amino acid
sequence comprising at least 15% amino acid identity to a Cas9 from
a bacterium (e.g., S. pyogenes), and two nucleic acid cleaving
domains (i.e., an HNH domain and a RuvC domain).
[0263] A site-directed polypeptide can comprise an amino acid
sequence comprising at least 15% amino acid identity to a Cas9 from
a bacterium (e.g., S. pyogenes), and two nucleic acid cleaving
domains, wherein one or both of the nucleic acid cleaving domains
comprise at least 50% amino acid identity to a nuclease domain from
Cas9 from a bacterium (e.g., S. pyogenes).
[0264] A site-directed polypeptide can comprise an amino acid
sequence comprising at least 15% amino acid identity to a Cas9 from
a bacterium (e.g., S. pyogenes), two nucleic acid cleaving domains
(i.e., an HNH domain and a RuvC domain), and a linker linking the
site-directed polypeptide to a non-native sequence.
[0265] A site-directed polypeptide can comprise an amino acid
sequence comprising at least 15% amino acid identity to a Cas9 from
a bacterium (e.g., S. pyogenes), two nucleic acid cleaving domains
(i.e., an HNH domain and a RuvC domain), wherein the site-directed
polypeptide comprises a mutation in one or both of the nucleic acid
cleaving domains that reduces the cleaving activity of the nuclease
domains by at least 50%.
[0266] A site-directed polypeptide can comprise an amino acid
sequence comprising at least 15% amino acid identity to a Cas9 from
a bacterium (e.g., S. pyogenes), and two nucleic acid cleaving
domains (i.e., an HNH domain and a RuvC domain), wherein one of the
nuclease domains comprises mutation of aspartic acid 10, and/or
wherein one of the nuclease domains comprises mutation of histidine
840, and wherein the mutation reduce the cleaving activity of the
nuclease domains by at least 50%.
[0267] Endoribonucleases
[0268] In some embodiments, a site-directed polypeptide can be an
endoribonuclease.
[0269] In some cases, the endoribonuclease can comprise an amino
acid sequence having at most about 20%, at most about 30%, at most
about 40%, at most about 50%, at most about 60%, at most about 70%,
at most about 75%, at most about 80%, at most about 85%, at most
about 90%, at most about 95%, at most about 99%, or 100%, amino
acid sequence identity and/or homology to a wild type reference
endoribonuclease. The endoribonuclease can comprise an amino acid
sequence having at least about 20%, at least about 30%, at least
about 40%, at least about 50%, at least about 60%, at least about
70%, at least about 75%, at least about 80%, at least about 85%, at
least about 90%, at least about 95%, at least about 99%, or 100%,
amino acid sequence identity and/or homology to a wild type
reference endoribonuclease (e.g., Csy4 from P. aeruginosa). The
reference endoribonuclease can be a Cas6 family member (e.g., Csy4,
Cas6). The reference endoribonuclease can be a Cas5 family member
(e.g., Cas5 from D. vulgaris). The reference endoribonuclease can
be a Type I CRISPR family member (e.g., Cas3). The reference
endoribonucleases can be a Type II family member. The reference
endoribonuclease can be a Type III family member (e.g., Cas6). A
reference endoribonuclease can be a member of the Repeat Associated
Mysterious Protein (RAMP) superfamily (e.g., Cas7).
[0270] The endoribonuclease can comprise amino acid modifications
(e.g., substitutions, deletions, additions etc). The
endoribonuclease can comprise one or more non-native sequences
(e.g., a fusion, an affinity tag). The amino acid modifications may
not substantially alter the activity of the endoribonuclease. An
endoribonuclease comprising amino acid modifications and/or fusions
can retain at least about 75%, at least about 80%, at least about
85%, at least about 90%, at least about 95%, at least about 97% or
100% activity of the wild-type endoribonuclease.
[0271] The modification can result alteration of the enzymatic
activity of the endoribonuclease. The modification can result in
less than 90%, less than 80%, less than 70%, less than 60%, less
than 50%, less than 40%, less than 30%, less than 20%, less than
10%, less than 5%, or less than 1% of the endoribonuclease. In some
instances, the modification occurs in the nuclease domain of an
endoribonuclease. Such modifications can result in less than 90%,
less than 80%, less than 70%, less than 60%, less than 50%, less
than 40%, less than 30%, less than 20%, less than 10%, less than
5%, or less than 1% of the nucleic acid-cleaving ability in one or
more of the plurality of nucleic acid-cleaving domains of the
wild-type endoribonuclease.
[0272] Conditionally Enzymatically Inactive Endoribonucleases
[0273] In some embodiments, an endoribonuclease can be
conditionally enzymatically inactive. A conditionally enzymatically
inactive endoribonuclease can bind to a polynucleotide in a
sequence-specific manner. A conditionally enzymatically inactive
endoribonuclease can bind a polynucleotide in a sequence-specific
manner, but cannot cleave the target polyribonucleotide.
[0274] In some cases, the conditionally enzymatically inactive
endoribonuclease can comprise an amino acid sequence having up to
about 20%, up to about 30%, up to about 40%, up to about 50%, up to
about 60%, up to about 70%, up to about 75%, up to about 80%, up to
about 85%, up to about 90%, up to about 95%, up to about 99%, or
100%, amino acid sequence identity and/or homology to a reference
conditionally enzymatically endoribonuclease (e.g., Csy4 from P.
aeruginosa). In some cases, the conditionally enzymatically
inactive endoribonuclease can comprise an amino acid sequence
having at least about 20%, at least about 30%, at least about 40%,
at least about 50%, at least about 60%, at least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 95%, at least about 99%, or 100%, amino
acid sequence identity and/or homology to a reference conditionally
enzymatically endoribonuclease (e.g., Csy4 from P. aeruginosa).
[0275] The conditionally enzymatically inactive endoribonuclease
can comprise a modified form of an endoribonuclease. The modified
form of the endoribonuclease can comprise an amino acid change
(e.g., deletion, insertion, or substitution) that reduces the
nucleic acid-cleaving activity of the endoribonuclease. For
example, the modified form of the conditionally enzymatically
inactive endoribonuclease can have less than less than 90%, less
than 80%, less than 70%, less than 60%, less than 50%, less than
40%, less than 30%, less than 20%, less than 10%, less than 5%, or
less than 1% of the nucleic acid-cleaving activity of the reference
(e.g., wild-type) conditionally enzymatically inactive
endoribonuclease (e.g., Csy4 from P. aeruginosa). The modified form
of the conditionally enzymatically inactive endoribonuclease can
have no substantial nucleic acid-cleaving activity. When a
conditionally enzymatically inactive endoribonuclease is a modified
form that has no substantial nucleic acid-cleaving activity, it can
be referred to as "enzymatically inactive."
[0276] The modified form of the conditionally enzymatically
inactive endoribonuclease can comprise a mutation that can result
in reduced nucleic acid-cleaving ability (i.e., such that the
conditionally enzymatically inactive endoribonuclease can be
enzymatically inactive in one or more of the nucleic acid-cleaving
domains). The mutation can result in less than 90%, less than 80%,
less than 70%, less than 60%, less than 50%, less than 40%, less
than 30%, less than 20%, less than 10%, less than 5%, or less than
1% of the nucleic acid-cleaving ability in one or more of the
plurality of nucleic acid-cleaving domains of the wild-type
endoribonuclease (e.g., Csy4 from P. aeruginosa). The mutation can
occur in the nuclease domain of the endoribonuclease. The mutation
can occur in a ferredoxin-like fold. The mutation can comprise the
mutation of a conserved aromatic amino acid. The mutation can
comprise the mutation of a catalytic amino acid. The mutation can
comprise the mutation of a histidine. For example, the mutation can
comprise a H29A mutation in Csy4 (e.g., Csy4 from P. aeruginosa),
or any corresponding residue to H29A as determined by sequence
and/or structural alignment. Other residues can be mutated to
achieve the same effect (i.e. inactivate one or more of the
plurality of nuclease domains).
[0277] Mutations of the invention can be produced by site-directed
mutation. Mutations can include substitutions, additions, and
deletions, or any combination thereof. In some instances, the
mutation converts the mutated amino acid to alanine. In some
instances, the mutation converts the mutated amino acid to another
amino acid (e.g., glycine, serine, threonine, cysteine, valine,
leucine, isoleucine, methionine, proline, phenylalanine, tyrosine,
tryptophan, aspartic acid, glutamic acid, asparagines, glutamine,
histidine, lysine, or arginine). The mutation can convert the
mutated amino acid to a non-natural amino acid (e.g.,
selenomethionine). The mutation can convert the mutated amino acid
to amino acid mimics (e.g., phosphomimics). The mutation can be a
conservative mutation. For example, the mutation can convert the
mutated amino acid to amino acids that resemble the size, shape,
charge, polarity, conformation, and/or rotamers of the mutated
amino acids (e.g., cysteine/serine mutation, lysine/asparagine
mutation, histidine/phenylalanine mutation).
[0278] A conditionally enzymatically inactive endoribonuclease can
be enzymatically inactive in the absence of a reactivation agent
(e.g., imidazole). A reactivation agent can be an agent that mimics
a histidine residue (e.g., may have an imidazole ring). A
conditionally enzymatically inactive endoribonuclease can be
activated by contact with a reactivation agent. The reactivation
agent can comprise imidazole. For example, the conditionally
enzymatically inactive endoribonuclease can be enzymatically
activated by contacting the conditionally enzymatically inactive
endoribonuclease with imidazole at a concentration of from about
100 mM to about 500 mM. The imidazole can be at a concentration of
about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300
mM, about 350 mM, about 400 mM, about 450 mM, about 500 mM, about
550 mM, or about 600 mM. The presence of imidazole (e.g., in a
concentration range of from about 100 mM to about 500 mM) can
reactivate the conditionally enzymatically inactive
endoribonuclease such that the conditionally enzymatically inactive
endoribonuclease becomes enzymatically active, e.g., the
conditionally enzymatically inactive endoribonuclease exhibits at
least about 50%, at least about 60%, at least about 70%, at least
about 80%, at least about 90%, at least about 95%, or more than
95%, of the nucleic acid cleaving ability of a reference
conditionally enzymatically inactive endoribonuclease (e.g., Csy4
from P. aeruginosa comprising H29A mutation).
[0279] A conditionally enzymatically inactive endoribonuclease can
comprise at least 20% amino acid identity to Csy4 from P.
aeruginosa, a mutation of histidine 29, wherein the mutation
results in at least 50% reduction of nuclease activity of the
endoribonuclease, and wherein at least 50% of the lost nuclease
activity can be restored by incubation of the endoribonuclease with
at least 100 mM imidazole.
[0280] Codon-Optimization
[0281] A polynucleotide encoding a site-directed polypeptide and/or
an endoribonuclease can be codon-optimized. This type of
optimization can entail the mutation of foreign-derived (e.g.,
recombinant) DNA to mimic the codon preferences of the intended
host organism or cell while encoding the same protein. Thus, the
codons can be changed, but the encoded protein remains unchanged.
For example, if the intended target cell was a human cell, a human
codon-optimized polynucleotide Cas9 could be used for producing a
suitable site-directed polypeptide. As another non-limiting
example, if the intended host cell were a mouse cell, then a mouse
codon-optimized polynucleotide encoding Cas9 could be a suitable
site-directed polypeptide. A polynucleotide encoding a
site-directed polypeptide can be codon optimized for many host
cells of interest. A host cell can be a cell from any organism
(e.g. a bacterial cell, an archaeal cell, a cell of a single-cell
eukaryotic organism, a plant cell, an algal cell, e.g.,
Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis
gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, and
the like, a fungal cell (e.g., a yeast cell), an animal cell, a
cell from an invertebrate animal (e.g. fruit fly, cnidarian,
echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g.,
fish, amphibian, reptile, bird, mammal), a cell from a mammal
(e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a
non-human primate, a human, etc.), etc. Codon optimization may not
be required. In some instances, codon optimization can be
preferable.
[0282] Nucleic Acid-Targeting Nucleic Acid
[0283] The present disclosure provides for a nucleic acid-targeting
nucleic acid that can direct the activities of an associated
polypeptide (e.g., a site-directed polypeptide) to a specific
target sequence within a target nucleic acid. The nucleic
acid-targeting nucleic acid can comprise nucleotides. The nucleic
acid-targeting nucleic acid can be RNA. A nucleic acid-targeting
nucleic acid can comprise a single guide nucleic acid-targeting
nucleic acid. An exemplary single guide nucleic acid is depicted in
FIG. 1A. The spacer extension 105 and the tracrRNA extension 135
can comprise elements that can contribute additional functionality
(e.g., stability) to the nucleic acid-targeting nucleic acid. In
some embodiments the spacer extension 105 and the tracrRNA
extension 135 are optional. A spacer sequence 110 can comprise a
sequence that can hybridize to a target nucleic acid sequence. The
spacer sequence 110 can be a variable portion of the nucleic
acid-targeting nucleic acid. The sequence of the spacer sequence
110 can be engineered to hybridize to the target nucleic acid
sequence. The CRISPR repeat 115 (i.e. referred to in this exemplary
embodiment as a minimum CRISPR repeat) can comprise nucleotides
that can hybridize to a tracrRNA sequence 125 (i.e. referred to in
this exemplary embodiment as a minimum tracrRNA sequence). The
minimum CRISPR repeat 115 and the minimum tracrRNA sequence 125 can
interact, the interacting molecules comprising a base-paired,
double-stranded structure. Together, the minimum CRISPR repeat 115
and the minimum tracrRNA sequence 125 can facilitate binding to the
site-directed polypeptide. The minimum CRISPR repeat 115 and the
minimum tracrRNA sequence 125 can be linked together to form a
hairpin structure through the single guide connector 120. The 3'
tracrRNA sequence 130 can comprise a protospacer adjacent motif
recognition sequence. The 3' tracrRNA sequence 130 can be identical
or similar to part of a tracrRNA sequence. In some embodiments, the
3' tracrRNA sequence 130 can comprise one or more hairpins.
[0284] In some embodiments, a nucleic acid-targeting nucleic acid
can comprise a single guide nucleic acid-targeting nucleic acid as
depicted in FIG. 1B. A nucleic acid-targeting nucleic acid can
comprise a spacer sequence 140. A spacer sequence 140 can comprise
a sequence that can hybridize to the target nucleic acid sequence.
The spacer sequence 140 can be a variable portion of the nucleic
acid-targeting nucleic acid. The spacer sequence 140 can be 5' of a
first duplex 145. The first duplex 145 comprises a region of
hybridization between a minimum CRISPR repeat 146 and minimum
tracrRNA sequence 147. The first duplex 145 can be interrupted by a
bulge 150. The bulge150 can comprise unpaired nucleotides. The
bulge 150 can be facilitate the recruitment of a site-directed
polypeptide to the nucleic acid-targeting nucleic acid. The bulge
150 can be followed by a first stem 155. The first stem 155
comprises a linker sequence linking the minimum CRISPR repeat 146
and the minimum tracrRNA sequence 147. The last paired nucleotide
at the 3' end of the first duplex 145 can be connected to a second
linker sequence 160. The second linker 160 can comprise a P-domain.
The second linker 160 can link the first duplex 145 to a
mid-tracrRNA 165. The mid-tracrRNA 165 can, in some embodiments,
comprise one or more hairpin regions. For example the mid-tracrRNA
165 can comprise a second stem 170 and a third stem 175.
[0285] In some embodiments, the nucleic acid-targeting nucleic acid
can comprise a double guide nucleic acid structure. FIG. 2 depicts
an exemplary double guide nucleic acid-targeting nucleic acid
structure. Similar to the single guide nucleic acid structure of
FIG. 1, the double guide nucleic acid structure can comprise a
spacer extension 205, a spacer 210, a minimum CRISPR repeat 215, a
minimum tracrRNA sequence 230, a 3' tracrRNA sequence 235, and a
tracrRNA extension 240. However, a double guide nucleic
acid-targeting nucleic acid may not comprise the single guide
connector 120. Instead the minimum CRISPR repeat sequence 215 can
comprise a 3' CRISPR repeat sequence 220 which can be similar or
identical to part of a CRISPR repeat. Similarly, the minimum
tracrRNA sequence 230 can comprise a 5' tracrRNA sequence 225 which
can be similar or identical to part of a tracrRNA. The double guide
RNAs can hybridize together via the minimum CRISPR repeat 215 and
the minimum tracrRNA sequence 230.
[0286] In some embodiments, the first segment (i.e., nucleic
acid-targeting segment) can comprise the spacer extension (e.g.,
105/205) and the spacer (e.g., 110/210). The nucleic acid-targeting
nucleic acid can guide the bound polypeptide to a specific
nucleotide sequence within target nucleic acid via the above
mentioned nucleic acid-targeting segment.
[0287] In some embodiments, the second segment (i.e., protein
binding segment) can comprise the minimum CRISPR repeat (e.g.,
115/215), the minimum tracrRNA sequence (e.g., 125/230), the 3'
tracrRNA sequence (e.g., 130/235), and/or the tracrRNA extension
sequence (e.g., 135/240). The protein-binding segment of a nucleic
acid-targeting nucleic acid can interact with a site-directed
polypeptide. The protein-binding segment of a nucleic
acid-targeting nucleic acid can comprise two stretches of
nucleotides that that can hybridize to one another. The nucleotides
of the protein-binding segment can hybridize to form a
double-stranded nucleic acid duplex. The double-stranded nucleic
acid duplex can be RNA. The double-stranded nucleic acid duplex can
be DNA.
[0288] In some instances, a nucleic acid-targeting nucleic acid can
comprise, in the order of 5' to 3', a spacer extension, a spacer, a
minimum CRISPR repeat, a single guide connector, a minimum
tracrRNA, a 3' tracrRNA sequence, and a tracrRNA extension. In some
instances, a nucleic acid-targeting nucleic acid can comprise, a
tracrRNA extension, a 3'tracrRNA sequence, a minimum tracrRNA, a
single guide connector, a minimum CRISPR repeat, a spacer, and a
spacer extension in any order.
[0289] A nucleic acid-targeting nucleic acid and a site-directed
polypeptide can form a complex. The nucleic acid-targeting nucleic
acid can provide target specificity to the complex by comprising a
nucleotide sequence that can hybridize to a sequence of a target
nucleic acid. In other words, the site-directed polypeptide can be
guided to a nucleic acid sequence by virtue of its association with
at least the protein-binding segment of the nucleic acid-targeting
nucleic acid. The nucleic acid-targeting nucleic acid can direct
the activity of a Cas9 protein. The nucleic acid-targeting nucleic
acid can direct the activity of an enzymatically inactive Cas9
protein.
[0290] Methods of the disclosure can provide for a genetically
modified cell. A genetically modified cell can comprise an
exogenous nucleic acid-targeting nucleic acid and/or an exogenous
nucleic acid comprising a nucleotide sequence encoding a nucleic
acid-targeting nucleic acid.
[0291] Spacer Extension Sequence
[0292] A spacer extension sequence can provide stability and/or
provide a location for modifications of a nucleic acid-targeting
nucleic acid. A spacer extension sequence can have a length of from
about 1 nucleotide to about 400 nucleotides. A spacer extension
sequence can have a length of more than 1, 5, 10, 15, 20, 25, 30,
35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220,
240, 260, 280, 300, 320, 340, 360, 380, 40, 1000, 2000, 3000, 4000,
5000, 6000, or 7000 or more nucleotides. A spacer extension
sequence can have a length of less than 1, 5, 10, 15, 20, 25, 30,
35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220,
240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000,
4000, 5000, 6000, 7000 or more nucleotides. A spacer extension
sequence can be less than 10 nucleotides in length. A spacer
extension sequence can be between 10 and 30 nucleotides in length.
A spacer extension sequence can be between 30-70 nucleotides in
length.
[0293] The spacer extension sequence can comprise a moiety (e.g., a
stability control sequence, an endoribonuclease binding sequence, a
ribozyme). A moiety can influence the stability of a nucleic acid
targeting RNA. A moiety can be a transcriptional terminator segment
(i.e., a transcription termination sequence). A moiety of a nucleic
acid-targeting nucleic acid can have a total length of from about
10 nucleotides to about 100 nucleotides, from about 10 nucleotides
(nt) to about 20 nt, from about 20 nt to about 30 nt, from about 30
nt to about 40 nt, from about 40 nt to about 50 nt, from about 50
nt to about 60 nt, from about 60 nt to about 70 nt, from about 70
nt to about 80 nt, from about 80 nt to about 90 nt, or from about
90 nt to about 100 nt, from about 15 nucleotides (nt) to about 80
nt, from about 15 nt to about 50 nt, from about 15 nt to about 40
nt, from about 15 nt to about 30 nt or from about 15 nt to about 25
nt. The moiety can be one that can function in a eukaryotic cell.
In some cases, the moiety can be one that can function in a
prokaryotic cell. The moiety can be one that can function in both a
eukaryotic cell and a prokaryotic cell.
[0294] Non-limiting examples of suitable moieties can include: 5'
cap (e.g., a 7-methylguanylate cap (m7 G)), a riboswitch sequence
(e.g., to allow for regulated stability and/or regulated
accessibility by proteins and protein complexes), a sequence that
forms a dsRNA duplex (i.e., a hairpin), a sequence that targets the
RNA to a subcellular location (e.g., nucleus, mitochondria,
chloroplasts, and the like), a modification or sequence that
provides for tracking (e.g., direct conjugation to a fluorescent
molecule, conjugation to a moiety that facilitates fluorescent
detection, a sequence that allows for fluorescent detection, etc.),
a modification or sequence that provides a binding site for
proteins (e.g., proteins that act on DNA, including transcriptional
activators, transcriptional repressors, DNA methyltransferases, DNA
demethylases, histone acetyltransferases, histone deacetylases, and
the like) a modification or sequence that provides for increased,
decreased, and/or controllable stability, or any combination
thereof. A spacer extension sequence can comprise a primer binding
site, a molecular index (e.g., barcode sequence). The spacer
extension sequence can comprise a nucleic acid affinity tag.
[0295] Spacer
[0296] The nucleic acid-targeting segment of a nucleic
acid-targeting nucleic acid can comprise a nucleotide sequence
(e.g., a spacer) that can hybridize to a sequence in a target
nucleic acid. The spacer of a nucleic acid-targeting nucleic acid
can interact with a target nucleic acid in a sequence-specific
manner via hybridization (i.e., base pairing). As such, the
nucleotide sequence of the spacer may vary and can determine the
location within the target nucleic acid that the nucleic
acid-targeting nucleic acid and the target nucleic acid can
interact.
[0297] The spacer sequence can hybridize to a target nucleic acid
that is located 5' of spacer adjacent motif (PAM). Different
organisms may comprise different PAM sequences. For example, in S.
pyogenes, the PAM can be a sequence in the target nucleic acid that
comprises the sequence 5'-XRR-3', where R can be either A or G,
where X is any nucleotide and X is immediately 3' of the target
nucleic acid sequence targeted by the spacer sequence.
[0298] The target nucleic acid sequence can be 20 nucleotides. The
target nucleic acid can be less than 20 nucleotides. The target
nucleic acid can be at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 30 or more nucleotides. The target nucleic acid can be
at most 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or
more nucleotides. The target nucleic acid sequence can be 20 bases
immediately 5' of the first nucleotide of the PAM. For example, in
a sequence comprising 5'-XRR-3', the target nucleic acid can be the
sequence that corresponds to the N's, wherein N is any
nucleotide.
[0299] The nucleic acid-targeting sequence of the spacer that can
hybridize to the target nucleic acid can have a length at least
about 6 nt. For example, the spacer sequence that can hybridize the
target nucleic acid can have a length at least about 6 nt, at least
about 10 nt, at least about 15 nt, at least about 18 nt, at least
about 19 nt, at least about 20 nt, at least about 25 nt, at least
about 30 nt, at least about 35 nt or at least about 40 nt, from
about 6 nt to about 80 nt, from about 6 nt to about 50 nt, from
about 6 nt to about 45 nt, from about 6 nt to about 40 nt, from
about 6 nt to about 35 nt, from about 6 nt to about 30 nt, from
about 6 nt to about 25 nt, from about 6 nt to about 20 nt, from
about 6 nt to about 19 nt, from about 10 nt to about 50 nt, from
about 10 nt to about 45 nt, from about 10 nt to about 40 nt, from
about 10 nt to about 35 nt, from about 10 nt to about 30 nt, from
about 10 nt to about 25 nt, from about 10 nt to about 20 nt, from
about 10 nt to about 19 nt, from about 19 nt to about 25 nt, from
about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from
about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from
about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from
about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from
about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from
about 20 nt to about 45 nt, from about 20 nt to about 50 nt, or
from about 20 nt to about 60 nt. In some cases, the spacer sequence
that can hybridize the target nucleic acid can be 20 nucleotides in
length. The spacer that can hybridize the target nucleic acid can
be 19 nucleotides in length.
[0300] The percent complementarity between the spacer sequence the
target nucleic acid can be at least about 30%, at least about 40%,
at least about 50%, at least about 60%, at least about 65%, at
least about 70%, at least about 75%, at least about 80%, at least
about 85%, at least about 90%, at least about 95%, at least about
97%, at least about 98%, at least about 99%, or 100%. The percent
complementarity between the spacer sequence the target nucleic acid
can be at most about 30%, at most about 40%, at most about 50%, at
most about 60%, at most about 65%, at most about 70%, at most about
75%, at most about 80%, at most about 85%, at most about 90%, at
most about 95%, at most about 97%, at most about 98%, at most about
99%, or 100%. In some cases, the percent complementarity between
the spacer sequence and the target nucleic acid can be 100% over
the six contiguous 5'-most nucleotides of the target sequence of
the complementary strand of the target nucleic acid. In some cases,
the percent complementarity between the spacer sequence and the
target nucleic acid can be at least 60% over about 20 contiguous
nucleotides. In some cases, the percent complementarity between the
spacer sequence and the target nucleic acid can be 100% over the
fourteen contiguous 5'-most nucleotides of the target sequence of
the complementary strand of the target nucleic acid and as low as
0% over the remainder. In such a case, the spacer sequence can be
considered to be 14 nucleotides in length. In some cases, the
percent complementarity between the spacer sequence and the target
nucleic acid can be 100% over the six contiguous 5'-most
nucleotides of the target sequence of the complementary strand of
the target nucleic acid and as low as 0% over the remainder. In
such a case, the spacer sequence can be considered to be 6
nucleotides in length. The target nucleic acid can be more than
about 50%, 60%, 70%, 80%, 90%, or 100% complementary to the seed
region of the crRNA. The target nucleic acid can be less than about
50%, 60%, 70%, 80%, 90%, or 100% complementary to the seed region
of the crRNA.
[0301] The spacer segment of a nucleic acid-targeting nucleic acid
can be modified (e.g., by genetic engineering) to hybridize to any
desired sequence within a target nucleic acid. For example, a
spacer can be engineered (e.g., designed, programmed) to hybridize
to a sequence in target nucleic acid that is involved in cancer,
cell growth, DNA replication, DNA repair, HLA genes, cell surface
proteins, T-cell receptors, immunoglobulin superfamily genes, tumor
suppressor genes, microRNA genes, long non-coding RNA genes,
transcription factors, globins, viral proteins, mitochondrial
genes, and the like.
[0302] A spacer sequence can be identified using a computer program
(e.g., machine readable code). The computer program can use
variables such as predicted melting temperature, secondary
structure formation, and predicted annealing temperature, sequence
identity, genomic context, chromatin accessibility, % GC, frequency
of genomic occurrence, methylation status, presence of SNPs, and
the like.
[0303] Minimum CRISPR Repeat Sequence
[0304] A minimum CRISPR repeat sequence can be a sequence at least
about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or
100% sequence identity and/or sequence homology with a reference
CRISPR repeat sequence (e.g., crRNA from S. pyogenes). A minimum
CRISPR repeat sequence can be a sequence with at most about 30%,
40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence
identity and/or sequence homology with a reference CRISPR repeat
sequence (e.g., crRNA from S. pyogenes). A minimum CRISPR repeat
can comprise nucleotides that can hybridize to a minimum tracrRNA
sequence. A minimum CRISPR repeat and a minimum tracrRNA sequence
can form a base-paired, double-stranded structure. Together, the
minimum CRISPR repeat and the minimum tracrRNA sequence can
facilitate binding to the site-directed polypeptide. A part of the
minimum CRISPR repeat sequence can hybridize to the minimum
tracrRNA sequence. A part of the minimum CRISPR repeat sequence can
be at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, or 100% complementary to the minimum tracrRNA sequence. A part
of the minimum CRISPR repeat sequence can be at most about 30%,
40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%
complementary to the minimum tracrRNA sequence.
[0305] The minimum CRISPR repeat sequence can have a length of from
about 6 nucleotides to about 100 nucleotides. For example, the
minimum CRISPR repeat sequence can have a length of from about 6
nucleotides (nt) to about 50 nt, from about 6 nt to about 40 nt,
from about 6 nt to about 30 nt, from about 6 nt to about 25 nt,
from about 6 nt to about 20 nt, from about 6 nt to about 15 nt,
from about 8 nt to about 40 nt, from about 8 nt to about 30 nt,
from about 8 nt to about 25 nt, from about 8 nt to about 20 nt or
from about 8 nt to about 15 nt, from about 15 nt to about 100 nt,
from about 15 nt to about 80 nt, from about 15 nt to about 50 nt,
from about 15 nt to about 40 nt, from about 15 nt to about 30 nt or
from about 15 nt to about 25 nt. In some embodiments, the minimum
CRISPR repeat sequence has a length of approximately 12
nucleotides.
[0306] The minimum CRISPR repeat sequence can be at least about 60%
identical to a reference minimum CRISPR repeat sequence (e.g., wild
type crRNA from S. pyogenes) over a stretch of at least 6, 7, or 8
contiguous nucleotides. The minimum CRISPR repeat sequence can be
at least about 60% identical to a reference minimum CRISPR repeat
sequence (e.g., wild type crRNA from S. pyogenes) over a stretch of
at least 6, 7, or 8 contiguous nucleotides. For example, the
minimum CRISPR repeat sequence can be at least about 65% identical,
at least about 70% identical, at least about 75% identical, at
least about 80% identical, at least about 85% identical, at least
about 90% identical, at least about 95% identical, at least about
98% identical, at least about 99% identical or 100% identical to a
reference minimum CRISPR repeat sequence over a stretch of at least
6, 7, or 8 contiguous nucleotides.
[0307] Minimum tracrRNA Sequence
[0308] A minimum tracrRNA sequence can be a sequence with at least
about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or
100% sequence identity and/or sequence homology to a reference
tracrRNA sequence (e.g., wild type tracrRNA from S. pyogenes). A
minimum tracrRNA sequence can be a sequence with at most about 30%,
40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence
identity and/or sequence homology to a reference tracrRNA sequence
(e.g., wild type tracrRNA from S. pyogenes). A minimum tracrRNA
sequence can comprise nucleotides that can hybridize to a minimum
CRISPR repeat sequence. A minimum tracrRNA sequence and a minimum
CRISPR repeat sequence can form a base-paired, double-stranded
structure. Together, the minimum tracrRNA sequence and the minimum
CRISPR repeat can facilitate binding to the site-directed
polypeptide. A part of the minimum tracrRNA sequence can hybridize
to the minimum CRISPR repeat sequence. A part of the minimum
tracrRNA sequence can be 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, or 100% complementary to the minimum CRISPR repeat
sequence.
[0309] The minimum tracrRNA sequence can have a length of from
about 6 nucleotides to about 100 nucleotides. For example, the
minimum tracrRNA sequence can have a length of from about 6
nucleotides (nt) to about 50 nt, from about 6 nt to about 40 nt,
from about 6 nt to about 30 nt, from about 6 nt to about 25 nt,
from about 6 nt to about 20 nt, from about 6 nt to about 15 nt,
from about 8 nt to about 40 nt, from about 8 nt to about 30 nt,
from about 8 nt to about 25 nt, from about 8 nt to about 20 nt or
from about 8 nt to about 15 nt, from about 15 nt to about 100 nt,
from about 15 nt to about 80 nt, from about 15 nt to about 50 nt,
from about 15 nt to about 40 nt, from about 15 nt to about 30 nt or
from about 15 nt to about 25 nt. In some embodiments, the minimum
tracrRNA sequence has a length of approximately 14 nucleotides.
[0310] The minimum tracrRNA sequence can be at least about 60%
identical to a reference minimum tracrRNA (e.g., wild type,
tracrRNA from S. pyogenes) sequence over a stretch of at least 6,
7, or 8 contiguous nucleotides. The minimum tracrRNA sequence can
be at least about 60% identical to a reference minimum tracrRNA
(e.g., wild type, tracrRNA from S. pyogenes) sequence over a
stretch of at least 6, 7, or 8 contiguous nucleotides. For example,
the minimum tracrRNA sequence can be at least about 65% identical,
at least about 70% identical, at least about 75% identical, at
least about 80% identical, at least about 85% identical, at least
about 90% identical, at least about 95% identical, at least about
98% identical, at least about 99% identical or 100% identical to a
reference minimum tracrRNA sequence over a stretch of at least 6,
7, or 8 contiguous nucleotides.
[0311] The duplex (i.e., first duplex in FIG. 1B) between the
minimum CRISPR RNA and the minimum tracrRNA can comprise a double
helix. The first base of the first strand of the duplex (e.g., the
minimum CRISPR repeat in FIG. 1B) can be a guanine. The first base
of the first strand of the duplex (e.g., the minimum CRISPR repeat
in FIG. 1B) can be an adenine. The duplex (i.e., first duplex in
FIG. 1B) between the minimum CRISPR RNA and the minimum tracrRNA
can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or
more nucleotides. The duplex (i.e., first duplex in FIG. 1B)
between the minimum CRISPR RNA and the minimum tracrRNA can
comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more
nucleotides.
[0312] The duplex can comprise a mismatch. The duplex can comprise
at least about 1, 2, 3, 4, or 5 or mismatches. The duplex can
comprise at most about 1, 2, 3, 4, or 5 or mismatches. In some
instances, the duplex comprises no more than 2 mismatches.
[0313] Bulge
[0314] A bulge can refer to an unpaired region of nucleotides
within the duplex made up of the minimum CRISPR repeat and the
minimum tracrRNA sequence. The bulge can be important in the
binding to the site-directed polypeptide. A bulge can comprise, on
one side of the duplex, an unpaired 5'-XXXY-3' where X is any
purine and Y can be a nucleotide that can form a wobble pair with a
nucleotide on the opposite strand, and an unpaired nucleotide
region on the other side of the duplex.
[0315] For example, the bulge can comprise an unpaired purine
(e.g., adenine) on the minimum CRISPR repeat strand of the bulge.
In some embodiments, a bulge can comprise an unpaired 5'-AAGY-3' of
the minimum tracrRNA sequence strand of the bulge, where Y can be a
nucleotide that can form a wobble pairing with a nucleotide on the
minimum CRISPR repeat strand.
[0316] A bulge on a first side of the duplex (e.g., the minimum
CRISPR repeat side) can comprise at least 1, 2, 3, 4, or 5 or more
unpaired nucleotides. A bulge on a first side of the duplex (e.g.,
the minimum CRISPR repeat side) can comprise at most 1, 2, 3, 4, or
5 or more unpaired nucleotides. A bulge on the first side of the
duplex (e.g., the minimum CRISPR repeat side) can comprise 1
unpaired nucleotide.
[0317] A bulge on a second side of the duplex (e.g., the minimum
tracrRNA sequence side of the duplex) can comprise at least 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10 or more unpaired nucleotides. A bulge on
a second side of the duplex (e.g., the minimum tracrRNA sequence
side of the duplex) can comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 or more unpaired nucleotides. A bulge on a second side of the
duplex (e.g., the minimum tracrRNA sequence side of the duplex) can
comprise 4 unpaired nucleotides.
[0318] Regions of different numbers of unpaired nucleotides on each
strand of the duplex can be paired together. For example, a bulge
can comprise 5 unpaired nucleotides from a first strand and 1
unpaired nucleotide from a second strand. A bulge can comprise 4
unpaired nucleotides from a first strand and 1 unpaired nucleotide
from a second strand. A bulge can comprise 3 unpaired nucleotides
from a first strand and 1 unpaired nucleotide from a second strand.
A bulge can comprise 2 unpaired nucleotides from a first strand and
1 unpaired nucleotide from a second strand. A bulge can comprise 1
unpaired nucleotide from a first strand and 1 unpaired nucleotide
from a second strand. A bulge can comprise 1 unpaired nucleotide
from a first strand and 2 unpaired nucleotides from a second
strand. A bulge can comprise 1 unpaired nucleotide from a first
strand and 3 unpaired nucleotides from a second strand. A bulge can
comprise 1 unpaired nucleotide from a first strand and 4 unpaired
nucleotides from a second strand. A bulge can comprise 1 unpaired
nucleotide from a first strand and 5 unpaired nucleotides from a
second strand.
[0319] In some instances a bulge can comprise at least one wobble
pairing. In some instances, a bulge can comprise at most one wobble
pairing. A bulge sequence can comprise at least one purine
nucleotide. A bulge sequence can comprise at least 3 purine
nucleotides. A bulge sequence can comprise at least 5 purine
nucleotides. A bulge sequence can comprise at least one guanine
nucleotide. A bulge sequence can comprise at least one adenine
nucleotide.
[0320] P-Domain (P-DOMAIN)
[0321] A P-domain can refer to a region of a nucleic acid-targeting
nucleic acid that can recognize a protospacer adjacent motif (PAM)
in a target nucleic acid. A P-domain can hybridize to a PAM in a
target nucleic acid. As such, a P-domain can comprise a sequence
that is complementary to a PAM. A P-domain can be located 3' to the
minimum tracrRNA sequence. A P-domain can be located within a 3'
tracrRNA sequence (i.e., a mid-tracrRNA sequence).
[0322] A p start at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,
or 20 or more nucleotides 3' of the last paired nucleotide in the
minimum CRISPR repeat and minimum tracrRNA sequence duplex. A
P-domain can start at most about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or
more nucleotides 3' of the last paired nucleotide in the minimum
CRISPR repeat and minimum tracrRNA sequence duplex.
[0323] A P-domain can comprise at least about 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 15, or 20 or more consecutive nucleotides. A P-domain can
comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or
more consecutive nucleotides.
[0324] In some instances, a P-domain can comprise a CC dinucleotide
(i.e., two consecutive cytosine nucleotides). The CC dinucleotide
can interact with the GG dinucleotide of a PAM, wherein the PAM
comprises a 5'-XGG-3' sequence.
[0325] A P-domain can be a nucleotide sequence located in the 3'
tracrRNA sequence (i.e., mid-tracrRNA sequence). A P-domain can
comprise duplexed nucleotides (e.g., nucleotides in a hairpin,
hybridized together. For example, a P-domain can comprise a CC
dinucleotide that is hybridized to a GG dinucleotide in a hairpin
duplex of the 3' tracrRNA sequence (i.e., mid-tracrRNA sequence).
The activity of the P-domain (e.g., the nucleic acid-targeting
nucleic acid's ability to target a target nucleic acid) may be
regulated by the hybridization state of the P-DOMAIN. For example,
if the P-domainis hybridized, the nucleic acid-targeting nucleic
acid may not recognize its target. If the P-domainis unhybridized
the nucleic acid-targeting nucleic acid may recognize its
target.
[0326] The P-domain can interact with P-domain interacting regions
within the site-directed polypeptide. The P-domain can interact
with an arginine-rich basic patch in the site-directed polypeptide.
The P-domain interacting regions can interact with a PAM sequence.
The P-domain can comprise a stem loop. The P-domain can comprise a
bulge.
[0327] 3'tracrRNA Sequence
[0328] A 3'tracr RNA sequence can be a sequence with at least about
30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%
sequence identity and/or sequence homology with a reference
tracrRNA sequence (e.g., a tracrRNA from S. pyogenes). A 3'tracr
RNA sequence can be a sequence with at most about 30%, 40%, 50%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity
and/or sequence homology with a reference tracrRNA sequence (e.g.,
tracrRNA from S. pyogenes).
[0329] The 3' tracrRNA sequence can have a length of from about 6
nucleotides to about 100 nucleotides. For example, the 3' tracrRNA
sequence can have a length of from about 6 nucleotides (nt) to
about 50 nt, from about 6 nt to about 40 nt, from about 6 nt to
about 30 nt, from about 6 nt to about 25 nt, from about 6 nt to
about 20 nt, from about 6 nt to about 15 nt, from about 8 nt to
about 40 nt, from about 8 nt to about 30 nt, from about 8 nt to
about 25 nt, from about 8 nt to about 20 nt or from about 8 nt to
about 15 nt, from about 15 nt to about 100 nt, from about 15 nt to
about 80 nt, from about 15 nt to about 50 nt, from about 15 nt to
about 40 nt, from about 15 nt to about 30 nt or from about 15 nt to
about 25 nt. In some embodiments, the 3' tracrRNA sequence has a
length of approximately 14 nucleotides.
[0330] The 3' tracrRNA sequence can be at least about 60% identical
to a reference 3' tracrRNA sequence (e.g., wild type 3' tracrRNA
sequence from S. pyogenes) over a stretch of at least 6, 7, or 8
contiguous nucleotides. For example, the 3' tracrRNA sequence can
be at least about 60% identical, at least about 65% identical, at
least about 70% identical, at least about 75% identical, at least
about 80% identical, at least about 85% identical, at least about
90% identical, at least about 95% identical, at least about 98%
identical, at least about 99% identical, or 100% identical, to a
reference 3' tracrRNA sequence (e.g., wild type 3' tracrRNA
sequence from S. pyogenes) over a stretch of at least 6, 7, or 8
contiguous nucleotides.
[0331] A 3' tracrRNA sequence can comprise more than one duplexed
region (e.g., hairpin, hybridized region). A 3' tracrRNA sequence
can comprise two duplexed regions.
[0332] The 3' tracrRNA sequence can also be referred to as the
mid-tracrRNA (See FIG. 1B). The mid-tracrRNA sequence can comprise
a stem loop structure. In other words, the mid-tracrRNA sequence
can comprise a hairpin that is different than a second or third
stems, as depicted in FIG. 1B. A stem loop structure in the
mid-tracrRNA (i.e., 3' tracrRNA) can comprise at least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 15 or 20 or more nucleotides. A stem loop
structure in the mid-tracrRNA (i.e., 3' tracrRNA) can comprise at
most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides. The stem
loop structure can comprise a functional moiety. For example, the
stem loop structure can comprise an aptamer, a ribozyme, a
protein-interacting hairpin, a CRISPR array, an intron, and an
exon. The stem loop structure can comprise at least about 1, 2, 3,
4, or 5 or more functional moieties. The stem loop structure can
comprise at most about 1, 2, 3, 4, or 5 or more functional
moieties.
[0333] The hairpin in the mid-tracrRNA sequence can comprise a
P-domain. The P-domain can comprise a double stranded region in the
hairpin.
[0334] tracrRNA Extension Sequence
[0335] A tracrRNA extension sequence can provide stability and/or
provide a location for modifications of a nucleic acid-targeting
nucleic acid. A tracrRNA extension sequence can have a length of
from about 1 nucleotide to about 400 nucleotides. A tracrRNA
extension sequence can have a length of more than 1, 5, 10, 15, 20,
25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,
200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 or more
nucleotides. A tracrRNA extension sequence can have a length from
about 20 to about 5000 or more nucleotides. A tracrRNA extension
sequence can have a length of more than 1000 nucleotides. A
tracrRNA extension sequence can have a length of less than 1, 5,
10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140,
160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400
nucleotides. A tracrRNA extension sequence can have a length of
less than 1000 nucleotides. A tracrRNA extension sequence can be
less than 10 nucleotides in length. A tracrRNA extension sequence
can be between 10 and 30 nucleotides in length. A tracrRNA
extension sequence can be between 30-70 nucleotides in length.
[0336] The tracrRNA extension sequence can comprise a moiety (e.g.,
stability control sequence, ribozyme, endoribonuclease binding
sequence). A moiety can influence the stability of a nucleic acid
targeting RNA. A moiety can be a transcriptional terminator segment
(i.e., a transcription termination sequence). A moiety of a nucleic
acid-targeting nucleic acid can have a total length of from about
10 nucleotides to about 100 nucleotides, from about 10 nucleotides
(nt) to about 20 nt, from about 20 nt to about 30 nt, from about 30
nt to about 40 nt, from about 40 nt to about 50 nt, from about 50
nt to about 60 nt, from about 60 nt to about 70 nt, from about 70
nt to about 80 nt, from about 80 nt to about 90 nt, or from about
90 nt to about 100 nt, from about 15 nucleotides (nt) to about 80
nt, from about 15 nt to about 50 nt, from about 15 nt to about 40
nt, from about 15 nt to about 30 nt or from about 15 nt to about 25
nt. The moiety can be one that can function in a eukaryotic cell.
In some cases, the moiety can be one that can function in a
prokaryotic cell. The moiety can be one that can function in both a
eukaryotic cell and a prokaryotic cell.
[0337] Non-limiting examples of suitable tracrRNA extension
moieties include: a 3' polyadenylated tail, a riboswitch sequence
(e.g., to allow for regulated stability and/or regulated
accessibility by proteins and protein complexes), a sequence that
forms a dsRNA duplex (i.e., a hairpin), a sequence that targets the
RNA to a subcellular location (e.g., nucleus, mitochondria,
chloroplasts, and the like), a modification or sequence that
provides for tracking (e.g., direct conjugation to a fluorescent
molecule, conjugation to a moiety that facilitates fluorescent
detection, a sequence that allows for fluorescent detection, etc.),
a modification or sequence that provides a binding site for
proteins (e.g., proteins that act on DNA, including transcriptional
activators, transcriptional repressors, DNA methyltransferases, DNA
demethylases, histone acetyltransferases, histone deacetylases, and
the like) a modification or sequence that provides for increased,
decreased, and/or controllable stability, or any combination
thereof. A tracrRNA extension sequence can comprise a primer
binding site, a molecular index (e.g., barcode sequence). In some
embodiments of the disclosure, the tracrRNA extension sequence can
comprise one or more affinity tags.
[0338] Single Guide Nucleic Acid
[0339] The nucleic acid-targeting nucleic acid can be a single
guide nucleic acid. The single guide nucleic acid can be RNA. A
single guide nucleic acid can comprise a linker (i.e. item 120 from
FIG. 1A) between the minimum CRISPR repeat sequence and the minimum
tracrRNA sequence that can be called a single guide connector
sequence.
[0340] The single guide connector of a single guide nucleic acid
can have a length of from about 3 nucleotides to about 100
nucleotides. For example, the linker can have a length of from
about 3 nucleotides (nt) to about 90 nt, from about 3 nt to about
80 nt, from about 3 nt to about 70 nt, from about 3 nt to about 60
nt, from about 3 nt to about 50 nt, from about 3 nt to about 40 nt,
from about 3 nt to about 30 nt, from about 3 nt to about 20 nt or
from about 3 nt to about 10 nt. For example, the linker can have a
length of from about 3 nt to about 5 nt, from about 5 nt to about
10 nt, from about 10 nt to about 15 nt, from about 15 nt to about
20 nt, from about 20 nt to about 25 nt, from about 25 nt to about
30 nt, from about 30 nt to about 35 nt, from about 35 nt to about
40 nt, from about 40 nt to about 50 nt, from about 50 nt to about
60 nt, from about 60 nt to about 70 nt, from about 70 nt to about
80 nt, from about 80 nt to about 90 nt, or from about 90 nt to
about 100 nt. In some embodiments, the linker of a single guide
nucleic acid is between 4 and 40 nucleotides. A linker can have a
length at least about 100, 500, 1000, 1500, 2000, 2500, 3000, 3500,
4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides. A
linker can have a length at most about 100, 500, 1000, 1500, 2000,
2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 or
more nucleotides.
[0341] The linker sequence can comprise a functional moiety. For
example, the linker sequence can comprise an aptamer, a ribozyme, a
protein-interacting hairpin, a CRISPR array, an intron, and an
exon. The linker sequence can comprise at least about 1, 2, 3, 4,
or 5 or more functional moieties. The linker sequence can comprise
at most about 1, 2, 3, 4, or 5 or more functional moieties.
[0342] In some embodiments, the single guide connector can connect
the 3' end of the minimum CRISPR repeat to the 5' end of the
minimum tracrRNA sequence. Alternatively, the single guide
connector can connect the 3' end of the tracrRNA sequence to the 5'
end of the minimum CRISPR repeat. That is to say, a single guide
nucleic acid can comprise a 5' DNA-binding segment linked to a 3'
protein-binding segment. A single guide nucleic acid can comprise a
5' protein-binding segment linked to a 3' DNA-binding segment.
[0343] A nucleic acid-targeting nucleic acid can comprise a spacer
extension sequence from 10-5000 nucleotides in length; a spacer
sequence of 12-30 nucleotides in length, wherein the spacer is at
least 50% complementary to a target nucleic acid; a minimum CRISPR
repeat comprising at least 60% identity to a crRNA from a
prokaryote (e.g., S. pyogenes) or phage over 6, 7, or 8 contiguous
nucleotides and wherein the minimum CRISPR repeat has a length from
5-30 nucleotides; a minimum tracrRNA sequence comprising at least
60% identity to a tracrRNA from a bacterium (e.g., S. pyogenes)
over 6, 7, or 8 contiguous nucleotides and wherein the minimum
tracrRNA sequence has a length from 5-30 nucleotides; a linker
sequence that links the minimum CRISPR repeat and the minimum
tracrRNA and comprises a length from 3-5000 nucleotides; a 3'
tracrRNA that comprises at least 60% identity to a tracrRNA from a
prokaryote (e.g., S. pyogenes) or phage over 6, 7, or 8 contiguous
nucleotides and wherein the 3' tracrRNA comprises a length from
10-20 nucleotides, and comprises a duplexed region; and/or a
tracrRNA extension comprising 10-5000 nucleotides in length, or any
combination thereof. This nucleic acid-targeting nucleic acid can
be referred to as a single guide nucleic acid-targeting nucleic
acid.
[0344] A nucleic acid-targeting nucleic acid can comprise a spacer
extension sequence from 10-5000 nucleotides in length; a spacer
sequence of 12-30 nucleotides in length, wherein the spacer is at
least 50% complementary to a target nucleic acid; a duplex
comprising 1) a minimum CRISPR repeat comprising at least 60%
identity to a crRNA from a prokaryote (e.g., S. pyogenes) or phage
over 6 contiguous nucleotides and wherein the minimum CRISPR repeat
has a length from 5-30 nucleotides, 2) a minimum tracrRNA sequence
comprising at least 60% identity to a tracrRNA from a bacterium
(e.g., S. pyogenes) over 6 contiguous nucleotides and wherein the
minimum tracrRNA sequence has a length from 5-30 nucleotides, and
3) a bulge wherein the bulge comprises at least 3 unpaired
nucleotides on the minimum CRISPR repeat strand of the duplex and
at least 1 unpaired nucleotide on the minimum tracrRNA sequence
strand of the duplex; a linker sequence that links the minimum
CRISPR repeat and the minimum tracrRNA and comprises a length from
3-5000 nucleotides; a 3' tracrRNA that comprises at least 60%
identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or
phage over 6 contiguous nucleotides, wherein the 3' tracrRNA
comprises a length from 10-20 nucleotides and comprises a duplexed
region; a P-domain that starts from 1-5 nucleotides downstream of
the duplex comprising the minimum CRISPR repeat and the minimum
tracrRNA, comprises 1-10 nucleotides, comprises a sequence that can
hybridize to a protospacer adjacent motif in a target nucleic acid,
can form a hairpin, and is located in the 3' tracrRNA region;
and/or a tracrRNA extension comprising 10-5000 nucleotides in
length, or any combination thereof.
[0345] Double Guide Nucleic Acid
[0346] A nucleic acid-targeting nucleic acid can be a double guide
nucleic acid. The double guide nucleic acid can be RNA. The double
guide nucleic acid can comprise two separate nucleic acid molecules
(i.e. polynucleotides). Each of the two nucleic acid molecules of a
double guide nucleic acid-targeting nucleic acid can comprise a
stretch of nucleotides that can hybridize to one another such that
the complementary nucleotides of the two nucleic acid molecules
hybridize to form the double stranded duplex of the protein-binding
segment. If not otherwise specified, the term "nucleic
acid-targeting nucleic acid" can be inclusive, referring to both
single-molecule nucleic acid-targeting nucleic acids and
double-molecule nucleic acid-targeting nucleic acids.
[0347] A double-guide nucleic acid-targeting nucleic acid can
comprise 1) a first nucleic acid molecule comprising a spacer
extension sequence from 10-5000 nucleotides in length; a spacer
sequence of 12-30 nucleotides in length, wherein the spacer is at
least 50% complementary to a target nucleic acid; and a minimum
CRISPR repeat comprising at least 60% identity to a crRNA from a
prokaryote (e.g., S. pyogenes) or phage over 6 contiguous
nucleotides and wherein the minimum CRISPR repeat has a length from
5-30 nucleotides; and 2) a second nucleic acid molecule of the
double-guide nucleic acid-targeting nucleic acid can comprise a
minimum tracrRNA sequence comprising at least 60% identity to a
tracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides and wherein the minimum tracrRNA sequence
has a length from 5-30 nucleotides; a 3' tracrRNA that comprises at
least 60% identity to a tracrRNA from a bacterium (e.g., S.
pyogenes) over 6 contiguous nucleotides and wherein the 3' tracrRNA
comprises a length from 10-20 nucleotides, and comprises a duplexed
region; and/or a tracrRNA extension comprising 10-5000 nucleotides
in length, or any combination thereof.
[0348] In some instances, a double-guide nucleic acid-targeting
nucleic acid can comprise 1) a first nucleic acid molecule
comprising a spacer extension sequence from 10-5000 nucleotides in
length; a spacer sequence of 12-30 nucleotides in length, wherein
the spacer is at least 50% complementary to a target nucleic acid;
a minimum CRISPR repeat comprising at least 60% identity to a crRNA
from a prokaryote (e.g., S. pyogenes) or phage over 6 contiguous
nucleotides and wherein the minimum CRISPR repeat has a length from
5-30 nucleotides, and at least 3 unpaired nucleotides of a bulge;
and 2) a second nucleic acid molecule of the double-guide nucleic
acid-targeting nucleic acid can comprise a minimum tracrRNA
sequence comprising at least 60% identity to a tracrRNA from a
prokaryote (e.g., S. pyogenes) or phage over 6 contiguous
nucleotides and wherein the minimum tracrRNA sequence has a length
from 5-30 nucleotides and at least 1 unpaired nucleotide of a
bulge, wherein the 1 unpaired nucleotide of the bulge is located in
the same bulge as the 3 unpaired nucleotides of the minimum CRISPR
repeat; a 3' tracrRNA that comprises at least 60% identity to a
tracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides and wherein the 3' tracrRNA comprises a
length from 10-20 nucleotides, and comprises a duplexed region; a
P-domain that starts from 1-5 nucleotides downstream of the duplex
comprising the minimum CRISPR repeat and the minimum tracrRNA,
comprises 1-10 nucleotides, comprises a sequence that can hybridize
to a protospacer adjacent motif in a target nucleic acid, can form
a hairpin, and is located in the 3' tracrRNA region; and/or a
tracrRNA extension comprising 10-5000 nucleotides in length, or any
combination thereof.
[0349] Complex of a Nucleic Acid-Targeting Nucleic Acid and a
Site-Directed Polypeptide
[0350] A nucleic acid-targeting nucleic acid can interact with a
site-directed polypeptide (e.g., a nucleic acid-guided nucleases,
Cas9), thereby forming a complex. The nucleic acid-targeting
nucleic acid can guide the site-directed polypeptide to a target
nucleic acid.
[0351] In some embodiments, a nucleic acid-targeting nucleic acid
can be engineered such that the complex (e.g., comprising a
site-directed polypeptide and a nucleic acid-targeting nucleic
acid) can bind outside of the cleavage site of the site-directed
polpeptide. In this case, the target nucleic acid may not interact
with the complex and the target nucleic acid can be excised (e.g.,
free from the complex).
[0352] In some embodiments, a nucleic acid-targeting nucleic acid
can be engineered such that the complex can bind inside of the
cleavage site of the site-directed polpeptide. In this case, the
target nucleic acid can interact with the complex and the target
nucleic acid can be bound (e.g., bound to the complex).
[0353] In some instances, a complex can comprise a site-directed
polypeptide, wherein the site-directed polypeptide can comprise an
amino acid sequence comprising at least 15% amino acid identity to
a Cas9 from S. pyogenes, and two nucleic acid cleaving domains
(i.e., an HNH domain and a RuvC domain); and a double-guide nucleic
acid-targeting nucleic acid comprising 1) a first nucleic acid
molecule comprising a spacer extension sequence from 10-5000
nucleotides in length; a spacer sequence of 12-30 nucleotides in
length, wherein the spacer is at least 50% complementary to a
target nucleic acid; and a minimum CRISPR repeat comprising at
least 60% identity to a crRNA from a prokaryote (e.g., S. pyogenes)
or phage over 6 contiguous nucleotides and wherein the minimum
CRISPR repeat has a length from 5-30 nucleotides; and 2) a second
nucleic acid molecule of the double-guide nucleic acid-targeting
nucleic acid can comprise a minimum tracrRNA sequence comprising at
least 60% identity to a tracrRNA from a prokaryote (e.g., S.
pyogenes) or phage over 6 contiguous nucleotides and wherein the
minimum tracrRNA sequence has a length from 5-30 nucleotides; a 3'
tracrRNA that comprises at least 60% identity to a tracrRNA from a
bacterium (e.g., S. pyogenes) over 6 contiguous nucleotides and
wherein the 3' tracrRNA comprises a length from 10-20 nucleotides,
and comprises a duplexed region; and/or a tracrRNA extension
comprising 10-5000 nucleotides in length, or any combination
thereof.
[0354] In some instances, a complex can comprise a site-directed
polypeptide, wherein the site-directed polypeptide can comprise an
amino acid sequence comprising at least 15% amino acid identity to
a Cas9 from S. pyogenes, and two nucleic acid cleaving domains
(i.e., an HNH domain and a RuvC domain); and, a double-guide
nucleic acid-targeting nucleic acid comprising 1) a first nucleic
acid molecule comprising a spacer extension sequence from 10-5000
nucleotides in length; a spacer sequence of 12-30 nucleotides in
length, wherein the spacer is at least 50% complementary to a
target nucleic acid; a minimum CRISPR repeat comprising at least
60% identity to a crRNA from a prokaryote (e.g., S. pyogenes) or
phage over 6 contiguous nucleotides and wherein the minimum CRISPR
repeat has a length from 5-30 nucleotides, and at least 3 unpaired
nucleotides of a bulge; and 2) a second nucleic acid molecule of
the double-guide nucleic acid-targeting nucleic acid can comprise a
minimum tracrRNA sequence comprising at least 60% identity to a
tracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides and wherein the minimum tracrRNA sequence
has a length from 5-30 nucleotides and at least 1 unpaired
nucleotide of a bulge, wherein the 1 unpaired nucleotide of the
bulge is located in the same bulge as the 3 unpaired nucleotides of
the minimum CRISPR repeat; a 3' tracrRNA that comprises at least
60% identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or
phage over 6 contiguous nucleotides and wherein the 3' tracrRNA
comprises a length from 10-20 nucleotides, and comprises a duplexed
region; a P-domain that starts from 1-5 nucleotides downstream of
the duplex comprising the minimum CRISPR repeat and the minimum
tracrRNA, comprises 1-10 nucleotides, comprises a sequence that can
hybridize to a protospacer adjacent motif in a target nucleic acid,
can form a hairpin, and is located in the 3' tracrRNA region;
and/or a tracrRNA extension comprising 10-5000 nucleotides in
length, or any combination thereof.
[0355] In some instances, a complex can comprise a site-directed
polypeptide, wherein the site-directed polypeptide can comprise an
amino acid sequence comprising at least 15% amino acid identity to
a Cas9 from S. pyogenes and, two nucleic acid cleaving domains
(i.e., an HNH domain and a RuvC domain); and, a nucleic
acid-targeting nucleic acid comprising a spacer extension sequence
from 10-5000 nucleotides in length; a spacer sequence of 12-30
nucleotides in length, wherein the spacer is at least 50%
complementary to a target nucleic acid; a minimum CRISPR repeat
comprising at least 60% identity to a crRNA from a prokaryote
(e.g., S. pyogenes) or phage over 6, 7, or 8 contiguous nucleotides
and wherein the minimum CRISPR repeat has a length from 5-30
nucleotides; a minimum tracrRNA sequence comprising at least 60%
identity to a tracrRNA from a bacterium (e.g., S. pyogenes) over 6,
7, or 8 contiguous nucleotides and wherein the minimum tracrRNA
sequence has a length from 5-30 nucleotides; a linker sequence that
links the minimum CRISPR repeat and the minimum tracrRNA and
comprises a length from 3-5000 nucleotides; a 3' tracrRNA that
comprises at least 60% identity to a tracrRNA from a prokaryote
(e.g., S. pyogenes) or phage over 6, 7, or 8 contiguous nucleotides
and wherein the 3' tracrRNA comprises a length from 10-20
nucleotides, and comprises a duplexed region; and/or a tracrRNA
extension comprising 10-5000 nucleotides in length, or any
combination thereof. This nucleic acid-targeting nucleic acid can
be referred to as a single guide nucleic acid-targeting nucleic
acid.
[0356] In some instances, a complex can comprise a site-directed
polypeptide, wherein the site-directed polypeptide can comprise an
amino acid sequence comprising at least 15% amino acid identity to
a Cas9 from S. pyogenes, and two nucleic acid cleaving domains
(i.e., an HNH domain and a RuvC domain); and, a nucleic
acid-targeting nucleic acid can comprising a spacer extension
sequence from 10-5000 nucleotides in length; a spacer sequence of
12-30 nucleotides in length, wherein the spacer is at least 50%
complementary to a target nucleic acid; a duplex comprising 1) a
minimum CRISPR repeat comprising at least 60% identity to a crRNA
from a prokaryote (e.g., S. pyogenes) or phage over 6 contiguous
nucleotides and wherein the minimum CRISPR repeat has a length from
5-30 nucleotides, 2) a minimum tracrRNA sequence comprising at
least 60% identity to a tracrRNA from a bacterium (e.g., S.
pyogenes) over 6 contiguous nucleotides and wherein the minimum
tracrRNA sequence has a length from 5-30 nucleotides, and 3) a
bulge wherein the bulge comprises at least 3 unpaired nucleotides
on the minimum CRISPR repeat strand of the duplex and at least 1
unpaired nucleotide on the minimum tracrRNA sequence strand of the
duplex; a linker sequence that links the minimum CRISPR repeat and
the minimum tracrRNA and comprises a length from 3-5000
nucleotides; a 3' tracrRNA that comprises at least 60% identity to
a tracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides, wherein the 3' tracrRNA comprises a length
from 10-20 nucleotides and comprises a duplexed region; a P-domain
that starts from 1-5 nucleotides downstream of the duplex
comprising the minimum CRISPR repeat and the minimum tracrRNA,
comprises 1-10 nucleotides, comprises a sequence that can hybridize
to a protospacer adjacent motif in a target nucleic acid, can form
a hairpin, and is located in the 3' tracrRNA region; and/or a
tracrRNA extension comprising 10-5000 nucleotides in length, or any
combination thereof. In some instances, a complex can comprise a
site-directed polypeptide, wherein the site-directed polypeptide
comprises an amino acid sequence comprising at least 15% amino acid
identity to a Cas9 from S. pyogenes, two nucleic acid cleaving
domains (i.e., an HNH domain and a RuvC domain), and a linker
linking the site-directed polypeptide to a non-native sequence; and
a double-guide nucleic acid-targeting nucleic acid comprising 1) a
first nucleic acid molecule comprising a spacer extension sequence
from 10-5000 nucleotides in length; a spacer sequence of 12-30
nucleotides in length, wherein the spacer is at least 50%
complementary to a target nucleic acid; and a minimum CRISPR repeat
comprising at least 60% identity to a crRNA from a prokaryote
(e.g., S. pyogenes) or phage over 6 contiguous nucleotides and
wherein the minimum CRISPR repeat has a length from 5-30
nucleotides; and 2) a second nucleic acid molecule of the
double-guide nucleic acid-targeting nucleic acid can comprise a
minimum tracrRNA sequence comprising at least 60% identity to a
tracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides and wherein the minimum tracrRNA sequence
has a length from 5-30 nucleotides; a 3' tracrRNA that comprises at
least 60% identity to a tracrRNA from a bacterium (e.g., S.
pyogenes) over 6 contiguous nucleotides and wherein the 3' tracrRNA
comprises a length from 10-20 nucleotides, and comprises a duplexed
region; and/or a tracrRNA extension comprising 10-5000 nucleotides
in length, or any combination thereof.
[0357] In some instances, a complex can comprise a site-directed
polypeptide, wherein the site-directed polypeptide comprises an
amino acid sequence comprising at least 15% amino acid identity to
a Cas9 from S. pyogenes, two nucleic acid cleaving domains (i.e.,
an HNH domain and a RuvC domain), and a linker linking the
site-directed polypeptide to a non-native sequence; and a
double-guide nucleic acid-targeting nucleic acid comprising 1) a
first nucleic acid molecule comprising a spacer extension sequence
from 10-5000 nucleotides in length; a spacer sequence of 12-30
nucleotides in length, wherein the spacer is at least 50%
complementary to a target nucleic acid; a minimum CRISPR repeat
comprising at least 60% identity to a crRNA from a prokaryote
(e.g., S. pyogenes) or phage over 6 contiguous nucleotides and
wherein the minimum CRISPR repeat has a length from 5-30
nucleotides, and at least 3 unpaired nucleotides of a bulge; and 2)
a second nucleic acid molecule of the double-guide nucleic
acid-targeting nucleic acid can comprise a minimum tracrRNA
sequence comprising at least 60% identity to a tracrRNA from a
prokaryote (e.g., S. pyogenes) or phage over 6 contiguous
nucleotides and wherein the minimum tracrRNA sequence has a length
from 5-30 nucleotides and at least 1 unpaired nucleotide of a
bulge, wherein the 1 unpaired nucleotide of the bulge is located in
the same bulge as the 3 unpaired nucleotides of the minimum CRISPR
repeat; a 3' tracrRNA that comprises at least 60% identity to a
tracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides and wherein the 3' tracrRNA comprises a
length from 10-20 nucleotides, and comprises a duplexed region; a
P-domain that starts from 1-5 nucleotides downstream of the duplex
comprising the minimum CRISPR repeat and the minimum tracrRNA,
comprises 1-10 nucleotides, comprises a sequence that can hybridize
to a protospacer adjacent motif in a target nucleic acid, can form
a hairpin, and is located in the 3' tracrRNA region; and/or a
tracrRNA extension comprising 10-5000 nucleotides in length, or any
combination thereof.
[0358] In some instances, a complex can comprise a site-directed
polypeptide, wherein the site-directed polypeptide comprises an
amino acid sequence comprising at least 15% amino acid identity to
a Cas9 from S. pyogenes, two nucleic acid cleaving domains (i.e.,
an HNH domain and a RuvC domain), and a linker linking the
site-directed polypeptide to a non-native sequence; and a nucleic
acid-targeting nucleic acid comprising a spacer extension sequence
from 10-5000 nucleotides in length; a spacer sequence of 12-30
nucleotides in length, wherein the spacer is at least 50%
complementary to a target nucleic acid; a minimum CRISPR repeat
comprising at least 60% identity to a crRNA from a prokaryote
(e.g., S. pyogenes) or phage over 6, 7, or 8 contiguous nucleotides
and wherein the minimum CRISPR repeat has a length from 5-30
nucleotides; a minimum tracrRNA sequence comprising at least 60%
identity to a tracrRNA from a bacterium (e.g., S. pyogenes) over 6,
7, or 8 contiguous nucleotides and wherein the minimum tracrRNA
sequence has a length from 5-30 nucleotides; a linker sequence that
links the minimum CRISPR repeat and the minimum tracrRNA and
comprises a length from 3-5000 nucleotides; a 3' tracrRNA that
comprises at least 60% identity to a tracrRNA from a prokaryote
(e.g., S. pyogenes) or phage over 6, 7, or 8 contiguous nucleotides
and wherein the 3' tracrRNA comprises a length from 10-20
nucleotides, and comprises a duplexed region; and/or a tracrRNA
extension comprising 10-5000 nucleotides in length, or any
combination thereof. This nucleic acid-targeting nucleic acid can
be referred to as a single guide nucleic acid-targeting nucleic
acid.
[0359] In some instances, a complex can comprise a site-directed
polypeptide, wherein the site-directed polypeptide comprises an
amino acid sequence comprising at least 15% amino acid identity to
a Cas9 from S. pyogenes, two nucleic acid cleaving domains (i.e.,
an HNH domain and a RuvC domain), and a linker linking the
site-directed polypeptide to a non-native sequence; and nucleic
acid-targeting nucleic acid can comprising a spacer extension
sequence from 10-5000 nucleotides in length; a spacer sequence of
12-30 nucleotides in length, wherein the spacer is at least 50%
complementary to a target nucleic acid; a duplex comprising 1) a
minimum CRISPR repeat comprising at least 60% identity to a crRNA
from a prokaryote (e.g., S. pyogenes) or phage over 6 contiguous
nucleotides and wherein the minimum CRISPR repeat has a length from
5-30 nucleotides, 2) a minimum tracrRNA sequence comprising at
least 60% identity to a tracrRNA from a bacterium (e.g., S.
pyogenes) over 6 contiguous nucleotides and wherein the minimum
tracrRNA sequence has a length from 5-30 nucleotides, and 3) a
bulge wherein the bulge comprises at least 3 unpaired nucleotides
on the minimum CRISPR repeat strand of the duplex and at least 1
unpaired nucleotide on the minimum tracrRNA sequence strand of the
duplex; a linker sequence that links the minimum CRISPR repeat and
the minimum tracrRNA and comprises a length from 3-5000
nucleotides; a 3' tracrRNA that comprises at least 60% identity to
a tracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides, wherein the 3' tracrRNA comprises a length
from 10-20 nucleotides and comprises a duplexed region; a P-domain
that starts from 1-5 nucleotides downstream of the duplex
comprising the minimum CRISPR repeat and the minimum tracrRNA,
comprises 1-10 nucleotides, comprises a sequence that can hybridize
to a protospacer adjacent motif in a target nucleic acid, can form
a hairpin, and is located in the 3' tracrRNA region; and/or a
tracrRNA extension comprising 10-5000 nucleotides in length, or any
combination thereof.
[0360] In some instances, a complex can comprise a site-directed
polypeptide, wherein the site-directed polypeptide comprises at
least 15% amino acid identity to a Cas9 from S. pyogenes, two
nucleic acid cleaving domains (i.e., an HNH domain and a RuvC
domain), wherein the site-directed polypeptide comprises a mutation
in one or both of the nucleic acid cleaving domains that reduces
the cleaving activity of the nuclease domains by at least 50%; and
a double-guide nucleic acid-targeting nucleic acid comprising 1) a
first nucleic acid molecule comprising a spacer extension sequence
from 10-5000 nucleotides in length; a spacer sequence of 12-30
nucleotides in length, wherein the spacer is at least 50%
complementary to a target nucleic acid; and a minimum CRISPR repeat
comprising at least 60% identity to a crRNA from a prokaryote
(e.g., S. pyogenes) or phage over 6 contiguous nucleotides and
wherein the minimum CRISPR repeat has a length from 5-30
nucleotides; and 2) a second nucleic acid molecule of the
double-guide nucleic acid-targeting nucleic acid can comprise a
minimum tracrRNA sequence comprising at least 60% identity to a
tracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides and wherein the minimum tracrRNA sequence
has a length from 5-30 nucleotides; a 3' tracrRNA that comprises at
least 60% identity to a tracrRNA from a bacterium (e.g., S.
pyogenes) over 6 contiguous nucleotides and wherein the 3' tracrRNA
comprises a length from 10-20 nucleotides, and comprises a duplexed
region; and/or a tracrRNA extension comprising 10-5000 nucleotides
in length, or any combination thereof.
[0361] In some instances, a complex can comprise a site-directed
polypeptide, wherein the site-directed polypeptide comprises at
least 15% amino acid identity to a Cas9 from S. pyogenes, two
nucleic acid cleaving domains (i.e., an HNH domain and a RuvC
domain), wherein the site-directed polypeptide comprises a mutation
in one or both of the nucleic acid cleaving domains that reduces
the cleaving activity of the nuclease domains by at least 50%; and
a double-guide nucleic acid-targeting nucleic acid comprising 1) a
first nucleic acid molecule comprising a spacer extension sequence
from 10-5000 nucleotides in length; a spacer sequence of 12-30
nucleotides in length, wherein the spacer is at least 50%
complementary to a target nucleic acid; a minimum CRISPR repeat
comprising at least 60% identity to a crRNA from a prokaryote
(e.g., S. pyogenes) or phage over 6 contiguous nucleotides and
wherein the minimum CRISPR repeat has a length from 5-30
nucleotides, and at least 3 unpaired nucleotides of a bulge; and 2)
a second nucleic acid molecule of the double-guide nucleic
acid-targeting nucleic acid can comprise a minimum tracrRNA
sequence comprising at least 60% identity to a tracrRNA from a
prokaryote (e.g., S. pyogenes) or phage over 6 contiguous
nucleotides and wherein the minimum tracrRNA sequence has a length
from 5-30 nucleotides and at least 1 unpaired nucleotide of a
bulge, wherein the 1 unpaired nucleotide of the bulge is located in
the same bulge as the 3 unpaired nucleotides of the minimum CRISPR
repeat; a 3' tracrRNA that comprises at least 60% identity to a
tracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides and wherein the 3' tracrRNA comprises a
length from 10-20 nucleotides, and comprises a duplexed region; a
P-domain that starts from 1-5 nucleotides downstream of the duplex
comprising the minimum CRISPR repeat and the minimum tracrRNA,
comprises 1-10 nucleotides, comprises a sequence that can hybridize
to a protospacer adjacent motif in a target nucleic acid, can form
a hairpin, and is located in the 3' tracrRNA region; and/or a
tracrRNA extension comprising 10-5000 nucleotides in length, or any
combination thereof.
[0362] In some instances, a complex can comprise a site-directed
polypeptide, wherein the site-directed polypeptide comprises at
least 15% amino acid identity to a Cas9 from S. pyogenes, two
nucleic acid cleaving domains (i.e., an HNH domain and a RuvC
domain), wherein the site-directed polypeptide comprises a mutation
in one or both of the nucleic acid cleaving domains that reduces
the cleaving activity of the nuclease domains by at least 50%; and
nucleic acid-targeting nucleic acid comprising a spacer extension
sequence from 10-5000 nucleotides in length; a spacer sequence of
12-30 nucleotides in length, wherein the spacer is at least 50%
complementary to a target nucleic acid; a minimum CRISPR repeat
comprising at least 60% identity to a crRNA from a prokaryote
(e.g., S. pyogenes) or phage over 6, 7, or 8 contiguous nucleotides
and wherein the minimum CRISPR repeat has a length from 5-30
nucleotides; a minimum tracrRNA sequence comprising at least 60%
identity to a tracrRNA from a bacterium (e.g., S. pyogenes) over 6,
7, or 8 contiguous nucleotides and wherein the minimum tracrRNA
sequence has a length from 5-30 nucleotides; a linker sequence that
links the minimum CRISPR repeat and the minimum tracrRNA and
comprises a length from 3-5000 nucleotides; a 3' tracrRNA that
comprises at least 60% identity to a tracrRNA from a prokaryote
(e.g., S. pyogenes) or phage over 6, 7, or 8 contiguous nucleotides
and wherein the 3' tracrRNA comprises a length from 10-20
nucleotides, and comprises a duplexed region; and/or a tracrRNA
extension comprising 10-5000 nucleotides in length, or any
combination thereof. This nucleic acid-targeting nucleic acid can
be referred to as a single guide nucleic acid-targeting nucleic
acid.
[0363] In some instances, a complex can comprise a site-directed
polypeptide, wherein the site-directed polypeptide comprises at
least 15% amino acid identity to a Cas9 from S. pyogenes, two
nucleic acid cleaving domains (i.e., an HNH domain and a RuvC
domain), wherein the site-directed polypeptide comprises a mutation
in one or both of the nucleic acid cleaving domains that reduces
the cleaving activity of the nuclease domains by at least 50%; and
nucleic acid-targeting nucleic acid can comprising a spacer
extension sequence from 10-5000 nucleotides in length; a spacer
sequence of 12-30 nucleotides in length, wherein the spacer is at
least 50% complementary to a target nucleic acid; a duplex
comprising 1) a minimum CRISPR repeat comprising at least 60%
identity to a crRNA from a prokaryote (e.g., S. pyogenes) or phage
over 6 contiguous nucleotides and wherein the minimum CRISPR repeat
has a length from 5-30 nucleotides, 2) a minimum tracrRNA sequence
comprising at least 60% identity to a tracrRNA from a bacterium
(e.g., S. pyogenes) over 6 contiguous nucleotides and wherein the
minimum tracrRNA sequence has a length from 5-30 nucleotides, and
3) a bulge wherein the bulge comprises at least 3 unpaired
nucleotides on the minimum CRISPR repeat strand of the duplex and
at least 1 unpaired nucleotide on the minimum tracrRNA sequence
strand of the duplex; a linker sequence that links the minimum
CRISPR repeat and the minimum tracrRNA and comprises a length from
3-5000 nucleotides; a 3' tracrRNA that comprises at least 60%
identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or
phage over 6 contiguous nucleotides, wherein the 3' tracrRNA
comprises a length from 10-20 nucleotides and comprises a duplexed
region; a P-domain that starts from 1-5 nucleotides downstream of
the duplex comprising the minimum CRISPR repeat and the minimum
tracrRNA, comprises 1-10 nucleotides, comprises a sequence that can
hybridize to a protospacer adjacent motif in a target nucleic acid,
can form a hairpin, and is located in the 3' tracrRNA region;
and/or a tracrRNA extension comprising 10-5000 nucleotides in
length, or any combination thereof.
[0364] In some instances, a complex can comprise a site-directed
polypeptide, wherein the site-directed polypeptide comprises at
least 15% amino acid identity to a Cas9 from S. pyogenes, two
nucleic acid cleaving domains (i.e., an HNH domain and a RuvC
domain), wherein the site-directed polypeptide comprises a mutation
in one or both of the nucleic acid cleaving domains that reduces
the cleaving activity of the nuclease domains by at least 50%; and
a double-guide nucleic acid-targeting nucleic acid comprising 1) a
first nucleic acid molecule comprising a spacer extension sequence
from 10-5000 nucleotides in length; a spacer sequence of 12-30
nucleotides in length, wherein the spacer is at least 50%
complementary to a target nucleic acid; and a minimum CRISPR repeat
comprising at least 60% identity to a crRNA from a prokaryote
(e.g., S. pyogenes) or phage over 6 contiguous nucleotides and
wherein the minimum CRISPR repeat has a length from 5-30
nucleotides; and 2) a second nucleic acid molecule of the
double-guide nucleic acid-targeting nucleic acid can comprise a
minimum tracrRNA sequence comprising at least 60% identity to a
tracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides and wherein the minimum tracrRNA sequence
has a length from 5-30 nucleotides; a 3' tracrRNA that comprises at
least 60% identity to a tracrRNA from a bacterium (e.g., S.
pyogenes) over 6 contiguous nucleotides and wherein the 3' tracrRNA
comprises a length from 10-20 nucleotides, and comprises a duplexed
region; and/or a tracrRNA extension comprising 10-5000 nucleotides
in length, or any combination thereof.
[0365] In some instances, a complex can comprise a site-directed
polypeptide, wherein the site-directed polypeptide comprises at
least 15% amino acid identity to a Cas9 from S. pyogenes, two
nucleic acid cleaving domains (i.e., an HNH domain and a RuvC
domain), wherein the site-directed polypeptide comprises a mutation
in one or both of the nucleic acid cleaving domains that reduces
the cleaving activity of the nuclease domains by at least 50%; and
a double-guide nucleic acid-targeting nucleic acid comprising 1) a
first nucleic acid molecule comprising a spacer extension sequence
from 10-5000 nucleotides in length; a spacer sequence of 12-30
nucleotides in length, wherein the spacer is at least 50%
complementary to a target nucleic acid; a minimum CRISPR repeat
comprising at least 60% identity to a crRNA from a prokaryote
(e.g., S. pyogenes) or phage over 6 contiguous nucleotides and
wherein the minimum CRISPR repeat has a length from 5-30
nucleotides, and at least 3 unpaired nucleotides of a bulge; and 2)
a second nucleic acid molecule of the double-guide nucleic
acid-targeting nucleic acid can comprise a minimum tracrRNA
sequence comprising at least 60% identity to a tracrRNA from a
prokaryote (e.g., S. pyogenes) or phage over 6 contiguous
nucleotides and wherein the minimum tracrRNA sequence has a length
from 5-30 nucleotides and at least 1 unpaired nucleotide of a
bulge, wherein the 1 unpaired nucleotide of the bulge is located in
the same bulge as the 3 unpaired nucleotides of the minimum CRISPR
repeat; a 3' tracrRNA that comprises at least 60% identity to a
tracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides and wherein the 3' tracrRNA comprises a
length from 10-20 nucleotides, and comprises a duplexed region; a
P-domain that starts from 1-5 nucleotides downstream of the duplex
comprising the minimum CRISPR repeat and the minimum tracrRNA,
comprises 1-10 nucleotides, comprises a sequence that can hybridize
to a protospacer adjacent motif in a target nucleic acid, can form
a hairpin, and is located in the 3' tracrRNA region; and/or a
tracrRNA extension comprising 10-5000 nucleotides in length, or any
combination thereof.
[0366] In some instances, a complex can comprise a site-directed
polypeptide, wherein the site-directed polypeptide comprises at
least 15% amino acid identity to a Cas9 from S. pyogenes, two
nucleic acid cleaving domains (i.e., an HNH domain and a RuvC
domain), wherein the site-directed polypeptide comprises a mutation
in one or both of the nucleic acid cleaving domains that reduces
the cleaving activity of the nuclease domains by at least 50%; and
nucleic acid-targeting nucleic acid comprising a spacer extension
sequence from 10-5000 nucleotides in length; a spacer sequence of
12-30 nucleotides in length, wherein the spacer is at least 50%
complementary to a target nucleic acid; a minimum CRISPR repeat
comprising at least 60% identity to a crRNA from a prokaryote
(e.g., S. pyogenes) or phage over 6, 7, or 8 contiguous nucleotides
and wherein the minimum CRISPR repeat has a length from 5-30
nucleotides; a minimum tracrRNA sequence comprising at least 60%
identity to a tracrRNA from a bacterium (e.g., S. pyogenes) over 6,
7, or 8 contiguous nucleotides and wherein the minimum tracrRNA
sequence has a length from 5-30 nucleotides; a linker sequence that
links the minimum CRISPR repeat and the minimum tracrRNA and
comprises a length from 3-5000 nucleotides; a 3' tracrRNA that
comprises at least 60% identity to a tracrRNA from a prokaryote
(e.g., S. pyogenes) or phage over 6, 7, or 8 contiguous nucleotides
and wherein the 3' tracrRNA comprises a length from 10-20
nucleotides, and comprises a duplexed region; and/or a tracrRNA
extension comprising 10-5000 nucleotides in length, or any
combination thereof. This nucleic acid-targeting nucleic acid can
be referred to as a single guide nucleic acid-targeting nucleic
acid.
[0367] In some instances, a complex can comprise a site-directed
polypeptide, wherein the site-directed polypeptide comprises at
least 15% amino acid identity to a Cas9 from S. pyogenes, two
nucleic acid cleaving domains (i.e., an HNH domain and a RuvC
domain), wherein the site-directed polypeptide comprises a mutation
in one or both of the nucleic acid cleaving domains that reduces
the cleaving activity of the nuclease domains by at least 50%; and
a nucleic acid-targeting nucleic acid can comprising a spacer
extension sequence from 10-5000 nucleotides in length; a spacer
sequence of 12-30 nucleotides in length, wherein the spacer is at
least 50% complementary to a target nucleic acid; a duplex
comprising 1) a minimum CRISPR repeat comprising at least 60%
identity to a crRNA from a prokaryote (e.g., S. pyogenes) or phage
over 6 contiguous nucleotides and wherein the minimum CRISPR repeat
has a length from 5-30 nucleotides, 2) a minimum tracrRNA sequence
comprising at least 60% identity to a tracrRNA from a bacterium
(e.g., S. pyogenes) over 6 contiguous nucleotides and wherein the
minimum tracrRNA sequence has a length from 5-30 nucleotides, and
3) a bulge wherein the bulge comprises at least 3 unpaired
nucleotides on the minimum CRISPR repeat strand of the duplex and
at least 1 unpaired nucleotide on the minimum tracrRNA sequence
strand of the duplex; a linker sequence that links the minimum
CRISPR repeat and the minimum tracrRNA and comprises a length from
3-5000 nucleotides; a 3' tracrRNA that comprises at least 60%
identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or
phage over 6 contiguous nucleotides, wherein the 3' tracrRNA
comprises a length from 10-20 nucleotides and comprises a duplexed
region; a P-domain that starts from 1-5 nucleotides downstream of
the duplex comprising the minimum CRISPR repeat and the minimum
tracrRNA, comprises 1-10 nucleotides, comprises a sequence that can
hybridize to a protospacer adjacent motif in a target nucleic acid,
can form a hairpin, and is located in the 3' tracrRNA region;
and/or a tracrRNA extension comprising 10-5000 nucleotides in
length, or any combination thereof.
[0368] In some instances, a complex can comprise a site-directed
polypeptide, wherein the site-directed polypeptide comprises at
least 15% amino acid identity to a Cas9 from S. pyogenes, two
nucleic acid cleaving domains (i.e., an HNH domain and a RuvC
domain), wherein the site-directed polypeptide comprises a mutation
in one or both of the nucleic acid cleaving domains that reduces
the cleaving activity of the nuclease domains by at least 50%; and
a double-guide nucleic acid-targeting nucleic acid comprising 1) a
first nucleic acid molecule comprising a spacer extension sequence
from 10-5000 nucleotides in length; a spacer sequence of 12-30
nucleotides in length, wherein the spacer is at least 50%
complementary to a target nucleic acid; and a minimum CRISPR repeat
comprising at least 60% identity to a crRNA from a prokaryote
(e.g., S. pyogenes) or phage over 6 contiguous nucleotides and
wherein the minimum CRISPR repeat has a length from 5-30
nucleotides; and 2) a second nucleic acid molecule of the
double-guide nucleic acid-targeting nucleic acid can comprise a
minimum tracrRNA sequence comprising at least 60% identity to a
tracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides and wherein the minimum tracrRNA sequence
has a length from 5-30 nucleotides; a 3' tracrRNA that comprises at
least 60% identity to a tracrRNA from a bacterium (e.g., S.
pyogenes) over 6 contiguous nucleotides and wherein the 3' tracrRNA
comprises a length from 10-20 nucleotides, and comprises a duplexed
region; and/or a tracrRNA extension comprising 10-5000 nucleotides
in length, or any combination thereof.
[0369] In some instances, a complex can comprise a site-directed
polypeptide, wherein the site-directed polypeptide comprises at
least 15% amino acid identity to a Cas9 from S. pyogenes, and two
nucleic acid cleaving domains, wherein one or both of the nucleic
acid cleaving domains comprise at least 50% amino acid identity to
a nuclease domain from Cas9 from S. pyogenes; and a double-guide
nucleic acid-targeting nucleic acid comprising 1) a first nucleic
acid molecule comprising a spacer extension sequence from 10-5000
nucleotides in length; a spacer sequence of 12-30 nucleotides in
length, wherein the spacer is at least 50% complementary to a
target nucleic acid; a minimum CRISPR repeat comprising at least
60% identity to a crRNA from a prokaryote (e.g., S. pyogenes) or
phage over 6 contiguous nucleotides and wherein the minimum CRISPR
repeat has a length from 5-30 nucleotides, and at least 3 unpaired
nucleotides of a bulge; and 2) a second nucleic acid molecule of
the double-guide nucleic acid-targeting nucleic acid can comprise a
minimum tracrRNA sequence comprising at least 60% identity to a
tracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides and wherein the minimum tracrRNA sequence
has a length from 5-30 nucleotides and at least 1 unpaired
nucleotide of a bulge, wherein the 1 unpaired nucleotide of the
bulge is located in the same bulge as the 3 unpaired nucleotides of
the minimum CRISPR repeat; a 3' tracrRNA that comprises at least
60% identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or
phage over 6 contiguous nucleotides and wherein the 3' tracrRNA
comprises a length from 10-20 nucleotides, and comprises a duplexed
region; a P-domain that starts from 1-5 nucleotides downstream of
the duplex comprising the minimum CRISPR repeat and the minimum
tracrRNA, comprises 1-10 nucleotides, comprises a sequence that can
hybridize to a protospacer adjacent motif in a target nucleic acid,
can form a hairpin, and is located in the 3' tracrRNA region;
and/or a tracrRNA extension comprising 10-5000 nucleotides in
length, or any combination thereof.
[0370] In some instances, a complex can comprise a site-directed
polypeptide, wherein the site-directed polypeptide comprises at
least 15% amino acid identity to a Cas9 from S. pyogenes, and two
nucleic acid cleaving domains, wherein one or both of the nucleic
acid cleaving domains comprise at least 50% amino acid identity to
a nuclease domain from Cas9 from S. pyogenes; and nucleic
acid-targeting nucleic acid comprising a spacer extension sequence
from 10-5000 nucleotides in length; a spacer sequence of 12-30
nucleotides in length, wherein the spacer is at least 50%
complementary to a target nucleic acid; a minimum CRISPR repeat
comprising at least 60% identity to a crRNA from a prokaryote
(e.g., S. pyogenes) or phage over 6, 7, or 8 contiguous nucleotides
and wherein the minimum CRISPR repeat has a length from 5-30
nucleotides; a minimum tracrRNA sequence comprising at least 60%
identity to a tracrRNA from a bacterium (e.g., S. pyogenes) over 6,
7, or 8 contiguous nucleotides and wherein the minimum tracrRNA
sequence has a length from 5-30 nucleotides; a linker sequence that
links the minimum CRISPR repeat and the minimum tracrRNA and
comprises a length from 3-5000 nucleotides; a 3' tracrRNA that
comprises at least 60% identity to a tracrRNA from a prokaryote
(e.g., S. pyogenes) or phage over 6, 7, or 8 contiguous nucleotides
and wherein the 3' tracrRNA comprises a length from 10-20
nucleotides, and comprises a duplexed region; and/or a tracrRNA
extension comprising 10-5000 nucleotides in length, or any
combination thereof. This nucleic acid-targeting nucleic acid can
be referred to as a single guide nucleic acid-targeting nucleic
acid.
[0371] In some instances, a complex can comprise a site-directed
polypeptide, wherein the site-directed polypeptide comprises at
least 15% amino acid identity to a Cas9 from S. pyogenes, and two
nucleic acid cleaving domains, wherein one or both of the nucleic
acid cleaving domains comprise at least 50% amino acid identity to
a nuclease domain from Cas9 from S. pyogenes; and a nucleic
acid-targeting nucleic acid can comprising a spacer extension
sequence from 10-5000 nucleotides in length; a spacer sequence of
12-30 nucleotides in length, wherein the spacer is at least 50%
complementary to a target nucleic acid; a duplex comprising 1) a
minimum CRISPR repeat comprising at least 60% identity to a crRNA
from a prokaryote (e.g., S. pyogenes) or phage over 6 contiguous
nucleotides and wherein the minimum CRISPR repeat has a length from
5-30 nucleotides, 2) a minimum tracrRNA sequence comprising at
least 60% identity to a tracrRNA from a bacterium (e.g., S.
pyogenes) over 6 contiguous nucleotides and wherein the minimum
tracrRNA sequence has a length from 5-30 nucleotides, and 3) a
bulge wherein the bulge comprises at least 3 unpaired nucleotides
on the minimum CRISPR repeat strand of the duplex and at least 1
unpaired nucleotide on the minimum tracrRNA sequence strand of the
duplex; a linker sequence that links the minimum CRISPR repeat and
the minimum tracrRNA and comprises a length from 3-5000
nucleotides; a 3' tracrRNA that comprises at least 60% identity to
a tracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides, wherein the 3' tracrRNA comprises a length
from 10-20 nucleotides and comprises a duplexed region; a P-domain
that starts from 1-5 nucleotides downstream of the duplex
comprising the minimum CRISPR repeat and the minimum tracrRNA,
comprises 1-10 nucleotides, comprises a sequence that can hybridize
to a protospacer adjacent motif in a target nucleic acid, can form
a hairpin, and is located in the 3' tracrRNA region; and/or a
tracrRNA extension comprising 10-5000 nucleotides in length, or any
combination thereof.
[0372] Any nucleic acid-targeting nucleic acid of the disclosure, a
site-directed polypeptide of the disclosure, an effector protein, a
multiplexed genetic targeting agent, a donor polynucleotide, a
tandem fusion protein, a reporter element, a genetic element of
interest, a component of a split system and/or any nucleic acid or
proteinaceous molecule necessary to carry out the embodiments of
the methods of the disclosure may be recombinant, purified and/or
isolated.
[0373] Nucleic Acids Encoding a Nucleic Acid-Targeting Nucleic Acid
and/or a Site-Directed Polypeptide
[0374] The present disclosure provides for a nucleic acid
comprising a nucleotide sequence encoding a nucleic acid-targeting
nucleic acid of the disclosure, a site-directed polypeptide of the
disclosure, an effector protein, a multiplexed genetic targeting
agent, a donor polynucleotide, a tandem fusion protein, a reporter
element, a genetic element of interest, a component of a split
system and/or any nucleic acid or proteinaceous molecule necessary
to carry out the embodiments of the methods of the disclosure. In
some embodiments, the nucleic acid encoding a nucleic
acid-targeting nucleic acid of the disclosure, a site-directed
polypeptide of the disclosure, an effector protein, a multiplexed
genetic targeting agent, a donor polynucleotide, a tandem fusion
protein, a reporter element, a genetic element of interest, a
component of a split system and/or any nucleic acid or
proteinaceous molecule necessary to carry out the embodiments of
the methods of the disclosure can be a vector (e.g., a recombinant
expression vector).
[0375] In some embodiments, the recombinant expression vector can
be a viral construct, (e.g., a recombinant adeno-associated virus
construct), a recombinant adenoviral construct, a recombinant
lentiviral construct, a recombinant retroviral construct, etc.
[0376] Suitable expression vectors can include, but are not limited
to, viral vectors (e.g. viral vectors based on vaccinia virus,
poliovirus, adenovirus, adeno-associated virus, SV40, herpes
simplex virus, human immunodeficiency virus, a retroviral vector
(e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors
derived from retroviruses such as Rous Sarcoma Virus, Harvey
Sarcoma Virus, avian leukosis virus, a lentivirus, human
immunodeficiency virus, myeloproliferative sarcoma virus, and
mammary tumor virus), plant vectors (e.g., T-DNA vector), and the
like. The following vectors can be provided by way of example, for
eukaryotic host cells: pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40
(Pharmacia). Other vectors may be used so long as they are
compatible with the host cell.
[0377] In some instances, a vector can be a linearized vector. A
linearized vector can comprise a site-directed polypeptide and/or a
nucleic acid-targeting nucleic acid. A linearized vector may not be
a circular plasmid. A linearized vector can comprise a
double-stranded break. A linearized vector may comprise a sequence
encoding a fluorescent protein (e.g., orange fluorescent protein
(OFP)). A linearized vector may comprise a sequence encoding an
antigen (e.g., CD4). A linearized vector can be linearized (e.g.,
cut) in a region of the vector encoding parts of the nucleic
acid-targeting nucleic acid. For example a linearized vector can be
linearized (e.g., cut) in a region of the nucleic acid-targeting
nucleic acid 5' to the crRNA portion of the nucleic acid-targeting
nucleic acid. A linearized vector can be linearized (e.g., cut) in
a region of the nucleic acid-targeting nucleic acid 3' to the
spacer extension sequence of the nucleic acid-targeting nucleic
acid. A linearized vector can be linearized (e.g., cut) in a region
of the nucleic acid-targeting nucleic acid encoding the crRNA
sequence of the nucleic acid-targeting nucleic acid. In some
instances, a linearized vector or a closed supercoiled vector
comprises a sequence encoding a site-directed polypeptide (e.g.,
Cas9), a promoter driving expression of the sequence encoding the
site-directed polypeptide (e.g., CMV promoter), a sequence encoding
a linker (e.g., 2A), a sequence encoding a marker (e.g., CD4 or
OFP), a sequence encoding portion of a nucleic acid-targeting
nucleic acid, a promoter driving expression of the sequence
encoding a portion of the nucleic acid-targeting nucleic acid, and
a sequence encoding a selectable marker (e.g., ampicillin), or any
combination thereof.
[0378] A vector can comprise a transcription and/or translation
control element. Depending on the host/vector system utilized, any
of a number of suitable transcription and translation control
elements, including constitutive and inducible promoters,
transcription enhancer elements, transcription terminators, etc.
may be used in the expression vector.
[0379] In some embodiments, a nucleotide sequence encoding a
nucleic acid-targeting nucleic acid of the disclosure, a
site-directed polypeptide of the disclosure, an effector protein, a
multiplexed genetic targeting agent, a donor polynucleotide, a
tandem fusion protein, a reporter element, a genetic element of
interest, a component of a split system and/or any nucleic acid or
proteinaceous molecule necessary to carry out the embodiments of
the methods of the disclosure can be operably linked to a control
element (e.g., a transcriptional control element), such as a
promoter. The transcriptional control element may be functional in
a eukaryotic cell, (e.g., a mammalian cell), a prokaryotic cell
(e.g., bacterial or archaeal cell). In some embodiments, a
nucleotide sequence encoding a nucleic acid-targeting nucleic acid
of the disclosure, a site-directed polypeptide of the disclosure,
an effector protein, a multiplexed genetic targeting agent, a donor
polynucleotide, a tandem fusion protein, a reporter element, a
genetic element of interest, a component of a split system and/or
any nucleic acid or proteinaceous molecule necessary to carry out
the embodiments of the methods of the disclosure can be operably
linked to multiple control elements. Operable linkage to multiple
control elements can allow expression of the nucleotide sequence
encoding a nucleic acid-targeting nucleic acid of the disclosure, a
site-directed polypeptide of the disclosure, an effector protein, a
multiplexed genetic targeting agent, a donor polynucleotide, a
tandem fusion protein, a reporter element, a genetic element of
interest, a component of a split system and/or any nucleic acid or
proteinaceous molecule necessary to carry out the embodiments of
the methods of the disclosure in either prokaryotic or eukaryotic
cells.
[0380] Non-limiting examples of suitable eukaryotic promoters (i.e.
promoters functional in a eukaryotic cell) can include those from
cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV)
thymidine kinase, early and late SV40, long terminal repeats (LTRs)
from retrovirus, human elongation factor-1 promoter (EF1), a hybrid
construct comprising the cytomegalovirus (CMV) enhancer fused to
the chicken beta-active promoter (CAG), murine stem cell virus
promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK) and
mouse metallothionein-I. The promoter can be a fungi promoter. The
promoter can be a plant promoter. A database of plant promoteres
can be found (e.g., PlantProm). The expression vector may also
contain a ribosome binding site for translation initiation and a
transcription terminator. The expression vector may also include
appropriate sequences for amplifying expression. The expression
vector may also include nucleotide sequences encoding non-native
tags (e.g., 6.times.His tag, hemagglutinin tag, green fluorescent
protein, etc.) that are fused to the site-directed polypeptide,
thus resulting in a fusion protein.
[0381] In some embodiments, a nucleotide sequence or sequences
encoding a nucleic acid-targeting nucleic acid of the disclosure, a
site-directed polypeptide of the disclosure, an effector protein, a
multiplexed genetic targeting agent, a donor polynucleotide, a
tandem fusion protein, a reporter element, a genetic element of
interest, a component of a split system and/or any nucleic acid or
proteinaceous molecule necessary to carry out the embodiments of
the methods of the disclosure can be operably linked to an
inducible promoter (e.g., heat shock promoter,
tetracycline-regulated promoter, steroid-regulated promoter,
metal-regulated promoter, estrogen receptor-regulated promoter,
etc.). In some embodiments, a nucleotide sequence encoding a
nucleic acid-targeting nucleic acid of the disclosure, a
site-directed polypeptide of the disclosure, an effector protein, a
multiplexed genetic targeting agent, a donor polynucleotide, a
tandem fusion protein, a reporter element, a genetic element of
interest, a component of a split system and/or any nucleic acid or
proteinaceous molecule necessary to carry out the embodiments of
the methods of the disclosure can be operably linked to a
constitutive promoter (e.g., CMV promoter, UBC promoter). In some
embodiments, the nucleotide sequence can be operably linked to a
spatially restricted and/or temporally restricted promoter (e.g., a
tissue specific promoter, a cell type specific promoter, etc.).
[0382] A nucleotide sequence or sequences encoding a nucleic
acid-targeting nucleic acid of the disclosure, a site-directed
polypeptide of the disclosure, an effector protein, a multiplexed
genetic targeting agent, a donor polynucleotide, a tandem fusion
protein, a reporter element, a genetic element of interest, a
component of a split system and/or any nucleic acid or
proteinaceous molecule necessary to carry out the embodiments of
the methods of the disclosure can be packaged into or on the
surface of biological compartments for delivery to cells.
Biological compartments can include, but are not limited to,
viruses (lentivirus, adenovirus), nanospheres, liposomes, quantum
dots, nanoparticles, polyethylene glycol particles, hydrogels, and
micelles.
[0383] Introduction of the complexes, polypeptides, and nucleic
acids of the disclosure into cells can occur by viral or
bacteriophage infection, transfection, conjugation, protoplast
fusion, lipofection, electroporation, calcium phosphate
precipitation, polyethyleneimine (PEI)-mediated transfection,
DEAE-dextran mediated transfection, liposome-mediated transfection,
particle gun technology, calcium phosphate precipitation, direct
micro-injection, nanoparticle-mediated nucleic acid delivery, and
the like.
[0384] Transgenic Cells and Organisms
[0385] The disclosure provides for transgenic cells and organisms.
The nucleic acid of a genetically modified host cell and/or
transgenic organism can be targeted for genome engineering.
[0386] Exemplary cells that can be used to generate transgenic
cells according to the methods of the disclosure can include, but
are not limited to, HeLa cell, Chinese Hamster Ovary cell, 293-T
cell, a pheochromocytoma, a neuroblastomas fibroblast, a
rhabdomyosarcoma, a dorsal root ganglion cell, a NSO cell, Tobacco
BY-2, CV-I (ATCC CCL 70), COS-I (ATCC CRL 1650), COS-7 (ATCC CRL
1651), CHO-K1 (ATCC, CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL
1658), HeLa (ATCC CCL 2), C 1271 (ATCC CRL 1616), BS-C-I (ATCC CCL
26), MRC-5 (ATCC CCL 171), L-cells, HEK-293 (ATCC CRL1 573) and PC
12 (ATCC CRL-1721), HEK293T (ATCC CRL-11268), RBL (ATCC CRL-1378),
SH-SY5Y (ATCC CRL-2266), MDCK (ATCC CCL-34), SJ-RH30 (ATCC
CRL-2061.), HepG2 (ATCC ND7/23 (ECACC 92090903), CHO (ECACC
85050302), Vera (ATCC CCL 81), Caco-2 (ATCC HTB 37), K562 (ATCC CCL
243), Jurkat (ATCC TIB-152), Per.C{dot over (o)}, Huvec (ATCC Human
Primary PCS 100-010, Mouse CRL 2514, CRL 2515, CRL 2516), HuH-7D12
(ECACC 01042712), 293 (ATCC CRL 10852), A549 (ATCC CCL 185), IMR-90
(ATCC CCL 186), MCF-7 (ATC HTB-22), U-2 OS (ATCC HTB-96), and T84
(ATCC CCL 248), or any cell available at American Type Culture
Collection (ATCC), or any combination thereof.
[0387] Organisms that can be transgenic can include bacteria,
archaea, single-cell eukaryotes, plants, algae, fungi (e.g.,
yeast), invertebrates (e.g., fruit fly, cnidarian, echinoderm,
nematode, etc), vertebrates (e.g., fish, amphibian, reptile, bird,
mammal), mammals mammal (e.g., a pig, a cow, a goat, a sheep, a
rodent, a rat, a mouse, a non-human primate, a human, etc.),
etc.
[0388] Transgenic organisms can comprise genetically modified
cells. Transgenic organisms and/or genetically modified cells can
comprise organisms and/or cells that have been genetically modified
with an exogenous nucleic acid comprising a nucleotide sequence
encoding nucleic acid-targeting nucleic acid of the disclosure, an
effector protein, and/or a site-directed polypeptide, or any
combination thereof.
[0389] A genetically modified cell can comprise an exogenous
site-directed polypeptide and/or an exogenous nucleic acid
comprising a nucleotide sequence encoding a site-directed
polypeptide. Expression of the site-directed polypeptide in the
cell may take 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, or more days. Cells,
introduced with the site-directed polypeptide, may be grown for
0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more days
before the cells can be removed from cell culture and/or host
organism.
[0390] Subjects
[0391] The disclosure provides for performing the methods of the
disclosure in a subject. A subject can be a human. A subject can be
a mammal (e.g., rat, mouse, cow, dog, pig, sheep, horse). A subject
can be a vetebrate or an invertebrate. A subject can be a
laboratory animal. A subject can be a patient. A subject can be
suffering from a disease. A subject can display symptoms of a
disease. A subject may not display symptoms of a disease, but still
have a disease. A subject can be under medical care of a caregiver
(e.g., the subject is hospitalized and is treated by a physician).
A subject can be a plant or a crop.
[0392] Kits
[0393] The present disclosure provides kits for carrying out the
methods of the disclosure. A kit can include one or more of: A
nucleic acid-targeting nucleic acid of the disclosure, a
polynucleotide encoding a nucleic acid-targeting nucleic acid, a
site-directed polypeptide of the disclosure, a polynucleotide
encoding a site-directed polypeptide, an effector protein, a
polynucleotide encoding an effector protein, a multiplexed genetic
targeting agent, a polynucleotide encoding a multiplexed genetic
targeting agent, a donor polynucleotide, a tandem fusion protein, a
polynucleotide encoding a tandem fusion protein, a reporter
element, a genetic element of interest, a component of a split
system and/or any nucleic acid or proteinaceous molecule necessary
to carry out the embodiments of the methods of the disclosure, or
any combination thereof.
[0394] A nucleic acid-targeting nucleic acid of the disclosure, a
polynucleotide encoding a nucleic acid-targeting nucleic acid, a
site-directed polypeptide of the disclosure, a polynucleotide
encoding a site-directed polypeptide, an effector protein, a
polynucleotide encoding an effector protein, a multiplexed genetic
targeting agent, a polynucleotide encoding a multiplexed genetic
targeting agent, a donor polynucleotide, a tandem fusion protein, a
polynucleotide encoding a tandem fusion protein, a reporter
element, a genetic element of interest, a component of a split
system and/or any nucleic acid or proteinaceous molecule necessary
to carry out the embodiments of the methods of the disclosure are
described in detail above.
[0395] A kit can comprise: (1) a vector comprising a nucleotide
sequence encoding a nucleic acid-targeting nucleic acid, and (2) a
vector comprising a nucleotide sequence encoding the site-directed
polypeptide and (2) a reagent for reconstitution and/or dilution of
the vectors.
[0396] A kit can comprise: (1) a vector comprising (i) a nucleotide
sequence encoding a nucleic acid-targeting nucleic acid, and (ii) a
nucleotide sequence encoding the site-directed polypeptide and (2)
a reagent for reconstitution and/or dilution of the vector.
[0397] A kit can comprise: (1) a vector comprising a nucleotide
sequence encoding a nucleic acid-targeting nucleic acid, (2) a
vector comprising a nucleotide sequence encoding the site-directed
polypeptide, (3) a vector comprising a nucleotide sequence encoding
an effector protein, a multiplexed genetic targeting agent, a donor
polynucleotide, a tandem fusion protein, a reporter element, a
genetic element of interest, a component of a split system and/or
any nucleic acid or proteinaceous molecule necessary to carry out
the embodiments of the methods of the disclosure, and (4) a reagent
for reconstitution and/or dilution of the vectors.
[0398] A kit can comprise: (1) a vector comprising (i) a nucleotide
sequence encoding a nucleic acid-targeting nucleic acid, (ii) a
nucleotide sequence encoding the site-directed polypeptide, (2) a
vector comprising a nucleotide sequence encoding an effector
protein, a multiplexed genetic targeting agent, a donor
polynucleotide, a tandem fusion protein, a reporter element, a
genetic element of interest, a component of a split system and/or
any nucleic acid or proteinaceous molecule necessary to carry out
the embodiments of the methods of the disclosure, and (3) a reagent
for reconstitution and/or dilution of the recombinant expression
vectors.
[0399] In some instances, a kit can comprise a site-directed
polypeptide (and/or a nucleic acid encoding the same), wherein the
site-directed polypeptide can comprise an amino acid sequence
comprising at least 15% amino acid identity to a Cas9 from S.
pyogenes, and two nucleic acid cleaving domains (i.e., an HNH
domain and a RuvC domain); and a double-guide nucleic
acid-targeting nucleic acid (and/or a nucleic acid encoding the
same) comprising 1) a first nucleic acid molecule comprising a
spacer extension sequence from 10-5000 nucleotides in length; a
spacer sequence of 12-30 nucleotides in length, wherein the spacer
is at least 50% complementary to a target nucleic acid; and a
minimum CRISPR repeat comprising at least 60% identity to a crRNA
from a prokaryote (e.g., S. pyogenes) or phage over 6 contiguous
nucleotides and wherein the minimum CRISPR repeat has a length from
5-30 nucleotides; and 2) a second nucleic acid molecule of the
double-guide nucleic acid-targeting nucleic acid can comprise a
minimum tracrRNA sequence comprising at least 60% identity to a
tracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides and wherein the minimum tracrRNA sequence
has a length from 5-30 nucleotides; a 3' tracrRNA that comprises at
least 60% identity to a tracrRNA from a bacterium (e.g., S.
pyogenes) over 6 contiguous nucleotides and wherein the 3' tracrRNA
comprises a length from 10-20 nucleotides, and comprises a duplexed
region; and/or a tracrRNA extension comprising 10-5000 nucleotides
in length, or any combination thereof.
[0400] In some instances, a kit can comprise a site-directed
polypeptide (and/or a nucleic acid encoding the same), wherein the
site-directed polypeptide can comprise an amino acid sequence
comprising at least 15% amino acid identity to a Cas9 from S.
pyogenes, and two nucleic acid cleaving domains (i.e., an HNH
domain and a RuvC domain); and, a double-guide nucleic
acid-targeting nucleic acid (and/or a nucleic acid encoding the
same) comprising 1) a first nucleic acid molecule comprising a
spacer extension sequence from 10-5000 nucleotides in length; a
spacer sequence of 12-30 nucleotides in length, wherein the spacer
is at least 50% complementary to a target nucleic acid; a minimum
CRISPR repeat comprising at least 60% identity to a crRNA from a
prokaryote (e.g., S. pyogenes) or phage over 6 contiguous
nucleotides and wherein the minimum CRISPR repeat has a length from
5-30 nucleotides, and at least 3 unpaired nucleotides of a bulge;
and 2) a second nucleic acid molecule of the double-guide nucleic
acid-targeting nucleic acid can comprise a minimum tracrRNA
sequence comprising at least 60% identity to a tracrRNA from a
prokaryote (e.g., S. pyogenes) or phage over 6 contiguous
nucleotides and wherein the minimum tracrRNA sequence has a length
from 5-30 nucleotides and at least 1 unpaired nucleotide of a
bulge, wherein the 1 unpaired nucleotide of the bulge is located in
the same bulge as the 3 unpaired nucleotides of the minimum CRISPR
repeat; a 3' tracrRNA that comprises at least 60% identity to a
tracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides and wherein the 3' tracrRNA comprises a
length from 10-20 nucleotides, and comprises a duplexed region; a
P-domain that starts from 1-5 nucleotides downstream of the duplex
comprising the minimum CRISPR repeat and the minimum tracrRNA,
comprises 1-10 nucleotides, comprises a sequence that can hybridize
to a protospacer adjacent motif in a target nucleic acid, can form
a hairpin, and is located in the 3' tracrRNA region; and/or a
tracrRNA extension comprising 10-5000 nucleotides in length, or any
combination thereof.
[0401] In some instances, a kit can comprise a site-directed
polypeptide (and/or a nucleic acid encoding the same), wherein the
site-directed polypeptide can comprise an amino acid sequence
comprising at least 15% amino acid identity to a Cas9 from S.
pyogenes and, two nucleic acid cleaving domains (i.e., an HNH
domain and a RuvC domain); and, a nucleic acid-targeting nucleic
acid (and/or a nucleic acid encoding the same) comprising a spacer
extension sequence from 10-5000 nucleotides in length; a spacer
sequence of 12-30 nucleotides in length, wherein the spacer is at
least 50% complementary to a target nucleic acid; a minimum CRISPR
repeat comprising at least 60% identity to a crRNA from a
prokaryote (e.g., S. pyogenes) or phage over 6, 7, or 8 contiguous
nucleotides and wherein the minimum CRISPR repeat has a length from
5-30 nucleotides; a minimum tracrRNA sequence comprising at least
60% identity to a tracrRNA from a bacterium (e.g., S. pyogenes)
over 6, 7, or 8 contiguous nucleotides and wherein the minimum
tracrRNA sequence has a length from 5-30 nucleotides; a linker
sequence that links the minimum CRISPR repeat and the minimum
tracrRNA and comprises a length from 3-5000 nucleotides; a 3'
tracrRNA that comprises at least 60% identity to a tracrRNA from a
prokaryote (e.g., S. pyogenes) or phage over 6, 7, or 8 contiguous
nucleotides and wherein the 3' tracrRNA comprises a length from
10-20 nucleotides, and comprises a duplexed region; and/or a
tracrRNA extension comprising 10-5000 nucleotides in length, or any
combination thereof. This nucleic acid-targeting nucleic acid can
be referred to as a single guide nucleic acid-targeting nucleic
acid.
[0402] In some instances, a kit can comprise a site-directed
polypeptide (and/or a nucleic acid encoding the same), wherein the
site-directed polypeptide can comprise an amino acid sequence
comprising at least 15% amino acid identity to a Cas9 from S.
pyogenes, and two nucleic acid cleaving domains (i.e., an HNH
domain and a RuvC domain); and, a nucleic acid-targeting nucleic
acid (and/or a nucleic acid encoding the same) can comprising a
spacer extension sequence from 10-5000 nucleotides in length; a
spacer sequence of 12-30 nucleotides in length, wherein the spacer
is at least 50% complementary to a target nucleic acid; a duplex
comprising 1) a minimum CRISPR repeat comprising at least 60%
identity to a crRNA from a prokaryote (e.g., S. pyogenes) or phage
over 6 contiguous nucleotides and wherein the minimum CRISPR repeat
has a length from 5-30 nucleotides, 2) a minimum tracrRNA sequence
comprising at least 60% identity to a tracrRNA from a bacterium
(e.g., S. pyogenes) over 6 contiguous nucleotides and wherein the
minimum tracrRNA sequence has a length from 5-30 nucleotides, and
3) a bulge wherein the bulge comprises at least 3 unpaired
nucleotides on the minimum CRISPR repeat strand of the duplex and
at least 1 unpaired nucleotide on the minimum tracrRNA sequence
strand of the duplex; a linker sequence that links the minimum
CRISPR repeat and the minimum tracrRNA and comprises a length from
3-5000 nucleotides; a 3' tracrRNA that comprises at least 60%
identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or
phage over 6 contiguous nucleotides, wherein the 3' tracrRNA
comprises a length from 10-20 nucleotides and comprises a duplexed
region; a P-domain that starts from 1-5 nucleotides downstream of
the duplex comprising the minimum CRISPR repeat and the minimum
tracrRNA, comprises 1-10 nucleotides, comprises a sequence that can
hybridize to a protospacer adjacent motif in a target nucleic acid,
can form a hairpin, and is located in the 3' tracrRNA region;
and/or a tracrRNA extension comprising 10-5000 nucleotides in
length, or any combination thereof.
[0403] In some instances, a kit can comprise a site-directed
polypeptide (and/or a nucleic acid encoding the same), wherein the
site-directed polypeptide comprises an amino acid sequence
comprising at least 15% amino acid identity to a Cas9 from S.
pyogenes, two nucleic acid cleaving domains (i.e., an HNH domain
and a RuvC domain), and a linker linking the site-directed
polypeptide to a non-native sequence; and a double-guide nucleic
acid-targeting nucleic acid (and/or a nucleic acid encoding the
same) comprising 1) a first nucleic acid molecule comprising a
spacer extension sequence from 10-5000 nucleotides in length; a
spacer sequence of 12-30 nucleotides in length, wherein the spacer
is at least 50% complementary to a target nucleic acid; and a
minimum CRISPR repeat comprising at least 60% identity to a crRNA
from a prokaryote (e.g., S. pyogenes) or phage over 6 contiguous
nucleotides and wherein the minimum CRISPR repeat has a length from
5-30 nucleotides; and 2) a second nucleic acid molecule of the
double-guide nucleic acid-targeting nucleic acid can comprise a
minimum tracrRNA sequence comprising at least 60% identity to a
tracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides and wherein the minimum tracrRNA sequence
has a length from 5-30 nucleotides; a 3' tracrRNA that comprises at
least 60% identity to a tracrRNA from a bacterium (e.g., S.
pyogenes) over 6 contiguous nucleotides and wherein the 3' tracrRNA
comprises a length from 10-20 nucleotides, and comprises a duplexed
region; and/or a tracrRNA extension comprising 10-5000 nucleotides
in length, or any combination thereof.
[0404] In some instances, a kit can comprise a site-directed
polypeptide (and/or a nucleic acid encoding the same), wherein the
site-directed polypeptide comprises an amino acid sequence
comprising at least 15% amino acid identity to a Cas9 from S.
pyogenes, two nucleic acid cleaving domains (i.e., an HNH domain
and a RuvC domain), and a linker linking the site-directed
polypeptide to a non-native sequence; and a double-guide nucleic
acid-targeting nucleic acid (and/or a nucleic acid encoding the
same) comprising 1) a first nucleic acid molecule comprising a
spacer extension sequence from 10-5000 nucleotides in length; a
spacer sequence of 12-30 nucleotides in length, wherein the spacer
is at least 50% complementary to a target nucleic acid; a minimum
CRISPR repeat comprising at least 60% identity to a crRNA from a
prokaryote (e.g., S. pyogenes) or phage over 6 contiguous
nucleotides and wherein the minimum CRISPR repeat has a length from
5-30 nucleotides, and at least 3 unpaired nucleotides of a bulge;
and 2) a second nucleic acid molecule of the double-guide nucleic
acid-targeting nucleic acid can comprise a minimum tracrRNA
sequence comprising at least 60% identity to a tracrRNA from a
prokaryote (e.g., S. pyogenes) or phage over 6 contiguous
nucleotides and wherein the minimum tracrRNA sequence has a length
from 5-30 nucleotides and at least 1 unpaired nucleotide of a
bulge, wherein the 1 unpaired nucleotide of the bulge is located in
the same bulge as the 3 unpaired nucleotides of the minimum CRISPR
repeat; a 3' tracrRNA that comprises at least 60% identity to a
tracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides and wherein the 3' tracrRNA comprises a
length from 10-20 nucleotides, and comprises a duplexed region; a
P-domain that starts from 1-5 nucleotides downstream of the duplex
comprising the minimum CRISPR repeat and the minimum tracrRNA,
comprises 1-10 nucleotides, comprises a sequence that can hybridize
to a protospacer adjacent motif in a target nucleic acid, can form
a hairpin, and is located in the 3' tracrRNA region; and/or a
tracrRNA extension comprising 10-5000 nucleotides in length, or any
combination thereof.
[0405] In some instances, a kit can comprise a site-directed
polypeptide (and/or a nucleic acid encoding the same), wherein the
site-directed polypeptide comprises an amino acid sequence
comprising at least 15% amino acid identity to a Cas9 from S.
pyogenes, two nucleic acid cleaving domains (i.e., an HNH domain
and a RuvC domain), and a linker linking the site-directed
polypeptide to a non-native sequence; and a nucleic acid-targeting
nucleic acid (and/or a nucleic acid encoding the same) comprising a
spacer extension sequence from 10-5000 nucleotides in length; a
spacer sequence of 12-30 nucleotides in length, wherein the spacer
is at least 50% complementary to a target nucleic acid; a minimum
CRISPR repeat comprising at least 60% identity to a crRNA from a
prokaryote (e.g., S. pyogenes) or phage over 6, 7, or 8 contiguous
nucleotides and wherein the minimum CRISPR repeat has a length from
5-30 nucleotides; a minimum tracrRNA sequence comprising at least
60% identity to a tracrRNA from a bacterium (e.g., S. pyogenes)
over 6, 7, or 8 contiguous nucleotides and wherein the minimum
tracrRNA sequence has a length from 5-30 nucleotides; a linker
sequence that links the minimum CRISPR repeat and the minimum
tracrRNA and comprises a length from 3-5000 nucleotides; a 3'
tracrRNA that comprises at least 60% identity to a tracrRNA from a
prokaryote (e.g., S. pyogenes) or phage over 6, 7, or 8 contiguous
nucleotides and wherein the 3' tracrRNA comprises a length from
10-20 nucleotides, and comprises a duplexed region; and/or a
tracrRNA extension comprising 10-5000 nucleotides in length, or any
combination thereof. This nucleic acid-targeting nucleic acid can
be referred to as a single guide nucleic acid-targeting nucleic
acid.
[0406] In some instances, a kit can comprise a site-directed
polypeptide (and/or a nucleic acid encoding the same), wherein the
site-directed polypeptide comprises an amino acid sequence
comprising at least 15% amino acid identity to a Cas9 from S.
pyogenes, two nucleic acid cleaving domains (i.e., an HNH domain
and a RuvC domain), and a linker linking the site-directed
polypeptide to a non-native sequence; and nucleic acid-targeting
nucleic acid (and/or a nucleic acid encoding the same) comprising a
spacer extension sequence from 10-5000 nucleotides in length; a
spacer sequence of 12-30 nucleotides in length, wherein the spacer
is at least 50% complementary to a target nucleic acid; a duplex
comprising 1) a minimum CRISPR repeat comprising at least 60%
identity to a crRNA from a prokaryote (e.g., S. pyogenes) or phage
over 6 contiguous nucleotides and wherein the minimum CRISPR repeat
has a length from 5-30 nucleotides, 2) a minimum tracrRNA sequence
comprising at least 60% identity to a tracrRNA from a bacterium
(e.g., S. pyogenes) over 6 contiguous nucleotides and wherein the
minimum tracrRNA sequence has a length from 5-30 nucleotides, and
3) a bulge wherein the bulge comprises at least 3 unpaired
nucleotides on the minimum CRISPR repeat strand of the duplex and
at least 1 unpaired nucleotide on the minimum tracrRNA sequence
strand of the duplex; a linker sequence that links the minimum
CRISPR repeat and the minimum tracrRNA and comprises a length from
3-5000 nucleotides; a 3' tracrRNA that comprises at least 60%
identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or
phage over 6 contiguous nucleotides, wherein the 3' tracrRNA
comprises a length from 10-20 nucleotides and comprises a duplexed
region; a P-domain that starts from 1-5 nucleotides downstream of
the duplex comprising the minimum CRISPR repeat and the minimum
tracrRNA, comprises 1-10 nucleotides, comprises a sequence that can
hybridize to a protospacer adjacent motif in a target nucleic acid,
can form a hairpin, and is located in the 3' tracrRNA region;
and/or a tracrRNA extension comprising 10-5000 nucleotides in
length, or any combination thereof.
[0407] In some instances, a kit can comprise a site-directed
polypeptide (and/or a nucleic acid encoding the same), wherein the
site-directed polypeptide comprises at least 15% amino acid
identity to a Cas9 from S. pyogenes, two nucleic acid cleaving
domains (i.e., an HNH domain and a RuvC domain), wherein the
site-directed polypeptide comprises a mutation in one or both of
the nucleic acid cleaving domains that reduces the cleaving
activity of the nuclease domains by at least 50%; and a
double-guide nucleic acid-targeting nucleic acid (and/or a nucleic
acid encoding the same) comprising 1) a first nucleic acid molecule
comprising a spacer extension sequence from 10-5000 nucleotides in
length; a spacer sequence of 12-30 nucleotides in length, wherein
the spacer is at least 50% complementary to a target nucleic acid;
and a minimum CRISPR repeat comprising at least 60% identity to a
crRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides and wherein the minimum CRISPR repeat has a
length from 5-30 nucleotides; and 2) a second nucleic acid molecule
of the double-guide nucleic acid-targeting nucleic acid can
comprise a minimum tracrRNA sequence comprising at least 60%
identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or
phage over 6 contiguous nucleotides and wherein the minimum
tracrRNA sequence has a length from 5-30 nucleotides; a 3' tracrRNA
that comprises at least 60% identity to a tracrRNA from a bacterium
(e.g., S. pyogenes) over 6 contiguous nucleotides and wherein the
3' tracrRNA comprises a length from 10-20 nucleotides, and
comprises a duplexed region; and/or a tracrRNA extension comprising
10-5000 nucleotides in length, or any combination thereof.
[0408] In some instances, a kit can comprise a site-directed
polypeptide (and/or a nucleic acid encoding the same), wherein the
site-directed polypeptide comprises at least 15% amino acid
identity to a Cas9 from S. pyogenes, two nucleic acid cleaving
domains (i.e., an HNH domain and a RuvC domain), wherein the
site-directed polypeptide comprises a mutation in one or both of
the nucleic acid cleaving domains that reduces the cleaving
activity of the nuclease domains by at least 50%; and a
double-guide nucleic acid-targeting nucleic acid (and/or a nucleic
acid encoding the same) comprising 1) a first nucleic acid molecule
comprising a spacer extension sequence from 10-5000 nucleotides in
length; a spacer sequence of 12-30 nucleotides in length, wherein
the spacer is at least 50% complementary to a target nucleic acid;
a minimum CRISPR repeat comprising at least 60% identity to a crRNA
from a prokaryote (e.g., S. pyogenes) or phage over 6 contiguous
nucleotides and wherein the minimum CRISPR repeat has a length from
5-30 nucleotides, and at least 3 unpaired nucleotides of a bulge;
and 2) a second nucleic acid molecule of the double-guide nucleic
acid-targeting nucleic acid can comprise a minimum tracrRNA
sequence comprising at least 60% identity to a tracrRNA from a
prokaryote (e.g., S. pyogenes) or phage over 6 contiguous
nucleotides and wherein the minimum tracrRNA sequence has a length
from 5-30 nucleotides and at least 1 unpaired nucleotide of a
bulge, wherein the 1 unpaired nucleotide of the bulge is located in
the same bulge as the 3 unpaired nucleotides of the minimum CRISPR
repeat; a 3' tracrRNA that comprises at least 60% identity to a
tracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides and wherein the 3' tracrRNA comprises a
length from 10-20 nucleotides, and comprises a duplexed region; a
P-domain that starts from 1-5 nucleotides downstream of the duplex
comprising the minimum CRISPR repeat and the minimum tracrRNA,
comprises 1-10 nucleotides, comprises a sequence that can hybridize
to a protospacer adjacent motif in a target nucleic acid, can form
a hairpin, and is located in the 3' tracrRNA region; and/or a
tracrRNA extension comprising 10-5000 nucleotides in length, or any
combination thereof.
[0409] In some instances, a kit can comprise a site-directed
polypeptide (and/or a nucleic acid encoding the same), wherein the
site-directed polypeptide comprises at least 15% amino acid
identity to a Cas9 from S. pyogenes, two nucleic acid cleaving
domains (i.e., an HNH domain and a RuvC domain), wherein the
site-directed polypeptide comprises a mutation in one or both of
the nucleic acid cleaving domains that reduces the cleaving
activity of the nuclease domains by at least 50%; and nucleic
acid-targeting nucleic acid (and/or a nucleic acid encoding the
same) comprising a spacer extension sequence from 10-5000
nucleotides in length; a spacer sequence of 12-30 nucleotides in
length, wherein the spacer is at least 50% complementary to a
target nucleic acid; a minimum CRISPR repeat comprising at least
60% identity to a crRNA from a prokaryote (e.g., S. pyogenes) or
phage over 6, 7, or 8 contiguous nucleotides and wherein the
minimum CRISPR repeat has a length from 5-30 nucleotides; a minimum
tracrRNA sequence comprising at least 60% identity to a tracrRNA
from a bacterium (e.g., S. pyogenes) over 6, 7, or 8 contiguous
nucleotides and wherein the minimum tracrRNA sequence has a length
from 5-30 nucleotides; a linker sequence that links the minimum
CRISPR repeat and the minimum tracrRNA and comprises a length from
3-5000 nucleotides; a 3' tracrRNA that comprises at least 60%
identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or
phage over 6, 7, or 8 contiguous nucleotides and wherein the 3'
tracrRNA comprises a length from 10-20 nucleotides, and comprises a
duplexed region; and/or a tracrRNA extension comprising 10-5000
nucleotides in length, or any combination thereof. This nucleic
acid-targeting nucleic acid can be referred to as a single guide
nucleic acid-targeting nucleic acid.
[0410] In some instances, a kit can comprise a site-directed
polypeptide (and/or a nucleic acid encoding the same), wherein the
site-directed polypeptide comprises at least 15% amino acid
identity to a Cas9 from S. pyogenes, two nucleic acid cleaving
domains (i.e., an HNH domain and a RuvC domain), wherein the
site-directed polypeptide comprises a mutation in one or both of
the nucleic acid cleaving domains that reduces the cleaving
activity of the nuclease domains by at least 50%; and nucleic
acid-targeting nucleic acid (and/or a nucleic acid encoding the
same) comprising a spacer extension sequence from 10-5000
nucleotides in length; a spacer sequence of 12-30 nucleotides in
length, wherein the spacer is at least 50% complementary to a
target nucleic acid; a duplex comprising 1) a minimum CRISPR repeat
comprising at least 60% identity to a crRNA from a prokaryote
(e.g., S. pyogenes) or phage over 6 contiguous nucleotides and
wherein the minimum CRISPR repeat has a length from 5-30
nucleotides, 2) a minimum tracrRNA sequence comprising at least 60%
identity to a tracrRNA from a bacterium (e.g., S. pyogenes) over 6
contiguous nucleotides and wherein the minimum tracrRNA sequence
has a length from 5-30 nucleotides, and 3) a bulge wherein the
bulge comprises at least 3 unpaired nucleotides on the minimum
CRISPR repeat strand of the duplex and at least 1 unpaired
nucleotide on the minimum tracrRNA sequence strand of the duplex; a
linker sequence that links the minimum CRISPR repeat and the
minimum tracrRNA and comprises a length from 3-5000 nucleotides; a
3' tracrRNA that comprises at least 60% identity to a tracrRNA from
a prokaryote (e.g., S. pyogenes) or phage over 6 contiguous
nucleotides, wherein the 3' tracrRNA comprises a length from 10-20
nucleotides and comprises a duplexed region; a P-domain that starts
from 1-5 nucleotides downstream of the duplex comprising the
minimum CRISPR repeat and the minimum tracrRNA, comprises 1-10
nucleotides, comprises a sequence that can hybridize to a
protospacer adjacent motif in a target nucleic acid, can form a
hairpin, and is located in the 3' tracrRNA region; and/or a
tracrRNA extension comprising 10-5000 nucleotides in length, or any
combination thereof.
[0411] In some instances, a kit can comprise a site-directed
polypeptide (and/or a nucleic acid encoding the same), wherein the
site-directed polypeptide comprises at least 15% amino acid
identity to a Cas9 from S. pyogenes, two nucleic acid cleaving
domains (i.e., an HNH domain and a RuvC domain), wherein the
site-directed polypeptide comprises a mutation in one or both of
the nucleic acid cleaving domains that reduces the cleaving
activity of the nuclease domains by at least 50%; and a
double-guide nucleic acid-targeting nucleic acid (and/or a nucleic
acid encoding the same) comprising 1) a first nucleic acid molecule
comprising a spacer extension sequence from 10-5000 nucleotides in
length; a spacer sequence of 12-30 nucleotides in length, wherein
the spacer is at least 50% complementary to a target nucleic acid;
and a minimum CRISPR repeat comprising at least 60% identity to a
crRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides and wherein the minimum CRISPR repeat has a
length from 5-30 nucleotides; and 2) a second nucleic acid molecule
of the double-guide nucleic acid-targeting nucleic acid can
comprise a minimum tracrRNA sequence comprising at least 60%
identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or
phage over 6 contiguous nucleotides and wherein the minimum
tracrRNA sequence has a length from 5-30 nucleotides; a 3' tracrRNA
that comprises at least 60% identity to a tracrRNA from a bacterium
(e.g., S. pyogenes) over 6 contiguous nucleotides and wherein the
3' tracrRNA comprises a length from 10-20 nucleotides, and
comprises a duplexed region; and/or a tracrRNA extension comprising
10-5000 nucleotides in length, or any combination thereof.
[0412] In some instances, a kit can comprise a site-directed
polypeptide (and/or a nucleic acid encoding the same), wherein the
site-directed polypeptide comprises at least 15% amino acid
identity to a Cas9 from S. pyogenes, two nucleic acid cleaving
domains (i.e., an HNH domain and a RuvC domain), wherein the
site-directed polypeptide comprises a mutation in one or both of
the nucleic acid cleaving domains that reduces the cleaving
activity of the nuclease domains by at least 50%; and a
double-guide nucleic acid-targeting nucleic acid (and/or a nucleic
acid encoding the same) comprising 1) a first nucleic acid molecule
comprising a spacer extension sequence from 10-5000 nucleotides in
length; a spacer sequence of 12-30 nucleotides in length, wherein
the spacer is at least 50% complementary to a target nucleic acid;
a minimum CRISPR repeat comprising at least 60% identity to a crRNA
from a prokaryote (e.g., S. pyogenes) or phage over 6 contiguous
nucleotides and wherein the minimum CRISPR repeat has a length from
5-30 nucleotides, and at least 3 unpaired nucleotides of a bulge;
and 2) a second nucleic acid molecule of the double-guide nucleic
acid-targeting nucleic acid can comprise a minimum tracrRNA
sequence comprising at least 60% identity to a tracrRNA from a
prokaryote (e.g., S. pyogenes) or phage over 6 contiguous
nucleotides and wherein the minimum tracrRNA sequence has a length
from 5-30 nucleotides and at least 1 unpaired nucleotide of a
bulge, wherein the 1 unpaired nucleotide of the bulge is located in
the same bulge as the 3 unpaired nucleotides of the minimum CRISPR
repeat; a 3' tracrRNA that comprises at least 60% identity to a
tracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides and wherein the 3' tracrRNA comprises a
length from 10-20 nucleotides, and comprises a duplexed region; a
P-domain that starts from 1-5 nucleotides downstream of the duplex
comprising the minimum CRISPR repeat and the minimum tracrRNA,
comprises 1-10 nucleotides, comprises a sequence that can hybridize
to a protospacer adjacent motif in a target nucleic acid, can form
a hairpin, and is located in the 3' tracrRNA region; and/or a
tracrRNA extension comprising 10-5000 nucleotides in length, or any
combination thereof.
[0413] In some instances, a kit can comprise a site-directed
polypeptide (and/or a nucleic acid encoding the same), wherein the
site-directed polypeptide comprises at least 15% amino acid
identity to a Cas9 from S. pyogenes, two nucleic acid cleaving
domains (i.e., an HNH domain and a RuvC domain), wherein the
site-directed polypeptide comprises a mutation in one or both of
the nucleic acid cleaving domains that reduces the cleaving
activity of the nuclease domains by at least 50%; and nucleic
acid-targeting nucleic acid (and/or a nucleic acid encoding the
same) comprising a spacer extension sequence from 10-5000
nucleotides in length; a spacer sequence of 12-30 nucleotides in
length, wherein the spacer is at least 50% complementary to a
target nucleic acid; a minimum CRISPR repeat comprising at least
60% identity to a crRNA from a prokaryote (e.g., S. pyogenes) or
phage over 6, 7, or 8 contiguous nucleotides and wherein the
minimum CRISPR repeat has a length from 5-30 nucleotides; a minimum
tracrRNA sequence comprising at least 60% identity to a tracrRNA
from a bacterium (e.g., S. pyogenes) over 6, 7, or 8 contiguous
nucleotides and wherein the minimum tracrRNA sequence has a length
from 5-30 nucleotides; a linker sequence that links the minimum
CRISPR repeat and the minimum tracrRNA and comprises a length from
3-5000 nucleotides; a 3' tracrRNA that comprises at least 60%
identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or
phage over 6, 7, or 8 contiguous nucleotides and wherein the 3'
tracrRNA comprises a length from 10-20 nucleotides, and comprises a
duplexed region; and/or a tracrRNA extension comprising 10-5000
nucleotides in length, or any combination thereof. This nucleic
acid-targeting nucleic acid can be referred to as a single guide
nucleic acid-targeting nucleic acid.
[0414] In some instances, a kit can comprise a site-directed
polypeptide (and/or a nucleic acid encoding the same), wherein the
site-directed polypeptide comprises at least 15% amino acid
identity to a Cas9 from S. pyogenes, two nucleic acid cleaving
domains (i.e., an HNH domain and a RuvC domain), wherein the
site-directed polypeptide comprises a mutation in one or both of
the nucleic acid cleaving domains that reduces the cleaving
activity of the nuclease domains by at least 50%; and a nucleic
acid-targeting nucleic acid (and/or a nucleic acid encoding the
same) comprising a spacer extension sequence from 10-5000
nucleotides in length; a spacer sequence of 12-30 nucleotides in
length, wherein the spacer is at least 50% complementary to a
target nucleic acid; a duplex comprising 1) a minimum CRISPR repeat
comprising at least 60% identity to a crRNA from a prokaryote
(e.g., S. pyogenes) or phage over 6 contiguous nucleotides and
wherein the minimum CRISPR repeat has a length from 5-30
nucleotides, 2) a minimum tracrRNA sequence comprising at least 60%
identity to a tracrRNA from a bacterium (e.g., S. pyogenes) over 6
contiguous nucleotides and wherein the minimum tracrRNA sequence
has a length from 5-30 nucleotides, and 3) a bulge wherein the
bulge comprises at least 3 unpaired nucleotides on the minimum
CRISPR repeat strand of the duplex and at least 1 unpaired
nucleotide on the minimum tracrRNA sequence strand of the duplex; a
linker sequence that links the minimum CRISPR repeat and the
minimum tracrRNA and comprises a length from 3-5000 nucleotides; a
3' tracrRNA that comprises at least 60% identity to a tracrRNA from
a prokaryote (e.g., S. pyogenes) or phage over 6 contiguous
nucleotides, wherein the 3' tracrRNA comprises a length from 10-20
nucleotides and comprises a duplexed region; a P-domain that starts
from 1-5 nucleotides downstream of the duplex comprising the
minimum CRISPR repeat and the minimum tracrRNA, comprises 1-10
nucleotides, comprises a sequence that can hybridize to a
protospacer adjacent motif in a target nucleic acid, can form a
hairpin, and is located in the 3' tracrRNA region; and/or a
tracrRNA extension comprising 10-5000 nucleotides in length, or any
combination thereof.
[0415] In some instances, a kit can comprise a site-directed
polypeptide (and/or a nucleic acid encoding the same), wherein the
site-directed polypeptide comprises at least 15% amino acid
identity to a Cas9 from S. pyogenes, two nucleic acid cleaving
domains (i.e., an HNH domain and a RuvC domain), wherein the
site-directed polypeptide comprises a mutation in one or both of
the nucleic acid cleaving domains that reduces the cleaving
activity of the nuclease domains by at least 50%; and a
double-guide nucleic acid-targeting nucleic acid (and/or a nucleic
acid encoding the same) comprising 1) a first nucleic acid molecule
comprising a spacer extension sequence from 10-5000 nucleotides in
length; a spacer sequence of 12-30 nucleotides in length, wherein
the spacer is at least 50% complementary to a target nucleic acid;
and a minimum CRISPR repeat comprising at least 60% identity to a
crRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides and wherein the minimum CRISPR repeat has a
length from 5-30 nucleotides; and 2) a second nucleic acid molecule
of the double-guide nucleic acid-targeting nucleic acid can
comprise a minimum tracrRNA sequence comprising at least 60%
identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or
phage over 6 contiguous nucleotides and wherein the minimum
tracrRNA sequence has a length from 5-30 nucleotides; a 3' tracrRNA
that comprises at least 60% identity to a tracrRNA from a bacterium
(e.g., S. pyogenes) over 6 contiguous nucleotides and wherein the
3' tracrRNA comprises a length from 10-20 nucleotides, and
comprises a duplexed region; and/or a tracrRNA extension comprising
10-5000 nucleotides in length, or any combination thereof.
[0416] In some instances, a kit can comprise a site-directed
polypeptide (and/or a nucleic acid encoding the same), wherein the
site-directed polypeptide comprises at least 15% amino acid
identity to a Cas9 from S. pyogenes, and two nucleic acid cleaving
domains, wherein one or both of the nucleic acid cleaving domains
comprise at least 50% amino acid identity to a nuclease domain from
Cas9 from S. pyogenes; and a double-guide nucleic acid-targeting
nucleic acid (and/or a nucleic acid encoding the same) comprising:
1) a first nucleic acid molecule comprising a spacer extension
sequence from 10-5000 nucleotides in length; a spacer sequence of
12-30 nucleotides in length, wherein the spacer is at least 50%
complementary to a target nucleic acid; a minimum CRISPR repeat
comprising at least 60% identity to a crRNA from a prokaryote
(e.g., S. pyogenes) or phage over 6 contiguous nucleotides and
wherein the minimum CRISPR repeat has a length from 5-30
nucleotides, and at least 3 unpaired nucleotides of a bulge; and 2)
a second nucleic acid molecule of the double-guide nucleic
acid-targeting nucleic acid can comprise a minimum tracrRNA
sequence comprising at least 60% identity to a tracrRNA from a
prokaryote (e.g., S. pyogenes) or phage over 6 contiguous
nucleotides and wherein the minimum tracrRNA sequence has a length
from 5-30 nucleotides and at least 1 unpaired nucleotide of a
bulge, wherein the 1 unpaired nucleotide of the bulge is located in
the same bulge as the 3 unpaired nucleotides of the minimum CRISPR
repeat; a 3' tracrRNA that comprises at least 60% identity to a
tracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6
contiguous nucleotides and wherein the 3' tracrRNA comprises a
length from 10-20 nucleotides, and comprises a duplexed region; a
P-domain that starts from 1-5 nucleotides downstream of the duplex
comprising the minimum CRISPR repeat and the minimum tracrRNA,
comprises 1-10 nucleotides, comprises a sequence that can hybridize
to a protospacer adjacent motif in a target nucleic acid, can form
a hairpin, and is located in the 3' tracrRNA region; and/or a
tracrRNA extension comprising 10-5000 nucleotides in length, or any
combination thereof.
[0417] In some instances, a kit can comprise a site-directed
polypeptide (and/or a nucleic acid encoding the same), wherein the
site-directed polypeptide comprises at least 15% amino acid
identity to a Cas9 from S. pyogenes, and two nucleic acid cleaving
domains, wherein one or both of the nucleic acid cleaving domains
comprise at least 50% amino acid identity to a nuclease domain from
Cas9 from S. pyogenes; and nucleic acid-targeting nucleic acid
(and/or a nucleic acid encoding the same) comprising a spacer
extension sequence from 10-5000 nucleotides in length; a spacer
sequence of 12-30 nucleotides in length, wherein the spacer is at
least 50% complementary to a target nucleic acid; a minimum CRISPR
repeat comprising at least 60% identity to a crRNA from a
prokaryote (e.g., S. pyogenes) or phage over 6, 7, or 8 contiguous
nucleotides and wherein the minimum CRISPR repeat has a length from
5-30 nucleotides; a minimum tracrRNA sequence comprising at least
60% identity to a tracrRNA from a bacterium (e.g., S. pyogenes)
over 6, 7, or 8 contiguous nucleotides and wherein the minimum
tracrRNA sequence has a length from 5-30 nucleotides; a linker
sequence that links the minimum CRISPR repeat and the minimum
tracrRNA and comprises a length from 3-5000 nucleotides; a 3'
tracrRNA that comprises at least 60% identity to a tracrRNA from a
prokaryote (e.g., S. pyogenes) or phage over 6, 7, or 8 contiguous
nucleotides and wherein the 3' tracrRNA comprises a length from
10-20 nucleotides, and comprises a duplexed region; and/or a
tracrRNA extension comprising 10-5000 nucleotides in length, or any
combination thereof. This nucleic acid-targeting nucleic acid can
be referred to as a single guide nucleic acid-targeting nucleic
acid.
[0418] In some instances, a kit can comprise a site-directed
polypeptide (and/or a nucleic acid encoding the same), wherein the
site-directed polypeptide comprises at least 15% amino acid
identity to a Cas9 from S. pyogenes, and two nucleic acid cleaving
domains, wherein one or both of the nucleic acid cleaving domains
comprise at least 50% amino acid identity to a nuclease domain from
Cas9 from S. pyogenes; and a nucleic acid-targeting nucleic acid
(and/or a nucleic acid encoding the same) comprising a spacer
extension sequence from 10-5000 nucleotides in length; a spacer
sequence of 12-30 nucleotides in length, wherein the spacer is at
least 50% complementary to a target nucleic acid; a duplex
comprising: 1) a minimum CRISPR repeat comprising at least 60%
identity to a crRNA from a prokaryote (e.g., S. pyogenes) or phage
over 6 contiguous nucleotides and wherein the minimum CRISPR repeat
has a length from 5-30 nucleotides, 2) a minimum tracrRNA sequence
comprising at least 60% identity to a tracrRNA from a bacterium
(e.g., S. pyogenes) over 6 contiguous nucleotides and wherein the
minimum tracrRNA sequence has a length from 5-30 nucleotides, and
3) a bulge wherein the bulge comprises at least 3 unpaired
nucleotides on the minimum CRISPR repeat strand of the duplex and
at least 1 unpaired nucleotide on the minimum tracrRNA sequence
strand of the duplex; a linker sequence that links the minimum
CRISPR repeat and the minimum tracrRNA and comprises a length from
3-5000 nucleotides; a 3' tracrRNA that comprises at least 60%
identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or
phage over 6 contiguous nucleotides, wherein the 3' tracrRNA
comprises a length from 10-20 nucleotides and comprises a duplexed
region; a P-domain that starts from 1-5 nucleotides downstream of
the duplex comprising the minimum CRISPR repeat and the minimum
tracrRNA, comprises 1-10 nucleotides, comprises a sequence that can
hybridize to a protospacer adjacent motif in a target nucleic acid,
can form a hairpin, and is located in the 3' tracrRNA region;
and/or a tracrRNA extension comprising 10-5000 nucleotides in
length, or any combination thereof.
[0419] In some embodiments of any of the above kits, the kit can
comprise a single guide nucleic acid-targeting nucleic acid. In
some embodiments of any of the above kits, the kit can comprise a
double guide nucleic acid-targeting nucleic acid. In some
embodiments of any of the above kits, the kit can comprise two or
more double guide or single guide nucleic acid-targeting nucleic
acids. In some embodiments, a vector may encode for a nucleic acid
targeting nucleic acid.
[0420] In some embodiments of any of the above kits, the kit can
further comprise a donor polynucleotide, or a polynucleotide
sequence encoding the donor polynucleotide, to effect the desired
genetic modification. Components of a kit can be in separate
containers; or can be combined in a single container.
[0421] A kit described above further comprise one or more
additional reagents, where such additional reagents can be selected
from: a buffer, a buffer for introducing the a polypeptide or
polynucleotide item of the kit into a cell, a wash buffer, a
control reagent, a control vector, a control RNA polynucleotide, a
reagent for in vitro production of the polypeptide from DNA,
adaptors for sequencing and the like. A buffer can be a
stabilization buffer, a reconstituting buffer, or a diluting
buffer.
[0422] In some instances, a kit can comprise one or more additional
reagents specific for plants and/or fungi. One or more additional
reagents for plants and/or fungi can include, for example, soil,
nutrients, plants, seeds, spores, Agrobacterium, T-DNA vector, and
a pBINAR vector.
[0423] In addition to above-mentioned components, a kit can further
include instructions for using the components of the kit to
practice the methods. The instructions for practicing the methods
are generally recorded on a suitable recording medium. For example,
the instructions may be printed on a substrate, such as paper or
plastic, etc. The instructions may be present in the kits as a
package insert, in the labeling of the container of the kit or
components thereof (i.e., associated with the packaging or
subpackaging) etc. The instructions can be present as an electronic
storage data file present on a suitable computer readable storage
medium, e.g. CD-ROM, diskette, flash drive, etc. In some instances,
the actual instructions are not present in the kit, but means for
obtaining the instructions from a remote source (e.g. via the
Internet), can be provided. An example of this embodiment is a kit
that includes a web address where the instructions can be viewed
and/or from which the instructions can be downloaded. As with the
instructions, this means for obtaining the instructions can be
recorded on a suitable substrate.
[0424] In some embodiments, a kit can comprise a linearized vector.
A linearized vector can comprise a plasmid comprising a
site-directed polypeptide and/or a nucleic acid-targeting nucleic
acid that is linearized (e.g., it is not circular). A linearized
vector can be stored in a buffer comprising 10 mM Tris-HCl, pH 8.0
and 1 mM EDTA, pH 8.0. A kit can comprise about 20 microliters of
the linearized CRISPR nuclease vector. In some embodiments, a kit
can comprise one or more circular vectors.
[0425] In some embodiments a kit can comprise an oligonucleotide
annealing buffer. An oligonucleotide annealing buffer can be a
buffer used to anneal DNA oligos together to generate a
double-stranded DNA that encode a nucleic acid-targeting nucleic
acid. A oligonucleotide annealing buffer can be at least about, 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 or more concentrated than the
concentration of use. An oligonucleotide annealing buffer can be 10
times more concentrated than the concentration when used. An
oligonucleotide annealing buffer can comprise 100 mM Tris-HCl, pH
8.0, 10 mM EDTA, pH 8.0 and 1M NaCl. A kit can comprise 250
microliters of the oligonucleotide annealing buffer.
[0426] A kit can comprise DNase-free water. A kit can comprise
RNAse-free water. A kit can comprise at least 1.5 milliliters of
RNase-free and/or DNAse-free water.
[0427] A kit can comprise a ligation buffer. A ligation buffer can
be used to ligate oligonucleotides to the linearized CRISPR
nuclease vector. A ligation buffer can be at least about, 1, 2, 3,
4, 5, 6, 7, 8, 9, or 10 or more concentrated than the concentration
of use. A ligation buffer can be 5 times as concentrated as the
concentration of use. A 5.times. ligation buffer can comprise 250
mM Tris-HCl, pH 7.6, 50 mM MgCl.sub.2, 5 mM ATP, 5 mM DTT, and 25%
(w/v) polyethylene glycol-8000. A kit can comprise about 80
microliters of a ligation buffer.
[0428] A kit can comprise a DNA ligase. A DNA ligase can be used to
ligate the oligonucleotides to the linearized CRISPR nuclease
vector. A DNA ligase can comprise 10 mM Tris-HCl, pH 7.5, 50 mM
KCl, 1 mM DTT, and 50% (v/v) glycerol. A kit can comprise 20
microliters of a DNA ligase.
[0429] A kit can comprise a sequencing primer. The sequencing
primer can be used to sequence the vector once the oligonucleotides
have been ligated into a linearized vector. A sequencing primer can
be diluted in Tris-EDTA buffer pH 8.0. A kit can comprise 20
microliters of a sequencing primer.
[0430] A kit can comprise a control oligonucleotide. A control
oligonucleotide can be an oligonucleotide to be ligated into a
linearized vector but does not encode for a nucleic acid-targeting
nucleic acid. A control oligonucleotide can be diluted in 1.times.
concentration of the oligonucleotide annealing buffer. A kit can
comprise 10 microliters of a control oligonucleotide.
[0431] In some instances, a kit can comprise a linearized vector
comprising a site-directed polypeptide and a nucleic acid-targeting
nucleic acid, an oligonucleotide annealing buffer, DNAse/RNAse free
water, a ligation buffer, a ligase enzyme, a sequencing primer and
a control oligonucleotide, or any combination thereof.
[0432] Pharmaceutical Compositions
[0433] Molecules, such as a nucleic acid-targeting nucleic acid of
the disclosure as described herein, a polynucleotide encoding a
nucleic acid-targeting nucleic acid, a site-directed polypeptide of
the disclosure, a polynucleotide encoding a site-directed
polypeptide, an effector protein, a polynucleotide encoding an
effector protein, a multiplexed genetic targeting agent, a
polynucleotide encoding a multiplexed genetic targeting agent, a
donor polynucleotide, a tandem fusion protein, a polynucleotide
encoding a tandem fusion protein, a reporter element, a genetic
element of interest, a component of a split system and/or any
nucleic acid or proteinaceous molecule necessary to carry out the
embodiments of the methods of the disclosure, can be formulated in
a pharmaceutical composition.
[0434] A pharmaceutical composition can comprise a combination of
any molecules described herein with other chemical components, such
as carriers, stabilizers, diluents, dispersing agents, suspending
agents, thickening agents, and/or excipients. The pharmaceutical
composition can facilitate administration of the molecule to an
organism. Pharmaceutical compositions can be administered in
therapeutically-effective amounts as pharmaceutical compositions by
various forms and routes including, for example, intravenous,
subcutaneous, intramuscular, oral, rectal, aerosol, parenteral,
ophthalmic, pulmonary, transdermal, vaginal, optic, nasal, and
topical administration.
[0435] A pharmaceutical composition can be administered in a local
or systemic manner, for example, via injection of the molecule
directly into an organ, optionally in a depot or sustained release
formulation. Pharmaceutical compositions can be provided in the
form of a rapid release formulation, in the form of an extended
release formulation, or in the form of an intermediate release
formulation. A rapid release form can provide an immediate release.
An extended release formulation can provide a controlled release or
a sustained delayed release.
[0436] For oral administration, pharmaceutical compositions can be
formulated readily by combining the molecules with
pharmaceutically-acceptable carriers or excipients. Such carriers
can be used to formulate tablets, powders, pills, dragees,
capsules, liquids, gels, syrups, elixirs, slurries, suspensions and
the like, for oral ingestion by a subject.
[0437] Pharmaceutical preparations for oral use can be obtained by
mixing one or more solid excipient with one or more of the
molecules described herein, optionally grinding the resulting
mixture, and processing the mixture of granules, after adding
suitable auxiliaries, if desired, to obtain tablets or dragee
cores. Cores can be provided with suitable coatings. For this
purpose, concentrated sugar solutions can be used, which can
contain an excipient such as gum arabic, talc,
polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or
titanium dioxide, lacquer solutions, and suitable organic solvents
or solvent mixtures. Dyestuffs or pigments can be added to the
tablets or dragee coatings, for example, for identification or to
characterize different combinations of active compound doses.
[0438] Pharmaceutical preparations which can be used orally can
include push-fit capsules made of gelatin, as well as soft, sealed
capsules made of gelatin and a plasticizer, such as glycerol or
sorbitol. In some embodiments, the capsule comprises a hard gelatin
capsule comprising one or more of pharmaceutical, bovine, and plant
gelatins. A gelatin can be alkaline-processed. The push-fit
capsules can comprise the active ingredients in admixture with
filler such as lactose, binders such as starches, and/or lubricants
such as talc or magnesium stearate and, stabilizers. In soft
capsules, the molecule can be dissolved or suspended in suitable
liquids, such as fatty oils, liquid paraffin, or liquid
polyethylene glycols. Stabilizers can be added. All formulations
for oral administration are provided in dosages suitable for such
administration.
[0439] For buccal or sublingual administration, the compositions
can be tablets, lozenges, or gels.
[0440] Parental injections can be formulated for bolus injection or
continuous infusion. The pharmaceutical compositions can be in a
form suitable for parenteral injection as a sterile suspension,
solution or emulsion in oily or aqueous vehicles, and can contain
formulatory agents such as suspending, stabilizing and/or
dispersing agents. Pharmaceutical formulations for parenteral
administration can include aqueous solutions of the active
compounds in water-soluble form.
[0441] Suspensions of molecules can be prepared as oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate or triglycerides, or liposomes. Aqueous injection
suspensions can contain substances which increase the viscosity of
the suspension, such as sodium carboxymethyl cellulose, sorbitol,
or dextran. The suspension can also contain suitable stabilizers or
agents which increase the solubility of the molecules to allow for
the preparation of highly concentrated solutions. Alternatively,
the active ingredient can be in powder form for constitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0442] The active compounds can be administered topically and can
be formulated into a variety of topically administrable
compositions, such as solutions, suspensions, lotions, gels,
pastes, medicated sticks, balms, creams, and ointments. Such
pharmaceutical compositions can comprise solubilizers, stabilizers,
tonicity enhancing agents, buffers and preservatives.
[0443] Formulations suitable for transdermal administration of the
molecules can employ transdermal delivery devices and transdermal
delivery patches, and can be lipophilic emulsions or buffered
aqueous solutions, dissolved and/or dispersed in a polymer or an
adhesive. Such patches can be constructed for continuous,
pulsatile, or on demand delivery of molecules. Transdermal delivery
can be accomplished by means of iontophoretic patches and the like.
Additionally, transdermal patches can provide controlled delivery.
The rate of absorption can be slowed by using rate-controlling
membranes or by trapping the compound within a polymer matrix or
gel. Conversely, absorption enhancers can be used to increase
absorption. An absorption enhancer or carrier can include
absorbable pharmaceutically acceptable solvents to assist passage
through the skin. For example, transdermal devices can be in the
form of a bandage comprising a backing member, a reservoir
containing compounds and carriers, a rate controlling barrier to
deliver the compounds to the skin of the subject at a controlled
and predetermined rate over a prolonged period of time, and
adhesives to secure the device to the skin.
[0444] For administration by inhalation, the molecule can be in a
form as an aerosol, a mist, or a powder. Pharmaceutical
compositions can be delivered in the form of an aerosol spray
presentation from pressurized packs or a nebuliser, with the use of
a suitable propellant, for example, dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide
or other suitable gas. In the case of a pressurized aerosol, the
dosage unit can be determined by providing a valve to deliver a
metered amount. Capsules and cartridges of, for example, gelatin
for use in an inhaler or insufflator can be formulated containing a
powder mix of the compounds and a suitable powder base such as
lactose or starch.
[0445] The molecules can also be formulated in rectal compositions
such as enemas, rectal gels, rectal foams, rectal aerosols,
suppositories, jelly suppositories, or retention enemas, containing
conventional suppository bases such as cocoa butter or other
glycerides, as well as synthetic polymers such as
polyvinylpyrrolidone and PEG. In suppository forms of the
compositions, a low-melting wax such as a mixture of fatty acid
glycerides or cocoa butter can be used.
[0446] In practicing the methods of the disclosure,
therapeutically-effective amounts of the compounds described herein
can be administered in pharmaceutical compositions to a subject
having a disease or condition to be treated. A
therapeutically-effective amount can vary widely depending on the
severity of the disease, the age and relative health of the
subject, the potency of the compounds used, and other factors. The
compounds can be used singly or in combination with one or more
therapeutic agents as components of mixtures.
[0447] Pharmaceutical compositions can be formulated using one or
more physiologically-acceptable carriers comprising excipients and
auxiliaries, which facilitate processing of the molecule into
preparations that can be used pharmaceutically. Formulation can be
modified depending upon the route of administration chosen.
Pharmaceutical compositions comprising a molecule described herein
can be manufactured, for example, by mixing, dissolving,
granulating, dragee-making, levigating, emulsifying, encapsulating,
entrapping, or compression processes.
[0448] The pharmaceutical compositions can include at least one
pharmaceutically acceptable carrier, diluent, or excipient and
molecule described herein as free-base or
pharmaceutically-acceptable salt form. The methods and
pharmaceutical compositions described herein include the use
crystalline forms (also known as polymorphs), and active
metabolites of these compounds having the same type of
activity.
[0449] Methods for the preparation of compositions comprising the
compounds described herein can include formulating the molecule
with one or more inert, pharmaceutically-acceptable excipients or
carriers to form a solid, semi-solid, or liquid composition. Solid
compositions can include, for example, powders, tablets,
dispersible granules, capsules, cachets, and suppositories. Liquid
compositions can include, for example, solutions in which a
compound is dissolved, emulsions comprising a compound, or a
solution containing liposomes, micelles, or nanoparticles
comprising a compound as disclosed herein. Semi-solid compositions
can include, for example, gels, suspensions and creams. The
compositions can be in liquid solutions or suspensions, solid forms
suitable for solution or suspension in a liquid prior to use, or as
emulsions. These compositions can also contain minor amounts of
nontoxic, auxiliary substances, such as wetting or emulsifying
agents, pH buffering agents, and other pharmaceutically-acceptable
additives.
[0450] Non-limiting examples of dosage forms can include feed,
food, pellet, lozenge, liquid, elixir, aerosol, inhalant, spray,
powder, tablet, pill, capsule, gel, geltab, nanosuspension,
nanoparticle, microgel, suppository troches, aqueous or oily
suspensions, ointment, patch, lotion, dentifrice, emulsion, creams,
drops, dispersible powders or granules, emulsion in hard or soft
gel capsules, syrups, phytoceuticals, and nutraceuticals, or any
combination thereof.
[0451] Non-limiting examples of pharmaceutically-acceptable
excipients can include granulating agents, binding agents,
lubricating agents, disintegrating agents, sweetening agents,
glidants, anti-adherents, anti-static agents, surfactants,
anti-oxidants, gums, coating agents, coloring agents, flavouring
agents, coating agents, plasticizers, preservatives, suspending
agents, emulsifying agents, plant cellulosic material, and
spheronization agents, or any combination thereof.
[0452] A composition can be, for example, an immediate release form
or a controlled release formulation. An immediate release
formulation can be formulated to allow the molecules to act
rapidly. Non-limiting examples of immediate release formulations
can include readily dissolvable formulations. A controlled release
formulation can be a pharmaceutical formulation that has been
adapted such that drug release rates and drug release profiles can
be matched to physiological and chronotherapeutic requirements or,
alternatively, has been formulated to effect release of a drug at a
programmed rate. Non-limiting examples of controlled release
formulations can include granules, delayed release granules,
hydrogels (e.g., of synthetic or natural origin), other gelling
agents (e.g., gel-forming dietary fibers), matrix-based
formulations (e.g., formulations comprising a polymeric material
having at least one active ingredient dispersed through), granules
within a matrix, polymeric mixtures, granular masses, and the
like.
[0453] A controlled release formulation can be a delayed release
form. A delayed release form can be formulated to delay a
molecule's action for an extended period of time. A delayed release
form can be formulated to delay the release of an effective dose of
one or more molecules, for example, for about 4, about 8, about 12,
about 16, or about 24 hours.
[0454] A controlled release formulation can be a sustained release
form. A sustained release form can be formulated to sustain, for
example, the molecule's action over an extended period of time. A
sustained release form can be formulated to provide an effective
dose of any molecule described herein (e.g., provide a
physiologically-effective blood profile) over about 4, about 8,
about 12, about 16 or about 24 hours.
[0455] Methods of Administration and Treatment Methods.
[0456] Pharmaceutical compositions containing molecules described
herein can be administered for prophylactic and/or therapeutic
treatments. In therapeutic applications, the compositions can be
administered to a subject already suffering from a disease or
condition, in an amount sufficient to cure or at least partially
arrest the symptoms of the disease or condition, or to cure, heal,
improve, or ameliorate the condition. Amounts effective for this
use can vary based on the severity and course of the disease or
condition, previous therapy, the subject's health status, weight,
and response to the drugs, and the judgment of the treating
physician.
[0457] Multiple therapeutic agents can be administered in any order
or simultaneously. If simultaneously, the multiple therapeutic
agents can be provided in a single, unified form, or in multiple
forms, for example, as multiple separate pills. The molecules can
be packed together or separately, in a single package or in a
plurality of packages. One or all of the therapeutic agents can be
given in multiple doses. If not simultaneous, the timing between
the multiple doses may vary to as much as about a month.
[0458] Molecules described herein can be administered before,
during, or after the occurrence of a disease or condition, and the
timing of administering the composition containing a compound can
vary. For example, the pharmaceutical compositions can be used as a
prophylactic and can be administered continuously to subjects with
a propensity to conditions or diseases in order to prevent the
occurrence of the disease or condition. The molecules and
pharmaceutical compositions can be administered to a subject during
or as soon as possible after the onset of the symptoms. The
administration of the molecules can be initiated within the first
48 hours of the onset of the symptoms, within the first 24 hours of
the onset of the symptoms, within the first 6 hours of the onset of
the symptoms, or within 3 hours of the onset of the symptoms. The
initial administration can be via any route practical, such as by
any route described herein using any formulation described herein.
A molecule can be administered as soon as is practicable after the
onset of a disease or condition is detected or suspected, and for a
length of time necessary for the treatment of the disease, such as,
for example, from about 1 month to about 3 months. The length of
treatment can vary for each subject.
[0459] A molecule can be packaged into a biological compartment. A
biological compartment comprising the molecule can be administered
to a subject. Biological compartments can include, but are not
limited to, viruses (lentivirus, adenovirus), nanospheres,
liposomes, quantum dots, nanoparticles, microparticles,
nanocapsules, vesicles, polyethylene glycol particles, hydrogels,
and micelles.
[0460] For example, a biological compartment can comprise a
liposome. A liposome can be a self-assembling structure comprising
one or more lipid bilayers, each of which can comprise two
monolayers containing oppositely oriented amphipathic lipid
molecules. Amphipathic lipids can comprise a polar (hydrophilic)
headgroup covalently linked to one or two or more non-polar
(hydrophobic) acyl or alkyl chains. Energetically unfavorable
contacts between the hydrophobic acyl chains and a surrounding
aqueous medium induce amphipathic lipid molecules to arrange
themselves such that polar headgroups can be oriented towards the
bilayer's surface and acyl chains are oriented towards the interior
of the bilayer, effectively shielding the acyl chains from contact
with the aqueous environment.
[0461] Examples of preferred amphipathic compounds used in
liposomes can include phosphoglycerides and sphingolipids,
representative examples of which include phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,
phosphatidic acid, phoasphatidylglycerol, palmitoyloleoyl
phosphatidylcholine, lysophosphatidylcholine,
lysophosphatidylethanolamine, dimyristoylphosphatidylcholine
(DMPC), dipalmitoylphosphatidylcholine (DPPC),
dioleoylphosphatidylcholine, distearoylphosphatidylcholine (DSPC),
dilinoleoylphosphatidylcholine and egg sphingomyelin, or any
combination thereof.
[0462] A biological compartment can comprise a nanoparticle. A
nanoparticle can comprise a diameter of from about 40 nanometers to
about 1.5 micrometers, from about 50 nanometers to about 1.2
micrometers, from about 60 nanometers to about 1 micrometer, from
about 70 nanometers to about 800 nanometers, from about 80
nanometers to about 600 nanometers, from about 90 nanometers to
about 400 nanometers, from about 100 nanometers to about 200
nanometers.
[0463] In some instances, as the size of the nanoparticle
increases, the release rate can be slowed or prolonged and as the
size of the nanoparticle decreases, the release rate can be
increased.
[0464] The amount of albumin in the nanoparticles can range from
about 5% to about 85% albumin (v/v), from about 10% to about 80%,
from about 15% to about 80%, from about 20% to about 70% albumin
(v/v), from about 25% to about 60%, from about 30% to about 50%, or
from about 35% to about 40%. The pharmaceutical composition can
comprise up to 30, 40, 50, 60, 70 or 80% or more of the
nanoparticle. In some instances, the nucleic acid molecules of the
disclosure can be bound to the surface of the nanoparticle.
[0465] A biological compartment can comprise a virus. The virus can
be a delivery system for the pharmaceutical compositions of the
disclosure. Exemplary viruses can include lentivirus, retrovirus,
adenovirus, herpes simplex virus I or II, parvovirus,
reticuloendotheliosis virus, and adeno-associated virus (AAV).
Pharmaceutical compositions of the disclosure can be delivered to a
cell using a virus. The virus can infect and transduce the cell in
vivo, ex vivo, or in vitro. In ex vivo and in vitro delivery, the
transduced cells can be administered to a subject in need of
therapy.
[0466] Pharmaceutical compositions can be packaged into viral
delivery systems. For example, the compositions can be packaged
into virions by a HSV-1 helper virus-free packaging system.
[0467] Viral delivery systems (e.g., viruses comprising the
pharmaceutical compositions of the disclosure) can be administered
by direct injection, stereotaxic injection,
intracerebroventricularly, by minipump infusion systems, by
convection, catheters, intravenous, parenteral, intraperitoneal,
and/or subcutaneous injection, to a cell, tissue, or organ of a
subject in need. In some instances, cells can be transduced in
vitro or ex vivo with viral delivery systems. The transduced cells
can be administered to a subject having a disease. For example, a
stem cell can be transduced with a viral delivery system comprising
a pharmaceutical composition and the stem cell can be implanted in
the patient to treat a disease. In some instances, the dose of
transduced cells given to a subject can be about 1.times.10.sup.5
cells/kg, about 5.times.10.sup.5 cells/kg, about 1.times.10.sup.6
cells/kg, about 2.times.10.sup.6 cells/kg, about 3.times.10.sup.6
cells/kg, about 4.times.10.sup.6 cells/kg, about 5.times.10.sup.6
cells/kg, about 6.times.10.sup.6 cells/kg, about 7.times.10.sup.6
cells/kg, about 8.times.10.sup.6 cells/kg, about 9.times.10.sup.6
cells/kg, about 1.times.10.sup.7 cells/kg, about 5.times.10.sup.7
cells/kg, about 1.times.10.sup.8 cells/kg, or more in one single
dose.
[0468] Pharmaceutical compositions in biological compartments can
be used to treatinflammatory diseases such as arthritis, cancers,
such as, for example, bone cancer, breast cancer, skin cancer,
prostate cancer, liver cancer, lung cancer, throat cancer and
kidney cancer, bacterial infections, to treat nerve damage, lung,
liver and kidney diseases, eye treatment, spinal cord injuries,
heart disease, arterial disease.
[0469] Introduction of the biological compartments into cells can
occur by viral or bacteriophage infection, transfection,
conjugation, protoplast fusion, lipofection, electroporation,
calcium phosphate precipitation, polyethyleneimine (PEI)-mediated
transfection, DEAE-dextran mediated transfection, liposome-mediated
transfection, particle gun technology, calcium phosphate
precipitation, direct micro-injection, nanoparticle-mediated
nucleic acid delivery, and the like.
[0470] Dosage
[0471] Pharmaceutical compositions described herein can be in unit
dosage forms suitable for single administration of precise dosages.
In unit dosage form, the formulation can be divided into unit doses
containing appropriate quantities of one or more compounds. The
unit dosage can be in the form of a package containing discrete
quantities of the formulation. Non-limiting examples can include
packaged tablets or capsules, and powders in vials or ampoules.
Aqueous suspension compositions can be packaged in single-dose
non-reclosable containers. Multiple-dose reclosable containers can
be used, for example, in combination with a preservative.
Formulations for parenteral injection can be presented in unit
dosage form, for example, in ampoules, or in multi-dose containers
with a preservative.
[0472] A molecule described herein can be present in a composition
in a range of from about 1 mg to about 2000 mg; from about 5 mg to
about 1000 mg, from about 10 mg to about 25 mg to 500 mg, from
about 50 mg to about 250 mg, from about 100 mg to about 200 mg,
from about 1 mg to about 50 mg, from about 50 mg to about 100 mg,
from about 100 mg to about 150 mg, from about 150 mg to about 200
mg, from about 200 mg to about 250 mg, from about 250 mg to about
300 mg, from about 300 mg to about 350 mg, from about 350 mg to
about 400 mg, from about 400 mg to about 450 mg, from about 450 mg
to about 500 mg, from about 500 mg to about 550 mg, from about 550
mg to about 600 mg, from about 600 mg to about 650 mg, from about
650 mg to about 700 mg, from about 700 mg to about 750 mg, from
about 750 mg to about 800 mg, from about 800 mg to about 850 mg,
from about 850 mg to about 900 mg, from about 900 mg to about 950
mg, or from about 950 mg to about 1000 mg.
[0473] A molecule described herein can be present in a composition
in an amount of about 1 mg, about 2 mg, about 3 mg, about 4 mg,
about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg,
about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg,
about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg,
about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg,
about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 250
mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about
500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg,
about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950
mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1150 mg,
about 1200 mg, about 1250 mg, about 1300 mg, about 1350 mg, about
1400 mg, about 1450 mg, about 1500 mg, about 1550 mg, about 1600
mg, about 1650 mg, about 1700 mg, about 1750 mg, about 1800 mg,
about 1850 mg, about 1900 mg, about 1950 mg, or about 2000 mg.
[0474] A molecule (e.g., site-directed polypeptide, nucleic
acid-targeting nucleic acid and/or complex of a site-directed
polypeptide and a nucleic acid-targeting nucleic acid) described
herein can be present in a composition that provides at least 0.1,
0.5, 1, 1.5, 2, 2.5 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 10 or more
units of activity/mg molecule. In some embodiments, the total
number of units of activity of the molecule delivered to a subject
is at least 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000,
70,000, 80,000, 90,000, 110,000, 120,000, 130,000, 140,000,
150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 210,000,
220,000, 230,000, or 250,000 or more units. In some embodiments,
the total number of units of activity of the molecule delivered to
a subject is at most 25,000, 30,000, 35,000, 40,000, 45,000,
50,000, 60,000, 70,000, 80,000, 90,000, 110,000, 120,000, 130,000,
140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000,
210,000, 220,000, 230,000, or 250,000 or more units.
[0475] In some embodiments, at least about 10,000 units of activity
is delivered to a subject, normalized per 50 kg body weight. In
some embodiments, at least about 10,000, 15,000, 25,000, 30,000,
35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000,
110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000,
180,000, 190,000, 200,000, 210,000, 220,000, 230,000, or 250,000
units or more of activity of the molecule is delivered to the
subject, normalized per 50 kg body weight. In some embodiments, a
therapeutically effective dose comprises at least 5.times.10.sup.5,
1.times.10.sup.6, 2.times.10.sup.6, 3.times.10.sup.6, 4, 10.sup.6,
5.times.10.sup.6, 6.times.10.sup.6, 7.times.10.sup.6,
8.times.10.sup.6, 9.times.10.sup.6, 1.times.10.sup.7,
1.1.times.10.sup.7, 1.2.times.10.sup.7, 1.5.times.10.sup.7,
1.6.times.10.sup.7, 1.7.times.10.sup.7, 1.8.times.10.sup.7,
1.9.times.10.sup.7, 2.times.10.sup.7, 2.1.times.10.sup.7, or
3.times.10.sup.7 or more units of activity of the molecule. In some
embodiments, a therapeutically effective dose comprises at most
5.times.10.sup.5 1.times.10.sup.6, 2.times.10.sup.6,
3.times.10.sup.6, 4, 10.sup.6, 5.times.10.sup.6, 6.times.10.sup.6,
7.times.10.sup.6, 8.times.10.sup.6, 9.times.10.sup.6,
1.times.10.sup.7, 1.1.times.10.sup.7, 1.2.times.10.sup.7,
1.5.times.10.sup.7, 1.6.times.10.sup.7, 1.7.times.10.sup.7,
1.8.times.10.sup.7, 1.9.times.10.sup.7, 2.times.10.sup.7,
2.1.times.10.sup.7, or 3.times.10.sup.7 or more units of activity
of the molecule.
[0476] In some embodiments, a therapeutically effective dose is at
least about 10,000, 15,000, 20,000, 22,000, 24,000, 25,000, 30,000,
40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 125,000,
150,000, 200,000, or 500,000 units/kg body weight. In some
embodiments, a therapeutically effective dose is at most about
10,000, 15,000, 20,000, 22,000, 24,000, 25,000, 30,000, 40,000,
50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 125,000, 150,000,
200,000, or 500,000 units/kg body weight.
[0477] In some embodiments, the activity of the molecule delivered
to a subject is at least 10,000, 11,000, 12,000, 13,000, 14,000,
20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000,
28,000, 30,000, 32,000, 34,000, 35,000, 36,000, 37,000, 40,000,
45,000, or 50,000 or more U/mg of molecule. In some embodiments,
the activity of the molecule delivered to a subject is at most
10,000, 11,000, 12,000, 13,000, 14,000, 20,000, 21,000, 22,000,
23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 30,000, 32,000,
34,000, 35,000, 36,000, 37,000, 40,000, 45,000, or 50,000 or more
U/mg of molecule.
[0478] Pharmacokinetic and Pharmacodynamic Measurements
[0479] Pharmacokinetic and pharmacodynamic data can be obtained by
various experimental techniques. Appropriate pharmacokinetic and
pharmacodynamic profile components describing a particular
composition can vary due to variations in drug metabolism in human
subjects. Pharmacokinetic and pharmacodynamic profiles can be based
on the determination of the mean parameters of a group of subjects.
The group of subjects includes any reasonable number of subjects
suitable for determining a representative mean, for example, 5
subjects, 10 subjects, 15 subjects, 20 subjects, 25 subjects, 30
subjects, 35 subjects, or more. The mean can be determined by
calculating the average of all subject's measurements for each
parameter measured. A dose can be modulated to achieve a desired
pharmacokinetic or pharmacodynamics profile, such as a desired or
effective blood profile, as described herein.
[0480] The pharmacokinetic parameters can be any parameters
suitable for describing a molecule. For example, the C.sub.max can
be, for example, not less than about 25 ng/mL; not less than about
50 ng/mL; not less than about 75 ng/mL; not less than about 100
ng/mL; not less than about 200 ng/mL; not less than about 300
ng/mL; not less than about 400 ng/mL; not less than about 500
ng/mL; not less than about 600 ng/mL; not less than about 700
ng/mL; not less than about 800 ng/mL; not less than about 900
ng/mL; not less than about 1000 ng/mL; not less than about 1250
ng/mL; not less than about 1500 ng/mL; not less than about 1750
ng/mL; not less than about 2000 ng/mL; or any other C.sub.max
appropriate for describing a pharmacokinetic profile of a molecule
described herein.
[0481] The T.sub.max of a molecule described herein can be, for
example, not greater than about 0.5 hours, not greater than about 1
hours, not greater than about 1.5 hours, not greater than about 2
hours, not greater than about 2.5 hours, not greater than about 3
hours, not greater than about 3.5 hours, not greater than about 4
hours, not greater than about 4.5 hours, not greater than about 5
hours, or any other T.sub.max appropriate for describing a
pharmacokinetic profile of a molecule described herein.
[0482] The AUC.sub.(0-inf) of a molecule described herein can be,
for example, not less than about 50 nghr/mL, not less than about
100 ng/hr/mL, not less than about 150 ng/hr/mL, not less than about
200 nghr/mL, not less than about 250 ng/hr/mL, not less than about
300 ng/hr/mL, not less than about 350 ng/hr/mL, not less than about
400 ng/hr/mL, not less than about 450 ng/hr/mL, not less than about
500 ng/hr/mL, not less than about 600 ng/hr/mL, not less than about
700 ng/hr/mL, not less than about 800 ng/hr/mL, not less than about
900 ng/hr/mL, not less than about 1000 nghr/mL, not less than about
1250 ng/hr/mL, not less than about 1500 ng/hr/mL, not less than
about 1750 ng/hr/mL, not less than about 2000 ng/hr/mL, not less
than about 2500 ng/hr/mL, not less than about 3000 ng/hr/mL, not
less than about 3500 ng/hr/mL, not less than about 4000 ng/hr/mL,
not less than about 5000 ng/hr/mL, not less than about 6000
ng/hr/mL, not less than about 7000 ng/hr/mL, not less than about
8000 ng/hr/mL, not less than about 9000 ng/hr/mL, not less than
about 10,000 ng/hr/mL, or any other AUC.sub.(0-inf) appropriate for
describing a pharmacokinetic profile of a molecule described
herein.
[0483] The plasma concentration of a molecule described herein
about one hour after administration can be, for example, not less
than about 25 ng/mL, not less than about 50 ng/mL, not less than
about 75 ng/mL, not less than about 100 ng/mL, not less than about
150 ng/mL, not less than about 200 ng/mL, not less than about 300
ng/mL, not less than about 400 ng/mL, not less than about 500
ng/mL, not less than about 600 ng/mL, not less than about 700
ng/mL, not less than about 800 ng/mL, not less than about 900
ng/mL, not less than about 1000 ng/mL, not less than about 1200
ng/mL, or any other plasma concentration of a molecule described
herein.
[0484] The pharmacodynamic parameters can be any parameters
suitable for describing pharmaceutical compositions of the
disclosure. For example, the pharmacodynamic profile can exhibit
decreases in factors associated with inflammation after, for
example, about 2 hours, about 4 hours, about 8 hours, about 12
hours, or about 24 hours.
[0485] Pharmaceutically-Acceptable Salts
[0486] The disclosure provides the use of
pharmaceutically-acceptable salts of any molecule described herein.
Pharmaceutically-acceptable salts can include, for example,
acid-addition salts and base-addition salts. The acid that is added
to the compound to form an acid-addition salt can be an organic
acid or an inorganic acid. A base that is added to the compound to
form a base-addition salt can be an organic base or an inorganic
base. In some embodiments, a pharmaceutically-acceptable salt is a
metal salt. In some embodiments, a pharmaceutically-acceptable salt
is an ammonium salt.
[0487] Metal salts can arise from the addition of an inorganic base
to a compound of the invention. The inorganic base consists of a
metal cation paired with a basic counterion, such as, for example,
hydroxide, carbonate, bicarbonate, or phosphate. The metal can be
an alkali metal, alkaline earth metal, transition metal, or main
group metal. In some embodiments, the metal is lithium, sodium,
potassium, cesium, cerium, magnesium, manganese, iron, calcium,
strontium, cobalt, titanium, aluminum, copper, cadmium, or
zinc.
[0488] In some embodiments, a metal salt is a lithium salt, a
sodium salt, a potassium salt, a cesium salt, a cerium salt, a
magnesium salt, a manganese salt, an iron salt, a calcium salt, a
strontium salt, a cobalt salt, a titanium salt, an aluminum salt, a
copper salt, a cadmium salt, or a zinc salt, or any combination
thereof.
[0489] Ammonium salts can arise from the addition of ammonia or an
organic amine to a compound of the invention. In some embodiments,
the organic amine is triethyl amine, diisopropyl amine, ethanol
amine, diethanol amine, triethanol amine, morpholine,
N-methylmorpholine, piperidine, N-methylpiperidine,
N-ethylpiperidine, dibenzylamine, piperazine, pyridine, pyrrazole,
pipyrrazole, imidazole, pyrazine, or pipyrazine, or any combination
thereof.
[0490] In some embodiments, an ammonium salt is a triethyl amine
salt, a diisopropyl amine salt, an ethanol amine salt, a diethanol
amine salt, a triethanol amine salt, a morpholine salt, an
N-methylmorpholine salt, a piperidine salt, an N-methylpiperidine
salt, an N-ethylpiperidine salt, a dibenzylamine salt, a piperazine
salt, a pyridine salt, a pyrrazole salt, a pipyrrazole salt, an
imidazole salt, a pyrazine salt, or a pipyrazine salt, or any
combination thereof.
[0491] Acid addition salts can arise from the addition of an acid
to a molecule of the disclosure. In some embodiments, the acid is
organic. In some embodiments, the acid is inorganic. In some
embodiments, the acid is hydrochloric acid, hydrobromic acid,
hydroiodic acid, nitric acid, nitrous acid, sulfuric acid,
sulfurous acid, a phosphoric acid, isonicotinic acid, lactic acid,
salicylic acid, tartaric acid, ascorbic acid, gentisinic acid,
gluconic acid, glucaronic acid, saccaric acid, formic acid, benzoic
acid, glutamic acid, pantothenic acid, acetic acid, propionic acid,
butyric acid, fumaric acid, succinic acid, methanesulfonic acid,
ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid,
citric acid, oxalic acid, or maleic acid, or any combination
thereof.
[0492] In some embodiments, the salt is a hydrochloride salt, a
hydrobromide salt, a hydroiodide salt, a nitrate salt, a nitrite
salt, a sulfate salt, a sulfite salt, a phosphate salt,
isonicotinate salt, a lactate salt, a salicylate salt, a tartrate
salt, an ascorbate salt, a gentisinate salt, a gluconate salt, a
glucaronate salt, a saccarate salt, a formate salt, a benzoate
salt, a glutamate salt, a pantothenate salt, an acetate salt, a
propionate salt, a butyrate salt, a fumarate salt, a succinate
salt, a methanesulfonate salt, an ethanesulfonate salt, a
benzenesulfonate salt, a p-toluenesulfonate salt, a citrate salt,
an oxalate salt, or a maleate salt, or any combination thereof.
[0493] Engineered Site-Directed Polypeptides
[0494] General Overview
[0495] The disclosure describes methods, compositions, systems,
and/or kits for modifying site-directed polypeptides (e.g., Cas9,
Csy4, Cas5, Cas6, Argonaut, etc.) and/or related enzymes.
Modifications may include any covalent or non-covalent modification
to site-directed polypeptides. In some cases, this may include
chemical modifications to one or more regions of the site-directed
polypeptide. In some cases, modifications may include conservative
or non-conservative amino acid substitutions of the site-directed
polypeptide. In some cases, modifications may include the addition,
deletion or substitution of any portion of the site-directed
polypeptide with amino acids, peptides, or domains that are not
found in the native site-directed polypeptide. In some cases, one
or more non-native domains may be added, deleted or substituted in
the site-directed polypeptide. In some cases the site-directed
polypeptide may exist as a fusion protein.
[0496] In some cases, the present disclosure provides for the
engineering of site-directed polypeptides to recognize a desired
target nucleic acid sequence with desired enzyme specificity and/or
activity. Modifications to a site-directed polypeptide can be
performed through protein engineering. Protein engineering can
include fusing functional domains to such engineered site-directed
polypeptide which can be used to modify the functional state of the
overall site-directed polypeptide or the actual target nucleic acid
sequence of an endogenous cellular locus. The site-directed
polypeptide of the disclosure can be used to regulate endogenous
gene expression, both through activation and repression of
endogenous gene transcription.
[0497] The site-directed polypeptide-fusions can also be linked to
other regulatory or functional domains, for example nucleases,
transposases or methylases, to modify endogenous chromosomal
sequences. In some cases, the site-directed polypeptide may be
linked to at least one or more regulatory domains, described
herein. Non-limiting examples of regulatory or functional domains
include transcription factor repressor or activator domains such as
KRAB and VP16, co-repressor and co-activator domains, DNA methyl
transferases, histone acetyltransferases, histone deacetylases, and
DNA cleavage domains such as the cleavage domain from the
endonuclease FokI.
[0498] In some instances, one or more specific domains, regions or
structural elements of the site-directed polypeptide can be
modified together. Modifications to the site-directed polypeptide
may occur, but are not limited to site-directed polypeptide
elements such as regions that recognize or bind to the
spacer-adjacent motif (PAM), and/or regions that bind or recognize
the nucleic acid-targeting nucleic acid. Such binding or
recognition elements may include a conserved bridging helix, highly
basic region, N-terminal region, C-terminal region, RuvC motifs
(e.g., RuvC and/or RuvC-like nuclease domains) and one or more
nuclease domains, such as HNH and/or HNH-like domains.
Modifications may be made to additional domains, structural
elements, sequence or amino acids within the site-directed
polypeptide.
[0499] Modifications to one or more region of the site-directed
polypeptide may be performed to alter various properties of the
site-directed polypeptide. In some cases, modifications may alter
binding recognition for certain nucleic acid target sequences. This
may include but is not limited to increasing binding affinity
and/or specificity to certain sequences or preferentially targeting
of certain target nucleic acid sequences/recognition elements. In
some cases, modifications may be used to alter native nuclease
function. In some cases, modifications to the site-directed
polypeptide may alter PAM specificity, tracrRNA specificity, crRNA
specificity, or specificity for additional nucleic acid elements,
such as a nucleic acid-targeting nucleic acid.
[0500] Described herein are also compositions and methods including
fusion proteins comprising a site-directed polypeptide (e.g., Cas9)
and one or more domains or regions engineered for genomic editing
(e.g., cleaving of genes; alteration of genes, for example by
cleavage followed by insertion (physical insertion or insertion via
homology-directed repair) of an exogenous sequence and/or cleavage
followed by NHEJ; partial or complete inactivation of one or more
genes; generation of alleles with altered functional states of
endogenous genes, insertion of regulatory elements; etc.) and
alterations of the genome which are carried into the germline. Also
disclosed are methods of making and using these compositions (i.e.
reagents), for example to edit (i.e. alter) one or more genes in a
target cell. Thus, the methods and compositions described herein
provide highly efficient methods for targeted gene alteration
(e.g., knock-in) and/or knockout (partial or complete) of one or
more genes and/or for randomized mutation of the sequence of any
target allele, and, therefore, allow for the generation of animal
models of human diseases. One skilled in the art will recognize
that although the term "genome engineering" or "genomic editing" is
often used to describe the methods herein, the methods and
compositions described herein can also be used to alter any target
nucleic acid that may not be strictly speaking in the genome of a
cell (e.g. can be used on a synthetic nucleic acid, a plasmid, a
vector, a viral nucleic acid, a recombinant nucleic acid,
etc.).
[0501] The methods and compositions described herein allow for
novel therapeutic applications, (e.g., prevention and/or treatment
of: genetic diseases, cancer, fungal, protozoal, bacterial, and
viral infection, ischemia, vascular disease, arthritis,
immunological disorders, etc.), novel diagnostics (e.g. prediction
and/or diagnosis of a condition) as well as providing for research
tools (e.g. kits, functional genomics assays, and generating
engineered cell lines and animal models for research and drug
screening), and means for developing plants with altered
phenotypes, including but not limited to, increased disease
resistance, and altering fruit ripening characteristics, sugar and
oil composition, yield, and color. The methods and compositions
described herein allow for novel epigenetic studies.
[0502] Protein Modifications and Engineering
[0503] Amino Acid Alterations
[0504] Site-directed polypeptides, as disclosed herein, can be
modified. The modification can comprise modifications to an amino
acid of the site-directed polypeptide. The modifications can alter
the primary amino acid sequence and/or the secondary, tertiary, and
quaternary amino acid structure. In some cases some amino acid
sequences of site-directed polypeptide of the invention can be
varied without a significant effect on the structure or function of
the protein. The type of mutation may be completely unimportant if
the alteration occurs in some regions (e.g. a non-critical) region
of the protein. In some cases, depending upon the location of the
replacement, the mutation may not have a major effect on the
biological properties of the resulting variant. For example,
properties and functions of the Cas9 variants can be of the same
type as wild-type Cas9. In some cases, the mutation can critically
impact the structure and/or function of the site-directed
polypeptide.
[0505] The location of where to modify a site-directed polypeptide
(e.g., a Cas9 variant) can be determined using sequence and/or
structural alignment. Sequence alignment can identify regions of a
polypeptide that similar and/or dissimilar (e.g., conserved, not
conserved, hydrophobic, hydrophilic, etc). In some instances, a
region in the sequence of interest that is similar to other
sequences is suitable for modification. In some instances, a region
in the sequence of interest that is dissimilar from other sequences
is suitable for modification. For example, sequence alignment can
be performed by database search, pairwise alignment, multiple
sequence alignment, genomic analysis, motif finding, benchmarking,
and/or programs such as BLAST, CS-BLAST, HHPRED, psi-BLAST, LALIGN,
PyMOL, and SEQALN. Structural alignment can be performed by
programs such as Dali, PHYRE, Chimera, COOT, O, and PyMOL.
Alignment can be performed by database search, pairwise alignment,
multiple sequence alignment, genomic analysis, motif finding, or
bench marking, or any combination thereof.
[0506] A site-directed polypeptide can be modified to increase
binding specificity to a nucleic acid-targeting nucleic acid and/or
a target nucleic acid. A site-directed polypeptide can be modified
to increase binding to specific regions of a nucleic acid-targeting
nucleic acid (e.g., the spacer extension, the spacer, the minimum
CRISPR repeat, the minimum tracrRNA sequence, the 3' tracrRNA
sequence, the tracrRNA extension) and/or a target nucleic acid.
[0507] In some cases, the modification can comprise a conservative
modification. A conservative amino acid change can involve
substitution of one of a family of amino acids which are related in
their side chains (e.g, cysteine/serine)
[0508] In some cases amino acid changes in the Cas9 protein
disclosed herein are non-conservative amino acid changes, (i.e.,
substitutions of dissimilar charged or uncharged amino acids). A
non-conservative amino acid change can involve substitution of one
of a family of amino acids which may be unrelated in their side
chains or a substitution that alters biological activity of the
site-directed polypeptide.
[0509] The mutation of amino acids can also change the selectivity
of binding to a target nucleic acid. The mutation may result in a
change that may comprise a change in the dissociation constant (Kd)
of binding between a mutated site-directed polypeptide and a target
nucleic acid. The change in Kd of binding between a mutated
site-directed polypeptide and a target nucleic acid may be more
than 1000-fold, more than 500-fold, more than 100-fold, more than
50-fold, more than 25-fold, more than 10-fold, more than 5-fold,
more than 4-fold, more than 3-fold, more than 2-fold higher or
lower than the Kd of binding of binding between a non-mutated
site-directed polypeptide and a target nucleic acid. The change in
Kd of binding between a mutated site-directed polypeptide and a
target nucleic acid may be less than 1000-fold, less than 500-fold,
less than 100-fold, less than 50-fold, less than 25-fold, less than
10-fold, less than 5-fold, less than 4-fold, less than 3-fold, less
than 2-fold higher or lower than the Kd of binding of binding
between a non-mutated site-directed polypeptide and a target
nucleic acid.
[0510] The mutation may result in a change that may comprise a
change in K.sub.d of binding between a mutated site-directed
polypeptide and a PAM motif. The change in K.sub.d of binding
between a mutated site-directed polypeptide and a PAM motif may be
more than 1000-fold, more than 500-fold, more than 100-fold, more
than 50-fold, more than 25-fold, more than 10-fold, more than
5-fold, more than 4-fold, more than 3-fold, more than 2-fold higher
or lower than the K.sub.d of binding between a non-mutated
site-directed polypeptide and a PAM motif. The change in K.sub.d of
binding between a mutated site-directed polypeptide and a PAM motif
may be less than 1000-fold, less than 500-fold, less than 100-fold,
less than 50-fold, less than 25-fold, less than 10-fold, less than
5-fold, less than 4-fold, less than 3-fold, less than 2-fold higher
or lower than the K.sub.d of binding of binding between a
non-mutated site-directed polypeptide and a PAM motif.
[0511] The mutation may result in a change that may comprise a
change in K.sub.d of the binding between a mutated site-directed
polypeptide and a nucleic acid-targeting nucleic acid. The change
in K.sub.d of binding between a mutated site-directed polypeptide
and a nucleic acid-targeting nucleic acid may be more than
1000-fold, more than 500-fold, more than 100-fold, more than
50-fold, more than 25-fold, more than 10-fold, more than 5-fold,
more than 4-fold, more than 3-fold, more than 2-fold higher or
lower than the K.sub.d of binding between a non-mutated
site-directed polypeptide and a nucleic acid-targeting nucleic
acid. The change in K.sub.d of binding between a mutated
site-directed polypeptide and a nucleic acid-targeting nucleic acid
acid may be less than 1000-fold, less than 500-fold, less than
100-fold, less than 50-fold, less than 25-fold, less than 10-fold,
less than 5-fold, less than 4-fold, less than 3-fold, less than
2-fold higher or lower than the K.sub.d of binding between a
non-mutated site-directed polypeptide and a nucleic acid-targeting
nucleic acid.
[0512] The mutation of a site-directed polypeptide can also change
the kinetics of the enzymatic action of the site-directed
polypeptide. The mutation may result in a change that may comprise
a change in the K.sub.m of the mutated site-directed polypeptide.
The change in K.sub.m of the mutated site-directed polypeptide may
be more than 1000-fold, more than 500-fold, more than 100-fold,
more than 50-fold, more than 25-fold, more than 10-fold, more than
5-fold, more than 4-fold, more than 3-fold, more than 2-fold higher
or lower than the K.sub.m of a non-mutated site-directed
polypeptide. The change in K.sub.m of a mutated site-directed
polypeptide may be less than 1000-fold, less than 500-fold, less
than 100-fold, less than 50-fold, less than 25-fold, less than
10-fold, less than 5-fold, less than 4-fold, less than 3-fold, less
than 2-fold higher or lower than the K.sub.m of a non-mutated
site-directed polypeptide.
[0513] The mutation of a site-directed polypeptide may result in a
change that may comprise a change in the turnover of the
site-directed polypeptide. The change in the turnover of the
mutated site-directed polypeptide may be more than 1000-fold, more
than 500-fold, more than 100-fold, more than 50-fold, more than
25-fold, more than 10-fold, more than 5-fold, more than 4-fold,
more than 3-fold, more than 2-fold higher or lower than the
turnover of a non-mutated site-directed polypeptide. The change in
the turnover of a mutated site-directed polypeptide may be less
than 1000-fold, less than 500-fold, less than 100-fold, less than
50-fold, less than 25-fold, less than 10-fold, less than 5-fold,
less than 4-fold, less than 3-fold, less than 2-fold higher or
lower than the turnover of a non-mutated site-directed
polypeptide.
[0514] The mutation may result in a change that may comprise a
change in the .DELTA.G of the enzymatic action of the site-directed
polypeptide. The change in the .DELTA.G of the mutated
site-directed polypeptide may be more than 1000-fold, more than
500-fold, more than 100-fold, more than 50-fold, more than 25-fold,
more than 10-fold, more than 5-fold, more than 4-fold, more than
3-fold, more than 2-fold higher or lower than the .DELTA.G of a
non-mutated site-directed polypeptide. The change in the turnover
of a mutated site-directed polypeptide may be less than 1000-fold,
less than 500-fold, less than 100-fold, less than 50-fold, less
than 25-fold, less than 10-fold, less than 5-fold, less than
4-fold, less than 3-fold, less than 2-fold higher or lower than the
.DELTA.G of a non-mutated site-directed polypeptide.
[0515] The mutation may result in a change that may comprise a
change in the V.sub.max of the enzymatic action of the
site-directed polypeptide. The change in the V.sub.max of the
mutated site-directed polypeptide may be more than 1000-fold, more
than 500-fold, more than 100-fold, more than 50-fold, more than
25-fold, more than 10-fold, more than 5-fold, more than 4-fold,
more than 3-fold, more than 2-fold higher or lower than the
V.sub.max of a non-mutated site-directed polypeptide. The change in
the turnover of a mutated site-directed polypeptide may be less
than 1000-fold, less than 500-fold, less than 100-fold, less than
50-fold, less than 25-fold, less than 10-fold, less than 5-fold,
less than 4-fold, less than 3-fold, less than 2-fold higher or
lower than the V.sub.max of a non-mutated site-directed
polypeptide.
[0516] The mutation may result in a change that may comprise a
change in any kinetic parameter of the site-directed polypeptide.
The mutation may result in in a change that may comprise a change
in any thermodynamic parameter of the site-directed polypeptide.
The mutation may result in in a change that may comprise a change
in the surface charge, surface area buried, and/or folding kinetics
of the site-directed polypeptide and/or enzymatic action of the
site-directed polypeptide.
[0517] Amino acids in the site-directed polypeptide of the present
invention that are essential for function can be identified by
methods such as site-directed mutagenesis, alanine-scanning
mutagenesis, protein structure analysis, nuclear magnetic
resonance, photoaffinity labeling, and electron tomography,
high-throughput screening, ELISAs, biochemical assays, binding
assays, cleavage assays (e.g., Surveyor assay), reporter assays,
and the like.
[0518] Other amino acid alterations may also include amino acids
with glycosylated forms, aggregative conjugates with other
molecules, and covalent conjugates with unrelated chemical moieties
(e.g., pegylated molecules). Covalent variants can be prepared by
linking functionalities to groups which are found in the amino acid
chain or at the N- or C-terminal residue. In some cases mutated
site-directed polypeptides may also include allelic variants and
species variants.
[0519] Truncations of regions which do not affect functional
activity of the Cas9 proteins may be engineered. Truncations of
regions which do affect functional activity of the Cas9 protein may
be engineered. A truncation may comprise a truncation of less than
5, less than 10, less than 15, less than 20, less than 25, less
than 30, less than 35, less than 40, less than 45, less than 50,
less than 60, less than 70, less than 80, less than 90, less than
100 or more amino acids. A truncation may comprise a truncation of
more than 5, more than 10, more than 15, more than 20, more than
25, more than 30, more than 35, more than 40, more than 45, more
than 50, more than 60, more than 70, more than 80, more than 90,
more than 100 or more amino acids. A truncation may comprise
truncation of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the
site-directed polypeptide.
[0520] Deletions of regions which do not affect functional activity
of the Cas9 proteins may be engineered. Deletions of regions which
do affect functional activity of the Cas9 protein may be
engineered. A deletion can comprise a deletion of less than 5, less
than 10, less than 15, less than 20, less than 25, less than 30,
less than 35, less than 40, less than 45, less than 50, less than
60, less than 70, less than 80, less than 90, less than 100 or more
amino acids. A deletion may comprise a deletion of more than 5,
more than 10, more than 15, more than 20, more than 25, more than
30, more than 35, more than 40, more than 45, more than 50, more
than 60, more than 70, more than 80, more than 90, more than 100 or
more amino acids. A deletion may comprise deletion of about 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95% or 100% of the site-directed polypeptide. A
deletion can occur at the N-terminus, the C-terminus, or at any
region in the polypeptide chain.
[0521] Screens
[0522] The disclosure provides for methods for engineering a
site-directed polypeptide. Screens can be used to engineering a
site-directed polypeptide. For example, a screen can be set up to
screen for the effect of mutations in a region of the site-directed
polypeptide. For example, a screen can be set up to test
modifications of the highly basic patch on the affinity for RNA
structure (e.g., nucleic acid-targeting nucleic acid structure), or
processing capability (e.g., target nucleic acid cleavage).
Exemplary screening methods can include but are not limited to,
cell sorting methods, mRNA display, phage display, and directed
evolution.
[0523] Fusions
[0524] In some instances, the site-directed polypeptide is modified
such that it comprises a non-native sequence (i.e. the polypeptide
has a modification that alters it from the allele or sequence it
was derived from) (e.g., the polypeptide can be referred to as a
fusion). The non-native sequence can also include one or more
additional proteins, protein domains, subdomains or polypeptides.
For example, Cas9 may be fused with any suitable additional
nonnative nucleic acid binding proteins and/or domains, including
but not limited to transcription factor domains, nuclease domains,
nucleic acid polymerizing domains. The non-native sequence can
comprise a sequence of Cas9 and/or a Cas9 homologue.
[0525] The non-native sequence can confer new functions to the
fusion protein. These functions can include for example, DNA
cleavage, DNA methylation, DNA damage, DNA repair, modification of
a target polypeptide associated with target DNA (e.g., a histone, a
DNA-binding protein, etc.), leading to, for example, histone
methylation, histone acetylation, histone ubiquitination, and the
like. Other functions conferred by a fusion protein can include
methyltransferase activity, demethylase activity, deamination
activity, dismutase activity, alkylation activity, depurination
activity, oxidation activity, pyrimidine dimer forming activity,
integrase activity, transposase activity, recombinase activity,
polymerase activity, ligase activity, helicase activity, photolyase
activity or glycosylase activity, acetyltransferase activity,
deacetylase activity, kinase activity, phosphatase activity,
ubiquitin ligase activity, deubiquitinating activity, adenylation
activity, deadenylation activity, SUMOylating activity,
deSUMOylating activity, ribosylation activity, deribosylation
activity, myristoylation activity, remodelling activity, protease
activity, oxidoreductase activity, transferase activity, hydrolase
activity, lyase activity, isomerase activity, synthase activity,
synthetase activity, and demyristoylation activity, or any
combination thereof.
[0526] Modifications to the Bridge Helix
[0527] In some cases, the bridge helix region of Cas9 may be
modified (e.g., to alter PAM specificity). In some cases, the
bridge helix may share homology with the bridge helix identified in
the Cas9 protein of S. pyogenes (residues 551-566). In some cases,
the bridge helix may share at least 5%, 10%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
98%, 99%, or 100% homology with residues 551-556 of S. pyogenes
Cas9 bridge helix. In some cases, the bridge helix may share at
most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% homology with
residues 551-556 of S. pyogenes Cas9 bridge helix.
[0528] In some cases, modifications to the bridge helix may include
but are not limited to individual amino acid modifications, as
described herein. In some cases, modification to the bridge helix
may include but are not limited to insertions, deletions or
substitution of individual amino acids, or polypeptides, such as
other protein elements (e.g domains, structural motifs,
proteins).
[0529] Modifications may include modifications to at least 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or
more amino acids of the bridge helix. Modifications may include
modifications to at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20 or more amino acids of the bridge helix.
Modifications may also include at least 5%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 98%, 99%, or 100% of the bridge helix. Modifications may also
include at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of
the bridge helix.
[0530] In some cases, modifications to site-directed polypeptide
bridge helix sequences may include particular polypeptide
structural motifs, including but not limited to alpha helix, beta
strand, beta sheet, 310-helix, pi-helix, polyproline I motif,
polyproline II motif, polyproline III motif, beta turn,
alpha-turn-alpha, or helix kinks or hinges. For example,
substitutions to the site-directed polypeptide bridge helix may
include substitution or addition with one or more proline amino
acid residues. Insertion of proline residues may introduce kinks in
the bridge helix which may alter the binding specificity of the
bridge helix for the PAM. In another example, substitution or
addition may include one or more glycine amino acid residues.
Insertion or substitution of glycine residues may introduce
increased flexibility in the bridge helix, or "hinges" which may
also alter the binding specificity of the bridge helix for the PAM.
Altering binding specificity may or may not affect enzymatic
activity of the Cas9 protein.
[0531] In some cases, modifications to site-directed polypeptide
bridge helix sequences may include deletion of at least 1%, 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the bridge helix. In
some cases, modifications to site-directed polypeptide bridge helix
sequences may include deletion of at most 1%, 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 98%, 99%, or 100% of the bridge helix.
[0532] In some cases, modifications to site-directed polypeptide
bridge helix sequences may include addition or substitution of at
least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of a
homologous site-directed polypeptide bridge helix. In some cases,
modifications to site-directed polypeptide bridge helix sequences
may include addition or substitution of at most 1%, 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 98%, 99%, or 100% of a homologous site-directed
polypeptide bridge helix.
[0533] For example, nonnative Cas9 bridge helices may be derived
from any suitable organism. In some cases, the Cas9 protein and
bridge helix may be derived from prokaryotic organisms, including
but not limited to archea, bacteria, protists (e.g., E. coli, S.
pyogenes, S. thermophilus, P. furiosus, and etc.).
[0534] For example, the bridge helix of the S. pyogenes Cas9 enzyme
may be substituted or inserted with the bridge helix, or fragment
thereof, derived from another Cas9 enzyme from a different
species.
[0535] In some instances, a site-directed polypeptide comprises an
amino acid sequence comprising at least 15% amino acid identity to
a Cas9 from S. pyogenes, two nucleic acid-cleaving domains (i.e., a
HNH domain and RuvC domain), and a modified bridge helix.
[0536] Modifications to the Highly Basic Patch
[0537] PAM binding and specificity may also be affected by
additional regions within the Cas9 protein. In some cases, a highly
basic patch or region, comprising basic amino acid residues
adjacent to the PAM binding site, may also be modified to alter PAM
specificity. In some cases, the highly basic patch or region may
share homology with the highly basic patch identified in the Cas9
of S. pyogenes contained within N-terminal region, or amino acid
residues 1-270 of the S. pyogenes Cas9. In some cases, the highly
basic patch may share at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%,
99%, or 100% homology with the S. pyogenes Cas9 highly basic patch.
In some cases, the highly basic patch may share at most 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 98%, 99%, or 100% homology with the S. pyogenes
Cas9 highly basic patch.
[0538] In some cases, modifications to the highly basic patch may
include but are not limited to individual amino acid modifications,
as described herein. In some cases, modification to the highly
basic patch may include but are not limited to insertions,
deletions or substitution of individual amino acids, or
polypeptides, such as other protein elements (e.g domains,
structural motifs, proteins).
[0539] Modifications may include modifications to at least 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 21, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acids
of the highly basic patch. Modifications may include modifications
to at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 21, 42, 43, 44, 45, 46, 47, 48, 49, 50
or more amino acids of the highly basic patch. Modifications may
also include at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%
of the highly basic patch. Modifications may also include at most
5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the highly basic
patch.
[0540] In some cases, modifications to highly basic patch sequence
of the site-directed polypeptide may include particular polypeptide
structural motifs, including but not limited to alpha helix, beta
strand, beta sheet, 310-helix, pi-helix, polyproline I motif,
polyproline II motif, polyproline III motif, beta turn,
alpha-turn-alpha, or helix kinks or hinges.
[0541] Substitutions to the highly basic patch of the site-directed
polypeptide may include substitution or addition with one or more
acidic amino acid residues. Insertion of acidic residues may
decrease the overall basic charge of this area of the site-directed
polypeptide and may alter the binding specificity of the highly
basic patch for the PAM. In another example, substitution or
addition may include one or more basic amino acid residues.
Insertion or substitution of basic residues may increase the charge
area or ionic strength of intereraction between the polypeptide and
the nucleic acid and may also alter the binding specificity of the
highly basic patch for the PAM. Altering binding specificity may or
may not affect enzymatic activity of the site-directed
polypeptide.
[0542] In some cases, modifications to the site-directed
polypeptide highly basic patch sequences may include deletion of at
least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the
highly basic patch. In some cases, modifications to site-directed
polypeptide highly basic patch sequences may include deletion of at
most 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the highly
basic patch.
[0543] In some cases, modifications to site-directed polypeptide
highly basic patch sequences may include addition or substitution
of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of a
homologous Cas9 highly basic patch. In some cases, modifications to
the site-directed polypeptide highly basic patch sequences may
include addition or substitution of at most 1%, 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 98%, 99%, or 100% of a homologous Cas9 highly basic
patch.
[0544] Homologous Cas9 highly basic patch sequences may be derived
from any suitable organism. In some cases, the Cas9 protein may be
derived from prokaryotic organisms such as archea, bacteria,
protists (e.g., E. coli, S. pyogenes, S. thermophilus, P. furiosus,
and etc.). For example, the highly basic patch of the S. pyogenes
Cas9 enzyme may be substituted or inserted with the highly basic
patch, or fragment thereof, derived from a Cas9 of another
species.
[0545] In some instances, a site-directed polypeptide comprises an
amino acid sequence comprising at least 15% amino acid identity to
a Cas9 from S. pyogenes, two nucleic acid-cleaving domains (i.e., a
HNH domain and RuvC domain), and a modified highly basic patch.
[0546] Modifications to the HNH Domain
[0547] In some cases, the HNH domain in a site-directed polypeptide
may be modified to alter PAM specificity. In some cases, the HNH
domain may share homology with the HNH domain identified in the C
terminal domain of the Cas9 protein of S. pyogenes (residues
860-1100). In some cases, the HNH domain may share at least 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% homology with residues
551-556 of S. pyogenes Cas9 HNH domain. In some cases, the HNH
domain may share at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or
100% homology with residues 860-1100 of S. pyogenes Cas9 HNH
domain.
[0548] In some cases, modifications to the HNH domain may include
but are not limited to individual amino acid modifications, as
described herein. In some cases, modification to the HNH domain may
include but are not limited to insertions, deletions or
substitution of individual amino acids, or polypeptides, such as
other protein elements (e.g domains, structural motifs,
proteins).
[0549] Modifications may include modifications to at least 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or
more amino acids of the HNH domain. Modifications may include
modifications to at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20 or more amino acids of the HNH domain.
Modifications may also include at least 5%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 98%, 99%, or 100% of the HNH domain. Modifications may also
include at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of
the HNH domain.
[0550] In some cases, modifications to site-directed polypeptide
HNH domain sequences may include particular polypeptide structural
motifs, including but not limited to alpha helix, beta strand, beta
sheet, 310-helix, pi-helix, polyproline I motif, polyproline II
motif, polyproline III motif, beta turn, alpha-turn-alpha, or helix
kinks or hinges.
[0551] Substitutions to the HNH domain of the site-directed
polypeptide may include substitution or addition with one or more
amino acid residues. In some cases, the HNH domain may be replaced
or fused with other suitable nucleic acid binding domains. A
nucleic acid-binding domain can comprise RNA. There can be a single
nucleic acid-binding domain. Examples of nucleic acid-binding
domains can include, but are not limited to, a helix-turn-helix
domain, a zinc finger domain, a leucine zipper (bZIP) domain, a
winged helix domain, a winged helix turn helix domain, a
helix-loop-helix domain, a HMG-box domain, a Wor3 domain, an
immunoglobulin domain, a B3 domain, a TALE domain, a Zinc-finger
domain, a RNA-recognition motif domain, a double-stranded
RNA-binding motif domain, a double-stranded nucleic acid binding
domain, a single-stranded nucleic acid binding domains, a KH
domain, a PUF domain, a RGG box domain, a DEAD/DEAH box domain, a
PAZ domain, a Piwi domain, and a cold-shock domain, a RNAseH
domain, a HNH domain, a RuvC-like domain, a RAMP domain, a Cas5
domain, a Cas6 domain.
[0552] In some cases, modifications to site-directed polypeptide
HNH domain sequences may include deletion of at least 1%, 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 98%, 99%, or 100% of the HNH domain. In some
cases, modifications to site-directed polypeptide HNH domain
sequences may include deletion of at most 1%, 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 98%, 99%, or 100% of the HNH domain.
[0553] In some cases, modifications to site-directed polypeptide
HNH domain sequences may include addition or substitution of at
least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of a
homologous Cas9 HNH domain. In some cases, modifications to
site-directed polypeptide HNH domain sequences may include addition
or substitution of at most 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%,
99%, or 100% of a homologous Cas9 HNH domain.
[0554] Homologous Cas9 HNH domains may be derived from any suitable
organism. In some cases, the Cas9 protein may be derived from
prokaryotic organisms such as archea, bacteria, protists (e.g., E.
coli, S. pyogenes, S. thermophilus, P. furiosus, and etc.). For
example, the HNH domain of the S. pyogenes Cas9 enzyme may be
substituted or inserted with the HNH domain, or fragment thereof,
derived from a Cas9 enzyme of another species. In some cases, at
least one homologous Cas9 HNH domain may be inserted into the HNH
domain. In some cases, the at least one homologous Cas9 HNH domain
may form an HNH domain array, comprising at least two HNH domains.
In some cases, an HNH domain array can comprise at least one Cas9
HNH domain and at least one second HNH domain.
[0555] In some instances, the modification to the HNH or HNH-like
domain can comprise insertion of the same or similar HNH or
HNH-like domain in tandem (e.g., adjacent) to the HNH domain of
Cas9. The HNH or HNH-like domain can be inserted N-terminal and/or
C-terminal of the HNH domain in Cas9. Insertion of one or more HNH
or HNH-like domains in Cas9 can be useful in extending specificity
in a target nucleic acid. Insertion of one or more HNH or HNH-like
domains in Cas9 can be useful in duplicating specificity in a
target nucleic acid. For example, insertion of one or more HNH or
HNH-like domains can configure Cas9 to recognize a longer stretch
of target nucleic acid, recognize a different RNA-DNA hybrid,
and/or recognize a target nucleic acid with higher binding
affinity.
[0556] In some instances, a site-directed polypeptide comprises an
amino acid sequence comprising at least 15% amino acid identity to
a Cas9 from S. pyogenes, two nucleic acid-cleaving domains (i.e., a
HNH domain and RuvC domain), and a modified HNH domain.
[0557] Modifications to the RuvC or RuvC-Like Domains
[0558] In some cases, the RuvC or RuvC-like domain in the
site-directed polypeptide may be modified to alter PAM specificity.
In some cases, the RuvC or RuvC-like domain may share homology with
the RuvC or RuvC-like domain identified in the Cas9 protein of S.
pyogenes (residues 1-270). In some cases, the RuvC or RuvC-like
domain may share at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or
100% homology with residues 551-556 of S. pyogenes Cas9 RuvC or
RuvC-like domain. In some cases, the RuvC or RuvC-like domain may
share at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% homology
with residues 1-270 of S. pyogenes Cas9 RuvC or RuvC-like
domain.
[0559] In some cases, modifications to the RuvC or RuvC-like domain
may include but are not limited to individual amino acid
modifications, as described herein. In some cases, modification to
the RuvC or RuvC-like domain may include but are not limited to
insertions, deletions or substitution of individual amino acids, or
polypeptides, such as other protein elements (e.g domains,
structural motifs, proteins).
[0560] Modifications may include modifications to at least 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or
more amino acids of the RuvC or RuvC-like domain. Modifications may
include modifications to at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids of the RuvC
or RuvC-like domain. Modifications may also include at least 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the RuvC or RuvC-like
domain. Modifications may also include at most 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 98%, 99%, or 100% of the RuvC or RuvC-like domain.
[0561] In some cases, modifications to site-directed polypeptide
RuvC or RuvC-like domain sequences may include particular
polypeptide structural motifs, including but not limited to alpha
helix, beta strand, beta sheet, 310-helix, pi-helix, polyproline I
motif, polyproline II motif, polyproline III motif, beta turn,
alpha-turn-alpha, or helix kinks or hinges.
[0562] Substitutions to the site-directed polypeptide RuvC or
RuvC-like domain may include substitution or addition with one or
more amino acid residues. In some cases, the RuvC or RuvC-like
domain may be replaced or fused with other suitable nucleic acid
binding domains. A nucleic acid-binding domain can comprise RNA.
There can be a single nucleic acid-binding domain. Examples of
nucleic acid-binding domains can include, but are not limited to, a
helix-turn-helix domain, a zinc finger domain, a leucine zipper
(bZIP) domain, a winged helix domain, a winged helix turn helix
domain, a helix-loop-helix domain, a HMG-box domain, a Wor3 domain,
an immunoglobulin domain, a B3 domain, a TALE domain, a Zinc-finger
domain, a RNA-recognition motif domain, a double-stranded
RNA-binding motif domain, a double-stranded nucleic acid binding
domain, a single-stranded nucleic acid binding domains, a KH
domain, a PUF domain, a RGG box domain, a DEAD/DEAH box domain, a
PAZ domain, a Piwi domain, a cold-shock domain, a RNAseH domain, a
HNH domain, a RuvC-like domain, a RAMP domain, a Cas5 domain, and a
Cas6 domain.
[0563] In some cases, modifications to site-directed polypeptide
RuvC or RuvC-like domain sequences may include deletion of at least
1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the RuvC or
RuvC-like domain. In some cases, modifications to site-directed
polypeptide RuvC or RuvC-like domain sequences may include deletion
of at most 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of
the RuvC or RuvC-like domain.
[0564] In some cases, modifications to site-directed polypeptide
RuvC or RuvC-like domain sequences may include addition or
substitution of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or
100% of a homologous Cas9 RuvC or RuvC-like domain. In some cases,
modifications to the site-directed polypeptide RuvC or RuvC-like
domain sequences may include addition or substitution of at most
1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of a homologous
Cas9 RuvC or RuvC-like domain.
[0565] Homologus Cas9 RuvC or RuvC-like domains may be derived from
any suitable organism. In some cases, the Cas9 protein may be
derived from prokaryotic organisms such as archea, bacteria,
protists (e.g., E. coli, S. pyogenes, S. thermophilus, P. furiosus
and etc.). For example, the RuvC or RuvC-like domain of the S.
pyogenes Cas9 enzyme may be substituted or inserted with the RuvC
or RuvC-like domain, or fragment thereof, derived from another Cas9
enzyme, such as one from another species.
[0566] In some instances, the modification to the RuvC or RuvC-like
domain can comprise insertion of the same or similar RuvC or
RuvC-like domain in tandem (e.g., adjacent) to the RuvC or
RuvC-like domain of Cas9. The RuvC or RuvC-like domain can be
inserted N-terminal and/or C-terminal of the RuvC or RuvC-like
domain in Cas9. Insertion of one or more RuvC or RuvC-like domains
in Cas9 can be useful in extending specificity in a target nucleic
acid. Insertion of one or more RuvC or RuvC-like domains in Cas9
can be useful in duplicating specificity in a target nucleic acid.
For example, insertion of one or more RuvC or RuvC-like domains can
configure Cas9 to recognize a longer stretch of target nucleic
acid, recognize a different RNA-DNA hybrid, and/or recognize a
target nucleic acid with higher binding affinity.
[0567] In some instances, a site-directed polypeptide comprises an
amino acid sequence comprising at least 15% amino acid identity to
a Cas9 from S. pyogenes, two nucleic acid-cleaving domains (i.e., a
HNH domain and RuvC domain), and a modified RuvC domain.
[0568] Modifications to Cas9 Domains Containing RNA Polymerase
Homologous Regions
[0569] In some cases, a site-directed polypeptide may be share
homology with RNA polymerase. Both proteins may share similar
functionally homologous domains that are involved in catalysis of
binding and manipulation of nucleic acids. For example, RNA
polymerase can comprise regions of polypeptide sequence that is
involved in binding RNA-DNA duplexes. In some cases, these regions
may aid in melting the duplex.
[0570] In some cases, a site-directed polypeptide may also comprise
certain regions that affect the binding specificity of the enzyme
for nucleic acids. In some cases, these regions may share either
sequence or functional homology with domains or regions as found in
RNA polymerase. In some cases, the basic region of the N-terminus
of the site-directed polypeptide may be bind to the tracrRNA and
crRNA or a single RNA (sgRNA). In S. pyogenes, this may correspond
to a region of residues 50-100.
[0571] Generally, the present disclosure provides for any suitable
modification to this region or adjacent regions. In some cases, the
tracrRNA/crRNA binding region (e.g., the nucleic acid-targeting
nucleic acid binding region) in the site-directed polypeptide may
be modified to alter specificity for the nucleic acid. In some
cases, the tracrRNA/crRNA binding region may share homology with
the tracrRNA/crRNA binding region identified in the Cas9 protein of
S. pyogenes (residues 50-100). In some cases, the tracrRNA/crRNA
binding region may share at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%,
99%, or 100% homology with residues 5-100 of S. pyogenes Cas9
tracrRNA/crRNA binding region. In some cases, the tracrRNA/crRNA
binding region may share at most 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%,
99%, or 100% homology with residues 50-100 of S. pyogenes Cas9
tracrRNA/crRNA binding region.
[0572] In some cases, modifications to the tracrRNA/crRNA binding
region may include but are not limited to individual amino acid
modifications, as described herein. In some cases, modification to
the tracrRNA/crRNA binding region may include but are not limited
to insertions, deletions or substitution of individual amino acids,
or polypeptides, such as other protein elements (e.g domains,
structural motifs, proteins).
[0573] Modifications may include modifications to at least 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or
more amino acids of the tracrRNA/crRNA binding region.
Modifications may include modifications to at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more
amino acids of the tracrRNA/crRNA binding region. Modifications may
also include at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%
of the tracrRNA/crRNA binding region. Modifications may also
include at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of
the tracrRNA/crRNA binding region.
[0574] In some cases, modifications to the site-directed
polypeptide tracrRNA/crRNA binding region sequences may include
particular polypeptide structural motifs, including but not limited
to alpha helix, beta strand, beta sheet, 310-helix, pi-helix,
polyproline I motif, polyproline II motif, polyproline III motif,
beta turn, alpha-turn-alpha, or helix kinks or hinges.
[0575] For example, substitutions to the site-directed polypeptide
tracrRNA/crRNA binding region may include substitution or addition
with one or more proteins or fragments thereof. For example, the
tracrRNA/crRNA binding region could be substituted with the
RNA-binding domain from any of the known RNA-binding Type I, Type
II, or Type III CRISPR system member. The tracrRNA/crRNA binding
region could be substituted with the RNA-binding domain from any of
the known RNA-binding member of the RAMP superfamily. The
tracrRNA/crRNA binding region could be substituted with the
RNA-binding domain from any of the known RNA-binding member of the
Cas7, Cas6, Cas5 families. In one example, the tracr RNA
requirement may be replaced with the requirement for a 5' hairpin
sequence with the spacer sequence placed downstream of the hairpin
for DNA recognition.
[0576] In some cases, modifications to site-directed polypeptide
tracrRNA/crRNA binding region sequences may include deletion of at
least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the
tracrRNA/crRNA binding region. In some cases, modifications to
site-directed polypeptide tracrRNA/crRNA binding region sequences
may include deletion of at most 1%, 5%, 10%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
98%, 99%, or 100% of the tracrRNA/crRNA binding region.
[0577] In some cases, modifications to site-directed polypeptide
tracrRNA/crRNA binding region sequences may include addition or
substitution of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or
100% of a homologous Cas9 tracrRNA/crRNA binding region. In some
cases, modifications to site-directed polypeptide tracrRNA/crRNA
binding region sequences may include addition or substitution of at
most 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of a
homologous Cas9 tracrRNA/crRNA binding region.
[0578] Homologous site-directed polypeptide tracrRNA/crRNA binding
regions may be derived from any suitable organism. In some cases,
the tracrRNA/crRNA binding region may be derived from prokaryotic
organisms, including but not limited to archea, bacteria, protists
(e.g., E. coli, S. pyogenes, S. thermophilus, P. furiosus and
etc.). For example, the tracrRNA/crRNA binding region of the S.
pyogenes Cas9 may be substituted or inserted with the
tracrRNA/crRNA binding region, or fragment thereof, derived from
another Cas9, such as one derived from another species.
[0579] In some instances, a site-directed polypeptide comprises an
amino acid sequence comprising at least 15% amino acid identity to
a Cas9 from S. pyogenes, two nucleic acid-cleaving domains (i.e., a
HNH domain and RuvC domain), and a modified polymerase-like
domain.
[0580] Modifications to Alter PAM Specificity
[0581] In some instances, a site-directed polypeptide can recognize
a protospacer adjacent motif (PAM). A PAM can be any sequence in a
target nucleic acid that is recognized by a site-directed
polypeptide and is immediately 3' of the target nucleic acid
sequence targeted by the spacer of a nucleic acid-targeting nucleic
acid. For example, a PAM can comprise 5'-NGG-3' or 5'-NGGNG-3',
5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3', 5'-NNNACA-3' where N
is any nucleotide and N is immediately 3' of the target nucleic
acid sequence targeted by the spacer sequence.
[0582] A site-directed polypeptide can be modified to alter PAM
specificity. For example, a site-directed polypeptide can be
modified such that prior to the modifying the polypeptide targets a
first protospacer adjacent motif and after the modifying the
site-directed polypeptide targets a second protospacer adjacent
motif. In some instances, altered PAM specificity can comprise a
change in binding specificity (e.g., increased binding, decreased
binding), and/or a change in the binding constant (e.g., increase
Kd, decrease Kd).
[0583] A site-directed polypeptide can be modified such that the
site-directed polypeptide can recognize a new type of PAM different
from the the type the wild-type site-directed polypeptide
recognizes. For example, a site-directed polypeptide that
recognizes the 5'-NGG-3' PAM can be modified such that it can
recognize the 5'-NGGNG-3' PAM, 5'-NNAAAAW-3', 5'-NNNNGATT-3',
5'-GNNNCNNA-3', or 5'-NNNACA-3'.
[0584] Any region of a site-directed polypeptide can be engineered
(e.g., bridge helix, HNH and/or HNH-like domain, RuvC and/or
RuvC-like domain, basic patch) to alter PAM specificity according
to the methods of the disclosure.
[0585] Regions corresponding to residues 445-507, 446-497,
1096-1225, 1105-1138 of a wild-type site-directed polypeptide
(e.g., Cas9 from S. pyogenes, SEQ ID NO: 8) can be engineered to
modify PAM recognition. Engineering of these regions can comprise
introducing mutations, replacing with corresponding regions from
other Cas9 orthologues, deletions, insertions etc. Regions
corresponding to residues 718-757, 22-49, 65-95, 445-507, 446-497,
1096-1225, 1105-1138 can be engineered to modify recognition of the
nucleic-acid targeting nucleic acid. Regions corresponding to
residues 445-507 and 1105-1138 can be engineered to modify P-domain
recognition.
[0586] In some instances, a site-directed polypeptide comprises an
amino acid sequence comprising at least 15% amino acid identity to
a Cas9 from S. pyogenes, two nucleic acid-cleaving domains (i.e., a
HNH domain and RuvC domain), and a modification, wherein prior to
introduction of the modification the site-directed polypeptide is
adapted to bind a first PAM and after introduction of the
modification, the site-directed polypeptide is adapted to bind to a
different PAM.
[0587] Modifications to Alter Nucleic Acid-Targeting Nucleic Acid
Specificity
[0588] In some instances, a site-directed polypeptide can recognize
a nucleic acid-targeting nucleic acid. A site-directed polypeptide
can be modified to alter a nucleic acid-targeting nucleic acid
specificity. For example, a site-directed polypeptide can be
modified such that prior to the modifying the polypeptide targets a
first a nucleic acid-targeting nucleic acid and after the modifying
the site-directed polypeptide targets a second a nucleic
acid-targeting nucleic acid. In some instances, altered nucleic
acid-targeting nucleic acid specificity can comprise a change in
binding specificity (e.g., increased binding, decreased binding),
and/or a change in the binding constant (e.g., increase Kd,
decrease Kd).
[0589] A site-directed polypeptide can be modified such that the
site-directed polypeptide can recognize a new type of a nucleic
acid-targeting nucleic acid different from the the type the
wild-type site-directed polypeptide recognizes Any region of a
site-directed polypeptide can be engineered (e.g., bridge helix,
HNH and/or FINE-like domain, RuvC and/or RuvC-like domain, basic
patch) to alter PAM specificity according to the methods of the
disclosure.
[0590] In some instances, a site-directed polypeptide comprises an
amino acid sequence comprising at least 15% amino acid identity to
a Cas9 from S. pyogenes, two nucleic acid-cleaving domains (i.e., a
HNH domain and RuvC domain), and a modification, wherein prior to
introduction of the modification the site-directed polypeptide is
adapted to bind a first a nucleic acid-targeting nucleic acid and
after introduction of the modification, the site-directed
polypeptide is adapted to bind to a different a nucleic
acid-targeting nucleic acid.
[0591] Modifications to Alter Hybridization Requirements
[0592] Insertions
[0593] A site-directed polypeptide can be modified to increase
binding specificity to a target nucleic acid. A sequence may be
inserted into the site-directed polypeptide. In some instances, a
HNH and/or HNH-like domain may be inserted in a site-directed
polypeptide. The non-native sequence (e.g., HNH and/or FINE-like
domain) may originate from any species. The insertion may take
place at any location in the site-directed polypeptide. The
insertion may occur in tandem (e.g., adjacent) to the native HNH
and/or HNH-like domain of the site-directed polypeptide. The
inserted HNH and/or HNH-like domain may comprise a mutation. The
inserted HNH and/or HNH-like domain may comprise a mutation that
reduces the nuclease activity of the domain. In some instances, a
RuvC and/or RuvC-like domain may be inserted in a site-directed
polypeptide. The insertion may take place at any location in the
site-directed polypeptide. The insertion may occur in tandem (e.g.,
adjacent) to the native RuvC and/or RuvC-like domain of the
site-directed polypeptide. The inserted RuvC and/or RuvC-like
domain may comprise a mutation. The inserted RuvC and/or RuvC-like
domain may comprise a mutation that reduces the nuclease activity
of the domain.
[0594] A site-directed polypeptide can be modified to increase
binding specificity to a nucleic acid-targeting nucleic acid. A
sequence may be inserted into the site-directed polypeptide. A HNH
and/or HNH-like domain may be inserted in a site-directed
polypeptide. The non-native sequence (e.g., HNH and/or HNH-like
domain) may originate from any species. The insertion may take
place at any location in the site-directed polypeptide. The
insertion may occur in tandem (e.g., adjacent) to the native HNH
and/or FINE-like domain of the site-directed polypeptide. The
inserted HNH and/or HNH-like domain may comprise a mutation. The
inserted HNH and/or HNH-like domain may be comprise a mutation that
reduces the nuclease activity of the domain. A RuvC and/or
RuvC-like domain may be inserted in a site-directed polypeptide.
The insertion may take place at any location in the site-directed
polypeptide. The insertion may occur in tandem (e.g., adjacent) to
the native RuvC and/or RuvC-like domain of the site-directed
polypeptide. The inserted RuvC and/or RuvC-like domain may comprise
a mutation. The inserted RuvC and/or RuvC-like domain may comprise
a mutation that reduces the nuclease activity of the domain.
[0595] A site-directed polypeptide can be engineered to comprise a
polypeptide domain that can bind to RNA-DNA hybrids (e.g., RNase
domain, zinc finger domain). For example, a site-directed
polypeptide can be engineered to comprise an RNaseH domain. The
inserted RNaseH domain may comprise a mutation. The inserted RNaseH
domain may be comprise a mutation that reduces the nuclease
activity of the domain.
[0596] A site-directed polypeptide can be engineered to comprise a
polypeptide domain that can bind to double-stranded DNA (e.g.,
domains comprising helix-turn-helix motifs, domains comprising
leucine zipper motifs, domains comprising helix-loop-helix motifs,
domains comprising zinc finger motifs). For example, a
site-directed polypeptide can be engineered to comprise a
helix-turn-helix motif. Non-limiting exemplary helix-turn-helix
motifs include those from dnaB, TetR, MuB, P2R, CysB, BirA, the
bacteriophage lambda repressor, Engrailed, Myb, LuxR, MarR, ETS,
ZNF10a, Kox-1. The helix-loop-helix motif can be di-helical,
tri-helical, tetrahelical, a winged helix-turn-helix, or other
modified helix-loop-helix. The inserted domain may be comprise a
mutation. The inserted domain may be comprise a mutation that
reduces the nuclease activity of the domain.
[0597] Compensatory Mutations
[0598] A site-directed polypeptide can comprise a mutation and/or
be engineered such that it may preferentially bind to a mutated
and/or engineered nucleic acid-targeting nucleic acid. Such
mutation of a site-directed polypeptide and nucleic acid-targeting
nucleic acid pair can be referred to as a compensatory mutation.
For example, a site-directed polypeptide can be engineered such
that its nuclease domain (e.g., HNH and/or FINE-like, RuvC and/or
RuvC-like) is replaced by a nucleic acid binding domain (e.g.,
Csy4, Cas5, Cas6 nucleic acid binding domain). A site-directed
polypeptide can be engineered such that a nucleic acid binding
domain (e.g., Csy4, Cas5, Cas6 nucleic acid binding domain) is
inserted into the site-directed polypeptide. The resulting
site-directed polypeptide can bind to a nucleic acid-targeting
nucleic acid that is mutated and/or engineered to comprise a
nucleic acid binding domain binding site (e.g., binding site for
Csy4, Cas5, Casio nucleic acid binding domains). The nucleic
acid-targeting nucleic acid can be mutated and/or engineered to
comprise a nucleic acid-binding domain binding site in the minimum
tracrRNA sequence. The nucleic acid-targeting nucleic acid can be
mutated and/or engineered to comprise a nucleic acid binding domain
binding site in the 3' tracrRNA sequence. The nucleic
acid-targeting nucleic acid can be mutated and/or engineered to
comprise a nucleic acid binding domain binding site in the tracrRNA
extension.
[0599] In some instances, a site-directed polypeptide comprises an
amino acid sequence comprising at least 15% amino acid identity to
a Cas9 from S. pyogenes, two nucleic acid-cleaving domains (i.e., a
HNH domain and RuvC domain), and a compensatory mutation, in which
the site-directed polypeptide is such that it can bind to an
engineered nucleic acid-targeting nucleic acid but not an
unmodified nucleic acid-targeting nucleic acid.
[0600] Methods to Generate Sticky Ends and Blunt Cuts
[0601] In some instances, one or more nickases (i.e., a
site-directed polypeptides comprising one substantially inactive
nuclease domain) can be used to generate targeted double stranded
cuts in target nucleic acid. Each nickase of the one or more
nickases can target one strand of the double-stranded target
nucleic acid. In some instances, two nickases can be used to
generate a targeted double stranded cut.
[0602] The two nickases can cut the target nucleic acid generating
a blunt end cut (wherein the cut sites of the target nucleic acid
are the same location on each strand). The two nickases can cut the
target nucleic acid at different locations within each strand such
that some single stranded nucleotides remain, thereby generating a
sticky end.
[0603] Cleavage of target nucleic acid by two modified
site-directed polypeptides having nickase activity may be used to
incur deletions or insertions of nucleic acid material from a
target nucleic acid by cleaving the target nucleic acid and
allowing the cell to repair the sequence in the absence of an
exogenously provided donor polynucleotide. In some instances, the
methods of the disclosure can be used to knock out a gene. If a
nucleic acid-targeting nucleic acid and two modified site-directed
polypeptides having nickase activity are co-administered to cells
with a donor polynucleotide sequence that includes at least a
segment with homology to the target nucleic acid, new nucleic acid
material may be inserted/copied into the site. Such methods may be
used to add, i.e. insert or replace, nucleic acid material to a
target nucleic acid (e.g., to "knock in" a nucleic acid that
encodes a protein, an siRNA, an miRNA, etc.), to add a tag (e.g.,
6.times.His, a fluorescent protein (e.g., a green fluorescent
protein; a yellow fluorescent protein, etc.), hemagglutinin (HA),
FLAG, etc.), to add a regulatory sequence to a gene (e.g. promoter,
polyadenylation signal, internal ribosome entry sequence (IRES), 2A
peptide, start codon, stop codon, splice signal, localization
signal, etc.), to modify a nucleic acid sequence (e.g., introduce a
mutation), and the like.
[0604] FIG. 32 depicts a method for generating blunt ends by
nickases. The target nucleic acid duplex 3210 can comprise a
plurality of PAM sequences 3215 (boxed), wherein one PAM is on one
strand of the target nucleic acid 3210 and one PAM is on the other
strand of the target nucleic acid 3210. A nucleic acid-targeting
nucleic acid 3205 as part of a complex with the nickase (nickase
not shown) can hybridize to the spacer sequence adjacent to the PAM
3215 on each strand of the target nucleic acid 3210. The nickase
can cleave one strand of the target nucleic acid 3210. Cleavage is
indicated by the triangles. If the PAMs are appropriately spaced
the nickases can cut the target nucleic acid in substantially the
same place on each strand, thereby resulting in a blunt end. The
PAM sequences may be separated by at least about 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 20, 30, 40, or 50 or more nucleotides. The PAM
sequences may be separated by at most about 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 20, 30, 40, or 50 or more nucleotides. In some instances,
the PAMs are spaced by 6 nucleotides (i.e., there are 6 nucleotides
in between each PAM). In some instances, the nucleic acid-targeting
nucleic acid cleaves about 3 nucleotides 5' of the PAM.
[0605] In some embodiments, two or more nickases can be employed to
generate sticky ends. FIG. 33 illustrates how two nickases targeted
to overlapping regions on a target nucleic acid can result in a
staggered double-stranded break resulting in sticky ends. The
target nucleic acid duplex 3310 can comprise a plurality of PAM
sequences 3315 (boxed). A nucleic acid-targeting nucleic acid 3305
as part of a complex with the nickase (nickase not shown) can
hybridize to the spacer sequence adjacent to the PAM 3315 on each
strand of the target nucleic acid 3310. The nickase can cleave one
strand of the target nucleic acid 3310. Cleavage is indicated by
the triangles. If the PAMs are appropriately spaced the nickases
can cut the target nucleic acid in staggered locations, thereby
resulting in a sticky end. The PAM sequences may be separated by at
least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides.
The PAM sequences may be separated by at most about 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10 or more nucleotides. The distance of the PAM
sequences can be related to the length of the sticky end generated.
For example, the farther the PAMs are away from each other, the
longer the sticky end will be.
[0606] A method for generating sticky ends using two or more
nickases can involve PAM sequences substantially adjcent to one
another (though on opposite strands). In some instances, the PAM
sequences are separated by at least about 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10 or more nucleotides. In some instances, the PAM sequences
are separated by at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or
more nucleotides. In some instances, the PAM sequences are
separated by one nucleotide. In some instances, the PAM sequences
are separated by no nucleotides.
[0607] Methods for Enrichment and Sequencing of Target Nucleic
Acids
[0608] General Overview
[0609] Sequencing can be useful for diagnosing disease by
identifying mutations and/or other sequence variants (e.g.,
polymorphisms). The methods of the disclosure provide for methods,
kits, and compositions for enriching a target nucleic acid sequence
without the use of amplification methodologies. A target nucleic
acid can be enriched with the use of a site-directed polypeptide
and a nucleic acid-targeting nucleic acid.
[0610] FIG. 3 depicts an exemplary embodiment of the methods of the
disclosure. A site-directed polypeptide 305 can bind a nucleic
acid-targeting nucleic acid 310, thereby forming a complex 306. The
nucleic acid-targeting nucleic acid 310 can comprise a nucleic acid
affinity tag 311. The site-directed polypeptide 305 can comprise a
nuclease domain. The site-directed polypeptide 305 can be
enzymatically active. The site-directed polypeptide 305 can
comprise an affinity tag 315. The nucleic acid-targeting nucleic
acid 310 can hybridize to a target nucleic acid 320. In some
embodiments, a plurality of complexes 306 can hybridize to a
plurality of locations within a target nucleic acid 320. In a
cleavage step 325, the nuclease domain of a site-directed
polypeptide 305 can cleave, or cut 330 the target nucleic acid 320.
The excised target nucleic acid 340 can be purified in a
purification step 335. Adaptors 345 can be ligated to the excised
target nucleic acid. The adaptors can facilitate sequencing of the
excised target nucleic acid.
[0611] FIG. 4 depicts an exemplary embodiment of the methods of the
disclosure. A site-directed polypeptide 405 can interact with a
nucleic acid-targeting nucleic acid 410, thereby forming a complex
406. The site-directed polypeptide 405 can comprise a nuclease
domain. In some embodiments, the nuclease domain of the
site-directed polypeptide 405 can be enzymatically inactive. The
site-directed polypeptide 405 can comprise an affinity tag 415. The
nucleic acid-targeting nucleic acid 410 can hybridize to a target
nucleic acid 420. The nucleic acid-targeting nucleic acid 410 can
comprise a nucleic acid affinity tag 411. The affinity tag 411 of
the nucleic acid-targeting nucleic acid can comprise a hairpin
structure. A plurality of complexes 406 can hybridize to a
plurality of locations within a target nucleic acid 420. In a
fragmenting step 225, the target nucleic acid 420 can be fragmented
into target nucleic acid fragment 445 (also herein referred to as a
"target nucleic acid"). The site-directed polypeptide 405 can be
purified by a capture agent 440 that can bind to the affinity tag
415 of the site-directed polypeptide 405. The fragmented target
nucleic acid 445 can be eluted from the complex 406 in a
purification step 450. In the same step, or optionally, in a
different step, adaptors 455 can be ligated to the target nucleic
acid. The adaptors can facilitate sequencing of the target nucleic
acid.
[0612] Complex of a Nucleic Acid-Targeting Nucleic Acid and a
Site-Directed Polypeptide
[0613] A nucleic acid-targeting nucleic acid can interact with a
site-directed polypeptide (e.g., a nucleic acid-guided nuclease,
e.g. Cas9), thereby forming a complex. The nucleic acid-targeting
nucleic acid can guide the site-directed polypeptide to a target
nucleic acid.
[0614] In some embodiments, a nucleic acid-targeting nucleic acid
can be engineered such that the complex (e.g., comprising a
site-directed polypeptide and a nucleic acid-targeting nucleic
acid) can bind outside of the cleavage site of the site-directed
polpeptide. In this case, the target nucleic acid may not interact
with the complex and the target nucleic acid can be excised (e.g.,
free from the complex).
[0615] In some embodiments, a nucleic acid-targeting nucleic acid
can be engineered such that the complex can bind inside of the
cleavage site of the site-directed polpeptide. In this case, the
target nucleic acid can interact with the complex and the target
nucleic acid can be bound (e.g., bound to the complex).
[0616] The nucleic acid-targeting nucleic acid can be engineered in
such a way that the complex (e.g., comprising a site-directed
polypeptide and/or a nucleic acid-targeting nucleic acid) can
hybridize to a plurality of locations within a nucleic acid
sample.
[0617] A plurality of complexes can be contacted to a nucleic acid
sample. The plurality of complexes can comprise nucleic
acid-targeting nucleic acids engineered to hybridize to the same
sequence. The plurality of complexes can comprise nucleic
acid-targeting nucleic acids engineered to hybridize to the
different sequences.
[0618] The sequences can be at different locations within a target
nucleic acid. The locations can comprise the same, or similar,
target nucleic acid sequences. The locations can comprise different
target nucleic acid sequences. The locations can be a defined
distance from each other. The locations can be less than 10
kilobases (Kb) apart, less than 8 Kb apart, less than 6 Kb apart,
less than 4 Kb apart, less than 2 Kb apart, less than 1 Kb apart,
less than 900 nucleotides apart, less than 800 nucleotides apart,
less than 700 nucleotides apart, less than 600 nucleotides apart,
less than 500 nucleotides apart, less than 400 nucleotides apart,
less than 300 nucleotides apart, less than 200 nucleotides apart,
less than 100 nucleotides apart.
[0619] The complexes can cleave the target nucleic acid which can
result in an excised target nucleic acid that can be less than 10
kilobases (Kb) long, less than 8 Kb long, less than 6 Kb long, less
than 4 Kb long, less than 2 Kb long, less than 1 Kb long, less than
900 nucleotides long, less than 800 nucleotides long, less than 700
nucleotides long, less than 600 nucleotides long, less than 500
nucleotides long, less than 400 nucleotides long, less than 300
nucleotides long, less than 200 nucleotides long, less than 100
nucleotides long.
[0620] The complexes can be bound to a fragmented target nucleic
acid that can be be less than 10 kilobases (Kb) long, less than 8
Kb long, less than 6 Kb long, less than 4 Kb long, less than 2 Kb
long, less than 1 Kb long, less than 900 nucleotides long, less
than 800 nucleotides long, less than 700 nucleotides long, less
than 600 nucleotides long, less than 500 nucleotides long, less
than 400 nucleotides long, less than 300 nucleotides long, less
than 200 nucleotides long, less than 100 nucleotides long.
[0621] Methods for Detecting Off-Target Binding Sites of
Site-Directed Polypeptides
[0622] General Overview
[0623] This disclosure describes methods, compositions, systems,
and/or kits for determining off target binding sites of
site-directed polypeptides. In some embodiments of the disclosure a
site-directed polypeptide can comprise a nucleic acid-targeting
nucleic acid, thereby forming a complex. The complex can be
contacted with a target nucleic acid. The target nucleic acid can
be captured with capture agents that can bind to the affinity tags
of the complex. The identity of the target nucleic acid can be
determined through sequencing. Sequencing (e.g., high throughput
sequencing, e.g., Illumina, Ion Torrent) can also identify the
frequency of off-target binding sites of the site-directed
polypeptide and/or complex, by counting the number of times a
particular binding site is read. The methods, compositions,
systems, and/or kits of the disclosure can facilitate the
development of more accurately and specifically targeted
site-directed polypeptides.
[0624] FIG. 5 depicts an exemplary embodiment of the methods of the
disclosure. A site-directed polypeptide 505 can comprise an
affinity tag 510. The site-directed polypeptide can comprise a
nucleic acid-binding domain 515. The nucleic acid-binding domain
515 can be a nucleic acid. In some embodiments, the nucleic
acid-binding domain 515, and the site-directed polypeptide 505 form
a complex 531. The complex 131 can be contacted 525 with a target
nucleic acid 530. In a preferred embodiment, the target nucleic
acid 530 is DNA (e.g. genomic DNA or gDNA). The complex can be
affinity purified 535 with a capture agent 540. The capture agent
540 can bind to the affinity tag 510 from the site-directed
polypeptide 505. The capture agent 540 can comprise a second
affinity tag 545. The capture agent 540 can be affinity purified
550 by binding to a solid support 555. In some embodiments, the
solid support 555 is a bead coated with an affinity reagent that
can bind to the affinity tag 545 of the capture agent. Optionally,
the solid support 555 can bind to the affinity tag 510 of the
site-directed polypeptide 505 to facilitate purification. In some
embodiments, one or more rounds of purification can occur. Each
round can comprise contacting a solid support 555 with the affinity
tags of the site-directed polypeptide 510 and/or the capture agent
545. The affinity purified complex can be eluted from the target
nucleic acid 530. The target nucleic acid can subsequently be
prepared for further processing. Processing can include downstream
analysis methods, e.g. sequencing.
[0625] FIG. 6 depicts an exemplary embodiment of the methods of the
disclosure. A site-directed polypeptide 605 can comprise an
affinity tag 610. The site-directed polypeptide 605 can comprise a
nucleic acid-binding domain 615. The nucleic acid-binding domain
615 can be a nucleic acid. In some embodiments, the nucleic
acid-binding domain 615 can comprise an affinity tag 620. In some
embodiments, the nucleic acid-binding domain 615 and the
site-specific polypeptide 605 can form a complex 631. The complex
631 may be contacted 625 with a target nucleic acid 630. In a
preferred embodiment, the target nucleic acid 630 is DNA. The
complex 631 can be affinity purified 635 with a capture agent 640.
The capture agent 640 may be a conditionally enzymatically inactive
site-directed polypeptide. The capture agent 640 can be a
conditionally enzymatically inactive variant of Csy4. The capture
agent 640 can bind to the affinity tag 620. The capture agent 640
can comprise an affinity tag 645. The capture agent 640 can be
affinity purified 650 by binding to a solid support 655. In some
embodiments, the solid support is a bead coated with an affinity
reagent that can bind to the affinity tag 645 of the capture agent
640. Optionally, the solid support 655 can bind to the affinity tag
610 of the site-directed polypeptide 605 to facilitate
purification. In some embodiments, two rounds of purification can
occur, each comprising contacting a solid support 655 with the
affinity tags of the site-directed polypeptide 610 and/or the
capture agent 640. Cleavage of the affinity tag 620 can facilitate
elution 660 of the target nucleic acid 630 from the solid support
655. The target nucleic acid 630 can subsequently be prepared for
further downstream analysis methods such as sequencing.
[0626] Methods
[0627] The disclosure provides methods for nuclease
immunoprecipitation and sequencing (NIP-Seq). In some embodiments,
the method can comprise a) contacting a nucleic acid sample with a
complex comprising an enzymatically inactive site-directed
polypeptide, a site-directed polypeptide, and a nucleic
acid-targeting nucleic acid. The complex can hybridize to a target
nucleic acid. The complex can be captured with a capture agent, and
the target nucleic acid bound to the complex can be sequenced. In
some embodiments, the method can further comprise determining the
identity of the off-target binding site. The method can be
performed using any of the site-directed polypeptides, nucleic
acid-targeting nucleic acids, and complexes of site-directed
polypeptides and nucleic acid-targeting nucleic acids as described
herein.
[0628] The methods can be performed outside of a cell. For example,
a sample can comprise purified genomic DNA, cell lysate,
homogenized tissue, plasma, and the like. The methods can be
performed in cells.
[0629] The site-directed polypeptide-target nucleic acid complexes
can be fixed or cross-linked to form complexes. The cells can be
crosslinked before they are lysed. Fixed or cross-linking cells can
stabilize protein-DNA complexes in the cell. Suitable fixatives and
cross-linkers can include, formaldehyde, glutaraldehyde,
ethanol-based fixatives, methanol-based fixatives, acetone, acetic
acid, osmium tetraoxide, potassium dichromate, chromic acid,
potassium permanganate, mercurials, picrates, formalin,
paraformaldehyde, amine-reactive NHS-ester crosslinkers such as
bis[sulfosuccinimidyl] suberate (BS3),
3,3'-dithiobis[sulfosuccinimidylpropionate] (DTSSP), ethylene
glycol bis[sulfosuccinimidylsuccinate (sulfo-EGS), disuccinimidyl
glutarate (DSG), dithiobis[succinimidyl propionate] (DSP),
disuccinimidyl suberate (DSS), ethylene glycol
bis[succinimidylsuccinate] (EGS), NHS-ester/diazirine crosslinkers
such as NHS-diazirine, NHS-LC-diazirine, NHS-SS-diazirine,
sulfo-NHS-diazirine, sulfo-NHS-LC-diazirine, and
sulfo-NHS-SS-diazirine.
[0630] The nucleic acid (e.g., genomic DNA) can be treated to
fragment the DNA before affinity purification. Fragmentation can be
performed through physical, mechanical or enzymatic methods.
Physical fragmentation can include exposing a target polynucleotide
to heat or to ultraviolet (UV) light. Mechanical disruption may be
used to mechanically shear a target polynucleotide into fragments
of the desired range. Mechanical shearing may be accomplished
through a number of methods known in the art, including repetitive
pipetting of the target polynucleotide, sonication and
nebulization. Target polynucleotides may also be fragmented using
enzymatic methods. In some cases, enzymatic digestion may be
performed using enzymes such as using restriction enzymes.
Restriction enzymes may be used to perform specific or non-specific
fragmentation of target polynucleotides. The methods may use one or
more types of restriction enzymes, generally described as Type I
enzymes, Type II enzymes, and/or Type III enzymes. Type II and Type
III enzymes are generally commercially available and well known in
the art. Type II and Type III enzymes recognize specific sequences
of nucleotide nucleotides within a double stranded polynucleotide
sequence (a "recognition sequence" or "recognition site"). Upon
binding and recognition of these sequences, Type II and Type III
enzymes cleave the polynucleotide sequence. In some cases, cleavage
will result in a polynucleotide fragment with a portion of
overhanging single stranded DNA, called a "sticky end." In other
cases, cleavage will not result in a fragment with an overhang,
creating a "blunt end." The methods may comprise use of restriction
enzymes that generate either sticky ends or blunt ends. Fragments
of nucleic acids can also be generated via amplification techniques
(e.g. polymerase chain reaction, long range polymerase chain
reaction, linear polymerase chain reaction, and etc.).
[0631] Once fragmented, the complexes comprising the site-directed
polypeptide can be purified by incubation with a solid support. For
example, if the site-directed polypeptide comprises a biotin tag,
the solid support can be coated with avidin or streptavidin to bind
to the biotin tag.
[0632] In some embodiments, once fragmented, the complexes
comprising the site-directed polypeptide, the target nucleic acid,
and/or the nucleic acid-targeting nucleic acid are purified by
incubation with a capture agent. A capture agent can refer to any
agent that can bind to an affinity tag fused to the site-directed
polypeptide. Exemplary capture agents can include, biotin,
streptavidin, and antibodies. For example, if the affinity tag
fused to the site-directed polypeptide is a FLAG tag, then the
capture agent will be an anti-FLAG-tag antibody. In some
embodiments, the capture agent can comprise an affinity tag (e.g.,
biotin, streptavidin).
[0633] In some instances, the capture agent is an enzymatically
inactive endoribonuclease. For example, a capture agent can be an
enzymatically inactive site-directed polypeptide, an enzymatically
inactive Csy4, Cas5, or Cas6.
[0634] The capture agent can be purified with a solid support. For
example, if the capture agent comprises a biotin tag, the bead can
be coated with avidin or streptavidin to bind the biotinylated
capture agent.
[0635] In some embodiments of the method, two or more rounds of
purification can be performed. At least 1, 2, 3, 4, 5, 6, 7 or more
rounds of purification can be performed. At most 1, 2, 3, 4, 5, 6,
7 or more rounds of purification can be performed. A first round of
purification can comprise purification with a solid support that
can bind to the affinity tag of the capture agent and a second
round of purification can comprise purification with a solid
support that can bind to the affinity tag of the site-directed
polypeptide. A first round of purification can comprise
purification with a solid support that can bind to the affinity tag
of the site-directed polypeptide and a second round of purification
can comprise purification with a solid support that will bind to
the affinity tag of the capture agent. The method can be used to
optimize the binding specificity of a site-directed polypeptide by
performing the method more than once.
[0636] The captured complex can comprise site-directed polypeptide
and a target nucleic acid. The target nucleic acid can be eluted
from the site-directed polypeptide complex by methods such as high
salt washing, ethanol precipitation, boiling, and gel
purification.
[0637] The eluted DNA can be prepared for sequencing analysis
(e.g., shearing, ligation of adaptors). Preparation for sequencing
analysis can include the generation of sequencing libraries of the
eluted target nucleic acid. Sequencing analysis can determine the
identity and frequency of off-target binding sites of site-directed
polypeptides. Sequence determination will also be performed using
methods that determine many (typically thousands to billions)
nucleic acid sequences in an intrinsically parallel manner, where
many sequences are read out preferably in parallel using a high
throughput serial process. Such methods include but are not limited
to pyrosequencing (for example, as commercialized by 454 Life
Sciences, Inc., Branford, Conn.); sequencing by ligation (for
example, as commercialized in the SOLiD.TM. technology, Life
Technology, Inc., Carlsbad, Calif.); sequencing by synthesis using
modified nucleotides (such as commercialized in TruSeq.TM. and
HiSeg.TM. technology by Illumina, Inc., San Diego, Calif.,
HeliScope.TM. by Helicos Biosciences Corporation, Cambridge, Mass.,
and PacBio RS by Pacific Biosciences of California, Inc., Menlo
Park, Calif.), sequencing by ion detection technologies (Ion
Torrent, Inc., South San Francisco, Calif.); sequencing of DNA
nanoballs (Complete Genomics, Inc., Mountain View, Calif.);
nanopore-based sequencing technologies (for example, as developed
by Oxford Nanopore Technologies, LTD, Oxford, UK), and other known
highly parallelized sequencing methods.
[0638] In some embodiments, the method further comprises collecting
data and storing data. The data can be machine readable and can be
stored and/or collected on a computer server (e.g. FIG. 31 and
Example 27).
[0639] Methods for Detecting Sequence Variants in Nucleic Acids
[0640] General Overview
[0641] In some embodiments, the methods of the disclosure provide
for detecting sequence variants in nucleic acids. The method can be
performed using any of the site-directed polypeptides, nucleic
acid-targeting nucleic acids, and complexes of site-directed
polypeptides and nucleic acid-targeting nucleic acids as described
herein. As depicted in FIG. 7, a nucleic acid sample 705 can be
ligated 720 with a nucleic acid tag 710. The nucleic acid tag can
be a single guide RNA. The nucleic acid tag can comprise a crRNA.
The nucleic acid tag can comprise a detectable label 715. Together,
the nucleic acid sample 705 ligated to the nuclei acid tag 710 can
be referred to as a tagged test sample 721. The tagged test sample
721 can be contacted 725 to an array 740 comprising immobilized
oligonucleotides 735. The immobilized oligonucleotides 735 can be
referred to as a nucleic acid library. The oligonucleotides 735 can
be double stranded DNA. The oligonucleotides 735 can comprise a
detectable label 730. The individual members of the tagged test
sample 721 can hybridize 745 to the oligonucleotides 735 to which
they share enough complementarity to facilitate hybridization. The
amount of hybridization can be quantified by comparing the
intensities of the two detectable labels 715 and 730. For example,
hybridized oligonucleotides can display two detectable labels.
Unhybridized oligonucleotides can display one detectable label 730.
The hybridized sample can be contacted with a site-directed
polypeptide 750. The site-directed polypeptide can cleave 755 the
oligonucleotides 735 in the array 740 that have hybridized with
members of the tagged test sample 721. Cleavage by the
site-directed polypeptide can allow the hybridized members of the
tagged test sample 721 to be removed. After cleavage by the
site-directed polypeptide 750, only unhybridized oligonucleotide
detectable labels 760 will remain on the array. The remaining
detectable label 760 can be quantified. The quantification of the
remaining detectable labels 760 can be correlated to which
sequences were represented in the nucleic acid sample 705 and which
were not. Oligonucleotides that do not display a remaining
detectable label 760 correspond to sequences that were represented
in the nucleic acid sample 705. Oligonucleotides that display a
remaining detectable label 760 correspond to sequences that were
not represented in the nucleic acid sample 705.
[0642] In some embodiments, a nucleic acid sample 805 can be
ligated 820 with a nucleic acid tag 810. The nucleic acid tag can
be a single guide RNA. The nucleic acid tag can comprise a crRNA.
The nucleic acid tag can comprise a detectable label 815. Together,
the nucleic acid sample ligated to the nuclei acid tag can be
referred to as a tagged test sample 821. The tagged test sample 821
can be contacted 825 to an array 840 comprising immobilized
oligonucleotides 835. The immobilized oligonucleotides can be
referred to as a nucleic acid library. The oligonucleotides 835 can
be double stranded DNA. The individual members of the tagged test
sample 821 can hybridize 845 to the oligonucleotides 835 to which
they share enough complementarity to facilitate hybridization. The
hybridized sample can be contacted with a site-directed polypeptide
850. The site-directed polypeptide can cleave 855 the
oligonucleotides 835 in the array 840 that have hybridized with
members of the tagged test sample 821. Cleavage by the
site-directed polypeptide 850 can allow the hybridized members of
the tagged test sample 821 to be removed. Cleavage by the
site-directed polypeptide 850 can allow a portion of the
immobilized olignucleotide to be cleaved and separated from the
array 860. The separated cleaved oligonucleotides 860 can be
ligated 865 to appropriate adaptors 870 for sequencing. Sequencing
of the cleaved oligonucleotides 860 can determine the sequences
represented in the nucleic acid sample 805.
[0643] In some embodiments, a nucleic acid library can be generated
for sequencing analysis using commercially available high
throughput sequencing platforms. The library can comprise nucleic
acids that can comprise one or more sequencing tags 930 and a
target sequence 945. The target sequence 945 can be a sequence that
may be represented in a nucleic acid sample 905. A target sequence
945 can comprise a protospacer adjacent motif (PAM) sequence.
Optionally, nucleic acids in a nucleic acid library can comprise
one or more identifying polynucleotide sequences 935, and one or
more extension sequences 940. In this embodiment, a nucleic acid
sample 905 can be ligated 920 with a nucleic acid tag 910. The
nucleic acid tag can be a single guide RNA. The nucleic acid tag
can comprise a crRNA. Optionally, the nucleic acid tag can comprise
an affinity tag 915. Together, the nucleic acid sample ligated to
the nuclei acid tag can be referred to as a tagged test sample 921.
The tagged test sample 921 can be contacted 925 to a nucleic acid
library. The tagged test sample 921 can hybridize to a nucleic acid
in the nucleic acid library, forming a complex 946. The hybridized
tagged test sample and nucleic acid library can be contacted with a
site-directed polypeptide 950. The site-directed polypeptide 950
can cleave the hybridized nucleic acid library members. The cleaved
nucleic acid library members 965 can be separated from the
uncleaved members. The uncleaved members can be subjected to
sequencing analysis. Sequencing analysis can determine which
sequences were represented in the nucleic acid sample 905. For
example, the sequences of the uncleaved members can correspond to
sequences that were not represented in the nucleic acid sample 905.
These sequences can be removed from the known sequences in the
nucleic acid library. The resulting sequences can be the sequences
of the cleaved members 965 of the nucleic acid library which can
correspond to sequences that were represented in the nucleic acid
sample 905.
[0644] The site-directed polypeptide 950 can comprise an affinity
tag 955. Optionally, the site-directed polypeptide 950 can be an
enzymatically inactive variant of a site-directed polypeptide. In
some embodiments, an enzymatically inactive site-directed
polypeptide can be contacted to a hybridized nucleic acid library
(e.g., complex 946). The site-directed polypeptide can bind but
cannot cleave the hybridized nucleic acid library members. The
site-directed polypeptide can be affinity purified 970 with a
capture agent 975 that can bind to the affinity tag 955.
Optionally, the complex 946 can be affinity purified with a capture
agent that can bind to the affinity tag 915. The affinity purified
nucleic acid library members can be subjected to sequencing
analysis. In this embodiment, the sequenced nucleic acid library
members can correspond to sequences that are represented in the
nucleic acid sample 905.
[0645] Sequencing
[0646] Methods for detecting sequence variants can comprise
sequencing the variants. Sequence determination can be performed
using methods that determine many (typically thousands to billions)
nucleic acid sequences in an intrinsically parallel manner, where
many sequences are read out preferably in parallel using a high
throughput serial process. Such methods can include but are not
limited to pyrosequencing (for example, as commercialized by 454
Life Sciences, Inc., Branford, Conn.); sequencing by ligation (for
example, as commercialized in the SOLiD.TM. technology, Life
Technology, Inc., Carlsbad, Calif.); sequencing by synthesis using
modified nucleotides (such as commercialized in TruSeq.TM. and
HiSeg.TM. technology by Illumina, Inc., San Diego, Calif.,
HeliScope.TM. by Helicos Biosciences Corporation, Cambridge, Mass.,
and PacBio RS by Pacific Biosciences of California, Inc., Menlo
Park, Calif.), sequencing by ion detection technologies (Ion
Torrent, Inc., South San Francisco, Calif.); sequencing of DNA
nanoballs (Complete Genomics, Inc., Mountain View, Calif.);
nanopore-based sequencing technologies (for example, as developed
by Oxford Nanopore Technologies, LTD, Oxford, UK), capillary
sequencing (e.g, such as commercialized in MegaBACE by Molecular
Dynamics), electronic sequencing, single molecule sequencing (e.g.,
such as commercialized in SMRT.TM. technology by Pacific
Biosciences, Menlo Park, Calif.), droplet microfluidic sequencing,
sequencing by hybridization (such as commercialized by Affymetrix,
Santa Clara, Calif.), bisulfate sequencing, and other known highly
parallelized sequencing methods.
[0647] Real Time PCR
[0648] Methods for detecting sequence variants can comprise
detecting the variants using real time PCR. Sequence determination
can be performed by real time polymerase chain reaction (RT-PCR,
also referred to as quantitative-PCR (QPCR)) can detect an amount
of amplifiable nucleic acid present in a sample. QPCR is a
technique based on the polymerase chain reaction, and can be used
to amplify and simultaneously quantify a target nucleic acid. QPCR
can allow for both detection and quantification of a specific
sequence in a target nucleic acid sample. The procedure can follow
the general principle of polymerase chain reaction, with the
additional feature that the amplified target nucleic acid can be
quantified as it accumulates in the reaction in real time after
each amplification cycle. Two methods of quantification can be: (1)
use of fluorescent dyes that intercalate with double-stranded
target nucleic acid, and (2) modified DNA oligonucleotide probes
that fluoresce when hybridized with a complementary target nucleic
acid. In the first method, a target nucleic acid-binding dye can
bind to all double-stranded (ds) nucleic acid in PCR, resulting in
fluorescence of the dye. An increase in nucleic acid product during
PCR therefore can lead to an increase in fluorescence intensity and
can be measured at each cycle, thus allowing nucleic acid
concentrations to be quantified. The reaction can be prepared
similarly to a standard PCR reaction, with the addition of
fluorescent (ds) nucleic acid dye. The reaction can be run in a
thermocycler, and after each cycle, the levels of fluorescence can
be measured with a detector; the dye can only fluoresce when bound
to the (ds)nucleic acid (i.e., the PCR product). With reference to
a standard dilution, the (ds) nucleic acid concentration in the PCR
can be determined. The values obtained can not have absolute units
associated with it. A comparison of a measured DNA/RNA sample to a
standard dilution can give a fraction or ratio of the sample
relative to the standard, allowing relative comparisons between
different tissues or experimental conditions. To ensure accuracy in
the quantification, the expression of a target gene can be
normalized to a stably expressed gene. This can allow for
correction of possible differences in nucleic acid quantity or
quality across samples. The second method can use a
sequence-specific RNA or DNA-based probe to quantify only the
nucleic acid containing the probe sequence; therefore, use of the
reporter probe can increase specificity, and can allow
quantification even in the presence of some non-specific nucleic
acid amplification. This can allow for multiplexing, (i.e.,
assaying for several genes in the same reaction by using specific
probes with differently colored labels), provided that all genes
are amplified with similar efficiency. This method can be carried
out with a nucleic acid-based probe with a fluorescent reporter
(e.g. 6-carboxyfluorescein) at one end and a quencher (e.g.,
6-carboxy-tetramethylrhodamine) of fluorescence at the opposite end
of the probe. The close proximity of the reporter to the quencher
can prevent detection of its fluorescence. Breakdown of the probe
by the 5' to 3' exonuclease activity of a polymerase (e.g., Taq
polymerase) can break the reporter-quencher proximity and thus can
allow unquenched emission of fluorescence, which can be detected.
An increase in the product targeted by the reporter probe at each
PCR cycle can result in a proportional increase in fluorescence due
to breakdown of the probe and release of the reporter
[0649] The reaction can be prepared similarly to a standard PCR
reaction, and the reporter probe can be added. As the reaction
commences, during the annealing stage of the PCR both probe and
primers can anneal to the target nucleic acid. Polymerization of a
new DNA strand can be initiated from the primers, and once the
polymerase reaches the probe, its 5'-3'-exonuclease can degrade the
probe, physically separating the fluorescent reporter from the
quencher, resulting in an increase in fluorescence. Fluorescence
can be detected and measured in a real-time PCR thermocycler, and
geometric increase of fluorescence can correspond to exponential
increase of the product is used to determine the threshold cycle in
each reaction. Relative concentrations of DNA present during the
exponential phase of the reaction can be determined by plotting
fluorescence against cycle number on a logarithmic scale (so an
exponentially increasing quantity can give a straight line). A
threshold for detection of fluorescence above background can be
determined. The cycle at which the fluorescence from a sample
crosses the threshold can be called the cycle threshold, Ct. Since
the quantity of DNA can double every cycle during the exponential
phase, relative amounts of DNA can be calculated, (e.g. a sample
with a Ct of 3 cycles earlier than another has 23=8 times more
template). Amounts of nucleic acid (e.g., RNA or DNA) can be
determined by comparing the results to a standard curve produced by
a real-time PCR of serial dilutions (e.g. undiluted, 1:4, 1:16,
1:64) of a known amount of nucleic acid. The QPCR reaction can
involve a dual fluorophore approach that takes advantage of
fluorescence resonance energy transfer (FRET), (e.g., LIGHTCYCLER
hybridization probes, where two oligonucleotide probes can anneal
to the amplicon). The oligonucleotides can be designed to hybridize
in a head-to-tail orientation with the fluorophores separated at a
distance that is compatible with efficient energy transfer. Other
examples of labeled oligonucleotides that are structured to emit a
signal when bound to a nucleic acid or incorporated into an
extension product include: SCORPIONS probes, Sunrise (or
AMPLIFLOUR) primers, and LUX primers and MOLECULAR BEACONS probes.
The QPCR reaction can use fluorescent Taqman methodology and an
instrument capable of measuring fluorescence in real time (e.g.,
ABI Prism 7700 Sequence Detector). The Taqman reaction can use a
hybridization probe labeled with two different fluorescent dyes.
One dye can be a reporter dye (6-carboxyfluorescein), the other can
be a quenching dye (6-carboxy-tetramethylrhodamine). When the probe
is intact, fluorescent energy transfer can occur and the reporter
dye fluorescent emission can be absorbed by the quenching dye.
During the extension phase of the PCR cycle, the fluorescent
hybridization probe can be cleaved by the 5'-3' nucleolytic
activity of the DNA polymerase. On cleavage of the probe, the
reporter dye emission can no longer transferred efficiently to the
quenching dye, resulting in an increase of the reporter dye
fluorescent emission spectra. Any nucleic acid quantification
method, including real-time methods or single-point detection
methods can be use to quantify the amount of nucleic acid in the
sample. The detection can be performed several different
methodologies (e.g., staining, hybridization with a labeled probe;
incorporation of biotinylated primers followed by avidin-enzyme
conjugate detection; incorporation of 32P-labeled deoxynucleotide
triphosphates, such as dCTP or dATP, into the amplified segment.
The quantification can or can not include an amplification step.
The quantitation can not be experimental.
[0650] Microarray
[0651] Methods for detecting sequence variants can comprise
sequencing and/or detecting the variants using a microarray.
Microarrays can be used for determining the expression level of a
plurality of genes in a nucleic acid sample. Microarrays can be
used for determining sequence identity of a plurality of sequences
in a nucleic acid sample.
[0652] A microarray can comprise a substrate. Substrates can
include, but are not limited to, glass and modified or
functionalized glass, plastics (including acrylics, polystyrene and
copolymers of styrene and other materials, polypropylene,
polyethylene, polybutylene, polyurethanes, Teflon.TM., and the
like), polysaccharides, nylon or nitrocellulose, resins, silica or
silica-based materials including silicon and modified silicon,
carbon, metals, inorganic glasses, and plastics.
[0653] Microarrays can comprise a plurality of polynucleotide
probes. A microarray can comprise about 1, 10, 100, 1000, 5000,
10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000,
100000, 110000, 120000 or more probes.
[0654] Probes can be can be at least 10, 20, 30, 40, 50, 60, 70,
80, 90, 100, 110, 120, 130, 140 nucleotides or more in length.
[0655] In some embodiments, probes can comprise sequence
information for a specific set of genes and/or species. A probe can
be complementary to a nucleic acid sequence encoding a host
protein. A probe can be complementary to a non-coding nucleic acid
sequence. A probe can be complementary to a DNA sequence. A probe
can be complementary to an RNA sequence.
[0656] Probes can be immobilized on a microarray. The
immobilization of polynucleotides on a solid substrate can be
achieved by direct synthesis (e.g., photolithographic synthesis) of
polynucleotides on a solid substrate or by immobilization
(spotting) of previously synthesized polynucleotides on
predetermined regions of a solid substrate. Polynucleotides can be
immobilized on a microarray substrate by activating a surface of a
solid substrate with a nucleophilic functional group (e.g., an
amino group), coupling biomolecules (e.g., polynucleotides)
activated with a good leaving group to the surface-activated solid
substrate, and removing unreacted reactants. Probes can be
immobilized to a bead further conjugated through a covalent or
ionic attachment to a solid support. Probes can be immobilized onto
a substrate using a specific film having a low conductivity and a
low melting temperature, namely a gold film. An applied
electromagnetic radiation can melt and can ablate the film at the
impingement site. The film can be in contact with a colloidal
dispersion and upon melting can generate a convective flow at the
reaction site, thereby leading to adhering of an insoluble particle
in the dispersion to the specifically melted site.
[0657] A microarray can analyze a nucleic acid sample comprising
nucleic acids of unknown identity (e.g., test sample) by comparing
the nucleic acid sample of unknown identity with a reference
sample. A nucleic acid sample can be prepared from DNA (e.g.,
isolated DNA, genomic DNA, extrachromasomal DNA). A nucleic acid
sample can be prepared from RNA. RNA can be reverse transcribed
into DNA with a gene-specific primer or a universal primer. The
reverse transcribed DNA (e.g., cDNA), can be treated with Rnase or
base (e.g., NaOH) to hydrolyze the RNA. The cDNA can be labelled
with a dye (e.g, Cy3, Cy5) with N-hydroxysuccinimide chemistry or
similar labeling chemistries. Suitable fluorescent dyes can include
a variety of commercial dyes and dye derivatives such as those that
are denoted Alexa, Fluorescein, Rhodamine, FAM, TAMRA, Joe, ROX,
Texas Red, BODIPY, FITC, Oregon Green, Lissamine and others. The
reference sample can be labeled with a different dye than the test
sample.
[0658] The test sample and the reference sample can be applied to a
microarray to contact multiple spots simultaneously. The test
sample and the reference sample can be applied to the microarray
under hybridizing conditions that can allow the nucleic acids in
the nucleic acid sample to bind to a complement probe on the
microarray. Various reaction steps can be performed with the bound
molecules in the microarray, including exposure of bound reactant
molecules to washing steps. The progress or outcome of the reaction
can be monitored at each spot (e.g., probe) in the microarray in
order to characterize the nucleic acid sample immobilized on the
chip. Microarray analysis usually can require an incubation period
that can range from minutes to hours. The duration of the
incubation period can be assay dependent and can be determined by a
variety of factors, such as the type of reactant, degree of mixing,
sample volume, target copy number, and density of the array. During
the incubation period, nucleic acids in the nucleic acid sample can
be in intimate contact with the microarray probes.
[0659] Detection can be performed using a confocal scanning
instrument with laser excitation and photomultiplier tube
detection, such as the ScanArray 3000 provided by GSI Lumonics
(Billerica, Mass.). Confocal and non-confocal fluorescent detection
systems can be used to implement the method such as those provided
by Axon Instruments (Foster City, Calif.), Genetic MicroSystems
(Santa Clara, Calif.), Molecular Dynamics (Sunnyvale, Calif.) and
Virtek (Woburn, Mass.). Alternative detection systems can include
scanning systems that use gas, diode and solid state lasers as well
as those that use a variety of other types of illumination sources
such as xenon and halogen bulbs. In addition to photomultiplier
tubes, detectors can include cameras that use charge coupled device
(CCD) and complementary metal oxide silicon (CMOS) chips.
[0660] The ratio of the intensities of the two dyes from the test
sample and the reference sample can be compared for each probe. The
strength of the signal detected from a given microarray spot can be
directly proportional to the degree of hybridization of a nucleic
acid in the sample to the probe at a given spot (e.g., a spot
comprises a probe). Analysis of the fluorescence intensities of
hybridized microarrays can include spot segmentation, background
determination (and possible subtraction), elimination of bad spots,
followed by a method of normalization to correct for any remaining
noise. Normalization techniques can include global normalization on
all spots or a subset of the spots such as housekeeping genes,
prelog shifting to obtain better baseline matches, or in the case
of two (or more) channel hybridizations finding the best fit that
helps to give an M vs. A plot that is centered about M=0 and/or
that helps to give a log(Red) vs. log(Green) plot that is centered
about the diagonal with the smallest spread. The M vs. A plot can
also be referred to as the R vs. I plot, where R is a ratio, such
as R=log.sub.2(Red/Green) and I is an intensity, such as I=log
VRed*Green. Scaling, shifting, best fits through scatter plots,
etc. can be techniques utilized to normalize microarray datasets
and to give better footing for subsequent analysis. Most of these
normalization methods can have some underlying hypothesis behind
them (such as "most genes within the study do not vary much").
[0661] Tagged Nucleic Acids and Methods of Use
[0662] General Overview
[0663] The disclosure provides for kits, methods, and compositions
for tagged nucleic acid-targeting nucleic acids, as described
herein. FIG. 10 depicts an exemplary embodiment of nucleic
acid-targeting nucleic acid 1005 of the disclosure. A nucleic
acid-targeting nucleic acid can comprise one or more non-native
sequences (e.g., tags) 1010/1015. A nucleic acid-targeting nucleic
acid can comprise a non-native sequence 1010/1015 at either the 3'
end, the 5' end, or both the 3' and 5' end of the nucleic
acid-targeting nucleic acid.
[0664] In some instances, a nucleic acid-targeting nucleic acid can
be nucleic acid-targeting nucleic acids as described herein, and
comprise one or more non-native sequences, such as either at the
3'end, the 5' end or both the 3' and 5' ends of the nucleic
acid-targeting nucleic acid.
[0665] In some instances, the nucleic acid-targeting nucleic acid
is a double-guide nucleic acid-targeting nucleic acid as described
herein, and as depicted in FIG. 11. A double-guide nucleic
acid-targeting nucleic acid can comprise two nucleic acid molecules
1101/1110. A double-guide nucleic acid-targeting nucleic acid can
comprise a plurality of non-native sequences 1115/1120/1125/1130.
The non-native sequence can be located at the 3'end, 5' end or both
3' and 5' ends of each molecule of the nucleic acid-targeting
nucleic acid. For example, the non-native sequence can be located
at the 3'end, 5' end or both 3' and 5' ends of the first nucleic
acid molecule 1105. The non-native sequence can be located at the
3'end, 5' end or both 3' and 5' ends of the second molecule 1110. A
nucleic acid-targeting nucleic acid can comprise a one or more
non-native sequences in any of the enumerated configurations in
FIG. 11.
[0666] The disclosure provides for methods of use of tagged nucleic
acid-targeting nucleic acids. The method can be performed using any
of the site-directed polypeptides, nucleic acid-targeting nucleic
acids, and complexes of site-directed polypeptides and nucleic
acid-targeting nucleic acids as described herein. In some
instances, a plurality of tagged nucleic acid-targeting nucleic
acids can be contacted to a plurality of target nucleic acids. FIG.
12 depicts an exemplary method of use for tagged nucleic
acid-targeting nucleic acids. A tagged nucleic acid-targeting
nucleic acid can comprise a spacer 1210 that can hybridize with a
target nucleic acid 1205. The nucleic acid-targeting nucleic acid
can comprise a non-native sequence (e.g., tag) 1220. The non-native
sequence 1220 can be an RNA-binding protein binding sequence. In
some instances, the non-native sequence 1220 can be a CRISPR
RNA-binding protein binding sequence. The non-native sequence 1220
can be bound by a RNA-binding protein 1215. The RNA-binding protein
1215 can comprise a non-native sequence 1225 (e.g., a fusion, i.e.,
the RNA-binding protein 1215 can be a fusion polypeptide). The
non-native sequence (e.g., fusion) 1225 can alter the transcription
of the target nucleic acid and/or an exogenous nucleic acid. The
non-native sequence (e.g., fusion) 1225 can comprise a first
portion of a split system.
[0667] In some embodiments, a second nucleic acid-targeting nucleic
acid, comprising a second spacer 1240 that can hybridize to a
second target nucleic acid 1245, can comprise a second non-native
sequence (e.g., tag) 1250. The second non-native sequence (e.g.,
tag) 1250 can be an RNA-binding protein binding sequence. The
second non-native sequence (e.g., tag) 1250 can be a CRISPR
RNA-binding protein binding sequence. The second non-native
sequence 1250 can be bound by a RNA-binding protein 1235. The
RNA-binding protein can comprise a non-native sequence 1230 (e.g.,
fusion, i.e., the RNA-binding protein 1235 can be a fusion). The
non-native sequence 1230 (e.g., fusion) can be a second portion of
a split system.
[0668] In some instances, the first portion of the split system
1225 and the second portion of the split system 1230 can be close
together in space, such that the first portion of the split system
1225 and the second portion of the split system 1230 interact 1255
to form an active split system 1260. An active split system 1260
can refer to an unsplit system, wherein the first portion and the
second portion form a whole piece of the split system. Activation
of the split system can indicate that two target nucleic acids
1205/1245 are close together in space.
[0669] Methods
[0670] The disclosure provides for methods for contacting a target
nucleic acid with a complex comprising a site-directed polypeptide
and a nucleic acid-targeting nucleic acid, and introducing one or
more effector proteins, wherein the one or more effector proteins
comprises a non-native sequence and can bind to the modified
nucleic acid-targeting nucleic acid. An effector protein can refer
to any protein with a functional effect. For example, an effector
protein can comprise enzymatic activity, remodel biological
molecules (e.g., folding chaperones), be a scaffolding protein,
and/or bind a small molecule or metabolite. The effector protein
can modify the target nucleic acid (e.g., cleavage, enzymatic
modification, transcriptional modification). The methods of the
disclosure provide for using the compositions of the disclosure as
biosensors. For example, the complexes (e.g., comprising a modified
nucleic acid-targeting nucleic acid, a site-directed polypeptide
and/or an effector protein) can be used to monitor genetic mobility
events, sense when sequences are close together in
three-dimensional space, and conditionally alter transcription.
[0671] Genetic Mobility Event
[0672] The disclosure provides for methods for determining the
occurrence of a genetic mobility event. The method can be performed
using any of the site-directed polypeptides, nucleic acid-targeting
nucleic acids, and complexes of site-directed polypeptides and
nucleic acid-targeting nucleic acids as described herein. A genetic
mobility event can comprise, for example, a translocation, a
recombination, an integration, a transposition, a horizontal gene
transfer event, a transformation, a transduction, a conjugation, a
gene conversion event, a duplication, a translocation, an
inversion, a deletion, a substitution, or any combination
thereof.
[0673] A genetic mobility event can comprise a recombination
between genes. The recombination can lead to deleterious gene
products (e.g., the BCR-ABL recombination which can contribute to
breast cancer). Recombination can include, for example, homologous
recombination, non-homologous recombination (e.g., non-homologous
end joining), and V(D)J recombination. Recombination can refer to
chromosomal crossover. Recombination can occur during prophase I of
meiosis (e.g., synapsis). Recombination can comprise
double-stranded breakage of nucleic acid strands of DNA, followed
by formation of a holiday junction by recombinases which can
catalyze swapping of the DNA strands.
[0674] Genetic mobility events can cause disease. For example,
chronic myelogenous leukemia can result from a genetic mobility
event. Translocation between chromosome 9 and 22 can result in a
fusion BCR-Abl1 gene, which can result in the lengthening of one
chromosome (e.g., 9), and the shortening of another chromosome
(e.g., 22, i.e., Philadelphia chromosome). The BCR-Abl1
translocation can lead to the production of a BCR-Abl fusion
protein which can interact with receptors (e.g., interleukin-3
receptor) to promote cell division, leading to chronic myelogenous
leukemia (CML). Other non-limiting exemplary genetic mobility
events include BRD3-NUT, BRD4-NUT, KIAA1549-BRAF, FIG/GOPC-ROS1,
ETV6-NTRK3, BCAS4-BCAS3, TBL1XR1-RGS17, ODZ4-NRG1, MALAT1-TFEB,
APSCR1-TFE3, PRCC-TFE3, CLTC-TFE3, NONO-TFE3, SFPQ-TFE3,
ETV6-NRTK3, EML4-ALK, EWSR1-ATF1, MN1-ETV6, CTNNB1-PLAG1,
LIFR-PLAG1, TCEA1-PLAG1, FGFrl-PLAG1, CHCHD7-PLAG1, HMGA2-FHIT,
HMGA-NFIB, CRTC1-MAM12, CRCT3-MAML2, EWSR1-POUF5F1, TMPRSS1-ERG,
TMPRSS2-ETV4, TMPRSS2-ETV5, HNRNPA2B1-ETV1, HERV-K-ETV1,
C150RF21-ETV1, SLC45A3-ETV1, SLC45A3-ETV5, SLC45A3-ELK4, KLK2-ETV4,
CANT1-ETV4, RET-PTC1/CCDC6, RET-PTC2/PRKAR1A, RET-PTC3,4/NCOA4,
RET-PTC5/GOLGA5, RET-PTC6/TRIM24, RET-PTC7/TRIM33, RET-PTC8/KTN1,
RET-PTC9/RFG9, RET-PTCM1, TFG-NTRK1, TPM3-NRTK1, TPR-NRTK1,
RET-D10S170, ELKS-RET, HOOKS3-RET, RFP-RET, AKAP9-BRAF, and
PAX8-PPARG.
[0675] Diseases that can be caused by genetic mobility events can
include Charcot-Marie-Tooth disease type 1A (CMT1A), juvenile
nephronophtisis (NPH), X-linked icthyosis, familial growth hormone
deficiency type 1A, fascioscapulohumeral muscular dystrophy (FSHD),
.alpha.-thalassemia, hemophilia A, Hunter syndrome (i.e.,
mucopolysaccharidosis II), Emery-Dreifuss musclar dystrophy,
Hemoglobin Lepore, steroid 21-hydroxylase deficiency,
glucocorticoid-suppressible hyperaldosteronism (GSH),
color-blindness (e.g., visual dichromacy), autosomal recessive
spinal muscular atrophy (SMA), cancer, T-cell acute lymphoblastic
leukemia (T-ALL), aggressive midline carcinoma, Astrocytoma,
Secretory breast carcinoma, Breast cancer, Kidney carcinoma,
Mesoblastic nephroma, Lung adenocarcinoma, Melanoma, Meningioma,
pleomorphic adenoma, mucoepidermoid cancer, Prostate carcinoma,
Thyroid carcinoma, and acute promyelocytic leukemia.
[0676] The methods of the disclosure provide for determining the
occurrence of a genetic mobility event in which a target nucleic
acid can be contacted with two complexes, each complex comprising a
site-directed polypeptide and a modified nucleic acid-targeting
nucleic acid, and two or more effector proteins can be introduced,
wherein the two or more effector proteins can bind to the modified
nucleic acid-targeting nucleic acids, wherein one of the two or
more effector proteins comprises a non-native sequence that is a
first piece of a split system and one of the two or more effector
proteins comprises a non-native sequence that is a second piece of
the split system. A split system can refer to a protein complex
composed of two or more protein fragments that individually are not
fluorescent, but, when formed into a complex, result in a
functional (that is, fluorescing) fluorescent protein complex.
Individual protein fragments of a split system (e.g., split
fluorescent protein) can be referred to as "complementing
fragments" or "complementary fragments". Complementing fragments
which can spontaneously assemble into a functional fluorescent
protein complex are known as self-complementing, self-assembling,
or spontaneously-associating complementing fragments. For example,
a split system can comprise GFP. In a GFP split system,
complementary fragments are derived from the three dimensional
structure of GFP, which includes eleven anti-parallel outer beta
strands and one inner alpha strand. A first fragment can comprise
one of the eleven beta-strands of the GFP molecule (e.g., GFP S11),
and a second fragment can comprise the remaining strands (e.g., GFP
S1-10).
[0677] Prior to the genetic mobility event the target nucleic acid
sequence targetable by one complex can be far apart from the target
nucleic acid sequence targetable by another sequence. The distance
between the two target nucleic acid sequences can comprise at least
about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more Kb. The
distance between the two target nucleic acid sequences can comprise
at most about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more Kb.
The two target nucleic acid sequences can be located on different
chromosomes. The two target nucleic acid sequences can be located
on the same chromosome.
[0678] Prior to the genetic mobility event the effector proteins
that comprise pieces of the split system may not be able to
interact with each other (e.g., the split system can be inactive).
After the genetic mobility event, the target nucleic acid sequence
targetable by one complex may be located in close proximity to the
target nucleic acid sequence targetable by the other complex. After
the genetic mobility event, the effector proteins that comprise
pieces of the split system may be able to interact with each other,
thereby activating the split system.
[0679] The activated split system can indicate the occurrence of
the genetic mobility event. For example, if the activated split
system is a fluorescent protein split system, then prior to the
genetic mobility event fluorescence may not be detected in the
sample. In some instances, the levels of fluorescence of the
inactive split system (e.g., background levels) may be 0.01, 0.05,
0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5 or more fold less fluorescent
compared to a control sample (e.g., cell) that does not comprise
the split system. In some instances, the levels of fluorescence of
the inactive split system (e.g., background level) may be 0.01,
0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5 or more fold more
fluorescent than a control sample (e.g., cell) that does not
comprise the split system.
[0680] After the genetic mobility event, the two split pieces can
unite to form an active fluorescent protein, and fluorescence can
be detected in the sample. An active split system can result in at
least about a 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more fold
increase in fluorescence. An active split system can result in at
most about a 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more fold
increase in fluorescence.
[0681] Detection of a genetic mobility can be used to genotype a
subject (e.g., a patient). A genotype can be indicative of a
disease. The detection of a genetic mobility event can be used to
diagnose a subject. The genetic and diagnostic information obtained
from the methods described herein can be communicated to a subject.
The genetic and diagnostic information obtained from the methods
described herein can be used to develop a subject-specific
treatment plan. For example, if the data obtained from the methods
of the disclosure indicate that a patient has a genotype that makes
them resistant to a particular therapeutic regimen, a new treatment
plan can be made for the subject.
[0682] Altering Transcription
[0683] The methods of the disclosure can provide for altering the
transcription of a nucleic acid. The method can be performed using
any of the site-directed polypeptides, nucleic acid-targeting
nucleic acids, and complexes of site-directed polypeptides and
nucleic acid-targeting nucleic acids as described herein. The
method provides for contacting a target nucleic acid with two
complexes, each complex comprising a site-directed polypeptide and
a modified nucleic acid-targeting nucleic acid, and introducing two
or more effector proteins, wherein the two or more effector
proteins can bind to the modified nucleic acid-targeting nucleic
acids, wherein the one of the two or more effector proteins
comprises a non-native sequence that is a first piece of a split
transcription factor system and one of the two or more effector
proteins comprises a non-native sequence that is a second piece of
the split transcription factor system, and wherein an interaction
between the first piece and the second piece of the split system
forms a transcription factor that alters transcription of the
nucleic acid.
[0684] The transcription factor can alter transcription levels of a
nucleic acid and/or a target nucleic acid. Altered transcription
can include increased transcription levels and/or decreased
transcription levels. A transcription factor can alter
transcription levels more than 2-fold, 3-fold, 5-fold, 10-fold,
50-fold, 100-fold, 1000-fold or more higher or lower than unaltered
transcription levels. A transcription factor can alter
transcription levels less than 2-fold, 3-fold, 5-fold, 10-fold,
50-fold, 100-fold, 1000-fold or more higher or lower than unaltered
transcription levels.
[0685] The transcription factor can alter the transcription of a
target nucleic acid and/or an exogenous nucleic acid. A target
nucleic acid can be the nucleic acid that is contacted by the
complex comprising the site-directed polypeptide and the nucleic
acid-targeting nucleic acid. An exogenous nucleic acid can comprise
a donor polynucleotide, a plasmid, and/or a target nucleic
acid.
[0686] An exogenous nucleic acid can comprise a polynucleotide
encoding genes involved in apoptosis. Suitable genes involved in
apoptosis can include tumor necrosis factor (TNF), TNF-R1, TNF-R2,
TNF receptor-associated death domain (TRADD), Fas receptor and Fas
ligand, caspases (e.g., caspase-3, caspase-8, caspase-10), APAF-1,
FADD, and apoptosis inducing factor (AIF). An exogenous nucleic
acid can comprise a polynucleotide encoding genes that result in
cell lysis. Suitable genes can include the Adenovirus death protein
(ADP), defensins, membrane-permeabilizing lytic peptides derived
from c-FLIP, procaspases, cell-penetrating peptides e.g. HIV TAT.
An exogenous nucleic acid can comprise a polynucleotide encoding an
antigen that can result in recruitment of immune cells to the cell
location (e.g., MHC class peptides). An exogenous nucleic acid can
comprise a polynucleotide encoding a nucleic-acid targeting nucleic
acid that targets sequences that occur many times within the genome
(e.g., microsatellites, tandem repeats), resulting in large scale
genome fragmentation and cell-death.
[0687] Modification of Target Nucleic Acid
[0688] The disclosure provides for methods to modify a target
nucleic acid using the nucleic acid-targeting nucleic acid of the
disclosure. The method can be performed using any of the
site-directed polypeptides, nucleic acid-targeting nucleic acids,
and complexes of site-directed polypeptides and nucleic
acid-targeting nucleic acids as described herein. For example, a
target nucleic acid can be contacted with a complex comprising a
site-directed polypeptide, a nucleic acid-targeting nucleic acid,
and one or more effector proteins, wherein the one or more effector
proteins comprises a non-native sequence and can bind to the
modified nucleic acid-targeting nucleic acid. The non-native
sequence can confer an enzymatic activity and/or transcriptional
activity of the effector protein can modify the target nucleic
acid. For example, if the effector protein comprises a non-native
sequence corresponding to a methyltransferase, then the
methyltransferase may be able to methylate the target nucleic acid.
The modification of the target nucleic acid may occur at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or
more nucleotides away from the either the 5' or 3' end of the
target nucleic acid. The modification of the target nucleic acid
may occur at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,
60, 70, 80, 90, 100 or more nucleotides away from the either the 5'
or 3' end of the target nucleic acid. The modification can occur on
a separate nucleic acid that does not comprise the target nucleic
acid (e.g., another chromosome).
[0689] Exemplary modifications can comprise methylation,
demethylation, acetylation, deacetylation, ubiquitination,
deubiquitination, deamination, alkylation, depurination, oxidation,
pyrimidine dimer formation, transposition, recombination, chain
elongation, ligation, glycosylation. Phosphorylation,
dephosphorylation, adenylation, deadenylation, SUMOylation,
deSUMOylation, ribosylation, deribosylation, myristoylation,
remodelling, cleavage, oxidoreduction, hydrolation, and
isomerization.
[0690] Determining a Genotype and Treatment
[0691] The disclosure provides for methods for treating a disease
using the nucleic acid-targeting nucleic acid of the disclosure.
The method can be performed using any of the site-directed
polypeptides, nucleic acid-targeting nucleic acids, and complexes
of site-directed polypeptides and nucleic acid-targeting nucleic
acids as described herein. For example, using the split system
described herein, the presence of two or more target nucleic acids
close together in space (e.g., in a genetic mobility event, in
chromatin structure, or on a linear nucleic acid) can be indicative
of a genotype (e.g, of a subject). A genotype can refer to the
presence or absence of a particular sequence of nucleic acid, a
nucleotide polymorphism (i.e., either a single nucleotide
polymorphism, or a multi-nucleotide polymorphism), an allelic
variant, or any other indication of the sequence of a nucleic acid.
The genotype can indicate whether a patient suffers from a disease
and/or is predisposed to contract a disease.
[0692] Determining a genotype can include, for example, determining
if a subject comprises a mutant sequence (e.g., nucleic acid
sequence comprising a mutation). In some instances, a first nucleic
acid-targeting nucleic acid comprising the appropriate components
as described herein to comprise a first part of a split system can
be designed to target a region near a predicted mutant sequence. In
some instances, a second nucleic acid-targeting nucleic acid
comprising the appropriate components as described herein to
comprise a second part of the split system can be designed to
target a region comprising the predicted mutant sequence. If the
mutant sequence does exist, the second nucleic acid-targeting
nucleic acid can bind to it, and the two parts of the split system
can interact. The interaction can generate a signal which can be
indicative of the presence of a mutant sequence.
[0693] A genotype can be identified by a biomarker. A biomarker can
be indicative of any physiological process. A biomarker can serve
as a indicator of efficacy of a treatment (e.g., drug treatment). A
biomarker can be a nucleic acid, a polypeptide, an analyte, a
solute, a small molecule, an ion, an atom, a modification to a
nucleic acid and/or polypeptide, and/or a degradation product. A
biomarker can refer to relative expression levels of a nucleic acid
and/or a polypeptide.
[0694] A subject-specific treatment plan may be identified from
determining the genotype of the subject using the methods of the
disclosure. For example, if a subject comprises a certain genotype
known to be unresponsive to a particular therapy, then the subject
can be treated with a different therapy. Determining of genotype
can allow a subject to be selected or deselected for a clinical
trial.
[0695] Determination of the genotype can be communicated from a
caregiver to a subject (e.g., from a doctor to a patient, or from a
person performing the genotype analysis to a customer). The
communication can occur in person (e.g., in a doctor's office),
over the phone, in writing, or electronically. The communication
can further inform the subject of a subject-specific treatment
regimen determined from the genotype of the subject.
[0696] The method can be performed more than once (e.g.,
iteratively) in a subject. For example, the genotype of a subject
can be determined, a course of treatment can be prescribed for the
subject, the genotype of the subject can be determined again. The
two genotypes can be compared to determine the effectiveness of the
course of treatment. The treatment plan can be altered based on the
comparison of the genotypes.
[0697] Location of Sequences in Three-Dimensional Space
[0698] In some instances, the disclosure provide for a method for
determining the location of sequences in three-dimensional space in
a cell. The method can be performed using any of the site-directed
polypeptides, nucleic acid-targeting nucleic acids, and complexes
of site-directed polypeptides and nucleic acid-targeting nucleic
acids as described herein. Determining the three-dimensional
organization of chromatin and nucleic acid can be important for
understanding gene regulation such as transcriptional activation
and/or repression of genes. In some instances, the method comprises
contacting a target nucleic acid with two complexes, wherein each
complex binds to its cognate target nucleic acid. The complexes can
comprise a site-directed polypeptide and a nucleic acid-target
nucleic acid of the disclosure. Two or more effector proteins can
be introduced, wherein the each of the two or more effector
proteins binds to a complex. The effector proteins can be similar
to the split system described above, wherein each effector protein
can comprise an inactive fragment of a whole polypeptide. When the
effector proteins are far apart in space, the effector proteins are
inactive (e.g., no signal is detected). When the effector proteins
are close enough in space to interact, they can form a detectable
active polypeptide.
[0699] The effector proteins can be part of a split affinity tag
system. In a split affinity tag system, the two inactive
polypeptide fragments of the system can correspond to two inactive
fragments of an affinity tag. When the two fragments bind together,
the whole affinity tag is restored, such that the affinity tag can
be detectable by a binding agent. A binding agent can refer to a
molecule that can bind and purify the affinity tag. Examples of
binding agents can include antibodies, antibody-conjugated beads,
and small-molecule conjugated beads.
[0700] Introduction of the complexes and polypeptides of the
disclosure can occur by viral or bacteriophage infection,
transfection, conjugation, protoplast fusion, lipofection,
electroporation, calcium phosphate precipitation, polyethyleneimine
(PEI)-mediated transfection, DEAE-dextran mediated transfection,
liposome-mediated transfection, particle gun technology, calcium
phosphate precipitation, direct micro injection,
nanoparticlemediated nucleic acid delivery, and the like.
[0701] The cells can be cultured with the complexes for at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 or more days. The cells can be cultured
with the complexes for at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more days. After an appropriate period of time (e.g, a period of
time to allow the complexes to bind to their target nucleic acid),
the cells can be lysed.
[0702] The cells can be crosslinked before they are lysed. Fixed or
cross-linking cells can stabilize protein-DNA complexes in the
cell. Suitable fixatives and cross-linkers can include,
formaldehyde, glutaraldehyde, ethanol-based fixatives,
methanol-based fixatives, acetone, acetic acid, osmium tetraoxide,
potassium dichromate, chromic acid, potassium permanganate,
mercurials, picrates, formalin, paraformaldehyde, amine-reactive
NHS-ester crosslinkers such as bis[sulfosuccinimidyl] suberate
(BS3), 3,3'-dithiobis[sulfosuccinimidylpropionate] (DTSSP),
ethylene glycol bis[sulfosuccinimidylsuccinate (sulfo-EGS),
disuccinimidyl glutarate (DSG), dithiobis[succinimidyl propionate]
(DSP), disuccinimidyl suberate (DSS), ethylene glycol
bis[succinimidylsuccinate] (EGS), NHS-ester/diazirine crosslinkers
such as NHS-diazirine, NHS-LC-diazirine, NHS-SS-diazirine,
sulfo-NHS-diazirine, sulfo-NHS-LC-diazirine, and
sulfo-NHS-SS-diazirine.
[0703] Lysed cells can be contacted with a binding agent (e.g, an
antibody) that is directed to bind to the affinity tag. The
contacting can occur in a test-tube. The contacting can occur in a
chromatographic setting (e.g., an affinity chromatography column).
Contacting with the binding agent can occur for at least 1 minute,
5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30
minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7
hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30
hours, 35 hours, 40 hours, 45 or more hours. Contacting with the
binding agent can occur for at most 1 minute, 5 minutes, 10
minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 1 hour, 2
hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9
hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours,
40 hours, 45 or more hours. In some instances, contacting with a
binding agent occurs prior to cell lysis.
[0704] The complexes can be purified with the binding agent. The
purified complexes can be subjected to nucleic acid purification
techniques to separate the target nucleic acid from the complexes.
Nucleic acid purification techniques can include spin column
separation, precipitation, and electrophoresis.
[0705] The nucleic acid (e.g., nucleic acid comprising the target
nucleic acid) can be subjected to sequencing methodologies. The
nucleic acid can be prepared for sequencing analysis by ligation of
one or more adaptors. Sequenced nucleic acids can be analyzed to
identify polymorphisms, diagnose a disease, determine a course of
treatment for a disease, and/or determine the three-dimensional
structure of the genome.
[0706] Tagged Nucleic Acid-Targeting Nucleic Acids with Linkers
[0707] The disclosure provides for compositions and methods for
generating and using tagged nucleic acid-targeting nucleic acids.
FIG. 13A depicts an exemplary untagged nucleic acid-targeting
nucleic acid. An untagged nucleic acid-targeting nucleic acid can
comprise a protospacer (PS), a minimum CRISPR repeat (MCR), a
single guide connector (SGC), a minimum tracrRNA sequence (MtS), a
3' tracrRNA sequence (3TS), and tracrRNA extension (TE). A tagged
nucleic acid-targeting nucleic acid comprising a linker can refer
to any of the nucleic acid-target nucleic acids described herein,
and comprising a linker at either the 5' end, the 3' end, or both
the 5' and 3' end of the nucleic acid-targeting nucleic acid.
[0708] A nucleic acid-targeting nucleic acid can comprise a
non-native sequence as depicted in FIG. 13B. The non-native
sequence can be referred to as a tag. The tag can be fused to
either the 5' end, the 3' end or both the 5' and 3' end of the
nucleic acid-targeting nucleic acid. The non-native sequence can
comprise a binding sequence for an RNA-binding protein. The
RNA-binding protein can be Csy4. The non-native sequence can be
fused to the protospacer sequence of the nucleic acid-targeting
nucleic acid.
[0709] The non-native fusion can be separated from the nucleic
acid-targeting nucleic acid by a linker. FIG. 14 depicts an
exemplary linker (e.g., Tag linker), separating the non-native
sequence (e.g, Csy4 hairpin) from the protospacer of the nucleic
acid-targeting nucleic acid. The linker sequence can be
complementary to the target nucleic acid. The linker sequence can
comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mismatches
with the target nucleic acid. The linker sequence can comprise at
most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mismatches with the
target nucleic acid. In some instances, the fewer mismatches
between the linker and target nucleic acid, the better cleavage
efficiency of the Cas9:nucleic acid-targeting nucleic acid
complex.
[0710] The linker sequence can be at least 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45,
50, 55, 60 or more nucleotides in length. The linker sequence can
be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more nucleotides
in length.
[0711] Multiplexed Genetic Targeting Agents
[0712] General Overview
[0713] This disclosure describes methods, compositions, systems,
and/or kits for multiplexed genome engineering. In some embodiments
of the disclosure a site-directed polypeptide can comprise a
nucleic acid-targeting nucleic acid, thereby forming a complex. The
complex can be contacted with a target nucleic acid. The target
nucleic acid can be cleaved, and/or modified by the complex. The
methods, compositions, systems, and/or kits of the disclosure can
be useful in modifying multiple target nucleic acids quickly,
efficiently, and/or simultaneously. The method can be performed
using any of the site-directed polypeptides, nucleic acid-targeting
nucleic acids, and complexes of site-directed polypeptides and
nucleic acid-targeting nucleic acids as described herein.
[0714] FIG. 15 depicts an exemplary embodiment of the methods of
the disclosure. A nucleic acid (e.g., a nucleic acid-targeting
nucleic acid) 1505 can be fused to a non-native sequence (e.g., a
moiety, an endoribonuclease binding sequence, ribozyme) 1510,
thereby forming a nucleic acid module 1512. The nucleic acid module
1512 (e.g., comprising the nucleic acid fused to a non-native
sequence) can be conjugated in tandem, thereby forming a
multiplexed genetic targeting agent (e.g., polymodule, e.g., array)
1511. The multiplexed genetic targeting agent 1511 can comprise
RNA. The multiplexed genetic targeting agent can be contacted 1515
with one or more endoribonucleases 1520. The endoribonucleases can
bind to the non-native sequence 1510. The bound endoribonuclease
can cleave a nucleic acid module 1512 of the multiplexed genetic
targeting agent 1511 at a prescribed location defined by the
non-native sequence 1510. The cleavage 1525 can process (e.g.,
liberate) individual nucleic acid modules 1512. In some
embodiments, the processed nucleic acid modules 1512 can comprise
all, some, or none, of the non-native sequence 1510. The processed
nucleic acid modules 1512 can be bound by a site-directed
polypeptide 1530, thereby forming a complex 1531. The complex 1531
can be targeted 1535 to a target nucleic acid 1540. The target
nucleic acid 1540 can by cleaved and/or modified by the complex
1531.
[0715] Multiplexed Genetic Targeting Agents
[0716] A multiplexed genetic targeting agent can be used in
modifying multiple target nucleic acids at the same time, and/or in
stoichiometric amounts. A multiplexed genetic targeting agent can
be any nucleic acid-targeting nucleic acid as described herein in
tandem. A multiplexed genetic targeting agent can refer to a
continuous nucleic acid molecule comprising one or more nucleic
acid modules. A nucleic acid module can comprise a nucleic acid and
a non-native sequence (e.g., a moiety, endoribonuclease binding
sequence, ribozyme). The nucleic acid can be non-coding RNA such as
microRNA (miRNA), short interfering RNA (siRNA), long non-coding
RNA (lncRNA, or lincRNA), endogenous siRNA (endo-siRNA),
piwi-interacting RNA (piRNA), trans-acting short interfering RNA
(tasiRNA), repeat-associated small interfering RNA (rasiRNA), small
nucleolar RNA (snoRNA), small nuclear RNA (snRNA), transfer RNA
(tRNA), and ribosomal RNA (rRNA), or any combination thereof. The
nucleic acid can be a coding RNA (e.g., a mRNA). The nucleic acid
can be any type of RNA. In some embodiments, the nucleic acid can
be a nucleic acid-targeting nucleic acid.
[0717] The non-native sequence can be located at the 3' end of the
nucleic acid module. The non-native sequence can be located at the
5' end of the nucleic acid module. The non-native sequence can be
located at both the 3' end and the 5' end of the nucleic acid
module. The non-native sequence can comprise a sequence that can
bind to a endoribonuclease (e.g., endoribonuclease binding
sequence). The non-native sequence can be a sequence that is
sequence-specifically recognized by an endoribonuclease (e.g.,
RNase T1 cleaves unpaired G bases, RNase T2 cleaves 3'end of As,
RNase U2 cleaves 3'end of unpaired A bases). The non-native
sequence can be a sequence that is structurally recognized by an
endoribonuclease (e.g., hairpin structure, single-stranded-double
stranded junctions, e.g., Drosha recognizes a
single-stranded-double stranded junction within a hairpin). The
non-native sequence can comprise a sequence that can bind to a
CRISPR system endoribonuclease (e.g., Csy4, Cas5, and/or Cas6
protein). The non-native sequence can comprise a nucleotide
sequence having at least or at most about about 40%, 50%, 60%, 70%,
80%, 85%, 90%, 95%, 98%, 99%, or 100%, nucleotide sequence identity
and/or sequence similarity to one of the following sequences:
TABLE-US-00004 5'-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3';
5'-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3';
5'-GUUGCAAACCUCGUUAGCCUCGUAGAGGAUUGAAAC-3';
5'-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3';
5'-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3';
5'-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3';
5'-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3';
5'-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3'.
5'-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3';
5'-GUUGCAAACCUCGUUAGCCUCGUAGAGGAUUGAAAC-3';
5'-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3';
5'-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3';
5'-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3';
5'-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3';
5'-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3',
5'-GUCGCCCCCCACGCGGGGGCGUGGAUUGAAAC-3';
5'-CCAGCCGCCUUCGGGCGGCUGUGUGUUGAAAC-3';
5'-GUCGCACUCUACAUGAGUGCGUGGAUUGAAAU-3';
5'-UGUCGCACCUUAUAUAGGUGCGUGGAUUGAAAU-3'; and
5'-GUCGCGCCCCGCAUGGGGCGCGUGGAUUGAAA-3'.
[0718] In some embodiments, wherein the non-native sequence
comprises an endoribonuclease binding sequence, the nucleic acid
modules can be bound by the same endoribonuclease. The nucleic acid
modules may not comprise the same endoribonuclease binding
sequence. The nucleic acid modules may comprise different
endoribonuclease binding sequences. The different endoribonuclease
binding sequences can be bound by the same endoribonuclease. In
some embodiments, the nucleic acid modules can be bound by
different endoribonucleases.
[0719] The moiety can comprise a ribozyme. The ribozyme can cleave
itself, thereby liberating each module of the multiplexed genetic
targeting agent. Suitable ribozymes can include peptidyl
transferase 23S rRNA, RnaseP, Group I introns, Group II introns,
GIR1 branching ribozyme, Leadzyme, hairpin ribozymes, hammerhead
ribozymes, HDV ribozymes, CPEB3 ribozymes, VS ribozymes, glmS
ribozyme, CoTC ribozyme, an synthetic ribozymes.
[0720] The nucleic acids of the nucleic acid modules of the
multiplexed genetic targeting agent can be identical. The nucleic
acid modules can differ by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,
25, 30, 35, 40, 45, 50 or more nucleotides. For example, different
nucleic acid modules can differ in the spacer region of the nucleic
acid module, thereby targeting the nucleic acid module to a
different target nucleic acid. In some instances, different nucleic
acid modules can differ in the spacer region of the nucleic acid
module, yet still target the same target nucleic acid. The nucleic
acid modules can target the same target nucleic acid. The nucleic
acid modules can target one or more target nucleic acids.
[0721] A nucleic acid module can comprise a regulatory sequence
that can allow for appropriate translation or amplification of the
nucleic acid module. For example, an nucleic acid module can
comprise a promoter, a TATA box, an enhancer element, a
transcription termination element, a ribosome-binding site, a 3'
un-translated region, a 5' un-translated region, a 5' cap sequence,
a 3' poly adenylation sequence, an RNA stability element, and the
like.
[0722] Methods
[0723] The disclosure provides for methods for the modification of
multiple target nucleic acids, simulataneously, through the use of
a multiplexed genetic targeting agent. A site-directed polypeptide,
an endoribonuclease, and a multiplexed genetic targeting agent can
be introduced into a host cell. A vector of the disclosure (e.g.,
comprising a multiplexed genetic targeting agent, an
endoribonuclease and/or a site-directed polypeptide) can be
introduced into a host cell. In some instances, more than one
endoribonuclease and/or multiplexed genetic targeting agent can be
introduced into cells. If a multiplexed genetic targeting agent
comprises different types of moieties, where the moieties are
different endoribonuclease binding sequences, then one or more
endoribonucleases corresponding to the types of binding sequences
in the multiplexed genetic targeting agent may be introduced into
cells.
[0724] Introduction can occur by any means to introduce a nucleic
acid into a cell such viral or bacteriophage infection,
transfection, conjugation, protoplast fusion, lipofection,
electroporation, calcium phosphate precipitation, polyethyleneimine
(PEI)-mediated transfection, DEAE-dextran mediated transfection,
liposome-mediated transfection, particle gun technology, calcium
phosphate precipitation, direct micro injection,
nanoparticle-mediated nucleic acid delivery, and the like. The
vector can be transiently expressed in the host cell. The vector
can be stably expressed in the host cell (e.g., by stably
integrating into the genome of the host cell).
[0725] In instances where a moiety comprises an endoribonuclease
binding sequence, an endoribonuclease can be expressed and can bind
to the endoribonuclease binding site on the multiplexed genetic
targeting agent. The endoribonuclease can cleave the multiplexed
genetic targeting agent into individual nucleic acid modules.
[0726] In instances where a moiety comprises a ribozyme, an
endoribonuclease may not be required to be expressed in a host
cell. The ribozyme can cleave itself, thereby resulting in cleavage
of the multiplexed genetic targeting agent into individual nucleic
acid modules.
[0727] Individual (e.g., cleaved) nucleic acid modules can comprise
all, some, or none, of the moiety (e.g., endoribonuclease binding
sequence). For example, the liberated (e.g., processed) nucleic
acid module can be subjected to exonuclease trimming and/or
degradation that may result in removal of the 5' and/or 3' end of
the nucleic acid module. In such instances, exonuclease trimming
and/or degradation may result in the removal of all, part, or none
of the moiety (e.g., endoribonuclease binding sequence).
[0728] The liberated (e.g., processed) nucleic acid module can bind
to a site-directed polypeptide thereby forming a complex. The
complex can be guided to a target nucleic acid by the nucleic
acid-targeting nucleic acid which can hybridize with the target
nucleic acid in a sequence-specific manner. Once hybridized, the
site-directed polypeptide of the complex can modify the target
nucleic acid (e.g., cleave the target nucleic acid). In some
instances, the modification comprises introduction of a
double-stranded break in the target nucleic acid. In some
instances, the modification comprises introduction of a
single-stranded break in the target nucleic acid.
[0729] In some embodiments, one or more donor polynucleotides
and/or vectors encoding the same can introduced into the cell. One
or more donor polynucleotides can be incorporated into the modified
(e.g., cleaved) target nucleic acids, thereby resulting in an
insertion. The same donor polynucleotide can be incorporated into
multiple cleavage sites of target nucleic acids. One or more donor
polynucleotides can be incorporated into one or more cleavage sites
of target nucleic acids. This can be referred to as multiplex
genome engineering. In some instances, no donor polynucleotide
and/or vector encoding the same may be introduced into the cells.
In these instances, the modified target nucleic acid can comprise a
deletion.
[0730] Stoichiometric Delivery of Nucleic Acids
[0731] General Overview
[0732] The disclosure provides for compositions, methods, and kits
for stoichiometric delivery of a nucleic acid to a cell and/or
subcellular localization. The stoichiometric delivery may be
mediated by a complex. FIG. 16 depicts an exemplary complex for
stoichiometric delivery of a plurality of nucleic acids to a cell
and/or subcellular location. The complex can comprise a plurality
of nucleic acids 1605. Each nucleic acid can comprise a nucleic
acid-binding protein binding site 1610. The nucleic acid-binding
protein binding sites 1610 can all be the same sequences, different
sequences, or some can be same sequences and some can be different
sequences. In some embodiments, the nucleic acid-binding protein
binding sites can bind a Cas6, Cas5, or Csy4 family member. The
complex can comprise a tandem fusion polypeptide 1630. The tandem
fusion polypeptide can comprise nucleic acid-binding proteins 1625
fused together in tandem. The nucleic acid-binding proteins can be
separated by a linker 1620. The nucleic acid-binding proteins 1625
can be the same protein, can be different proteins, or some can be
the same proteins and some can be different proteins. The nucleic
acid-binding proteins 1625 can be Csy4 proteins. The nucleic
acid-binding proteins 1625 can bind the nucleic acid-binding
protein binding site 1610 on the nucleic acid 1605. The tandem
fusion polypeptide 1630 can comprise a non-native sequence 1615. In
some instances, the non-native sequence is a subcellular (e.g.,
nuclear) localization sequence. In some embodiments, the nucleic
acid 1605 can encode a non-native sequence (e.g. a subcellular,
(e.g., nuclear) localization sequence). The complex can be
introduced 1635 into cells, wherein one or more of the nucleic
acids 105 can be translated into polypeptides 1640. A translated
polypeptide 1640 can bind and cleave the nucleic acid-binding
protein binding site 1610 on the nucleic acid 1605. The cleavage
1645 can liberate the nucleic acid 1650 which can be a nucleic
acid-targeting nucleic acid. The liberated nucleic acid 1650 can
bind to a translated polypeptide 1645 (e.g., a site-directed
polypeptide), thereby forming a unit. The translated polypeptide
1645 can comprise a nuclear localization signal. The unit can
translocate to the nucleus, wherein the unit can be guided to a
target nucleic acid hybridizable with the spacer of the liberated
nucleic acid 1650. The unit can be hybridized to a target nucleic
acid. The site-directed polypeptide of the unit can cleave the
target nucleic acid. The cleavage of the target nucleic acid can be
referred to as genome engineering. The method can be performed
using any of the site-directed polypeptides, nucleic acid-targeting
nucleic acids, and complexes of site-directed polypeptides and
nucleic acid-targeting nucleic acids as described herein.
[0733] In some embodiments, multiple nucleic acid-targeting nucleic
acids can be stoichiometrically delivered to a cell and/or
subcellular location. FIG. 17 depicts an exemplary complex for
stoichiometric delivery of a plurality of nucleic acids. The
complex can comprise a plurality of nucleic acids 1705. Each
nucleic acid can comprise a plurality of nucleic acid-binding
protein binding sites 1710/1711. The nucleic acid-binding protein
binding sites 1710/1711 can all be the same sequences, different
sequences, or some can be same sequences and some can be different
sequences. In some embodiments, the nucleic acid-binding protein
binding sites 1710/1711 can bind a Cas6, Cas5, or Csy4 family
member. The complex can comprise a tandem fusion polypeptide 1730.
The tandem fusion polypeptide can comprise nucleic acid-binding
proteins 1725 fused together in tandem. The nucleic acid-binding
proteins can be separated by a linker 1720. The nucleic
acid-binding proteins 1725 can be the same protein, can be
different proteins, or some can be the same proteins and some can
be different proteins. The RNA-binding proteins 1725 can be a
combination of Csy4, Cas5, and Cas6 polypeptides. The nucleic
acid-binding proteins 1725 can bind the nucleic acid-binding
protein binding site 1710 on the nucleic acid 1705. The tandem
fusion polypeptide 1730 can comprise a non-native sequence 1715. In
some instances, the non-native sequence is a subcellular (e.g.,
nuclear) localization sequence. In some embodiments, the nucleic
acid 1705 can encode for a non-native sequence (e.g. a subcellular,
(e.g., nuclear) localization sequence). The complex can be
introduced into cells 1735, wherein one or more of the nucleic
acids can be translated into polypeptides 1740/1750. A translated
polypeptide 1740 can bind and cleave the nucleic acid-binding
protein binding site 1711 on the nucleic acid 1705. The cleavage
1745 can liberate the nucleic acid 1755, which can be a nucleic
acid-targeting nucleic acid and/or a donor polynucleotide. The
liberated nucleic acid 1755 can bind to a translated polypeptide
1750 (e.g., a site-directed polypeptide), thereby forming a unit.
In some instances, the translated polypeptide 1750 comprises a
nuclear localization signal. The unit can translocate to the
nucleus, wherein the unit can be guided to a target nucleic acid
hybridizable with the spacer of the liberated RNA 1755. The unit
can be hybridized to a target nucleic acid. The site-directed
polypeptide of the unit can cleave the target nucleic acid.
[0734] Methods
[0735] The disclosure provides for methods for stoichiometric
delivery of nucleic acids to a cell (e.g., stoichiometrically
deliverable nucleic acids). The method can comprise binding a
tandem fusion polypeptide to a plurality of stoichiometrically
deliverable nucleic acids, thereby forming a complex. The complex
can comprise stoichiometric amounts of the nucleic acids (e.g., the
complex can comprise the plurality of nucleic acids in a prescribed
ratio and/or amount). 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic
acids can be stoichiometrically delivered. In some instances, 3
stoichiometrically deliverable nucleic acids can be
stoichiometrically delivered. In some instances, 4
stoichiometrically deliverable nucleic acids can be
stoichiometrically delivered.
[0736] The stoichiometrically deliverable nucleic acid can encode
for a polypeptide or a non-coding RNA. The polypeptide may be a
CRISPR system polypeptide (e.g., a site-directed polypeptide, an
endoribonuclease). The stoichiometrically deliverable nucleic acid
can encode for more than one polypeptide. The stoichiometrically
deliverable nucleic acid can comprise a plurality of
stoichiometrically deliverable nucleic acids (e.g., in an array).
The stoichiometrically deliverable nucleic acid can encode for a
non-coding RNA. Examples of non-coding RNAs can include microRNA
(miRNA), short interfering RNA (siRNA), long non-coding RNA
(lncRNA, or lincRNA), endogenous siRNA (endo-siRNA),
piwi-interacting RNA (piRNA), trans-acting short interfering RNA
(tasiRNA), repeat-associated small interfering RNA (rasiRNA), small
nucleolar RNA (snoRNA), small nuclear RNA (snRNA), transfer RNA
(tRNA), and ribosomal RNA (rRNA). The stoichiometrically
deliverable nucleic acid can be RNA.
[0737] The stoichiometrically deliverable nucleic acid can encode
for a non-native sequence. In some instances, the
stoichiometrically deliverable nucleic acid encodes for a
non-native sequence such that when a polypeptide is translated from
a stoichiometrically deliverable nucleic acid encoding a
polypeptide, the polypeptide is fused to the non-native sequence
(e.g., thereby generating a fusion protein). The non-native
sequence can be a peptide affinity tag. The non-native sequence
(e.g., peptide affinity tag) can be located at the N-terminus of
the polypeptide, the C-terminus of the polypeptide, or any location
within the polypeptide (e.g., a surface accessible loop). In some
embodiments, the non-native sequence is a nuclear localization
signal (NLS). A NLS can be monopartite or bipartite sequence. The
NLS can be recognized by nuclear import machinery (e.g.,
importins). A NLS can be a small peptide (e.g., PKKKRKV of the SV40
large t-antigen). A NLS can be a polypeptide domain (e.g., acidic
M9 domain of hnRNP A1).
[0738] The non-native sequence can be a nucleic acid affinity tag
(e.g., nucleic acid localization signal). For example, a
stoichiometrically deliverable nucleic acid encoding a DNA (e.g., a
donor polynucleotide) can comprise a nucleic acid localization
signal which can localize the DNA to the nucleus. Such nucleic acid
localization signals can include, for example, peptide-nucleic acid
(PNA) sequences.
[0739] The stoichiometrically deliverable nucleic acids can
comprise regulatory sequences that can allow for appropriate
translation or amplification of the nucleic acid. For example, an
nucleic acid can comprise a promoter, a TATA box, an enhancer
element, a transcription termination element, a DNA stability
element, a ribosome-binding site, a 3' un-translated region, a 5'
un-translated region, a 5' cap sequence, a 3' poly adenylation
sequence, an RNA stability element, and the like.
[0740] The nucleic acid can comprise a nucleic acid-binding protein
binding site. The nucleic acid-binding protein binding site can be
bound by an nucleic acid-binding protein. The nucleic acid-binding
protein binding site can be bound by a CRISPR polypeptide (e.g., a
site-directed polypeptide, an endoribonuclease). The nucleic
acid-binding protein binding site can be bound by a Cas5 or Cas6
family polypeptide. The nucleic acid-binding protein binding site
can be bound by a Csy4, Cas5, or Cas6 polypeptide. Some examples of
nucleic acid-binding protein binding sites can include, for
example, sequences that can be bound by RNA-binding proteins such
as the MS2 binding sequence, the U1A binding sequence, the boxB
sequence, the eIF4A sequence, hairpins, sequences that can be bound
by RNA recognition motif (RRM) domains (e.g., U1A), sequences that
can be bound by double stranded RNA binding domains (dsRBD) (e.g.,
Staufen), sequences that can be bound PAZ domains (e.g., PAZ,
Argonaute), sequences that can be bound by PIWI domains (e.g.,
PIWI, MILI, MIWI, Argonaute), and the like. Some examples of
nucleic acid-binding protein binding sites can include, for
example, sequences that can be bound by DNA-binding proteins such
as zinc fingers, a helix-turn-helix domain, a zinc finger domain, a
leucine zipper (bZIP) domain, a winged helix domain, a winged helix
turn helix domain, a helix-loop-helix domain, a HMG-box domain, a
Wor3 domain, an immunoglobulin domain, a B3 domain, a TALE domain,
and the like.
[0741] The nucleic acid can comprise one or more nucleic
acid-binding protein binding sites. The nucleic acid can comprise
1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acid-binding protein
binding sites. The one or more nucleic acid-binding protein binding
sites may be the same. The one or more nucleic acid-binding protein
binding sites may be different. For example, the nucleic acid can
comprise a Csy4 binding site and a MS2 binding site. The one or
more nucleic acid-binding protein binding sites can be separated by
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,
110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350,
400, 450, 500 or more nucleotides. In some embodiments, the 3'-most
nucleic acid-binding protein binding site can be bound by a tandem
fusion polypeptide of the disclosure.
[0742] Tandem Fusion Polypeptide
[0743] In some embodiments, the method of the disclosure provides
for binding a plurality of nucleic acids to a tandem fusion
polypeptide. A tandem fusion polypeptide can comprise a plurality
of nucleic acid binding proteins fused together in one polypeptide
chain. A tandem fusion polypeptide can comprise 1, 2, 3, 4, 5, 6,
7, 8, 9, 10 or more nucleic acid-binding proteins. Nucleic
acid-binding proteins of the tandem fusion polypeptide can bind to
the nucleic acid-binding protein binding sites of the nucleic acids
of the disclosure. Examples of nucleic acid-binding proteins can
include MS2, U1A, boxB sequence binding proteins (e.g., zinc
fingers), eIF4A, Staufen, PAZ, Argonaute, PIWI, MILI, MIWI, zinc
fingers, a helix-turn-helix domain, a zinc finger domain, a leucine
zipper (bZIP) domain, a winged helix domain, a winged helix turn
helix domain, a helix-loop-helix domain, a HMG-box domain, a Wor3
domain, an immunoglobulin domain, a B3 domain, a TALE domain, and
the like. In some embodiments, the nucleic acid-binding protein is
an RNA-binding protein. The RNA-binding protein can be a member of
a CRISPR system. In some embodiments, the RNA-binding protein can
be a member of the Cas5 or Cas6 family of proteins. In some
embodiments, the RNA-binding protein can be Csy4, Cas5, Cas6, or
any combination thereof. In some embodiments, the nucleic
acid-binding protein is a DNA-binding protein (e.g., a zinc
finger).
[0744] In some instances, the nucleic acid-binding proteins are
separated by a linker. A linker can comprise about 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino
acids.
[0745] A tandem fusion polypeptide can comprise a non-native
sequence (e.g., peptide affinity tag). The non-native sequence can
comprise a nuclear localization signal (NLS) that can direct the
tandem fusion polypeptide to a subcellular location (e.g.,
nucleus).
[0746] Each nucleic acid-binding protein of the tandem fusion
polypeptide can comprise its own non-native sequence. The
non-native sequence of each nucleic acid-binding protein can be the
same. The non-native sequence of each nucleic acid-binding protein
can be different. The non-native sequence of some of the nucleic
acid-binding proteins of the tandem fusion polypeptide can be the
same and the non-native sequence of some of the nucleic
acid-binding proteins of the tandem fusion polypeptide can be
different.
[0747] In some instances, the methods of the disclosure can provide
for forming a complex comprising a tandem fusion polypeptide and a
plurality of nucleic acids of the disclosure. Formation of the
complex can comprise the nucleic acid-binding proteins of the
tandem fusion polypeptide binding to their cognate nucleic
acid-binding protein binding sequence in the nucleic acids of the
disclosure. For example, a stoichiometrically deliverable nucleic
acid comprising a Csy4 binding site, can bind to the Csy4 protein
subunit in the tandem fusion protein. The complex can be formed
outside of cells (e.g., in vitro). The complex can be formed in
cells (e.g., in vivo). When a complex is formed in vitro it can be
introduced into a cell by, for example, transfection,
transformation, viral transduction, electroporation, injection, and
the like.
[0748] The methods of the disclosure provide for therapeutic
delivery of multiple nucleic acids both in vivo, in vitro, and ex
vivo. The delivered nucleic acids can be used to treat a disease.
For example, the delivered nucleic acids can be used in gene
therapy and/or can integrate into the genome of the cell, thereby
providing a therapeutic outcome. A therapeutic outcome can refer to
increase or decrease in the levels of a protein, nucleic acid, or
any biological molecule related to a disease such as a degradation
product, small molecule, and/or ion. For example, a therapeutic
outcome can comprise increasing the levels of an anti-inflammatory
gene, or decreasing the levels of a protein in a pathway related to
a disease. A therapeutic outcome can refer to a physiological
effect. Physiological effects can include, morphological changes,
metabolic changes, and/or structural changes in a cell. A
therapeutic outcome can refer to changes in the modifications of a
protein and/or nucleic acid, such as glycosylation, acetylation,
methylation, demethylation, depurination, ubiquitinylation, and the
like.
[0749] A therapeutic outcome can be measured by changes in the
genetic makeup of the cell, the levels of biomolecules of interest
in the cell, and/or the physiological changes in the cell.
Measurements can be made using molecular biology techniques such as
spectroscopy, spectrometry, sequencing, ELISA, microscopy, and/or
x-ray crystallograhpy. Measurements can be made using animal
models, such as mouse, rats, dogs, and primates. For example,
genetically modified cells of the disclosure can be introduced into
mice and assessed for biological and physiological changes such as,
for example, the ability to metastasize and/or differentiate.
[0750] Spacers for Blood Disorders
[0751] This disclosure provides for compositions, methods and kits,
for genetic engineering of hematopoietic stem cells (HSCs).
[0752] Compositions
[0753] A HSC can comprise a site-directed polypeptide (e.g.,
Cas9).
[0754] A HSC can comprise a nucleic acid-targeting nucleic acid.
The nucleic acid-targeting nucleic acid of the disclosure can
target a gene involved in a genetic disorder. Table 1 lists
exemplary genes that are involved in disorders. The genes listed in
Table 1 can be genes that can be targeted by a nucleic
acid-targeting nucleic acid. The nucleic acid-targeting nucleic
acid of the disclosure can comprise a spacer that can target a gene
listed in Table 1.
[0755] Table 2 depicts exemplary spacers of the nucleic
acid-targeting nucleic acids of the disclosure. Each spacer of
Table 2 can be a spacer that can be inserted into a nucleic
acid-targeting nucleic acid. Exemplary spacers are companied by the
name of the disorder and the gene involved in the disorder that is
targeted by the spacers.
TABLE-US-00005 TABLE 2 Spacers of blood disorders. Gene Spacer
Disease Arylsulfatase A AGGGTTTATTTTCTTACGCT Metachromatic
Leukodystrophy Wiskott-Aldrich CCGAGGTCCCTAGTCCGGAA Wiskott-Aldrich
Syndrome Syndrome protein ATP-binding CGGAGGTGGGCGGAGCCTCC
Adrenoleukodystrophy cassette D1 C-C chemokine AGCTTTCTCGTCTGGGTATT
Human Immunodeficiency Virus receptor type 5 Hemoglobin beta
GAAAATAAATGTTTTTTATT Beta-thalassemia subunit Interleukin-2
ACAGAAACTTTATTTCTCAT X-linked Severe Combined ID recetor subunit
gamma Cystinosin CTTTGGGAGGCCGAGGCGGG MLSD cystinosis Ribosomal
protein TTTTAGAAACAGTATGAGAT Diamon-Blackfan anemia S19 Fanconi
anemia ATGCACAAAATAAACAGCAG Fanconi Anemia complimentation group B
Shwachman- GAGTTAGTTCACATCTACAG Shwachman-Bodian-Diamond
Bodian-Diamond syndrome syndrome gene
[0756] Methods
[0757] The disclosure provides for methods for introducing a
nucleic acid-targeting nucleic acid and site-directed polypeptide
into an HSC. In some embodiments, the HSC is extracted from a
patient prior to introduction. The extracted HSC can be purified
(e.g., by apheresis). The site-directed polypeptide and/or the
nucleic acid-targeting nucleic acid can be introduced into an HSC
that has been purified. The site-directed polypeptide and/or the
nucleic acid-targeting nucleic acid can be introduced into an HSC
that has not been purified. The introduction can occur in an HSC in
vitro (e.g., outside of the patient, extracted cell). In some
instances, the introduction occurs in an HSC in vivo (e.g., inside
of a patient, unextracted cell).
[0758] Introduction of the site-directed polypeptide and/or the
nucleic acid-targeting nucleic acid of the disclosure can occur by
for example, viral transduction, transfection, electroporation,
optical transfection and/or chemical transfection.
[0759] Once introduced into a HSC, the nucleic acid-targeting
nucleic acid of the disclosure and the site-directed polypeptide
can form a complex. The complex can be guided to a target nucleic
acid (e.g., the genes listed in Table 3) by the nucleic
acid-targeting nucleic acid. The nucleic acid-targeting nucleic
acid can hybridize with the target nucleic acid. The site-directed
polypeptide can modify the target nucleic acid (e.g., by cleaving
the target nucleic acid).
[0760] In some instances, the modified target nucleic acid
comprises a deletion. In some instances, the modified target
nucleic acid comprises an insertion of a donor polynucleotide. A
donor polynucleotide, a portion of a donor polynucleotide, a copy
of a donor polynucleotide or a portion of a copy of a donor
polynucleotide can be inserted into a target nucleic acid. The
method can be performed using any of the site-directed
polypeptides, nucleic acid-targeting nucleic acids, and complexes
of site-directed polypeptides and nucleic acid-targeting nucleic
acids as described herein.
TABLE-US-00006 TABLE 3 List of Genes Involved in Diseases Name of
Disease Gene(s) Metachromatic leukodystrophy (MLD) Arylsulfatase A
Wiskott-Aldrich syndrome (WAS) Wiskott-Aldrich Syndrome protein
Wiskott-Aldrich syndrome (WAS) Leukosialin Neutropenia
Wiskott-Aldrich Syndrome protein Adrenoleukodystrophy ATP-binding
cassette D1 Human Immunodeficiency Virus (HIV) C-C chemokine
receptor type 5 Beta-thalassemia hemoglobin subunit beta
Sickle-cell anemia hemoglobin subunit beta X-linked Severe Combined
Immunodeficiency (X- Interleukin-2 receptor subunit gamma SCID)
Multisystemic Lysosomal Storage Disorder Cystinosin cystinosis
Diamond-Blackfan anemia Ribosomal protein S19 Fanconi Anemia
Fanconi anemia complementation groups A, B and C
Shwachman-Bodian-Diamond syndrome SBDS gene Gaucher's disease
Glucocerebrosidase Hemophilia A Anti-hemophiliac factor OR Factor
VIII Hemophilia B Christmas factor, Serine protease, Factor IX
Adenosine deaminase deficiency (ADA-SCID) Adenosine deaminase GM1
gangliosidoses beta-galactosidease Glycogen storage disease type
II, Pompe disease, acid alpha-glucosidase acid maltase deficiency
Niemann-Pick disease, SMPD1-associated (Types Sphingomyelin
phosphodiesterase 1 OR A and B) acid sphingomyelinase Krabbe
disease, globoid cell leukodystrophy, Galactosylceramidase OR
galactosylceramide lipidosis galactercerebrosidease Multiple
Sclerosis (MS) Human leukocyte angitens DR-15, DQ-6, DRB1
[0761] Computational Methods
[0762] The disclosure provides for computational methods to
identify spacers for nucleic acid-targeting nucleic acids. The
computational method can comprise scanning the nucleic acid
sequence of a genome for a protospacer adjecent motif. Upon finding
a protospacer adjacent motif, the program can automatically count
between 10-30 nucleotides upstream of the protospacer adjacent
motif. The 10-30 nucleotides upstream of the protospacer adjacent
motif can constitute a putative spacer sequence. In other words,
the 10-30 nucleotides upstream of the protospacer adjcent motif in
the genome can correspond to a target nucleic acid, and a sequence
complementary to the target nucleic acid can be referred to as a
spacer.
[0763] The program can test every sequence iteration of the
putative spacer sequence to ascertain how effective the sequence
will be as a spacer in a nucleic acid-targeting nucleic acid. For
example, the program can take each iteration of the putative spacer
sequence and perform an in silico secondary structure prediction on
the sequence. The secondary structure prediction can comprise
appending the putative spacer sequences to a nucleic acid-targeting
nucleic acid backbone (e.g., the nucleic acid-targeting nucleic
acid without the spacer). The secondary structure prediction can
perform a secondary structure prediction analysis of the docked
putative spacer sequence in the nucleic acid-targeting nucleic acid
backbone. Secondary structure prediction analysis can comprise, for
example, predicting which nucleotides may form duplexes, hairpins,
which nucleotides are unstructured, and/or which nucleotides may be
unpaired.
[0764] The computational method can comprise implementing a folding
test on each putative spacer sequence that has undergone secondary
structure prediction analysis. The folding test can comprise in
silico folding of the nucleic acid-targeting nucleic acid
comprising the putative spacer sequence. The nucleic acid-targeting
nucleic acid and putative spacer sequence can either pass or fail
the folding test.
[0765] To pass the folding test, the secondary structure of the
backbone nucleic acid-targeting nucleic acid may need to be
conserved, less than 5, 4, 3, 2, or 1 nucleotide in the spacer
hybridize with nucleotides outside of the spacer, and other second
structure in the spacer is contained within the spacer.
[0766] Seamless Reporter Selection
[0767] General Overview
[0768] This disclosure describes methods, compositions, systems and
kits for genetic modification of cells and selection of such
genetically modified cells by seamless incorporation, detection and
excision of a reporter element. In some embodiments of the
disclosure, a donor polynucleotide can comprise a nucleic acid to
be introduced to a cell genome (here called the genetic element of
interest) as well as a nucleic acid sequence encoding a reporter
element (e.g. GFP), a site-directed polypeptide and two nucleic
acid-targeting nucleic acids. Either the site-directed polypeptide,
the nucleic acid-targeting nucleic acids, and/or all three may be
controlled by an inducible promoter. A site-directed polypeptide
and a nucleic acid-targeting nucleic acid may form a complex which
can target a site in the cell genome by hybridization of the
nucleic acid targeting nucleic acid to a target nucleic acid in the
genome. The site-directed polypeptide of the complex may cleave the
target nucleic acid. The donor polynucleotide can be inserted into
the cleaved target nucleic acid. After introduction of a double
strand break (or single strand break) at the target site in the
presence of the donor polynucleotide, the population of recipient
cells may be screened for the presence of the reporter molecule as
a proxy for the presence of the genetic element of interest. After
isolation of reporter molecule-containing cells, the reporter
element can be excised by induction of the site-directed
polypeptide and/or nucleic acid-targeting nucleic acid expression.
The nucleic acid-targeting nucleic acids can target the 5' and 3'
ends of the reporter element and can result in the excision of the
reporter element. The method can be performed using any of the
site-directed polypeptides, nucleic acid-targeting nucleic acids,
and complexes of site-directed polypeptides and nucleic
acid-targeting nucleic acids as described herein.
[0769] FIG. 18 depicts an exemplary embodiment of the methods of
the disclosure. A nucleic acid can comprise a plurality of genetic
elements 1805/1810. The genetic elements 1805 and 1810 can be, for
example, genes, non-coding nucleic acids, introns, exons, DNA
and/or RNA. The genetic elements 1805 and 1810 can be parts of the
same gene. In between the genetic elements can be a target nucleic
acid 106 suitable for genetic engineering. A site-directed
polypeptide and a nucleic acid-targeting nucleic acid of the
disclosure can form a complex which can target 1815 the target
nucleic acid 1806. The site-directed polypeptide of the complex can
cleave 1820 the target nucleic acid 1806. A donor polynucleotide
can be inserted 1825 into the cleaved target nucleic acid 1806. The
donor polynucleotide can comprise a genetic element of interest
1830. The genetic element of interest 1830 can be a gene. The donor
polynucleotide can also comprise a reporter element 1835. The donor
polynucleotide can also comprise a polynucleotide sequence encoding
a site-directed polypeptide and one or more nucleic acid-acid
targeting nucleic acid. In some instances, the polynucleotide
encoding the site-directed polypeptide and the one or more nucleic
acid-targeting nucleic acids encodes two nucleic acid-targeting
nucleic acids. The polynucleotide sequence encoding the
site-directed polypeptide and the nucleic acid targeting nucleic
acid can be operably linked to an inducible promoter. Insertion of
the donor polynucleotide into the target nucleic acid 1806 can
result in the expression of the reporter element 1835. The reporter
element 1835 can be used as a way to select cells that comprise the
donor polynucleotide.
[0770] FIG. 19 depicts an exemplary embodiment for the removal of
the reporter element 1915 from the target nucleic acid. A target
nucleic acid can comprise a plurality of genetic elements
1905/1920. The reporter element 1915 can be fused to a genetic
element of interest 1910. Expression of the reporter gene 1915 can
be induced which can result in the production of a site-directed
polypeptide and one or more nucleic acid-targeting nucleic acids.
The site-directed polypeptide can form complexes with the nucleic
acid-targeting nucleic acids. The complexes can be guided to the
reporter element 1915 by the nucleic acid-targeting nucleic acid of
the complex. One of the two nucleic acid-targeting nucleic acids
can target 1925 the 5' end of the reporter element 1915. One of the
two nucleic acid-targeting nucleic acids can target 1930 the 3' end
of the reporter element 1915. The targeted ends of the reporter
element 1915 can be cleaved by the site-directed polypeptide of the
complex, thereby excising 1935 the reporter element 1915. The
target nucleic acid can comprise the genetic element of interest
1910 portion of the donor polynucleotide. The nucleic
acid-targeting nucleic acids can be designed such that the donor
polynucleotide is excised (including the genetic element of
interest).
[0771] Methods
[0772] The present disclosure provides for methods of selecting
cells using a reporter element and excision of the reporter
element. A site-directed polypeptide, an endoribonuclease, a
nucleic acid targeting nucleic acid, a donor polynucleotide and/or
a nucleic acid-targeting nucleic acid can be introduced into a
cell. The donor polynucleotide may include one or more genetic
elements of interest. The donor polynucleotide may include one or
more reporter elements. The donor polynucleotide includes one or
more genetic elements of interest and one or more reporter
elements. More than one site-directed polypeptide,
endoribonuclease, donor polynucleotide and and/or nucleic
acid-targeting nucleic acid can be introduced into a cell. In some
instances, the cell already expresses a site-directed polypeptide,
and/or a nucleic acid-targeting nucleic acid. In some instances,
the site-directed polypeptide, and/or nucleic acid targeting
nucleic acid are encoded on a plasmid. In some instances, the
site-directed polypeptide, and/or nucleic acid targeting nucleic
acid is encoded on more than one plasmid. In some instances, more
than one site-directed polypeptide or nucleic acid encoding a
site-directed polypeptide is introduced into the cell. In some
instances, the cell is a cell lysate.
[0773] Introduction can occur by any means to introduce a nucleic
acid into a cell such as viral or bacteriophage infection,
transfection, conjugation, protoplast fusion, lipofection,
electroporation, calcium phosphate transfection, polyethyleneimine
(PEI)-mediated transfection, DEAE-dextran mediated transfection,
liposome-mediated transfection, particle gun technology, calcium
phosphate precipitation, direct micro injection,
nanoparticle-mediated nucleic acid delivery, and the like. The
vector can be transiently expressed in the host cell. The vector
can be stably expressed in the host cell (e.g., by stably
integrating into the genome of the host cell).
[0774] A nucleic acid targeting nucleic acid can bind to a nucleic
acid characterized by a particular target sequence and/or any
sequence homologous to a particular sequence. The target sequence
can be part or all of a gene, a 5' end of a gene, a 3' end of a
gene, a regulatory element (e.g. promoter, enhancer), a pseudogene,
non-coding DNA, a microsatellite, an intron, an exon, chromosomal
DNA, mitrochondrial DNA, sense DNA, antisense DNA, nucleoid DNA,
chloroplast DNA or RNA among other nucleic acid entities.
[0775] The site-directed polypeptide can cleave the target nucleic
acid bound by a nucleic acid targeting nucleic acid. The
site-directed polypeptide may not cleave the target nucleic acid.
In some instances, an endoribonuclease cleaves the target nucleic
acid. The endoribonuclease can be encoded by the vector. The
endoribonuclease can be encoded by the donor polynucleotide. The
endoribonuclease can be present in the cell. Expression of the
endoribonuclease and/or site-directed polypeptide can be induced by
a conditional promoter. A donor polynucleotide can be incorporated
in the target nucleic acid at the site where it was cleaved.
[0776] Excision
[0777] The methods disclosed herein may further comprise excision
of all, some or none of the reporter element. A first nucleic
acid-targeting nucleic acids of the reporter element can target the
5' end of the reporter element. A second nucleic acid-targeting
nucleic acids of the reporter element can target the 3' end of the
reporter element. A nucleic acid-targeting nucleic acid can target
both the 5' and 3' ends of the reporter element. A nucleic
acid-targeting nucleic acid can target two sequences in the
reporter element and/or donor polynucleotide. The two target
sequences can be at least about 70, 75, 80, 85, 90, 91, 92, 93, 94,
95, 96, 97, 98, 99 or 100% identical. The two target sequences can
be at most about 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99 or 100% identical. When the nucleic acid-targeting nucleic
acids of the reporter element are expressed, they may form a
complex with a site-directed polypeptide and target the 5' and 3'
ends of the reporter element by hybridizing to a complementary
region on the 5' and 3' ends of the reporter element. Hybridization
of the complex with the reporter element can result in cleavage of
all, some or none of the reporter element. The cleaved nucleic acid
can be rejoined by, for example, non-homologous end joining. The
rejoined nucleic acid may not introduce a deletion or insertion.
The rejoined nucleic acid may introduce a deletion or insertion.
The cleaved nucleic acid can be rejoined by, for example,
homologous recombination. Homologous recombination can be used to
rejoin a cleaved nucleic acid when the target nucleic acid sites
are substantially identical.
[0778] Screening
[0779] The methods disclosed herein may further comprise excising a
reporter element from a selected cell, thereby forming a second
cell; and screening the second cell. Screening may comprise
screening for the absence of all or some of the reporter element.
Screening can include fluorescene activate cell-sorting (FACS),
wherein cells expressing a fluorescent protein encoded for by the
reporter element are separated from cells that do not express a
fluorescent protein. Cells may be contacted with fluorescent
protein, fluorescent probe or fluorochrome conjugated antibodies
that bind proteins encoded for by the reporter element or genetic
element and subsequently selected by FACS. Fluorochromes can
include but are not limited to Cascade Blue, Pacific Blue, Pacific
Orange, Lucifer yellow, NBD, R-Phycoerythrin (PE), PE-Cy5
conjugates, PE-Cy7 conjugates, Red 613, PerCP, TruRed, FluorX,
Fluorescein, BODIPY-FL, TRITC, Texas Red, Allophycocyanin, APC-Cy7
conjugates (PharRed), various Alexa Fluor dyes, Cy2, Cy3, Cy3B,
Cy3.5, Cy5, Cy5.5, Cy7, various DyLights, Y66H, Y66F, EBFP, EBFP2,
Azurite, GFPuv, T-Sapphire, TagBFP, Cerulean, mCFP, ECFP, CyPet,
Y66W, dKeima-Red, mKeima-Red, TagCFP, AmCyanl, mTFP1, S65A,
Midoriishi-Cyan, GFP, Turbo GFP, TagGFP, TagGFP2, AcGFP1, S65L,
Emerald, S65T, S65C, EGFP, Azami-Green, ZaGreenl, Dronpa-Green,
TagYFP, EYFP, Topaz, Venus, mCitirine, YPet, Turbo YFP, PhiYFP,
PhiYFPm, ZaYellowl, mBanana, Kusabira-Orange, mOrange, mOrane2,
mKO, TurboRFP, tdTomato, DsRed-Express2, TagRFP, DsRed monomer,
DsRed2, mStrawberry, Turbo FP602, AsRed2, mRFP1, J-Red, mCherry,
HcRedl, mKate2, Katushka, mKate, TurboFP635, mPlum, mRaspberry,
mNeptune, E2-Crimson.
[0780] Cells may be contacted with antibodies that bind peptide
affinity tags encoded for by the reporter element or genetic
element and subsequently can be selected by immunomagnetic beads
which recognize the antibodies. Screening may comprise staining
cells by adding X-gal when the reporter element or genetic element
encodes b-galactosidase. Screening may comprise manual sorting
(e.g. diluting cell suspensions) and microscopy (e.g. fluorescence
microscopy). Screening may comprise high-content screening.
[0781] Reporter elements may encode drug resistance genes, thereby
allowing for selection of cells containing the reporter element by
the addition of drugs, the drugs killing the cells that do not
express the reporter element. Such drug can include, but are not
limited to erythromycin, clindamycin, chloramphenicol, gentamicin,
kanamycin, streptomycin, tetracycline, the combination
quinupristin-dalfopristin, enrofloxacin, vancomycin, oxacillin,
penicillin, sulfonamide sulfisoxazole, trimethoprim, methoinine
sulphoximine, methotrexate, puromycine, blasticidin, histidinol,
hygromycin, zeocin, bleomycin and neomycin.
[0782] Libraries
[0783] The present disclosure provides for a library of expression
vectors comprising donor polynucleotides. In some embodiments, the
library can comprise expression vectors comprising polynucleotide
sequences encoding for differing genetic elements of interest but
the same reporter elements. In some embodiments, the library can
comprise expression vectors comprising polynucleotide sequences
encoding for differing genetic elements of interest and differing
reporter elements. Reporter elements may differ in their nucleic
acid targeting sequences (crRNA and tracrRNA). Reporter elements
may differ in their reporter genes (e.g. genes encoding fluorescent
proteins). The present disclosure provides for methods of using the
library to generate a plurality of genetically modified cells. The
present disclosure provides for methods of using the library for a
high throughput genetic screen. These libraries can allow for
analyzing large numbers of individual genes to infer gene function.
Libraries can comprise from about 10 individual members to about
10.sup.12 individual members; e.g. a library can comprise from
about 10 individual members to about 10.sup.2 individual members,
from about 10.sup.2 individual members to about 10.sup.3 individual
members, from about 10.sup.3 individual members to about 10.sup.5
individual members, from about 10.sup.5 individual members to about
10.sup.7 individual members, from about 10.sup.7 individual members
to about 10.sup.9 individual members, or from about 10.sup.9
individual members to about 10.sup.12 individual members.
[0784] Modifying Cells (Transfection/Infection)
[0785] Methods of disclosure provide for selection of cells
comprising the donor polynucleotide. In some embodiments, a method
can involve contacting a target nucleic acid or introducing into a
cell (or a population of cells) one or more nucleic acids
comprising nucleotide sequences encoding a nucleic acid-targeting
nucleic acid, a site-directed polypeptide, and/or donor
polynucleotide of the disclosure. Methods for introducing a nucleic
acid into a cell can include viral or bacteriophage infection,
transfection, conjugation, protoplast fusion, lipofection,
electroporation, calcium phosphate precipitation, polyethyleneimine
(PEI)-mediated transfection, DEAE-dextran mediated transfection,
liposome-mediated transfection, particle gun technology, calcium
phosphate precipitation, direct micro injection,
nanoparticle-mediated nucleic acid delivery, and the like. In some
embodiments, contacting a target nucleic acid or introducing into a
cell (or a population of cells) one or more nucleic acids may not
comprise viral infection. In some embodiments, contacting a target
nucleic acid or introducing into a cell (or a population of cells)
one or more nucleic acids may not comprise bacteriophage infection.
In some embodiments, contacting a target nucleic acid or
introducing into a cell (or a population of cells) one or more
nucleic acids may not comprise transfection.
[0786] Engineered Nucleic Acid-Targeting Nucleic Acids
[0787] Engineered P-Domains
[0788] A nucleic acid-targeting nucleic acid can be engineered
(e.g., comprise modifications). An engineered nucleic
acid-targeting nucleic acid can refer to any of the engineered
nucleic acid-targeting nucleic acids as described herein. For
example, an engineered nucleic acid-targeting nucleic acid can
comprise a minimum CRISPR repeat, a minimum tracrRNA, and a 3'
tracrRNA. A P-domain of a nucleic acid-targeting nucleic acid can
interact with region of a site-directed polypeptide. A P-domain can
interact with a plurality of regions of a site-directed
polypeptide. A P-domain can interact with a plurality of regions of
a site-directed polypeptide wherein at least one of the regions
interacts with a PAM in a protospacer adjacent motif. Examples of
these regions can include amino acids 1096-1225, and 1105-1138 of
Cas9 in S. pyogenes.
[0789] A modification can be introduced into the P-domain. A
P-domain can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25, or 30 or more adjacent nucleotides. A P-domain can
comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,
or 30 or more adjacent nucleotides. A P-domain can start one
nucleotide 3' of the last paired nucleotide in the duplex
comprising the minimum CRISPR repeat and the minimum tracrRNA
sequence. A P-domain can start at least about 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 15, 20, 25, or 30 or more nucleotides 3' of the last
paired nucleotide in the duplex comprising the minimum CRISPR
repeat and the minimum tracrRNA sequence. A P-domain can start at
most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 or more
nucleotides 3' of the last paired nucleotide in the duplex
comprising the minimum CRISPR repeat and the minimum tracrRNA
sequence.
[0790] An engineered P-domain can comprise at least about 1, 2, 3,
4, 5, 6, 7, 8, 9, or 10 or more mutations. An engineered P-domain
can comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more
mutations. The mutations can be adjacent to one another (e.g.,
sequential). The mutations can be separated from one another. The
mutations can be separated by at least about 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 or more nucleotides. The mutations can be separated by
at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more
nucleotides. Mutations to a nucleic acid-targeting nucleic acid can
comprise insertions, deletions, and substitutions of nucleotides in
the nucleic acid-targeting nucleic acid.
[0791] In some instances, an engineered nucleic acid-targeting
nucleic acid comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30% or more nucleotide identity and/or similarity to a
wildtype nucleic acid-targeting nucleic acid. In some instances, an
engineered nucleic acid-targeting nucleic acid comprises at least
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30% or more
nucleotide identity and/or similarity to a wildtype nucleic
acid-targeting nucleic acid.
[0792] In some instances, a CRISPR nucleic acid portion of the
engineered nucleic acid-targeting nucleic acid comprises at most
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30% or more
nucleotide identity and/or similarity to a wildtype CRISPR nucleic
acid. In some instances, a CRISPR nucleic acid portion of the
engineered nucleic acid-targeting nucleic acid comprises at least
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30% or more
nucleotide identity and/or similarity to a wildtype CRISPR nucleic
acid.
[0793] A tracrRNA nucleic acid portion of the engineered nucleic
acid-targeting nucleic acid can comprise at most about 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30% or more nucleotide identity and/or
similarity to a wildtype tracrRNA nucleic acid. A tracrRNA nucleic
acid portion of the engineered nucleic acid-targeting nucleic acid
can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30% or more nucleotide identity and/or similarity to a wildtype
tracrRNA nucleic acid.
[0794] The modifications in the P-domain can be such that the
engineered nucleic acid-targeting nucleic acid is newly configured
to hybridize to a new PAM sequence in a target nucleic acid. The
modification to the P-domain can be complementary to the PAM in the
target nucleic acid. The modification to the P-domain can comprise
the reverse complement of the PAM in the target nucleic acid. The
new PAM can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
or more nucleotides. The new PAM can comprise at most about 1, 2,
3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides. Modifications in the
P domain can occur in concert with modifications to a P-domain
binding and PAM-binding region of a site-directed polypeptide.
These modifications can be compensatory, wherein a modified
P-domain is specifically modified to bind to a modified
site-directed polypeptide, wherein the modification enables the
site-directed polypeptide to bind to the engineered P-domain with
greater specificity.
[0795] An engineered P-domain can be engineered to bind to a new
PAM (e.g., the engineered P-domain can hybridize to a new PAM). The
new PAM (e.g., different PAM), can be bimodal (i.e., a bimodal PAM
can comprise two separate regions of the PAM). The two separate
regions of a bimodal PAM can be separated by at least about 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides. The two separate
regions of a bimodal PAM can be separated by at most about 1, 2, 3,
4, 5, 6, 7, 8, 9, or 10 or more nucleotides.
[0796] An engineered P-domain can be be engineered to bind to a new
PAM (e.g., different PAM), wherein the new PAM is trimodal. A
trimodal PAM can comprise three separate regions of a PAM sequence
(e.g., three separate regions that can be used in targeting a
nucleic acid-targeting nucleic acid to a target nucleic acid). The
three separate regions of a trimodal PAM can be separated by at
least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides.
The three separate regions of a trimodal PAM can be separated by at
most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more
nucleotides.
[0797] An engineered nucleic acid-targeting nucleic acid can
comprise at least two hairpins. A first hairpin can comprise a
duplex between the minimum CRISPR repeat and the minimum tracrRNA
sequence. The second hairpin can be downstream of the first
hairpin. The second hairpin can start at least 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 or more nucleotides downstream of the last paired
nucleotide of the first duplex. The second hairpin can start at
most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides
downstream of the last paired nucleotide of the first duplex. The
second hairpin can comprise an engineered P-domain.
[0798] The engineered P-domain of the second hairpin can be located
on one side of the duplex hairpin. The engineered P-domain of the
second hairpin can be located on both sides of the duplex hairpin.
The engineered P-domain of the second hairpin can comprise at least
about 1, 2, 3, 4, 5, 10, or 20% of the nucleotides in the second
hairpin. The engineered P-domain of the second hairpin can comprise
at most about 1, 2, 3, 4, 5, 10, or 20% of the nucleotides in the
second hairpin.
[0799] The second hairpin can comprise a tracrRNA (e.g., the
mid-tracrRNA, or 3' tracrRNA of a nucleic acid-targeting nucleic
acid). The second hairpin can comprise at least about 1, 5, 10, 15,
20, 25, 30, 35, 40, 45, or 50% or more identity to a tracrRNA. The
second hairpin can comprise at most about 1, 5, 10, 15, 20, 25, 30,
35, 40, 45, or 50% or more identity to a tracrRNA.
[0800] The second hairpin comprising the engineered P-domain can be
configured to de-duplex (e.g., melt, unwind). The second hairpin
can de-duplex when in contact with a target nucleic acid. The
second hairpin can de-duplex when in contact with a protospacer
adjacent motif of a target nucleic acid.
[0801] In some instances, an engineered P-domain can be configured
to hybridize to a region in a nucleic acid-targeting nucleic acid
(e.g., the same nucleic acid-targeting nucleic acid comprising the
engineered P-domain), and the engineered P-domain can be configured
to hybridize to a target nucleic acid. In other words, the
engineered P-domain can comprise a switchable sequence, in which in
some instances, the P-domainis hybridized to the nucleic
acid-targeting nucleic acid, thereby forming a hairpin, and in some
instances the P-domain is hybridized to a PAM in a target nucleic
acid.
[0802] An engineered nucleic acid-targeting nucleic acid comprising
a modified P-domain can be engineered to bind to a PAM with a lower
dissociation constant than a nucleic acid-targeting nucleic acid
that does not comprise a modified P-domain (e.g., wild-type nucleic
acid-targeting nucleic acid). An engineered nucleic acid-targeting
nucleic acid comprising a modified P-domain can be engineered to
bind to a PAM with a dissociation constant at least about 10, 50,
100, 150, 200, 250, 300, 350, 400, 450, 500, 550 or 600% or more
lower or higher than a nucleic acid-targeting nucleic acid that
does not comprise a modified P-domain.
[0803] An engineered nucleic acid-targeting nucleic acid comprising
a modified P-domain can be engineered to bind to a PAM with a
dissociation constant at most about 10, 50, 100, 150, 200, 250,
300, 350, 400, 450, 500, 550 or 600% or more lower or higher than a
nucleic acid-targeting nucleic acid that does not comprise a
modified P-domain. An engineered nucleic acid-targeting nucleic
acid comprising a modified P-domain can be engineered to bind to a
PAM with a dissociation constant at least about 1-fold, 2-fold,
3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, or
50-fold or more lower or higher than a nucleic acid-targeting
nucleic acid that does not comprise a modified P-domain. An
engineered nucleic acid-targeting nucleic acid comprising a
modified P-domain can be engineered to bind to a PAM with a
dissociation constant at most about 1-fold, 2-fold, 3-fold, 4-fold,
5-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold or more
lower or higher than a nucleic acid-targeting nucleic acid that
does not comprise a modified P-domain.
[0804] An engineered nucleic acid-targeting nucleic acid comprising
a modified P-domain can be engineered to bind to a PAM with greater
specificity than a nucleic acid-targeting nucleic acid that does
not comprise a modified P-domain (e.g., wild-type nucleic
acid-targeting nucleic acid). Greater specificity can refer to a
reduction in off-target binding (e.g., binding of the nucleic
acid-targeting nucleic acid to an incorrect PAM or PAM-like
sequence). For example, an engineered nucleic acid-targeting
nucleic acid comprising a modified P-domain can be engineered to
reduce non-specific binding by at least about 10, 50, 100, 150,
200, 250, 300, 350, 400, 450, 500, 550 or 600% or more than a
nucleic acid-targeting nucleic acid that does not comprise a
modified P-domain. An engineered nucleic acid-targeting nucleic
acid comprising a modified P-domain can be engineered to reduce
non-specific binding by at most about 10, 50, 100, 150, 200, 250,
300, 350, 400, 450, 500, 550 or 600% or more than a nucleic
acid-targeting nucleic acid that does not comprise a modified
P-domain. An engineered nucleic acid-targeting nucleic acid
comprising a modified P-domain can be engineered to reduce
non-specific binding by at least about 1-fold, 2-fold, 3-fold,
4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold or
than a nucleic acid-targeting nucleic acid that does not comprise a
modified P-domain. An engineered nucleic acid-targeting nucleic
acid comprising a modified P-domain can be engineered to reduce
non-specific binding by at most about 1-fold, 2-fold, 3-fold,
4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold or
more than a nucleic acid-targeting nucleic acid that does not
comprise a modified P-domain.
[0805] Engineered Bulges
[0806] An engineered nucleic acid-targeting nucleic acid can be
engineered such that a modification can be introduced into the
bulge region of the nucleic acid-targeting nucleic acid. A bulge is
a typical nucleic acid feature that comprises unpaired nucleotides.
A bulge can comprise unpaired nucleotides on each strand of the
duplex that comprises the bulge. In other words, a bulge can
comprise an unpaired nucleotide on the minimum CRISPR repeat strand
of the duplex and an unpaired nucleotide on the minimum tracrRNA
sequence strand of the duplex.
[0807] A bulge can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8,
9 or 10 or more unpaired nucleotides on a first strand of a duplex
in a nucleic acid-targeting nucleic acid (i.e., the minimum CRISPR
repeat strand of the duplex comprising the minimum CRISPR repeat
and minimum tracrRNA sequence). A bulge can comprise at most about
1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more unpaired nucleotides on a
first strand of a duplex in a nucleic acid-targeting nucleic acid
(i.e., the minimum CRISPR repeat strand of the duplex comprising
the minimum CRISPR repeat and minimum tracrRNA sequence). A bulge
can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more
unpaired nucleotides on a second strand of a duplex in a nucleic
acid-targeting nucleic acid (i.e., the minimum tracrRNA sequence
strand of the duplex comprising the minimum CRISPR repeat and
minimum tracrRNA sequence). A bulge can comprise at most about 1,
2, 3, 4, 5, 6, 7, 8, 9 or 10 or more unpaired nucleotides on a
second strand of a duplex in a nucleic acid-targeting nucleic acid
(i.e., the minimum tracrRNA sequence strand of the duplex
comprising the minimum CRISPR repeat and minimum tracrRNA
sequence). A bulge can comprise one unpaired nucleotide on the
minimum CRISPR RNA sequence and 3 unpaired nucleotides on the
minimum tracrRNA sequence strand.
[0808] The nucleotides adjacent to an unpaired nucleotide can be a
nucleotide that forms a wobble base pairing interaction. Wobble
base pairing interactions can include guanine-uracil,
hypoanthine-uracil, hypoxanthine-adenine, and
hypoxanthine-cytosine. At least 1, 2, 3, 4, or 5 or more
nucleotides adjacent to an unpaired nucleotide can form a wobble
pairing. At most 1, 2, 3, 4, or 5 or more nucleotides adjacent to
an unpaired nucleotide can form a wobble pairing.
[0809] An engineered bulge can comprise at least about 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10 or more mutations. An engineered bulge can
comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more
mutations. The mutations can be adjacent to one another (e.g.,
sequential). The mutations can be separated from one another. The
mutations can be separated by at least about 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 or more nucleotides. The mutations can be separated by
at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more
nucleotides. Mutations to a nucleic acid-targeting nucleic acid can
comprise insertions, deletions, and substitutions of nucleotides in
the nucleic acid-targeting nucleic acid.
[0810] A bulge of an engineered nucleic acid-targeting nucleic acid
can comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30% or more nucleotide identity and/or similarity to a wildtype
nucleic acid-targeting nucleic acid. A bulge of an engineered
nucleic acid-targeting nucleic acid can comprise at least about 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30% or more nucleotide identity
and/or similarity to a wildtype nucleic acid-targeting nucleic
acid.
[0811] One strand of the bulge can be mutated and the other strand
is not mutated. In other words, in some instances, the sequence of
the bulge on the minimum CRISPR RNA strand is the same as a
wild-type nucleic acid-targeting nucleic acid, and the sequence of
the bulge on the minimum tracrRNA sequence is mutated. In other
words, the sequence of the bulge on the minimum CRISPR RNA strand
is mutated, and the sequence of the bulge on the minimum tracrRNA
sequence is the same as a wild-type nucleic acid-targeting nucleic
acid.
[0812] The modifications in the bulge can be such that the
engineered nucleic acid-targeting nucleic acid is newly configured
to bind to a new site-directed polypeptide. The new site-directed
polypeptide can comprise at least about 5, 10, 15, 20, 25, 30, 35,
40, 45 or 50% or more amino acid sequence identity to a wild-type
site-directed polypeptide. The new site-directed polypeptide can
comprise at most about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50% or
more amino acid sequence identity to a wild-type site-directed
polypeptide. The new site-directed polypeptide can be a homologue
of Cas9. The new site-directed polypeptide can be an orthologue of
Cas9. The new site-directed polypeptide can be a chimera of two
different site-directed polypeptides. The new site-directed
polypeptide can comprise a mutation as disclosed herein.
[0813] An engineered nucleic acid-targeting nucleic acid comprising
a modified bulge can be engineered to bind to a site-directed
polypeptide with a lower or higher dissociation constant than a
nucleic acid-targeting nucleic acid that does not comprise a
modified bulge (e.g., wild-type nucleic acid-targeting nucleic
acid). An engineered nucleic acid-targeting nucleic acid comprising
a modified bulge can be engineered to bind to a site-directed
polypeptide with a dissociation constant at least about 10, 50,
100, 150, 200, 250, 300, 350, 400, 450, 500, 550 or 600% or more
lower or higher than a nucleic acid-targeting nucleic acid that
does not comprise a modified bulge. An engineered nucleic
acid-targeting nucleic acid comprising a modified bulge can be
engineered to bind to a site-directed polypeptide with a
dissociation constant at most about 10, 50, 100, 150, 200, 250,
300, 350, 400, 450, 500, 550 or 600% or more lower or higher than a
nucleic acid-targeting nucleic acid that does not comprise a
modified bulge. An engineered nucleic acid-targeting nucleic acid
comprising a modified bulge can be engineered to bind to a
site-directed polypeptide with a dissociation constant at least
about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold,
30-fold, 40-fold, or 50-fold or more lower or higher than a nucleic
acid-targeting nucleic acid that does not comprise a modified
bulge. An engineered nucleic acid-targeting nucleic acid comprising
a modified bulge can be engineered to bind to a site-directed
polypeptide with a dissociation constant at most about 1-fold,
2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold,
or 50-fold or more lower or higher than a nucleic acid-targeting
nucleic acid that does not comprise a modified bulge.
[0814] Methods
[0815] The disclosure provides for methods for engineering nucleic
acid-targeting nucleic acids. The methods can comprise modifying a
nucleic acid-targeting nucleic acid. The modifying can comprise
inserting, deleting, substituting, and mutating the nucleotides in
the nucleic acid-targeting nucleic acid. The modifying can comprise
modifying the nucleic acid-targeting nucleic acid such that the
nucleic acid-targeting nucleic acid can bind to new protospacer
adjacent motifs and/or new site-directed polypeptides as compared
to a wild-type nucleic acid-targeting nucleic acid. The method can
be performed using any of the site-directed polypeptides, nucleic
acid-targeting nucleic acids, and complexes of site-directed
polypeptides and nucleic acid-targeting nucleic acids as described
herein.
[0816] An engineered nucleic acid-targeting nucleic acid can be
used to cleave a target nucleic acid. An engineered nucleic
acid-targeting nucleic acid can be introduced into cells with a
site-directed polypeptide, thereby forming a complex. The complex
can hybridize to a target nucleic acid, wherein the target nucleic
acid comprises a protospacer adjacent motif. The site-directed
polypeptide of the complex can cleave the target nucleic acid.
[0817] Complementary portions of the nucleic acid sequences of
pre-CRISPR nucleic acid and tracr nucleic acid sequences from
Streptococcus pyogenes SF370 are shown in FIG. 20.
[0818] FIG. 21 depicts exemplary structure of the duplex (e.g.,
hairpin) comprising the minimum CRISPR repeat and the minimum
tracrRNA sequence and a portion of the 3' tracrRNA sequence. The
duplex comprises a bulge region.
[0819] Table 4 contains the sequences of DNA templates used to
synthesize the single guide nucleic acid-targeting nucleic acids of
the disclosure.
TABLE-US-00007 TABLE 4 DNA templates of the guide nucleic acids of
the disclosure Duplex variants group 1 TEMP3-
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC FL
TATGCTGTTTTGGAAACAAAACAGCATAGCAAGTTAAAATAAGGCTAGTCCGT
TATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v2
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGAAAAAGAG
CTAGAAATAGCAAGTTTTTTTAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
CACCGAGTCGGTGCTTTTTTT SGR-v3
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGATATAGAG
CTAGAAATAGCAAGTTATATTAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
CACCGAGTCGGTGCTTTTTTT SGR-v4
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGG
ATGAAAATCCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
ACCGAGTCGGTGCTTTTTTT SGR-v5
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGAAAATGAG
GATGAAAATCCAAGTATTTTTAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
CACCGAGTCGGTGCTTTTTTT SGR-v6
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGATTATGAG
GATGAAAATCCAAGTATAATTAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
CACCGAGTCGGTGCTTTTTTT SGR-v7
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTAATTGAG
GATGAAAATCCAAGTAATTATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
CACCGAGTCGGTGCTTTTTTT SGR-v8
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGAAAATCAA
GTGATGAAAATCGAGATTTTTAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
CACCGAGTCGGTGCTTTTTTT SGR-v9
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGAAAATGAA
GGATGAAAATCCAGTATTTTTAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
CACCGAGTCGGTGCTTTTTTT SGR-v10
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGATTTAGAG
CTAGAAATAGCAAGTTAAATTAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
CACCGAGTCGGTGCTTTTTTT SGR-v11
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTCTCAGAG
CTAGAAATAGCAAGTTGAGATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
CACCGAGTCGGTGCTTTTTTT SGR-v12
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTCCCAGAG
CTAGAAATAGCAAGTTGGGATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
CACCGAGTCGGTGCTTTTTTT SGR-v13
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGACT
CAGAAATCAGAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
ACCGAGTCGGTGCTTTTTTT Duplex variants group 2 SGR-v14
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC
TAGAAATAGCTCTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCAC
CGAGTCGGTGCTTTTTTT SGR-v15
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGG
AAACTCTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC GGTGCTTTTTTT
SGR-v16 AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAAA
TAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCT TTTTTT
SGR-v17 AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATATTTTAGAGC
TAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
ACCGAGTCGGTGCTTTTTTT SGR-v18
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATATTTTAGAGC
TAGAAATAGCAAGTTAAAACAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
ACCGAGTCGGTGCTTTTTTT SGR-v19
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGACGATAGA
ACGGAAACGTTGGACATCGTTAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
CACCGAGTCGGTGCTTTTTTT SGR-v20
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGACGATGAG
ACGGAAACGTCAAGTATCGTTAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
CACCGAGTCGGTGCTTTTTTT SGR-v21
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAAGAC
TAGAAATAGTGGACTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
ACCGAGTCGGTGCTTTTTTT SGR-v22
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATCGTTTAGAGC
TAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
ACCGAGTCGGTGCTTTTTTT SGR-v23
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTGGTAGAG
CTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
CACCGAGTCGGTGCTTTTTTT SGR-v24
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTGCGAGC
TAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
ACCGAGTCGGTGCTTTTTTT SGR-v25
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGT
GAGAAATAGCAAGTTCACATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
ACCGAGTCGGTGCTTTTTTT SGR-v26
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC
TAGAAATAGCAAGTTACACTAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
ACCGAGTCGGTGCTTTTTTT SGR-v27
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC
TAGAAATAGCAAGTTAACAGAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
ACCGAGTCGGTGCTTTTTTT SGR-v28
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC
TAGAAATAGCAAGTTAAAATAACTGGCTAGTCCGTTATCAACTTGAAAAAGTG
GCACCGAGTCGGTGCTTTTTTT SGR-v29
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC
TAGAAATAGCAAGTTAAAATGCTAGTCCGTTATCAACTTGAAAAAGTGGCACC
GAGTCGGTGCTTTTTTT Tracr-Variant Group SGR-v30
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC
TAGAAATAGCAAGTTAAAATGGAACTAGTCCGTTATCAACTTGAAAAAGTGGC
ACCGAGTCGGTGCTTTTTTT SGR-v31
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC
TAGAAATAGCAAGTTAAAATTTCGCTAGTCCGTTATCAACTTGAAAAAGTGGC
ACCGAGTCGGTGCTTTTTTT SGR-v32
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC
TAGAAATAGCAAGTTAAAATAAGCGAAGTCCGTTATCAACTTGAAAAAGTGG
CACCGAGTCGGTGCTTTTTTT SGR-v33
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC
TAGAAATAGCAAGTTAAAATAAGGCTTCACCGTTATCAACTTGAAAAAGTGGC
ACCGAGTCGGTGCTTTTTTT SGR-v34
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC
TAGAAATAGCAAGTTAAAATAAGGCTAGTGGCTTATCAACTTGAAAAAGTGGC
ACCGAGTCGGTGCTTTTTTT SGR-v35
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC
TAGAAATAGCAAGTTAAAATAAGGCTAGTCCGAATTCAACTTGAAAAAGTGG
CACCGAGTCGGTGCTTTTTTT SGR-v36
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC
TAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTAAGTACTTGAAAAAGTGGC
ACCGAGTCGGTGCTTTTTTT SGR-v37
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC
TAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCATGATGAAAAAGTGGC
ACCGAGTCGGTGCTTTTTTT SGR-v38-
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC MMO
TAGAAATAGCAAGTTAAAATAAGAATGATACATCACAAAAAAAAGGCTTTAT
GCCGTAACTACTACTTATTTTCAAAATAAGTAGTTTTTTTT SGR-v39-
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC ST2
TAGAAATAGCAAGTTAAAATAAGGCTTCATGCCGAAATCAACACCCTGTCATT
TTATGGCAGGGTGTTTTCGTTATTTTTTT SGR-v40-
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC CJ
TAGAAATAGCAAGTTAAAATAAAGAGTTTGCGGGACTCTGCGGGGTTACAATC
CCCTAAAACCGCTTTTTTT SGR-v41-
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC NM
TAGAAATAGCAAGTTAAAATAAGGCCGTCTGAAAAGATGTGCCGCAACGCTCT
GCCCCTTAAAGCTTCTGCTTTAAGGGGCATTTTTT Csy4-tag-group SGR-v42
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC
TAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTATACTGCCGTA
TAGGCAGAGATTTTTT SGR-v43
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC
TAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTATACTGCCGTA
TAGGCAGAGAAATGGACTCGGAATACTGCCGTATAGGCAGAGATTTTTT SGR-v44
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC
TATCACTGCCGTATAGGCAGTGATAGCAAGTTAAAATAAGGCTAGTCCGTTAT
CAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v45
AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC
TATCACTGCCGTATAGGCAGTGATAGCAAGTTAAAATAAGGCTAGTCCGTTAT
CAACTTGAAAAAGTGGCACCGAGTCGGTGCTAATGGACTCGATACTGCCGTAT
AGGCAGAGATTTTTT
[0820] Table 2 shows the RNA sequences of the DNA templates in
Table 1.
TABLE-US-00008 TABLE 2 RNA sequences of single guide nucleic
acid-targeting nucleic acids of the disclosure Caribou Single guide
P/N nucleic acid-targeting nucleic acid sequence Duplex variants
group 1 TEMP3- GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAUGCUGUUUUGGAAACA FL
AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAA
AAGUGGCACCGAGUCGGUGCUUUUUUU SGR-v2
GGGGCCACUAGGGACAGGAUGAAAAAGAGCUAGAAAUAGCAAGUUUUU
UUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v3
GGGGCCACUAGGGACAGGAUGAUAUAGAGCUAGAAAUAGCAAGUUAUA
UUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v4
GGGGCCACUAGGGACAGGAUGUUUUAGAGGAUGAAAAUCCAAGUUAAA
AUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v5
GGGGCCACUAGGGACAGGAUGAAAAUGAGGAUGAAAAUCCAAGUAUUU
UUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v6
GGGGCCACUAGGGACAGGAUGAUUAUGAGGAUGAAAAUCCAAGUAUAA
UUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v7
GGGGCCACUAGGGACAGGAUGUAAUUGAGGAUGAAAAUCCAAGUAAUU
AUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v8
GGGGCCACUAGGGACAGGAUGAAAAUCAAGUGAUGAAAAUCGAGAUUU
UUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v9
GGGGCCACUAGGGACAGGAUGAAAAUGAAGGAUGAAAAUCCAGUAUUU
UUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v10
GGGGCCACUAGGGACAGGAUGAUUUAGAGCUAGAAAUAGCAAGUUAAA
UUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v11
GGGGCCACUAGGGACAGGAUGUCUCAGAGCUAGAAAUAGCAAGUUGAG
AUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v12
GGGGCCACUAGGGACAGGAUGUCCCAGAGCUAGAAAUAGCAAGUUGGG
AUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v13
GGGGCCACUAGGGACAGGAUGUUUUAGACUCAGAAAUCAGAAGUUAAA
AUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU Duplex
variants group 2 SGR-v14
GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCUCUAAAAU
AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUU UUUUU SGR-v15
GGGGCCACUAGGGACAGGAUGUUUUAGAGGAAACUCUAAAAUAAGGCU
AGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU SGR-v16
GGGGCCACUAGGGACAGGAUGUUUUAGAAAUAAAAUAAGGCUAGUCCG
UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU SGR-v17
GGGGCCACUAGGGACAGGAUAUUUUAGAGCUAGAAAUAGCAAGUUAAA
AUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v18
GGGGCCACUAGGGACAGGAUAUUUUAGAGCUAGAAAUAGCAAGUUAAA
ACAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v19
GGGGCCACUAGGGACAGGAUGACGAUAGAACGGAAACGUUGGACAUCG
UUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v20
GGGGCCACUAGGGACAGGAUGACGAUGAGACGGAAACGUCAAGUAUCG
UUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v21
GGGGCCACUAGGGACAGGAUGUUUUAAGACUAGAAAUAGUGGACUAAA
AUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v22
GGGGCCACUAGGGACAGGAUCGUUUAGAGCUAGAAAUAGCAAGUUAAA
AUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v23
GGGGCCACUAGGGACAGGAUGUGGUAGAGCUAGAAAUAGCAAGUUAAA
AUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v24
GGGGCCACUAGGGACAGGAUGUUUGCGAGCUAGAAAUAGCAAGUUAAA
AUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v25
GGGGCCACUAGGGACAGGAUGUUUUAGAGUGAGAAAUAGCAAGUUCAC
AUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v26
GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUACA
CUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v27
GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAC
AGAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v28
GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAA
AUAACUGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCUUUUUUU SGR-v29
GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAA
AUGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU UUUU Tracr-Variant
Group SGR-v30 GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAA
AUGGAACUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v31
GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAA
AUUUCGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v32
GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAA
AUAAGCGAAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v33
GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAA
AUAAGGCUUCACCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v34
GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAA
AUAAGGCUAGUGGCUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v35
GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAA
AUAAGGCUAGUCCGAAUUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v36
GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAA
AUAAGGCUAGUCCGUUAAGUACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v37
GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAA
AUAAGGCUAGUCCGUUAUCAUGAUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v38-
GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAA MMO
AUAAGAAUGAUACAUCACAAAAAAAAGGCUUUAUGCCGUAACUACUAC
UUAUUUUCAAAAUAAGUAGUUUUUUU SGR-v39-
GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAA ST2
AUAAGGCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUG UUUUCGUUAUUUUUUU
SGR-v40- GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAA CJ
AUAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCU UUUUUU SGR-v41-
GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAA NM
AUAAGGCCGUCUGAAAAGAUGUGCCGCAACGCUCUGCCCCUUAAAGCU
UCUGCUUUAAGGGGCAUUUUUU Csy4-tag-group SGR-v42
GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAA
AUAAGGCUAGUCCGUUAUCAACUUAUACUGCCGUAUAGGCAGAGAUUU UUU SGR-v43
GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAA
AUAAGGCUAGUCCGUUAUCAACUUAUACUGCCGUAUAGGCAGAGAAAU
GGACUCGGAAUACUGCCGUAUAGGCAGAGAUUUUUU SGR-v44
GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAUCACUGCCGUAUAGGC
AGUGAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAG
UGGCACCGAGUCGGUGCUUUUUUU SGR-v45
GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAUCACUGCCGUAUAGGC
AGUGAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAG
UGGCACCGAGUCGGUGCUAAUGGACUCGAUACUGCCGUAUAGGCAGAG AUUUUUU
[0821] Table 5 indicates the activity and purpose of additional
nucleic acid-targeting nucleic acid variants. + refers to active, -
refers to inactive. The experimental data of these variants is
shown in FIG. 37.
TABLE-US-00009 Nucleic acid-targeting nucleic acid variants and
their activity name dCB ### Actvity Purpose Spacer sgBackbone SGR-
- Linker GGGGCC GTTTTAGAGCTAGAAATAGC v80 length ACTAGG
AAGTTAAAATAACTCGGCTA GACAG GTCCGTTATCAACTTGAAAA GAT
AGTGGCACCGAGTCGGTGCT SGR- - Linker GGGGCC GTTTTAGAGCTAGAAATAGC v81
length ACTAGG AAGTTAAAATAACTCTGGCT GACAG AGTCCGTTATCAACTTGAAA GAT
AAGTGGCACCGAGTCGGTGC T SGR- - Linker GGGGCC GTTTTAGAGCTAGAAATAGC
v82 length ACTAGG AAGTTAAAATAACTCTCTGG GACAG CTAGTCCGTTATCAACTTGA
GAT AAAAGTGGCACCGAGTCGGT GCT SGR- ++ Control GGGGCC
GTTTTAGAGCTAGAAATAGC v83 ACTAGG AAGTTAAAATAAGGCTAGTC GACAG
CGTTATCAACTTGAAAAAGT GAT GGCACCGAGTCGGTGCT SGR- ++ Minimal GGGGCC
GTTTTAGAGGAAACAAGTTA v84 sgRNA ACTAGG AAATAAGGCTAGTCCGTTAT GACAG
CAACTTGAAAAAGTGGCACC GAT GAGTCGGTGCT SGR- ++ Minimal GGGGCC
GTTTTAGAGACAAGTTAAAA v85 sgRNA ACTAGG TAAGGCTAGTCCGTTATCAA GACAG
CTTGAAAAAGTGGCACCGAG GAT TCGGTGCT SGR- ++ Minimal GGGGCC
GTTTTAGGAGAAACTTTAAA v86 sgRNA ACTAGG ATAAGGCTAGTCCGTTATCA GACAG
ACTTGAAAAAGTGGCACCGA GAT GTCGGTGCT SGR- - Minimal GGGGCC
GTTTTATCGAAATCTAAAAT v87 sgRNA ACTAGG AAGGCTAGTCCGTTATCAAC GACAG
TTGAAAAAGTGGCACCGAGT GAT CGGTGCT SGR- - Minimal GGGGCC
GTTTTACTTCGGTAAAATAA v88 sgRNA ACTAGG GGCTAGTCCGTTATCAACTT GACAG
GAAAAAGTGGCACCGAGTCG GAT GTGCT SGR- ++ Minimal GGGGCC
GTTTTAGATACTTAAAATAA v89 sgRNA ACTAGG GGCTAGTCCGTTATCAACTT GACAG
GAAAAAGTGGCACCGAGTCG GAT GTGCT SGR- - Minimal GGGGCC
GTTTTATGAAACTAAAATAA v90 sgRNA ACTAGG GGCTAGTCCGTTATCAACTT GACAG
GAAAAAGTGGCACCGAGTCG GAT GTGCT SGR- - Minimal GGGGCC
GTTTCTTCGGAAATAAGGCT v91 sgRNA ACTAGG AGTCCGTTATCAACTTGAAA GACAG
AAGTGGCACCGAGTCGGTGC GAT T SGR- ++ Change GGGGCC
GTTTTAGGCTAGAAATAGCA v92 bulge ACTAGG AGTTAAAATAAGGCTAGTCC angle
GACAG GTTATCAACTTGAAAAAGTG GAT GCACCGAGTCGGTGCT SGR- ++ Change
GGGGCC GTTTTAGCTAGAAATAGCAA v93 bulge ACTAGG GTTAAAATAAGGCTAGTCCG
angle GACAG TTATCAACTTGAAAAAGTGG GAT CACCGAGTCGGTGCT SGR- ++ Change
GGGGCC GTTTTACTAGAAATAGCAAG v94 bulge ACTAGG TTAAAATAAGGCTAGTCCGT
angle GACAG TATCAACTTGAAAAAGTGGC GAT ACCGAGTCGGTGCT SGR- - Change
GGGGCC GTTTTAGAGCTAGAAATAGC v95 bulge ACTAGG AGTTAAAATAAGGCTAGTCC
angle GACAG GTTATCAACTTGAAAAAGTG GAT GCACCGAGTCGGTGCT SGR- - Change
GGGGCC GTTTTAGAGCTAGAAATAGA v96 bulge ACTAGG GTTAAAATAAGGCTAGTCCG
angle GACAG TTATCAACTTGAAAAAGTGG GAT CACCGAGTCGGTGCT SGR- ++ Change
GGGGCC GTTTTAGAGCTAGAAATAAG v97 bulge ACTAGG TTAAAATAAGGCTAGTCCGT
angle GACAG TATCAACTTGAAAAAGTGGC GAT ACCGAGTCGGTGCT SGR- - Change
GGGGCC GTTTTAGAGCTAGAAATAGT v98 bulge ACTAGG TAAAATAAGGCTAGTCCGTT
angle GACAG ATCAACTTGAAAAAGTGGCA GAT CCGAGTCGGTGCT SGR- - Control
GGGGCC GTTTTAGAGCTAGAAATAGC v99 ACTAGG AAGTTAAAATAAGGCTAGTC GACAG
CGTTATCAACTTGAAAAAGT GAT GGCACCGAGTCGGTGCT SGR- dCB154 - nexus/h
GGGGCC GTTTTAGAGCTAGAAATAGC v124 airpin ACTAGG AAGTTAAAATAACCCTAGTC
variant GACAG CGTTATCAACTTGAAAAAGT GAT GGCACCGAGTCGGTGCT SGR-
dCB155 +/- nexus/h GGGGCC GTTTTAGAGCTAGAAATAGC v125 airpin ACTAGG
AAGTTAAAATAACCCTAGTG variant GACAG GGTTATCAACTTGAAAAAGT GAT
GGCACCGAGTCGGTGCT SGR- dCB156 + nexus/h GGGGCC GTTTTAGAGCTAGAAATAGC
v126 airpin ACTAGG AAGTTAAAATAAGGCTAGTG variant GACAG
GGTTATCAACTTGAAAAAGT GAT GGCACCGAGTCGGTGCT SGR- dCB157 ++ nexus/h
GGGGCC GTTTTAGAGCTAGAAATAGC v46 airpin ACTAGG AAGTTAAAATAAGGCTAGTA
variant GACAG AGTTATCAACTTGAAAAAGT GAT GGCACCGAGTCGGTGCT SGR-
dCB158 ++ nexus/h GGGGCC GTTTTAGAGCTAGAAATAGC v47 airpin ACTAGG
AAGTTAAAATAAGGCTAGTT variant GACAG TGTTATCAACTTGAAAAAGT GAT
GGCACCGAGTCGGTGCT SGR- dCB159 ++ nexus/h GGGGCC
GTTTTAGAGCTAGAAATAGC v48 airpin ACTAGG AAGTTAAAATAAGACTAGTT variant
GACAG CGTTATCAACTTGAAAAAGT GAT GGCACCGAGTCGGTGCT SGR- dCB160 -
nexus/h GGGGCC GTTTTAGAGCTAGAAATAGC v49 airpin ACTAGG
AAGTTAAAATAAGGCTAGGA variant GACAG AACTAGCCTTCTCAA GAT SGR- dCB161
- nexus/h GGGGCC GTTTTAGAGCTAGAAATAGC v50 airpin ACTAGG
AAGTTAAAATAAGGCTAGGA variant GACAG AACTAGCCCTCAATA GAT SGR- dCB162
+/- nexus/h GGGGCC GTTTTAGAGCTAGAAATAGC v51 airpin ACTAGG
AAGTTAAAATAAGGCTAGTC variant GACAG CGTAGAAAATGCC GAT SGR- dCB163 +
nexus/h GGGGCC GTTTTAGAGCTAGAAATAGC v52 airpin ACTAGG
AAGTTAAAATAAGGCTAGTC variant GACAG CGTAGAAATACGG GAT SGR- dCB164 +
nexus/h GGGGCC GTTTTAGAGCTAGAAATAGC v53 airpin ACTAGG
AAGTTAAAATAAGGCTAGTC variant GACAG CGTAGAAATACTTAT GAT SGR- dCB165
+ nexus/h GGGGCC GTTTTAGAGCTAGAAATAGC v54 airpin ACTAGG
AAGTTAAAATAAGGCTAGTC variant GACAG CGTTATTATTAGGGGGTTA GAT SGR-
dCB166 - nexus GGGGCC GTTTTAGAGCTAGAAATAGC v55 trunc- ACTAGG
AAGTTAAAATAAGG ation GACAG GAT SGR- dCB167 - nexus GGGGCC
GTTTTAGAGCTAGAAATAGC v56 trunc- ACTAGG AAGTTAAAATAAGGCTAG ation
GACAG GAT SGR- dCB168 +/- nexus GGGGCC GTTTTAGAGCTAGAAATAGC v57
trunc- ACTAGG AAGTTAAAATAAGGCTAGTC ation GACAG CG GAT SGR- dCB169
+/- nexus GGGGCC GTTTTAGAGCTAGAAATAGC v58 trunc- ACTAGG
AAGTTAAAATAAGGCTAGTC ation GACAG CGTTAT GAT SGR- +/- nexus GGGGCC
GTTTTAGAGCTAGAAATAGC v100 stem ACTAGG AAGTTAAAATAAGGGCTAGT length
GACAG CCCGTTATCAACTTGAAAAA GAT GTGGCACCGAGTCGGTGCT SGR- ++ nexus
GGGGCC GTTTTAGAGCTAGAAATAGC v101 stem ACTAGG AAGTTAAAATAAGGGGCTAG
length GACAG TCCCCGTTATCAACTTGAAA GAT AAGTGGCACCGAGTCGGTGC T SGR- +
nexus GGGGCC GTTTTAGAGCTAGAAATAGC v102 stem ACTAGG
AAGTTAAAATAAGGGGGCTA length GACAG GTCCCCCGTTATCAACTTGA GAT
AAAAGTGGCACCGAGTCGGT GCT SGR- ++ nexus GGGGCC GTTTTAGAGCTAGAAATAGC
v103 stem ACTAGG AAGTTAAAATAAAACTAGTT length GACAG
TGTTATCAACTTGAAAAAGT GAT GGCACCGAGTCGGTGCT SGR- ++ nexus GGGGCC
GTTTTAGAGCTAGAAATAGC v104 stem ACTAGG AAGTTAAAATAAGGACTAGT length
GACAG TCCGTTATCAACTTGAAAAA GAT GTGGCACCGAGTCGGTGCT SGR- ++ Control
GGGGCC GTTTTAGAGCTAGAAATAGC v105 ACTAGG AAGTTAAAATAAGGCTAGTC GACAG
CGTTATCAACTTGAAAAAGT GAT GGCACCGAGTCGGTGCT SGR- +/- nexus GGGGCC
GTTTTAGAGCTAGAAATAGC v106 stem ACTAGG AAGTTAAAATAAGGCTAGTC length
GACAG CTGTTATCAACTTGAAAAAG GAT TGGCACCGAGTCGGTGCT SGR- ++ nexus
GGGGCC GTTTTAGAGCTAGAAATAGC v107 stem ACTAGG AAGTTAAAATAAAGCTAGTC
length GACAG TGTTATCAACTTGAAAAAGT GAT GGCACCGAGTCGGTGCT SGR- -
nexus GGGGCC GTTTTAGAGCTAGAAATAGC v108 loop ACTAGG
AAGTTAAAATAAGGTCTAGT size GACAG CCCGTTATCAACTTGAAAAA GAT
GTGGCACCGAGTCGGTGCT SGR- + nexus GGGGCC GTTTTAGAGCTAGAAATAGC v109
loop ACTAGG AAGTTAAAATAAGGTACTAG size GACAG TCGCCGTTATCAACTTGAAA
GAT AAGTGGCACCGAGTCGGTGC T SGR- - nexus GGGGCC GTTTTAGAGCTAGAAATAGC
v110 loop ACTAGG AAGTTAAAATAAGGTATCTA size GACAG
GTGCGCCGTTATCAACTTGA GAT AAAAGTGGCACCGAGTCGGT GCT SGR- ++ Control
GGGGCC GTTTTAGAGCTAGAAATAGC v111 ACTAGG AAGTTAAAATAAGGCTAGTC GACAG
CGTTATCAACTTGAAAAAGT GAT GGCACCGAGTCGGTGCT
SGR- - Tracr GGGGCC GTTTTAGAGCTAGAAATAGC v112 hybrid- ACTAGG
AAGTTAAAATCAAACAAAGC LRH GACAG TTCAGCTGAGTTTCAATTTCT GAT
GGCCCATGTTGGGCACATAC ATATGCCACCGAG SGR- ++ Tracr GGGGCC
GTTTTAGAGCTAGAAATAGC v113 hybrid- ACTAGG AAGTTAAAATAAGGCAGTGA SM159
GACAG TTTTTAATCCAGTCCGTACAC GAT AACTTGAAAAAGTGCGCACC
GATTCGGTGCTTTTTT SGR- ++ Tracr GGGGCC GTTTTAGAGCTAGAAATAGC v114
hybrid- ACTAGG AAGTTAAAATAAGGCTTAGA ST3 GACAG CCGTACTCAACTTGAAAAGG
GAT TGGCACCGATTCGGTGTTTTT TTT SGR- ++ Tracr GGGGCC
GTTTTAGAGCTAGAAATAGC v115 hybrid- ACTAGG AAGTTAAAATCAAAGCGCTT LBU
GACAG TGCGCGGAGTTTCAACTTTT GAT SGR- - Major GGGGCC
GAGAATCTCCTAGAAATAGC v120 variant ACTAGG TCTTATTCTTAAGGGATCACC
GACAG GAATACAACTTGAAAAAGTG GAT GCACCGAGTCGGTGCT SGR- ++ Major
GGGGCC GACGATGAGACGGAAACGTC v121 variant ACTAGG
AAGTATCGTTAAGGGATCAC GACAG CGAATACAACTTGAAAAAGT GAT
GGCACCGAGTCGGTGCT SGR- - Major GGGGCC AACGATGAGACGGAAACGTC v122
variant ACTAGG AAGTATCGTCAAGGGATCAC GACAG CCAATACAACTTGAAAAAGT GAT
GGCACCGAGTCGGTGCT SGR- - Major GGGGCC AACGGTGAGGTGGAAACACC v123
variant ACTAGG AAGTACCGTCAAGGTAGCAC GACAG CCGACAAGTC GAT
[0822] Methods for the Generation of Tagged Cell Lines Using a
Nucleic Acid-Targeting Nucleic Acid
[0823] The methods of the disclosure provide for tagging a cell
with a donor polynucleotide, wherein the donor polynucleotide can
divide and/or differentiate, and the donor polynucleotide can be
transmitted to each daughter cell during cell division. The method
can be performed using any of the site-directed polypeptides,
nucleic acid-targeting nucleic acids, and complexes of
site-directed polypeptides and nucleic acid-targeting nucleic acids
as described herein.
[0824] A tagged cell can be generated by contacting the cell with a
donor polynucleotide, and a complex comprising a site-directed
polypeptide and a nucleic acid-targeting nucleic acid. The donor
polynucleotide can be inserted into the cleaved target nucleic
acid, thereby generating a tagged cell. The tagged cell can be
propagated such as in a cell line, or to produce a propagated
population of cells.
[0825] A donor polynucleotide can be introduced into the cut site
by use of a donor cassette for homologous recombination that
comprises ends homologous to sequences on either side of the
double-strand break. The donor polynucleotide can comprise an
additional sequence between the two ends. The additional sequence
can be a nucleic acid sequence. The additional sequence can encode
for a gene. The additional sequence can encode for a non-coding
nucleic acid element.
[0826] The donor polynucleotide (e.g., the additional sequence of a
donor polynucleotide between two homologous ends) can comprise a
marker. A marker can comprise a visualization marker (e.g., a
fluorescent marker such as GFP). A marker can comprise a random
polynucleotide sequence (e.g., such as a random hexamer sequence).
A marker can be a barcode.
[0827] NHEJ can introduce unique sequence signature at each cut
site. The repair mechanism can result in the introduction of
insertions (e.g., insertion of a donor polynucleotide), deletions
or mutations into a cut site. A cell that undergoes NHEJ to repair
a double-strand break can comprise a unique sequence after repair
has taken place (e.g., a unique sequence can be inserted into the
double-strand break). If more than one site is cut within a cell,
repair can introduce the donor polynucleotide at each site, thereby
adding sequence diversity to that cell. The repaired site can
provide a unique barcode sequence to the cell that can be preserved
during cell division and passed on to all progeny of the modified
cell. A donor polynucleotide can be inserted into at least about 1,
2, 3, 4, 5, 6, 7, 8, 9 or 10 or more sites (e.g, cleaved target
nucleic acids). A donor polynucleotide can be inserted into at most
about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more sites (e.g, cleaved
target nucleic acids).
[0828] Homologous recombination (HR) can be used to introduce
barcode sequences into a cell and/or a cell population (e.g., a
human cell, a mammalian cell, a yeast, a fungi, a protozoa, an
archaea). A library of donor plasmids (e.g., comprising the donor
polynucleotide) can be prepared with randomized sequences in the
donor cassette. The library can be made from oligonucleotides, a
piece of double-stranded DNA, a plasmid, and/or a minicircle.
[0829] Donor polynucleotide sequences can be introduced into the
genomes of individual cells for the purpose of tracking cell
lineage. Sites can be chosen for modification in silent or
"safe-harbor" regions of the genome, distant from genes and
regulatory elements, to minimize potentially deleterious effects on
cellular function. Sites within functional genetic elements can
also be used to track cell fate.
[0830] For example, donor polynucleotides can be introduced into
stem cell and/or stem cell populations. The methods of the
disclosure can be used for tracking cell lineage development in
animal models. For example, cell fate development and/or
differentiation in hematopoiesis can be tracked using the methods
of the disclosure. The methods of the disclosure can be used for
therapeutic cell engineering-based therapies. For example, a cell
can be tagged with a donor polynucleotide encoding a therapeutic
protein. The cell can be propagated. The propagated cell can be
introduced into a subject. As another example, a differentiated
cell can be removed from a subject. The differentiated cell can be
tagged with two markers: one expressed when the cell is
differentiated, one expressed when the cell is de-differentiated.
Identifying the markers can be useful in determining
differentiation events. In another example, a differentiated cell
can be obtained from a subject. The differentiated cell can be
de-differentiated into a pluripotent cell. The pluripotent cell can
be tagged with a donor polynucleotide encoding a therapeutic
protein. The cell can be re-differentiated into a new cell type
while expressing the therapeutic protein, thereby creating a
patient-specific therapeutic cell. Tagged cells can divide and
differentiate, and the modification(s) to their genome can be
transmitted to each daughter cell during cell division.
[0831] In some instances, two cells can be tagged with two
different donor polynucleotide markers. The two cells can be
combined. The combined mixture can be assayed simultaneously. The
donor polynucleotides can allow the multiplex analysis of the two
cells because the donor polynucleotide can be used to distinguish
the two cells.
[0832] A cell population can be chosen for introducing
double-stranded breaks, or generating cellular signatures. Cells
may be purified or selected. For example, a population of
hematopoetic stem cells (CD45positive) may be selected by FACS or
magnetic bead purification. Bone marrow may be treated ex vivo with
the nuclease. Cells may be targeted in vivo by the use of viruses
with a particular tropism. Cells may be selected by using viruses
engineered to target cells bearing a particular receptor.
[0833] Tagged cells can be analyzed by high-throughput sequencing
either at the population level or at the single-cell level. At the
population level, a collection of cells can be lysed. The genomic
DNA can be extracted. PCR primers can be designed to amplify the
genomic region that has been modified by the nuclease. Sequences
can be enriched by hybridization. A sequence library can be
prepared from the genomic DNA and enriched. The region of interest
can be enriched, and a sequence library can be prepared. A sequence
library can be prepared simultaneously during enrichment using
primers comprising appropriate sequence tags to be used with
nucleic acid sequencing technologies. If the double-stranded break
is made within a region that can be transcribed, RNA can be used to
prepare sequence libraries.
[0834] Once nucleic acid sequence data has been obtained, the
sequences can be analyzed to determine the clonal structure. This
can be carried out by gathering common sequences together and
counting those sequences.
[0835] Cells can be sub-selected by sorting schemes based on cell
surface markers using flow cytometry or affinity purification
methods. Cell surface markers can be used to define cell states,
and by comparing cell states with clonal structure, the fate of
modified cell populations can be determined.
[0836] At the single-cell level, cells can be isolated. PCR
products can be generated from each individual cell. This can be
achieved in microwell arrays, microfluidic devices, and/or
emulsions. Where more than one genomic modification is carried out
per cell, PCR products can be coupled together, either physically,
or chemically, to ensure their relationship to the parent cell.
[0837] Methods for Quantifying Genome-Editing Events
[0838] For RNA-dependent nucleases, such as Cas9, the nucleic acid
recognition functionality and nuclease activities can be linked. In
some instances, nucleic acid recognition functionality and nuclease
activities may not be linked. The nuclease sites can be located
within the specific sequence recognized by the nuclease.
[0839] Non-Homologous End-Joining can be an imperfect repair
process that can result in the insertion of multiple bases at the
site of the double stranded break. NHEJ can result in the
introduction of insertions, deletions and/or mutations into a cut
site. NHEJ can significantly disrupt the original sequence. The
disruption of the native sequence as a consequence of repair
mechanisms can be used to assess the efficiency of genome editing
approaches.
[0840] Homologous recombination can enable more complete repair of
the target nucleic acid break by exchanging nucleotide sequences
between similar or identical molecules of nucleic acid. An
additional sequence can be introduced into the target nucleic acid
at the cut site by use of a donor cassette (e.g., donor
polynucleotide) that comprises ends homologous to sequences either
side of the double-strand break and additional sequence between the
two ends.
[0841] This disclosure describes an approach for assessing
double-stranded break activity and NHEJ-mediated
insertions/deletions introduced by nucleic acid-dependent
nucleases, such as Cas9. The method takes advantage of the fact
that the sites in a target nucleic acid recognized by Cas9 during
the initial nuclease recognition and nucleic acid cleavage activity
can be destroyed during the NHEJ process, either by the
introduction of insertions or deletions.
[0842] In some instances, the method provides for the design of a
nucleic acid-targeting nucleic acid to target a site of interest in
a target nucleic acid (e.g., genome). A nucleic acid template
encoding the nucleic acid-targeting nucleic acid can be designed
with a promoter sequence appended at the 5' end of the nucleic
acid-targeting nucleic acid to enable in vitro synthesis of the
nucleic acid-targeting nucleic acid.
[0843] Primers can be designed at positions that flank the cleavage
site. The cleavage site (and/or nucleic acid regions around the
cleavage site) can be amplified (e.g., from genomic nucleic acid),
thereby generating a product (e.g., amplified PCR product). The
product (e.g., amplified PCR product) can be at least about 100,
200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200 or more
bases in length. The product (e.g., amplified PCR product) can be
at most about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,
1100, 1200 or more bases in length. The product (e.g., amplified
PCR product) can be about 200-600 base pairs in length.
[0844] The products can be purified. The products can be incubated
with an RNA-dependent nuclease (e.g. Cas9) and the nucleic
acid-targeting nucleic acid. Those molecules that have been
amplified from genomic nucleic acid that have not be modified by
NHEJ can comprise the correct sequence that can be recognized and
cleaved by Cas9. The molecules that have been amplified from
genomic nucleic acid that has been modified by NHEJ may not
comprise sites that can be recognized and/or cut by Cas9.
[0845] The digested products can then be analyzed by methods such
as gel electrophoresis, capillary electrophoresis, high-throughput
sequencing and/or quantitative PCR (e.g., qPCR). In the case of gel
electrophoresis, a gel can be imaged. Once a gel has been imaged,
the percentage of cells modified by NHEJ can be estimated by
measuring the intensity of bands corresponding to digested
products, and comparing to the intensity of bands corresponding to
undigested products.
[0846] Methods for Delivering Donor Polynucleotide to a
Double-Stranded Break for Insertion into the Double-Stranded
Break
[0847] This disclosure describes methods for bringing a donor
polynucleotide into close proximity to a site-directed target
nucleic acid break to enhance insertion (e.g., homologous
recombination) of the donor polynucleotide into the site of the
double-stranded break. The method can be performed using any of the
site-directed polypeptides, nucleic acid-targeting nucleic acids,
and complexes of site-directed polypeptides and nucleic
acid-targeting nucleic acids as described herein.
[0848] In some instances, the methods of the disclosure provide for
for bringing a donor polynucleotide in close proximity to the site
of a double-stranded break in a target nucleic acid, by binding it
to the nuclease that generates the double-stranded break (e.g.,
Cas9).
[0849] A complex comprising a site-directed polypeptide, a nucleic
acid-targeting nucleic acid, and a donor polynucleotide can be
delivered to a target nucleic acid. FIG. 30 illustrates exemplary
methods for bringing a donor polynucleotide into proximity to the
site of a double-stranded break in a target nucleic acid. For
example, a nucleic acid-targeting nucleic acid can comprise a 3'
hybridizing extension sequence, which can be part of a tracrRNA
extension sequence (shown in the light dotted line attached to the
nucleic acid-targeting nucleic acid). A 3' hybridizing extension
sequence can be a non-native sequence. FIG. 30A illustrates that
the tracrRNA extension sequence at the 3' end of the nucleic
acid-targeting nucleic acid can include a sequence that can
hybridize to an end of the donor polynucleotide (e.g., the 3' end)
(the donor polynucleotide is shown in bold thicker dashed line).
The 3' hybridizing extension sequence can be at least about 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length. The 3'
hybridizing extension sequence can be at most about 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10 or more nucleotides in length. The 3' hybridizing
sequence can hybridize to at least about 1, 2, 3, 4, 5, 5, 6, 7, 8,
9, or 10 or more nucleotides of the donor polynucleotide. The 3'
hybridizing sequence can hybridize to at most about 1, 2, 3, 4, 5,
5, 6, 7, 8, 9, or 10 or more nucleotides of the donor
polynucleotide. The 3' hybridizing sequence can hybridize to the
donor polynucleotide with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
or more mismatches. The 3' hybridizing sequence can hybridize to
the donor polynucleotide with at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10 or more mismatches.
[0850] The 3' hybridizing extension can hybridize to the 3' end of
the donor polynucleotide. The 3' hybridizing extension can
hybridize to at least the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more
3' most nucleotides of the donor polynucleotide. The 3' hybridizing
extension can hybridize to at most the 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 or more 3' most nucleotides of the donor polynucleotide.
[0851] As depicted in FIG. 30B, the tracr nucleic acid extension at
the 3' end of the nucleic acid-targeting nucleic acid can include a
sequence that can hybridize to the 5' end of the donor DNA. The 3'
hybridizing extension can hybridize to at least the 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10 or more 5' most nucleotides of the donor
polynucleotide. The 3' hybridizing extension can hybridize to at
most the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more 5' most
nucleotides of the donor polynucleotide.
[0852] The tracr nucleic acid extension at the 3' end of the
nucleic acid-targeting nucleic acid can include a sequence that can
hybridize to a region between the 3' end and 5' end of the donor
polynucleotide, as shown in FIG. 30C. The 3' hybridizing extension
can hybridize to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more
nucleotides between the 3' and 5' end of the donor polynucleotide.
The 3' hybridizing extension can hybridize to at most the 1, 2, 3,
4, 5, 6, 7, 8, 9, or 10 or more nucleotides between the 3' and 5'
end of the donor polynucleotide.
[0853] The tracr nucleic acid extension at the 3' end of the
nucleic acid-targeting nucleic acid can include a sequence that can
hybridize along the full length of the donor polynucleotide, as
shown in FIG. 30D. The nucleic acid-targeting nucleic acid can
hybridize along at least about 20, 30, 40, 50, 60, 70, 80, 90, or
100% of the donor polynucleotide. The nucleic acid-targeting
nucleic acid can hybridize along at most about 20, 30, 40, 50, 60,
70, 80, 90, or 100% of the donor polynucleotide. The 3' hybridizing
extension sequence can hybridize along the full length of the donor
polynucleotide with at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
or more mismatches. The 3' hybridizing extension sequence can
hybridize along the full length of the donor polynucleotide with at
most about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more mismatches.
[0854] The tracr nucleic acid extension at the 3' end of the
nucleic acid-targeting nucleic acid (e.g., 3' hybridizing
extension) can comprise a sequence that can be used as a template
and converted by, for example, a reverse transcriptase to generate
hybrid nucleic acid (e.g., the resulting nucleic acid is an RNA-DNA
hybrid, wherein the newly transcribed nucleic acid can be DNA), as
shown in FIG. 30E. Exemplary reverse transcriptases include
SuperScript, ThermoScript, HIV reverse transcriptase, and MMLV
reverse transcriptase. The reverse transcriptase can extend the
donor polynucleotide sequence from the 3' hybridizing extension
template.
[0855] The tracr nucleic acid extension at the 3' end of the
nucleic acid-targeting nucleic acid can incorporate a nucleic acid
sequence that can binds an RNA binding protein (RBP). The
RNA-binding protein can be fused to a DNA binding protein (DBP), as
shown in FIG. 30F. The DNA-binding protein can bind to the donor
polynucleotide.
[0856] The sequences used to bring the donor polynucleotide into
close proximity with a double-stranded break can be appended to the
5' end of the nucleic acid-targeting nucleic acid (e.g., the spacer
extension). The sequences sequences used to bring the donor
polynucleotide into close proximity with a double-stranded break
can be appended to both the 5' end and the 3' end of the nucleic
acid-targeting nucleic acid.
[0857] The nuclease used in the methods of the disclosure (e.g.,
Cas9) can comprise nickase activity in which the nuclease can
introduce single-stranded breaks in a target nucleic acid. Pairs of
nucleases with nickase activity can be targeted to regions in close
proximity to each other. A first nuclease can bind to a first
nucleic acid-targeting nucleic acid that can interact with a first
donor polynucleotide. A second nuclease can bind to a second
nucleic acid-targeting nucleic acid that can interact with a second
donor polynucleotide. The first and second donor polynucleotides
can be designed to hybridize with each other to make a
double-stranded donor polynucleotide. Two separate donor
polynucleotides can be brought to the nuclease site.
[0858] In some embodiments, the donor polynucleotide can be single
stranded. In some embodiments, the donor polynucleotide can be
double stranded. In some embodiments, the donor DNA can be a
minicircle. In some embodiments, the donor polynucleotide can be a
plasmid. In some embodiments, the plasmid can be supercoiled. In
some embodiments, the donor polynucleotide can be methylated. In
some embodiments, the donor polynucleotide can be unmethylated. The
donor polynucleotide can comprise a modification. Modifications can
include those described here including, but not limited to,
biotinylation, chemical conjugate, and synthetic nucleotides.
[0859] Methods for Cloning and Expressing a Vector Comprising a
Site-Directed Polypeptide and a Nucleic Acid-Targeting Nucleic
Acid
[0860] The disclosure provides for methods for cloning an
engineered nucleic acid-targeting nucleic acid into a vector (e.g.,
a linearized vector). The method can be performed using any of the
site-directed polypeptides, nucleic acid-targeting nucleic acids,
and complexes of site-directed polypeptides and nucleic
acid-targeting nucleic acids as described herein.
[0861] A user (e.g., a scientist) can design single-stranded DNA
oligonucleotides. The single-stranded DNA oligonucleotides can,
when hybridized together encode a spacer sequence to target a
target nucleic acid. The single-stranded DNA oligonucleotides can
be at least about 5, 10, 15, 20, 25, 30 or more nucleotides in
length. The single-stranded DNA oligonucleotides can be at most
about 5, 10, 15, 20, 25, 30 or more nucleotides in length. The
single-stranded DNA oligonucleotides can be 19-20 nucleotides in
length.
[0862] A single-stranded DNA oligonucleotide can be designed such
that it can hybridize to a target nucleic acid (e.g., a sequence
adjacent to a protospacer adjacent motif, such as the 3' or 5' end
of the protospacer adjacent motif). The DNA oligonucleotide can
encode a sequence corresponding to the sense or antisense strand of
the target nucleic acid sequence.
[0863] The single-stranded oligonucleotides can comprise a first
portion that can hybridize and/or is complementary to a target
nucleic acid. The single-stranded oligonucleotides can comprise a
first portion that can hybridize and/or is complementary another
single-stranded oligonucleotide. The single-stranded
oligonucleotide can comprise a second portion that can hybridize to
a sequence in the linearized vector. In other words, a pair of
single-stranded oligonucleotides can comprise a first portion that
hybridizes to each other and a second portion that comprise
single-stranded overhangs, wherein the overhangs can hybridize to
sticky ends in the linearized vector. In some instances, an
overhang comprises 5'-GTTTT-3'. In some instances, an overhang
comprises 5'-CGGTG-3'
[0864] The single stranded DNA nucleotides can be annealed together
to generate a double-stranded oligonucleotide. The single-stranded
DNA nucleotides can be annealed together in an oligonucleotide
annealing buffer (e.g., comprising Tris-HCl, EDTA and NaCl). The
double-stranded oligonucleotide can be diluted to a working
concentration (e.g., a concentration suitable for ligation into a
linearized plasmid). The diluted double-stranded oligonucleotide
can be ligated into a linearized vector. Ligation can be performed
in a ligation buffer (e.g., comprising Tris-HCl, MgCl.sub.2, ATP)
and with a ligase (e.g., T4 DNA ligase). The double-stranded
oligonucleotide can be ligated into a linearized vector at a region
within the sequence encoding the nucleic acid-targeting nucleic
acid. In other words, the linearized vector can be linearized at a
point within the region encoding the nucleic acid-targeting nucleic
acid, wherein the linearization generates sticky ends that are
complementary to the sticky ends of the double-stranded
oligonucleotide. When the double-stranded oligonucleotide is
ligated into the vector, it can generate a sequence encoding for an
engineered nucleic acid-targeting nucleic acid comprising a spacer
sequence corresponding to the double-stranded oligonucleotide
sequence.
[0865] The ligated vector can be transformed into chemically
competent cells (e.g., DH5-alpha, Top10) and selected for
expression of the correctly ligated vector (e.g., by antibotic
screening). The selected transformants can be analyzed for the
presence of an insert by sequencing. Sequencing can be performed
using a sequencing primer that can hybridize to a portion of the
vector.
[0866] Correctly ligated vector can be prepared (e.g., by large
scale DNA preparation, maxiprep), and purified. The vector,
comprising a site-directed polypeptide, a nucleic acid-targeting
nucleic acid, wherein the nucleic acid-targeting nucleic acid
comprise the double-stranded DNA oligonucleotides can be introduced
(e.g., transfected) into a cell line of choice (e.g., mammalian
cell line).
[0867] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
EXAMPLES
Example 1
Modification of a Site-Directed Polypeptide for Altered PAM
Specificity
[0868] In some embodiments, the disclosure provides for a modified
site-directed polypeptide that is modified to alter PAM
specificity. A nucleic acid encoding a modified site-directed
polypeptide is introduced into cells by transfection. The modified
site-directed polypeptide comprises an inserted HNH or RuvC
nuclease domain. The modified site-directed polypeptide comprises a
modified highly basic patch. A nucleic acid-targeting nucleic acid
that comprises a spacer that can hybridize with the target nucleic
acid is also introduced into cells by transfection. The modified
site-directed polypeptide and the nucleic acid-targeting nucleic
acid form a complex. The complex is guided to the target nucleic
acid by the nucleic acid-targeting nucleic acid. Once hybridized
with the nucleic acid-targeting nucleic acid, the target nucleic
acid is cleaved by the nuclease domains of the site-directed
polypeptide. In some embodiments, the modified site-directed
polypeptide binds to the target nucleic acid with a lower Kd.
[0869] In some embodiments, a donor polynucleotide is also
introduced into the cells. In some instances, the donor
polynucleotide, a portion of the donor polynucleotide, a copy of
the donor polynucleotide, or a portion of a copy of the donor
polynucleotide is inserted into the cleaved target nucleic
acid.
Example 2
Modification of a Site-Directed Polypeptide for Altered Target
Nucleic Acid Specificity
[0870] In some embodiments, the disclosure provides for a modified
site-directed polypeptide that is modified to alter target nucleic
acid specificity. A modified site-directed polypeptide is
introduced into cells. The modified site-directed polypeptide
comprises a modification in the highly basic patch and/or the
HNH-like domain. A nucleic acid-targeting nucleic acid that
comprises a spacer that can hybridize with the target nucleic acid
is also introduced into cells. The modified site-directed
polypeptide and the nucleic acid-targeting nucleic acid form a
complex. The complex is guided to the target nucleic acid by the
nucleic acid-targeting nucleic acid. Once hybridized with the
nucleic acid-targeting nucleic acid, the target nucleic acid is
cleaved by the site-directed polypeptide. In some embodiments, the
modified site-directed polypeptide binds to the target nucleic acid
with a lower Kd.
[0871] In some embodiments, a donor polynucleotide is also
introduced into the cells. In some instances, the donor
polynucleotide, a portion of the donor polynucleotide, a copy of
the donor polynucleotide, or a portion of a copy of the donor
polynucleotide is inserted into the cleaved target nucleic
acid.
Example 3
Recombinant Expression of a Site-Directed Polypeptide
[0872] A recombinant DNA sequence can be assembled that encodes for
a modified site-directed polypeptide, and enables the expression of
the modified site-directed polypeptide in a host organism. The
recombinant DNA sequence comprises a promoter sequence, and may
additionally comprise an affinity tag for purification, or an
epitope tag. In a non-limiting example, a plasmid comprises the
recombinant DNA sequence for expression of the modified
site-directed polypeptide.
[0873] Production of Recombinant Protein.
[0874] A plasmid encoding the site directed modified polypeptide is
introduced into bacterial cells (e.g., E coli). The polypeptide is
expressed in bacterial cells, and then purified from cell lysate
using chromatography methods. The activity of the modified
site-directed polypeptide is measured using assay methods designed
to determine the specificity of the modified polypeptide, the PAM
sequence, the specificity profile of the site-directed polypeptide
and the nucleic acid preference (for example, DNA or RNA, or
modified nucleic acids.) for the polypeptide.
[0875] Software is designed to choose sites that can be cut using
the modified site-directed polypeptide. Guide RNA sequences are
designed to direct the activity of the site-directed polypeptide.
Once designed, the site-directed polypeptide is used to cleave
nucleic acids.
[0876] Introduction of the modified site-directed polypeptide into
cells
[0877] The modified peptide is introduced into cells to target
nucleic acid sites. A polypeptide that retains nuclease activity is
used to introduce single-stranded or double-stranded DNA breaks
into target DNA. A polypeptide with DNA-binding but not DNA cutting
activity, is used to bind double stranded DNA to a cell. This can
be used to effect transcriptional activation or repression.
Example 4
Selection of Sites for Modification within Cas9 Sequences
[0878] As described above Type II CRISPR systems containing Cas9
orthologues can be classified into three groups (Type II-A, Type
II-B, and Type II-C) based on analysis of their CRISPR-Cas loci.
Cas9 orthologues within these groups can be broadly defined by two
clades comprising shorter (Cas9/Csn1-type) and longer
(Cas9/Csx12-type) sequences. In addition to these larger groups,
there can be two additional families of homologues that comprise
HNH and RuvC domains arranged with similar topology to Cas9, but
with significant differences in the length and sequence of
insertions between conserved sequence elements. Secondary structure
predictions and sequence alignments are used to define regions of
the polypeptide for modification. Regions that fall between
secondary structure elements or between regions of high sequence
conservation are selected as candidates for insertions or
deletions. Regions that have similarity to domains of known
structure are analyzed to identify specific regions for inserting
or deleting sequences.
[0879] FIG. 35 shows the CDD sequence alignment TIGR0185 for a
small number of diverse Cas9 orthologs. Amino acids with a "X"
below them are considered to be similar. Amino acids with a "Y"
below them can be considered to be highly conserved or identical in
all sequences. Amino acids residues without an "X" or a "Y" are not
conserved. This alignment does not include the C-terminal region
(approximately corresponding to amino acid residues 1100-1350) of
longer Cas9 orthologs. The sequences listed in FIG. 35 are listed
in Table 6.
TABLE-US-00010 TABLE 6 Sequences listed in FIG. 35. Genbank
Accession Species gi 22533915 Streptococcus agalactiae 2603V/R gi
34483507 Wolinella succinogenes gi 12721472 Pasteurella multocida
subsp. multocida str. Pm70 gi 24377777 Streptococcus mutans UA159
gi 13622193 Streptococcus pyogenes M1 GAS gi 41815893 Treponema
denticola ATCC 35405 gi 218767588 Neisseria meningitidis Z2491 gi
157150687 Streptococcus gordonii str. Challis substr. CH1 gi
294660600 Mycoplasma gallisepticum str. R(low) gi 218563121
Campylobacter jejuni subsp. jejuni NCTC 11168 = ATCC 700819 gi
370792169 Listeria innocua ATCC 33091
[0880] Locations for insertion of new functional domains, regions
for insertion of alternative sequences, or for deletion or
reduction in region size that modify Cas9 activity can include, but
are not limited to, the regions highlighted in Table 7. The numbers
represent the amino-acid sequence numbers based on the Cas9
sequence from Streptococcus pyogenes M1 GAS.
TABLE-US-00011 TABLE 7 Exemplary locations for modifying Cas9
gi|13622193 Streptococcus Insertion/Deletion pyogenes M1 GAS site
start finish Length 1 22 42 20 2 97 134 37 3 170 312 142 4 350 400
50 5 426 444 18 6 455 492 37 7 528 541 13 8 578 589 11 9 600 612 12
10 628 634 6 11 654 658 4 12 684 692 8 13 710 727 17 14 753 756 3
15 792 794 2 16 801 804 3 17 826 834 8 18 873 881 8 19 893 915 22
20 939 952 13 21 971 974 3 22 1005 1029 24 23 1096 1225 129 24 1105
1138 33
[0881] Once a region has been identified as a potential location
for inserting a new polypeptide sequence into the protein, or
deleting a region of the protein, the DNA sequence that codes for
the protein is modified to incorporate the modification.
Example 5
Sequence Enrichment of Site-Directed Polypeptide-Bound Target
Nucleic Acid
[0882] The disclosure provides methods for sequence enrichment
without amplification using site-directed polypeptides.
[0883] In some embodiments, the method will comprise a) contacting
a target nucleic acid with a complex comprising a nucleic
acid-targeting nucleic acid and a site-directed polypeptide, b)
cleaving the target nucleic acid c) purifying the target nucleic
acid, and d) sequencing the target nucleic acid, wherein said
target nucleic acid is enriched.
[0884] In some embodiments, the site-directed polypeptide will be
enzymatically inactive. Use of an enzymatically inactive
site-directed polypeptide will facilitate binding of the target
nucleic acid to the site-directed polypeptide complex. In some
embodiments, the site-directed polypeptide will be enzymatically
active.
[0885] In some embodiments, sequence enrichment will be performed
outside of cells (e.g., cell-free sample). For example, a sample
will comprise purified genomic DNA. In some embodiments, sequence
enrichment will be performed on a cellular sample (e.g. cells, cell
lysate).
[0886] In some instances, the site-directed polypeptide-target
nucleic acid complexes will be fixed or cross-linked to form
complexes. If the method is being performed on cells, cells will be
lysed. Lysis conditions will be chosen to maintain intact
protein-DNA complexes.
[0887] The nucleic acid sample will be treated to fragment the
target nucleic acid before affinity purification. Fragmentation can
be performed through physical, mechanical or enzymatic methods.
Physical fragmentation will include exposing a target
polynucleotide to heat or to ultraviolet (UV) light. Mechanical
disruption will be used to mechanically shear a target
polynucleotide into fragments of the desired range. Mechanical
shearing will be accomplished through a number of methods,
including repetitive pipetting of the target polynucleotide,
sonication and nebulization. Target nucleic acids will also be
fragmented using enzymatic methods. In some cases, enzymatic
digestion will be performed using enzymes such as using restriction
enzymes. Restriction enzymes will be used to perform specific or
non-specific fragmentation of target polynucleotides. The methods
will use one or more types of restriction enzymes, generally
described as Type I enzymes, Type II enzymes, and/or Type III
enzymes. Type II and Type III enzymes recognize specific sequences
of nucleotides within a double stranded polynucleotide sequence (a
"recognition sequence" or "recognition site"). Upon binding and
recognition of these sequences, Type II and Type III enzymes cleave
the polynucleotide sequence. In some cases, cleavage will result in
a polynucleotide fragment with a portion of overhanging single
stranded nucleic acid, called a "sticky end." In other cases,
cleavage will not result in a fragment with an overhang, creating a
"blunt end." The methods may comprise use of restriction enzymes
that generate either sticky ends or blunt ends.
[0888] Once fragmented, the complexes comprising the site-directed
polypeptide will be purified by incubation with a solid support.
For example, if the site-directed polypeptide comprises a biotin
tag, the solid support will be coated with avidin or streptavidin
to bind to the biotin tag.
[0889] In an alternative embodiment, once fragmented, the complexes
comprising the site-directed polypeptide, the target nucleic acid,
and/or the nucleic acid-targeting nucleic acid, will be purified by
incubation with a capture agent. The capture agent will bind to the
affinity tag fused to the site-directed polypeptide. The capture
agent will comprise an antibody. For example, if the affinity tag
fused to the site-directed polypeptide is a FLAG tag, then the
capture agent will be an anti-FLAG-tag antibody.
[0890] In some embodiments, the capture agent will be purified with
a solid support. For example, if the capture agent comprises a
biotin tag, the solid support will be coated with avidin or
streptavidin to bind the biotinylated capture agent.
[0891] In some embodiments, the nucleic acid-targeting nucleic acid
will comprise an affinity tag. The affinity tag will comprise a
sequence that can bind to an endoribonuclease. In some instances,
the affinity tag will comprise a sequence that can bind to a
conditionally enzymatically inactive endoribonuclease. The
conditionally enzymatically inactive endoribonuclease will bind,
but not cleave, the affinity tag.
[0892] In some embodiments, the endoribonuclease and/or the
conditionally enzymatically inactive endoribonuclease will comprise
an affinity tag.
[0893] The conditionally enzymatically inactive endoribonuclease
will be purified with a solid support. The solid support will bind
to the affinity tag of the conditionally enzymatically inactive
endoribonuclease. For example, if the conditionally enzymatically
inactive endoribonuclease comprises a biotin tag, the solid support
will be coated with avidin or streptavidin to bind the biotinylated
capture agent.
[0894] In some embodiments, the conditionally enzymatically
inactive endoribonuclease will be immobilized on any of a variety
of insoluble support.
[0895] In some embodiments of the method, two rounds of
purification will be performed. In some instances, a first round
will comprise purification with a solid support that will bind to
the affinity tag of the capture agent and a second round will
comprise purification with a solid support that will bind to the
affinity tag of the site-directed polypeptide. In some instances, a
first round will comprise purification with a solid support that
will bind to the affinity tag of the site-directed polypeptide and
a second round will comprise purification with a solid support that
will bind to the affinity tag of the capture agent.
[0896] In some embodiments, the methods of the disclosure will be
used for multiplex sequence enrichment. In this embodiment, a
plurality of nucleic acid-targeting nucleic acids can be contacted
with a nucleic acid sample, wherein each nucleic acid-targeting
nucleic acid is engineered to target a different target nucleic
acid (e.g., sequence in a genome) within the nucleic acid
sample.
[0897] The captured complexes will comprise a target nucleic acid.
The target nucleic acid will be eluted from the site-directed
polypeptide complex by standard methods including high salt
washing, ethanol precipitation, boiling, gel purification, and the
like.
[0898] The eluted DNA will be prepared for sequencing analysis by
ligation of one or more adaptors.
[0899] The sequencing libraries will be sequenced as described
herein. Sequenced libraries will be analyzed to identify
polymorphisms, diagnose a disease, determine a course of treatment
for a disease, and/or generate antibody libraries.
Example 6
Sequence Enrichment of Target Nucleic Acid not Bound to a Complex
Comprising a Site-Directed Polypeptide
[0900] In some embodiments, sequence enrichment will be performed
with an enzymatically active site-directed polypeptide. In some
instances, the site-directed polypeptide will be enzymatically
active. In this instance, the target nucleic acid will not be bound
to the site-directed polypeptide, but will be excised.
[0901] A target nucleic acid will be identified, and nucleic
acid-targeting nucleic acids will be designed to direct the
site-directed polypeptide to sequences that flank the target
nucleic acid. The sample will be incubated with a complex
comprising a designed nucleic acid-targeting nucleic acid and the
site-directed polypeptide such that the site-directed polypeptide
will cleave the DNA at both ends of the target nucleic acid. Upon
cleavage of the target nucleic acid, the target nucleic acid will
be cleaved from the parent nucleic acid. The cleaved target nucleic
acid will be purified (e.g., by gel electrophoresis, size-selective
elution from beads, or other carboxylate-derivatized beads, or by
precipitation with appropriate concentrations of salt and PEG to
preferentially precipitate larger or smaller DNA).
[0902] In some embodiments, sequence enrichment will be performed
outside of cells (e.g., cell-free sample). For example, a sample
will comprise purified genomic DNA. In some embodiments, sequence
enrichment will be performed on a cellular sample (e.g. cells, cell
lysate).
[0903] If the method is being performed on cells, cells will be
lysed. Lysis conditions will be chosen to maintain intact
protein-DNA complexes.
[0904] In some embodiments, the target nucleic acid to be sequenced
will not be bound to a nucleic acid-targeting nucleic acid and/or a
site-directed polypeptide. In this embodiment, the nucleic acid
bound to the site-directed polypeptide and/or the nucleic
acid-targeting nucleic acid will be purified away. The purification
of the site-directed polypeptide will proceed as previously
described herein. Briefly, the complexes comprising the
site-directed polypeptide will be purified by incubation with a
solid support. For example, if the site-directed polypeptide
comprises a biotin tag, the solid support will be coated with
avidin or streptavidin to bind to the biotin tag.
[0905] In an alternative embodiment, once fragmented, the complexes
comprising the site-directed polypeptide, the nucleic
acid-targeting nucleic acid, and non-target nucleic acid, will be
purified by incubation with a capture agent. The capture agent will
bind to the affinity tag fused to the site-directed polypeptide.
The capture agent will comprise an antibody. For example, if the
affinity tag fused to the site-directed polypeptide is a FLAG tag,
then the capture agent will be an anti-FLAG-tag antibody.
[0906] The capture agent will be purified with a solid support. For
example, if the capture agent comprises a biotin tag, the solid
support will be coated with avidin or streptavidin to bind the
biotinylated capture agent.
[0907] In some embodiments, the methods of the disclosure will be
used for multiplex sequence enrichment. In this embodiment, a
plurality of nucleic acid-targeting nucleic acids can be introduced
into a cell, wherein each nucleic acid-targeting nucleic acid is
engineered to target a different target nucleic acid (e.g.,
sequence in a genome).
[0908] The captured complex will not comprise a target nucleic
acid.
[0909] The target nucleic acid will comprise the nucleic acid that
is not bound to the complexes comprising the site-directed
polypeptide. The target nucleic acid can be collected by standard
nucleic acid purification methods (e.g., a commercially available
PCR purification kit, an agarose gel).
[0910] The collected target nucleic acid will be prepared for
sequencing analysis (e.g., deep sequencing) by ligation of one or
more adapters as described herein.
[0911] Sequenced target nucleic acid will be analyzed to identify
polymorphisms, diagnose a disease, determine a course of treatment
for a disease, and/or generate antibody libraries.
Example 7
Sequencing Target Nucleic Acids
[0912] The eluted target nucleic acids will be prepared for
sequencing analysis. Preparation for sequencing analysis will
include the generation of sequencing libraries of the eluted target
nucleic acid. Sequencing analysis will determine the identity and
frequency of off-target binding sites of site-directed
polypeptides.
[0913] Sequence determination will be performed using methods that
determine many (typically thousands to billions) nucleic acid
sequences in an intrinsically parallel manner, where many sequences
are read out preferably in parallel using a high throughput serial
process. Such methods can include but are not limited to
pyrosequencing (for example, as commercialized by 454 Life
Sciences, Inc., Branford, Conn.); sequencing by ligation (for
example, as commercialized in the SOLiD.TM. technology, Life
Technology, Inc., Carlsbad, Calif.); sequencing by synthesis using
modified nucleotides (such as commercialized in TruSeq.TM. and
HiSeg.TM. technology by Illumina, Inc., San Diego, Calif.,
HeliScope.TM. by Helicos Biosciences Corporation, Cambridge, Mass.,
and PacBio RS by Pacific Biosciences of California, Inc., Menlo
Park, Calif.), sequencing by ion detection technologies (Ion
Torrent, Inc., South San Francisco, Calif.); sequencing of DNA
nanoballs (Complete Genomics, Inc., Mountain View, Calif.);
nanopore-based sequencing technologies (for example, as developed
by Oxford Nanopore Technologies, LTD, Oxford, UK), capillary
sequencing (e.g, such as commercialized in MegaBACE by Molecular
Dynamics), electronic sequencing, single molecule sequencing (e.g.,
such as commercialized in SMRT.TM. technology by Pacific
Biosciences, Menlo Park, Calif.), droplet microfluidic sequencing,
sequencing by hybridization (such as commercialized by Affymetrix,
Santa Clara, Calif.), bisulfate sequencing, and other known highly
parallelized sequencing methods.
[0914] In some embodiments, sequencing will be performed by
microarray analysis.
Example 8
Generation of Antibody Libraries
[0915] The methods disclosed herein will be used to generate
protein libraries (e.g., antibody libraries). Protein libraries
will be useful for preparing expression libraries, which will be
used for screening proteins (e.g. antibodies) for use in
therapeutics, reagents, and/or diagnostics. Protein libraries will
also be useful for synthesizing and/or cloning additional
antibodies.
[0916] Protein libraries will be generated by engineering a nucleic
acid-targeting nucleic acid to hybridize to target nucleic acid
sequences encoding immunoglobulins. The complexes comprising a
site-directed polypeptide and the nucleic acid-targeting nucleic
acid will be purified using methods described herein. In some
embodiments, the nucleic acid hybridizing to the nucleic
acid-targeting nucleic acid will be the target nucleic acid and
will be eluted and sequenced, using methods described herein. In
some embodiments, the nucleic acid hybridizing to the nucleic-acid
targeting nucleic acid will not be the target nucleic acid. The
target nucleic acid will be the nucleic acid that is excised
between the cleavage sites of a plurality of complexes (e.g.,
complexes comprising a site-directed polypeptide and nucleic
acid-targeting nucleic acid). The excised target nucleic acid will
be purified and sequenced, using methods described herein.
Example 9
Genotyping
[0917] The methods disclosed herein will be used to perform Human
Leukocyte Antigen (HLA) typing. HLA genes are some of the most
polymorphic genes in humans. Understanding the genotypes of these
regions will be important for obtaining a good match for tissue and
organ transplants.
[0918] To perform HLA typing, a nucleic acid-targeting nucleic acid
will be engineered to hybridize to target nucleic acid sequences in
HLA genes. The complexes comprising a site-directed polypeptide and
the nucleic acid-targeting nucleic acid will be purified using
methods described herein. In some embodiments, the nucleic acid
hybridizing to the nucleic acid-targeting nucleic acid will be the
target nucleic acid and will be eluted and sequenced, using methods
described herein. In some embodiments, the nucleic acid hybridizing
to the nucleic-acid targeting nucleic acid will not be the target
nucleic acid. The target nucleic acid will be the nucleic acid that
is excised between the cleavage sites of a plurality of complexes
(e.g., complexes comprising a site-directed polypeptide and nucleic
acid-targeting nucleic acid). The excised target nucleic acid will
be purified and sequenced, using methods described herein.
Example 10
Site-Directed Polypeptide Immunoprecipitation
[0919] The disclosure provides methods for nuclease
immunoprecipitation and sequencing (NIP-Seq). In some embodiments,
the method will comprise a) contacting a nucleic acid sample with
an enzymatically inactive site-directed polypeptide, wherein the
enzymatically inactive site-directed polypeptide binds a target
nucleic acid, thereby forming a complex, b) capturing the complex
with a capture agent, and c) sequencing the target nucleic acid. In
some embodiments, the method will further comprise d) determining
the identity of the off-target binding site.
[0920] In some embodiments, the methods of the disclosure will be
performed outside of cells. For example, a sample will comprise
purified genomic DNA.
[0921] The site-directed polypeptide-target nucleic acid complexes
will be fixed or cross-linked to form complexes.
[0922] The nucleic acid (e.g., genomic DNA) will be treated to
fragment the DNA before affinity purification. Fragmentation can be
performed through physical, mechanical or enzymatic methods.
Physical fragmentation can include exposing a target polynucleotide
to heat or to ultraviolet (UV) light. Mechanical disruption may be
used to mechanically shear a target polynucleotide into fragments
of the desired range. Mechanical shearing may be accomplished
through a number of methods known in the art, including repetitive
pipetting of the target polynucleotide, sonication and
nebulization. Target polynucleotides may also be fragmented using
enzymatic methods. In some cases, enzymatic digestion may be
performed using enzymes such as using restriction enzymes.
Restriction enzymes may be used to perform specific or non-specific
fragmentation of target polynucleotides. The methods may use one or
more types of restriction enzymes, generally described as Type I
enzymes, Type II enzymes, and/or Type III enzymes. Type II and Type
III enzymes are generally commercially available and well known in
the art. Type II and Type III enzymes recognize specific sequences
of nucleotide nucleotides within a double stranded polynucleotide
sequence (a "recognition sequence" or "recognition site"). Upon
binding and recognition of these sequences, Type II and Type III
enzymes cleave the polynucleotide sequence. In some cases, cleavage
will result in a polynucleotide fragment with a portion of
overhanging single stranded DNA, called a "sticky end." In other
cases, cleavage will not result in a fragment with an overhang,
creating a "blunt end." The methods may comprise use of restriction
enzymes that generate either sticky ends or blunt ends.
[0923] Once fragmented, the complexes comprising the site-directed
polypeptide will be purified by incubation with a solid support For
example, if the site-directed polypeptide comprises a biotin tag,
the solid support will be coated with avidin or streptavidin to
bind to the biotin tag.
[0924] In an alternative embodiment, once fragmented, the complexes
comprising the site-directed polypeptide, the target nucleic acid,
and/or the nucleic acid-targeting nucleic acid, will be purified by
incubation with a capture agent. The capture agent will bind to the
affinity tag fused to the site-directed polypeptide. The capture
agent will comprise an antibody. For example, if the affinity tag
fused to the site-directed polypeptide is a FLAG tag, then the
capture agent will be an anti-FLAG-tag antibody.
[0925] The capture agent will be purified with a solid support. For
example, if the capture agent comprises a biotin tag, the bead will
be coated with avidin or streptavidin to bind the biotinylated
capture agent.
[0926] In some embodiments of the method, two or more rounds of
purification will be performed. A first round will comprise
purification with a solid support that can bind to the affinity tag
of the capture agent and a second round will comprise purification
with a solid support that can bind to the affinity tag of the
site-directed polypeptide. A first round will comprise purification
with a solid support that will bind to the affinity tag of the
site-directed polypeptide and a second round will comprise
purification with a solid support that will bind to the affinity
tag of the capture agent.
[0927] In some embodiments, the method will be used to optimize the
binding specificity of a site-directed polypeptide by performing
the method more than once.
[0928] The captured complex will comprise site-directed polypeptide
and a target nucleic acid. The target nucleic acid will be eluted
from the site-directed polypeptide complex by standard methods
including high salt washing, ethanol precipitation, boiling, gel
purification, and the like.
[0929] The eluted DNA will be prepared for sequencing analysis
using standard methods. The sequencing libraries will be sequenced
and analyzed to identify the sequence, and frequency of
nuclease-binding sites.
[0930] In some embodiments, the method will be performed a
plurality of times. In some embodiments, the method further
comprises collecting data and storing data. The data can be stored
collected and stored on a computer server.
Example 11
In Vivo Site-Directed Polypeptide Immunoprecipitation
[0931] In some embodiments, the method will comprise: a)
introducing an enzymatically inactive site-directed polypeptide
into a cell, wherein the enzymatically inactive site-directed
polypeptide binds a target nucleic acid, thereby forming a complex,
b) capturing the complex with a capture agent, and c) sequencing
the target nucleic acid. In some embodiments, the method will
further comprise d) determining the identity of the off-target
binding site.
[0932] In some instances, the site-directed polypeptide will
comprise an affinity tag. Polypeptides comprising an affinity tag
have been described herein.
[0933] Cells will be fixed or cross-linked. Fixed and/or
cross-linked cells will be lysed. Lysis conditions will be chosen
to maintain intact protein-DNA complexes. The lysate will be
treated to fragment the DNA before affinity purification. Suitable
fragmentation techniques are described herein.
[0934] Once fragmented, the complexes comprising the site-directed
polypeptide, the target nucleic acid and/or the nucleic
acid-targeting nucleic acid, will be purified from the lysate by
incubation with a solid support. For example, if the site-directed
polypeptide comprises a biotin tag, the solid support will be
coated with avidin or streptavidin to bind to the biotin tag.
[0935] In an alternative embodiment, once fragmented, the complexes
comprising the site-directed polypeptide, will be purified from the
lysate by incubation with a capture agent. The capture agent will
bind to the affinity tag fused to the site-directed polypeptide.
The capture agent will comprise an antibody. For example, if the
affinity tag fused to the site-directed polypeptide is a FLAG tag,
then the capture agent will be a FLAG-tag antibody.
[0936] In some embodiments, the capture agent will comprise an
affinity tag. The capture agent will be purified with a solid
support. The solid support will bind to the affinity tag of the
capture agent. For example, if the capture agent comprises a biotin
tag, the bead will be coated with avidin or streptavidin to bind
the biotinylated capture agent.
[0937] In some embodiments of the method, two rounds of
purification will be performed. In some instances, a first round
will comprise purification with a solid support that will bind to
the affinity tag of the capture agent and a second round will
comprise purification with a solid support that will bind to the
affinity tag of the site-directed polypeptide. In some instances, a
first round will comprise purification with a solid support that
will bind to the affinity tag of the site-directed polypeptide, and
a second round will comprise purification with a solid support that
will bind to the affinity tag of the capture agent.
[0938] In some embodiments, the method will be used to optimize the
binding specificity of a site-directed polypeptide by performing
the method more than once.
[0939] The captured complex will comprise site-directed polypeptide
and a target nucleic acid. The target nucleic acid will be eluted
from the site-directed polypeptide complex by standard methods
including high salt washing, ethanol precipitation, boiling, gel
purification, and the like.
[0940] The eluted DNA will be prepared for sequencing analysis.
Sequencing libraries will be made from the eluted target nucleic
acid. The sequencing libraries will be sequenced and analyzed to
identify the sequence, and frequency of nuclease-binding sites.
Example 12
Immunoprecipitation with an Enzymatically Inactive Endoribonuclease
Capture Agent
[0941] A method for determining the identity of an off-target
binding site of a nuclease will comprise: a) contacting a nucleic
acid sample with a enzymatically inactive site-directed polypeptide
and a nucleic acid-targeting nucleic acid, wherein the
enzymatically inactive site-directed polypeptide and nucleic
acid-targeting nucleic acid binds a target nucleic acid, thereby
forming a complex, b) capturing the complex with a capture agent,
wherein the capture agent comprises a conditionally enzymatically
inactive site-directed polypeptide, c) sequencing the target
nucleic acid, and d) determining the identity of the off-target
binding site. This method is designed to be performed on a
cell-free nucleic acid sample, and/or a nucleic acid sample
originating from a cell.
[0942] The site-directed polypeptide-target nucleic acid complexes
will be fixed or cross-linked to form complexes.
[0943] If the nucleic acid sample originates from cells, the fixed
and/or cross-linked complexes will be lysed. Lysis conditions can
be chosen to maintain intact protein-DNA complexes. The lysate will
be treated to fragment the DNA before affinity purification. If the
nucleic acid sample originates from a cell-free sample, the
cell-free nucleic acid will be treated to fragment the DNA.
Suitable fragmentation techniques are described herein.
[0944] In some embodiments, the nucleic acid-targeting nucleic acid
will comprise an affinity tag. The affinity tag will comprise a
sequence that can bind to a conditionally enzymatically inactive
site-directed polypeptide. In some instances, the affinity tag will
comprise a sequence that can bind to a conditionally enzymatically
inactive endoribonuclease. In some instances, the affinity tag will
comprise a sequence that can bind to a conditionally enzymatically
inactive Csy4 protein. The conditionally enzymatically inactive
site-directed polypeptide will bind, but not cleave, the affinity
tag. The affinity tag will comprise the nucleotide sequence
5'-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3'. The affinity tag will be
introduced into a nucleic acid using standard recombinant
methods.
[0945] Once fragmented, the complexes comprising the site-directed
polypeptide, the target nucleic acid, and/or the nucleic
acid-targeting nucleic acid will be purified by incubation with a
conditionally enzymatically inactive site-directed polypeptide
(e.g., variant Csy4).
[0946] In some embodiments, the conditionally enzymatically
inactive site-directed polypeptide will comprise an affinity
tag.
[0947] The conditionally enzymatically inactive site-directed
polypeptide will be purified with a solid support. The solid
support will bind to the affinity tag of the conditionally
enzymatically inactive site-directed polypeptide. For example, if
the conditionally enzymatically inactive site-directed polypeptide
comprises a biotin tag, the bead will be coated with avidin or
streptavidin to bind the biotinylated capture agent.
[0948] In some embodiments, the enzymatically inactive
site-directed polypeptide will be immobilized on any of a variety
of insoluble support.
[0949] In some embodiments of the method, two rounds of
purification will be performed. In some instances, a first round
will comprise purification with a solid support that will bind to
the affinity tag of the conditionally enzymatically inactive
site-directed polypeptide (e.g., variant Csy4) and a second round
will comprise purification with a solid support that will bind to
the affinity tag of the site-directed polypeptide. In some
instances, a first round will comprise purification with a solid
support that will bind to the affinity tag of the site-directed
polypeptide and a second round will comprise purification with a
solid support that will bind to the affinity tag of the
conditionally enzymatically inactive site-directed polypeptide
(e.g., variant Csy4).
[0950] In some embodiments, the method will be used to optimize the
binding specificity of a site-directed polypeptide by performing
the method more than once.
[0951] The captured complex will comprise a site-directed
polypeptide and a target nucleic acid. The target nucleic acid will
be eluted from the site-directed polypeptide complex by standard
methods including high salt washing, ethanol precipitation,
boiling, gel purification, and the like.
[0952] The eluted DNA will be prepared for sequencing analysis. The
sequencing libraries will be sequenced and analyzed to identify the
sequence, and frequency of nuclease-binding sites.
Example 13
Sequencing
[0953] The eluted target nucleic acids will be prepared for
sequencing analysis. Preparation for sequencing analysis will
include the generation of sequencing libraries of the eluted target
nucleic acid. Sequencing analysis will determine the identity and
frequency of off-target binding sites of site-directed
polypeptides.
[0954] Sequence determination will also be performed using methods
that determine many (typically thousands to billions) nucleic acid
sequences in an intrinsically parallel manner, where many sequences
are read out preferably in parallel using a high throughput serial
process. Such methods include but are not limited to pyrosequencing
(for example, as commercialized by 454 Life Sciences, Inc.,
Branford, Conn.); sequencing by ligation (for example, as
commercialized in the SOLiD.TM. technology, Life Technology, Inc.,
Carlsbad, Calif.); sequencing by synthesis using modified
nucleotides (such as commercialized in TruSeq.TM. and HiSeg.TM.
technology by Illumina, Inc., San Diego, Calif., HeliScope.TM. by
Helicos Biosciences Corporation, Cambridge, Mass., and PacBio RS by
Pacific Biosciences of California, Inc., Menlo Park, Calif.),
sequencing by ion detection technologies (Ion Torrent, Inc., South
San Francisco, Calif.); sequencing of DNA nanoballs (Complete
Genomics, Inc., Mountain View, Calif.); nanopore-based sequencing
technologies (for example, as developed by Oxford Nanopore
Technologies, LTD, Oxford, UK), and other known highly parallelized
sequencing methods.
Example 14
Modification of a Target Nucleic Acid with an Effector Protein
[0955] A vector comprising a site-directed polypeptide, a nucleic
acid-targeting nucleic acid, and/or an effector protein is
introduced into a cell. Once inside the cell a complex is formed
comprising the elements encoded in the vector. The nucleic
acid-targeting nucleic acid is modified with a Csy4 protein binding
sequence. The effector protein, Csy4, binds to the modified nucleic
acid-targeting nucleic acid. Csy4 comprises a non-native sequence
(e.g., a fusion), that modifies a target nucleic acid. The
non-native sequence is a sequence that modifies the transcription
of the target nucleic acid. The non-native sequence is a
transcription factor. The transcription factor increases the level
of transcription of the target nucleic acid. In some cases, the
non-native sequence is a methylase. The methylase results in
increases in methylation of the target nucleic acid. In some cases
the non-native sequence is a demethylase. The demethylase results
in decreases in methylation of the target nucleic acid. In some
cases, the non-native sequence is a Rad51-recruiting peptide. The
Rad51-recruiting peptide increases the level of homologous
recombination at the target site. In some cases, the non-native
sequence is a BCRA-2 recruiting peptide. The BRCA-2-recruiting
peptide increases the level of homologous recombination at the
target site.
Example 15
Use of a Site-Directed Polypeptide as a Biosensor for a Genetic
Mobility Event
[0956] A vector(s) comprising a site-directed polypeptide, a
nucleic acid-targeting nucleic acid, and/or an effector protein is
introduced into a cell. The site-directed polypeptide and effector
proteins are fused to cellular localization sequences (e.g. a
nuclear localization signal). Once inside the cell a complex is
formed comprising the elements encoded in the vector(s). In some
instances, two vectors are introduced into the cell. The vector(s)
encodes for a first effector protein (Csy4) that comprises a first
inactive portion of a split green fluorescent protein (GFP) and
binds to a first nucleic acid-targeting nucleic acid and a second
effector protein (Csy4, Cas5, or Cas6) that comprises a second
inactive portion of the split GFP and binds to a second nucleic
acid-targeting nucleic acid. The first nucleic acid-targeting
nucleic acid is modified with a first Csy4, Cas5 or Cas6 protein
binding sequence that can be bound by a first Csy4, Cas5 or Cas6
protein. The second nucleic acid-targeting nucleic acid is modified
with a second Csy4, Cas5 or Cas6 protein binding sequence that can
be bound by a second Csy4, Cas5 or Cas6 protein. In some
embodiments, the first Csy4, Cas5 or Cas6 protein interacts with
the first Csy4, Cas5 or Cas6 protein binding sequence, and the
second Csy4, Cas5 or Cas6 protein interacts with the second Csy4,
Cas5 or Cas6 protein binding sequence. When the first and second
nucleic acid-targeting nucleic acids direct the site-directed
polypeptide to bind to two sequences that are in close proximity,
the first effector protein and the second effector protein will
bring the first inactive portion of the split GFP into contact with
the second inactive portion of the split GFP, to generate an active
GFP. The nucleic acid-targeting nucleic acids of the complex are
designed such that one nucleic acid-targeting nucleic acid guides
the complex to, for example, a region at or near the Bcr gene, and
another nucleic acid-targeting nucleic acid guides the complex to,
for example, a region at or near the Abl gene. If a translocation
event has not occurred the Bcr gene is on chromosome 22 and the Abl
gene is on chromosome 9, and the target nucleic acid sequences are
sufficiently far enough apart such that the two inactive portions
of the split GFP system are unable to interact, thereby not
generating a signal. If a translocation event has occurred, the Bcr
gene and the Abl gene are translocated such that the genes are
close together. In this instance, the target nucleic acid sequences
are sufficiently close enough together such that the two inactive
portions of the split GFP system come together to form an active
GFP. A GFP signal can be detected by a fluorometer. The signal is
indicative of a particular genotype resulting from the genetic
mobility event.
Example 16
Use of a Site-Directed Polypeptide as a Biosensor for a Genetic
Mutation
[0957] The system described in Example 2 can also be used to detect
the presence of specific mutation within a cell. In this example, a
first nucleic-acid targeting nucleic acid is chosen to direct the
site directed polynucleotide to a native sequence located near a
mutation site. The second nucleic-acid targeting nucleic acid is
chosen to recognize a mutant sequence (e.g., the mutant sequence
having been identified by DNA sequencing). The nucleic-acid
targeting nucleic acid is chosen such that the mutant sequence
occurs within the first 12 nucleic acids immediately 5' to the PAM
sequence in the site. In this instance, the target nucleic acid
sequences are sufficiently close enough together such that the two
inactive portions of the split GFP system come together to form an
active GFP. A GFP signal can be detected by a fluorometer. The
signal is indicative of a particular genotype
Example 17
Use of a Site-Directed Polypeptide as as a Therapeutic for Diseases
that Comprise a Genetic Mobility Event
[0958] A vector (s) comprising a site-directed polypeptide, a
nucleic acid-targeting nucleic acid, and/or an effector protein, a
nucleic acid comprising a cell-lysis inducing peptide (e.g.
Adenovirus death protein) operably linked to a first promoter will
also be introduced into the cell. Once inside the cell a complex is
formed comprising the elements encoded in the vector(s). In some
instances, two vectors are introduced into the cell. The vector (s)
encodes for a first effector protein (comprising a first Csy4, Cas5
or Cas6 protein sequence) that binds to a first nucleic
acid-targeting nucleic acid and comprises an activator domain for a
first transcription factor that binds to the first promoter and a
second effector protein (comprising a second Csy4, Cas5 or Cas6
protein sequence) that binds to a second nucleic acid-targeting
nucleic acid and comprises the DNA binding domain for the first
transcription factor. The first nucleic acid-targeting nucleic acid
is modified with a first Csy4, Cas5 or Cas6 protein binding
sequence that can be bound by a first Csy4, Cas5 or Cas6 protein
sequence. The second nucleic acid-targeting nucleic acid is
modified with a second Csy4, Cas5 or Cas6 protein binding sequence
that can be bound by a second Csy4, Cas5 or Cas6 protein. In some
embodiments, the first Csy4, Cas5 or Cas6 protein interacts
preferentially with the first Csy4, Cas5 or Cas6 protein binding
sequence, and the second Csy4, Cas5 or Cas6 protein interacts
preferentially with the second Csy4, Cas5 or Cas6 protein binding
sequence. If a diseased cell comprises a genome containing a
genetic mobility event, when the first and second nucleic
acid-targeting nucleic acids direct the site-directed polypeptide
to bind to two sequences that are in close proximity, the first
effector protein and the second effector protein will bring the
activator domain and the DNA-binding domain of the first
transcription factor into close proximity. The DNA-binding domain
of the first transcription factor can bind to the first promoter
operably linked to the cell-lysis inducing peptide, and the
proximal activator domain will induce transcription of RNA encoding
the cell-lysis inducing peptide. In a non-diseased cell, that does
not comprise the genetic mobility event, the DNA-binding domain and
the activator domains of the first transcription factor will not be
brought into close proximity, and there will be no transcription of
the cell-lysis inducing peptide.
[0959] The nucleic acid-targeting nucleic acids of the complex are
designed such that one nucleic acid-targeting nucleic acid guides
the complex to, for example, a region at or near the Bcr gene, and
another nucleic acid-targeting nucleic acid guides the complex to,
for example, a region at or near the Abl gene. In a non-diseased
cell, a translocation event has not occurred, the Bcr gene is on
chromosome 22 and the Abl gene is on chromosome 9, and the target
nucleic acid sequences are sufficiently far enough apart such that
the two inactive portions of the transcription factor system are
unable to interact, and cannot induce transcription of the
cell-lysis inducing peptide. In a diseased cell, in which a
translocation event has occurred, the Bcr gene and the Abl gene are
translocated such that the genes are close together. In this
instance, the target nucleic acid sequences are sufficiently close
enough together such that the two inactive portions of the
transcription factor system come together to induce transcription
of the cell-death inducing peptide. Cell-lysis will be dependent
upon a particular genotype resulting from the genetic mobility
event.
Example 18
Use of a Site-Directed Polypeptide as a Therapeutic for Diseases
that Comprise a Genetic Mutation
[0960] The system described in Example 4 can also be used to detect
the presence of specific mutation within a cell. In this example,
the first nucleic-acid targeting nucleic acid is chosen to direct
the site directed polynucleotide to a native sequence located near
a mutation site. The second nucleic-acid targeting nucleic acid is
chosen to recognize a mutant sequence (e.g., the mutant sequence
having been identified by DNA sequencing). The nucleic-acid
targeting nucleic acid is chosen such that the mutant sequence
occurs within the first 12 nucleic acids immediately 5' to the PAM
sequence in the site. In this instance, the target nucleic acid
sequences are sufficiently close enough together such that the two
inactive portions of the transcription factor come together to
enable transcription of a cell-lysis inducing peptide.
Example 19
Recruiting the Immune System to Attack Diseased Tissue Containing a
Genetic Mobility Event or a Genetic Mutation
[0961] The system described in Example 4 and/or 5 can also be used
to direct transcription by the split transcription factor system
that will result in the display of an antigen on the cell surface.
In some instances, the antigen is a peptide displayed by an MHC
class II molecules. In some instances, the antigen is a
cell-surface protein that recruits immune effector cells to the
site.
Example 20
Detecting Three-Dimensional Position of Nucleic Acids
[0962] A vector (s) comprising a site-directed polypeptide, a
nucleic acid-targeting nucleic acid, and/or an effector protein is
introduced into a cell. Once inside the cell a complex is formed
comprising the elements encoded in the vector(s). Two vectors are
introduced into the cell. One vector encodes for an effector
protein (Csy4) that comprises a first inactive portion of a split
affinity tag system. A second vector encodes for an effector
protein (Csy4, Cas5, or Cas6) that comprises a second inactive
portion of the split affinity tag. The nucleic acid-targeting
nucleic acid of the complexes is modified with a Csy4, Cas5 or Cas6
protein binding sequence. The effector proteins bind to the
modified nucleic acid-targeting nucleic acid. The nucleic
acid-targeting nucleic acids are designed to guide the complexes to
regions of interest in a three-dimensional nucleic acid structure
(e.g., chromatin). If the target sequences are not close together
in space, the two inactive portions of the split affinity tag are
unable to interact. If the target sequences are close together in
space, then the two inactive portions of the split affinity tag can
come together to form the whole affinity tag.
[0963] The cells are lysed and the cell lysis is incubated with an
antibody that binds to the affinity tag. The antibody is purified,
thereby purifying the affinity tag and the nucleic acid to which
the complexes are bound. The purified nucleic acid is dissociated
from the complexes using high salt wash. The dissociated purified
nucleic acid is prepared for sequencing analysis, and sequenced.
The sequencing results correspond to regions of chromatin that are
close together in three-dimensional space. The sequencing results
can be used to further understand gene expression and treat
disease.
Example 21
Multiplex Genome Engineering
[0964] A vector comprising a multiplexed genetic targeting agent
comprising nucleic acid modules which comprise a nucleic
acid-targeting nucleic acid and an endoribonuclease binding
sequence is introduced into a cell. In some embodiments, the cell
already comprises a site-directed polypeptide and an
endoribonuclease. In some instances, the cell is contacted with a
vector comprising a polynucleotide sequence encoding a
site-directed polypeptide and a vector comprising a polynucleotide
sequence encoding an endoribonuclease. In some instances, the cell
is contacted with a vector comprising a polynucleotide sequence
encoding both the site-directed polypeptide and the
endoribonuclease. In some embodiments, the vector comprises a
polynucleotide sequence encoding one or more endoribonucleases. In
some embodiments, the vector comprises a polynucleotide sequence
encoding a multiplexed genetic targeting agent, a site-directed
polypeptide, and one or more endoribonucleases. The array is
transcribed into RNA. The one or more endoribonucleases binds to
the one or more endoribonuclease binding sequences in the
multiplexed genetic targeting agent. The one or more
endoribonucleases cleaves the one or more endoribonuclease binding
sequences in the multiplexed genetic targeting agent, thus
liberating the individual nucleic acid modules. In some
embodiments, the nucleic acid modules comprise all, some, or none,
of the endoribonuclease binding sequence.
[0965] The liberated nucleic acid modules bind to site-directed
polypeptides, thereby forming complexes. The complexes are targeted
to one or more target nucleic acids. The one or more nucleic acid
modules hybridizes to the one or more target nucleic acids. The one
or more site-directed polypeptides cleaves the one or more target
nucleic acids at a cleavage site defined by the nucleic acid
module, thus resulting in one or more modified target nucleic
acids.
[0966] In some embodiments, one or more donor polynucleotides
and/or a vectors encoding the same are introduced into the cell.
One or more donor polynucleotides are incorporated into the one or
more cleaved target nucleic acids, thereby resulting in one or more
modified target nucleic acids (e.g., addition). In some instances,
the same donor polynucleotide is incorporated into multiple
cleavage sites. In some instances, one or more donor
polynucleotides are incorporated into multiple cleavage sites. In
some instances, no donor polynucleotide and/or vector encoding the
same are introduced into the cells. In these instances, the
modified target nucleic acid can comprise a deletion.
Example 22
Method of Stoichiometric Delivery of RNA to a Cell
[0967] In some embodiments, the disclosure provides for a method
for stoichiometric delivery of nucleic acids to the nucleus of a
cell. In some embodiments, three stoichiometrically deliverable
nucleic acid are used: one encoding for Cas9, one encoding for a
nucleic-acid targeting nucleic acid, and one encoding Csy4. Each of
the three nucleic acids comprises a Csy4-binding site.
[0968] In some embodiments, the method provides for a tandem fusion
polypeptide. The fusion polypeptide comprises three Csy4
polypeptides. The three Csy4 polypeptides are separated by a
linker. The three Csy4 polypeptides bind to the Csy4-binding sites
on each of the three nucleic acid molecules, thereby forming a
complex.
[0969] In some embodiments, the complex is formed outside of a cell
and introduced into the cell. The complex is formed by mixing the
three stoichiometrically deliverable nucleic acids and the fusion
protein and letting the reaction occur to allow binding between the
tandem fusion polypeptide and three Csy4-binding sites. The complex
is introduced by injection, electroporation, transfection,
transformation, viral transduction, and the like. Inside the cell,
some of the nucleic acids of the complex are translated. In some
embodiments, the resulting translation products are Csy4 and
NLS-Cas9 (e.g., Cas9 comprising an NLS. The NLS may not have to be
at the N-terminus). The Csy4 cleaves the Csy4-binding site on the
nucleic acid encoding the nucleic acid-targeting nucleic acid,
thereby liberating the nucleic acid-targeting nucleic acid from the
tandem fusion polypeptide. NLS-Cas9 binds the liberated nucleic
acid-targeting nucleic acid, thereby forming a unit. This unit
translocates to the nucleus. Inside the nucleus, the unit is guided
to a target nucleic acid that hybridizes with the spacer of the
nucleic acid-targeting nucleic acid. The Cas9 of the unit cleaves
the target nucleic acid. The cleavage of the target nucleic acid by
Cas9 is referred to as genome engineering.
[0970] In some embodiments, the complex is formed inside of a cell.
A vector encoding the three stoichiometrically deliverable nucleic
acids is introduced into the cell. Three different vectors encoding
one of each of the three stoichiometrically deliverable nucleic
acids is introduced the cell. Two vectors are introduced into the
cell, wherein one of the two vectors encodes for two
stoichiometrically deliverable nucleic acids and one of the two
vectors encodes for one stoichiometrically deliverable nucleic
acid. Any of the vectors can encode the tandem fusion
polypeptide.
[0971] Inside the cell, vector nucleic acid encoding RNA or a
polypeptide is transcribed into RNA. A complex comprising the three
nucleic acids and the tandem fusion polypeptide is formed, whereby
each of the Csy4-binding proteins binds to the Csy4-binding site on
each of the three nucleic acids. The nucleic acids of the complex
are translated. In some embodiments, the resulting translation
products are Csy4 and NLS-Cas9 (e.g., Cas9 comprising an NLS. The
NLS may not have to be at the N-terminus). The Csy4 cleaves the
Csy4-binding site on the nucleic acid encoding the nucleic
acid-targeting nucleic acid, thereby liberating the nucleic
acid-targeting nucleic acid from the tandem fusion polypeptide.
NLS-Cas9 binds the liberated nucleic acid-targeting nucleic acid,
thereby forming a unit. This unit translocates to the nucleus.
Inside the nucleus, the unit is guided to a target nucleic acid
that hybridizes with the spacer of the nucleic acid-targeting
nucleic acid. The Cas9 of the unit cleaves the target nucleic
acid.
Example 23
Method of Stoichiometric Delivery of Multiple Nucleic
Acid-Targeting Nucleic Acids to a Cell
[0972] In some embodiments, the disclosure provides for a method
for stoichiometric delivery of a plurality of nucleic acids to a
cell, wherein some of the plurality of nucleic acids are nucleic
acid-targeting nucleic acids. In some embodiments, the plurality of
nucleic acids comprises four nucleic acids: one encoding for Cas9,
two encoding for nucleic-acid targeting nucleic acids, and one
encoding for Csy4.
[0973] The nucleic acids comprise two or more nucleic acid-binding
protein binding sites. In some instances, the first nucleic
acid-binding protein binding site (e.g., the more 5' site)
comprises a Csy4 binding site. In some instances, the second
nucleic acid-binding protein binding site (e.g., the more 3' site)
comprises a different nucleic acid-binding protein binding site
(e.g., MS2 binding site). In some instances, the second nucleic
acid-binding protein binding site from each of the
stoichiometrically deliverable nucleic acids is different. For
example, the second nucleic acid-binding protein binding site can
be a site that binds one of a CRISPR polypeptide (e.g., Cas5, Cas6)
In some instances, the nucleic acid encoding Cas9 also comprises a
nuclear localization signal (NLS).
[0974] In some embodiments, the tandem fusion polypeptide comprises
three nucleic acid-binding proteins. The three nucleic acid-binding
proteins are Csy4, Cas5, Cas6. The tandem fusion polypeptide
comprises a nuclear localization signal. The tandem fusion
polypeptides comprises more than one copy of a nucleic acid-binding
protein (e.g., 2 copies of Csy4, one copy of Cas5, one copy of
Cas6).
[0975] In some embodiments, a complex comprising the tandem fusion
protein and the four nucleic acids is formed outside of a cell. The
complex is formed by mixing the four nucleic acids and the tandem
fusion protein and letting the reaction occur to allow binding
between the tandem fusion polypeptide and four nucleic acid-binding
protein binding sites. The complex is introduced into the cell.
Introduction occurs by either transformation, transfection, viral
transduction, microinjection, or electroporation, or any technique
capable of introducing biomolecules across a cell membrane. The
complex is formed inside the cell (e.g., after introduction of
vectors comprising nucleic acid sequences encoding the nucleic
acids and tandem fusion protein).
[0976] Inside the cell, the nucleic acids encoding Csy4 and Cas9
are translated, resulting in Csy4 and NLS-Cas9 (e.g., Cas9
comprising an NLS. The NLS may not have to be at the N-terminus).
Csy4 cleaves the Csy4-binding site on the nucleic acids encoding
the nucleic acid-targeting nucleic acids, thereby liberating them
from the tandem fusion polypeptide.
[0977] NLS-Cas9 binds the liberated nucleic acid-targeting nucleic
acids, thereby forming a plurality units. The units translocate to
the nucleus. Inside the nucleus, the units are guided to a target
nucleic acid that hybridizes with the spacer of the nucleic
acid-targeting nucleic acid of the unit. The Cas9 of the unit
cleaves the target nucleic acid.
Example 24
Method of Stoichiometric Delivery of RNA and a Donor
Polynucleotide
[0978] The disclosure provides for a method of stoichiometric
delivery of RNA components that can be used in genome engineering.
The method can also comprise delivery of a donor polynucleotide
that can be inserted into a site of genome engineering.
[0979] The disclosure provides for a method for stoichiometric
delivery of a plurality of RNAs and a donor polynucleotide to a
cell. In some embodiments, the plurality of RNAs comprises three
RNAs and a DNA. In some instances, the three RNAs are: one encoding
for Cas9, one encoding for Csy4, and one encoding for a nucleic
acid-targeting nucleic acid. The DNA is a DNA encoding a donor
polynucleotide.
[0980] In some instances, the RNAs comprise a plurality of nucleic
acid-binding protein binding sites (e.g., two nucleic acid-binding
protein binding sites). The first nucleic acid-binding protein
binding site (e.g., the more 5' site) comprises a Csy4 binding
site. The second nucleic acid-binding protein binding site (e.g.,
the more 3' site comprises a different nucleic acid-binding protein
binding site). The second nucleic acid-binding protein binding site
in each of the nucleic acids of the disclosure is different. For
example, the second nucleic acid-binding protein binding site binds
a CRISPR polypeptide (e.g., Cas5, Cas6) and/or a DNA binding
protein (e.g., a zinc finger protein). The nucleic acids of the
method also comprise a sequence encoding for a nuclear localization
signal (e.g., the RNA encoding Cas9, and the DNA encoding a donor
polynucleotide).
[0981] In some embodiments, the tandem fusion polypeptide comprises
four nucleic acid-binding proteins (e.g., RNA-binding proteins and
DNA-binding proteins). In some instances, three nucleic
acid-binding proteins are Csy4, Cas5, Cas6, and the fourth nucleic
acid-binding protein is a DNA-binding protein (e.g., zinc finger
protein). In some instances, the tandem fusion polypeptide
comprises a nuclear localization signal.
[0982] In some embodiments, a complex comprising the tandem fusion
protein and the nucleic acids (e.g., three RNAs and one DNA) is
formed outside of a cell. The complex is formed by mixing the
nucleic acids (e.g., three RNAs and one DNA) and the tandem fusion
protein and letting the reaction occur to allow binding between the
tandem fusion polypeptide and four RNA-binding protein binding
sites. The complex can be introduced into the cell. The complex is
formed inside the cell (e.g., after introduction of vectors
comprising nucleic acid sequences encoding the nucleic acids and
tandem fusion protein).
[0983] Inside the cell, the RNAs encoding Csy4 and Cas9 are
translated, resulting in Csy4 and NLS-Cas9 (e.g., Cas9 comprising
an NLS. The NLS may not have to be at the N-terminus as written
here). Csy4 can cleave the Csy4-binding site on the RNAs encoding
the nucleic acid-targeting nucleic acid and the DNA, thereby
liberating them from the tandem fusion polypeptide. In some
instances, the liberated donor polynucleotide translocates to the
nucleus by its nuclear localization signal.
[0984] NLS-Cas9 binds the liberated nucleic acid-targeting nucleic
acid, thereby forming a unit. The unit translocates to the nucleus.
Inside the nucleus, the unit is guided to a target nucleic acid
that hybridizes with the spacer of the nucleic acid-targeting
nucleic acid of the unit. The Cas9 of the unit cleaves the target
nucleic acid. The donor polynucleotide, a portion of the donor
polynucleotide, a copy of the donor polynucleotide, or a portion of
a copy of the donor polynucleotide can be inserted into the cleaved
target nucleic acid.
Example 25
Seamless Selection of Genetically Modified Cells
[0985] A plurality of cells is contacted with a vector comprising
sequences encoding a polypeptide homologous to Cas9, a nucleic
acid-targeting nucleic acid and a donor polynucleotide. In some
cases, one or more of the sequences encoding the polypeptide
homologous to Cas9, the nucleic acid-targeting nucleic acid and the
donor polynucleotide are located on different vectors. e cells are
transfected with the vector. In some instances, the cells are
infected with a virus carrying the vector. In some instances, the
cell already comprises a protein homologous to Cas9 and the vector
does not encode this polypeptide. In some instances, the cell
already comprises a CRISPR system (e.g. Cas proteins, crRNA and
tracrRNA) and the vector only encodes the donor polynucleotide. The
donor polynucleotide comprises sequences encoding a genetic element
of interest and a reporter element. The reporter element comprises
nucleic acid-targeting nucleic acid sequences, a protein homologous
to Cas 9 and a fluorescent protein. The nucleic acid-targeting
nucleic acid guide Cas9 to a target nucleic acid (e.g. a site in
the host cell genome), resulting in a double stranded DNA break of
the target nucleic acid and insertion of the donor polynucleotide.
Insertion of the donor polynucleotide is screened for by screening
for the reporter. In some cases, screening comprises
fluorescence-activated cell sorting. Screening comprises multiple
selection methods. Cas9 and/or the nucleic acid-targeting nucleic
acids are controlled by an inducible promoter. After selecting a
population of cells that comprise the reporter signal, the reporter
element is removed by activating the inducible promoter, which
transcribes the nucleic acid-targeting nucleic acids and the
site-directed polypeptide of the donor polynucleotide. The
transcribed nucleic acid-targeting nucleic acids and the
transcribed site-directed polypeptide can form complexes. One
complex can be targeted to the 3' end of the reporter element of
the donor polynucleotide. One complex can be targeted to the 5' end
of the reporter element of the donor polynucleotide. The 3' and 5'
ends of the reporter element can be cleaved. The cleaved target
nucleic acid can be rejoined by cellular mechanisms, thereby
resulting in an in-frame nucleic acid sequence encoding the same
nucleic acid sequence as prior to insertion of the donor
polynucleotide. In this way, the reporter element is seamlessly
inserted and removed from cells.
Example 26
Method for Cas9 Cleavage Using Engineered Nucleic Acid-Targeting
Nucleic Acids
[0986] Reagents
[0987] pCB002 plasmid containing temp3 target DNA sequence was
digested with 1U of AscI per 1 ug of DNA to linearize the vector.
The reaction was stopped by incubating reaction mix at 80 C for 20
min. Reaction was then purified using Qiagen PCR clean up kit.
[0988] Single guide nucleic acid were generated using T7 High Yield
RNA Synthesis Kit (Cat No. E2040S), using half the volume of
reagents recommended by the manufactures for RNA of >300
nucleotides. Generally between 200-350 ng of template were used in
a 20 uL reaction that incubates for 16 hours. Samples were treated
with DNase, and purified using Thermo GeneJet RNA purification Kit
(Cat No. K0732), and eluted in 20 uL. Typical yields range from
1.4-2 ug/uL.
[0989] At the start of cleavage assay set-up, all sgRNA were
diluted to 3500 nM concentration and heat shocked at 80 C for 15
minutes. Samples were removed from heating element and allowed to
equilibrate to room temperature. An aliquot of 4 uL of single guide
nucleic acid-targeting nucleic acid can be run out on an agarose
gel to confirm RNA integrity.
[0990] Aliquots of Cas9 at 2-2.5 mg/mL were removed from freezer
and thawed as quickly as possible and then diluted in 1.times.
cleavage buffer to appropriate stock concentration.
[0991] Cleavage Assay
[0992] A master mix of water, 5.times. cleavage buffer of (100 mM
HEPES, 500 mM KCl, 25 mM MgCl2, 5 mM DTT, and 25% glycerol at pH
7.4), and Cas9 to 250 nM was aliquoted into thin wall PCR tubes.
sgRNA was added to appropriate tubes at a final concentration of
250 nM.
[0993] The reaction was incubated at 37 C for 10 minutes, 10 nM
linearized plasmid was added and reactions (final reaction volume
20 uL) were incubated at 37 C for and hour. The reaction was
terminated by heating reaction to 60 C for 20 minutes. 10 uL
aliquots were mixed with 2 uL 6.times.DNA loading dye and analyzed
by electrophoresis on a 1.5% agarose gel stained with SYBR safe.
The appearance of a .about.2800 bp and .about.1300 bp fragments was
indicative of Cas9 mediated cleavage. The results of the
experiments are shown in FIGS. 6, 10 and 11. All synthetic guide
RNA sequences designed and shown in FIG. 5 supported sgRNA
cleavage, except for SGRv8, in which the entire complementary
region was inverted. These results indicate that different regions
of the sgRNA can be amenable to engineering and still retain
function.
[0994] Engineered nucleic acid-targeting nucleic acids were tested
in the assay described above. FIGS. 22A and 22B shows the designs
of an initial set of duplex variants in single-guide nucleic
acid-targeting nucleic acid backbone variants used to test
targeting and cleavage activity.
[0995] FIGS. 23A and 23B illustrate a second set of duplex variants
with smaller modifications in the duplex. V28 comprises a 2 base
insertion 3' to the complementary region; V29 comprises a 3 base
deletion 3' to the complementary region.
[0996] FIGS. 24A and 24B illustrate tracr variants which comprise
mutations in the tracrRNA portion of the nucleic acid-targeting
nucleic acid (i.e., minimum tracrRNA sequence and 3' tracrRNA
sequence). V38-V41 comprise fusions between the complementary
region/duplex and the 3' ends of the tracr nucleic acid sequences
from M. mobile 163K (v38), S. thermophilus LMD-9 (V39), C. jejuni
(V40), and N. meningitides.
[0997] FIGS. 25A and 25B depict variants comprising modifications
to enable Csy4 binding to the nucleic acid-targeting nucleic acid.
The additional hairpin sequences are derived from the CRISPR repeat
in Pseudomonas aeruginosa PA14.
[0998] FIG. 26A-C shows data from the in vitro cleavage assay
demonstrating the activity of the nucleic acid-targeting nucleic
acid variants on Cas9 cleavage. Variant SGRv8 failed to support
target nucleic acid cleavage (FIG. 26B lane 9).
[0999] FIG. 27 and FIG. 28 shows two independent repeats of the
Cas9 cleavage assay testing the variants depicted in FIGS.
23-25.
[1000] Additional engineered nucleic acid-targeting nucleic acids
were made as listed in Table 2, and tested in the same assay. The
results of the assay are shown in FIG. 37 and listed in the
activity column of Table 2.
[1001] The results of these experiments indicate the importance of
the bulge and P-domain regions in effecting target nucleic acid
cleavage. The functionality of variants 42-45 indicates that the
addition of a Csy4 binding sequence to a nucleic acid-targeting
nucleic acid does not disrupt target nucleic acid cleavage.
Example 27
Sequencing Analysis Systems
[1002] FIG. 30 depicts a system that is configured to implement the
methods of the disclosure. The system can include a computer server
("server") that is programmed to implement the methods described
herein. FIG. 30 depicts a system 3000 adapted to enable a user to
detect, analyze, and communicate sequencing results of for example,
nuclease-targeted enriched nucleic acids, sequenced target nucleic
acids, data concerning the methods of the disclosure, diagnose a
disease, genotype a patient, make a patient-specific treatment
decision, or any combination thereof. The system 3000 includes a
central computer server 3001 that is programmed to implement
exemplary methods described herein. The server 3001 includes a
central processing unit (CPU, also "processor") 3005 which can be a
single core processor, a multi core processor, or plurality of
processors for parallel processing. The server 3001 also includes
memory 3010 (e.g. random access memory, read-only memory, flash
memory); electronic storage unit 3015 (e.g. hard disk);
communications interface 3020 (e.g. network adaptor) for
communicating with one or more other systems; and peripheral
devices 3025 which may include cache, other memory, data storage,
and/or electronic display adaptors. The memory 3010, storage unit
3015, interface 3020, and peripheral devices 3025 are in
communication with the processor 3005 through a communications bus
(solid lines), such as a motherboard. The storage unit 3015 can be
a data storage unit for storing data. The server 3001 is
operatively coupled to a computer network ("network") 3030 with the
aid of the communications interface 3020. The network 3030 can be
the Internet, an intranet and/or an extranet, an intranet and/or
extranet that is in communication with the Internet, a
telecommunication or data network. The network 3030 in some cases,
with the aid of the server 3001, can implement a peer-to-peer
network, which may enable devices coupled to the server 3001 to
behave as a client or a server. The microscope and micromanipulator
can be peripheral devices 3025 or remote computer systems 3040.
[1003] The storage unit 3015 can store files, such as sequencing
results, target binding sites, personalized genetic data,
genotypes, images, data analysis of images and/or sequencing
results, or any aspect of data associated with the disclosure.
[1004] The server can communicate with one or more remote computer
systems through the network 3030. The one or more remote computer
systems may be, for example, personal computers, laptops, tablets,
telephones, Smart phones, or personal digital assistants.
[1005] In some situations the system 3000 includes a single server
3001. In other situations, the system includes multiple servers in
communication with one another through an intranet, extranet and/or
the Internet.
[1006] The server 3001 can be adapted to store sequencing results,
target binding sites, personalized genetic data, and/or other
information of potential relevance. Such information can be stored
on the storage unit 3015 or the server 3001 and such data can be
transmitted through a network.
[1007] Methods as described herein can be implemented by way of
machine (or computer processor) executable code (or software)
stored on an electronic storage location of the server 3001, such
as, for example, on the memory 3010, or electronic storage unit
3015. During use, the code can be executed by the processor 3005.
In some cases, the code can be retrieved from the storage unit 3015
and stored on the memory 3010 for ready access by the processor
3005. In some situations, the electronic storage unit 3015 can be
precluded, and machine-executable instructions are stored on memory
3010. Alternatively, the code can be executed on a second computer
system 3040.
[1008] Aspects of the systems and methods provided herein, such as
the server 3001, can be embodied in programming. Various aspects of
the technology may be thought of as "products" or "articles of
manufacture" typically in the form of machine (or processor)
executable code and/or associated data that is carried on or
embodied in a type of machine readable medium. Machine-executable
code can be stored on an electronic storage unit, such memory
(e.g., read-only memory, random-access memory, flash memory) or a
hard disk. "Storage" type media can include any or all of the
tangible memory of the computers, processors or the like, or
associated modules thereof, such as various semiconductor memories,
tape drives, disk drives and the like, which may provide
non-transitory storage at any time for the software programming.
All or portions of the software may at times be communicated
through the Internet or various other telecommunication networks.
Such communications, for example, may enable loading of the
software from one computer or processor into another, for example,
from a management server or host computer into the computer
platform of an application server. Thus, another type of media that
may bear the software elements includes optical, electrical, and
electromagnetic waves, such as used across physical interfaces
between local devices, through wired and optical landline networks
and over various air-links. The physical elements that carry such
waves, such as wired or wireless likes, optical links, or the like,
also may be considered as media bearing the software. As used
herein, unless restricted to non-transitory, tangible "storage"
media, terms such as computer or machine "readable medium" refer to
any medium that participates in providing instructions to a
processor for execution.
[1009] Hence, a machine readable medium, such as
computer-executable code, may take many forms, including but not
limited to, tangible storage medium, a carrier wave medium, or
physical transmission medium. Non-volatile storage media can
include, for example, optical or magnetic disks, such as any of the
storage devices in any computer(s) or the like, such may be used to
implement the system. Tangible transmission media can include:
coaxial cables, copper wires, and fiber optics (including the wires
that comprise a bus within a computer system). Carrier-wave
transmission media may take the form of electric or electromagnetic
signals, or acoustic or light waves such as those generated during
radio frequency (RF) and infrared (IR) data communications. Common
forms of computer-readable media therefore include, for example: a
floppy disk, a flexible disk, hard disk, magnetic tape, any other
magnetic medium, a CD-ROM, DVD, DVD-ROM, any other optical medium,
punch cards, paper tame, any other physical storage medium with
patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM,
any other memory chip or cartridge, a carrier wave transporting
data or instructions, cables, or links transporting such carrier
wave, or any other medium from which a computer may read
programming code and/or data. Many of these forms of computer
readable media may be involved in carrying one or more sequences of
one or more instructions to a processor for execution.
Example 28
Array-Based Sequencing Using a Site-Directed Polypeptide
[1010] A nucleic acid sample is ligated with a nucleic acid tag
comprising a single guide RNA and a detectable label. Together, the
nucleic acid sample ligated to the nuclei acid tag is referred to
as a tagged test sample. The tagged test sample is contacted to a
microarray comprising immobilized oligonucleotides. The immobilized
oligonucleotides are a double stranded nucleic acid library. The
oligonucleotides comprise a detectable label (e.g., fluorescent
label. The individual members of the tagged test sample hybridize
to the oligonucleotides to which they share enough complementarity
to facilitate hybridization. The amount of hybridization can be
quantified by comparing the intensities of the two detectable
labels from the sample library and the immobilized
oligonucleotides. For example, hybridized oligonucleotides can
display two detectable labels (that from the sample library and the
oligonucleotide). Unhybridized oligonucleotides can display one
detectable label (that from the oligonucleotide). The hybridized
probes are contacted with Cas9. Cas9 cleaves the oligonucleotides
in the microarray that have hybridized with members of the tagged
test sample. Cleavage by the site-directed polypeptide allows the
hybridized members of the tagged test sample to be removed. After
cleavage by the site-directed polypeptide, only unhybridized
oligonucleotide detectable labels remain on the microarray. The
remaining detectable label is quantified. The quantification of the
remaining detectable labels is correlated to which sequences were
represented in the nucleic acid sample and which were not (e.g., by
position mapping). Oligonucleotides that do not display a remaining
detectable label correspond to sequences that were represented in
the nucleic acid sample. Oligonucleotides that display a remaining
detectable label correspond to sequences that were not represented
in the nucleic acid sample.
Example 29
Cleavage of a Target Nucleic Acid with a Tagged Nucleic
Acid-Targeting Nucleic Acid
[1011] This example describes the results of target nucleic acid
cleavage with a nucleic acid-targeting nucleic acid comprising a
Csy4 binding sequence on the 5' end of the nucleic acid-targeting
nucleic acid. Cas9 was incubated with or without a guide RNA
targeting a single site in a linear double stranded DNA sequence.
After 1 hour, cleavage products were separated and visualized on an
agarose gel. FIG. 13D illustrates that Cas9 cleavage mediated by a
tagged nucleic acid-targeting nucleic acid (lane 3) was less
efficient than Cas9 cleavage mediated by an untagged nucleic
acid-targeting nucleic acid (lane 1). After 1 hour, .about.100% of
the target was cleaved by Cas9 guided by the untagged nucleic
acid-targeting nucleic acid, where as only a small fraction of the
target was cleaved in the same time by Cas9 guided by the tagged
nucleic acid-targeting nucleic acid. These experiments indicate the
location of the non-native sequence can be used to tune cleavage
effectiveness of cleavage of the Cas9: nucleic acid-targeting
nucleic acid complex. For example, FIG. 27 and FIG. 29 show the
functionality of the addition of a Csy4 binding sequence to various
locations in the nucleic acid-targeting nucleic acid that retain
activity.
Example 30
Genome Engineering Genes in Blood Disorders
[1012] A nucleic acid-targeting nucleic acid comprising a spacer
sequence described in Figure XX is introduced into a cell with a
site-directed polypeptide, thereby forming a complex. The complex
targets the gene involved in a blood disorder that has substantial
complementarity to the spacer sequence of the nucleic
acid-targeting nucleic acid. Once the nucleic acid-targeting
nucleic acid is hybridized to the target nucleic acid the
site-directed polypeptide cleaves the target nucleic acid. The
cleaved target nucleic acid is can be engineered with a donor
polynucleotide.
Example 31
Protocol to Determine Target Nucleic Acid Cleavage and
Modification
[1013] This protocol can be used to determine if a target nucleic
acid has been cleaved or if the target nucleic acid has been
modified such as with an insertion or deletion. Primers surrounding
the target site are used to PCR amplify, in a 25 .mu.L reaction, a
500-600 nt product from gDNA. The primers comprise at least 100 nt
on either side of the cut site. The resulting products from the
cleavage assay are about greater than 100 nt.
[1014] About 5 .mu.L of PCR product is run on an agarose gel to
determine if amplification is clean. With the remaining PCR product
the melt and annealing protocol is as follows:
TABLE-US-00012 95.degree. C. 10 min 95.degree. C. to 85.degree. C.
(-2.0.degree. C./s) 85.degree. C. 1 min 85.degree. C. to 75.degree.
C. (-0.3.degree. C./s) 75.degree. C. 1 min 75.degree. C. to
65.degree. C. (-0.3.degree. C./s) 65.degree. C. 1 min 65.degree. C.
to 55.degree. C. (-0.3.degree. C./s) 55.degree. C. 1 min 55.degree.
C. to 45.degree. C. (-0.3.degree. C./s) 45.degree. C. 1 min
45.degree. C. to 35.degree. C. (-0.3.degree. C./s) 35.degree. C. 1
min 35.degree. C. to 25.degree. C. (-0.3.degree. C./s) 25.degree.
C. 1 min 4.degree. C. Hold
[1015] A T7E1 Master Mix of water, NEB2 buffer, and T7E1 enzyme is
prepared. Multiply for each reaction, plus extra. Table 8 shows the
components of the T7E1 master mix.
TABLE-US-00013 TABLE 8 Reaction components for T7E1 Master Mix. 1X
reaction Water 7.5 .mu.L NEB 2 2 .mu.L buffer T7E1 0.5 .mu.L enzyme
PCR 10 .mu.L product Total 20 .mu.L
[1016] To each reaction, in a 200 .mu.L, strip cap tube, the
following reagents are added: T7E1 master mix (10 .mu.L), and PCR
sample (10 .mu.L). The reaction is incubated at 37.degree. C. for
25 minutes.
[1017] Loading buffer is added to the sample and the entire sample
is run on a 3% gel at 120 V for 20 minutes. The gel may be run
longer if more resolution is required. Image and save the gel
image.
[1018] The image is quantified to determine the amount of cleavage
of the target nucleic acid.
Example 32
Cellular Testing of Nucleic Acid-Targeting Nucleic Acid
Variants
[1019] This example shows that nucleic acid-targeting nucleic acid
variants depicted in FIGS. 22, 24 and 25 and Example 26 were tested
in a cell-based assay to determine if the in vitro functionality
determined in Example 26 matched in vivo functionality. HEK293
cells were grown to 60-70% confluence in 10 cm dishes. Cells were
removed by trypsinization, counted using a hemocytometer and then
separated in to aliquots of 7.times.10.sup.4 cells for each well to
be transfected. For each well, 250 ng pCB045 plasmid expressing
mammalian codon-optimized Cas9 was mixed with 30 ng guide RNA and
40 ng copGFP DNA and 0.5u1 Lipofectamine 2000 in a final volume of
50u1 DMEM. DNA and lipid were incubated for 15 minutes prior to
transfection. Transfections were carried out at the time of
plating, by adding the lipid:DNA mix to 450u1 DMEM+10% Fetal Bovine
Serum containing 7.times.10.sup.4 cells. The transfection cell mix
was added to 96 well tissue culture plates coated with Rat Tail
Collagen I. Cells were incubated at 37 C in an incubator with 5%
CO2 for 40 hours.
[1020] Media was removed from each of the wells and cells were
lysed using Quickextract solution (Epicentre) according to
manufacturer's instructions. DNA harvested from QuickExtract lysis
was diluted 1:10 and used as a template in PCR reactions for T7E1
assays as described in Example 30.
[1021] FIG. 36 indicates that all variants except v8 and v9 were
able to cleave target nucleic acid. Nucleic acid-targeting nucleic
acid variant v8 was also substantially inactive in in vitro assays
as depicted in FIG. 23B. Nucleic acid-targeting nucleic acid
variant v9 was very weakly active in the in vitro assay depicted in
FIG. 23B.
Example 33
Determining a Cell Fate with a Tagged Cell
[1022] This example describes how to track a cell developing from a
cell lineage. A hematopoietic stem cell (e.g., a hemocytoblast) is
contacted with a site-directed polypeptide, a nucleic
acid-targeting nucleic acid, and a donor polynucleotide. The
site-directed polypeptide and nucleic acid-targeting nucleic acid
form a complex and target a region of the hematopoietic genome for
cleavage. Once cleaved, the donor polynucleotide is inserted into
the cleaved site in the hepatopoietic cell's genome. The
hematopoietic stem cell is induced to differentiate through normal
differentiation processes. At different stages of differentiation
the sample comprising the differentiated hematopoietic cells can be
assayed for the presence of the donor polynucleotide. In this way,
the differentiation process of a cell can be tracked.
Example 34
Clone Double-Stranded Oligonucleotide Encoding a Nucleic
Acid-Targeting Nucleic Acid into a Linearized Vector
[1023] This example describes how to generate a double-stranded
oligonucleotide encoding a portion of nucleic acid-targeting
nucleic acid (e.g., a spacer) and insert it into a linearized
vector. The linearized vector or a closed supercoiled vector
comprises a sequence encoding a site-directed polypeptide (e.g.,
Cas9), a promoter driving expression of the sequence encoding the
site-directed polypeptide (e.g., CMV promoter), a sequence encoding
a linker (e.g., 2A), a sequence encoding a marker (e.g., CD4 or
OFP), a sequence encoding portion of a nucleic acid-targeting
nucleic acid, a promoter driving expression of the sequence
encoding a portion of the nucleic acid-targeting nucleic acid, and
a sequence encoding a selectable marker (e.g., ampicillin), or any
combination thereof.
[1024] Equal amounts of two single-stranded oligonucleotides are
annealed together (e.g., 50 micromolar). The two single-stranded
oligonucleotides can hybridize together. At least one of the two
single-stranded oligonucleotides is complementary to a target
nucleic acid (e.g., a 10-30 nucleotide region adjacent to a
protospacer adjacent motif in a target). At least one of the two
single-stranded nucleotides comprises a 3' overhang sequence
comprising the sequence 5'-GTTT-3'. At least one of the two
single-stranded oligonucleotides comprises a 3' overhang comprising
the sequence 5'-CGGTG-3'. In some instances, one of the two
single-stranded oligonucleotides comprises a 5'-GTTT-3' overhang
and the other of the two single-stranded oligonucleotides comprises
a 5'-CGGTG-3'. Annealing is performed in an annealing buffer
comprising at least 10 mM tris HCl pH 8.0, 1 mM EDTA, pH 8.0, and
100 mM NaCl. Annealing is performed by heating the oligonucleotide
mixture at 95 C for 3-5 minutes, removing the oligonucleotide
mixture from the heating source, and allowing the mixture to cool
to room temperature for 5-10 minutes. The double-stranded
oligonucleotide mixture is centrifuged gently. After annealing the
mixture may be stored at 4 C or -20 C. The mixture, now of
double-stranded oligonucleotides, is diluted to prepare two stock
solutions of 500 nanomolar and 5 nanomolar. The stock solutions are
prepared by diluting the oligonucleotide mixture in water.
[1025] The double-stranded oligonucleotide (dsOligonucleotide) is
ligated into a linearized vector. The linearized vector comprises a
sequence encoding a site-directed polypeptide (e.g., Cas9), a
marker protein (e.g., orange fluorescent protein), and/or a
sequence encoding a nucleic acid-targeting nucleic acid, wherein
the linearized vector is linearized at a region of the sequence
encoding the nucleic acid-targeting nucleic acid, such that the
sticky ends generated match the overhang ends of the
dsOligonucleotide. The ligation reaction can comprise 1.times.
ligation buffer (e.g., 50 mM Tris-HCl pH 7.6, 5 mM MgCl.sub.2, 1 mM
ATP, 1 mM DTT, and/or 5% PEG 8000), 30 nanogram linearized vector,
5 nM dsOligonucleotide, and DNA ligase (e.g., 4 microliters
5.times. ligation buffer, 2 microliters linearized vector at 15
nanogram/microliter, 2 microliters 5 nanomolar dsOligonucleotide,
11 microliters water, 1 microliter T4 DNA ligase). The reaction is
mixed. The reaction is incubated at room temperature for 10
minutes-2 hours. The reaction is placed on ice and transformed into
competent cells.
[1026] Transformation into competent cells comprises transforming
into chemically competent TOP10 E. coli cells. Competent cells are
thawed on ice. 3 microliters of the reaction mixture is added to
the competent cells and mixed gently. The cells are incubated on
ice for 10-30 minutes. The cells are heat-shocked for 30 seconds at
42 C. The cells are transferred to ice for 2 minutes. 250
microliters of medium (SOC or LB) is added to teh cells. The cells
are shaked at 200 rpm for 1 hour at 37 C. The cells are then spread
on an agar plate comprising 100 micrograms/milliliter ampicillin
and stored overnight at 37 C.
[1027] The transformants are analyzed. For example, the
transformants are analyzed to determine the identity of the
dsOligonucleotide ligated into the vector, and/or confirm the
ligation is not a false positive. To analyze transformants,
colonies are picked and cultured overnight in LB medium comprising
100 micrograms/milliliter ampicillin at 37 C. The plasmid
comprising the site-directed polypeptide and dsOligonucleotide is
isolated (e.g., by miniprep kit). A sequencing reaction is
performed on the isolated plasmid. The sequencing reaction utilizes
a sequencing primer that is designed to sequence the
dsOligonucleotide (e.g., the sequencing primer is a U6 sequencing
primer that binds to the U6 promoter which is located just upstream
of the sequence encoding the dsOligonucleotide.
[1028] Once a desired dsOligonucleotide insertion is identified,
the plasmid can be stored at -20 C or in a glycerol stock at -80 C.
To make a glycerol stock, the original colony comprising the
desired plasmid is streaked on an agar plate comprising 100
micrograms/milliliter ampicillin and incubated overnight at 37 C. A
single colony is isolated grown in LB comprising 100
micrograms/milliliter ampicillin until the culture reaches
stationary phase. The culture is mixed with glycerol and flash
frozen in liquid nitrogen (e.g., 0.85 mL culture is mixed with 0.15
mL glycerol).
[1029] The purified plasmid comprising the desired
dsOligonucleotide is inserted into a cell line (e.g., mammalian
cell line, HeLa) by transfection. To transfect the plasmid, the
plasmid is purified at high concentrations using for example, a
maxi prep kit. The plasmid is transfected with lipid-based buffer
(e.g., Lipofectamine 2000) into cells which are plated at 70%
confluency. 3 micrograms of the vector is transfected into the
cells.
Example 35
Nanoparticle Delivery of an Engineered Nucleic Acid-Targeting
Nucleic Acid
[1030] A nanoparticle encapsulating a nucleic acid encoding an
engineered nucleic acid-targeting nucleic acid and a site-directed
polypeptide will be prepared. Nanoparticles will be prepared by
mixing DOPE, Chol, DSPE-PEG and C.sub.16mPEG-Ceramide at a molar
ratio of 18:60:20:1:1 in 10 mL of 90% ethanol (total lipid 30
.mu.mole). The nucleic acid will be dissolved in 10 mL of 20 mM
Tris buffer (pH 7.4-7.6). After being heated to 37.degree. C., the
two solutions will be mixed together through a duel syringe pump
and the mixed solution will be subsequently diluted with 20 mL of
20 mM Tris buffer (300 mM NaCl, pH 7.4-7.6). The mixture will be
incubated at 37.degree. C. for 30 minutes and dialyzed in 10 mM PBS
buffer (138 mM NaCl, 2.7 mM KCl, pH 7.4). Stable particles will be
obtained after the removal of ethanol from the mixture by dialysis.
The nanoparticle solution will be concentrated by centrifugation at
3,000 rpm and a temperature of 4.degree. C. The concentrated
suspension will be collected after a given time and will be
sterilized by filtration through a 0.22 .mu.m syringe filter
(Millex-GV, Millipore, USA). A homogeneous suspension of the
nanoparticles comprising the nucleic acid encoding the engineered
nucleic acid-targeting nucleic acid and the site-directed
polypeptide will be obtained.
[1031] The nanoparticles will be contacted to a cell. The
nanoparticle will enter the cell. Inside the cell, the nanoparticle
will release the nucleic acid encoding the engineered nucleic
acid-targeting nucleic acid and the site-directed polypeptide. The
nucleic acid will be transcribed and/or translated to produce an
engineered nucleic acid-targeting nucleic acid that binds to a
site-directed polypeptide protein, thereby forming a complex. The
complex will target a target nucleic acid that hybridizes with the
engineered nucleic acid-targeting nucleic acid. The complex will
cleave the target nucleic acid.
[1032] In some instances, the nanoparticle will further comprise a
nucleic acid encoding a donor polynucleotide. When the target
nucleic acid is cleaved by the site-directed polypeptide, the donor
polynucleotide will be inserted into the site of the cleaved target
nucleic acid.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20160046949A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20160046949A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References