U.S. patent application number 14/835675 was filed with the patent office on 2017-02-09 for compositions and methods of engineered crispr-cas9 systems using split-nexus cas9-associated polynucleotides.
The applicant listed for this patent is Caribou Biosciences, Inc.. Invention is credited to Paul Daniel Donohoue, Andrew Paul May.
Application Number | 20170037432 14/835675 |
Document ID | / |
Family ID | 56694254 |
Filed Date | 2017-02-09 |
United States Patent
Application |
20170037432 |
Kind Code |
A1 |
Donohoue; Paul Daniel ; et
al. |
February 9, 2017 |
COMPOSITIONS AND METHODS OF ENGINEERED CRISPR-CAS9 SYSTEMS USING
SPLIT-NEXUS CAS9-ASSOCIATED POLYNUCLEOTIDES
Abstract
The present specification discloses engineered Type II
CRISPR-Cas9 systems comprising split-nexus Cas9-associated
polynucleotides (sn-casPNs), including systems comprising three
split-nexus Cas9-associated polynucleotides
(sn1-casPN/sn2-casPN/sn3-casPN) and systems comprising two
split-nexus Cas9-associated polynucleotides (sn1-casPN/sn2-casPN).
Together with a Cas9 protein, the sn-casPNs facilitate
site-specific modifications, including cleavage and mutagenesis, of
a target polynucleotide in vitro and in vivo. Furthermore, the
engineered Type II CRISPR-Cas9 systems comprising sn-casPNs are
useful in methods of regulating expression of a target nucleic
acid. Methods are described herein for the creation of a variety of
engineered Type II CRISPR-Cas9 systems comprising two or more
sn-casPNs. Polynucleotide sequences, expression cassettes, vectors,
compositions, and kits for carrying out a variety of methods are
also described. Furthermore, the present specification provides
genetically modified cells, compositions of modified cells,
transgenic organisms, pharmaceutical compositions, as well as a
variety of compositions and methods involving the engineered Type
II CRISPR-Cas9 systems.
Inventors: |
Donohoue; Paul Daniel;
(Berkeley, CA) ; May; Andrew Paul; (San Francisco,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Caribou Biosciences, Inc. |
Berkeley |
CA |
US |
|
|
Family ID: |
56694254 |
Appl. No.: |
14/835675 |
Filed: |
August 25, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62202715 |
Aug 7, 2015 |
|
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|
62209334 |
Aug 24, 2015 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/113 20130101;
C12N 2310/3513 20130101; C12N 15/87 20130101; C12N 2310/531
20130101; C12N 9/96 20130101; C12N 2310/10 20130101; C12N 9/22
20130101; C12N 2310/20 20170501; C12N 15/111 20130101 |
International
Class: |
C12N 15/87 20060101
C12N015/87; C12N 9/22 20060101 C12N009/22 |
Claims
1. A Type II CRISPR-Cas9-associated polynucleotide composition
comprising: a first Type II CRISPR-Cas9-associated split-nexus
polynucleotide having a 5' end and a 3' end (sn1-casPN) comprising,
in the 5' to 3' direction, a first stem element nucleotide sequence
I and a nexus stem element nucleotide sequence I; a second Type II
CRISPR-Cas9-associated split-nexus polynucleotide having a 5' end
and a 3' end (sn2-casPN) comprising, in the 5' to 3' direction, a
nexus stem element nucleotide sequence II, wherein the nexus stem
element nucleotide sequence I of the sn1-casPN and the nexus stem
element nucleotide sequence II of the sn2-casPN are capable of
forming a nexus stem element by base-pair hydrogen bonding between
the nexus stem element nucleotide sequence I and the nexus stem
element nucleotide sequence II; and a third Type II
CRISPR-Cas9-associated polynucleotide having a 5' end and a 3' end
(sn3-casPN) comprising, in the 5' to 3' direction, a DNA target
binding sequence and a first stem element nucleotide sequence II,
wherein the first stem element nucleotide sequence I of the
sn1-casPN and the first stem element nucleotide sequence II of the
sn3-casPN are capable of forming a first stem element by base-pair
hydrogen bonding between the first stem element nucleotide sequence
I and the first stem element nucleotide sequence II.
2. (canceled)
3. (canceled)
4. The composition of claim 1, wherein the first stem element
nucleotide sequence I of the sn1-casPN further comprises, in the 5'
to 3' direction, an upper stem element nucleotide sequence I, a
bulge element nucleotide sequence I, and a lower stem element
nucleotide sequence I, and the first stem element nucleotide
sequence II of the sn3-casPN further comprises, in the 5' to 3'
direction, a lower stem element nucleotide sequence II, a bulge
element nucleotide sequence II, and an upper stem element
nucleotide sequence II, wherein the upper stem element nucleotide
sequence I of the sn1-casPN and the upper stem element nucleotide
sequence II of the sn2-casPN are capable of forming an upper stem
element by base-pair hydrogen bonding between the upper stem
element nucleotide sequence I and the upper stem element nucleotide
sequence II, and the lower stem element nucleotide sequence I of
the sn1-casPN and the lower stem element nucleotide sequence II of
the sn1-casPN are capable of forming a lower stem element by
base-pair hydrogen bonding between the lower stem element
nucleotide sequence I and the lower stem element nucleotide
sequence II.
5. The composition of claim 1, further comprising: a first adjunct
polynucleotide having a 5' end and a 3' end comprising a second
stem element nucleotide sequence II; wherein the sn2-casPN
comprises, in the 5' to 3' direction, the nexus stem element
nucleotide sequence II and a second stem element nucleotide
sequence I, and the second stem element nucleotide sequence I of
the sn2-casPN and the second stem element nucleotide sequence II of
the first adjunct polynucleotide are capable of forming a second
stem element by base-pair hydrogen bonding between the second stem
element nucleotide sequence I and the second stem element
nucleotide sequence II.
6. The composition of claim 5, wherein the first adjunct
polynucleotide further comprises, in the 5' to 3' direction, a loop
element nucleotide sequence and the second stem element nucleotide
sequence II, and wherein the 5' end of the first adjunct
polynucleotide is covalently bonded to the 3' end of the
sn2-casPN.
7. The composition of claim 6, further comprising: a second adjunct
polynucleotide having a 5' end and a 3' end comprising third stem
element nucleotide sequence II; wherein the first adjunct
polynucleotide comprises, in the 5' to 3' direction, the loop
element nucleotide sequence, the second stem element nucleotide
sequence II, and a third stem element nucleotide sequence I, and
the third stem element nucleotide sequence I of the first adjunct
polynucleotide and the third stem element nucleotide sequence II of
the second adjunct polynucleotide are capable of forming a third
stem element by base-pair hydrogen bonding between the third stem
element nucleotide sequence I and third stem element nucleotide
sequence II.
8. The composition of claim 7, wherein the second adjunct
polynucleotide further comprises, in the 5' to 3' direction, a loop
element nucleotide sequence and the third stem element nucleotide
sequence II, wherein 5' end of the second adjunct polynucleotide is
covalently bonded to the 3' end of the first adjunct
polynucleotide.
9. The composition of claim 1, wherein the sn1-casPN further
comprises a first auxiliary polynucleotide 3' adjacent the nexus
stem element nucleotide sequence I.
10. The composition of claim 9, wherein the sn2-casPN further
comprises a second auxiliary polynucleotide 5' adjacent the nexus
stem element nucleotide sequence II.
11. The composition of claim 1, wherein the sn2-casPN further
comprises an auxiliary polynucleotide 5' adjacent the nexus stem
element nucleotide sequence II.
12. The composition of claim 10, wherein the first auxiliary
polynucleotide further comprises an effector binding element
nucleotide sequence I, the second auxiliary polynucleotide further
comprises an effector binding element nucleotide sequence II, and
the effector binding element nucleotide sequence I of the first
auxiliary polynucleotide and the effector binding element
nucleotide sequence II of the second auxiliary polynucleotide are
capable of forming an effector binding element by base-pair
hydrogen bonding between the effector binding element nucleotide
sequence I and the effector binding element nucleotide sequence
II.
13. The composition of claim 12, wherein the first auxiliary
polynucleotide further comprises, in the 5' to 3' direction, a
linker element nucleotide sequence I and the effector binding
element nucleotide sequence I, the second auxiliary polynucleotide
comprises, in the 5' to 3' direction, the effector binding element
nucleotide sequence II and a linker element nucleotide sequence II,
and the linker element nucleotide sequence I of the first auxiliary
polynucleotide and the linker element nucleotide sequence II of the
second auxiliary polynucleotide are capable of forming a linker
element by base-pair hydrogen bonding between the linker element
nucleotide sequence I and the linker element nucleotide sequence
II.
14. The composition of claim 9, wherein the first auxiliary
polynucleotide comprises a hairpin.
15. The composition of claim 10, wherein the first auxiliary
polynucleotide comprises a hairpin and the second auxiliary
polynucleotide comprises a hairpin.
16. The composition of claim 11, wherein the auxiliary
polynucleotide comprises a hairpin.
17. The composition of claim 15, wherein the first auxiliary
polynucleotide further comprises, in the 5' to 3' direction, a
linker element nucleotide sequence I and the hairpin, the second
auxiliary polynucleotide further comprises, in the 5' to 3'
direction, the hairpin and a linker element nucleotide sequence II,
and the linker element nucleotide sequence I of the first auxiliary
polynucleotide and the linker element nucleotide sequence II of the
second auxiliary polynucleotide are capable of forming linker
element by base-pair hydrogen bonding between the linker element
nucleotide sequence I and the linker element nucleotide sequence
II.
18. A Type II CRISPR-Cas9-associated split-nexus polynucleotide
composition comprising: a first Type II CRISPR-Cas9-associated
split-nexus polynucleotide having a 5' end and a 3' end (sn1-casPN)
comprising, in the 5' to 3' direction, an upper stem element
nucleotide sequence I, a bulge element nucleotide sequence I, a
lower stem element nucleotide sequence I, and a nexus stem element
nucleotide sequence I; a second Type II CRISPR-Cas9-associated
split-nexus polynucleotide having a 5' end and a 3' end (sn2-casPN)
comprising, in the 5' to 3' direction, a nexus stem element
nucleotide sequence II, a second stem element comprising a hairpin,
and a third stem element comprising a hairpin, wherein the nexus
stem element nucleotide sequence I of the sn1-casPN and the nexus
stem element nucleotide sequence II of the sn2-casPN are capable of
forming a nexus stem element by base-pair hydrogen bonding between
the nexus stem element nucleotide sequence I and the nexus stem
element nucleotide sequence II; and a third Type II
CRISPR-Cas9-associated polynucleotide having a 5' end and a 3' end
(sn3-casPN) comprising, in the 5' to 3' direction, a DNA target
binding sequence, a lower stem element nucleotide sequence II, a
bulge element nucleotide sequence II, and an upper stem element
nucleotide sequence II, wherein the upper stem element nucleotide
sequence I of the sn1-casPN and the upper stem element nucleotide
sequence II of the sn3-PN are capable of forming an upper stem
element by base-pair hydrogen bonding between the upper stem
element nucleotide sequence I and the upper stem element nucleotide
sequence II, and the lower stem element nucleotide sequence I of
the sn1-PN and the lower stem element nucleotide sequence II of the
sn1-PN are capable of forming a lower stem element by base-pair
hydrogen bonding between the lower stem element nucleotide sequence
I and the lower stem element nucleotide sequence II.
19. (canceled)
20. The composition of claim 18, wherein the sn1-casPN further
comprises a first auxiliary polynucleotide 3' adjacent the nexus
stem element nucleotide sequence I, and the sn2-casPN further
comprises a second auxiliary polynucleotide 5' adjacent the nexus
stem element nucleotide sequence II.
21. A Type II CRISPR-Cas9 system, comprising: the Type II
CRISPR-Cas9-associated polynucleotide composition of claim 1; and a
Cas9 protein or a DNA sequence encoding a Cas9 protein.
22. The composition of claim 12, wherein the effector binding
element is a double-stranded RNA, and the effector binding element
comprises a Csy4 protein binding site.
23. The composition of claim 20, wherein the first auxiliary
polynucleotide further comprises an effector binding element
nucleotide sequence I, the second auxiliary polynucleotide further
comprises an effector binding element nucleotide sequence II, and
the effector binding element nucleotide sequence I of the first
auxiliary polynucleotide and the effector binding element
nucleotide sequence II of the second auxiliary polynucleotide are
capable of forming an effector binding element by base-pair
hydrogen bonding between the effector binding element nucleotide
sequence I and the effector binding element nucleotide sequence II.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 62/202,715, filed 7 Aug. 2015, now
pending, and U.S. Provisional Patent Application Ser. No.
62/209,334, filed 24 Aug. 2015, now pending, the contents of which
are herein incorporated by reference in their entireties.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not applicable.
SEQUENCE LISTING
[0003] The present application contains a Sequence Listing that has
been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. The ASCII copy, created
on 24 Aug. 2015, is named CB1017-10_ST25.txt and is 20 kb in
size.
TECHNICAL FIELD
[0004] The present invention relates to engineered Type II
CRISPR-Cas9 systems.
BACKGROUND OF THE INVENTION
[0005] Genome engineering includes altering the genome by deleting,
inserting, mutating, or substituting specific nucleic acid
sequences. The alteration can be gene or location specific. Genome
engineering can use nucleases to cut DNA, thereby generating a site
for alteration. In certain cases, the cleavage can introduce
double-stranded breaks in the target DNA. Double-stranded breaks
can be repaired, e.g., by endogenous non-homologous end joining
(NHEJ) or homology-directed repair (HDR). HDR relies on the
presence of a template for repair. In some examples of genome
engineering, a donor polynucleotide, or portion thereof, can be
inserted into the break.
[0006] Clustered regularly interspaced short palindromic repeats
(CRISPR) and associated Cas proteins constitute the CRISPR-Cas
system. This system provides adaptive immunity against foreign DNA
in bacteria (Barrangou, R., et al., "CRISPR provides acquired
resistance against viruses in prokaryotes," Science 315, 1709-1712
(2007); Makarova, K. S., et al., "Evolution and classification of
the CRISPR-Cas systems," Nat Rev Microbiol 9, 467-477 (2011);
Garneau, J. E., et al., "The CRISPR/Cas bacterial immune system
cleaves bacteriophage and plasmid DNA," Nature 468, 67-71 (2010);
Sapranauskas, R., et at., "The Streptococcus thermophilus
CRISPFt/Cas system provides immunity in Escherichia coli," Nucleic
Acids Res 39, 9275-9282 (2011)). The RNA-guided Cas9 endonuclease
specifically targets and cleaves DNA in a sequence-dependent manner
(Gasiunas, G., et at., "Cas9-crRNA ribonucleoprotein complex
mediates specific DNA cleavage for adaptive immunity in bacteria,"
Proc Natl Acad Sci USA 109, E2579-E2586 (2012); Jinek, M., et at.,
"A programmable dual-RNA-guided DNA endonuclease in adaptive
bacterial immunity," Science 337, 816-821 (2012); Sternberg, S. H.,
et al., "DNA interrogation by the CRISPR RNA-guided endonuclease
Cas9," Nature 507, 62 (2014); Deltcheva, E., et at., "CRISPR RNA
maturation by trans-encoded small RNA and host factor RNase III,"
Nature 471, 602-607 (2011)), and has been widely used for
programmable genome editing in a variety of organisms and model
systems (Cong, L., et al., "Multiplex genome engineering using
CRISPR/Cas systems," Science 339, 819-823 (2013); Jiang, W., et
al., "RNA-guided editing of bacterial genomes using CR1SPR-Cas
systems," Nat. Biotechnol. 31, 233-239 (2013); Sander, J. D. &
Joung, J. K., "CRISPR-Cas systems for editing, regulating and
targeting genomes," Nature Biotechnol. 32, 347-355. (2014)).
[0007] Jinek, M., et al., ("A programmable dual-RNA-guided DNA
endonuclease in adaptive bacterial immunity," Science
337(6096):816-21 (2012)) showed that in a subset of
CRISPR-associated (Cas) systems the mature CRISPR (crRNA) that is
base paired to trans-activating crRNA (tracrRNA) forms a two-part
RNA structure that directs the CRISPR-associated protein Cas9 to
introduce double-stranded breaks in target DNA. At sites
complementary to the crRNA-guide (spacer) sequence, the Cas9 HNH
nuclease domain cleaves the complementary strand and the Cas9
RuvC-like domain cleaves the non-complementary strand. Dual
crRNA/tracrRNA molecules were engineered into single-chain
crRNA/tracrRNA molecules. These single-chain crRNA/tracrRNA
directed target sequence-specific Cas9 double-strand DNA
cleavage.
[0008] Jinek, M., et al., designed two versions of single-chain
crRNA/tracrRNA containing a target recognition sequence (spacer) at
the 5' end followed by a hairpin structure retaining the
base-pairing interactions that normally occur between the tracrRNA
and the crRNA (see FIG. 5B of Jinek, M., et al.). For each
single-chain crRNA/tracrRNA, the 3' end of crRNA was covalently
attached to the 5' end of tracrRNA. In cleavage assays using
plasmid DNA, Jinek, M., et al., observed that a 3' truncated
single-chain crRNA/tracrRNA did not cleave target DNA as
efficiently in the assay as a longer single-chain crRNA/tracrRNA
that was not truncated at the 3' end (see FIG. 5B and FIG. S14 A,
B, and C of Jinek, M., et al.). These data confirmed that the "5 to
12 positions beyond the tracrRNA:crRNA base-pairing interaction are
important for efficient Cas9 binding and/or target recognition"
(Jinek, M., et al., Science 337(6096):820 (2012)).
[0009] Briner; A., et al., ("Guide RNA Functional Modules Direct
Cas9 Activity and Orthogonality," Molecular Cell 56(2), 2014, Pages
333-339) elucidated the molecular basis of selective Cas9/guide-RNA
interactions by identifying and characterizing distinct sequence
and structural modules within guide RNAs that direct Cas9
endonuclease activity and define orthogonality. They established
six modules within native crRNA:tracrRNA duplexes and single guide
RNAs (sg1RNAs) across forty-one systems from three distinct Cas9
families. The six identified modules are the spacer, the lower
stem, the bulge, the upper stem, the nexus hairpin, and 3'
hairpins. These modules are illustrated with reference to an sgRNA
in FIG. 2.
[0010] Using the sgRNA/Cas9 system from Streptococcus pyogenes,
Briner, A., et al., showed that a bulge within the sgRNA is
structurally necessary for DNA cleavage both in vitro and in vivo,
whereas sequence substitutions are tolerated in other regions.
Furthermore, expendable features can be removed to generate
functional miniature sgRNAs. They also identified a conserved
module "named the nexus; this feature exhibits sequence and
structural features important for cleavage" (Briner, A., et al.,
page 2). They stated that this module, the nexus, is "necessary for
DNA cleavage" (Briner, A., et al., Summary). The nexus hairpin
confers activity to its cognate Cas9. The location of this nexus
hairpin corresponds to the 5 to 12 positions beyond the
tracrRNA:crRNA base-pairing interaction that are important for
efficient Cas9 binding and/or target recognition as identified by
Jinek, M., et al. (see above).
[0011] Briner, A., et al., showed that the general nexus hairpin
shape with a GC-rich stem and an offset uracil was shared between
the two Streptococcus families. In contrast, the idiosyncratic
double stem of the nexus hairpin was unique to, and ubiquitous in,
Lactobacillus systems. Some bases within the nexus hairpin were
strictly conserved even between distinct families, including A52
and C55, further highlighting the important role of this module. In
the crystal structure of SpyCas9 A52 interacts with the backbone of
residues 1103-1107 close to the 5' end of the target strand in the
in the crystal structure of SpyCas9, suggesting that the
interaction of the nexus hairpin with the protein backbone may be
required for protospacer-adjacent motif (PAM) binding.
[0012] Wright, A. V., et al., ("Rational design of a split-Cas9
enzyme complex," PNAS 112(10), 2015, pages 2984-2989) determined
the RNA molecular determinants of sgRNA motifs that promote
heterodimerization of the .alpha.-helical and nuclease lobes to
form a ternary complex. Crystal structures of sgRNA/DNA-bound Cas9
showed that the spacer and the stem-loop motifs (i.e., the lower
stem, the bulge, and the upper stem modules described by Briner,
A., et al.) at the 5' end of the sgRNA primarily contact the
.alpha.-helical lobe, whereas the two hairpins (i.e., the hairpins
module described by Briner, A., et al.) at the 3' end bind the
outside face of the nuclease lobe. They noted that "the nexus
motif, recently shown to be critical for activity" (Wright, A. V.,
et al., page 2986, col. 1), occupies a central position between the
lobes and forms extensive interactions with the bridge helix. Based
on this interaction profile, Wright, et al., generated a
full-length sgRNA and two shorter sgRNA constructs that were
selectively truncated from either the 5' or 3' end (no
modifications were made to the critical nexus hairpin) and
determined their affinities for wild-type Cas9, the individual
.alpha.-helical and nuclease lobes, and split-Cas9.
[0013] Contrary to the above-described teachings of the prior art,
experiments performed in support of the present invention
unexpectedly demonstrated that Cas9 functions (e.g., binding and
cutting double-strand DNA) are supported by guide RNAs having a
split nexus, as well as guide RNAs having modifications of the
split nexus.
[0014] Results presented in the present specification open new
design and engineering avenues for CR1SPR technologies and set the
stage for the development of next-generation CRISPR-based
technologies.
SUMMARY OF THE INVENTION
[0015] Aspects of the present invention relate to engineered Type
II CRISPR-Cas9 system wherein at least two polynucleotides are
necessary to form a nexus stem element.
[0016] In one aspect, the present invention relates to an
engineered Type II CRISPR-Cas9 system comprising three
polynucleotides capable of forming a complex with a Cas9, protein
to cause the Cas9 protein to bind a first DNA sequence comprising a
DNA target sequence preferentially relative to a second DNA
sequence without the DNA target binding sequence. At least two of
the three polynucleotides are necessary to form a nexus stem
element. In some embodiments, the engineered Type II CRISPR-Cas9
system further comprises a Cas9 protein or a DNA sequence encoding
a Cas9 protein. In additional embodiments, the present invention
relates to the three polynucleotides in complex with a Cas9
protein.
[0017] In one embodiment, an engineered Type II CRISPR-Cas9 system
of the present invention comprises a first polynucleotide, a second
polynucleotide, and a third polynucleotide that are separate
polynucleotides each having a 5' end and a 3' end.
[0018] The first polynucleotide comprising in a 5' to 3' direction
a first stem element nucleotide sequence I and a nexus stem element
nucleotide sequence I. The second polynucleotide comprising a nexus
stem clement nucleotide sequence II, wherein the nexus stem element
nucleotide sequence I and the nexus stem element nucleotide
sequence II are capable of forming the nexus stem element by
base-pair hydrogen bonding between the nexus stem element
nucleotide sequence I and the nexus stem element nucleotide
sequence II. The third polynucleotide comprising in a 5' to 3'
direction a DNA target binding sequence and a first stem element
nucleotide sequence II, wherein the first stem element nucleotide
sequence I and the first stem element nucleotide sequence II are
capable of forming a first stem element by base-pair hydrogen
bonding between the first stem element nucleotide sequence I and
the first stem element nucleotide sequence II.
[0019] In another embodiment, an engineered Type II CRISPR-Cas9
system of the present invention comprises a first polynucleotide, a
second polynucleotide, and a third polynucleotide that are separate
polynucleotides each having a 5' end and a 3' end. The first
polynucleotide comprising in a 5' to 3' direction an upper stem
element nucleotide sequence I, a bulge element nucleotide sequence
I, a lower stem element nucleotide sequence I, and a nexus stem
element nucleotide sequence I. The second polynucleotide comprising
a nexus stem element nucleotide sequence II, wherein the nexus stem
element nucleotide sequence I and the nexus stem element nucleotide
sequence II are capable of forming the nexus stem element by
base-pair hydrogen bonding between the nexus stem element
nucleotide sequence I and the nexus stem element nucleotide
sequence II. The third polynucleotide comprising in a 5' to 3'
direction a DNA target binding sequence, a lower stem element
nucleotide sequence II, a bulge element nucleotide sequence II, and
an upper stem element nucleotide sequence II, wherein the upper
stem element nucleotide sequence I and the upper stem element
nucleotide sequence II are capable of forming an upper stem element
by base-pair hydrogen bonding between the upper stem element
nucleotide sequence I and the upper stem element nucleotide
sequence II, and the lower stem element nucleotide sequence I and
the lower stem element nucleotide sequence II are capable of
forming a lower stem element by base-pair hydrogen bonding between
the lower stem element nucleotide sequence I and the lower stem
element nucleotide sequence II.
[0020] In further embodiments the second polynucleotide comprises
first and/or second adjunct polynucleotides. The second
polynucleotide can further comprise in a 5' to 3' direction the
nexus stem element nucleotide sequence II and a second stem element
nucleotide sequence I, and a first adjunct polynucleotide that
comprises a second stem element nucleotide sequence II. The second
stem element nucleotide sequence I and the second stem element
nucleotide sequence II are capable of forming a second stem element
by base-pair hydrogen bonding between the second stem element
nucleotide sequence I and the second stem element nucleotide
sequence II. In some embodiments, the first adjunct polynucleotide
further comprises in a 5' to 3' direction a loop element nucleotide
sequence and the second stem element nucleotide sequence II,
wherein 5' end of the loop element nucleotide sequence is
covalently bonded to the 3' end of the second stem element
nucleotide sequence I, thus forming a hairpin. In yet further
embodiments, the first adjunct polynucleotide comprises in a 5' to
3' direction the second stem element nucleotide sequence II and a
third stem element nucleotide sequence 1, and a second adjunct
polynucleotide comprises in a 5' to 3' direction a third stem
element nucleotide sequence II. The third stem element nucleotide
sequence I and the third stem element nucleotide sequence II are
capable of forming a third stem clement by base-pair hydrogen
bonding between the third stem element nucleotide sequence I and
third stem element nucleotide sequence II. In some embodiments the
second adjunct polynucleotide further comprises in a 5' to 3'
direction a loop element nucleotide sequence and the third stem
element nucleotide sequence II, wherein 5' end of the loop element
nucleotide sequence is covalently bonded to the 3' end of the third
stem element nucleotide sequence I.
[0021] Additional embodiments of the present invention include the
first polynucleotide further comprising a first auxiliary
polynucleotide 3' adjacent the nexus stem element nucleotide
sequence I, the second polynucleotide further comprising a second
auxiliary polynucleotide 5' adjacent the nexus stem element
nucleotide sequence II, or both the first polynucleotide and the
second polynucleotide each comprising an auxiliary sequence.
[0022] In some embodiments of the present invention, the first
auxiliary polynucleotide comprises an effector binding element
nucleotide sequence I, and the second auxiliary polynucleotide
comprises an effector binding element nucleotide sequence II. The
effector binding element nucleotide sequence I and the effector
binding element nucleotide sequence II are capable of forming an
effector binding element by base-pair hydrogen bonding between the
effector binding element nucleotide sequence I and the effector
binding element nucleotide sequence I. The effector binding element
can be, for example, a double-stranded RNA and the effector protein
is a double-stranded RNA binding protein capable of binding the
effector binding element. In selected embodiments the effector
protein is a catalytically inactive variant of a protein selected
from the group consisting of Cas5, Cas6, and Csy4.
[0023] In additional embodiments, the first auxiliary
polynucleotide further comprises in a 5' to 3' direction a linker
element nucleotide sequence I and the effector binding element
nucleotide sequence I, and the second auxiliary polynucleotide
comprises in a 5' to 3' direction the effector binding clement
nucleotide sequence II and a linker element nucleotide sequence II.
The linker element nucleotide sequence I and the linker clement
nucleotide sequence II are capable of forming a linker element by
base-pair hydrogen bonding between the effector binding element
nucleotide sequence I and the effector binding element nucleotide
sequence I.
[0024] In yet further embodiments, the first auxiliary
polynucleotide, the second auxiliary polynucleotide, or both the
first auxiliary polynucleotide and the second auxiliary
polynucleotide each comprises a hairpin. Furthermore, the first
auxiliary polynucleotide can further comprises in a 5' to 3'
direction a linker clement nucleotide sequence I and the hairpin,
the second auxiliary polynucleotide comprises in a 5' to 3'
direction the hairpin and a linker element nucleotide sequence II,
or both the first auxiliary polynucleotide comprises in a 5' to 3'
direction a linker element nucleotide sequence I and the hairpin
and the second auxiliary polynucleotide comprises in a 5' to 3'
direction the hairpin and a linker element nucleotide sequence II.
The linker element nucleotide sequence land the linker element
nucleotide sequence II are capable of forming linker element by
base-pair hydrogen bonding between the effector binding element
nucleotide sequence I and the effector binding element nucleotide
sequence I.
[0025] In another aspect an engineered Type II CRISPR-Cas9 system
of the present invention comprises three polynucleotides. A first
polynucleotide comprises in a 5' to 3' direction an upper stem
element nucleotide sequence I, a bulge clement nucleotide sequence
I, a lower stem element nucleotide sequence I, and a nexus stem
element nucleotide sequence I. A second polynucleotide comprises in
a 5' to 3' direction a nexus stem element nucleotide sequence II, a
second stem element comprising a hairpin, and a third stem element
comprising a hairpin. The nexus stem element nucleotide sequence I
and the nexus stem element nucleotide sequence II are capable of
forming the nexus stem element by base-pair hydrogen bonding
between the nexus stem element nucleotide sequence I and the nexus
stem element nucleotide sequence II. A third polynucleotide
comprises in a 5' to 3' direction a DNA target binding sequence, a
lower stem element nucleotide sequence II, a bulge element
nucleotide sequence II, and an upper stem element nucleotide
sequence II. The upper stem element nucleotide sequence I and the
upper stem element nucleotide sequence II are capable of forming an
upper stem element by base-pair hydrogen bonding between the upper
stem element nucleotide sequence I and the upper stem element
nucleotide sequence II, and the lower stem element nucleotide
sequence I and the lower stem element nucleotide sequence II arc
capable of forming a lower stem element by base-pair hydrogen
bonding between the lower stem element nucleotide sequence I and
the lower stem element nucleotide sequence II. The engineered Type
II CRISPR-Cas9 system can further comprise a Cas9 protein or a DNA
sequence encoding a Cas9 protein.
[0026] In some embodiments of this aspect of the present invention
the first polynucleotide further comprises a first auxiliary
polynucleotide 3' adjacent the nexus stem element nucleotide
sequence I, and the second polynucleotide further comprises a
second auxiliary polynucleotide 5' adjacent the nexus stem element
nucleotide sequence II.
[0027] These aspects and other embodiments of the present invention
using the sn-casPNs/Cas9 protein systems of the present invention
will readily occur to those of ordinary skill in the art in view of
the disclosure herein.
BRIEF DESCRIPTION OF THE FIGURES
[0028] FIG. 1A and FIG. 1B present illustrative examples of dual
guide Type II CRISPR-Cas9 associated RNAs. FIG. 1A shows a two-RNA
component Type II CRISPR-Cas9 system comprising a crRNA (FIG. 1A,
101) and a tracrRNA (FIG. 1A, 102). FIG. 1B illustrates the
formation of base-pair hydrogen bonds between the crRNA and the
tracrRNA to form secondary structure (see U.S. Published Patent
Application No. 2014-0068797, published 6 Mar. 2014; see also Jinek
M., et al., "A programmable dual-RNA-guided DNA endonuclease in
adaptive bacterial immunity," Science, 2012; 337:816-21). The
figure presents an overview of and nomenclature for secondary
structural elements of the crRNA and tracrRNA of the Streptococcus
pyogenes Cas9 including the following: a spacer element (FIG. 1B,
103); a first stem element comprising a lower stem element (FIG.
1B, 104), a bulge element comprising unpaired nucleotides (FIG. 1B,
105), and an upper stem element (FIG. 1B, 106); a nexus element
(FIG. 1B, 107); a second hairpin element comprising a second stem
element (FIG. 1B, 108); and a third hairpin element comprising a
third stem element (FIG. 1B, 109). The figures are not
proportionally rendered nor are they to scale. The locations of
indicators are approximate.
[0029] FIG. 2 shows another example of a CRISPR-Cas9 associated
RNA. The figure illustrates a single guide RNA (sgRNA) wherein the
crRNA is covalently joined to the tracrRNA and forms a RNA
polynucleotide secondary structure through base-pair hydrogen
bonding (see, e.g., U.S. Published Patent Application No.
2014-0068797, published 6 Mar. 2014). The figure presents an
overview of and nomenclature for secondary structural elements of a
sgRNA of the Streptococcus pyogenes Cas9 including the following: a
spacer element (FIG. 2, 201); a first stem element comprising a
lower stem element (FIG. 2, 202), a bulge element comprising
unpaired nucleotides (FIG. 2, 205), and an upper stem element (FIG.
2, 203); a loop element (FIG. 2, 204) comprising unpaired
nucleotides; (a first hairpin element comprises the first stem
element and the loop element); a nexus element (FIG. 2, 206); a
second hairpin element comprising a second stem element (FIG. 2,
207); and a third hairpin element comprising a third stem element
(FIG. 2, 208). (See, e.g., FIGS. 1 and 3 of Briner, A. E., et al.,
"Guide RNA Functional Modules Direct Cas9 Activity and
Orthogonality," Molecular Cell Volume 56, Issue 2, 23 Oct. 2014,
Pages 333-339.) The figure is not proportionally rendered nor is it
to scale. The locations of indicators are approximate.
[0030] FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, FIG. 3F, FIG.
3G, and FIG. 3H present a variety of polynucleotides of the
engineered Type II CRISPR-Cas9 systems of the present invention.
The "split-nexus Cas9-associated polynucleotides" (sn-casPNs) of
the present invention comprise two or more polynucleotides, wherein
the polynucleotide backbone is broken within the nexus element.
These figures present exemplary sn-casPN structures. Other
modifications of sn-casPNs are described in the present
specification. The figures are not proportionally rendered nor are
they to scale. The indicators for locations corresponding to
elements are only illustrative to provide reference points in the
example polynucleotides.
[0031] Table 1 presents a series of indicators used consistently in
FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, FIG. 3F, FIG. 3G, and
FIG. 3H.
TABLE-US-00001 TABLE 1 Numerical Indicators Used to Illustrate
Regions of Nucleotide Sequence Associated with Example sn-casPNs
Indicators and Corresponding Elements Second Polynucleotide
(sn2-casPN) 304 to 305 corresponds to a split nexus stem element
nucleotide sequence II 305 to 306 corresponds to a second
connective nucleotide sequence 306 to 307 corresponds to a second
stem element nucleotide sequence I 307 to 308 corresponds to a loop
element nucleotide sequence 308 to 309 corresponds to a second stem
element nucleotide sequence II 309 to 310 corresponds to a third
connective nucleotide sequence 310 to 311 corresponds to a third
stem element nucleotide sequence I 311 to 312 corresponds to a loop
element nucleotide sequence 312 to 313 corresponds to a third stem
element nucleotide sequence II 313 to 314 corresponds to a 3'
nucleotide sequence First Polynucleotide (sn1-casPN) 315 to 316
corresponds to a split nexus stem element nucleotide sequence I 316
to 317 corresponds to a first connective nucleotide sequence 317 to
320 corresponds to a first stem element nucleotide sequence I 317
to 318 corresponds to a lower stem element nucleotide sequence I
318 to 319 corresponds to a bulge element nucleotide sequence I 319
to 320 corresponds to an upper stem element nucleotide sequence I
320 to 321 corresponds to a loop element nucleotide sequence
Additional Polynucleotides (sn3-casPN, sn4-casPN) 321 to 324
corresponds to a first stem element nucleotide sequence II 321 to
322 corresponds to an upper stem element nucleotide sequence II 322
to 323 corresponds to a bulge element nucleotide sequence II 323 to
324 corresponds to a lower stem element nucleotide sequence II 324
to 325 corresponds to a nucleic acid target binding sequence (a
spacer element)
[0032] FIG. 3A illustrates an example of a split-nexus
Cas9-associated three polynucleotide system. FIG. 3A, 301
illustrates a first polynucleotide (sn1-casPN) that comprises a
first portion of the split nexus element. FIG. 3A, 302 illustrates
a second polynucleotide (sn2-casPN) that comprises a second portion
of the split nexus element. FIG. 3A, 303 illustrates a third
polynucleotide (sn3-casPN) that comprises a spacer element.
Examples of polynucleotide secondary structures that form through
base-pair hydrogen bonding between indicated sequences include the
following: sn1-casPN split nexus stem element nucleotide sequence
I/sn2-casPN split nexus stem element nucleotide sequence II form a
split nexus stem element; sn2-casPN second stem element nucleotide
sequence I/sn2-casPN second stem element nucleotide sequence II
form a second stem element; sn2-casPN third stem element nucleotide
sequence I/sn2-casPN third stem element nucleotide sequence II form
a third stem element; sn1-casPN first stem element nucleotide
sequence I/sn3-casPN first stem element nucleotide sequence II form
a first stem element, the first stem element comprising sn1-casPN
lower stem element nucleotide sequence I/sn3-casPN lower stem
element nucleotide sequence II form a lower stem element, and
sn1-casPN upper stem element nucleotide sequence I/sn3-casPN upper
stem element nucleotide sequence II form an upper stem element.
[0033] FIG. 3B illustrates an example of a split-nexus
Cas9-associated two polynucleotide system. FIG. 3B, 326 illustrates
a first polynucleotide (sn1-casPN) that comprises a first portion
of the split nexus element. FIG. 3B, 302 illustrates a second
polynucleotide (sn2-casPN) that comprises a second portion of the
split nexus element. Examples of polynucleotide secondary
structures that form through base-pair hydrogen bonding between
indicated sequences include the following: sn1-casPN split nexus
stem element nucleotide sequence I/sn2-casPN split nexus stem
element nucleotide sequence II form a split nexus stem element;
sn2-casPN second stem element nucleotide sequence I/sn2-casPN
second stem element nucleotide sequence II form a second stem
element; sn2-casPN third stem element nucleotide sequence
I/sn2-casPN third stem element nucleotide sequence II form a third
stem element; sn1-casPN first stem element nucleotide sequence
I/sn1-casPN first stem element nucleotide sequence II form a first
stem element, the first stem element comprising sn1-casPN lower
stem clement nucleotide sequence I/sn1-casPN lower stem element
nucleotide sequence II forming a lower stem element, and sn1-casPN
upper stem element nucleotide sequence I/sn1-casPN upper stem
element nucleotide sequence II forming an upper stem element.
[0034] FIG. 3C illustrates an example of a split-nexus
Cas9-associated three polynucleotide system. FIG. 3C, 327
illustrates a first polynucleotide (sn1-casPN) that comprises a
first portion of the split nexus element. FIG. 3C, 302 illustrates
a second polynucleotide (sn2-casPN) that comprises a second portion
of the split nexus element. FIG. 3C, 328 illustrates a third
polynucleotide (sn3-casPN) that comprises a spacer element.
Examples of polynucleotide secondary structures that form through
base-pair hydrogen bonding between indicated sequences include the
following: sn1-casPN split nexus stem element nucleotide sequence
I/sn2-casPN split nexus stem clement nucleotide sequence II form a
split nexus stem element; sn2-casPN second stem element nucleotide
sequence I/sn2-casPN second stem element nucleotide sequence II
form a second stem element; sn2-casPN third stem element nucleotide
sequence I/sn2-casPN third stem clement nucleotide sequence II form
a third stem element; sn1-casPN first stem element nucleotide
sequence I/sn3-casPN first stem element nucleotide sequence II form
a first stem element.
[0035] FIG. 3D illustrates an example of a split-nexus
Cas9-associated two polynucleotide system. FIG. 3D, 329 illustrates
a first polynucleotide (sn1-casPN) that comprises a first portion
of the split nexus element. FIG. 3D, 302 illustrates a second
polynucleotide (sn2-casPN) that comprises a second portion of the
split nexus element. Examples of polynucleotide secondary
structures that form through base-pair hydrogen bonding between
indicated sequences include the following: sn1-casPN split nexus
stem element nucleotide sequence I/sn2-easPN split nexus stem
element nucleotide sequence II form a split nexus stem element;
sn2-casPN second stern element nucleotide sequence I/sn2-casPN
second stem element nucleotide sequence II form a second stem
element; sn2-casPN third stem element nucleotide sequence
I/sn2-casPN third stem element nucleotide sequence II form a third
stem element; sn1-casPN first stem element nucleotide sequence
I/sn1-casPN first stem element nucleotide sequence II form a first
stem element.,
[0036] FIG. 3E illustrates an example of a split-nexus
Cas9-associated four polynucleotide system. FIG. 3E, 301
illustrates a first polynucleotide (sn1-casPN) that comprises a
first portion of the split nexus element. FIG. 3E, 302 illustrates
a second polynucleotide (sn2-casPN) that comprises a second portion
of the split nexus element. FIG. 3E, 330 illustrates a third
polynucleotide (sn3-casPN). FIG. 3E, 331 illustrates a spacer
polynucleotide (sn4-casPN) that comprises a spacer element.
Examples of polynucleotide secondary structures that form through
base-pair hydrogen bonding between indicated sequences include the
following: sn1-casPN split nexus stem element nucleotide sequence
I/sn2-casPN split nexus stem element nucleotide sequence II form a
split nexus stem element; sn2-casPN second stem element nucleotide
sequence I/sn2-casPN second stem element nucleotide sequence II
form a second stem element; sn2-casPN third stem element nucleotide
sequence I/sn2-casPN third stem element nucleotide sequence II form
a third stem element; sn1-casPN first stem element nucleotide
sequence I/sn3-casPN first stem element nucleotide sequence II form
a first stem element, the first stem element comprising sn1-casPN
lower stem clement nucleotide sequence I/sn3-casPN lower stem
clement nucleotide sequence II forming a lower stem element, and
sn1-casPN upper stem element nucleotide sequence I/sn3-casPN upper
stem element nucleotide sequence II forming an upper stem
element.
[0037] FIG. 3F illustrates an example of a split-nexus
Cas9-associated three polynucleotide system. FIG. 3F, 332
illustrates a first polynucleotide (sn1-casPN) that comprises a
first portion of the split nexus element. FIG. 3F, 302 illustrates
a second polynucleotide (sn2-casPN) that comprises a second portion
of the split nexus element. FIG. 3F, 331 illustrates a spacer
polynucleotide (sn4-casPN) that comprises a spacer element.
Examples of polynucleotide secondary structures that form through
base-pair hydrogen bonding between indicated sequences include the
following: sn1-casPN split nexus stem element nucleotide sequence
I/sn2-casPN split nexus stem element nucleotide sequence II form a
split nexus stem element; sn2-casPN second stem element nucleotide
sequence I/sn2-casPN second stem element nucleotide sequence II
form a second stem element; sn2-casPN third stem element nucleotide
sequence I/sn2-casPN third stem element nucleotide sequence II form
a third stem element; sn1-casPN first stem element nucleotide
sequence I/sn1-casPN first stem element nucleotide sequence II form
a first stem element, the first stem element comprising sn1-casPN
lower stem element nucleotide sequence I/sn1-casPN lower stem
element nucleotide sequence II forming a lower stem element, and
sn1-casPN upper stem element nucleotide sequence I/sn1-casPN upper
stem element nucleotide sequence II forming an upper stem
element.
[0038] FIG. 3G illustrates an example of a split-nexus
Cas9-associated four polynucleotide system. FIG. 3G, 327
illustrates a first polynucleotide (sn1-casPN) that comprises a
first portion of the split nexus element. FIG. 3G, 302 illustrates
a second polynucleotide (sn2-casPN) that comprises a second portion
of the split nexus element. FIG. 3G, 333 illustrates a third
polynucleotide (sn3-casPN). FIG. 3G, 331 illustrates a spacer
polynucleotide (sp4-casPN) that comprises a spacer element.
Examples of polynucleotide secondary structures that form through
base-pair hydrogen bonding between indicated sequences include the
following: sn1-casPN split nexus stem element nucleotide sequence
I/sn2-casPN split nexus stem element nucleotide sequence II form a
split nexus stem clement; sn2-casPN second stem element nucleotide
sequence I/sn2-casPN second stem element nucleotide sequence II
form a second stem element; sn2-casPN third stem element nucleotide
sequence I/sn2-casPN third stem element nucleotide sequence II form
a third stem element; sn1-casPN first stem element nucleotide
sequence I/sn3-casPN first stem element nucleotide sequence II form
a first stem element.
[0039] FIG. 3H illustrates an example of a split-nexus
Cas9-associated three polynucleotide system. FIG. 3H, 334
illustrates a first polynucleotide (sn1-casPN) that comprises a
first portion of the split nexus element. FIG. 3H, 302 illustrates
a second polynucleotide (sn2-casPN) that comprises a second portion
of the split nexus element. FIG. 3H, 331 illustrates a spacer
polynucleotide (sn4-casPN) that comprises a spacer element.
Examples of polynucleotide secondary structures that form through
base-pair hydrogen bonding between indicated sequences include the
following: sn1-casPN split nexus stem element nucleotide sequence
I/sn2-casPN split nexus stem element nucleotide sequence II form a
split nexus stem element; sn2-casPN second stem element nucleotide
sequence I/sn2-casPN second stem element nucleotide sequence II
form a second stem element; sn2-casPN third stem element nucleotide
sequence I/sn2-casPN third stem element nucleotide sequence II form
a third stem element; sn1-casPN first stem element nucleotide
sequence I/sn1-casPN first stem element nucleotide sequence II form
a first stem element.
[0040] FIG. 4A presents modifications of Polynucleotide 1
(sn1-casPN) and Polynucleotide 2 (sn2-casPN) described above in
FIG. 3A to FIG. 3H. FIG. 4B presents examples of further
modifications to polynucleotide 1 (sn1-casRNA; described above in
FIG. 3A to FIG. 3H) and polynucleotide 3 (sn3-casRNA; described
above in FIG. 3A, FIG. C, FIG. E, and FIG. 3G) described above in
FIG. FIG. 4A and FIG. 4B present examples of sn1-casPN, sn2-casPN,
and sn3-casPN structures. Other modifications of sn 1 -casPN,
sn2-casPN, and sn3-casPN are described in the present
specification. The figures are not proportionally rendered nor are
they to scale. The indicators for locations corresponding to
elements are only illustrative to provide reference points in the
example polynucleotides. Table 2 presents a series of indicators
used consistently in FIG. 4A and FIG. 4B.
TABLE-US-00002 TABLE 2 Numerical Indicators Used to Illustrate
Regions of Nucleotide Sequences Associated with Examples of
sn1-casPNs, sn2-casPNs, and sn3-casPNs Indicators and Corresponding
Elements Second Polynucleotide (sn2-casPN; second auxiliary
polynucleotide; first adjunct polynucleotide; second adjunct
polynucleotide) 405 to 406 corresponds to a split nexus stem
element nucleotide sequence II 406 to 407 corresponds to a second
connective nucleotide sequence 407 to 408 corresponds to a second
stem element nucleotide sequence I 408 to 409 corresponds to a loop
element nucleotide sequence 409 to 410 corresponds to a second stem
element nucleotide sequence II 410 to 411 corresponds to a third
connective nucleotide sequence 411 to 412 corresponds to a third
stem element nucleotide sequence I 412 to 413 corresponds to a loop
element nucleotide sequence 413 to 414 corresponds to a third stem
element nucleotide sequence II 414 to 415 corresponds to a 3'
nucleotide sequence 405 to 418 corresponds to a second auxiliary
polynucleotide 405 to 416 corresponds to a linker element
nucleotide sequence II 416 to 417 corresponds to an affinity
nucleotide sequence II 417 to 418 corresponds to an effector
binding element nucleotide sequence II First Polynucleotide
(sn1-casPN; auxiliary polynucleotide) 419 to 420 corresponds to a
split nexus stem element nucleotide sequence I 420 to 421
corresponds to a first connective nucleotide sequence 419 to 424
corresponds to a first auxiliary polynucleotide 419 to 422
corresponds to a linker element nucleotide sequence I 422 to 423
corresponds to an affinity nucleotide sequence I 423 to 424
corresponds to an effector binding element nucleotide sequence I
421 to 425 corresponds to a lower stem element nucleotide sequence
I 425 to 426 corresponds to a bulge element nucleotide sequence I
426 to 427 corresponds to an upper stem element nucleotide sequence
I 427 to 428 corresponds to a first accessory polynucleotide Third
Polynucleotide (sn3-casPN; accessory polynucleotide) 429 to 430
corresponds to a second accessory polynucleotide 430 to 431
corresponds to an upper stem element nucleotide sequence II 431 to
432 corresponds to a bulge element nucleotide sequence II 432 to
433 corresponds to a lower stem element nucleotide sequence II 433
to 434 corresponds to a nucleic acid target binding sequence (a
spacer element)
[0041] FIG. 4A, 401 illustrates a first polynucleotide (sn1-casPN)
that comprises a first portion of the split nexus element and an
optional first auxiliary polynucleotide that is located 3' of the
split nexus element. FIG. 4A, 402 illustrates an example of a
second polynucleotide (sn2-casPN) that comprises a second portion
of the split nexus element, an optional second connective sequence,
and an optional second auxiliary polynucleotide that is located 5'
of the split nexus element. FIG. 4A, 402, 403 illustrates a
sn2-casPN comprising a first adjunct polynucleotide. FIG. 4A-402,
403, 404 illustrates a sn2-casPN further comprising second adjunct
polynucleotide. In FIG. 4A, the 5' three dots represent further
polynucleotide sequence.
[0042] In some embodiments, a sn2-casPN can comprises one or more
of the following: a first adjunct polynucleotide, a second adjunct
polynucleotide, a second auxiliary polynucleotide, or combinations
thereof. A first adjunct polynucleotide comprises one or more of
the following: a loop element nucleotide sequence, a second stem
element nucleotide sequence II, a third connective nucleotide
sequence, a third stem element nucleotide sequence I, or
combinations thereof. A second adjunct polynucleotide comprises one
or more of the following: a loop element nucleotide sequence, a
third stem element nucleotide sequence II, a 3' nucleotide
sequence, or combinations thereof.
[0043] In some embodiments, neither sn1-casPN nor sn2-casPN
comprise an auxiliary polynucleotide. Combinations of sn1-casPN
and/or sn2-casPN comprising an auxiliary polynucleotide include,
but are not limited to, the following: sn1-casPN-first auxiliary
polynucleotide/sn2-casPN; sn1-casPN/sn2-casPN-second auxiliary
polynucleotide; or sn1-casPN-first auxiliary
polynucleotide/sn2-casPN-second auxiliary polynucleotide.
Furthermore, the first auxiliary polynucleotide comprises one or
more of the following: a linker element nucleotide sequence I, an
affinity nucleotide sequence I, an effector binding element
nucleotide sequence I, or combinations thereof. In addition, the
second auxiliary polynucleotide comprises one or more of the
following: a linker element nucleotide sequence II, an affinity
nucleotide sequence II, an effector binding element nucleotide
sequence II, or combinations thereof.
[0044] Examples of polynucleotide secondary structures that are
capable of forming through base-pair hydrogen bonding between
indicated sequences (when the sequences are present) include the
following: sn1-casPN split nexus stem element nucleotide sequence
I/sn2-casPN split nexus stem element nucleotide sequence II form a
split nexus stem element; sn2-casPN second stem element nucleotide
sequence I/first adjunct polynucleotide second stem element
nucleotide sequence II form a second stem element; and first
adjunct polynucleotide third stem element nucleotide sequence
I/second adjunct polynucleotide third stem element nucleotide
sequence II form a third stem element.
[0045] Furthermore, in some embodiments the first auxiliary
polynucleotide and the second auxiliary polynucleotide arc capable
of forming secondary structure through base-pair hydrogen bonding
between indicated sequences, for example, including one or more of
the following: sn1-casPN first auxiliary polynucleotide/sn2-casPN
second auxiliary polynucleotide form; sn1-casPN affinity nucleotide
sequence I/sn2-casPN affinity nucleotide sequence II; sn1-casPN
effector binding element nucleotide sequence I/sn2-casPN effector
binding element nucleotide sequence II; and sn1-casPN linker
element nucleotide sequence I/sn2-casPN linker element nucleotide
sequence II.
[0046] However, in other embodiments base-pair hydrogen bonding
between one or more of these sequences is not required. In
addition, in some embodiments secondary structure forms through
base-pair hydrogen bonding within an indicated sequence, for
example, sn1-casPN first auxiliary polynucleotide can comprise a
hairpin and/or sn2-casPN second auxiliary polynucleotide can
comprise a hairpin.
[0047] Further modifications of the variations of sn2-casPN
described above in FIG. 4A include a second hairpin element
comprising a second stem element and a loop element, a third
hairpin element comprising a third stem element and a loop element,
and both the second hairpin element and the third hairpin element.
For example, by connecting the 3' end of the second stem element
nucleotide sequence I (FIG. 4, 408) to the 5' end of the second
stem element nucleotide sequence II (FIG. 4, 409) a second hairpin
element is formed. Similarly, by connecting the 3' end of the third
stem element nucleotide sequence I (FIG. 4, 412) to the 5' end of
the third stem element nucleotide sequence II (FIG. 4, 413) a third
hairpin element is formed.
[0048] In some embodiments, a sn2-casPN can comprises one or more
of the following: a first adjunct polynucleotide, a second adjunct
polynucleotide, a second auxiliary polynucleotide, or combinations
thereof. A first adjunct polynucleotide comprises one or more of
the following: a loop element nucleotide sequence, a second stem
element nucleotide sequence II, a third connective nucleotide
sequence, a third stem clement nucleotide sequence I, or
combinations thereof. A second adjunct polynucleotide comprises one
or more of the following: a loop element nucleotide sequence, a
third stem element nucleotide sequence II, a 3' nucleotide
sequence, or combinations thereof.
[0049] FIG. 4B, 401 illustrates a first polynucleotide (sn1-casPN)
that comprises a first portion of the split nexus element, an
optional first auxiliary polynucleotide that is located 3' of the
split nexus element, and an optional first accessory polynucleotide
that is located 5' of the upper stem element nucleotide sequence I.
FIG. 4B, 405 illustrates an example of a third polynucleotide
(sn3-casPN) that comprises an optional second accessory
polynucleotide that is located 3' of the upper stem element
nucleotide sequence II.
[0050] In some embodiments, neither sn1-casPN nor sn3-casPN
comprises an accessory polynucleotide. Combinations of sn1-casPN
and/or sn3-casPN comprising an auxiliary polynucleotide include,
but are not limited to, the following: sn1-casPN-first accessory
polynucleotide/sn3-casPN; sn1-casPN/sn3-casPN-second accessory
polynucleotide; or sn1-casPN-first accessory
polynucleotide/sn3-casPN-second accessory polynucleotide.
Furthermore, the first accessory polynucleotide can comprise one or
more of the following: a linker element, an affinity sequence (for
example a ligand or ligand-binding moiety), an effector binding
element, or combinations thereof. In addition, the second auxiliary
polynucleotide can comprise one or more of the following: a linker
element, an affinity sequence (e.g., a ligand or ligand-binding
moiety), an effector binding element, or combinations thereof.
[0051] FIG. 5A, FIG. 5B, and FIG. 5C relate to structural
information for an embodiment of a sn1-casRNA/sn2-casRNA/Cas9
protein complex, wherein the sn1-casRNA, sn2-casRNA correspond to
sn1-casPN and sn2-casPN of FIG. 3B. FIG. 5A and FIG. 5B provide a
close-up, open book view of SpyCas9. FIG. 5A presents a model of
the .alpha.-Helical lobe of SpyCas9 (FIG. 5A, 501) in complex with
sn1-casRNA (FIG. 5A, 502). The section of the sn1-casRNA
corresponding to the spacer element (i.e., a nucleic acid target
binding sequence) is indicated by a bracket (FIG. 5A, 503). The 5'
end of the sn1-casRNA (FIG. 5A, 504) is also indicated. The 3' end
of the sn1-casRNA is the location of the break in the nexus
element, that is the 3' end of the first portion of the split nexus
(FIG. 5A, 505). FIG. 5B presents a model of the Catalytic nuclease
lobe (FIG. 5B, 506) of SpyCas9 in complex with sn2-casRNA (FIG. 5B,
507). The 5' end of the sn2-casRNA is the location of the break in
the nexus element, that is the 5' end of the second portion of the
split nexus (FIG. 5A, 508). The 3' end of the sn2-casRNA (FIG. 5B,
509) is also indicated. The relative positions of the RuvC domain
(FIG. 5B, 510; RNase H domain) and the HNH domain (FIG. 5B, 511;
HNH nuclease domain) are indicated. FIG. 5C provide a view of an
assembled sn1-casRNA/sn2-casRNA/Cas9 protein complex. The relative
locations of the following elements are indicated: the
.alpha.-Helical lobe of SpyCas9 (FIG. 5C, 501); the Catalytic
nuclease lobe (FIG. 5C, 506) of SpyCas9; the sn1-casRNA (FIG. 5C,
502); the sn2-casRNA (FIG. 5C, 507); the 3' end of the sn2-casRNA
(FIG. 5C, 509); the 5' end of the sn1-casRNA (FIG. 5C, 504) is also
indicated; the relative position of the RuvC domain (FIG. 5C, 510);
and the area of the 5' and 3' ends of the split nexus element (FIG.
5C 508, 505).
[0052] FIG. 6A, FIG. 6B and FIG. 6C illustrate an example of a
split-nexus Cas9-associated two polynucleotide system. This system
corresponds to a first polynucleotide (sn1-casPN) that comprises a
first portion of the split nexus element (FIG. 3B, 326) and a
second polynucleotide (sn2-casPN) that comprises a second portion
of the split nexus element (FIG. 3B, 302). The figures are not
proportionally rendered nor are they to scale. The indicators for
locations corresponding to elements are only illustrative to
provide reference points in the example polynucleotides. Table 3
presents a series of indicators used in FIG. 6A and FIG. 6B.
TABLE-US-00003 TABLE 3 Numerical Indicators Used to Illustrate
Regions of Nucleotide Sequences Associated with Example sn1-casRNA
and sn2-casRNA Indicators and Corresponding Elements 601 to 602
corresponds to a split nexus stem element nucleotide sequence I 602
to 604 corresponds to a first auxiliary polynucleotide 602 to 603
corresponds to a linker element nucleotide sequence I 603 to 604
corresponds to an effector binding element nucleotide sequence I
605 to 606 corresponds to a split nexus stem element nucleotide
sequence II 606 to 608 corresponds to a second auxiliary
polynucleotide 606 to 607 corresponds to a linker element
nucleotide sequence II 607 to 608 corresponds to an effector
binding element nucleotide sequence II
[0053] FIG. 6A illustrates a sn1-casRNA comprising a first
auxiliary polynucleotide (FIG. 6A, 602 to 604) and a sn2-casRNA
comprising a second auxiliary polynucleotide (FIG. 6A, 606 to 608).
The figure shows the sn1-casRNA and sn2-casRNA before association
and formation of hydrogen bond base pairs (bp) between them. FIG.
6B illustrates the sn1-casRNA comprising a first auxiliary
polynucleotide and the sn2-casRNA comprising a second auxiliary
polynucleotide after formation of hydrogen bond base pairs between
them. A linker clement is formed between the linker clement
nucleotide sequence I (FIG. 6B, 602 to 603) and the linker element
nucleotide sequence II (FIG. 6B, 606 to 607). The bottom dash-lined
box (FIG. 6B, 609) shows formation of a nexus stem element. The top
dashed-line box (FIG. 6B, 610) shows formation of an effector
binding element, in this example a Csy4 RNA binding element. FIG.
6C illustrates the association of the sn2-casRNA with the catalytic
nuclease lobe (FIG. 6C, 613) of SpyCas9 and the association of the
sn1-casRNA with the .alpha.-Helical lobe (FIG. 6C, 614) of SpyCas9.
Also shown is an effector protein Csy4* (FIG. 6C, 615), which is a
variant of Csy4 without endoribonuclease activity. Furthermore, the
first portion of the split nexus (FIG. 6C, 616), the second portion
of the split nexus (FIG. 6C, 617), the 3' end of the sn2-casRNA
(FIG. 6C, 611), the 5' end of the sn1-casRNA (FIG. 6C, 612), the
first auxiliary polynucleotide (FIG. 6C, 602 to 604), and the
second auxiliary polynucleotide (FIG. 6C, 606 to 608) are
indicated. The thick downward pointing arrow indicates the assembly
of the sn2-casRNA/catalytic nuclease lobe (FIG. 6C, 613) of
SpyCas9, the sn1-casRNA/.alpha.-Helical lobe (FIG. 6C, 614) of
SpyCas9, and the Csy4* protein (FIG. 6C, 615) into a complex (FIG.
6C, 618). In the complex (FIG. 6C, 618) the sn2-casRNA/catalytic
nuclease lobe (FIG. 6C, 613) of SpyCas9 and the
sn1-casRNA/.alpha.-Helical lobe (FIG. 6C, 614) of SpyCas9 have
assembled into an active sn1-casRNA/sn2-casRNA/Cas9 complex (FIG.
6C, 619). The Csy4* protein (FIG. 6C, 615) has bound to Csy4 RNA
binding element (FIG. 6C, 610). The linker element (620) is also
indicated.
[0054] FIG. 7A and FIG. 7B illustrate an example of a split-nexus
Cas9-associated two polynucleotide system. This system corresponds
to a first polynucleotide (sn1-casPN) that comprises a first
portion of the split nexus element (FIG. 3B, 326) and a second
polynucleotide (sn2-casPN) that comprises a second portion of the
split nexus clement (FIG. 3B, 302). The figures are not
proportionally rendered nor are they to scale. The indicators for
locations corresponding to elements are only illustrative to
provide reference points in the example polynucleotides. Table 4
presents a series of indicators used in FIG. 7A and FIG. 7B.
TABLE-US-00004 TABLE 4 Numerical Indicators Used to Illustrate
Regions of Nucleotide Sequences Associated with Example sn1-casRNA
and sn2-casRNA Indicators and Corresponding Elements 701 to 702
corresponds to a split nexus stem element nucleotide sequence I 702
to 703 corresponds to a first auxiliary polynucleotide 704 a
hairpin element formed by hydrogen bond base pairing between bases
within the first auxiliary polynucleotide 705 to 706 corresponds to
a split nexus stem element nucleotide sequence II 706 to 707
corresponds to a second auxiliary polynucleotide 708 a hairpin
element formed by hydrogen bond base pairing between bases within
the second auxiliary polynucleotide
[0055] FIG. 7A illustrates a sn1-casRNA comprising a first
auxiliary polynucleotide (FIG. 7A, 702 to 703) and a sn2-casRNA
comprising a second auxiliary polynucleotide (FIG. 7A, 706 to 707).
The figure shows the sn1-casRNA and sn2-casRNA before association
and formation of hydrogen bond base pairs between them. The figure
shows a hairpin element formed by hydrogen bond base pairing
between bases within the first auxiliary polynucleotide (FIG. 7A,
704) and a hairpin element formed by hydrogen bond base pairing
between bases within the second auxiliary polynucleotide (FIG. 7A,
708). FIG. 7B illustrates the sn1-casRNA comprising a first
auxiliary polynucleotide and the sn2-casRNA comprising a second
auxiliary polynucleotide assembled into an active
sn1-casRNA/sn2-casRNA/Cas9 complex. In FIG. 7B the Cas9 protein
(FIG. 7B, 709), the first auxiliary polynucleotide comprising a
hairpin element (FIG. 7B, 704), and the second auxiliary
polynucleotide comprising a hairpin element (FIG. 7B, 708) are
indicated.
[0056] FIG. 8 presents the results of the Cas 9 cleavage assay
using the AAVS-1 target double-stranded DNA. In the figure,
replicates of three arc shown for each combination of
sn-casRNAs.sup.EX. At the top of each panel is a graphical
representation of the sn-casRNAs.sup.EX).sup.t used in the assay.
FIG. 8, Panel A shows the biochemical activity of
sn1-casRNA.sup.EX, sn2-casRNA.sup.EX, sn3-casRNA.sup.EX-AAVS1. FIG.
8, Panel B shows the biochemical activity of sn1-casRNA.sup.EX and
sn2-casRNA.sup.EX. FIG. 8, Panel C shows the biochemical activity
of sn2-casRNA.sup.EX and sn3-casRNA.sup.EX-AAVS1, FIG. 8, Panel D
shows the biochemical activity of sn1-casRNA.sup.EX and
sn3-casRNA.sup.EX-AAVS1. The last lane of FIG. 8, Panel D contains
molecular weight standards. Cleavage percentages are shown at the
bottom of each lane. For lanes indicated as LOD, any cleavage
activity was below the limit of detection.
[0057] FIG. 9 presents the results of the Cas9 cleavage assay using
the Csy4* protein to enhance the cleavage activity of the
sn-casRNAs comprising an additional Csy4 RNA binding sequence. The
cleavage assays used two different split-nexus Cas9-associated two
polynucleotide systems that were variants of the system present in
FIG. 3B. In the first system the sn 1 -casRNA further comprised a
first auxiliary polynucleotide comprising a Csy4 binding element
nucleotide sequence I and the sn2-casRNA comprised a second
auxiliary polynucleotide comprising a Csy4 binding element
nucleotide sequence II, wherein the first auxiliary polynucleotide
and the second auxiliary polynucleotide associate to form a Csy4
RNA binding element
(sn1-casRNA.sup.EXCsy-Csy/sn2-casRNA.sup.EXCsy-Csy). In the second
system the sn1-casRNA further comprised a first auxiliary
polynucleotide comprising a linker element nucleotide sequence I
and a Csy4 binding element nucleotide sequence I and the sn2-casRNA
comprised a second auxiliary polynucleotide comprising a linker
element nucleotide sequence H and a Csy4 binding element nucleotide
sequence II, wherein the first auxiliary polynucleotide and the
second auxiliary polynucleotide associate to form a linker element
and a Csy4 RNA binding element
(sn1-casRNA.sup.EXCsy-lnkCsy/sn2-casRNA.sup.EXCsy-lnkCsy). Each of
the two systems was used to cleave four different targets, where
the sn1-casRNAs each comprised a spacer complementary to one of the
four targets: AAVS-1, CD-34, CD-151, and JAK-1. In the figure, the
cleavage activity is shown at the bottom of each lane (except for
lanes 1 and 10, which are molecular weight standards). For lanes
indicated as LOD, any cleavage activity was below the limit of
detection. The systems used in each of the Cas9 cleavage assay
reactions were as shown in Table 5.
TABLE-US-00005 TABLE 5 Split Nexus Polynucleotide Components Used
in Cas9 Cleavage Assays Csy4* Protein Lane sn-casRNAs.sup.EXCsy
Added? 1 No (Molecular Weight Standard) n/a 2
sn1-casRNA.sup.EXCsy-Csy-AAVS1/sn2-casRNA.sup.EXCsy-Csy NO 3
sn1-casRNA.sup.EXCsy-lnkCsy-AAVS1/sn2-casRNA.sup.EXCsy- NO lnkCsy 4
sn1-casRNA.sup.EXCsy-Csy-AAVS1/sn2-casRNA.sup.EXCsy-Csy YES 5
sn1-casRNA.sup.EXCsy-lnkCsy-AAVS1/sn2-casRNA.sup.EXCsy- YES lnkCsy
6 sn1-casRNA.sup.EXCsy-Csy-CD34/sn2-casRNA.sup.EXCsy-Csy NO 7
sn1-casRNA.sup.EXCsy-lnkCsy-CD34/sn2-casRNA.sup.EXCsy- NO lnkCsy 8
sn1-casRNA.sup.EXCsy-Csy-CD34/sn2-casRNA.sup.EXCsy-Csy YES 9
sn1-casRNA.sup.EXcsy-lnkCsy-CD34/sn2-casRNA.sup.EXCsy- YES lnkCsy
10 No (Molecular Weight Standard) n/a 11
sn1-casRNA.sup.EXCsy-Csy-CD151/sn2-casRNA.sup.EXCsy-Csy NO 12
sn1-casRNA.sup.EXCsy-lnkCsy-CD151/sn2-casRNA.sup.EXCsy- NO lnkCsy
13 sn1-casRNA.sup.EXCsy-Csy-CD151/sn2-casRNA.sup.EXCsy-Csy YES 14
sn1-casRNA.sup.EXCsy-lnkCsy-CD151/sn2-casRNA.sup.EXCsy- YES lnkCsy
15 sn1-casRNA.sup.EXCsy-Csy-JAK-1/sn2-casRNA.sup.EXCsy-Csy NO 16
sn1-casRNA.sup.EXCsy-lnkCsy-JAK-1/sn2-casRNA.sup.EXCsy- NO lnkCsy
17 sn1-casRNA.sup.EXCsy-Csy-JAK-1/sn2-casRNA.sup.EXCsy-Csy YES 18
sn1-casRNA.sup.EXCsy-lnkCsy-JAK-1/sn2-casRNA.sup.EXCsy- YES
lnkCsy
[0058] FIG. 10 presents the result of the Cas9 cleavage assay using
sn1-casRNAs.sup.EX2 and sn2-casRNA.sup.EX2. Cleavage percentages
are shown at the bottom of each lane except for lane 1, which is a
molecular weight standard. FIG. 10, lane 2, presents cleavage
results for a sn1-casRNA.sup.EX2-AAVS1 and sn2-casRNA.sup.EX2
system. FIG. 10, lane 3, presents cleavage results for a
sn1-casRNA.sup.EX2-CD151 and sn2-casRNA.sup.EX2 system. FIG. 10,
lane 4, presents the results for a sn1-casRNA.sup.EX2-JAK1 and
sn2-casRNA.sup.EX2 system. At the top of the figure is a graphical
representation of the sn-casRNAs.sup.EX2 used in the assay.
[0059] FIG. 11 presents the results of Cas9 cleavage assays. The
cleavage assays used two different split-nexus Cas9-associated two
polynucleotide systems similar to the system illustrated in FIG.
7A. In the figure, the cleavage activity is shown at the bottom of
each lane (except for lanes 1 and 10, which are molecular weight
standards). For lanes indicated as LOD, any cleavage activity was
below the limit of detection. Representations of the sn-casRNA(s)
used in each assay are illustrated at the top of the figure. The
systems used in each of the Cas9 cleavage assay reactions were as
shown in Table 6.
TABLE-US-00006 TABLE 6 Split Nexus Polynucleotide Components Used
in Cas9 Cleavage Assays Csy4* Protein Lane sn-casRNAs.sup.EX3Csy
Added? 1 None (Molecular Weight Standard) n/a 2
sn1-casRNA.sup.EX3Csy-Csy-AAVS1 NO 3 sn2-casRNA.sup.EX3Csy-Csy NO 4
sn1-casRNA.sup.EX3Csy-Csy-AAVS1/sn2-casRNA.sup.EX3Csy-Csy NO 5
sn1-casRNA.sup.EX3Csy-Csy-AAVS1/sn2-casRNA.sup.EX3Csy-Csy YES 6
sn1-casRNA.sup.EX3Csy-lnkCsy-AAVS1 NO 7
sn2-casRNA.sup.EX3Csy-lnkCsy NO 8
sn1-casRNA.sup.EX3Csy-lnkCsy-AAVS1/sn2-casRNA.sup.EX3Csy- NO lnkCsy
9 sn1-casRNA.sup.EX3Csy-lnkCsy-AAVS1/sn2-casRNA.sup.EX3Csy- YES
lnkCsy 10 None (Molecular Weight Standard) n/a
[0060] FIG. 12 presents examples of putative split nexus
arrangements of known tracrRNA sequences from the bacterial species
listed in Table 7. In the figure, the first column is an
identifying number for the bacterial species (as shown in Table 7,
the second column is the sequence of the split nexus tracrRNA (an
example of sn1-casRNA/sn2-casRNA), and the third column is the SEQ
ID NO of the oligonucleotide. All bacterial species listed in Table
7 have at least one identified Type II CRISPR-Cas9 system.
TABLE-US-00007 TABLE 7 Bacterial Species and Putative Split-nexus
tracrRNA Sequences ID Genus/Species 1 Streptococcus pyogenes 2
Streptococcus thermophilus CRISPR-1 3 Listeria innocua 4 Neisseria
meningitidis 5 Streptococcus gallolyticus 6 Staphylococcus aureus 7
Corynebacterium diphtheriae 8 Parvibaculum lavamentivorans 9
Campylobacter lari 10 Neisseria cinerea 11 Streptococcus
pasteurianus
[0061] FIG. 13 is an oligonucleotide table that sets forth the
sequences of oligonucleotides used in the Examples of the present
specification. The first column is an identifying letter for the
oligonucleotide, the second column is the sequence of the
oligonucleotide, and the third column is the SEQ ID NO of the
oligonucleotide.
INCORPORATION BY REFERENCE
[0062] All patents, publications, and patent applications cited in
this specification are herein incorporated by reference as if each
individual patent, publication, or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety for all purposes.
DETAILED DESCRIPTION OF THE INVENTION
[0063] It is to be understood that the terminology used herein is
for the purpose of describing particular embodiments only, and is
not intended to be limiting. As used in this specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a primer" includes one or more
primer, reference to "a recombinant cell" includes one or more
recombinant cell, reference to "a cross-linking agent" includes one
or more cross-linking agent, and the like.
[0064] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains. Although
other methods and materials similar, or equivalent, to those
described herein can be used in the practice of the present
invention, preferred materials and methods are described
herein.
[0065] In view of the teachings of the present specification, one
of ordinary skill in the art can apply conventional techniques of
immunology, biochemistry, chemistry, molecular biology,
microbiology, cell biology, genomics, and recombinant
polynucleotides, as taught, for example, by the following standard
texts: Antibodies: A Laboratory Manual, Second edition, E. A.
Greenfield, 2014, Cold Spring Harbor Laboratory Press, ISBN
978-1-936113-81-1; Culture of Animal Cells: A Manual of Basic
Technique and Specialized Applications, 6th Edition, R. I.
Freshney, 2010, Wiley-Blackwell, ISBN 978-0-470-52812-9; Transgenic
Animal Technology, Third Edition: A Laboratory Handbook, 2014, C.
A. Pinkert, Elsevier, ISBN 978-0124104907; The Laboratory Mouse,
Second Edition, 2012, H. Hedrich, Academic Press, ISBN
978-0123820082; Manipulating the Mouse Embryo: A Laboratory Manual,
2013, R. Behringer, et al., Cold Spring Harbor Laboratory Press,
ISBN 978-1936113019; PCR 2: A Practical Approach, 1995, M. J.
McPherson, et al., IRL Press, ISBN 978-0199634248; Methods in
Molecular Biology (Series), J. M. Walker, ISSN 1064-3745, Humana
Press; RNA: A Laboratory Manual, 2010, D. C. Rio, et al., Cold
Spring Harbor Laboratory Press, ISBN 978-0879698911; Methods in
Enzymology (Series), Academic Press; Molecular Cloning: A
Laboratory Manual (Fourth Edition), 2012, M. R. Green, et al., Cold
Spring Harbor Laboratory Press, ISBN 978-1605500560; Bioconjugate
Techniques, Third Edition, 2013, G. T. Hermanson, Academic Press,
ISBN 978-0123822390; Methods in Plant Biochemistry and Molecular
Biology, 1997, W. V. Dashek, CRC Press, ISBN 978-0849394805; Plant
Cell Culture Protocols (Methods in Molecular Biology), 2012, V. M.
Loyola-Vargas, et al., Humana Press, ISBN 978-1617798177; Plant
Transformation Technologies, 2011, C. N. Stewart, et al.,
Wiley-Blackwell, ISBN 978-0813821955; Recombinant Proteins from
Plants (Methods in Biotechnology), 2010, C. Cunningham, et al.,
Humana Press, ISBN 978-1617370212; Plant Genomics: Methods and
Protocols (Methods in Molecular Biology), 2009, D. J. Somers, et
al., Humana Press, ISBN 978-1588299970; Plant Biotechnology:
Methods in Tissue Culture and Gene Transfer, 2008, R.
Keshavachandran, et al., Orient Blackswan, ISBN 978-8173716164.
[0066] As used herein and described in detail below, the term
"sn-casPNs" refers to split-nexus Cas9-associated polynucleotides
of the present invention. One distinguishing feature of the
sn-casPNs is that at least two of the two or more Cas associated
polynucleotides are necessary to form a nexus stem element.
[0067] The term "Cas protein" as used herein refers to Type II
CRISPR Cas proteins (as described, e.g., in Chylinski, K., (2013)
"The tracrRNA and Cas9 families of type II CRISPR-Cas immunity
systems," RNA Biol. 2013 10(5):726-737), including, but not limited
to Cas9, Cas9-like, Cas1, Cas2, Cas3, Csn2, Cas4, proteins encoded
by Cas9 orthologs, Cas9-like synthetic proteins, and variants and
modifications thereof.
[0068] The term "Cas9 protein" as used herein refers to Cas9
wild-type proteins derived from Type II CR1SPR-Cas9 systems,
modifications of Cas9 proteins, variants of Cas9 proteins, Cas9
orthologs, and combinations thereof.
[0069] As used herein "sn-casPNs/Cas9 protein system" and
"sn-casPNs/Cas9 system" are used interchangeably to refer to
engineered Type II CRISPR-Cas9 systems comprising at least
sn-casPNs and Cas9 protein components, expressible forms of the
components thereof, or combinations of the components and
expressible forms of the components. An engineered Type II
CRISPR-Cas9 system of the present invention comprises at least a
two polynucleotide system of sn-casPNs as described herein.
sn-casPNs/Cas9 systems can comprise further CRISPR Cas components,
such as additional Cas proteins.
[0070] As used herein, the terms "wild-type," "naturally-occurring"
and "unmodified" are used to mean the typical (or most common)
form, appearance, phenotype, or strain existing in nature; for
example, the typical form of cells, organisms, characteristics,
polynucleotides, proteins, macromolecular complexes, genes, RNAs,
DNAs, or genomes as they occur in and can be isolated from a source
in nature. The wild-type form, appearance, phenotype, or strain
serve as the original parent before an intentional modification.
Thus, mutant, variant, engineered, recombinant, and modified forms
are not wild-type forms.
[0071] As used herein, the terms "engineered," "genetically
engineered," "recombinant," "modified," and "non-naturally
occurring" are interchangeable and indicate intentional human
manipulation.
[0072] As used herein, the terms "nucleic acid," "nucleotide
sequence," "oligonucleotide," and "polynucleotide" are
interchangeable. All refer to a polymeric form of nucleotides. The
nucleotides may be deoxyribonucleotides (DNA) or ribonucleotides
(RNA), or analogs thereof, and they may be of any length.
Polynucleotides may perform any function and may have any secondary
structure and three-dimensional structure. The terms encompass
known analogs of natural nucleotides and nucleotides that are
modified in the base, sugar and/or phosphate moieties. Analogs of a
particular nucleotide have the same base-pairing specificity (e.g.,
an analog of A base pairs with T). A polynucleotide may comprise
one modified nucleotide or multiple modified nucleotides (e.g.,
many modified nucleotides are available from commercial providers
like TriLink (San Diego, Calif.) and Intregrated DNA Technologies
(Coralville, Iowa)). Examples of modified nucleotides include
methylated nucleotides and nucleotide analogs. Nucleotide structure
may be modified before or after a polymer is assembled. Following
polymerization, polynucleotides may be additionally modified via,
for example, conjugation with a labeling component or
target-binding component. A nucleotide sequence may incorporate
non-nucleotide components. The terms also encompasses nucleic acids
comprising modified backbone residues or linkages, that (i) are
synthetic, naturally occurring, and non-naturally occurring, and
(ii) have similar binding properties as a reference polynucleotide
(e.g., DNA or, RNA). Examples of such analogs include, but are not
limited to, phosphorothioates, phosphoramidates, methyl
phosphonates, chiral-methyl phosphonates, 2-O-methyl
ribonucleotides, peptide-nucleic acids, and morpholino
structures.
[0073] Peptide-nucleic acids (PNAs) are synthetic homologs of
nucleic acids wherein the polynucleotide phosphate-sugar backbone
is replaced by a flexible pseudo-peptide polymer. Nucleobases are
linked to the polymer. PNAs have the capacity to hybridize with
high affinity and specificity to complementary sequences of RNA and
DNA.
[0074] In phosphorothioate nucleic acids, the phosphorothioate (PS)
bond substitutes a sulfur atom for a non-bridging oxygen in the
polynucleotide phosphate backbone. This modification makes the
internucleotide linkage resistant to nuclease degradation. In some
embodiments, phosphorothioate bonds are introduced between the last
3-5 nucleotides at the 5'- or 3'-end of a polynucleotide sequence
to inhibit exonuclease degradation. Placement of phosphorothioate
bonds throughout an entire oligonucleotide helps reduce degradation
by endonucleases as well.
[0075] Threose nucleic acid (TNA) is an artificial genetic polymer.
TNA's backbone structure comprises repeating threose sugars linked
by phosphodiester bonds. TNA polymers are resistant to nuclease
degradation. TNA can self-assemble by base-pair hydrogen bonding
into duplex structures.
[0076] Linkage inversions can be introduced into polynucleotides
through use of "reversed phosphoramidites" (see, e.g.,
www.ucalgary.ca/dnalab/synthesis/modifications/linkages). Typically
such polynucleotides have phosphoramidite groups on the 5'-OH
position and a dimethoxytrityl (DMT) protecting group on the 3'-OH
position. Normally, the DMT protecting group is on the 5'-OH and
the phosphoramidite is on the 3'-OH. The most common use of linkage
inversion is to add a 3'-3' linkage to the end of a polynucleotide
with a phosphorothioate backbone. The 3'-3' linkage stabilizes the
polynucleotide to exonuclease degradation by creating an
oligonucleotide having two 5'-OH ends and no 3'-OH end.
[0077] Polynucleotide sequences are displayed herein in the
conventional 5' to 3' orientation.
[0078] As used herein, the term "complementarity" refers to the
ability of a nucleic acid sequence to form hydrogen bond(s) with
another nucleic acid sequence (e.g., through traditional
Watson-Crick base pairing). A percent complementarity indicates the
percentage of residues in a nucleic acid molecule that can form
hydrogen bonds with a second nucleic acid sequence. When two
polynucleotide sequences have 100% complementary, the two sequences
arc perfectly complementary, i.e., all of a first polynucleotide's
contiguous residues hydrogen bond with the same number of
contiguous residues in a second polynucleotide.
[0079] As used herein, the term "sequence identity" generally
refers to the percent identity of bases or amino comparing a first
polynucleotide or polypeptide to a second polynucleotide or
polypeptide using algorithms having various weighting parameters.
Sequence identity between two polypeptides or two polynucleotides
can be determined using sequence alignment by various methods and
computer programs (e.g., BLAST, CS-BLAST, FASTA, HMMER, L-ALIGN,
etc.), available through the worldwide web at sites including
GENBANK (www.ncbi.nlm.nih.gov/genbank/) and EMBL-EBI
(vvww.ebi.ac.uk.). Sequence identity between two polynucleotides or
two polypeptide sequences is generally calculated using the
standard default parameters of the various methods or computer
programs.
[0080] As used herein "hybridization" or "hybridize" or
"hybridizing" is the process of combining two complementary
single-stranded DNA or RNA molecules and allowing them to form a
single double-stranded molecule (DNA/DNA, DNA/RNA, RNA/RNA) through
hydrogen base pairing. Hybridization stringency is typically
determined by the hybridization temperature and the salt
concentration of the hybridization buffer, for example, high
temperature and low salt provide high stringency hybridization
conditions. Examples of salt concentration ranges and temperature
ranges for different hybridization conditions are as follows: high
stringency, approximately 0.01M to approximately 0.05M salt,
hybridization temperature 5.degree. C. to 10.degree. C. below Tm;
moderate stringency, approximately 0.16M to approximately 0.33M
salt, hybridization temperature 20.degree. C. to 29.degree. C.
below Tm; low stringency, approximately 0.33M to approximately
0.82M salt, hybridization temperature 40.degree. C. to 48.degree.
C. below Tm. Tm of duplex nucleic acids is calculated by standard
methods well-known in the art (Maniatis, T., et al (1982) Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press:
New York; Casey, J., et al., (1977) Nucleic Acids Res., 4: 1539;
Bodkin, D. K., et al., (1985) J. Virol. Methods, 10: 45; Wallace,
R. B., et al. (1979) Nucleic Acids Res. 6: 3545.) Algorithm
prediction tools to estimate Tm are also widely available. High
stringency conditions for hybridization typically refer to
conditions under which a nucleic acid having complementarity to a
target sequence predominantly hybridizes with the target sequence,
and substantially does not hybridize to non-target sequences.
Typically hybridization conditions are of moderate stringency,
preferably high stringency.
[0081] As used herein a "stem-loop structure" or "stem-loop
element" refers to a polynucleotide having a secondary structure
that includes a region of nucleotides that are known or predicted
to form a double strand region (the "stem element") that is linked
on one side by a region of predominantly single-stranded
nucleotides (the "loop element"). The term "hairpin" element is
also used herein to refer to stem-loop structures. Such structures
are well known in the art. The base pairing may be exact. However,
as is known in the art, that a stem element does not require exact
base pairing. Thus, the stem element may include one or more base
mismatches or non-paired bases.
[0082] As used herein, the term "recombination" refers to a process
of exchange of genetic information between two polynucicotidcs.
[0083] As used herein, the terms "donor polynucleotide," "donor
template" and "donor oligonucleotide" are used interchangeably and
refer to a polynucleotide that provides a nucleic acid sequence of
which at least a portion is intended to be integrated into a
selected nucleic acid target site. Typically, a donor
polynucleotide is a single-strand polynucleotide or a double-strand
polynucleotide. For example, an engineered Type IT CRISPR-Cas9
system of the present invention can be used in combination with a
donor DNA template to modify a DNA target sequence in a genomic DNA
wherein the genomic DNA is modified to comprise at least a portion
of the donor DNA template at the DNA target sequence. In some
embodiments, a vector comprises a donor polynucleotide (e.g., a
targeting vector). In other embodiments, a donor polynucleotide is
an oligonucleotide.
[0084] As used herein, the term "homology-directed repair (HDR)"
refers to DNA repair that takes place in cells, for example, during
repair of double-strand breaks in DNA. HDR requires nucleotide
sequence homology and uses a donor template (e.g., a donor DNA
template) or donor oligonucleotide to repair the sequence wherein
the double-strand break occurred (e.g., DNA target sequence). This
results in the transfer of genetic information from, for example,
the donor template DNA to the DNA target sequence. HDR may result
in alteration of the DNA target sequence (e.g., insertion,
deletion, mutation) if the donor template DNA sequence or
oligonucleotide sequence differs from the DNA target sequence and
part or all of the donor template DNA polynucleotide or
oligonucleotide is incorporated into the DNA target sequence. In
some embodiments, an entire donor template DNA polynucleotide, a
portion of the donor template DNA polynucleotide, or a copy of the
donor polynucleotide is integrated at the site of the DNA target
sequence.
[0085] The terms "vector" and "plasmid" are used interchangeably
and as used herein refer to a polynucleotide vehicle to introduce
genetic material into a cell. Vectors can be linear or circular.
Vectors can integrate into a target genome of a host cell or
replicate independently in a host cell. Vectors can comprise, for
example, an origin of replication, a multicloning site, and/or a
selectable marker. An expression vector typically comprises an
expression cassette. Vectors and plasmids include, but are not
limited to, integrating vectors, prokaryotic plasmids, eukaryotic
plasmids, plant synthetic chromosomes, episomes, viral vectors,
cosmids, and artificial chromosomes.
[0086] As used herein the term "expression cassette" is a
polynucleotide construct, generated recombinantly or synthetically,
comprising regulatory sequences operably linked to a selected
polynucleotide to facilitate expression of the selected
polynucleotide in a host cell. For example, the regulatory
sequences can facilitate transcription of the selected
polynucleotide in a host cell, or transcription and translation of
the selected polynucleotide in a host cell. An expression cassette
can, for example, be integrated in the genome of a host cell or be
present in an expression vector.
[0087] As used herein a "targeting vector" is a recombinant DNA
construct typically comprising tailored DNA arms homologous to
genomic DNA that flanks critical elements of a target gene or
target sequence. When introduced into a cell the targeting vector
integrates into the cell genome via homologous recombination.
Elements of the target gene can be modified in a number of ways
including deletions and/or insertions. A defective target gene can
be replaced by a functional target gene, or in the alternative a
functional gene can be knocked out. Optionally a targeting vector
comprises a selection cassette comprising a selectable marker that
is introduced into the target gene. Targeting regions adjacent or
sometimes within a target gene can be used to affect regulation of
gene expression.
[0088] As used herein, the terms "regulatory sequences,"
"regulatory elements," and "control elements" are interchangeable
and refer to polynucleotide sequences that are upstream (5'
non-coding sequences), within, or downstream (3' non-translated
sequences) of a polynucleotide target to be expressed. Regulatory
sequences influence, for example, the timing of transcription,
amount or level of transcription, RNA processing or stability,
and/or translation of the related structural nucleotide sequence.
Regulatory sequences may include activator binding sequences,
enhancers, introns, polyadenylation recognition sequences,
promoters, repressor binding sequences, stem-loop structures,
translational initiation sequences, translation leader sequences,
transcription termination sequences, translation termination
sequences, primer binding sites, and the like.
[0089] As used herein the term "operably linked" refers to
polynucleotide sequences or amino acid sequences placed into a
functional relationship with one another. For instance, a promoter
or enhancer is operably linked to a coding sequence if it
regulates, or contributes to the modulation of, the transcription
of the coding sequence. Operably linked DNA sequences encoding
regulatory sequences are typically contiguous to the coding
sequence. However, enhancers can function when separated from a
promoter by up to several kilobases or more. Accordingly, some
polynucleotide elements may be operably linked but not
contiguous.
[0090] As used herein, the term "expression" refers to
transcription of a polynucleotide from a DNA template, resulting
in, for example, an mRNA or other RNA transcript (e.g., non-coding,
such as structural or scaffolding RNAs). The term further refers to
the process through which transcribed mRNA is translated into
peptides, polypeptides, or proteins. Transcripts and encoded
polypeptides may be referred to collectively as "gene product."
Expression may include splicing the mRNA in a eukaryotic cell, if
the polynucleotide is derived from genomic DNA.
[0091] As used herein, the term "gene" comprises a DNA region
encoding a gene product (e.g., an RNA or a protein), as well as all
DNA regions that regulate the production of the gene product,
whether or not such regulatory sequences are adjacent to the DNA
region encoding the gene product. For example, in addition to the
DNA region encoding the gene product, a gene can include promoter
sequences, termination sequences, translational regulatory
sequences (e.g., ribosome binding sites and internal ribosome entry
sites), enhancers, silencers, insulators, boundary elements,
replication origins, matrix attachment sites, locus control
regions, and combinations thereof.
[0092] As used herein the term "modulate" refers to a change in the
quantity, degree or amount of a function. For example, the
sn-casPNs/Cas9 protein systems disclosed herein may modulate the
activity of a promoter sequence by binding at or near the promoter.
Depending on the action occurring after binding, the sn-casPNs/Cas9
protein systems can induce, enhance, suppress, or inhibit
transcription of a gene operatively linked to the promoter
sequence. Thus, "modulation" of gene expression includes both gene
activation and gene repression.
[0093] Modulation can be assayed by determining any characteristic
directly or indirectly affected by the expression of the target
gene. Such characteristics include, e.g., changes in RNA or protein
levels, protein activity, product levels, associated gene
expression, or activity level of reporter genes. Accordingly, the
terms "modulating expression," "inhibiting expression," and
"activating expression" of a gene can refer to the ability of a
sn-casPNs/Cas9 protein system to change, activate, or inhibit
transcription of a gene.
[0094] As used herein, the term "amino acid" refers to natural and
synthetic (unnatural) amino acids, including amino acid analogs,
modified amino acids, peptidomimetics, glycine, and D or L optical
isomers.
[0095] As used herein, the terms "peptide," "polypeptide," and
"protein" are interchangeable and refer to polymers of amino acids.
A polypeptide may be of any length. It may be branched or linear,
it may be interrupted by non-amino acids, and it may comprise
modified amino acids. The terms may be used to refer to an amino
acid polymer that has been modified through, for example,
acetylation, disulfide bond formation, glycosylation, lipidation,
phosphorylation, cross-linking, and/or conjugation (e.g., with a
labeling component or ligand). Polypeptide sequences are displayed
herein in the conventional N-terminal to C-terminal
orientation.
[0096] Polypeptides and polynucleotides can be made using routine
techniques in the field of molecular biology (see, e.g., standard
texts discussed above). Furthermore, essentially any polypeptide or
polynucleotide can be custom ordered from commercial sources.
[0097] As used herein, "non-native" refers to a nucleic acid
sequence or polypeptide sequence that is not found in the
corresponding native (or wild-type) nucleic acid sequence or
polypeptide sequence. Non-native can also refer to a naturally
occurring nucleic acid or polypeptide sequence that comprises
mutations, insertions, deletions, or other modifications. A
non-native nucleic acid sequence or polypeptide sequence may be
linked to a naturally occurring nucleic acid sequence or
polypeptide sequence by genetic engineering to generate a chimeric
nucleic acid sequence or polypeptide sequence.
[0098] As used herein, "fusion" refers to a polypeptide sequence
("fusion polypeptide") and/or nucleic acid sequence ("fusion
polynucleotide," "fusion nucleic acids") comprising one or more
non-native sequences. Fusion can also refer to the attachment of a
moiety to a polypeptide sequence or nucleic acid sequence, wherein
the moiety is not native to the corresponding nucleic acid sequence
or polypeptide sequence (i.e., the corresponding wild-type nucleic
acid sequence or polypeptide sequence does not comprise the
moiety). Examples of sequences and moieties that can be useful in
the generation of fusion polypeptides or fusion polynucleotides
include: a subcellular localization signal or coding sequences
therefore (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 small molecule such as biotin or a dye
(e.g., alexa fluor dyes, Cyanine3 dye, Cyanine5 dye); a detectable
label, including a moiety that can provide a detectable signal
(e.g., an enzyme, a radioisotope, a member of a specific binding
pair; a fluorophore; a fluorescent protein; a quantum dot; and the
like); a member of a FRET pair (donor/acceptor) (e.g.,
EDANS/fluorescein, IAEDANS/fluorescein,
fluorescein/tetramethylrhodamine, fluorescein/Cy5, EDANS/DABCYL,
fluorescein/QSY-7, fluorescein/LC Red 640, fluorescein/Cy 5.5 and
fluorescein/LC Red 705); a fluorophore/quantum dot donor/acceptor
pair; fluorescent labels (e.g., fluorescein, rhodamine,
tetramethylrhodamine, eosin, erythrosin, coumarin,
methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow,
Cascade Blue.TM., Texas Red, IAEDANS, EDANS, BODIPY.RTM. FL, LC Red
640, Cy 5, Cy 5.5, LC Red 705 and Oregon green); an enzyme (horse
radish peroxidase, luciferase, beta-galactosidase, and the like); a
fluorescent protein (e.g., a green fluorescent protein (GFP), a red
fluorescent protein, a yellow fluorescent protein, any of a variety
of fluorescent and colored proteins); a nanoparticle (e.g.,
fluorescent or luminescent nanoparticles, and magnetic
nanoparticles); 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; 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); and
radioisotopes.
[0099] The term "binding" as used herein refers to a non-covalent
interaction between macromolecules (e.g., between a protein and a
polynucleotide, between a polynucleotide and a polynucleotide, and
between a protein and a protein). Such non-covalent interaction is
also referred to as "associating" or "interacting" (e.g., when a
first macromolecule interacts with a second macromolecule, the
first macromolecule binds to second macromolecule in a non-covalent
manner). Some portions of a binding interaction may be
sequence-specific; however, all components of a binding interaction
do not need to be sequence-specific, such as the contact points of
the protein with phosphate residues in a DNA backbone. Binding
interactions can be characterized by a dissociation constant (Kd).
"Affinity" refers to the strength of binding. An increased binding
affinity is correlated with a lower Kd. An example of non-covalent
binding is hydrogen bond formation between base pairs.
[0100] As used herein, the term "effector protein" refers to any
polypeptide with a functional effect that selectively or
specifically binds to an effector protein binding element within a
polynucleotide. Such effector protein binding elements can be
single-stranded or double-stranded polynucleotides. For example, an
effector protein can comprise enzymatic activity, remodel
biological molecules (e.g., folding chaperones), or be a
scaffolding protein. In addition to binding a cognate effector
protein binding element, an effector protein can modify a
polynucleotide comprising a cognate effector binding element (e.g.,
cleavage, enzymatic modification, transcriptional modification).
Alternatively, an effector protein can just bind to its cognate
effector protein binding element. Effector proteins with enzymatic
activity can be modified to be enzymatically inactive, however,
they maintain their ability to bind an effector protein binding
element. For example, Csy4 binds a Csy4 double-strand RNA binding
element. Csy4 is normally an active endoribonuclease but Csy4 has
variants in which its endonuclease activity has been eliminated
(e.g., Csy4*). Cas 7, Cas5, and Cas6 are also examples of effector
proteins. Other examples of effector proteins include, but are not
limited to single-strand RNA binding proteins (e.g., p19 siRNA
Binding Protein), single-strand DNA binding proteins (e.g.,
adnovirus DBP,Extreme Thermostable Single-Stranded DNA Binding
Protein), double-strand RNA binding proteins (e.g., DICER),
double-strand DNA binding proteins (e.g., Zinc Finger proteins) and
double-strand RNA/DNA hybrids (e.g., Ribonuclease H).
[0101] 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 means 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 recombinant
cell.
[0102] As used herein, "organism" refers to any living biological
entity, such as a bacterium, protist, fungus, plant, or animal,
composed of one or more cells.
[0103] As used herein, a "host cell" generally refers 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. Examples of host cells
include, but are not limited to: 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 (such as soy, tomatoes, sugar beets,
pumpkin, hay, cannabis, tobacco, plantains, yams, sweet potatoes,
cassava, potatoes, wheat, sorghum, soybean, rice, wheat, corn,
oil-producing Brassica (e.g., oil-producing rapeseed and canola),
cotton, sugar cane, sunflower, millet, and alfalfa), fruits,
vegetables, grains, seeds, 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.). Furthermore, a cell can be a
stem cell or progenitor cell.
[0104] As used herein, the term "transgenic organism" refers to an
organism comprising a recombinantly introduced polynucleotide.
[0105] As used herein, the terms "transgenic plant cell" and
"transgenic plant" are interchangeable and refer to a plant cell or
a plant containing a recombinantly introduced polynucleotide.
Included in the term transgenic plant is the progeny (any
generation) of a transgenic plant or a seed such that the progeny
or seed comprises a DNA sequence encoding a recombinantly
introduced polynucleotide or a fragment thereof.
[0106] As used herein, the phrase "generating a transgenic plant
cell or a plant" refers to using recombinant DNA methods and
techniques to construct a vector for plant transformation to
transform the plant cell or the plant and to generate the
transgenic plant cell or the transgenic plant.
[0107] The term "excipient" as used herein typically refers to any
pharmacologically inactive substance used for in the formulation or
administration of pharmaceutical compositions of the present
invention, for example, a carrier or vehicle. Examples of
excipients useful in the practice of the present invention are
described herein.
[0108] The term "physiological conditions" as used herein refers to
conditions compatible with living cells, e.g., predominantly
aqueous conditions of a temperature, pH, salinity, etc.
[0109] The terms "therapeutic composition," "pharmaceutical
composition," "therapeutic preparation," and "pharmaceutical
preparation" are used interchangeably herein and encompass
compositions of the present invention suitable for application or
administration to a subject, typically a human. In general such
compositions are safe, sterile, and preferably free of contaminants
that are capable of eliciting undesirable responses in the subject
(i.e., the compound(s) comprising the composition are
pharmaceutically acceptable). Compositions can be formulated for
application or administration to a subject in need thereof by a
number of different routes of administration including oral (i.e.,
administered by mouth or alimentary canal) or parenteral (e.g.,
buccal, rectal, transdermal, transmucosal, subcutaneous,
intravenous, intraperitoneal, intradermal, intratracheal,
intrathecal, pulmonary, and the like).
[0110] The term "subject" as used herein refers to any member of
the subphylum chordata, including, without limitation, humans and
other primates, including non-human primates such as rhesus
macaque, chimpanzees and other apes and monkey species; farm
animals such as cattle, sheep, pigs, goats and horses; domestic
mammals such as dogs and cats; laboratory animals including rodents
such as mice, rats and guinea pigs; birds, including domestic, wild
and game birds such as chickens, turkeys and other gallinaceous
birds, ducks, geese; and the like. The term does not denote a
particular age. Thus, adult, young, and newborn individuals are
intended to be covered.
[0111] A CRISPR (Clustered Regularly Interspaced Short Palindromic
Repeats) is a genomic locus found in the gnomes of many prokaryotes
(e.g., bacteria and archaea). CRISPR loci 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
are 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.
[0112] A CRISPR locus includes 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 occur in clusters. Repeats frequently
diverge between species. Repeats are regularly interspaced with
unique intervening sequences, referred to as "spacers," resulting
in a repeat-spacer-repeat locus architecture. Spacers are identical
to or have high homology with known foreign invader sequences. A
spacer-repeat unit encodes a crisprRNA (crRNA). A crRNA refers to
the mature form of the spacer-repeat unit. A crRNA comprises a
"seed" sequence that is involved in targeting a target nucleic acid
(e.g., possibly as a surveillance mechanism against foreign nucleic
acid). A seed sequence is typically located towards the 5' end of a
crRNA (e.g. in the Cascade complex; for a description of the
Cascade complex see, e.g., Jore, M. M. et al., "Structural basis
for CRISPR RNA-guided DNA recognition by Cascade," Nature
Structural & Molecular Biology 18, 529-536 (2011)) or at the 3'
end of the spacer of a crRNA (e.g., in a Type II CRISPR-Cas9
system), directly adjacent to the first stem.
[0113] A CR1SPR locus comprises polynucleotide sequences encoding
for CRISPR Associated Genes (Cas) genes. Cas genes are involved in
the biogenesis and/or the interference stages of crRNA function.
Cas genes 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 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 are 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.
[0114] The integration stage of a CRISPR system refers 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 typically occurs
at the leader end of the CRISPR locus. Cas proteins (e.g., Cas1 and
Cas2) are involved in integration of new spacer sequences.
Integration proceeds similarly for some types of CRISPR systems
(e.g., Type I-III).
[0115] Mature crRNAs are processed from a longer polycistronic
CRISPR locus transcript (i.e., pre-crRNA array). A pre-crRNA array
comprises a plurality of crRNAs. The repeats in the pre-crRNA array
are recognized by Cas genes. Cas genes 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 exonuclease). A
crRNA may comprise all, some, or none of the CRISPR repeat
sequence.
[0116] Interference refers to the stage in the CRISPR system that
is functionally responsible for combating infection by a foreign
invader. CRISPR interference follows a similar mechanism to RNA
interference (RNAi: e.g., wherein a target RNA is targeted (e.g.,
hybridized) by a short interfering RNA (siRNA)), which results in
target RNA degradation and/or destabilization. CRISPR systems
perform interference of a target nucleic acid by coupling crRNAs
and Cas genes, thereby forming CRISPR ribonucleoproteins (crRNPs).
crRNA of the crRNP guides 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 are subjected to cleavage by Cas proteins. Target
nucleic acid interference typically requires a protospacer adjacent
motif (PAM) in a target nucleic acid.
[0117] There are 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. Modifications of the components of CRISPR-Type II
systems are extensively discussed in the present specification.
[0118] crRNA biogenesis in a Type II CRISPR system comprises a
trans-activating CRISPR RNA (tracrRNA). A tracrRNA is typically
modified by endogenous RNaseIII. The tracrRNA of the complex
hybridizes to a crRNA repeat in the pre-crRNA array. Endogenous
RNaseIII is recruited to cleave the pre-crRNA. Cleaved crRNAs is
subjected to exoribonuclease trimming to produce the mature crRNA
form (e.g., 5' trimming). The tracrRNA typically remains hybridized
to the crRNA. The tracrRNA and the crRNA 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 activates a wild-type, cognate
Cas9 for target nucleic acid cleavage. Target nucleic acid in a
Type II CRISPR system comprises 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.
[0119] Type II CRISPR-Cas9 systems can be further subdivided into
II-A (contains Csn2) and II-B (contains Cas4)-and Type II-C
(neither Csn2 nor Cas4, e.g. N. meningitides). A large number of
Cas9 orthologs are known in the art as well as their associated
tracrRNA and crRNA components (see, e.g., "Supplementary Table S2.
List of bacterial strains with identified Cas9 orthologs," Fonfara,
Ines, et al., "Phylogeny of Cas9 Determines Functional
Exchangeability of Dual-RNA and Cas9 among Orthologous Type II
CRISPR/Cas Systems," Nucleic Acids Research 42.4 (2014): 2577-2590,
including all Supplemental Data; Chylinski K., et al.,
"Classification and evolution of type II CRISPR-Cas systems,"
Nucleic Acids Research, 2014; 42(10):6091-6105, including all
Supplemental Data; Kevin M Esvelt, K. M., et al., (2013)
"Orthogonal Cas9 proteins for RNA-guided gene regulation and
editing," Nature Methods 10, 1116-1121, a number of orthogonal Cas9
proteins identified including a Cas9 protein from Neisseria
meningitidis).
[0120] In addition, variants and modifications of Cas9 protein are
known in the art. U.S. Published Patent Application 20140273226,
published Sep. 18, 2014, discusses the S. pyogenes Cas9 gene, Cas9
protein, variants of the Cas9 protein including host-specific codon
optimized Cas9 coding sequences (e.g., 0129-0137, U.S. Published
Patent Application 20140273226) and Cas9 fusion proteins (e.g.,
233-240, U.S. Published Patent Application 20140273226). U.S.
Published Patent Application 20140315985, published Oct. 23, 2014,
teaches a large number of exemplary wild-type Cas9 polypeptides
(e.g., SEQ ID NO: 1-256, SEQ ID NO: 795-1346, U.S. Published Patent
Application 2014031598) including the sequence of Cas9 from S.
pyogenes (SEQ ID NO: 8, U.S. Published Patent Application
2014031598). Modifications and variants of Cas9 proteins are also
discussed (e.g., 504-608, U.S. Published Patent Application
2014031598).
[0121] Aspects of the present invention can be practiced by one of
ordinary skill in the art following the guidance of the
specification to use Type II CRISPR Cas proteins and Cas-protein
encoding polynucleotides, including, but not limited to Cas9,
Cas9-like, Cas1, Cas2, Cas3, Csn2, Cas4, proteins encoded by Cas9
orthologs, Cas9-like synthetic proteins, and variants and
modifications thereof. The cognate RNA components of these Cas
proteins can be manipulated and modified for use in the practice of
the present invention by one of ordinary skill in the art following
the guidance of the present specification.
[0122] Cas9 is an exemplary Type II CRISPR Cas protein. Cas9 is an
endonuclease that can be programmed by the tracrRNA/crRNA to
cleave, site-specifically, target DNA using two distinct
endonuclease domains (HNH and RuvC/RNase H-like domains) (see U.S.
Published Patent Application No. 2014-0068797, published 6 Mar.
2014; see also Jinek M., et al., "A programmable dual-RNA-guided
DNA endonuclease in adaptive bacterial immunity," Science, 2012;
337:816-210. Two RNA components of a Type II CRISPR-Cas9 system are
illustrated in FIG. 1A. Typically each CRISPR-Cas9 system comprises
a tracrRNA and a crRNA. Cas9 is the signature protein
characteristic for Type II CRISPR systems.
[0123] The crRNA has a region of complementarity to a potential DNA
target sequence and a second region that forms base-pair hydrogen
bonds with the tracrRNA to form a secondary structure, typically to
form at least a stem structure. The region of complementarity to
the DNA target is the spacer. The tracrRNA and a crRNA interact
through a number of base-pair hydrogen bonds to form secondary RNA
structures, for example, as illustrated in FIG. 1B. Complex
formation between tracrRNA/crRNA and Cas9 protein results in
conformational change of the Cas9 protein that facilitates binding
to DNA, endonuclease activities of the Cas9 protein, and
crRNA-guided site-specific DNA cleavage by the endonuclease. For a
Cas9 protein/tracrRNA/crRNA complex to cleave a DNA target
sequence, the DNA target sequence is adjacent to a cognate
protospaccr adjacent motif (PAM).
[0124] The term sgRNA typically refers to a single guide RNA (i.e.,
a single, contiguous polynucleotide sequence) that essentially
comprises a crRNA connected at its 3' end to the 5' end of a
tracrRNA through a "loop" sequence (see, e.g., U.S. Published
Patent Application No. 2014-0068797, published 6 Mar. 2014). sgRNA
interacts with a cognate Cas9 protein essentially as described for
tracrRNA/crRNA polynucleotides, as discussed above. Similar to
crRNA, sgRNA has a spacer element (FIG. 2, 201), a region of
complementarity to a DNA target sequence, adjacent a second region
that forms base-pair hydrogen bonds that form a secondary
structure, typically a stem structure (e.g., in FIG. 2, 202, 203,
204, 205).
[0125] Using a sgRNA/Cas9 protein system, U.S. Published Patent
Application No. 2014-0315985, published 23 Oct. 2014, and later
published Briner, A. E., et al., ("Guide RNA Functional Modules
Direct Cas9 Activity and Orthogonality," Molecular Cell Volume 56,
Issue 2, 23 Oct. 2014, pages 333-339) demonstrated that expendable
features can be removed to generate functional miniature sgRNAs.
These publications discuss the importance of the "nexus," which is
located in the portion of sgRNA that corresponds to tracrRNA (not
crRNA), to confer cleavage activity to Cas9. The nexus confers the
ability of a sgRNA or a tracrRNA to bind to its cognate cas9
protein and confer an apoenzyme to haloenzyme conformational
transition.
[0126] The nexus is located immediately downstream of (i.e.,
located in the 3' direction from) the lower stem in Type II
CRISPR-Cas9 systems. An example of the relative location of the
nexus is illustrated in the sgRNA shown in FIG. 2, 206. U.S.
Published Patent Application No. 2014-0315985 and Briner, et al.,
also disclose consensus sequences and secondary structures of
predicted sgRNAs for several sgRNA/Cas9 families. These references
show that the general arrangement of secondary structures in the
predicted sgRNAs up to and including the nexus correspond to those
shown FIG. 2 herein, that is, in a 5' to 3' direction, a spacer, a
first stem, and the nexus. FIG. 2 presents an overview of and
nomenclature for elements of a sgRNA of the Streptococcus pyogenes
Cas9. Relative to FIG. 2, there are variations in the number and
arrangement of stem structures located 3' of the nexus in the
sgRNAs illustrated in U.S. Published Patent Application No.
2014-0315985 and Briner, et al.
[0127] Fonfara, et al., ("Phylogeny of Cas9 Determines Functional
Exchangeability of Dual-RNA and Cas9 among Orthologous Type II
CRISPR/Cas Systems," Nucleic Acids Research 42.4 (2014): 2577-2590,
including all Supplemental Data, in particular Supplemental FIG.
S11) present the crRNA/tracrRNA sequences and secondary structures
of eight Type II CRISPR-Cas9 systems. RNA duplex secondary
structures were predicted using RNAcofold of the Vienna RNA package
(Bernhart, S. H., et al., (2006) "Partition function and base
pairing probabilities of RNA heterodimers," Algorithms Mol. Biol.,
1, 3; Hofacker, I. L., et al., (2002) "Secondary structure
prediction for aligned RNA sequences. J. Mol. Biol., 319,
1059-1066) and RNAhybrid
(bibiserv.techfak.uni-bielefeld.de/rnahybrid/)). The structure
predictions were then visualized using VARNA (Darty, K., et al.,
(2009) VARNA: Interactive drawing and editing of the RNA secondary
structure Bioinformatics, 25, 1974-1975). Fonfara, et al., show
that the crRNA/tracrRNA complex for Campylobacter jejuni does not
have the bulge region illustrated in FIG. 1B, 105; however, it
retains the general arrangement of secondary structures up to and
including the nexus corresponding to those shown FIG. 1B herein,
that is, in a 5' to 3' direction, a spacer, a first stem, and the
nexus. Ran, F. A., et al., ("In vivo genome editing using
Staphylococcus aureus Cas9," Nature, 2015, Apr. 9;
520(7546):186-91, including all extended data) present the
crRNA/tracrRNA sequences and secondary structures of eight Type II
CRISPR-Cas9 systems (see Extended Data FIG. 1 of Ran, F. A., et
al.). Predicted tracrRNA structures were based on the Constraint
Generation RNA folding model (Zuker, M., "Mfold web server for
nucleic acid folding and hybridization prediction," Nucleic Acids
Res., 31, 3406-3415 (2003)). The crRNA/tracrRNA structures for the
eight bacterial species presented in FIG. 1 of Ran, et al., show
that the general arrangement of secondary structures in the
predicted crRNA/tracrRNAs up to and including the nexus correspond
to those shown FIG. 1B herein, that is, in a 5' to 3' direction, a
spacer, a first stem, and the nexus.
[0128] As discussed above and in the Background of the present
Specification, Jinek, M., et al., ("A programmable dual-RNA-guided
DNA endonuclease in adaptive bacterial immunity," Science
337(6096):816-21 (2012)), Briner, A., et al., ("Guide RNA
Functional Modules Direct Cas9 Activity and Orthogonality,"
Molecular Cell 56(2), 2014, Pages 333-339) and Wright, A. V., et
al., ("Rational design of a split-Cas9 enzyme complex," PNAS
112(10), 2015, pages 2984-2989) all noted the importance of the
nexus hairpin for guide RNA/Cas9 enzyme complex activity.
[0129] However, contrary to these teachings, experiments performed
in support of the present invention unexpectedly demonstrated that
the nexus hairpin structure can be broken and modified; thus
providing new design and engineering avenues for CRISPR
technologies as described herein.
[0130] In a first aspect, the present invention relates to an
engineered Type II CRISPR-Cas9 system comprising two or more
polynucleotides (sn-casPNs) capable of forming a complex with a
Cas9 protein to cause the Cas9 protein to bind a first DNA sequence
comprising a DNA target sequence preferentially relative to a
second DNA sequence without the DNA target binding sequence. In
some embodiments, the complex cuts the first DNA sequence. In the
system, at least two of the two or more polynucleotides are
necessary to form a nexus stem element. In addition to binding the
first DNA sequence the sn-casPNs/Cas9 complex can cause the Cas9
protein to bind and cleave the first DNA sequence. A preferred
embodiment comprises three sn-casPNs (sn1-casPN, sn2-casPN, and
sn3-casPN; two examples are shown in FIG. 3A, FIG. 3C), wherein
sn3-casPN comprises a spacer clement (i.e., a DNA target binding
sequence). Another preferred embodiment comprises two sn-casPNs
(sn1-casPN, sn2-casPN; two examples are shown in FIG. 3B, FIG. 3D),
wherein sn1-casPN comprises a spacer element (i.e., a DNA target
binding sequence) and a first portion of the nexus element. Two
variations of three sn-casPNs are presented in FIG. 3F, FIG. 3H.
Two variations of four sn-casPNs are presented in FIG. 3E, FIG.
3G).
[0131] In one embodiment of the first aspect of the present
invention, the two or more polynucleotides comprise a first
polynucleotide (e.g., FIG. 3A, 301; FIG. 3C, 327; FIG. 3E, 301;
FIG. 3G, 327) comprising a first nexus stem element nucleotide
sequence I and a second polynucleotide (e.g., FIG. 3A, 302; FIG.
3C, 302; FIG. 3E, 302; FIG. 3G, 302) comprising a first nexus stem
element nucleotide sequence II, wherein (i) the first nexus stem
element nucleotide sequence II and the first nexus stem element
nucleotide sequence II are capable of forming the nexus stem
element by base-pair hydrogen bonding between the first nexus stem
element nucleotide sequence I and the second nexus stem element
nucleotide sequence II, and (ii) the first polynucleotide and the
second polynucleotide are separate polynucleotides each having a 5'
end and a 3' end.
[0132] In some embodiments of the first aspect of the present
invention, the first polynucleotide (e.g., FIG. 3A, 301; FIG. 3C,
327) comprises in a 5' to 3' direction a first stem element
nucleotide sequence I and the nexus stem element nucleotide
sequence I and a third polynucleotide (e.g., FIG. 3A, 303; FIG. 3C,
328) comprises in a 5' to 3' direction a DNA target binding
sequence and a first stem element nucleotide sequence II, wherein
the first stem element nucleotide sequence I and the first stem
element nucleotide sequence II are capable of forming a first stem
element by base-pair hydrogen bonding between the first stem
element nucleotide sequence I and the first stem element nucleotide
sequence II, wherein the third polynucleotide is a separate
polynucleotide having a 5' end and a 3' end.
[0133] In other embodiments of the first aspect of the present
invention, the first polynucleotide (e.g., FIG. 3A, 301) comprises
in a 5' to 3' direction an upper stem element nucleotide sequence
I, a bulge element nucleotide sequence I, the first stem element
nucleotide sequence I, and the nexus stem element nucleotide
sequence I, and the third polynucleotide (e.g., FIG. 3A, 303)
comprises in a 5' to 3' direction the DNA target binding sequence,
the first stem clement nucleotide sequence II, a bulge element
nucleotide sequence II, and an upper stem element nucleotide
sequence II, wherein the upper stem element nucleotide sequence I
and the upper stem element nucleotide sequence II form an upper
stem element by base-pair hydrogen bonding between the upper stem
element nucleotide sequence I and the upper stem element nucleotide
sequence II, and the first stem element nucleotide sequence I and
the first stem element nucleotide sequence II form the first stem
element by base-pair hydrogen bonding between the first stem
element nucleotide sequence I and the first stem element nucleotide
sequence II.
[0134] In some embodiments of the first aspect of the present
invention, the first polynucleotide (e.g., FIG. 3E, 301; FIG. 3G,
327) further comprises in a 5' to 3' direction a first stem element
nucleotide sequence I and the nexus stem element nucleotide
sequence I, a third polynucleotide (e.g., FIG. 3E, 330; FIG. 3G,
333) comprises a first stem element nucleotide sequence II, and a
spacer polynucleotide (e.g., FIG. 3E, 331; FIG. 3G, 331) comprises
a DNA target binding sequence, wherein the first stem element
nucleotide sequence I and the first stem element nucleotide
sequence II form a first stem clement by base-pair hydrogen bonding
between the first stem element nucleotide sequence I and the first
stem element nucleotide sequence II. In this embodiment, the first
polynucleotide, the second polynucleotide, the third
polynucleotide, and the spacer polynucleotide are separate
polynucleotides each having a 5' end and a 3' end.
[0135] In further embodiments of the first aspect of the present
invention, the first polynucleotide (FIG. 3E, 301) comprises in a
5' to 3' direction an upper stem element nucleotide sequence I, a
bulge element sequence I, a lower stem element nucleotide sequence
I, and the nexus stem element nucleotide sequence I, a third
polynucleotide (FIG. 3E, 330) comprises in a 5' to 3' direction a
first lower stem element nucleotide sequence II, a bulge element
nucleotide sequence II, and an upper stem element nucleotide
sequence II, and a spacer polynucleotide (FIG. 3E, 331) comprises a
DNA target binding sequence, wherein the upper stem element
nucleotide sequence I and the upper stem element nucleotide
sequence II form an upper stem element by base-pair hydrogen
bonding between the upper stem element nucleotide sequence I and
the upper stem element nucleotide sequence II, and the lower stem
element nucleotide sequence I and the lower stem element nucleotide
sequence II form a lower stem clement by base-pair hydrogen bonding
between the lower stem element nucleotide sequence I and the lower
stem element nucleotide sequence II.
[0136] Additional embodiments will be clear to one of ordinary
skill in the art in view of the teachings of the present
specification.
[0137] In a second aspect of the present invention, an engineered
Type II CRISPR-Cas9 system comprises two or more polynucleotides.
The two or more polynucleotides comprise a tracr element that is
capable of forming a complex with a Cas9 protein to cause the Cas9
protein to bind DNA sequences containing protospacer adjacent motif
(PAM) sequences preferentially relative to DNA sequences without
PAM sequences. In some embodiments, the complex preferentially
binds and cuts DNA sequences containing PAM sequences. The tracr
element comprises a first polynucleotide (e.g., FIG. 3A, 301; FIG.
3C, 327; FIG. 3E, 301; FIG. 3G, 327) comprising a nexus stem
element nucleotide sequence 1 and a second polynucleotide (e.g.,
FIG. 3A, 302; FIG. 3C, 302; FIG. 3E, 302; FIG. 3G, 302) comprising
a nexus stem element nucleotide sequence II, wherein the nexus stem
element nucleotide sequence I and the nexus stem clement nucleotide
sequence II arc capable of forming a nexus stem element by
base-pair hydrogen bonding between the nexus stem element
nucleotide sequence I and the nexus stem element nucleotide
sequence II, and (ii) the first polynucleotide and the second
polynucleotide are separate polynucleotides each having a 5' end
and a 3' end.
[0138] In some embodiments of the second aspect of the present
invention, the first polynucleotide (e.g., FIG. 3A, 301; FIG. 3C,
327) comprises in a 5' to 3' direction a first stem element
nucleotide sequence I and the nexus stem element nucleotide
sequence I and a third polynucleotide (e.g., FIG. 3A, 303; FIG. 3C,
328) comprises in a 5' to 3' direction a DNA target binding
sequence and a first stem element nucleotide sequence II, wherein
the first stem element nucleotide sequence I and the first stem
element nucleotide sequence II are capable of forming a first stem
element by base-pair hydrogen bonding between the first stem
element nucleotide sequence land the first stem element nucleotide
sequence II, wherein the third polynucleotide is a separate
polynucleotide having a 5' end and a 3' end.
[0139] In other embodiments of the second aspect of the present
invention, the first polynucleotide (e.g., FIG. 3A, 301) comprises
in a 5' to 3' direction an upper stem element nucleotide sequence
I, a bulge element nucleotide sequence I, the first stem element
nucleotide sequence I, and the nexus stem element nucleotide
sequence I, and the third polynucleotide (e.g., FIG. 3A, 303)
comprises in a 5' to 3' direction the DNA target binding sequence,
the first stem element nucleotide sequence II, a bulge element
nucleotide sequence II, and an upper stem element nucleotide
sequence II, wherein the upper stem element nucleotide sequence I
and the upper stem element nucleotide sequence II form an upper
stem element by base-pair hydrogen bonding between the upper stem
element nucleotide sequence I and the upper stem element nucleotide
sequence II, and the first stem element nucleotide sequence I and
the first stem element nucleotide sequence II form the first stem
element by base-pair hydrogen bonding between the first stem
element nucleotide sequence I and the first stem element nucleotide
sequence II.
[0140] In some embodiments of the second aspect of the present
invention, the first polynucleotide (e.g., FIG. 3E, 301; FIG. 3G,
327) further comprises in a 5' to 3' direction a first stem element
nucleotide sequence I and the nexus stem element nucleotide
sequence I, a third polynucleotide (e.g., FIG. 3E, 330; FIG. 3G,
333) comprises a first stem element nucleotide sequence II, and a
spacer polynucleotide (e.g., FIG. 3E, 331; FIG. 3G, 331) comprises
a DNA target binding sequence, wherein the first stem element
nucleotide sequence I and the first stem element nucleotide
sequence II form a first stem element by base-pair hydrogen bonding
between the first stem element nucleotide sequence I and the first
stem element nucleotide sequence II. In this embodiment, the first
polynucleotide, the second polynucleotide, the third
polynucleotide, and the spacer polynucleotide are separate
polynucleotides each having a 5' end and a 3' end.
[0141] In further embodiments of the second aspect of the present
invention, the first polynucleotide (FIG. 3E, 301) comprises in a
5' to 3' direction an upper stem element nucleotide sequence I, a
bulge element sequence I, a lower stem element nucleotide sequence
I, and the nexus stem element nucleotide sequence I, a third
polynucleotide (FIG. 3E, 330) comprises in a 5' to 3' direction a
first lower stem element nucleotide sequence II, a bulge element
nucleotide sequence II, and an upper stem element nucleotide
sequence II, and a spacer polynucleotide (FIG. 3E, 331) comprises a
DNA target binding sequence, wherein the upper stem element
nucleotide sequence I and the upper stem element nucleotide
sequence II form an upper stem element by base-pair hydrogen
bonding between the upper stem element nucleotide sequence I and
the upper stem element nucleotide sequence II, and the lower stem
element nucleotide sequence I and the lower stem element nucleotide
sequence II form a lower stem element by base-pair hydrogen bonding
between the lower stem element nucleotide sequence I and the lower
stem element nucleotide sequence II.
[0142] Additional embodiments will be clear to one of ordinary
skill in the art in view of the teachings of the present
specification.
[0143] With reference to the term "tracr element," as used herein
the term refers to two or more sn-casPNs capable of forming a
complex with a Cas9 protein to cause the Cas9 protein to bind DNA
sequences containing PAM sequences preferentially relative to DNA
sequences without PAM sequences. Sternberg, S. H. et al., ("DNA
interrogation by the CRISPR RNA-guided endonuclease Cas9," Nature.
2014 Mar. 6; 507(7490): 62-67)) teach methods using double-tethered
DNA curtains to examine the locations and corresponding lifetimes
of all binding events for tracrRNA/crRNA/Cas with DNA. Following
the guidance of the present specification, one of ordinary skill in
the art can apply such methods to evaluate preferential binding
(higher binding affinity) of, for example, sn-casPNs/Cas9 complexes
to DNA sequences containing PAM sequences versus DNA sequences
without PAM sequences to confirm presence of a tracr element
comprising two or more of the sn-casPNs.
[0144] With reference to the sn-casPNs, a "spacer" or "spacer
clement" as used herein refers to a target binding sequence that
can specifically hybridize to a complementary target nucleic acid
sequence and a "spacer polynucleotide" refers to a polynucleotide
sequence comprising a spacer element. The spacer element interacts
with the target nucleic acid sequence through hydrogen bonding
between complimentary base pairs (i.e., paired bases). Typically, a
spacer element (a DNA target binding sequence) binds to a selected
DNA target sequence. The spacer element determines the location of
the Cas9 protein site-specific binding and endonucleolytic
cleavage. Spacer elements range from approximately 17- to
approximately 84 nucleotides long, depending on the Cas9 protein
with which they are associated, and have an average length of 36
nucleotides (Marraffini, L. A., et al., "CRISPR interference:
RNA-directed adaptive immunity in bacteria and archaea," Nature
reviews Genetics. 2010; 11(3):181-190). In a Type II CR1SPR-Cas9
system the spacer element typically comprises a "seed" sequence
that is involved in targeting a target nucleic acid. For example,
for SpyCas9 the functional length for a spacer element to direct
specific cleavage is typically about 12-25 nucleotides. Variability
of the functional length for a spacer element is known in the art
(see, e.g., U.S. Published Patent Application No. 2014-0315985,
published 23 Oct. 2014). Spacer polynucleotides in some embodiments
have polynucleotide sequences in addition to the spacer element and
such polynucleotide sequences are typically located at the 5' end
of the spacer element, the 3' end of the spacer element, internal
to the spacer element, or combinations thereof.
[0145] The creation of secondary structure between two
polynucleotides through base-pair hydrogen bonding (e.g., stem
elements and hairpins) can be determined by a number of methods
known to those of ordinary skill in the art (e.g., experimental
techniques, including but not limited to X-ray crystallography,
Nuclear Magnetic Resonance (NMR) spectroscopy, Cryo-electron
microscopy (Cryo-EM), Chemical/enzymatic probing, thermal
denaturation (melting studies), and Mass spectrometry; predictive
techniques, such as computational structure prediction; preferred
methods include Chemical/enzymatic probing, thermal denaturation
(melting studies)). Methods to predict secondary structures of
single-stranded RNA or DNA sequences are known in the art, for
example, the "RNAfold web server"
(rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) predicts secondary
structures of single-stranded RNA or DNA sequences (see, e.g.,
Gruber A R, et al., The Vienna RNA Websuite, Nucleic Acids Res.
2008; Lorenz, R., et al., (2011) "ViennaRNA Package 2.0",
Algorithms for Molecular Biology, 6, 26). A preferred method to
evaluate RNA secondary structure is to use the combined
experimental and computational SHAPE method (Low J. T., et al.,
"SHAPE-Directed RNA Secondary Structure Prediction," Methods (San
Diego, Calif.) 2010; 52(2):150-158).
[0146] In a third aspect of the present invention, an engineered
Type II CRISPR-Cas9 system comprises two or more polynucleotides
comprising a first polynucleotide and a second polynucleotide each
having 5' and 3' ends. The first polynucleotide (e.g., FIG. 3A,
301; FIG. 3C, 327; FIG. 3E, 301; FIG. 3G, 327) comprises a nexus
stem element nucleotide sequence I, the nexus stem element
nucleotide sequence I comprising in a 5' to 3' direction
Nw-N1-N2-Nx, where Nw is a first connective nucleotide sequence
wherein w is the length of the connective nucleotide sequence and w
is greater than or equal to two, N1 is a nucleotide, N2 is a
nucleotide, and Nx is a first auxiliary polynucleotide wherein x is
the length of the first auxiliary polynucleotide and x is greater
than or equal to zero. In some embodiments, for Nw, w is greater
than or equal to zero, preferably w is greater than or equal to 1,
more preferably w is greater than or equal to 2. The second
polynucleotide (e.g., FIG. 3A, 302; FIG. 3C, 302; FIG. 3E, 302;
FIG. 3G, 302) comprises a nexus stem element nucleotide sequence
II, the nexus stem element nucleotide sequence II comprising in a
5' to 3' direction Ny--Nc2-Nc1-Nz, where Ny is a second auxiliary
polynucleotide wherein y is the length of the second auxiliary
polynucleotide and y is greater than or equal to zero, Nc2 is a
nucleotide that is complementary to N2, Nc1 is a nucleotide that is
complementary to N1, and Nz is s second connective nucleotide
sequence wherein z is the length of the second connective
nucleotide sequence and z is greater than or equal to zero. In some
embodiments, for Nz, z is greater than or equal to 1, preferably z
is greater than or equal to 2. In this aspect, the first nexus stem
clement nucleotide sequence and the second nexus stem clement
nucleotide sequence are capable of forming a nexus stem element by
base-pair hydrogen bonding between at least N1/Nc1 and N2/Nc2 and
the first polynucleotide and the second polynucleotide are separate
polynucleotides.
[0147] In some embodiments of the third aspect of the present
invention, the first polynucleotide (e.g., FIG. 3A, 301; FIG. 3C,
327) comprises in a 5' to 3' direction a first stem element
nucleotide sequence I and the nexus stem element nucleotide
sequence I and a third polynucleotide (e.g., FIG. 3A, 303; FIG. 3C,
328) comprises in a5' to 3' direction a DNA target binding sequence
and a first stem element nucleotide sequence II, wherein the first
stem element nucleotide sequence I and the first stem element
nucleotide sequence II are capable of forming a first stem element
by base-pair hydrogen bonding between the first stem element
nucleotide sequence I and the first stem element nucleotide
sequence II, wherein the third polynucleotide is a separate
polynucleotide having a 5' end and a 3' end.
[0148] In other embodiments of the third aspect of the present
invention, the first polynucleotide (e.g., FIG. 3A, 301) comprises
in a 5' to 3' direction an upper stem element nucleotide sequence
I, a bulge element nucleotide sequence I, the first stem element
nucleotide sequence I, and the nexus stem element nucleotide
sequence I, and the third polynucleotide (e.g., FIG. 3A, 303)
comprises in a 5' to 3' direction the DNA target binding sequence,
the first stem element nucleotide sequence II, a bulge element
nucleotide sequence II, and an upper stem element nucleotide
sequence II, wherein the upper stem element nucleotide sequence I
and the upper stem element nucleotide sequence II form an upper
stem element by base-pair hydrogen bonding between the upper stem
element nucleotide sequence I and the upper stem element nucleotide
sequence II, and the first stem element nucleotide sequence I and
the first stem element nucleotide sequence II form the first stem
element by base-pair hydrogen bonding between the first stem
element nucleotide sequence I and the first stem element nucleotide
sequence II.
[0149] In some embodiments of the third aspect of the present
invention, the first polynucleotide (e.g., FIG. 3E, 301; FIG. 3G,
327) further comprises in a 5' to 3' direction a first stem element
nucleotide sequence I and the nexus stem element nucleotide
sequence I, a third polynucleotide (e.g., FIG. 3E, 330; FIG. 3G,
333) comprises a first stem element nucleotide sequence II, and a
spacer polynucleotide (e.g., FIG. 3E, 331; FIG. 3G, 331) comprises
a DNA target binding sequence, wherein the first stem element
nucleotide sequence I and the first stem element nucleotide
sequence II form a first stem element by base-pair hydrogen bonding
between the first stem element nucleotide sequence I and the first
stem element nucleotide sequence II. In this embodiment, the first
polynucleotide, the second polynucleotide, the third
polynucleotide, and the spacer polynucleotide are separate
polynucleotides each having a 5' end and a 3' end.
[0150] In further embodiments of the third aspect of the present
invention, the first polynucleotide (FIG. 3E, 301) comprises in a
5' to 3' direction an upper stem element nucleotide sequence I, a
bulge element sequence I, a lower stem element nucleotide sequence
I, and the nexus stem element nucleotide sequence I, a third
polynucleotide (FIG. 3E, 330) comprises in a 5' to 3' direction a
first lower stem element nucleotide sequence II, a bulge element
nucleotide sequence II, and an upper stem element nucleotide
sequence II, and a spacer polynucleotide (FIG. 3E, 331) comprises a
DNA target binding sequence, wherein the upper stem element
nucleotide sequence I and the upper stem element nucleotide
sequence II form an upper stem element by base-pair hydrogen
bonding between the upper stem element nucleotide sequence I and
the upper stem element nucleotide sequence II, and the lower stem
element nucleotide sequence I and the lower stem element nucleotide
sequence II form a lower stem element by base-pair hydrogen bonding
between the lower stem element nucleotide sequence I and the lower
stem element nucleotide sequence II.
[0151] Additional embodiments will be clear to one of ordinary
skill in the art in view of the teachings of the present
specification.
[0152] A fourth aspect of the present invention comprises a
modification of the first, second, and third aspects of the present
invention, wherein the 5' end of the first polynucleotide and the
3' end of the third polynucleotide are joined by a loop element.
Accordingly, in the fourth aspect of the invention there is no
"third polynucleotide" because it has been subsumed into "a first
polynucleotide comprising a first hairpin." In some embodiments,
this first polynucleotide comprises in a 5' to 3' direction a DNA
target binding sequence, a first stem element, and the nexus stem
element nucleotide sequence I, wherein the first stem element
comprises a first hairpin (e.g., FIG. 3D, 329). In further
embodiments the first stem clement further comprises a lower stem
clement, a bulge element, and an upper stem element, wherein the
lower stem element is adjacent the bulge element, the bulge element
is adjacent the upper stem element, the bulge element is interposed
between the lower stem element and the upper stem element, and the
upper stem element comprises the first hairpin (e.g., FIG. 3B,
326). In some embodiments the spacer element is separated from the
first polynucleotide comprising the first hairpin (e.g., FIG. 3F,
332; FIG. 3H, 334) and a spacer polynucleotide (e.g., FIG. 3F, 331;
FIG. 3H, 331) comprises the spacer element. The variations of
sn-casPNs described below that use a first accessory polynucleotide
or a second accessory polynucleotide do not apply to the sn-casPNs
comprising a first polynucleotide comprising a first hairpin.
[0153] Additional embodiments will be clear to one of ordinary
skill in the art in view of the teachings of the present
specification.
[0154] Components of a sn1-casRNA/sn2-casRNA/Cas9 system is
illustrated in FIG. 5A, FIG. 5B, and FIG. 5C. An example of
sn1-casRNA/sn2-casRNA is presented in FIG. 3B, wherein the first
polynucleotide is sn1-casRNA (FIG. 3B, 326) and the second
polynucleotide is sn2-casRNA (FIG. 3B, 302). FIG. 5A presents a
model of the .alpha.-Helical lobe of SpyCas9 (FIG. 5A, 501) in
complex with sn1-casRNA (FIG. 5A, 502). The section of the
sn1-casRNA corresponding to the spacer element (i.e., a nucleic
acid target binding sequence) is indicated by a bracket (FIG. 5A,
503). 5B presents a model of the Catalytic nuclease lobe (FIG. 5B,
506) of SpyCas9 in complex with sn2-casRNA (FIG. 5B, 507). The
relative positions of the RuvC domain (FIG. 5B, 510; RNase H
domain) and the HNH domain (FIG. 5B, 511; HNH nuclease domain) are
indicated. FIG. 5C provide a view of an assembled
sn1-casRNA/sn2-casRNA/Cas9 protein complex. The relative locations
of the 3' end of the sn1-casRNA split nexus element (FIG. 5C, 505)
and 3' end of the split nexus element of the sn2-casRNA (FIG. 5C
508) are indicated.
[0155] A fifth aspect of the present invention comprises a
modification of the first, second, and third aspects of the present
invention, wherein the modification is the addition of an optional
accessory polynucleotide to the first polynucleotide, the third
polynucleotide, or both the first polynucleotide and the third
polynucleotide. In some embodiments of the fifth aspect of the
invention, the first polynucleotide further comprises a first
accessory polynucleotide located 5' of the first stem clement
nucleotide sequence I. When the first stem element of the first
polynucleotide comprises, in a 5' to 3' direction, the upper stem
element nucleotide sequence I, the bulge element sequence I, the
lower stem element nucleotide sequence I, and the nexus stem
element nucleotide sequence I, then the first accessory
polynucleotide is located 5' of the upper stem element nucleotide
sequence I (e.g., FIG. 4B, 401-427 to 428).
[0156] In other embodiments of the fifth aspect of the present
invention, the third polynucleotide further comprises a second
accessory polynucleotide located 3' of the first stem element
nucleotide sequence II. When the first stem element of the third
polynucleotide comprises, in a 5' to 3' direction, the DNA target
binding sequence, the first stem element nucleotide sequence II,
the bulge element nucleotide sequence II, and the upper stem
element nucleotide sequence II, then the second accessory
polynucleotide is located 3' of the upper stem element nucleotide
sequence II (e.g., FIG. 4B, 405-429 to 430).
[0157] The accessory polynucleotide can comprise a variety of
moieties including, but not limited to, an affinity tag, a ligand,
a ligand-binding sequence, a linker sequence, a hairpin, an
affinity nucleotide sequences, an effector binding element, fused
effector proteins, a subcellular localization signal or coding
sequences therefore; a small molecule, a detectable label, a member
of a FRET pair, a fluorophore/quantum dot donor/acceptor pair,
fluorescent labels, an enzyme, a fluorescent protein, a
nanoparticle, a quantum dot.
[0158] A sixth aspect of the present invention is directed to
modifications of the second polynucleotide of sn-casPNs/Cas9
systems. In one embodiment, the second polynucleotide comprises, in
a 5' to 3' direction, the nexus stem element nucleotide sequence II
and a second stem element, wherein the second stem element
comprises a hairpin. The second polynucleotide can also comprise in
a 5' to 3' direction the nexus stem element nucleotide sequence II,
the second stem element, and a third stem element, wherein the
third stem element comprises a hairpin. Furthermore, the second
polynucleotide comprises, in a 5' to 3' direction, the nexus stem
element nucleotide sequence II and a second stem element, wherein
the second stem element comprises a hairpin, and a third stem
clement, wherein the third stem element comprises a hairpin (e.g.,
FIG. 3A, 302).
[0159] In another embodiment of the sixth aspect of the present
invention, the second polynucleotide further comprises in a 5' to
3' direction the nexus stem clement nucleotide sequence II, a
second connective sequence, and a second stem element nucleotide
sequence I (e.g., FIG. 4A, 402-406-407) and a first adjunct
polynucleotide comprising a second stem element nucleotide sequence
II (FIG. 4A, 403-409 to 410), wherein the second stem element
nucleotide sequence I and the second stem element nucleotide
sequence II form a second stem element by base-pair hydrogen
bonding between the second stem element nucleotide sequence I and
the second stem element nucleotide sequence II. In some
embodiments, the 5' end of the second stem element nucleotide
sequence II and the 3' end of the second stem element nucleotide
sequence I are connected by a loop element to create a second
hairpin.
[0160] Furthermore, the first adjunct polynucleotide can comprise
in a 5' to 3' direction the second stem element nucleotide sequence
II and a third stem element nucleotide sequence I (FIG. 4A,
403-411-412), and a second adjunct polynucleotide (FIG. 4A, 404)
comprises in a 5' to 3' direction a third stem element nucleotide
sequence II (FIG. 4A, 404-413 to 414), wherein the third stem
element nucleotide sequence I and the third stem element nucleotide
sequence II form a third stem element by base-pair hydrogen bonding
between the third stem element nucleotide sequence I and third stem
element nucleotide sequence II. In some embodiments, the 5' end of
the third stem element nucleotide sequence II and the 3' end of the
third stem element nucleotide sequence I are connected by a loop
element to create a third hairpin.
[0161] In other embodiments the 3' end of the second polynucleotide
comprises a 3' terminal sequence that can comprise a variety of
moieties including, but not limited to, an affinity tag, a ligand,
a ligand-binding sequence, a linker sequence, a hairpin, an
affinity nucleotide sequences, an effector binding element, fused
effector proteins, a subcellular localization signal or coding
sequences therefore; a small molecule, a detectable label, a member
of a FRET pair, a fluorophore/quantum dot donor/acceptor pair,
fluorescent labels, an enzyme, a fluorescent protein, a
nanoparticle, a quantum dot.
[0162] A seventh aspect of the present invention is directed to the
modification of the 3' end of the split nexus of the first
polynucleotide and the 5' end of the split nexus of the second
polynucleotide wherein the modification is the addition of an
optional auxiliary polynucleotide to the first polynucleotide, the
second polynucleotide, or both the first polynucleotide and the
second polynucleotide. In one embodiment, the first polynucleotide
further comprises a first auxiliary polynucleotide 3' adjacent the
nexus stem element nucleotide sequence I. In another embodiment,
the second polynucleotide further comprises a second auxiliary
polynucleotide 5' adjacent the nexus stem element nucleotide
sequence II. In yet another embodiment, the first polynucleotide
comprises a first auxiliary polynucleotide 3' adjacent the nexus
stem element nucleotide sequence I, and the second polynucleotide
comprises a second auxiliary polynucleotide 5' adjacent the nexus
stem element nucleotide sequence II. In some embodiments a linker
element polynucleotide is interposed between the nexus element
nucleotide sequence and the auxiliary polynucleotide. The first
auxiliary polynucleotide and/or second auxiliary polynucleotide can
comprise a binding site for a single-strand polynucleotide binding
protein, such as a single-strand RNA binding protein.
[0163] In a further embodiment of the seventh aspect of the
invention, the first polynucleotide comprises a first auxiliary
polynucleotide 3' adjacent the nexus stem element nucleotide
sequence I and the second polynucleotide comprises a second
auxiliary polynucleotide 5' adjacent the nexus stem element
nucleotide sequence II, and the first auxiliary polynucleotide
comprises an effector binding element nucleotide sequence I, and
the second auxiliary polynucleotide comprises an effector binding
element nucleotide sequence II, wherein the effector binding
element nucleotide sequence I and the effector binding element
nucleotide sequence II are capable of forming an effector binding
element by base-pair hydrogen bonding between the effector binding
element nucleotide sequence I and the effector binding element
nucleotide sequence I. Thus, providing a double-stranded
polynucleotide effector binding element to which an effector
protein can bind. In some embodiments the double-stranded
polynucleotide is an RNA and the effector protein is a
double-stranded RNA binding protein capable of binding the effector
binding element. Examples of double-stranded RNA binding effector
proteins include Cas5, Cas6, and Csy4. In some embodiments the
effector binding protein is catalytically inactive (e.g., Csy4*)
but still binds the effector binding element.
[0164] In some embodiments of the seventh aspect of the invention,
the first auxiliary polynucleotide and/or second auxiliary
polynucleotide further comprises one or more first affinity
nucleotide sequence. An affinity nucleotide sequence can be
covalently linked to a polypeptide. An affinity nucleotide sequence
can comprise a ligand. In some embodiments, one of the affinity
nucleotide sequences comprises a ligand and the other affinity
nucleotide sequence comprises a cognate ligand-binding moiety.
[0165] FIG. 4A illustrates an example of a first polynucleotide
(FIG. 4A, 401) comprising a first auxiliary polynucleotide (FIG.
4A, 401-419 to 424), wherein the first auxiliary polynucleotide
comprises a linker element nucleotide sequence I (FIG. 4A, 401-419
to 422), an affinity nucleotide sequence I (FIG. 4A, 401-422 to
423), and an effector binding element nucleotide sequence I (FIG.
4A, 401-423 to 424), and a second polynucleotide (FIG. 4A, 402)
comprising a second auxiliary polynucleotide (FIG. 4A, 402-405 to
418), wherein the second auxiliary polynucleotide comprises a
linker element nucleotide sequence II (FIG. 4A, 402-405 to 416), an
affinity nucleotide sequence II (FIG. 4A, 402-416 to 417), and an
effector binding element nucleotide sequence II (FIG. 4A, 402-417
to 418).
[0166] An example of use of an effector protein is Csy4* with a
cognate effector protein binding element can be given with
reference to this figure. Effector binding effector binding element
nucleotide sequence I (FIG. 4A, 401-423 to 424) and effector
binding element nucleotide sequence II (FIG. 4A, 402-417 to 418)
form a double-strand RNA structure through base-pair hydrogen
bonding to form a Csy4* double-strand binding element. After
formation of the double-strand RNA binding element Csy4* protein
binds the binding element and stabilizes the interaction of the
first auxiliary polynucleotide and the second auxiliary
polynucleotide. Csy* and its cognate binding element is used in
this manner in the Cas9 cleavage experiment presented in Example
5.
[0167] A related example of use of an effector protein is Csy4*
with a cognate effector protein binding element is presented in
FIG. 6A, FIG. 6B, and FIG. 6C for a two polynucleotide
sn-casPNs/Cas9 system. This system corresponds to a first
polynucleotide (sn1-casPN) that comprises a first portion of the
split nexus element (FIG. 3B, 326) and a second polynucleotide
(sn2-casPN) that comprises a second portion of the split nexus
element (FIG. 3B, 302).
[0168] The ability of Csy4* to facilitate sn-casRNAs/Cas9 cleavage
of four double-strand DNA targets is demonstrated in Example 5. The
data presented in FIG. 9 demonstrate an effector protein (here
Csy4*) enhanced cleavage of target double-stranded DNA by
split-nexus Cas9-associated polynucleotide systems of the present
invention comprising auxiliary polynucleotides having an effector
binding element (here the Csy RNA binding sequence).
[0169] Wright, A. V., et al., ("Rational design of a split-Cas9
enzyme complex," PNAS 112(10), 2015, pages 2984-2989) designed a
split-Cas9 enzyme in which the nuclease lobe and .alpha.-helical
lobe are expressed as separate polypeptides. In this example, FIG.
6A shows the sn1-casRNA and sn2-casRNA before association and
formation of hydrogen bond base pairs between them. FIG. 6B
illustrates the sn1-casRNA comprising a first auxiliary
polynucleotide and the sn2-casRNA comprising a second auxiliary
polynucleotide after formation of hydrogen bond base pairs between
them in order to illustrate formation of an effector binding
element. The top dashed-line box (FIG. 6B, 610) shows formation of
an effector binding element, in this example a Csy4* RNA binding
element. FIG. 6C illustrates the association of the sn2-casRNA with
the catalytic nuclease lobe (FIG. 6C, 613) of SpyCas9 and the
association of the sn1-casRNA with the .alpha.-Helical lobe (FIG.
6C, 614) of SpyCas9. Also shown is an effector protein Csy4* (FIG.
6C, 615), which is a variant of Csy4 without endoribonuclease
activity. The thick downward pointing arrow indicates the assembly
of the sn2-casRNA/catalytic nuclease lobe (FIG. 6C, 613) of
SpyCas9, the sn1-casRNA/.alpha.-Helical lobe (FIG. 6C, 614) of
SpyCas9, and the Csy4* protein (FIG. 6C, 615) into a complex (FIG.
6C, 618). This example illustrates sn1-casRNA recruiting the
.alpha.-Helical lobe and sn2-casRNA recruiting the catalytic
nuclease lobe into a ternary complex further stabilized by the
binding of the Csy4* protein to recapitulate the activity of Cas9
to catalyze site-specific DNA cleavage.
[0170] In further embodiments of the seventh aspect of the
invention, the effector protein comprises at least one zinc finger
domain.
[0171] The first auxiliary polynucleotide and/or second auxiliary
polynucleotide can also comprise one or more hairpins. FIG. 7A
illustrates a sn1-casRNA comprising a first auxiliary
polynucleotide (FIG. 7A, 702 to 703) and a sn2-casRNA comprising a
second auxiliary polynucleotide (FIG. 7A, 706 to 707). The figure
shows the sn1-casRNA and sn2-casRNA before association and
formation of hydrogen bond base pairs between them. The figure
shows a hairpin clement formed by hydrogen bond base pairing
between bases within the first auxiliary polynucleotide (FIG. 7A,
704) and a hairpin element formed by hydrogen bond base pairing
between bases within the second auxiliary polynucleotide (FIG. 7A,
708). FIG. 7B illustrates the sn1-casRNA/sn2-casRNA assembled into
an active complex with Cas9.
[0172] All aspects of the invention can employ a Cas9 protein (or
as needed nucleic acid sequences encoding a Cas9 protein) or a Cas9
fusion (or as needed nucleic acid sequences encoding a Cas9
fusion).
[0173] The term "affinity tag" as used herein refers to one or more
moiety that increases the binding affinity of one sn-casPN to
another sn-casPN and/or to a Cas9 protein. Some embodiments of the
present invention use an "affinity sequence," which is a
polynucleotide sequence comprising one or more affinity tag.
Examples of affinity sequences that can be used to modify a first
sn-casPN include using a MS2 binding sequence, U1A binding
sequence, stem-loop sequence, eIF4A binding sequence, Transcription
activator-like effector (TALE) binding sequence (Valton, J., et
al., "Overcoming Transcription Activator-like Effector (TALE) DNA
Binding Domain Sensitivity to Cytosine Methylation" J Biol Chem.
2012 Nov. 9; 287(46): 38427-38432), or zinc finger domain binding
sequence (Font, J., et al., "Beyond DNA: zinc finger domains as
RNA-binding modules," Methods Mol Biol. 2010; 649:479-91; Isalan,
M., et al., "A rapid, generally applicable method to engineer zinc
fingers illustrated by targeting the HIV-1 promoter," Nat
Biotechnol. 2001 July; 19(7): 656-660). Other sn-casPNs and/or the
Cas9 protein coding sequence can be modified to comprise a cognate
affinity tag: an MS2 coding sequence, U1A coding sequence,
stem-loop binding protein coding sequence, eIF4A coding sequence,
TALE coding sequence, or a zinc finger domain coding sequence,
respectively.
[0174] A wide variety of affinity tags are disclosed in U.S.
Published Patent Application No. 2014-0315985 (published 23 Oct.
2014).
[0175] The terms "ligand" and "ligand-binding moiety" as used
herein refer to moieties that facilitate the binding of one
sn-casPN to another sn-casPN or to a Cas9 protein. Ligands and
ligand-binding moieties are cognate affinity tags.
[0176] One embodiment of use of a ligand moiety is to build a
ligand-binding moiety into the Cas9 protein or attach a
ligand-binding moiety to a first sn-casPN and modify a
polynucleotide sequence of a different sn-casPN to contain the
ligand. A ligand/ligand-binding moiety useful in the practice of
the present invention is Avidin or Streptavidin/Biotin (see, e.g.,
Livnah, O, et al., "Three-dimensional structures of avidin and the
avidin-biotin complex," Proceedings of the National Academy of
Sciences of the United States of America, 1993; 90(11):5076-5080;
Airenne, K. J., et al., "Recombinant avidin and avidin-fusion
proteins.," Biomol Eng. 1999 Dec. 31; 16(1-4):87-92.). One example
of a Cas9 protein with a ligand-binding moiety is a Cas9 protein
fused to a ligand Avidin or Streptavidin designed to bind a
biotinylated sn-casPN, wherein the sn-casPN comprises an
polynucleotide sequence with which the biotin is associated. Biotin
is a high affinity and high specificity ligand for the Avidin or
Streptavidin protein. By fusing an Avidin or Streptavidin
polypeptide chain to the Cas9 protein, the Cas9 protein has a high
affinity and specificity for a biotinylated sn-casPN-biotin.
[0177] Biotinylation is preferably in close proximity to the 5' or
3' ends of a sn-casPN. The sequence of the sn-casPN and location of
the biotin is provided to commercial manufacturers for synthesis of
the sn-casPN-biotin. Changes to cleavage percentage and specificity
of a ligand-binding modified sn-casPNs/Cas9 system are evaluated as
described, for example, in Example 3 and/or Example 9.
[0178] Examples of other ligands and ligand-binding moieties that
can be similarly used include, but are not limited to
(ligand/ligand-binding moiety): estradiollestrogen receptor (see,
e.g., Zuo, J., et al., "Technical advance: An estrogen
receptor-based transactivator XVE mediates highly inducible gene
expression in transgenic plants," Plant J. 2000 October;
24(2):265-73), rapamycin/FKBP12, and FK506/FKKBP (see, e.g., B.
Setscrew,. et al., "A split-Cas9 architecture for inducible genome
editing and transcription modulation," Nature Biotechnology 33,
139-142 (2015); Chiu M. I., et al., "RAPTI, a mammalian homolog of
yeast Tor, interacts with the FKBP12/rapamycin complex," PNAS 1994;
91(26):12574-12578).
[0179] Another example of a ligand and ligand-binding moiety is to
provide one or more aptamer or modified aptamer in a polynucleotide
sequence of a sn-casPN that has a high affinity and binding
specificity for a selected region of a Cas9 protein. In one
embodiment, a ligand-binding moiety is a polynucleotide comprising
an aptamer (see, e.g., Navani, N. K., et al.,"In vitro Selection of
Protein-Binding DNA Aptamers as Ligands for Biosensing
Applications," Biosensors and Biodetection, Methods in Molecular
Biology.TM. Volume 504, 2009, pp 399-415; A. V. Kulbachinskiy,
"Methods for Selection of Aptamers to Protein Targets,"
Biochemistry (Moscow), 2007, Vol. 72, No. 13, pp. 1505-1518.).
Aptamers are single-stranded functional nucleic acids that possess
cognate ligand recognition capability. Typically, the aptamer is
located at the 5' or 3' end of a sn-casPN. In the practice of the
present invention one example of a ligand is a casPN/Cas9
complex.
[0180] In another embodiment, a ligand-binding moiety comprises a
modified polynucleotide wherein a nonnative functional group is
introduced at positions oriented away from the hydrogen bonding
face of the bases of the modified polynucleotide, such as the
5-position of pyrimidines and the 8-position of purines ("Slow
Off-rate Modified Aptamers or SOMAmers"; see, e.g., Rohloff, J. C.,
et al., "Nucleic Acid Ligands With Protein-like Side Chains:
Modified Aptamers and Their Use as Diagnostic and Therapeutic
Agents," Molecular Therapy Nucleic Acids (2014) 3, e201). An
aptamer with high specificity and affinity for Cas9 proteins could
be obtained by in vitro selection and screening of an aptamer
library.
[0181] In yet another embodiment, an established aptamer binding
sequence/aptamer is used by introducing the aptamer-binding region
into the Cas9 protein. For example, a biotin-binding aptamer can be
introduced into a sn-casPN and the Cas9 protein can be selectively
biotinylated to form a cognate binding site for the biotin-binding
aptamer.
[0182] The creation of a high affinity binding site for a selected
ligand on a Cas9 protein can be achieved using several protein
engineering methods known to those of ordinary skill in the art in
view of the guidance of the present specification. Examples of such
protein engineering methods include, rational protein design,
directed evolution using different selection and screening methods
for the library (e.g. phage display), DNA shuffling, computational
methods (e.g. ROSETTA, www.rosettacommons.org/software), or
introduction of a known high affinity ligand into Cas9. Libraries
obtained by these methods can be screened to select for Cas9
protein high affinity binders using, for example, a phage display
assay, a cell survival assay, or a binding assay.
[0183] In another aspect of the present invention, at least one of
the sn-casPNs of a sn-casPNs/Cas9 system is a circular
polynucleotide.
[0184] In yet another aspect of the present invention, at least one
linear sn-casPN of a sn-casPNs/Cas9 system comprises a 5' terminal
sequence and/or a 3' terminal sequence, and at least one 5'
terminal sequence and/or 3' terminal sequence comprises an
exonuclease resistance moiety associated with the 5' terminal
sequence and/or 3' terminal sequence. Examples of exonuclease
resistant moieties include, but are not limited to, a hairpin in
the terminal sequence, a single-stranded polynucleotide binding
sequence to which a single-stranded polynucleotide binds, and a
linkage inversion.
[0185] One aspect of the invention relates to methods of
manufacturing the sn-casPNs of the present invention. In one
embodiment, the method of manufacturing comprises chemically
synthesizing one or more of the sn-casPNs of a sn-casPNs/Cas9
system. In some embodiments, the sn-casPNs comprise RNA bases, DNA
bases, or a combination of RNA bases and DNA bases. Furthermore,
nucleobase backbones other than or in addition to a phosphodiester
backbone can be synthesized, for example, using nucleic acids,
peptide-nucleic acids, threose nucleic acid, or combinations
thereof. In some embodiments, the method of manufacturing comprises
producing one or more of the sn-casPNs of a sn-casPNs/Cas9 system
by in vitro transcription.
[0186] In one aspect, the present invention relates to expression
cassettes comprising polynucleotide coding sequences for two or
more sn-casPNs and/or a Cas9 protein. An expression cassette of the
present invention at least comprises a polynucleotide encoding a
sn-casPN of the present invention. Expression cassettes useful in
the practice of the present invention can further include Cas9
protein coding sequences. In one embodiment, an expression cassette
comprises a sn-casPN coding sequence. In another embodiment, one or
more expression cassette comprises sn-casPN coding sequence and a
cognate Cas9 protein coding sequence. Expression cassettes
typically comprise regulatory sequences that are involved in one or
more of the following: regulation of transcription,
post-transcriptional regulation, and regulation of translation.
Expression cassettes can be introduced into a wide variety of
organisms including bacterial cells, yeast cells, insect cells,
mammalian cells, and plant cells. Expression cassettes typically
comprise functional regulatory sequences corresponding to the host
cells or organism(s) into which they are being introduced.
[0187] One aspect of the present invention relates to vectors,
including expression vectors, comprising polynucleotide coding
sequences for a sn-casPN and/or a Cas9 protein. Vectors useful for
practicing the present invention include plasmids, viruses
(including phage), and integratable DNA fragments (e.g., fragments
integratable into the host genome by homologous recombination). A
vector replicates and functions independently of the host genome,
or may, in some instances, integrate into the genome itself.
Suitable replicating vectors will contain a replicon and control
sequences derived from species compatible with the intended
expression host cell. A vector can comprise one or more expression
cassette of polynucleotide coding sequences for sn-casPNs and/or a
Cas9 protein. Vectors include, but are not limited to, bacterial
vectors, yeast vectors, algal vectors, insect cell vectors,
mammalian vectors, and viral vectors.
[0188] Transformed host cells are cells that have been transformed
or transfected with the vectors constructed using recombinant DNA
techniques.
[0189] General methods for construction of expression vectors are
known in the art. Expression vectors for most host cells are
commercially available. There are several commercial software
products designed to facilitate selection of appropriate vectors
and construction thereof, such as bacterial plasmids for bacterial
transformation and gene expression in bacterial cells, yeast
plasmids for cell transformation and gene expression in yeast and
other fungi, algal expression systems for use in algae cells,
insect cell vectors for insect cell transformation and gene
expression in insect cells, mammalian vectors for mammalian cell
transformation and gene expression in mammalian cells or mammals,
viral vectors (including retroviral, lentiviral, and adenoviral
vectors) for cell transformation and gene expression, and methods
to easily enable cloning of such polynucleotides. SnapGene.TM. (GSL
Biotech LLC, Chicago, Ill.;
snapgene.com/resources/plasmid_files/your_time_is_valuable/), for
example, provides an extensive list of vectors, individual vector
sequences, and vector maps, as well as commercial sources for many
of the vectors.
[0190] Expression vectors can also include polynucleotides encoding
protein tags (e.g., poly-His tags, hemagglutinin tags, fluorescent
protein tags, bioluminescent tags). The coding sequences for such
protein tags can be fused to a Cas9 protein coding sequence or can
be included in an expression cassette, for example, in a targeting
vector.
[0191] In some embodiments, polynucleotides encoding sn-casPNs
and/or Cas9 protein arc operably linked to an inducible promoter, a
repressible promoter, or a constitutive promoter.
[0192] Aspects of the invention relate to vector systems comprising
one or more vectors for expression of sn-casPNs and Cas9 proteins
in prokaryotic or eukaryotic cells. Alternatively, sn-casPNs and
Cas9 proteins can be transcribed in vitro, for example using T7
promoter regulatory sequences and T7 polymerase. Translation of
Cas9 proteins can also be carried out in vitro.
[0193] Vectors comprising sn-casPNs/Cas9 systems can be introduced
into and propagated in a prokaryote. Prokaryotic vectors are well
known in the art. Typically a prokaryotic vector comprises an
origin of replication suitable for the target host cell (e.g., oriC
derived from E. coli, pUC derived from pBR322, pSC101 derived from
Salmonella), 15A origin (derived from p15A) and bacterial
artificial chromosomes). Vectors can include a selectable marker
(e.g., genes encoding resistance for ampicillin, chloramphenicol,
gentamicin, and kanamycin). Zeocin.TM. (Life Technologies, Grand
Island, N.Y.) can be used as a selection in bacteria, fungi
(including yeast), plants and mammalian cell lines. Accordingly,
vectors can be designed that carry only one drug resistance gene
for Zeocin for selection work in a number of organisms. Useful
promoters are known for expression of proteins in prokaryotes, for
example, T5, T7, Rhamnose (inducible), Arabinose (inducible), and
PhoA (inducible). Furthermore, T7 promoters are widely used in
vectors that also encode the T7 RNA polymerase. Prokaryotic vectors
can also include ribosome binding sites of varying strength, and
secretion signals (e.g., mal, sec, tat, ompC, and pelB). In
addition, vectors can comprise RNA polymerase promoters for the
expression of sn-casRNAs. Prokaryotic RNA polymerase transcription
termination sequences arc also well known (e.g., transcription
termination sequences from S. pyogenes).
[0194] Integrating vectors for stable transformation of prokaryotes
are also known in the art (see, e.g., Heap, J. T., et al., (2012)
"Integration of DNA into bacterial chromosomes from plasmids
without a counter-selection marker," Nucleic Acids Res. 2012 April;
40(8):e59).
[0195] Expression of proteins in prokaryotes is typically carried
out in Escherichia coli with vectors containing constitutive or
inducible promoters directing the expression of either fusion or
non-fusion proteins.
[0196] A wide variety of RNA polymerase promoters suitable for
expression of sn-casRNAs and Cas9 proteins are available in
prokaryotes (see, e.g., Jiang, Y., et at., (2015) "Multigene
editing in the Escherichia coli genome via the CRISPR-Cas9 system,"
Environ Microbiol. 81(7):2506-14); Estrem, S. T., et at., (1999)
"Bacterial promoter architecture: subsite structure of UP elements
and interactions with the carboxy-terminal domain of the RNA
polymerase alpha subunit," Genes Dev.15; 13(16):2134-47).
[0197] Fusion vectors add a number of amino acids to a protein
encoded therein (e.g., to the amino terminus of the recombinant
protein). Such fusion vectors serve one or more purposes. Examples
include, but are not limited to, the following: (i) to increase
expression of recombinant protein; (ii) to increase the solubility
of the recombinant protein; and (iii) to aid in the purification of
the recombinant protein by acting as a ligand in affinity
purification. In fusion-expression vectors, a proteolytic cleavage
site is sometimes introduced at the junction of the fusion moiety
and the recombinant protein. This enables the separation of the
recombinant protein from the fusion moiety following the
purification of the fusion protein. Such enzymes, and their cognate
proteolytic cleavage sites, include Factor Xa, thrombin and
enterokinase. Examples of fusion-expression vectors include, but
are not limited to, the following: pGEX, pMAL, and pRIT5 that fuse
glutathione S-transferase (GST), maltose E binding protein, or
protein A, respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression
vectors include, but are not limited to, pTrc and pET 11d.
[0198] In some embodiments, a vector is a yeast expression vector
comprising a sn-casPNs/Cas9 system. Examples of vectors for
expression in yeast Saccharomyces cerivisae include, but are not
limited to, the following: pYcpScc1, pMFa, pJRY88, pYES2, and picZ.
Methods for gene expression' in yeast cells are known in the art
(see, e.g., Methods in Enzymology, Volume 194, "Guide to Yeast
Genetics and Molecular and Cell Biology, Part A," (2004) Christine
Guthrie and Gerald R. Fink (eds.), Elsevier Academic Press, San
Diego, Calif.). Typically, expression of protein encoding genes in
yeast requires a promoter operably linked to a coding region of
interest plus a transcriptional terminator. Various yeast promoters
can be used to construct expression cassettes for expression of
genes in yeast. Examples of promoters include, but are not limited
to, promoters of genes encoding the following yeast proteins:
alcohol dehydrogenase 1 (ADH1) or alcohol dehydrogenase 2 (ADH2),
phosphoglycerate kinase (PGK), triose phosphate isomerase (TPI),
glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also known as
TDH3, or triose phosphate dehydrogenase), galactose-1-phosphate
uridyl-transferase (GAL7), UDP-galactose epimerase (GAL10),
cytochrome ci (CYC1), and acid phosphatase (PHO5). Hybrid
promoters, such as the ADH2/GAPDH, CYC1/GAL10 and the ADH2/GAPDH
promoter (which is induced at low cellular-glucose concentrations,
e.g., about 0.1 percent to about 0.2 percent) also may be used. In
S. pombe, suitable promoters include the thiamine-repressed nmtl
promoter and the constitutive cytomegalovirus promoter in
pTL2M.
[0199] Yeast RNA polymerase III promoters (e.g., promoters from 5S,
U6 or RPR1 genes) as well as polymerase III termination sequences
are known in the art (see, e.g., www.yeastgenome.org; Harismendy,
O., et al., (2003) "Genome-wide location of yeast RNA polymerase
III transcription machinery," The EMBO Journal.
22(18):4738-4747.)
[0200] A protein expression promoter may be inducible or
constitutive. In some embodiments, a preferred promoter is a
tightly regulated inducible promoter, such that a high copy number
can be achieved in the absence of expression. Examples include, but
are not limited to, the normally divergent GAL1p and GAL10 p
promoters, which are tightly suppressed in glucose media and highly
induced by galactose after catabolite repression has been relieved
by growth on a non-repressing carbon source such as glycerol or
lactate. An open reading frame that encodes a polypeptide may be
inserted into a GAL1p vector (see, e.g., Cartwright, et al.,(1994)
"Use of .beta.-lactamase as a secreted reporter of promoter
function in yeast," Yeast 10:497; and Harley, et al., (1998)
"Transmembrane Protein Insertion Orientation in Yeast Depends on
the Charge Difference across Transmembrane Segments, Their Total
Hydrophobicity, and Its Distribution," J. Biol. Chem. 273:24963).
Other vectors and promoters that can be used include the hybrid
GAL1-CYCp promoter in the Yep URA3 leu2D vector pPAP1488 in strain
PAP1502 (see, e.g., Pedersen, et al. (1996) "Expression in High
Yield of Pig .alpha.1.beta.1 Na,K-ATPase and Inactive Mutants D369N
and D807N in Saccharomyces cerevisiae," J. Biol. Chem. 1996 271:
2514-2522). This strain has plasmid pPAP1488 integrated at the Trpl
locus. This provides an additional copy of the GAL4 gene driven by
the GAL10 promoter, and when GAL expression is induced, high levels
of the Gal4p positive activator are produced.
[0201] In this vector system, growth in the absence of uracil
produces a vector copy number of 15 to 20, determined by 2-micron
replication functions. The copy number of the vector can be further
increased, by at least 10 fold, by culturing the yeast cells in
media lacking leucine, because of the very weak promoter associated
with the defective leu2d allele. A proportional increase in
GAL1p-driven expression requires the high galactose-induced levels
of the Gal4p activator provided in strain PAP1502 by the integrated
PAP1488 plasmid. Any other ura3 leu2 GaI+S. cerevisiae strain into
which this plasmid is inserted may be used instead of PAP1502.
[0202] Another yeast promoter that can be used is the promoter of
the glycerol-3-phosphate dehydrogenase gene (GPD1). Expression of
polypeptides using the GPD1 promoter can be regulated by the
presence (repressed) or absence (derepressed) of high levels of
glucose or sucrose in a fermentation medium. Alternatively, a
non-repressing carbon source, such as ethanol or glycerol, can be
added to the fermentation medium (see, e.g., U.S. Pat. No.
5,667,986).
[0203] Regulation of plasmid copy number can provide some control
over the level of RNA products expressed from RNA polymerase III
promoters. Furthermore, RNA polymerase III transcription can be
regulated in yeast (Dingermann, T., et al., (1992) " RNA polymerase
III catalysed transcription can be regulated in Saccharomyces
cerevisiae by the bacterial tetracycline repressor-operator
system," EMBO J. 11(4):1487-92).
[0204] In addition to a promoter, several upstream activation
sequences (UASs), also called enhancers, may be used to enhance
polypeptide expression. Exemplary upstream activation sequences for
expression in yeast include the UASs of genes encoding these
proteins: CYC1, ADH2, GAL1, GAL7, GAL10, and ADH2. Exemplary
transcription termination sequences for expression in yeast include
the termination sequences of the .alpha.-factor, CYC1, GAPDH, and
PGK genes. One or multiple termination sequences can be used.
[0205] Any protein coding regions expressed in yeast cells can be
codon-optimized for expression in the specific host yeast cell to
be engineered, as is well known in the art.
[0206] Suitable promoters, terminators, and coding regions may be
cloned into E. coli-yeast shuttle vectors and transformed into
yeast cells. These vectors allow strain propagation in both yeast
and E. coli strains. Typically, the vector contains a selectable
marker and sequences enabling autonomous replication or chromosomal
integration in each host. Examples of plasmids typically used in
yeast are the shuttle vectors pRS423, pRS424, pRS425, and pRS426
(American Type Culture Collection, Manassas, Va.). These plasmids
contain a yeast 2 micron origin of replication, an E. coli
replication origin (e.g., pMB1), and a selectable marker.
[0207] Example 15 presents an illustration of genome engineering in
Saccharomyces cerevisiae using the split-nexus Cas9-associated
polynucleotides (sn-casPNs). A Cas9 vector and two
sn1-casRNA/sn2-casRNA vector pairs are used to modifying the genome
of the yeast. This protocol provides data to verify that the Cas9
and sn1-casRNA/sn2-casRNA system provide specific RNA-mediated
endonuclease activity at targeted endogenous genomic loci in yeast.
The constructs are also used in experiments to verify that the Cas9
and sn1-casRNA/sn2-casRNA system provides specific RNA-mediated
endonuclease activity at targeted endogenous genomic loci in yeast
and can stimulate homologous recombination events at such loci
using donor DNA. Other chromosomal loci in S. cerevisiae can
similarly targeted for modification by selection of appropriate
spacer sequences and donor oligonucleotides. Functional genes can
be introduced into the S. cerevisiae genome without disruption of
endogenous genes. Furthermore, introduction of selectable markers
into endogenous target genes can be used to provide selectable
knock-out mutations of the target genes.
[0208] Integrating vectors are also widely available for stable
transformation of yeast (Stearns T., et al., (1990) "Manipulating
yeast genome using plasmid vectors," Methods Enzymol. 1990;
185:280-97).
[0209] For use of sn-casPNs/Cas9 systems in algal cells, suitable
vectors and expression control sequences are well known in the art
(see, e.g., Hallmann, A. (2007), "Algal Transgenics and
Biotechnology," Transgenic Plant Journal 1(1), 81-98; Oey, M., et
al., "Gateway-Assisted Vector Construction to Facilitate Expression
of Foreign Proteins in the Chloroplast of Single Celled Algae,"
Feb. 11, 2014 DOI: 10.1371/journal.pone.0086841) including RNA
polymerase III promoters (see, e.g., Dieci, G., et al., (2009)
"Eukaryotic snoRNAs: A paradigm for gene expression flexibility,"
Genomics 94(2):83-88). Furthermore, algal expression systems are
commercially available (Algae Expression & Engineering
Products, ThermoFisher Scientific, Grand Island, N.Y.).
[0210] For use of sn-casPNs/Cas9 systems in insects or insect
cells, suitable expression control sequences are well known in the
art. In some embodiments, it is desirable that the expression
control sequence comprises a constitutive promoter. Examples of
suitable strong promoters include, but are not limited to, the
following: the baculovirus promoters for the piO, polyhedrin
(polh), p 6.9, capsid, UAS (contains a Gal4 binding site), Ac5,
cathepsin-like genes, the B. mori actin gene promoter; Drosophila
melanogaster hsp70, actin, .alpha.-1-tubulin or ubiquitin gene
promoters, RSV or MMTV promoters, copia promoter, gypsy promoter,
and the cytomegalovirus IE gene promoter. Examples of weak
promoters that can be used include, but are not limited to, the
following: the baculovirus promoters for the id, ie2, ieO, etl, 39K
(aka pp31), and gp64 genes. If it is desired to increase the amount
of gene expression from a weak promoter, enhancer elements, such as
the baculovirus enhancer element, hr5, may be used in conjunction
with the promoter.
[0211] In some embodiments, the expression control sequence
comprises an organ- or tissue-specific promoter. Many such
expression control sequences. For example, suitable promoters that
direct expression in insect silk glands include the Bombyx mori p25
promoter, which directs organ-specific expression in the posterior
silk gland, and the silk fibroin heavy chain gene promoter, which
directs specific expression of genes in the median silk gland.
[0212] Examples of insect regulatable expression control sequences
(e.g., comprising an inducible promoter and/or enhancer element)
include, but are not limited to, the following: Drosophila hsp70
promoters, Drosophila metallothionein promoter, an
ecdysone-regulated promoter, and other well-known inducible
systems. A Tet-regulatable molecular switch may be used in
conjunction with any constitutive promoter (e.g., in conjunction
with the CMV-IE promoter or baculovirus promoters). Another type of
inducible promoter is a baculovirus late or very late promoter that
is only activated following infection by a baculovirus.
[0213] For the expression of sn-casPNs in insects, RNA polymerase
III promoters are known in the art, for example, the U6 promoter.
Conserved features of RNA polymerase III promoters in insects are
also known (see, e.g., Hernandez, G., (2007) "Insect small nuclear
RNA gene promoters evolve rapidly yet retain conserved features
involved in determining promoter activity and RNA polymerase
specificity," Nucleic Acids Res. 2007 January; 35(1):21-34).
[0214] Methods for designing and preparing constructs suitable for
generating transgenic insects or vectors for infection of an insect
are conventional. Methods for transformation, culturing, and
manipulation of insect cell lines are also conventional. Examples
of insect cell lines include, but are not limited to, the
following: Antheraea cells, Tn-368, Drosophila S2 Cells, High
Five.TM. Cells (Life Technologies, Grand Island N.Y.), Sf21 Cells,
and Sf9 cells. Insect cell lines are commercially available, for
example, from the American Type Culture Collection (Manassas
Va.).
[0215] A variety of immortalized lepidopteran insect cell lines are
suitable for infection by vectors/constructs comprising the
sn-casPNs/Cas9 proteins of the present invention. Examples of
immortalized lepidopteran insect cell lines that are suitable for
infection by the vectors/constructs of the invention include, but
are not limited to, the following: Sf9 and Tn 5B1-4.
[0216] In another embodiment, the vector is a transposon-based
vector. One transposon-based vector is a viral vector that further
comprises inverted terminal repeats of a suitable transposon
between which the gene of interest is cloned. One or more genes,
under the control of a suitable expression control sequences, are
cloned into the transposon-based vector. In some systems, the
transposon-based vector carries its own transposase. However,
typically the transposon-based vector does not encode a suitable
transposase. In this case, the vector is co-infected into an insect
(e.g., an insect larva) with a helper virus or plasmid that
provides a transposase. The recombinant vector, generally with a
helper, is introduced by conventional methods (e.g.,
microinjection) into an egg or early embryo. The transgenes become
integrated at a transposon site (e.g., sequences corresponding to
the inverted terminal repeat of the transposon) in the insect
genome. Examples of suitable types of transposon-based vectors
include, but are not limited to, the following: Minos, mariner,
Hermes, sleeping beauty, and piggyBac.
[0217] TTAA-specific, short repeat elements are a group of
transposons (Class II mobile elements) that have similar structures
and movement properties. piggyBac vectors are the most extensively
studied of these insertion elements. piggyBac is 2.4 kb long and
terminates in 13bp perfect inverted repeats, with additional
internal 19bp inverted repeats located asymmetrically with respect
to the ends. A piggyBac vector may encode a trans-acting
transposase that facilitates its own movement. Alternatively, the
transposase encoding sequences can be deleted and this function
supplied on a helper plasmid or virus. Some piggyBac vectors have
deleted non-essential genes to facilitate cloning of large inserts.
Inserts as large as 15 kB can be cloned into certain piggyBac
vectors. For example, this allows for the insertion of
approximately six or seven genes with their expression control
sequences. For example, a collection of sn-casPNs can be introduced
together via a single transposon vector into a single site in an
insect genome.
[0218] Several piggyBac vectors have been developed for insect
transgencsis. Two constructs were developed by analysis of deletion
mutations within and outside of the boundaries of the transposon.
Using such constructs, it is possible to increase the amount of
genetic material mobilized by the piggyBac transposase by
minimizing the size of the vector. The minimal requirements for
movement include the 5' and 3' terminal repeat domains and
attendant TTAA target sequences. A minimum of 50 bases separating
the TTAA target sites of the element is typically required for
efficient mobilization.
[0219] piggyBac can transpose in insect cells while carrying a
marker gene and movement of the piggyBac element can occur in cells
from lepidopteran species distantly related to the species from
which it originated. For example, piggyBac has been shown to
transform D. melanogaster, Anastrepha suspena (oriental fruit fly),
Bactrocera dorsalis, Bombyx man, Pectinophora glossypiella,
Tribolium castellani, and several mosquito species. At least three
lepidopteran species, P. gossypiella, T ni and B. mori, have been
successfully transformed using the piggyBac element.
[0220] Typically, a helper virus or plasmid that expresses a
transposase is co-infected with the transposon-based vector.
Expression of the transposase is determined by the choice of
promoter for the insect system being tested. Examples of
promoter-driven helper constructs that are useful for lepidopteran
transformation include, but are not limited to, the following:
Drosophila hsp70, baculovirus iel promoter, and Drosophila Actin 5C
promoter. For further guidance on the use of baculovirus-based
vectors, see, e.g., WO/2005/042753.
[0221] Methods for generating transgenic insects are conventional.
For example, one or more genes to be introduced are placed under
the control of a suitable expression control sequence and are
cloned into a vector (e.g., an attenuated baculovirus vector or a
non-permissive viral vector that is not infective for the target
insect). The sequences to be introduced into the insect are flanked
by genomic sequences from the insect. The construct is then
introduced into an insect egg (e.g., by microinjection). The
transgenes then integrate by homologous recombination of the
flanking sequences into complementary sequences in the insect
genome.
[0222] Methods for introducing constructs into an embryo to
generate a transgenic insect (e.g., by microinjection) are known.
Survivorship is typically high (up to 75%) for microinjected
embryos. In general, pre-blastoderm eggs are stuck with a fine
glass capillary holding a solution of the plasmid DNA and/or
recombinant virus. G0 larvae hatched from the virus-injected eggs
are screened for expression of the transfected genes. Breeding
transgenic G1 insects with normal insects results in Mendelian
inheritance.
[0223] Once a transgene is stably integrated into the genome of an
insect egg or early embryo, conventional methods can be used to
generate a transgenic insect, in which the transgene is present in
all of the insect somatic and germ cells. Methods for producing
homozygous transgenic insects (e.g., using suitable back-crosses)
are conventional.
[0224] By selecting appropriate expression control sequences for
each of the genes, a multiply transgenic insect that comprises
genomically integrated copies of sn-casPNs and Cas9 protein genes
can be designed such that the genes of are expressed at suitable
levels, at the desired time during insect growth.
[0225] In another aspect, the sn-casPNs/Cas9 systems are
incorporated into mammalian vectors for use in mammalian cells. A
large number of mammalian vectors suitable for use with the
sn-casPNs/Cas9 systems of the present invention are commercially
available (e.g., from Life Technologies, Grand Island, N.Y.;
NeoBiolab, Cambridge, Mass.; Promega, Madison, Wis.; DNA2.0, Menlo
Park, Calif.; Addgene, Cambridge, Mass.).
[0226] Vectors derived from mammalian viruses can be used for
expressing the sn-casPNs and Cas9 proteins of the present invention
in mammalian cells. These include vectors derived from viruses such
as adenovirus, papovirus, herpesvirus, polyomavirus,
cytomegalovirus, lentivirus, retrovirus and simian virus 40 (SV40)
(see, e.g., Kaufman, R. J., (2000) "Overview of vector design for
mammalian gene expression," Molecular Biotechnology, Volume 16,
Issue 2, pp 151-160; Cooray S., et al., (2012) "Retrovirus and
lentivirus vector design and methods of cell conditioning," Methods
Enzymo1.507:29-57). Regulatory sequences operably linked to the
sn-casPNs/Cas9 components can include activator binding sequences,
enhancers, introns, polyadenylation recognition sequences,
promoters, repressor binding sequences, stem-loop structures,
translational initiation sequences, translation leader sequences,
transcription termination sequences, translation termination
sequences, primer binding sites, and the like. Commonly used
promoters are constitutive mammalian promoters CMV, EF1a, SV40,
PGK1 (mouse or human), Ubc, CAG, CaMKIIa, and beta-Act. and others
known in the art (Khan, K. H. (2013) "Gene Expression in Mammalian
Cells and its Applications," Advanced Pharmaceutical Bulletin 3(2),
257-263). Furthermore, for expression of the sn-casPNs of the
present invention, mammalian RNA polymerase III promoters,
including H1 and U6, are used.
[0227] Adenovirus is a member of the Adenoviridae family.
Adenovirus vectors are derived from adenovirus. Adenovirus is
medium sized, non-enveloped icosahedral virus. It is composed of a
nucleocapsid and a double-stranded linear DNA genome that can be
used as a cloning vector. The extensive knowledge and data on
adenovirus transcription regulation favored the engineering of
adenovirus vectors modified for expression of inserted genes. For
this purpose, the early regions E1 and E3 were deleted, thus making
the virus incapable of replication, requiring the host cell to
provide this function in trans. An expression cassette comprising
protein coding sequences (e.g., a Cas9 protein) is typically
inserted to replace the deleted El region. In the cassette, a gene
is placed under control of an additional major late promoter or
under control of an exogenous promoter, such as cytomegalovirus or
selected regulatable promoter.
[0228] The genome of adenovirus can be manipulated in such a way
that it encodes and expresses a gene product of interest while at
the same time inactivating the adenovirus' ability to replicate via
a normal lytic cycle. Some such adenoviral vectors include those
derived from adenovirus strain Ad type 5 d1324 or other adenovirus
strains (e.g., Ad2, Ad3, and Ad7). In certain circumstances,
recombinant adenoviruses can be advantageous because they cannot
infect non-dividing cells, and they can be used to infect
epithelial cells and a variety of other cell types. In addition,
the virus particle is relatively stable, and it is amenable to
purification and concentration. The adenoviral genome's carrying
capacity for foreign DNA is up to approximately 8 kilobases, which
is large compared with other gene delivery vectors. Thr large
double-stranded DNA adenovirus does not integrate into the genome,
limiting its use to transient, episomal expression. Because it is
not integrated into the genome of a host cell (unlike retroviral
DNA) potential problems such as insertional mutagenesis are
avoided.
[0229] Adeno-associated virus (AAV), an single-strand DNA member of
the family Parvoviridae, is a naturally replication-deficient
virus. Like adenovirus, it can infect non-dividing cells; however,
it has the advantage of integration competence. AAV vectors are
among the viral vectors most frequently used for gene therapy.
Twelve human serotypes of AAV (AAV serotype 1 [AAV-1] to AAV-12)
and more than 100 serotypes from non-human are known. A number of
factors have increased AAV's potential as a delivery vehicle for
gene therapy applications, including the lack of pathogenicity of
the virus, the persistence of the virus, and the many available
serotypes. AAV is a small (25-nm), non-enveloped virus that
comprises a linear single-stranded DNA genome. Productive infection
by AAV typically occurs only in the presence of a helper virus, for
example, adenovirus or herpesvirus. In the absence of helper virus,
AAV (serotype 2) can become latent by integrating into human
chromosome 19q13.4 (locus AAVS-1) (see, e.g., Daya, S., et al.,
(2008) "Gene Therapy Using Adeno-Associated Virus Vectors,"
Clinical Microbiology Reviews, 21(4): 583-593).
[0230] Vaccinia virus is a member of the poxvirus family. Vaccinia
vectors are derived from vaccinia virus. The vaccinia virus genome
is comprised of a double stranded DNA of nearly 200,000 bp. It
replicates in the cytoplasm of the host cell. Cells infected with
the vaccinia virus produce up to 5000 virus particles per cell,
leading to high levels of expression for encoded gene products. The
vaccinia system has been efficiently used in very large scale
culture (1000 L) to produce several proteins, including HIV-1
rgp160 and human pro-thrombin.
[0231] Retrovirus is a member of the Retroviridac family.
Retroviral vectors arc derived from retrovirus. Retroviruses are
RNA viruses that replicate via a double-strand DNA intermediate.
One advantage of using a retrovirus as vector is that most
retroviruses do not kill the host, but instead produce progeny
virons over an indefinite period of time. Therefore, retroviral
vectors (i) can be used to make stably transformed cell lines, (ii)
provide viral gene expression driven by strong promoters, which can
be subverted to control the expression of transgenes; and (iii)
include those derived from retroviruses having a broad host range
(e.g., amphotropic strains of murine leukaemia virus (MLV)) thus
allowing the transfection of many cell types.
[0232] Exogenous gene-expression systems based on the retroviral
vector are also a method for generating stable, high-expressing
mammalian cell lines.
[0233] Lentivirus is a member of the Retroviridae family. A
single-strand RNA virus, it can infect both dividing and
nondividing cells, as well as provide stable expression through
integration into the genome. To increase the safety of lentivirus,
components necessary to produce a viral vector are split across
multiple plasmids. Transfer vectors are typically replication
incompetent and may additionally contain a deletion in the 3'LTR,
which renders the virus "self-inactivating" (SIN) after
integration. Packaging and envelope plasmids are typically used in
combination with a transfer vector. For example, a packaging
plasmid can encode the Gag, Pol, Rev, and Tat genes. A transfer
plasmid can comprise viral LTRs and the psi packaging signal.
Usually one or more suitable promoter operably linked to the genes
to be expressed (e.g., sn-casPNs and/or Cas9 protein coding
sequences) because the 5'LTR is a weak promoter and requires the
presence of Tat to activate expression. The envelope plasmid
comprises an envelope protein (usually VSVG because of its wide
infectivity range).
[0234] Lentiviral vectors based on human immunodeficiency virus
type-1 (HIV-1) have additional accessory proteins that enable
integration in the absence of cell division. HIV-1 vectors have
been designed to address a number of safety concerns. These include
separate expression of the viral genes in trans to prevent
recombination events leading to the generation of
replication-competent viruses. Furthermore, the development of
self-inactivating (SIN) vectors reduces the potential for
transactivation of neighboring genes and allows the incorporation
of regulatory elements to target gene expression to particular cell
types (see, e.g., Cooray, S., et al., (2012) " Retrovirus and
lentivirus vector design and methods of cell conditioning," Methods
Enzymol. 507:29-57).
[0235] In some embodiments, a recombinant mammalian expression
vector is capable of preferentially directing expression of the
nucleic acid in a particular cell type (e.g., using tissue-specific
regulatory elements to express a polynucleotide). Tissue-specific
regulatory elements are known in the art and include, but are not
limited to, the albumin promoter, lymphoid-specific promoters,
neuron-specific promoters (e.g., the neurofilament promoter),
pancreas-specific promoters, mammary gland-specific promoters
(e.g., milk whey promoter), and in particular promoters of T cell
receptors and immunoglobulins. Developmentally-regulated promoters
are also encompassed, e.g., the murine hox promoters and the
alpha-fetoprotein promoter.
[0236] A number of vectors for use in mammalian cells are
commercially available, for example: pcDNA3 (Life Technologies,
Grand Island N.Y.); customizable expression vectors, transient
vectors, stable vectors, and lentiviral vectors (DNA 2.0, Menlo
Park Calif.); and pFN10A (ACT) Flexi.RTM. Vector (Promega, Madison,
Wis.). Furthermore, the following elements can be incorporated into
vectors for use in mammalian cells: RNA polymerase II promoters
operatively linked to Cas9 coding sequences; RNA polymerase III
promoters operably linked to coding sequences for sn-casRNAs;
selectable markers (e.g., G418, gentamicin, kanamycin and
Zeocin.TM. (Life Technologies, Grand Island, N.Y.)). Nuclear
targeting sequences can also be added, for example, to Cas9 protein
coding sequences.
[0237] Regulatory elements may also direct expression in a
temporal-dependent manner, which may or may not also be tissue or
cell-type specific (e.g., in a cell-cycle dependent or
developmental stage-dependent manner). In some embodiments, vectors
comprise one or more RNA polymerase III promoter (e.g., operably
linked to sn-casPNs coding sequences), one or more RNA polymerase
II promoters (e.g., operably linked to a Cas9 protein coding
sequence), one or more RNA polymerase I promoters, or combinations
thereof. As noted above, examples of mammalian RNA polymerase III
promoters include, but are not limited to, the following: U6 and H1
promoters. Examples of RNA polymerase II promoters were discussed
above. RNA polymerase I promoters are well known in the art.
[0238] Numerous mammalian cell lines have been utilized for
expression of gene products including HEK 293 (Human embryonic
kidney) and CHO (Chinese Hamster Ovary). These cell lines can be
transfected by standard methods (e.g., using calcium phosphate or
polyethyleneimine (PEI), or electroporation). Other typical
mammalian cell lines include, but are not limited to,: HeLa, U2OS,
549, HT1080, CAD, P19, NIH 3T3, L929, N2a, Human embryonic kidney
293 cells, MCF-7, Y79, SO-Rb50, Hep G2, DUKX-X11, J558L, and Baby
hamster kidney (BHK) cells.
[0239] The sn-casPNs/Cas9 protein systems of the present invention
can be used to manipulate mammalian cell bioprocesses for
manufacturing. The Chinese Hamster Ovary (CHO) cells and mouse
myeloma cells (including Sp2/0 and NS0 cells) are the most widely
used host mammalian cells. Two derivatives of the CHO cell line,
CHO-K1 and CHO pro-3, have given rise to the two most commonly used
cell lines in bioprocessing today, DG44 and DUKX-X11 (both of these
cell lines were engineered to be deficient in
dihydrofolatereductase activity).
[0240] Example 14 describes the modification of CHO cells for
industrial applications. This example describes use of the
split-nexus Cas9-associated polynucleotides (sn-casPNs) of the
present invention for modifying the genome of a CHO cell. Also
described is an experimental for sequence validation and selection
of sn-casPN modified cells for future uses in industrial
applications (e.g., production of antibodies). The methods provide
for modification of chromosomal loci within CHO cells by selection
of appropriate spacer sequences for sn-casPNs. Selection is
specific to a specific gene target and the procedure outlined in
the example is readily modifiable by one of ordinary skill in the
art for other gene targets.
[0241] Methods of introducing polynucleotides (e.g., an expression
vector) into host cells are known in the art and are typically
selected based on the kind of host cell. Such methods include, for
example, viral or bacteriophage infection, transfection,
conjugation, electroporation, calcium phosphate precipitation,
polyethylencimine-mediated transfection, DEAE-dextran mediated
transfection, protoplast fusion, lipofection, liposome-mediated
transfection, particle gun technology, direct microinjection, and
nanoparticle-mediated delivery.
[0242] In some embodiments of the present invention it is useful to
express all components of a sn-casPNs/Cas9 system in a host cell.
Expression of sequences encoding sn-casRNAs and Cas9 protein in a
host cell can be accomplished through use of expression cassettes
as described above. However, expression of sn-casDNA in a target
cell is not accomplished with the use of standard cloning vectors.
Single-stranded DNA expression vectors, which can intracellularly
generate single-stranded DNA molecules, have been developed (Chen,
Y., et al.," Intracellular production of DNA enzyme by a novel
single-stranded DNA expression vector," Gene Ther. 2003 September;
10(20):1776-80; Miyata S., et al., "In vivo production of a stable
single-stranded cDNA in Saccharomyces cerevisiae by means of a
bacterial retron," Proc Natl Acad Sci USA 1992; 89: 5735-5739;
Mirochnitchenko, O., et al., "Production of single-stranded DNA in
mammalian cells by means of a bacterial retron," J Biol Chem 1994;
269: 2380-2383; Mao J., et al., "Gene regulation by antisense DNA
produced in vivo. J Biol Chem 1995; 270: 19684-19687). Typically
these single-stranded DNA expression vectors rely on transcription
of a selected single-stranded DNA sequence to form an RNA
transcript that is the substrate for a reverse transcriptase and
RNaseH to generate the selected single-stranded DNA in a host cell.
For example, components of single-stranded DNA expression vectors
often comprise, a reverse transcriptase coding sequence (e.g., a
mouse Moloney leukemia viral reverse transcriptase gene), a reverse
transcriptase primer binding site (PBS) as well as regions of the
promoter that are essential for the reverse transcription
initiation, the coding sequence of interest (e.g., a sn-casDNA
coding sequence), a stem loop structure designed for the
termination of the reverse transcription reaction, and an RNA
transcription promoter suitable for use in a host cell (used to
create a mRNA template comprising the previous components). Reverse
transcriptase expressed in cells uses endogenous tRNApro as a
primer. After reverse transcription, single-stranded DNA is
released when the template mRNA is degraded either by endogenous
RNase H or the RNase H activity of the reverse transcriptase (Chen,
Y., et al., "Expression of ssDNA in Mammalian Cells," BioTechniques
34:167-171 January 2003). Such expression vectors may be employed
for expression of a sn-casDNAs of the present invention in a host
cell.
[0243] The present invention also encompasses gene therapy methods
for preventing or treating diseases, disorders, and conditions
using the sn-casPNs/Cas9 systems described herein. In one
embodiment, a gene therapy method uses the introduction of nucleic
acid sequences into an organism or cells of an organism (e.g.,
patient) to achieve expression of sn-casPNs/Cas9 protein components
of the present invention to provide modification of a target
function. For example, cells from an organism may be engineered, ex
vivo, by (i) introduction of vectors comprising expression
cassettes expressing the sn-casPNs and Cas9 protein, (ii) direct
introduction of sn-casPNs (e.g., sn-casPNs: DNA polynucleotides,
RNA polynucleotides, RNA/DNA hybrid polynucleotides, nucleobases
connected with alternative backbones, or combinations thereof) and
Cas9 protein, or (iii) introduction of combinations of these
components. The engineered cells are provided to an organism (e.g.,
patient) to be treated.
[0244] Examples of gene therapy and delivery techniques for therapy
are known in the art (see, e.g., Kay, M. A., (2011)
"State-of-the-art gene-based therapies: the road ahead," Nature
Reviews Genetics 12, 316-328; Wang, D., et al., (2014)
"State-of-the-art human gene therapy: part I. Gene delivery
technologies," Discov Med. 18(97):67-77; Wang, D., et al., (2014)
"State-of-the-art human gene therapy: part II. Gene therapy
strategies and clinical applications," Discov Med. 18(98):151-61;
"The Clinibook: Clinical Gene Transfer State of the Art," Odile
Cohen-Haguenauer (Editor), EDP Sciences (Oct. 31, 2012), ISBN-10:
2842541715).
[0245] Example 11 illustrates the use of sn-casRNAs of the present
invention to modify targets present in human genomic DNA and
measure the level of cleavage activity at those sites. Target sites
are first selected from genomic DNA and then sn-casRNAs arc
designed to target those selected sequences. Measurements are then
carried out to determine the level of target cleavage that has
taken place. Cleavage percentage data and specificity data provide
criteria on which to base choices for a variety of applications.
For example, in some situations the activity of the sn-casRNA may
be the most important factor. In other situations, the specificity
of the cleavage site may be relatively more important than the
cleavage percentage.
[0246] In some aspects, components of the present invention are
delivered using nanoscale delivery systems. Components to be
delivered include, but are not limited to, polynucleotides encoding
sn-casPNs and/or Cas9 protein, expression cassettes comprising
sn-casPNs and/or Cas 9 proteins, sn-casPNs, Cas 9 protein, and
combinations thereof. The components of the invention can be
formulated as nanoparticles. Extensive libraries of nanoparticles,
composed of an assortment of different sizes, shapes, and
materials, and with various chemical and surface properties, are
widely available. Examples of nanoparticles particularly useful in
biotechnology and nanomedicine include: fullerenes (e.g.,
buckyballs and carbon tubes); liquid crystals; liposomes; silica
and silicon-based nanoparticles (e.g., mesoporous silica
nanoparticles); nanoshells; nanorods; metal and metal oxides
nanoparticles (e.g., spherical nucleic acids, densely packed
polynucleotides surrounding a gold core); polycations; and cationic
cyclodextrins.
[0247] One example of nanoparticle formation includes the use of
cationic cyclodextrins that can self-assemble into nanoparticles to
form colloidal particles (Draz, M. S., et al., (2014) "Nanopartic
le-Mediated Systemic Delivery of siRNA for Treatment of Cancers and
Viral Infections," Theranostics. 2014; 4(9):872-892). Example 18
describes production of Cas9 protein and
sn1-casRNA/sn2-casRNA/sn3-casRNA components. These sn-casPNs/Cas9
system components are formed into ribonucleoprotein complexes and
are also prepared as particles with a SC12CDClickpropylamine
vector. SC12CDClickpropylarnine vectors have been described for use
with siRNA (see, e.g., Aoife M. O'Mahony, A. M., et al., (2013)
"Cationic and PEGylated Amphiphilic Cyclodextrins: Co-Formulation
Opportunities for Neuronal Sirna Delivery," PLOSONE 8(6):e66413).
Characterization of the SC12CDClickpropylamine vector
sn-casRNAs/Cas9 particles is described in Example 18.
[0248] Cationic cyclodextrins include, but are not limited to,
carboxyethyl-.beta.-cyclodextrin, amphiphilic cyclodextrins (e.g.,
heptakis[2-(.omega.o-amino-oligo(ethylene
glycol))-6-deoxy-6-hexadecylthio]-.beta.-cyclodextrin and
heptakis[2-(.omega.-amino-oligo(ethylene
glycol))-6-deoxy-6-dodecylthio]-.beta.-cyclodextrin); and cationic
multi-armed .alpha.-cyclodextrin (.alpha.-CD):PEG polyrotaxane.
[0249] Liposomes are another example of nanoparticle formation.
sn-casPNs/Cas9 system component of the present invention can be
entrapped in liposomes. Liposomes for use with the sn-casPNs/Cas9
system components typically comprise a cationic lipid. Examples of
the cationic lipids include DODAC (dioctadecyldimethylammonium
chloride), DOTMA
(N-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium), DDAB
(didodecylammonium bromide), DOTAP
(1,2-dioleoyloxy-3-trimethylammonio propane), DC-Chol
(3-beta-N-(Ne,N%-dimethyl-aminoethane)-carbamol cholesterol), DMRIE
(1,2-dimyristoyloxypropyl-3-dimethylhydroxyethyl ammonium), DOSPA
(2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamin-
um trifluoroacetate), DSTAP (1,2-Distearoyl-3-Trimethylammonium
Propane), DODAP (dioleoyl-3-dimethylammonium-propane), DOGS
(dioctadecylamidoglycylcarboxyspermine), and the like. A single
type of cationic lipid may be used alone, or a combination of two
or more types of cationic lipids can be used. Cationic lipids are
typically combined with other lipids (e.g., phospholipids and
cholesterol) to form liposomes.
[0250] Examples of phospholipids for liposome formation include,
but are not limited to, the following: phosphatidylcholine;
L-.alpha.-phosphatidylcholine (egg phosphatidylcholine (EPC), or
hydrogenated soy phosphatidylcholine (HSPC));
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC);
phosphatidylserine (PS); phosphatidylinositol (PI);
phosphatidylglycerol (PG); phosphatidylethanolamine (PE); dioleoyl
phosphatidylglycerol (DOPG);
1,2-Dioleoyl-sn-glycero-3-phosphocholine (or dioleoyl
phosphatidylcholine) (DOPC); dioleoyl phosphatidylserine (DOPS);
1,2-dileoyl-sn-glycero-3-phosphoethanolamine (DOPE);
1,2-Dioleoyl-sn-glycero-3-phosphate (DOPA);
1-Myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC);
1,2-Dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DPPG);
1,2-Dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DMPG);
1,2-Dimyristoyl-sn-glycero-3-yhosphocholine (DMPC);
1,2-Distearoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DSPG);
1-Palmitoyl-2-olcoyl-sn-glyccro-3-phosphocholine (POPC);
diacylphosphatidylcholine; diacylphosphatidic acid; N-dodecanoyl
phosphatidylethanolamine: N-succinyl phosphatidylethanolamine:
N-glutaryl phosphatidylethanolamine: lysylphosphatidylglycerol;
sphingolipids (e.g., sphingomyelin); and mixtures thereof.
[0251] A variety of sterols and derivatives thereof (e.g.,
cholesterol) can be used to stabilize liposomes. Cholesterol can be
chemically modified with a ligand designed to be recognized by a
particular organ or cell type such as a long chain fatty acid, an
amino acid, an amino acid derivative, a protein, glycoprotein, an
oligosaccharide, a hormone, modified protein, or the like.
Liposomes containing such modified cholesterols are suitable for
being targeted to a specific organ or cell type (see, e.g., U.S.
Pat. No. 4,544,545).
[0252] Hydrophilic polymers such as polyethylene glycol (PEG) and
other polyethoxylated polymers can be used to shield liposomes to
enhance the circulatory half-life of the liposome. Such hydrophilic
polymers can be associated non-covalently with the liposomes or
conjugated or covalently linked to a particular component of the
liposome (e.g., PEG-derivatized lipids; such as
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (ammonium salt) (mPEG-DSPE), and stearylated
PEG2000). Additional exemplary hydrophilic polymers include, but
are not limited to, polyvinyl alcohols, polylactic acids,
polyglycolic acids, polyvinylpyrrolidones, polyacrylamides,
polyglycerols, polyaxozlines, polyaminoacids (PAAs), and mixtures
thereof.
[0253] Methods for the preparation of the liposomal compositions
include liposomes formed by a thin film hydration method wherein
rehydration uses an aqueous solution comprising a sn-casPN/Cas9
system of the present invention (see, e.g., Example 18).
[0254] Example 18 describes n-casR1NAs/Cas9 protein complexes in
non-viral delivery vectors comprising cationic molecules. In the
example, production of Cas9 mRNA and
sn1-casRNA/sn2-casRNA/sn3-casRNA components is described. These
components are then formed into ribonucleoprotein complexes as well
as ribonucleoprotein/SC12CDClickpropylamine particles. The
complexes and particles are entrapped in liposomes. These liposomes
are characterized using a number of criteria, including in vivo
activity. The example establishes criteria for selecting optimal
liposomal compositions for encapsulation of sn-casRNAs/Cas9
complexes of the present invention according to their advantages
and limitations.
[0255] In other embodiments, liposomes are formed by a lipid
solution injection method wherein a lipid solution is injected into
an aqueous solution comprising components of the sn-casPN/Cas9
systems of the present invention. The lipids are typically
dissolved in a solvent, for example, an organic solvent (such as an
alcohol; e.g., ethanol), followed by injection into the aqueous
solution comprising the sn-casPN/Cas9 system while stirring.
Liposome vesicles are formed upon injection into the aqueous
solution trapping small amounts of aqueous solution in the internal
aqueous compartment(s) of the vesicles. One advantage of this
method is that it is scalable.
[0256] Examples of sn-casPNs/Cas9 systems of the present invention
that can be entrapped in liposomes include, but are not limited to,
polynucleotides encoding sn-casPNs and/or Cas9 protein, expression
cassettes comprising sn-casPNs and/or Cas 9 proteins, sn-casPNs,
Cas9 protein, complexes of sn-casPNss and Cas9 protein, and
combinations thereof.
[0257] Aspects of the present invention include, but are not
limited to the following: one or more expression cassettes
comprising polynucleotides encoding sn-casPNs and/or Cas9 protein;
one or more vectors, including expression vectors, comprising
polynucleotides encoding sn-casPNs and/or Cas9 protein; methods of
manufacturing expression cassettes comprising production of
polynucleotides comprising expression cassettes encoding sn-casPNs
and/or Cas9 protein; methods of manufacturing vectors, including
expression vectors, comprising production of vectors comprising
polynucleotides encoding sn-casPNs and/or Cas9 protein; methods of
introducing one ore more expression cassettes, comprising
introducing polynucleotides encoding sn-casPNs and/or Cas9 protein
into a selected host cell; methods of introducing one or more
vectors, including expression vectors, comprising introducing
vector(s) comprising polynucleotides encoding sn-casPNs and/or Cas9
protein into a selected host cell; host cells comprising one or
more expression cassettes comprising polynucleotides encoding
sn-casPNs and/or Cas9 protein (recombinant cells); host cells
comprising one or more vectors, including expression vectors,
comprising polynucleotides encoding sn-casPNs and/or Cas9 protein
(recombinant cells); host cells comprising one or more
polynucleotides encoding sn-casPNs and/or Cas9 protein (recombinant
cells); host cells expressing the products of one or more
polynucleotides encoding sn-casPNs and/or Cas9 protein (recombinant
cells); methods for manufacturing sn-casPNs comprising producing
sn-casPNs by in vitro transcription and/or producing Cas9 protein
by in vitro translation; and methods for manufacturing sn-casPNs
and/or Cas9 protein, comprising isolating the sn casPNs and/or Cas9
protein from host cells (recombinant cells) expressing the products
of one or more polynucleotides encoding sn-casPNs and/or Cas9
protein.
[0258] Another aspect of the present invention relates to methods
to generate non-human genetically modified organisms. Generally, in
these methods expression cassettes comprising polynucleotide
sequences of the sn-casPNs and Cas9 protein, as well as a targeting
vector are introduced into zygote cells to site-specifically
introduce a selected polynucleotide sequence at a DNA target
sequence in the genome to generate a modification of the genomic
DNA. The selected polynucleotide sequence is present in the
targeting vector. Modifications of the genomic DNA typically
include, insertion of a polynucleotide sequence, deletion of a
polynucleotide sequence, or mutation of a polynucleotide sequence,
for example, gene correction, gene replacement, gene tagging,
transgenc insertion, gene disruption, gene mutation, mutation of
gene regulatory sequences, and so on. In one embodiment of methods
to generate non-human genetically modified organisms, the organism
is a mouse. One embodiment of this aspect of the invention is the
generation of genetically modified mice.
[0259] Generating transgenic mice involves five basic steps (Cho
A., et al., "Generation of Transgenic Mice," Current protocols in
cell biology, 2009; Chaper. Unit-19.11). First, purification of a
transgenic construct (e.g., expression cassettes comprising
polynucleotide sequences of the sn-casPNs and Cas9 protein, as well
as a targeting vector). Second, harvesting donor zygotes. Third,
microinjection of the transgenic construct into the mouse zygote.
Fourth, implantation of microinjected zygotes into pseudo-pregnant
recipient mice. Fifth, performing gcnotyping and analysis of the
modification of the genomic DNA established in founder mice.
[0260] Example 17 describes use of the split-nexus Cas9-associated
polynucleotides (sn-casPNs) of the present invention for creating
genomic modifications in non-human animals. The example describes
generation of transgenic mice using two-part sn-casRNA (sn1-casRNA
and sn2-casRNA) system (see, e.g., FIG. 3B). The production of Cas9
mRNA and sn1-casRNA/sn2-casRNA is described. The mRNAs are use for
one-cell embryo injection. The example describes the creation of
double-gene mutant mice as well as the evaluation of in vivo
off-target effects of the sn-casRNAs/Cas9 system. Furthermore, the
example includes evaluation of in vivo gene repair using a donor
oligonucleotide with the sn-casRNAs/Cas9 system. The results of
these analyses are to demonstrate that mice with genomic repair
modifications in multiple genes can be generated using the
sn-casPNs/Cas9 systems described herein.
[0261] In another embodiment of methods to generate non-human
genetically modified organisms, the organism is a plant. The
sn-casPNs/Cas9 protein systems described herein are used to effect
efficient, cost-effective gene editing and manipulation in plant
cells. It is generally preferable to insert a functional
recombinant DNA in a plant genome at a non-specific location.
However, in certain instances, it may be useful to use
site-specific integration to introduce a recombinant DNA construct
into the genome. Such introduction of recombinant DNA into plants
is facilitated using the sn-casPNs/Cas9 protein systems of the
present invention. Recombinant vectors for use in plant are known
in the art. The vectors can include, for example, scaffold
attachment regions (SARs), origins of replication, and/or
selectable markers.
[0262] For embodiments in which polynucleotides encoding sn-casPNs
and/or Cas9 protein are used to transform a plant, a promoter
demonstrating the ability to drive expression of the coding
sequence in that particular species of plant is selected. Promoters
that can be used effectively in different plant species are well
known in the art, as well. Inducible, viral, synthetic, or
constitutive promoters can be used in plants for expression of
polypeptides. Promoters that are spatially regulated, temporally
regulated, and spatio-temporally regulated can also be useful. A
list of preferred promoters includes, but is not limited to, the
FMV35S promoter, the enhanced CaMV35S promoters, CaMV 35S promoter,
opine promoters, monocot promoters, plant ubiquitin promoter (Ubi),
rice actin 1 promoter (Act-1), maize alcohol dehydrogenase 1
promoter (Adh-1).
[0263] Factors that determine which regulatory sequences to use in
a recombinant construct, include, but are not limited to, desired
expression level, and cell- or tissue-preferential expression,
inducibility, efficiency, and selectability. One of skill in the
art can modulate expression of a coding sequence by selecting and
positioning regulatory sequences relative to the coding
sequence.
[0264] Suitable regulatory sequences initiate mainly transcription
or only transcription in certain cell types. Methods for
identifying and characterizing regulatory sequences in plant
genomic DNA are known. U.S. Patent Application Publication No.
20110177228, published Jul. 21, 2011, describes a large number of
such regulatory sequences as follows.
[0265] Root-active promoters confer transcription in root tissue,
e.g., root vascular tissues, root epidermis, or root endodermis.
Some root-active promoters are root-preferential promoters and
confer transcription predominantly in root tissue. Examples of
root-preferential promoters include, but are not limited to, the
following: PT0625, PT0660, PT0683, PT0758, YP0128, and YP0275.
Other root-preferential promoters include the PT0613, PT0672,
PT0688, and PT0837, which promote transcription primarily in root
tissue but also to some extent in ovules and/or seeds. Other
root-preferential promoters include the root-specific subdomains of
the CaMV 35S promoter and the tobacco RD2 promoter.
[0266] In some embodiments, promoters specifically active in
maturing endosperm can be used. Transcription from a maturing
endosperm promoter generally begins after fertilization and occurs
primarily in endosperm tissue during seed development.
Transcription is commonly highest during the cellularization phase.
Examples of maturing endosperm promoters that can be used in
expression vector constructs include, but are not limited to, the
napin promoter, the soybean trypsin inhibitor promoter, the soybean
a' subunit of the beta-conglycinin promoter, the Arcelin-5
promoter, the ACP promoter, the phaseolin promoter, the
stearoyl-ACP desaturase promoter, the oleosin promoter, the zein
promoters (e.g., 15 kD, 16 kD, 19 kD, 22 kD, and 27 kD zein
promoters), the Osgt-1 promoter from the rice glutelin-1 gene, the
beta-amylase promoter, and the barley hordcin promoter. Other
maturing endosperm promoters include the PT0676, PT0708 and YP0092
promoters.
[0267] Examples of promoters active in ovary tissues include, but
are not limited to, the following: the polygalacturonidasc
promoter, the banana TRX promoter, the melon actin promoter,
YP0396, and PT0623. In addition, examples of promoters that are
active primarily in ovules include YP0007, YP0008, YP0028, YP0039,
YP0092, YP0103, YP0111, YP0115, YP0119, YP0120, YP0121, and
YP0374.
[0268] To achieve expression in embryo sac/early endosperm,
regulatory sequences are used that are active in polar nuclei
and/or the central cell, or in precursors to polar nuclei, but not
in egg cells or precursors to egg cells. A pattern of transcription
that extends from polar nuclei into early endosperm development can
also be found with embryo sac/early endosperm-preferential
promoters (although transcription typically decreases significantly
in later endosperm development during and after the cellularization
phase). Expression iti the zygote or developing embryo typically is
not present with embryo sac/early endosperm promoters. Examples of
such promoters include those derived from the following genes, but
are not limited to, the following: Arabidopsis viviparous-1,
Arabidopsis atmycl, Arabidopsis FIE, Arabidopsis MEA, Arabidopsis
F1S2, FIE 1.1, maize MAC1, and maize Cat3. Additional Arabidopsis
promoters include YP0039, YP0101, YP0102, YP0110, YP0117, YP0119,
YP0137, DME, YP0285, and YP0212. Examples of rice promoters include
p530c10, pOsFIE2-2, pOsMEA, pOsYp102, and pOsYp285.
[0269] Regulatory sequences that preferentially drive transcription
in zygotic cells following fertilization can provide
embryo-preferential expression. Examples of embryo-preferential
promoters include, but are not limited to, the following: the
barley lipid transfer protein (Ltp1) promoter, YP0088, YP0097,
YP0107, YP0143, YP0156, PT0650, PT0695, PT0723, PT0740, PT0838, and
PT0879.
[0270] Promoters active in photosynthetic tissue confer
transcription in green tissues such as leaves and stems. Examples
of photosynthetic tissue promoters include the
ribulose-1,5-bisphosphate carboxylase (RbcS) promoters such as the
RbcS promoter from eastern larch (Larix laricina), the pine cab6
promoter, the Cab-1 promoter from wheat, the CAB-1 promoter from
spinach, the cab1R promoter from rice, the pyruvate orthophosphate
dikinase (PPDK) promoter from corn, the tobacco Lhcbl*2 promoter,
the Arabidopsis thaliana SUC2 sucrose-H+ symporter promoter, the
thylakoid membrane protein promoters from spinach (psaD, psaF,
psaE, PC, FNR, atpC, atpD, cab, and rbcS), and the PT0668, PT0886,
YP0144, YP0380 and PT0585 promoters.
[0271] Examples of promoters that have high or preferential
activity in vascular bundles include YP0022, YP0080, YP0087,
YP0093, and YP0108. Other vascular tissue-preferential promoters
include the glycine-rich cell wall protein GRP 1.8 promoter, the
Commelina yellow mottle virus (CoYMV) promoter, and the rice tungro
bacilliform virus (RTBV) promoter.
[0272] Inducible promoters confer transcription in response to
external stimuli such as chemical agents or environmental stimuli.
For example, inducible promoters can confer transcription in
response to hormones such as giberellic acid or ethylene, or in
response to light or drought. Examples of drought-inducible
promoters include PD0901, PD1367, PT0710, PT0848, YP0286, YP0337,
YP0356, YP0374, YP0377, YP0380, YP0381, YP0384, YP0385, YP0388,
YP0396, PT0633, and PT0688. Examples of nitrogen-inducible
promoters include PT0863, PT0829, PT0665, and PT0886. Examples of
shade-inducible promoters include PR0924 and PT0678. An example of
a promoter induced by salt is rd29A.
[0273] A stem promoter may be specific to one or more stem tissues
or specific to stem and other plant parts. Stem promoters may have
high or preferential activity in, for example, epidermis and
cortex, vascular cambium, procambium, or xylem. Examples of stem
promoters include the following: YP0018, CryIA(b), and
CryIA(c).
[0274] Examples of other classes of promoters include
shoot-preferential, callus-preferential, trichome
cell-preferential, guard cell-preferential such as PT0678,
tuber-preferential, parenchyma cell-preferential, and
senescence-preferential promoters. In some embodiments, a promoter
may preferentially drive expression in reproductive tissues.
[0275] A 5' untranslated region (UTR) can be included in vector
constructs. A 5' UTR is transcribed, but is not translated, and
lies between the start site of the transcript and the translation
initiation codon and may include the +1 nucleotide. A 3' UTR can be
positioned between the translation termination codon and the end of
the transcript. UTRs can have particular functions such as
increasing mRNA stability or attenuating translation. Examples of
3' UTRs include, but are not limited to, the following:
polyadenylation signals and transcription termination sequences,
(e.g., a nopaline synthase termination sequence).
[0276] Additional regulatory sequences are described in U.S. Patent
Application Publication No. 2011017728, published Jul. 21,
2011.
[0277] RNA Polymerase III promoters that can be used in plant
vectors for the expression of sn-casPNs include 7SL, U6 (e.g., U6
snoRNA promoter) and U3 (e.g., U3 snoRNA promoter).
[0278] In any transformation experiment, DNA is introduced into a
small percentage of target cells only. Genes that encode selectable
markers are useful and efficient in identifying cells that are
stably transformed when they receive and integrate a transgenic DNA
construct into their genomes. Preferred marker genes provide
selective markers that confer resistance to a selective agent, such
as an antibiotic or herbicide. Any herbicide to which plants may be
resistant is a useful agent for a selective marker.
[0279] Selectable markers can be used to select for plants or plant
cells containing vectors comprising the sn-casPNs and/or Cas9
protein of the present invention. A selectable marker can provide a
selectable phenotype on a plant cell. For example, a marker can
provide resistance to an antibiotic (e.g., kanamycin, G418,
bleomycin, or hygromycin), to an herbicide (a bar gene that codes
for bialaphos resistance; a mutant EPSP synthase gene that encodes
glyphosate resistance; a nitrilasc gene that confers resistance to
bromoxynil; a mutant acetolactate synthase gene (ALS) that confers
imidazolinone or sulphonylurea resistance) or methotrexate (a
methotrexate-resistant DHFR gene). Expression vectors can also
include a tag sequence designed to promote detection or
manipulation of the expressed polypeptide. Commonly expressed as a
fusion with the encoded polypeptide are tag sequences. Examples of
tag sequences include, but are not limited to, the following:
luciferase, beta-glucuronidase (GUS), green fluorescent protein
(GFP), glutathione S-transferase (GST), polyhistidine, c-myc,
hemagglutinin, or epitope (e.g., a FLAG.RTM. epitope,
Sigma-Aldrich, St. Louis, Mo.). Such tags can be inserted anywhere
within the polypeptide, including at either the carboxyl or amino
terminus.
[0280] Potentially transformed cells are exposed to the selective
agent, and, among the surviving cells there will be cells in which
the resistance-conferring gene has been integrated and is expressed
at sufficient levels for cell survival. Cells may be tested further
to confirm stable integration of the exogenous DNA.
[0281] A screenable marker, which may be used to monitor
expression, may also be included in a recombinant vector or
construct of the present invention. Screenable markers include, but
are not limited to, a p-glucuronidase or uidA gene (GUS) that
encodes an enzyme for which various chromogenic substrates are
known; an R-locus gene, which encodes a product that regulates the
production of anthocyanin pigments (red color) in plant tissues; a
.beta.-lactamase gene, a gene that encodes an enzyme for which
various chromogenic substrates are known (e.g., PADAC, a
chromogenic cephalosporin); a luciferase gene; a xylE gene that
encodes a catechol dioxygenase that can convert chromogenic
catechols; an .alpha.-amylase gene; a tyrosinase that encodes an
enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which
in turn condenses to melanin; and an .alpha.-galactosidase, which
catalyzes a chromogenic .alpha.-galactose substrate.
[0282] Polynucleotides of the present invention may be introduced
into a plant cell, either permanently or transiently, together with
other genetic elements, for example, promoters, enhancers, introns,
and untranslated leader sequences.
[0283] Xing, H. L., et al., ((2014) "A CRISPR/Cas9 toolkit for
multiplex genome editing in plants," BMC Plant Biology 2014,
14:327) have described module vectors for expression of CR1SPR-Cas9
systems in dicots and monoeots. Binary vectors with two types of
backbones are utilized; the first based on pGreen vectors (Hellens,
R. P., et al., (2000) "pGreen: a versatile and flexible binary Ti
vector for agrobacterium-mediated plant transformation," Plant Mol
Biol 42:819-832); and the second based on pCAMB1A vectors. The
pGreen-like vectors are relatively small, allowing them to be used
for transient Cas9 and sn-casRNA expression in protoplasts to test
the effectiveness of target sites. The vectors can be directly used
to generate transgenic plants after validation in protoplasts. In
Agrobacterium, the pGreen-like vectors depend on their pSa origin
for propagation, and they require a helper plasmid to provide
replication protein (RepA). Agrobacterium containing pSoup helper
plasmid can be used as hosts for pGreen-like vectors.
[0284] Among the pCAMBIA-derived binary vectors, those with a
hygromycin-resistance gene as a selectable marker were derived from
pCAMBIA1300, while those with a kanamycin-resistance gene were
derived from pCAMBIA2300, and those with a Basta-resistance gene
were derived from pCAMBIA3300. The vectors pCAMBIA1300/2300/3300
(Curtis, M. D., et al., (2003) "A gateway cloning vector set for
high-throughput functional analysis of genes in plants," Plant
Physiol 133:462-469; Lee, L. Y., Gelvin, S. B. (2008) "T-DNA binary
vectors and systems," Plant Physiol 146:325-332.) and their
derivatives (including the Gateway-compatible pMDC series) are some
of the most widely used binary vectors for a variety of plant
species and with several plant transformation protocols
specifically optimized based on these vectors.
[0285] Such binary vector systems can be used with the expression
cassettes of the present invention to provide, for example,
multiple sn-casRNAs for multiplex genome editing. For example, in a
three polynucleotide split nexus system, sn1-casRNA and sn2-casRNA
DNA coding sequences are placed under the control of RNA Polymerase
III promoters in the first vector. Multiple sn3-casRNA each
comprising a different DNA targeting sequence are each placed under
the control of RNA Polymerase III promoters and cloned into the
same vector. A Cas9 protein coding sequence optimized for
expression in the selected plant is also included in the
vector.
[0286] Among preferred plant transformation vectors are those
derived from a Ti plasmid of Agrobacterium tumefaciens (Lee, L. Y.,
et al., "T-DNA Binary Vectors and Systems," Plant Physiol. 2008
February; 146(2): 325-332). Also useful and known in the art are
Agrobacterium rhizogenes plasmids. There are several commercial
software products designed to facilitate selection of appropriate
plant plasmids for plant cell transformation and gene expression in
plants and methods to easily enable cloning of such
polynucleotides. SnapGene.TM. (GSL Biotech LLC, Chicago, Ill.;
www.snapgene.com/resources/plasmid_files/your_time_is_valuable/),
for example, provides an extensive list of plant vectors including
individual vector sequences and vector maps, as well as commercial
sources for many of the vectors.
[0287] Methods and compositions for transforming plants by
introducing a recombinant DNA construct into a plant genome
includes any of a number of methods known in the art. One method
for constructing transformed plants is microprojectile bombardment.
Agrobacterium-mediated transformation is another method for
constructing transformed plants. Alternatively, other
non-Agrobacterium species (e.g., Rhizobium) and other prokaryotic
cells that are able to infect plant cells and introduce
heterologous nucleotide sequences into the infected plant cell's
genome can be used. Further transformation methods include
electroporation, liposomes, transformation using pollen or viruses,
chemicals that increase free DNA uptake, or free DNA delivery by
means of microprojectile bombardment. DNA constructs of the present
invention can be introduced into the genome of a plant host using
conventional transformation techniques that are well known to those
skilled in the art (see, e.g.,."Methods to Transfer Foreign Genes
to Plants," Y Narusaka, et al.,
cdn.intechopen.com/pdfs-wm/30876.pdf).
[0288] Typically, a transgenic plant formed using Agrobacterium
transformation methods contains one simple recombinant DNA sequence
inserted into one chromosome; this is referred to as a transgenic
event. Such transgenic plants are heterozygous because of the
inserted exogenous sequence. It is possible to form a transgenic
plant that is homozygous with respect to a transgene by sexually
mating (i.e., selfing) an independent segregant transgenic plant
containing a single exogenous gene sequence to itself, for example
an F0 plant, to produce F1 seed. One quarter of the F1 seeds will
be heterozygous for the transgene. Plants formed by germinating F1
seeds can be tested for heterozygosity. Typical zygosity assays
include, but are not limited to, SNP assays and thermal
amplification assays that distinguish between homozygotes and
heterozygotes. The progeny resulting from crossing a heterozygous
plant with itself or with another heterozygous plant are always
heterozygous.
[0289] As an alternative to using a recombinant DNA construct for
the direct transformation of a plant, transgenic plants can be
formed by crossing a first plant that has been transformed with a
recombinant DNA construct with a second plant that lacks the
construct. As an example, a first plant line into which has been
introduced a recombinant DNA construct for gene suppression can be
crossed with a second plant line to introgress the recombinant DNA
into the second plant line, thus forming a transgenic plant
line.
[0290] The sn-casPNs/Cas9 protein systems of the present invention
provide plant breeders with a new tool to induce mutations.
Accordingly, one skilled in the art can analyze genomic sources and
identify genes of interest having desired traits or characteristics
(e.g., herbicide resistance genes) and use the sn-casPNs/Cas9
systems of the present invention to introduce such genes into plant
varieties lacking the genes; this result can be achieved with more
precision than by using previous mutagenic agents, thereby
accelerating and enhancing plant breeding programs.
[0291] Example 16 describes targeted mutagenesis in Zea mays using
a three-part sn-casRNA system (sn1-casRNA, sn-2-casRNA and
sn3-casRNA) to create genomic modifications in plants. Three
different maize genomic target sequences are targeted for cleavage.
Vectors comprising expression cassettes of the
sn1-casRNA/sn2-casRNA/sn3-casRNA/Cas9 systems are described. The
generation of mutations at the targeted sites is used to
demonstrate that the sn-casPNs/Cas9 systems as described herein
cleave maize chromosomal DNA and can be used to generate genomic
mutations.
[0292] Another aspect of the present invention comprises methods of
modifying DNA using sn-casPNs and Cas9 proteins. Generally, a
method of modifying DNA involves contacting a target DNA with a
sn-casPNs/Cas9 protein complex (a "targeting complex"). In some
cases, the Cas9 protein component exhibits nuclease activity that
cleaves both strands of a double-stranded DNA target at a site in
the double-stranded DNA that is complementary to a DNA target
binding sequence in the sn-casPNs. With nuclease-active Type II
Cas9 proteins, site-specific cleavage of the target DNA occurs at
sites determined by (i) base-pair complementarity between the DNA
target binding sequence in the sn-casPNs and the target DNA, and
(ii) a protospacer adjacent motif (PAM) present in the target DNA.
The nuclease activity cleaves the target DNA to produce
double-strand breaks. In cells the double-strand breaks are
repaired cellular mechanisms including, but not limited to:
non-homologous end joining (NHEJ), and homology-directed repair
(HDR).
[0293] Repair of double-strand breaks by NHEJ occurs by direct
ligation of the break ends to one another. Typically no new
polynucleotide sequences are inserted at the site of the
double-strand break; however, insertions or deletions may occur
when a small number of nucleotides are either randomly inserted or
deleted at the site of the double-strand break. Furthermore, two
different sn-casPNs that comprise DNA target binding sequences
targeting two different DNA target sequences are used to provide
deletion of an intervening DNA sequence (i.e., the DNA sequence
between the two DNA target sequences). Deletion of the intervening
sequence occurs when NHEJ rejoins the ends of the two cleaved DNA
target sequences to each other. Similarly, NHEJ may be used to
direct insertion of donor template DNA or portion thereof using
donor template DNA, for example, containing compatible overhangs.
Accordingly, one embodiment of the present invention includes
methods of modifying DNA by introducing insertions and/or deletions
at a target DNA site.
[0294] Repair of double-strand breaks by HDR uses a donor
polynucleotide (donor template DNA) or oligonucleotide having
homology to the cleaved target DNA sequence. The donor template DNA
or oligonucleotide is used for repair of the double-strand break in
the target DNA sequence resulting in the transfer of genetic
information (i.e., polynucleotide sequences) from the donor
template DNA or oligonucleotide at the site of the double-strand
break in the DNA. Accordingly, new genetic information (i.e.,
polynucleotide sequences) may be inserted or copied at a target DNA
site.
[0295] One aspect of the present invention is directed to a method
of modifying a nucleic acid target binding sequence (e.g., DNA)
comprising, contacting nucleic acid target binding sequence (e.g.,
a DNA target sequence in a DNA polynucleotide) with a
sn-casPNs/Cas9 system of the present invention (e.g., an
sn1-casPN/sn2-casPN/Cas9 protein complex (such as shown in FIG. 3B,
sn1-casPN, FIG. 3B, 326, and sn2-casPN, FIG. 3B, 302; or FIG. 3A
sn1-casPN, FIG. 3A, 301, sn2-casPN, FIG. 3a, 302, and sn3-casPN,
FIG. 3A, 303), wherein the sn-casPNs/Cas9 protein form a complex
that binds and cuts the nucleic acid target sequence (e.g., a DNA
target sequence) resulting in a modification of the target nucleic
acid (e.g., a DNA polynucleotide comprising the DNA target
sequence). This method can be carried out in vitro or in vivo. The
method can, for example, be used to modify DNA derived from a cell
(e.g., a eukaryotic cell) isolated from an organism. Furthermore,
in some embodiments the method comprises contacting a DNA target
sequence in genomic DNA with a donor DNA template wherein the
genomic DNA is modified in that it comprises that at least a
portion of the donor DNA template integrated at the DNA target
sequence.
[0296] Methods for bringing a donor polynucleotide into proximity
to the site of a double-stranded break in a target nucleic acid arc
described in U.S. Published Patent Application No. 20140315985,
published Oct. 23, 2014 (see, e.g., 0121, 0851-0860).
[0297] Example 1 describes production of exemplary sn-casPN
components of the present invention. Example 2 describes production
of double-stranded DNA target regions for use in Cas9 cleavage
assays. Example 3 and Example 7 provide in vitro examples of a
method of modifying DNA using a sn-casPNs/Cas9 system
(sn1-casRNA/sn2-casRNA) of the present invention. Example 6
provides an in vitro example of a method of modifying DNA using a
different sn-casPNs/Cas9 system (sn1-casRNA/sn2-casRNA/sn3-casRNA).
Furthermore, the data presented in Example 4 demonstrate use of the
sn-casPNs/Cas9 systems of the present invention for deep sequencing
analysis for detection of target modifications in eukaryotic
cells.
[0298] In some methods of the present invention, cells comprise
polynucleotide sequences encoding a sn-casPNs and a Cas9 protein
comprising active RuvC and HNH nuclease domains. Expression of
these polynucleotide sequences is placed under the control of one
or more inducible promoter. When the DNA binding sequence of an
sn-casPN is complementary to a DNA target in, for example, a
promoter of a gene, upon inducing expression of the sn-casPNs and
Cas9 protein, expression from the gene is shut off (as a result of
the cleavage of the promoter sequence by the sn-casPNs/Cas9 protein
complex). The polynucleotides encoding the sn-casPNs and Cas9
protein can be integrated in the cellular genome, present on
vectors, or combinations thereof.
[0299] In methods of modifying a target DNA using the
sn-casPNs/Cas9 protein complexes of the present invention, repair
of a double-stranded break by either NHEJ and/or HDR can lead to,
for example, gene correction, gene replacement, gene tagging, gene
disruption, gene mutation, transgene insertion, or nucleotide
deletion. Methods of modifying a target DNA using the
sn-casPNs/Cas9 protein complexes of the present invention in
combination with a donor template DNA can be used to insert or
replace polynucleotide sequences in a DNA target sequence, for
example, to introduce a polynucleotide that encodes a protein or
functional RNA (e.g., siRNA), to introduce a protein tag, to modify
a regulatory sequence of a gene, or to introduce a regulatory
sequence to a gene (e.g. a promoter, an enhancer, an internal
ribosome entry sequence, a start codon, a stop codon, a
localization signal, or polyadenylation signal), to modify a
nucleic acid sequence (e.g., introduce a mutation), and the
like.
[0300] In some embodiments of the sn-casPNs/Cas9 protein systems of
the present invention, a mutated form of the Cas9 protein is used.
Modified versions of the Cas9 protein can contain a single inactive
catalytic domain (i.e., either inactive RuvC or inactive HNH). Such
modified Cas9 proteins cleave only one strand of a target DNA thus
creating a single-strand break. Modified Cas9 protein having a
single inactive catalytic domain can bind DNA based on
sn-casPN-conferred specificity; however, it will only cut one of
the double-stranded DNA strands. As an example, in the Cas9 protein
from Streptococcus pyogenes the RuvC domain can be inactivated by a
D10A mutation and the HNH domain can be inactivated by an H840A
mutation. When using a modified Cas9 protein having a single
inactive catalytic domain in the sn-casPNs/Cas9 protein complexes
of the present invention NHEJ is less likely to occur at the
single-strand break site.
[0301] In other modified versions of the Cas9 protein both
catalytic domains are inactive (i.e., inactive RuvC and inactive
HNH; "dCas"). Such dCas9 proteins have no substantial nuclease
activity; however, they can bind DNA based on sn-casPN-conferred
specificity. As an example, in the Cas9 protein from Streptococcus
pyogenes a D10A mutation and an H840A mutation result in a dCas 9
protein having no substantial nuclease activity.
[0302] The present invention also includes methods of modulating in
vitro or in vivo transcription using sn-casPNs/Cas9 protein
complexes described herein. In one embodiment, a sn-casPNs/Cas9
protein complex can repress gene expression by interfering with
transcription when a sn-casPN directs DNA target binding of the
sn-casPNs/Cas9 protein complex to the promoter region of a gene.
Use of sn-casPNs/Cas9 protein complexes to reduce transcription
also includes complexes wherein the dCas9 protein is fused to a
known down regulator of a target gene (e.g., a repressor
polypeptide). For example, expression of a gene is under the
control of regulatory sequences to which a repressor polypeptide
can bind. A sn-casPN can direct DNA target binding of a
sn-casPNs/Cas9 protein-repressor protein complex to the DNA
sequences encoding the regulatory sequences or adjacent the
regulatory sequences such that binding of the sn-casPNs/Cas9
protein-repressor protein complex brings the repressor protein into
operable contact with the regulatory sequences. This results in
repression of expression of the target gene. Similarly, dCas9 is
fused to an activator polypeptide to activate or increase
expression of a gene under the control of regulatory sequences to
which an activator polypeptide can bind.
[0303] In one aspect the present invention relates to a method of
modulating the expression of a gene comprising transcriptional
regulatory elements comprising, contacting a DNA target sequence in
the gene with a sn-casPNs/Cas9 system of the present invention,
wherein the sn-casPNs and the Cas9 protein form a complex that
binds to the DNA target sequence resulting in modulation of the
expression of the gene. In one embodiment, the Cas9 protein is a
Cas9 that is nuclease-deficient. In other embodiments, the
sn-casPNs/Cas9 complex further comprises a fusion protein.
[0304] Example 13 describes use of the split-nexus Cas9-associated
polynucleotides (sn-casPNs) of the present invention for the
repression or activation of endogenous genes in human cells. The
nuclease deficient S. pyogenes Cas9 (dCas9) with mutation D10A and
H840A is used. The sn1-casRNA-CD71 sequence comprises a 20
nucleotide spacer sequence that directs the sn-casRNAs/Cas9 protein
complex to the upstream untranslated region of the of the
transferrin receptor CD71. Activation of CD71 expression in
dCas9-VP64 transfected samples is measured by the increase in
detected fluorescence compared to the measured fluorescence of a
non-transfected control population of HeLa cells as detected by
FACS sorting. Repression of CD71 expression in dCas9-KRAB
transfected samples is measured by the decrease in detected
fluorescence compared to the measured fluorescence of a
non-transfected control population of HeLa cells as detected by
FACS sorting. This procedure provides data to verify that the
sn-casPNs/Cas9 protein systems of the present invention can be used
in the activation or repression of endogenous genes.
[0305] In some embodiments, a non-native sequence can confer new
functions to a fusion protein. Examples of fusion proteins
including a Cas9 protein (e.g., Cas9 protein) and other regulatory
or functional domains include, but are not limited to a nuclease, a
transposase, a methylase, a transcription factor repressor or
activator domain (e.g., such as KRAB and VP16), co-repressor and
co-activator domains, DNA methyl transferases, histone
acetyltransferases, histone dcacetylases, and DNA cleavage domains
(e.g., a cleavage domain from the endonuclease FokI). Further
examples include, but are not limited to the following:
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, 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, remodeling activity, protease
activity, oxidoreductase activity, transferase activity, hydrolase
activity, lyase activity, isomerase activity, synthase activity,
synthetase activity, demyristoylation activity, and any
combinations thereof.
[0306] In another aspect, the sn-casPNs/Cas9 systems of the present
invention are used in methods for high-throughput functional
genomics screening. Forward genetic screens are powerful tools for
the discovery and functional annotation of genetic elements (see,
e.g., Gilbert et al., (2013) "CRISPR-Mediated Modular RNA-Guided
Regulation of Transcription in Eukaryotes," Cell 18;
154(2):442-51). The sn-casPNs/Cas9 systems can be used to generate
genome-scale libraries of sn-casPNs for unbiased, phenotypic
screening. Approaches for genome-scale screening include knockout
approaches that inactivate genomic loci and approaches that
modulate transcriptional activity. In knockout screening,
loss-of-function mutations mediated by sn-casPNs/Cas9 systems are
generated by double-strand break induction and NHEJ-mediated
repair. Knockout screens are useful to identify essential gene
functions, for example, gene functions related to drug and toxin
sensitivities. One example of such functional genomics screening is
presented in Example 12. In the example, a two-part sn-casRNA (snl
-casRNA and sn2-casRNA) system is used to create a lentiviral
library of sn1-casRNAs. The library is used in a knockout method to
identify candidate genes important in resistance to drug treatment.
This procedure provides data to verify that the sn-casPNs/Cas9
systems of the present invention can be used in functional
screening to interrogate gene-function on a genome-wide scale.
[0307] Another method of the present invention is the use of
sn-casPNs/dCas systems to isolate or purify regions of genomic DNA
(gDNA). In an embodiment of the method, a dCas9 protein is fused to
an epitope (e.g., a FLAG.RTM. epitope, Sigma-Aldrich, St. Louis,
Mo.) and a sn-casPN directs DNA target binding of a sn-casPNs/dCas9
protein-epitope complex to DNA sequences within the region of
genomic DNA to be isolated or purified. An affinity agent is used
to bind the epitope and the associated gDNA bound to the
sn-casPNs/dCas9 protein-epitope complex.
[0308] In further aspect, the present invention includes kits
comprising sn-casPNs or polynucleotides encoding sn-casPNs and
instructions. Kits can comprise one or more of the following:
sn-casPNs and cognate Cas9 protein; polynucleotides encoding
sn-casPNs and cognate protein; recombinant cells comprising
sn-casPNs; recombinant cells comprising sn-casPNs and cognate
protein; and the like. Any kits of the present invention can
further comprise other components such as solutions, buffers,
substrates, cells, instructions, vectors (e.g., targeting vectors),
and so on.
[0309] The invention also includes the use of T7E1 assays to
evaluate and compare the percent cleavage in vivo of sn-casPNs/Cas9
systems relative to selected double-stranded DNA target sequences
(Example 9). Also, the invention also includes methods for
Identification and Screening of Trans-Activating CRISPR RNA
(Example 8), which can be modified for use in the sn-casPNs/Cas9
systems and methods of the present invention. Furthermore, the
invention includes methods of generating and testing split nexus
modifications in tracrRNAs (Example 10), for example, based on
crRNA/tracrRNAs know in the art or identified by methods described
in Example 8.
[0310] The present invention also includes pharmaceutical
compositions comprising sn-casPNs/Cas9 protein systems, or one or
more polynucleotides encoding sn-casPNs and a Cas9 protein.
Pharmaceutical composition, for example, the nanoparticle
compositions comprising sn-casPNs/Cas9 systems described above, may
further comprise pharmaceutically acceptable excipients.
[0311] A pharmaceutical composition can comprise a combination of
any of the sn-casPNs/Cas9 systems described herein with other
components, for example, excipients (e.g., carriers, stabilizers,
diluents, suspending agents, thickening agents, and others as
described herein). The compositions facilitate administration of
the sn-casPNs/Cas9 systems to a subject. Pharmaceutical
compositions can be administered in therapeutically effective
amounts by various forms and routes including, for example,
intravenous, subcutaneous, or inhalation.
[0312] Methods for the preparation of pharmaceutical compositions
comprising the sn-casPNs/Cas9 systems can include formulating them
with one or more inert, pharmaceutically acceptable excipient. For
example, the pharmaceutical compositions can be liquid solutions or
suspensions. Typical excipients useful in the practice of the
present invention include, but are not limited to, the following:
carrier or vehicle (e.g., water or buffered aqueous solutions);
buffer systems (e.g., comprising acetate, phosphate, citrate,
borate, tartrate, histidine, succinate, and mixtures thereof);
antioxidant g (e.g., sodium thiosulfate, ethylenediaminetetraacetic
acid, citric acid, cysteins, thioglycerol, thioglycolic acid,
thiosorbitol, butylated hydroxanisol, butylated hydroxyltoluene,
and propyl gallate, and mixtures thereof); agents to maintain
isotonicity (e.g., sodium chloride, sugars, polyols (sugar
alcohols), boric acid, sodium tartrate, propylene glycol, and
mixtures thereof); one or more sugar (e.g., trehalose, maltose,
sucrose, lactose, mannose, dextrose, fructose, etc.) or sugar
alcohol (e.g., sorbitol, maltitol, lactitol, mannitol, glycerol,
etc.), alcohol (e.g., ethanol, t-butanol, etc.); and preservatives
(alcohols, benzoic acid, salicylic acid, phenol and its derivatives
(e.g., cresol, p-cresol, m-cresol and o-cresol), cctrimide, BHA
(butylated hydroxytoluene), BHA (butylated hydroxyanisole); and
mixtures thereof).
[0313] Advantages of the sn-casPNs/Cas9 systems of the present
invention include, but are not limited to, the following. Use of a
multipart sn-casPNs/Cas9 system allows improved control of activity
for in vivo systems. Expression control of all parts of the system
provides further layers of regulation over assembly of the specific
components needed to constitute a functional sn-casPNs/Cas9 system,
for example, relative to an sgRNA/Cas9 system.
[0314] The split nexus element, accessory, auxiliary, and adjunct
polynucleotides of the sn-casPNs of the present invention provide
additional sites (relative to crRNA/tracrRNA/Cas9 and sgRNA/Cas9
complexes) for adding and/or tethering functional moieties (e.g.,
polypeptides, small molecules, labels, and the like).
[0315] In some embodiments of the present invention, for example, a
three polynucleotide engineered CRISPR-Cas9 system, the shorter
length of the sn-casPNs (relative to the longer lengths of
crRNA/tracrRNA and sgRNA) allows for higher quality and more rapid
chemical synthesis of the sn-casPNs. Furthermore, the shorter
length of the sn-casPNs facilitates packaging into virus-based
vectors.
[0316] Furthermore, a three polynucleotide engineered CRISPR-Cas9
system of the present invention (e.g., as illustrated in FIG. 3A)
can be used to provide partially preformed Cas9 complexes in an in
vivo system to allow rapid activation. For example, sn1-casRNA,
sn3-casRNA, and Cas9 protein are expressed in a cell. These
components form a sn1RNA/sn3-casRNA/Cas9 protein complex, which is
not active for binding or cleaving a target. When the sn2 component
is expressed or introduced into the cell, the
sn1-casRNA/sn2-casRNA/sn3-casRNA/Cas9 protein complex is rapidly
activated, which enables temporal control over site-specific
targeting.
[0317] Additional advantages of the present invention will be
apparent to one of ordinary skill in the art in view of the
teachings of the present specification.
[0318] 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. From the above description and the following
Examples, one skilled in the art can ascertain essential
characteristics of this invention, and without departing from the
spirit and scope thereof, can make changes, substitutions,
variations, and modifications of the invention to adapt it to
various usages and conditions. Such changes, substitutions,
variations, and modifications are also intended to fall within the
scope of the present disclosure.
[0319] Experimental
[0320] Aspects of the present invention are further illustrated in
the following Examples. Efforts have been made to ensure accuracy
with respect to numbers used (e.g., amounts, concentrations,
percent changes, etc.) but some experimental errors and deviations
should be accounted for. Unless indicated otherwise, temperature is
in degrees Centigrade and pressure is at or near atmospheric. It
should be understood that these Examples, while indicating some
embodiments of the invention, are given by way of illustration
only.
[0321] The following examples arc not intended to limit the scope
of what the inventors regard as various aspects of the present
invention.
[0322] Materials and Methods
[0323] Oligonucleotide sequences (e.g., the primer sequences shown
in FIG. 13) were provided to commercial manufacturers for synthesis
(Integrated DNA Technologies, Coralville, Iowa; or Eurofins,
Luxembourg).
[0324] sn-casPNs are assembled by PCR using 3' overlapping primers
containing the corresponding DNA sequences to the sn-casPNs.
Furthermore, DNA sequences encoding the sn-casPNs can be cloned in
a suitable vector for propagation and subsequent isolation of
sn-casPN sequences (e.g., using restriction enzyme cleavage of the
vector to yield the sn-casPN).
EXAMPLE 1
Production of sn-casRNA Components
[0325] This example described production of a split-nexus
Cas9-associated three polynucleotide system (e.g., similar to the
system illustrated in FIG. 3A).
[0326] RNA components were produced by in vitro transcription
(e.g., T7 Quick High Yield RNA Synthesis Kit, New England Biolabs,
Ipswich, Mass.) from double-stranded DNA template incorporating a
T7 promoter at the 5' end of the DNA sequences.
[0327] The double-stranded DNA template for the specific sn2-casRNA
component, used in the examples (referred to herein as
sn2-casRNA.sup.EX), was assembled by PCR using 3' overlapping
primers containing the corresponding DNA sequences to the
sn2-casRNA.sup.EX component. The oligonucleotides used in the
assembly are presented in Table 8.
TABLE-US-00008 TABLE 8 Overlapping Primers for Generation of
sn2-casRNA Component Templates Type of sn-casPN Target for
DNA-binding Component Sequence Oligonucleotides* sn2-casRNA.sup.EX
n/a A, B, C *DNA primer oligonucleotide sequences are shown in FIG.
13
[0328] The DNA primers were present at a concentration of 2 nM
each. Two outer DNA primers corresponding to the T7 promoter
(forward primer: Oligonucleotide A, Table 8), and the 3'end of the
RNA sequence (reverse primers: Oligonucleotides C, Table 8) were
used at 640 nM to drive the amplification reaction. PCR reactions
were performed using Q5 Hot Start High-Fidelity 2.times. Master Mix
(New England Biolabs, Ipswich, Mass.) as following the
manufacturer's instructions. PCR assembly reactions were carried
out using the following thermal cycling conditions: 98.degree. C.
for 2 minutes, 35 cycles of 15 seconds at 98.degree. C., 15 seconds
at 62.degree. C., 15 seconds at 72.degree. C., and a final
extension at 72.degree. C. for 2 min. DNA quality was evaluated by
agarose gel electrophoresis (1.5%, SYBR.RTM. Safe, Life
Technologies, Grand Island, N.Y.).
[0329] The double-stranded DNA templates for the specific
sn1-casRNA and sn3-casRNA components, used in the examples, were
assembled by complexing two complementary oligonucleotide sequences
(referred to as sn1-casRNA.sup.EX and sn3-casRNA.sup.EX) The
oligonucleotides used in the assembly are presented in Table 9.
TABLE-US-00009 TABLE 9 Overlapping Primers for Generation of
sn-casRNAs Component Templates Type of sn-casPN Target for
DNA-binding Component Sequence Oligonucleotide.sup.2
sn1-casRNA.sup.EX n/a D, E sn3-casRNA.sup.EX-AAVS1 AAVS-1.sup.1 F,
G .sup.1AAVS-1, Adeno-Associated Virus Integration Site 1 - Human
Genome; .sup.2DNA primer sequences are shown in FIG. 13.
[0330] The DNA primers were present at a concentration of 10 .mu.M
each, 10 uL of each primer were mixed together and incubated for 2
minutes at 95.degree. C., removed from thermocycler and allowed to
equilibrate to room temperature.
[0331] Between 0.25-0.5 mg of the DNA template for each sn-casRNA
component was transcribed using T7 High Yield RNA Synthesis Kit
(New England Biolabs, Ipswich, Mass.) for approximately 16 hours at
37.degree. C. Transcription reactions were treated with DNAse I
(New England Biolabs, Ipswich, Mass.) and purified using GeneJet
RNA Cleanup and Concentration Kit (Life Technologies, Grand Island,
N.Y.). RNA yield was quantified using the Nanodrop.TM. 2000 System
(Thermo Scientific, Wilmington Del.). The quality of the
transcribed RNA was checked by agarose gel electrophoresis (2%,
SYBRO Safe, Life Technologies, Grand Island, N.Y.). The sn-casRNA
sequences are as shown in Table 10.
TABLE-US-00010 TABLE 10 sn-casRNA Sequences sn2- 5'-GUCCGUUAUC
AACUUGAAA SEQ ID NO: 68 casRNA.sup.EX AGUGGCACCG AGUCGGUGCU U-3'
sn1- 5'-GCAGGACAGC AUAGCAAGUU SEQ ID NO: 69 casRNA.sup.EX
GAGAUAAGGC UA-3' sn3- 5'-GGGGCCACUA GGGACAGGAU SEQ ID NO: 70
casRNA.sup.EX- GUCUCAGAGC UAUGCUGU-3' AAVS1
[0332] This method for production of sn1-casRNA.sup.EX,
sn2-casRNA.sup.EX, and sn3-casRNA.sup.EX can be applied to the
production of other sn-casRNAs as described herein.
EXAMPLE 2
Production of Double-Stranded DNA Target Regions for Use in Cas9
Cleavage Assays
[0333] Target double stranded DNA for use in an in vitro Cas9
cleavage assays were produced using PCR amplification of the target
region from genomic DNA.
[0334] Double-stranded DNA target regions (e.g., AAVS-1) for
biochemical assays were amplified by PCR from phenol-chloroform
prepared human cell line K562 (ATCC, Manassas, Va.) genomic DNA
(gDNA). PCR reactions were carried out with Q5 Hot Start
High-Fidelity 2.times. Master Mix (New England Biolabs, Ipswich,
Mass.) following the manufacturer's instructions. 20 ng/.mu.L gDNA
in a final volume of 25 ml were used to amplify the selected target
region under the following conditions: 98.degree. C. for 2 minutes,
35 cycles of 20 s at 98.degree. C., 20 s at 60.degree. C., 20 s at
72.degree. C., and a final extension at 72.degree. C. for 2 mm. PCR
products were purified using Spin Smart.TM. PCR purification tubes
(Denville Scientific, South Plainfield N.J.) and quantified using
Nanodrop.TM. 2000 UV-Vis spectrophotometer (Thermo Scientific,
Wilmington Del.).
[0335] The forward and reverse primers used for amplification of
selected targeted sequences from gDNA were as follows: AAVS-1,
oligonucleotides H and I (FIG. 13). The amplified double-stranded
DNA target for AAVS-1 was 495 bp.
[0336] Other suitable double-stranded DNA target regions are
obtained using essentially the same method. For non-human target
regions, genomic DNA from the selected organism (e.g., plant,
bacteria, yeast, algae) is used instead of DNA derived from human
cells. Furthermore, polynucleotide sources other than genomic DNA
can be used (e.g., vectors and gel isolated DNA fragments).
EXAMPLE 3
Cas9 Cleavage Assays
[0337] This example illustrates the use of a split-nexus
Cas9-associated three polynucleotide system of the present
invention in in vitro Cas9 cleavage assays to evaluate and compare
the percent cleavage of selected sn-casRNAs/Cas9 protein complexes
relative to selected double-stranded DNA target sequences.
[0338] The cleavage of double-stranded DNA target sequences was
determined for sn-casRNA.sup.EX components of Example 2 against a
double-stranded DNA target (AAVS-1; Example 2).
[0339] All three sn-casPN.sup.EX components in equimolar amounts
were mixed in an annealing buffer (1.25 mM HEPES, 0.625 mM
MgCl.sub.2, 9.375 mM KCl at pH7.5), incubated for 2 minutes at
95.degree. C., removed from thermocycler and allowed to equilibrate
to room temperature. Additional combinations of two of the three
sn-casRNAs.sup.EX were tested as described below with reference to
the data presented in FIG. 8. When only two components of the
polynucleotide system were used water was added instead of the
third sn-casRNA.sup.EX component.
[0340] The sn-casRNAs.sup.EX were added to a Cas9 reaction mix. The
Cas9 reaction mix comprised Cas9 protein diluted to a final
concentration of 200 .mu.M in reaction buffer (20 mM HEPES, 100 mM
KCl, 5 mM MgCl.sub.2, 1 mM DTT, and 5% glycerol at pH 7.4). In the
reaction mix the final concentration of each sn-casRNA.sup.EX was
500 nM in each reaction mix. Each reaction mix was incubated at
37.degree. C. for 10 minutes. The cleavage reaction was initiated
by the addition of target DNA to a final concentration of 15 nM.
Samples were mixed and centrifuged briefly before being incubated
for 15 minutes at 37.degree. C. Cleavage reactions were terminated
by the addition of Protcinase K (Denville Scientific, South
Plainfield, N.J.) at a final concentration of 0.2 mg/mL and 0.44
mg/ml RNase A Solution (SigmaAldrich, St. Louis, Mo.).
[0341] Samples were incubated for 25 minutes at 37.degree. C. and
25 minutes at 55.degree. C. 12 .mu.L of the total reaction were
evaluated for cleavage activity by agarose gel electrophoresis (2%,
SYBRO Gold, Life Technologies, Grand Island, N.Y.). For the AAVS-1
double-stranded DNA target, the appearance of DNA bands at
approximately 316 bp and approximately 179 bp indicated that
cleavage of the target DNA had occurred. Cleavage percentages were
calculated using area under the curve values as calculated by FIJI
(ImageJ; an open source Java image processing program) for each
cleavage fragment and the target DNA, and dividing the sum of the
cleavage fragments by the sum of both the cleavage fragments and
the target DNA.
[0342] FIG. 8 presents the results of the Cas9 cleavage assay using
the AAVS-1 target double-stranded DNA. In the figure, replicates of
three are shown for each combination of sn-casRNAs.sup.EX . At the
top of each panel is a graphical representation of the
sn-casRNAs.sup.EX used in the assay. FIG. 8, Panel A shows the
biochemical activity of sn1-casRNA.sup.EX, sn2-casRNA.sup.EX,
sn3-casRNA.sup.EX-AAVS1. FIG. 8, Panel B shows the biochemical
activity of sn1-casRNA.sup.EX and sn2-casRNA.sup.EX. FIG. 8, Panel
C shows the biochemical activity of sn2-casRNA.sup.EX and
sn3-casRNA.sup.EX-AAVS1, FIG. 8, Panel D shows the biochemical
activity of sn1-casRNA.sup.EX and sn3-casRNA.sup.EX-AAVS1. The last
lane of FIG. 8, Panel D contains molecular weight standards.
Cleavage percentages are shown at the bottom of each lane. As can
be seen from the data in the FIG. 8, sn1-casRNA.sup.EX,
sn2-casRNA.sup.EX, and sn3-casRNA.sup.EX-AAVS1 had an average
percent cleavage of 46.9% (standard deviation of 0.3%). For all
reactions where only two sn-casRNA.sup.EX components were present
(e.g. FIG. 8, Panel B, FIG. 8, Panel C, FIG. 8, Panel D) no
cleavage activity was observed (for lanes indicated as LOD, any
cleavage activity was below the limit of detection.).
[0343] The data presented in FIG. 8 demonstrate that the
split-nexus Cas9-associated polynucleotide systems of the present
invention facilitate Cas mediated site-specific cleavage of target
double-stranded DNA. The data also show that all three sn-casRNA
components of the split-nexus Cas9-associated three polynucleotide
system are needed to support Cas mediated site-specific cleavage
activity.
[0344] Following the guidance of the present specification and
examples, the Cas9 cleavage assay described in this example can be
practiced by one of ordinary skill in the art with other Type II
CRISPR Cas9 proteins including, but not limited to, Cas9 and Cas9
fusions combined with their cognate polynucleotide components
modified as described herein to comprise a split nexus element.
EXAMPLE 4
Deep Sequencing Analysis for Detection of Target Modifications in
Eukaryotic Cells
[0345] This example illustrates the use of deep sequencing analysis
to evaluate and compare the percent cleavage in vivo of selected
sn-casRNA/Cas9 protein complexes relative to selected
double-stranded DNA target sequences.
[0346] A. Formation of RNP Complexes of sn1-casRNA.sup.EX,
sn2-casRNA.sup.EX, sn3-casRNA.sup.EX-AAVS1 and Cas9 protein.
[0347] S. pyogenes Cas9 was C-terminally tagged with two nuclear
localization sequences (NLS) and recombinantly expressed in E.
coli. Ribonucleoprotein (RNP) complexes were set up at two
concentrations, 50 pmol Cas9:150 pmols sn-casRNAs.sup.EX and 200
pmols Cas9:600 pmols sn-casRNAs.sup.EX, in triplicate. All three
sn-casRNAs.sup.EX (sn1-casRNA.sup.EX, sn2-casRNA.sup.EX,
sn3-casRNA.sup.EX-AAVS1) components in equimolar amounts were mixed
in an anndaling buffer (1.25 mM HEPES, 0.625 mM MgCl.sub.2, 9.375
mM KCl at pH7.5) to the desired concentration (150 pmols or 600
pmols) in a final volume of 5 .mu.L, incubated for 2 minutes at
95.degree. C., removed from the thermocycler and allowed to
equilibrate to room temperature. Cas9 protein was diluted to an
appropriate concentration in binding buffer (20 mM HEPES, 100 mM
KCl, 5 mM MgCl.sub.2, 1 mM DTT, and 5% glycerol at pH 7.4) to a
final volume of 5 .mu.L and mixed with the 5 .mu.L of
heat-denatured sn-casRNAs.sup.EX followed by incubation at
37.degree. C. for 30 minutes.
[0348] B. Cell Transfections Using sn-casRNAs.sup.EX/Cas9 Protein
RNPs
[0349] RNP complexes were transfected into K562 cells (ATCC,
Manassas Va.), using the Nucleofector.RTM. 96-well Shuttle System
(Lonza, Allendale, N.J.) and the following protocol. RNP complexes
were dispensed in a 10 .mu.L final volume into individual wells of
a 96-well plate. K562 cells suspended in media were transferred
from a culture flask to a 50 mL conical tube. Cells were pelleted
by centrifugation for 3 minutes at 200.times.g, the culture medium
aspirated, and the cells washed once with calcium and
magnesium-free PBS. K562 cells were then pelleted by centrifugation
for 3 minutes at 200.times.g, the PBS aspirated and cell pellet was
resuspended in 10 mL of calcium and magnesium-free PBS.
[0350] The cells were counted using the Countess.RTM. II Automated
Cell Counter (Life Technologies, Grand Island, N.Y.).
2.2.times.10.sup.7 cells were transferred to a 50 ml tube and
pelleted. The PBS was aspirated and the cells were resuspended in
Nucleofector.TM. SF (Lonza, Allendale, N.J.) solution to a density
of 1.times.10.sup.7 cells/mL. 20 .mu.L of the cell suspension are
then added to individual wells containing 10 .mu.L of RNP complexes
and the entire volume was transferred to the wells of a 96-well
Nucleocuvette.TM. Plate (Lonza, Allendale, N.J.). The plate was
loaded onto the Nucleofector.TM. 96-well Shuttle.TM. (Lonza,
Allendale, N.J.) and cells were nucleofected using the 96-FF-120
Nucleofector.TM. program (Lonza, Allendale, N.J.).
Post-nucleofection, 70 .mu.L Iscove's Modified Dulbecco's Media
(IMDM; Life Technologies, Grand Island, N.Y.), supplemented with
10% FBS (Fisher Scientific, Pittsburgh, Pa.), penicillin and
streptomycin (Life Technologies, Grand Island, N.Y.), was added to
each well and 50 .mu.L of the cell suspension were transferred to a
96-well cell culture plate containing 150 .mu.L pre-warmed IMDM
complete culture medium. The plate was then transferred to a tissue
culture incubator and maintained at 37.degree. C. in 5% CO.sub.2
for 48 hours.
[0351] C. Target Double-Stranded DNA Generation for Deep
Sequencing
[0352] gDNA was isolated from K562 cells 48 hours after RNP
transfection using 50 .mu.L QuickExtract DNA Extraction solution
(Epicentre, Madison, Wis.) per well followed by incubation at
37.degree. C. for 10 minutes, 65.degree. C. for 6 minutes and
95.degree. C. for 3 minutes to stop the reaction. The isolated gDNA
was then diluted with 50 .mu.L water and samples were stored at
-80.degree. C.
[0353] Using the isolated gDNA, a first PCR was performed using Q5
Hot Start High-Fidelity 2.times. Mix (New England Biolabs, Ipswich,
Mass.) at 1.times. concentration, primers at 0.5 .mu.M each (FIG.
13, oligonucleotides H & I), 3.75 .mu.L of gDNA in a final
volume of 10 L and amplified 98.degree. C. for 1 minute, 35 cycles
of 10 s at 98.degree. C., 20 s at 60.degree. C., 30 s at 72.degree.
C., and a final extension at 72.degree. C. for 2 min. PCR reaction
was diluted 1:100 in water.
[0354] A "barcoding" PCR was set up using unique primers for each
sample to facilitate multiplex sequencing. The primer pairs are
shown in Table 11.
TABLE-US-00011 TABLE 11 Barcoding Primers ID Sample Primers*
BARCODING PRIMER set-1 50 pmol Cas9:150 pmol L, M sn-casRNA rep-1
BARCODING PRIMER set-2 50 pmol Cas9:150 pmol L, N sn-casRNA rep-2
BARCODING PRIMER set-3 50 pmol Cas9:150 pmol L, O sn-casRNA rep-3
BARCODING PRIMER set-4 200 pmol Cas9:600 pmol L, P sn-casRNA rep-1
BARCODING PRIMER set-5 200 pmol Cas9:600 pmol L, Q sn-casRNA rep-2
BARCODING PRIMER set-6 200 pmol Cas9:600 pmol L, R sn-casRNA rep-3
*Primer sequences are shown in FIG. 13
[0355] The barcoding PCR was performed using Q5 Hot Start
High-Fidelity 2.times. Master Mix (New England Biolabs, Ipswich,
Mass.) at lx concentration, primers at 0.5 .mu.M each (Table 11), 1
.mu.L of 1:100 diluted first PCR, in a final volume of 10 .mu.L and
amplified 98.degree. C. for 1 minutes, 12 cycles of 10 s at
98.degree. C., 20 s at 60.degree. C., 30 s at 72.degree. C., and a
final extension at 72.degree. C. for 2 min.
[0356] D. SPRIselect Clean-Up
[0357] PCR reactions were pooled into a single microfuge tube for
SPRIselect (Beckman Coulter, Pasadena, Calif.) bead -based clean up
of amplicons for sequencing.
[0358] To the pooled amplicons, 0.9.times. volumes of SPRIselect
beads were added, and mixed and incubated at room temperature (RT)
for 10 minutes. The microfuge tube was placed on magnetic tube
stand (Beckman Coulter, Pasadena, Calif.) until solution had
cleared. Supernatant was removed and discarded, and the residual
beads were washed with 1 volume of 85% ethanol, and incubated at RT
for 30 s. After incubation ethanol was aspirated and beads were air
dried at RT for 10 mm. The microfuge tube was then removed from the
magnetic stand and 0.25.times. volumes of Qiagen EB buffer (Qiagen,
Venlo, Limburg) was added to the beads, mixed vigorously, and
incubated for 2 min. at room temperature. The microfuge tube was
returned to the magnet, incubated until solution had cleared, and
supernatant containing the purified amplicons was dispensed into a
clean microfuge tube. The purified amplicon library was quantified
using the Nanodrop.TM. 2000 System (Thermo Scientific, Wilmington
Del.) and library-quality analyzed using the Fragment Analyzer.TM.
System (Advanced Analytical Technologies, Inc., Ames, Iowa) and the
DNF-910 Double-stranded DNA Reagent Kit (Advanced Analytical
Technologies, Inc. Ames, Iowa).
[0359] E. Deep Sequencing Set-Up
[0360] The amplicon library was normalized to a 4 nmolar
concentration as calculated from Nanodrop values and size of the
amplicons. The library was analyzed on MiScq Sequencer (Illumina,
San Diego) with MiScq Reagent Kit v2 (Illumina, San Diego) for 300
cycles with two 151-cycle paired-end run plus two eight-cycle index
reads.
[0361] F. Deep Sequencing Data Analysis
[0362] The identity of products in the sequencing data was
determined based on the index barcode sequences adapted onto the
amplicons in the barcoding round of PCR. A computational script was
used to process the MiSeq data by executing the following tasks:
[0363] Reads were aligned to the human genome (build GRCh38/38)
using Bowtie (bowtie-bio.sourceforge.net/index.shtml) software.
[0364] Aligned reads were compared to the expected wild-type AAVS-1
locus sequence, reads not aligning to any part of the AAVS-1 locus
were discarded (Table 12, "other"). [0365] Reads matching wild-type
AAVS-1 sequence (Table 12, "WT") were tallied. [0366] Reads with
indels (insertion or the deletion of bases) were categorized by
indel type and tallied (Table 12, "indel"). [0367] Total indel
reads were divided by the sum of wild-type reads and indel reads
gave percent-mutated reads.
[0368] The results of this analysis are presented in Table 12.
TABLE-US-00012 TABLE 12 Deep Sequencing Data Sample Type.sup.1
Total.sup.2 Aligned.sup.3 WT.sup.4 indel.sup.5 Other.sup.6 50 pmol
33807 33680 18119 15561 3 Cas9:150 pmol sn-casRNAs.sup.EX rep 1 50
pmol 33070 32991 18225 14766 2 Cas9:150 pmol sn-casRNAs.sup.EX rep2
50 pmol 33062 32986 18580 14406 5 Cas9:150 pmol sn-casRNAs.sup.EX
rep3 200 pmol 34089 33993 9321 24672 1 Cas9:650 pmol
sn-casRNAs.sup.EX rep1 200 pmol 28691 28600 7100 26893 2 Cas9:650
pmol sn-casRNAs.sup.EX rep2 200 pmol 28573 28509 12184 16325 1
Cas9:650 pmol sn-casRNAs.sup.EX rep3 .sup.1Sample type; .sup.2Total
MiSeq reads; .sup.3Total reads aligns to target locus (AAVS-1);
.sup.4Total wt reads (i.e. unmodified sequence); .sup.5Mutated
reads (cas9 cleavaged); .sup.6Reads not aligning to AAVS-1
locus.
[0369] As can be seen from the measured indels across replicates in
Table 12, sn-casPNs/Cas9 systems are capable of in vivo
modification of a target locus. Additionally the increased indel
frequency as a result of increased transfected sn-casPNs/Cas9
concentration is indicative of dose dependent sn-casPNs/Cas9 system
mediated cleavage. The data presented in Table 12 demonstrate that
the split-nexus Cas9-associated polynucleotide systems of the
present invention facilitate in vivo Cas9-mediated site-specific
cleavage of a genomic locus.
[0370] Following the guidance of the present specification and
examples, the analysis described in this example can be practiced
by one of ordinary skill in the art with other Type II CRISPR Cas9
proteins including, but not limited to, Cas9 and Cas9 fusions
combined with their cognate polynucleotide components modified as
described herein to comprise a split nexus element.
EXAMPLE 5
Csy4* Facilitated sn-casRNA/Cas9 Cleavage
[0371] This example illustrates the use of sn-casRNAs of the
present invention and an effector protein, the nuclease deficient
P. aeruginosa Csy4 protein possessing the H29A mutation (Csy4*), to
increase association of two sn-casRNAs augmented with a Csy4 RNA
binding sequence.
[0372] A. Generation of sn-casRNA Components
[0373] The double-stranded DNA templates for the specific
sn-casRNA.sup.EXCsy components comprising a Csy4 binding sequence
were assembled by PCR using 3' overlapping primers containing the
corresponding DNA sequences to the sn-casRNA.sup.EXCsy components.
The oligonucleotide used in the assembly are presented in Table
13.
TABLE-US-00013 TABLE 13 Overlapping Primers for Generation of
sn-casRNA.sup.EXCsys with Csy4 RNA Binding Sequence DNA Target
Binding Type of Cas RNA Component Sequence Oligonucleotides* first
polynucleotide w/ Csy4 binding sequence 3' AAVS-1 A, Y, S, T of
split nexus (sn1-casRNA.sup.EXCsy-Csy) first polynucleotide w/ Csy4
binding sequence 3' CD34 A, Z, S, T of split nexus
(sn1-casRNA.sup.EXCsy-Csy) first polynucleotide w/ Csy4 binding
sequence 3' CD151 A, AA, S, T of split nexus
(sn1-casRNA.sup.EXCsy-Csy) first polynucleotide w/ Csy4 binding
sequence 3' JAK-1 A, AB, S, T of split nexus
(sn1-casRNA.sup.EXCsy-Csy) Second polynucleotide w/ Csy4 binding
sequence n/a A, U, AC 3' of split nexus (sn2-casRNA.sup.EXCsy-Csy)
AAVS first polynucleotide w/ linker + Csy4 binding AAVS-1 A, Y, V,
W sequence 3' of split nexus (sn1-casRNA.sup.EXCsy-lnkCsy) CD34
first polynucleotide w/ linker + Csy4 CD34 A, Z, V, W binding
sequence 3' of split nexus (sn1-casRNA.sup.EXCsy-lnkCsy) CD151
first polynucleotide w/ linker + Csy4 CD151 A, AA, V, W binding
sequence 3' of split nexus (sn1-casRNA.sup.EXCsy-lnkCsy) JAK-1
first polynucleotide w/ linker + Csy4 JAK-1 A, AB, V, W binding
sequence 3' of split nexus (sn1-casRNA.sup.EXCsy-lnkCsy) Second
polynucleotide w/ linker + Csy4 binding n/a A, X, AC sequence 3' of
split nexus (sn2-casRNA.sup.EXCsy-lnkCsy) *DNA primer sequences are
shown in FIG. 13
[0374] The DNA primers were present at a concentration of 2 nM
each. Two outer DNA primers corresponding to the T7 promoter
(forward primer: Oligonucleotide A, Table 13, and the 3'end of the
RNA sequence (reverse primers: Oligonucleotides T, AC, or W, Table
13) were used at 640 nM to drive the amplification reaction. PCR
and transcription was preformed as described in Example 1 described
in this specification. Transcribed sn-casRNA.sup.EXCsy sequences
are shown in Table 14.
TABLE-US-00014 TABLE 14 sn-casRNA.sup.EXCsy Sequences Type of Cas
RNA Component RNA sequence SEQ ID NO sn1-casRNA.sup.EXCsy-Csy-AAVS1
5'-GGGGCCACUA GGGACAGGAU GUCUCAGAGC SEQ ID NO: 71 UAUGCUGUCC
UGGAAACAGG ACAGCAUAGC AAGUUGAGAU AAGGCUACUG CC-3'
sn1-casRNA.sup.EXCsy-Csy-CD34 5'-GUUUGUGUUU CCAUAAACUG GUCUCAGAGC
SEQ ID NO: 72 UAUGCUGUCC UGGAAACAGG ACAGCAUAGC AAGUUGAGAU
AAGGCUACUG CC-3' sn1-casRNA.sup.EXCsy-Csy-CD151 5'-GCCCGCCACC
ACCAGGAUGU GUCUCAGAGC SEQ ID NO: 73 UAUGCUGUCC UGGAAACAGG
ACAGCAUAGC AAGUUGAGAU AAGGCUACUG CC-3'
sn1-casRNA.sup.EXCsy-Csy-JAK-1 5'-GGCAGCCAGC AUGAUGAGAC GUCUCAGAGC
SEQ ID NO: 74 UAUGCUGUCC UGGAAACAGG ACAGCAUAGC AAGUUGAGAU
AAGGCUACUG CC-3' sn2-casRNA.sup.EXCsy-Csy 5'-GGCAGGUCCG UUAUCAACUU
GAAAAAGUGG SEQ ID NO: 75 CACCGAGUCG GUGCUU-3'
sn1-casRNA.sup.EXCsy-InkCsy-AAVS1 5'-GGGGCCACUA GGGACAGGAU
GUCUCAGAGC SEQ ID NO: 76 UAUGCUGUCC UGGAAACAGG ACAGCAUAGC
AAGUUGAGAU AAGGCUAGUU CACUGCC-3' sn1-casRNA.sup.EXCsy-InkCsy-CD34
5'-GUUUGUGUUU CCAUAAACUG GUCUCAGAGC SEQ ID NO: 77 UAUGCUGUCC
UGGAAACAGG ACAGCAUAGC AAGUUGAGAU AAGGCUAGUU CACUGCC-3'
sn1-casRNA.sup.EXCsy-InkCsy- 5'-GCCCGCCACC ACCAGGAUGU GUCUCAGAGC
SEQ ID NO: 78 CD151 UAUGCUGUCC UGGAAACAGG ACAGCAUAGC AAGUUGAGAU
AAGGCUAGUU CACUGCC-3' sn1-casRNA.sup.EXCsy-InkCsy-JAK-1
5'-GGCAGCCAGC AUGAUGAGAC GUCUCAGAGC SEQ ID NO: 79 UAUGCUGUCC
UGGAAACAGG ACAGCAUAGC AAGUUGAGAU AAGGCUAGUU CACUGCC-3'
sn2-casRNA.sup.EXCsy-InkCsy 5'-GGGCAGUGAA CUAGCCUUAU CUCAACUUGC SEQ
ID NO: 80 UAUGCUGUCC UGUUUCCAGG ACAGCAUAGC UCUGAGAC-3'
[0375] B. Generation of Double-Stranded DNA Targets for Biochemical
Assay
[0376] Target double-stranded DNA for use in the in vitro Cas9
cleavage assays were produced using PCR amplification as described
in Example 2 herein. The forward and reverse primers used for
amplification from gDNA were as follows: AAVS-1 oligonucleotides
were J and K (FIG. 13), the amplified double-stranded DNA target
for AAVS-1 was 288 bp; CD34 (Hematopoietic Progenitor Cell Antigen)
oligonucleotides were AD and AE (FIG. 13), the amplified
double-stranded DNA target for CD34 was 258 bp; CD151
(Platelet-Endothelial Cell Tetraspanin Antigen) oligonucleotides
were AF and AG (FIG. 13), the amplified CD151 double-stranded DNA
target was 272 bp; and, JAK-1 (Janus Kinase 1) oligonucleotides
were AH and AI, the amplified JAK-1 double-stranded DNA target was
298 bp.
[0377] C. Csy4* Supported Cas9 Cleavage Biochemical Assay
[0378] sn-casRNAs.sup.EXCsy were prepared for use in the
biochemical assay as described in Example 3 herein. With the
modification that prior to the addition of Cas9, 250 nM of Csy*
protein was added to the reaction and sn-casRNAs.sup.EXCsy and
Csy4* were incubated at 37.degree. C. for 5 min. After the
incubation, Cas9 was added and biochemical reactions were carried
out as described in Example 3. A non-Csy4* control was
included.
[0379] For the AAVS-1 double-stranded DNA target, the appearance of
DNA bands at approximately 174 bp and approximately 114 bp
indicated that cleavage of the target DNA had occurred. For the
CD34 double-stranded DNA target, the appearance of DNA bands at
approximately 105 bp and approximately 153 bp indicated that
cleavage of the target DNA had occurred. For the CD151
double-stranded DNA target, the appearance of DNA bands at
approximately 109 bp and approximately 163 bp indicated that
cleavage of the target DNA had occurred. For the JAK-1
double-stranded DNA target, the appearance of DNA bands at
approximately 204 bp and approximately 94 bp indicated that
cleavage of the target DNA had occurred.
[0380] FIG. 9 presents the results of the Cas9 cleavage assay using
the Csy4* protein and the sn-casRNAs.sup.EXCsy. The cleavage assays
used two different split-nexus Cas9-associated two polynucleotide
systems that were variants of the system present in FIG. 3B. In the
first system the sn1-casRNAs.sup.EXCsy further comprised a first
auxiliary polynucleotide comprising a Csy4 binding element
nucleotide sequence I (sn1-casRNA.sup.EXCsy-Csy) and the sn2-casRNA
comprised a second auxiliary polynucleOtide comprising a Csy4
binding element nucleotide sequence II (sn2-casRNA.sup.EXCsy-Csy),
wherein the first auxiliary polynucleotide and the second auxiliary
polynucleotide associate to form a Csy4 RNA binding element
(sn1-casRNA/sn2-casRNA/Csy4RNA). In the second system the
sn1-casRNA further comprised a first auxiliary polynucleotide
comprising a linker element nucleotide sequence I and a Csy4
binding element nucleotide sequence I (sn 1
-casRNA.sup.EXCsy-lnkCsy) and the sn2-casRNA comprised a second
auxiliary polynucleotide comprising a linker element nucleotide
sequence II and a Csy4 binding element nucleotide sequence II
(sn2-casRNA.sup.EXCsy-lnkCsy), wherein the first auxiliary
polynucleotide and the second auxiliary polynucleotide associate to
form a linker element and a Csy4 RNA binding clement (see, e.g.,
the general representations in FIG. 6A and FIG. 6B). Each of the
two systems was used to target cleavage to four different targets,
where the sn-casRNAs.sup.EXCsy each comprised a spacer
complementary to one of the four targets: AAVS-1, CD-34, CD-151,
and JAK-1 (See Table 13 above). In the figure, the cleavage
activity is shown at the bottom of each lane (except for lanes 1
and 10, which are molecular weight standards). For lanes indicated
as LOD, any cleavage activity was below the limit of detection. The
systems used in each of the Cas9 cleavage assay reactions were as
shown in Table 5/F1G.9 (see Brief Description of the Figures, FIG.
9).
[0381] As can be seen from the data in the figure, the addition of
Csy4* enhanced the cleavage activity of the sn-casRNAs.sup.EXCsy
system for multiple double-stranded DNA target sequences: for
AAVS-1 compare lanes 2/3 (no Csy4* protein) to lanes 4/5,
respectively; for CD-34 compare lanes 6/7 (no Csy4* protein) to
lanes 8/9; for CD-151 compare lanes 11/12 (no Csy4* protein) to
13/14; and, for JAK-1 compare lanes 15/16 (no Csy4* protein) to
lanes 17/18.
[0382] The data presented in FIG. 9 demonstrate that an effector
protein (here Csy4*) enhanced cleavage of target double-stranded
DNA by split-nexus Cas9-associated polynucleotide systems of the
present invention comprising auxiliary polynucleotides having an
effector binding element (here the Csy RNA binding sequence).
[0383] Following the guidance in the present specification and
examples, increasing the association of two sn-casRNAs comprising a
Csy4 RNA binding sequence with a nuclease deficient P. aeruginosa
Csy4 protein as described in this example can be practiced by one
of ordinary skill in the art with other Type II CRISPR Cas9
proteins including, but not limited to, Cas9 and Cas9 fusions
combined with their cognate polynucleotide components modified as
described herein to comprise a split nexus element. Furthermore, in
view of the guidance in the present specification and examples one
of ordinary skill in the art can use other effector
protein/effector binding sequence combinations as exemplified
herein by the Csy* protein/Csy RNA binding sequence.
EXAMPLE 6
sn1-CasRNA/sn2-casRNA/Cas9 Cleavage Activity
[0384] This example illustrates the use of a split-nexus
Cas9-associated two polynucleotide system of the present invention
in in vitro Cas9 cleavage assays to evaluate and compare the
percent cleavage of selected sn1-casRNA/sn2-casRNA/Cas9 protein
complexes relative to selected double-stranded DNA target
sequences.
[0385] The double-stranded DNA templates for the sn-casRNA.sup.EX2
components used in this example were assembled by PCR using 3'
overlapping primers containing the corresponding DNA sequences to
the sn-casRNA.sup.EX2 components. A graphical representation of the
sn-casRNA.sup.EX2 components is presented in FIG. 10. The
oligonucleotide used in the assembly are presented in Table 15.
TABLE-US-00015 TABLE 15 Overlaping Primers for Generation of
sn1-casRNA and sn2-casRNA Type of sn-casRNA.sup.EX2 Component
Oligonucleotides* AAVS-1 sn1-casRNA Y, AJ CD151 sn1-casRNA AA, AJ
JAK-1 sn1-casRNA AB, AJ sn2-casRNA A, C, B *DNA primer sequences
are shown in FIG. 13
[0386] Generation of double-stranded DNA template for RNA
transcription was performed as described in Example 1 herein.
Transcribed sn-casRNAs.sup.EX2 sequences are shown in Table 16.
TABLE-US-00016 TABLE 16 sn-casRNA Sequences Type of Cas RNA
Component RNA sequence SEQ ID NO sn1-casRNAs.sup.EX2-AAVSA
5'-GGGGCCACUA GGGACAGGAU GUCUCAGAGC SEQ ID NO: 81 UAUGCUGUCC
UGGAAACAGG ACAGCAUAGC AAGUUGAGAU AAGGCUA-3'
sn1-casRNAs.sup.EX2-CD151 5'-GCCCGCCACC ACCAGGAUGU GUCUCAGAGC SEQ
ID NO: 82 UAUGCUGUCC UGGAAACAGG ACAGCAUAGC AAGUUGAGAU AAGGCUA-3'
sn1-casRNAs.sup.EX2-JAK-1 5'-GGCAGCCAGC AUGAUGAGAC GUCUCAGAGC SEQ
ID NO: 83 UAUGCUGUCC UGGAAACAGG ACAGCAUAGC AAGUUGAGAU AAGGCUA-3'
sn1-casRNAs.sup.EX2 5'-GUCCGUUAUC AACUUGAAAA AGUGGCACCG SEQ ID NO:
84 AGUCGGUGCU U-3'
[0387] Target double-stranded DNA for use in the in vitro Cas9
cleavage assays were produced using PCR amplification as described
in Example 2 herein. The forward and reverse primers used for
amplification from gDNA were as follows: AAVS-1 oligonucleotides
were J and K (FIG. 13), the amplified double-stranded DNA target
for AAVS-1 was 288 bp; CD151 oligonucleotides were AF and AG (FIG.
13), the amplified CD151 double-stranded DNA target was 272 bp;
and, JAK-1 oligonucleotides were AH and AI, the amplified JAK-1
double-stranded DNA target was 298 bp. In vitro cleavage was
performed as described in Example 3 herein.
[0388] FIG. 10 presents the result of the Cas9 cleavage assay using
the sn1-casRNAs.sup.EX2and sn2-casRNA.sup.EX2 described above.
Cleavage percentages arc shown at the bottom of each lane except
for lane 1, which is a molecular weight standard. FIG. 10, lane 2,
presents cleavage results for a sn1-casRNA.sup.EX2-AAVS1 and
sn2-casRNA.sup.EX2 system, which demonstrated a cleavage activity
of 97.6%. FIG. 10, lane 3, presents cleavage results for a
sn1-casRNA.sup.EX2-CD151 and sn2-casRNA.sup.EX2 system, which
demonstrated a cleavage activity of 48.8%. FIG. 10, lane 4,
presents the results for a sn1-casRNA.sup.EX2-JAK1 and
sn2-casRNA.sup.EX2 system, which demonstrated a cleavage activity
of 60.0%.
[0389] The data presented in FIG. 10 demonstrated that the
sn1-casRNA and sn2-casRNA constructs as described herein facilitate
the in vitro Cas mediated site-specific cleavage of a
double-stranded DNA target. These data support that the split-nexus
Cas9-associated polynucleotide systems of the present invention
facilitate in vivo Cas9-mediated site-specific cleavage of genomic
loci.
[0390] Following the guidance of the present specification and
examples, the Cas9 cleavage assay described in this example can be
practiced by one of ordinary skill in the art with other Type II
CRISPR Cas9 proteins including, but not limited to, Cas9 and Cas9
fusions combined with their cognate polynucleotide components
modified as described herein to comprise a split nexus element.
EXAMPLE 7
sn1-CasRNA.sup.EX3Csy/sn2-casRNA.sup.EX3Csy/Cas9 Cleavage
Activity
[0391] This example illustrates the use of two different
split-nexus Cas9-associated two polynucleotide systems of the
present invention to evaluate and compare their percent cleavage
activities relative to selected double-stranded DNA target
sequences.
[0392] The two different split-nexus Cas9-associated two
polynucleotide systems were as follows: one was the system
illustrated in FIG. 7A
(sn1-casRNA.sup.EX3Csy-Csy-AAVS1/sn2-casRNA.sup.EX3Csy-Csy); and
the second was a variant of the system present in FIG. 7A. In the
second system the sn1-casRNA.sup.EX3Csy-lnkCsy-AAVS1 comprised, 5'
to 3', a split nexus stem element nucleotide sequence I, a first
auxiliary polynucleotide (having a linker element nucleotide
sequence I and a hairpin forming polynucleotide), and the
sn2-casRNA.sup.EX3Csy-lnkCsy-AAVS1 comprised, 5' to 3', a second
auxiliary polynucleotide (having a hairpin forming polynucleotide
and a linker element nucleotide sequence II) and a split nexus stem
element nucleotide sequence II. Each of the two systems was used to
target cleavage of an AAVS-1 target, where the
sn1-casRNA.sup.ESC3sy-AAVS1 and sn1-casRNA.sup.EX3Csy-lnkCsy-AAVS1
each comprised a spacer complementary to the AAVS-1.
[0393] The double-stranded DNA templates for sn-casRNA.sup.EX-Cys
components used in this example were assembled by PCR using 3'
overlapping primers containing the corresponding DNA sequences to
the sn-casRNA.sup.EX3-Cys components. The oligonucleotides used in
the assemblies are presented in Table 17.
TABLE-US-00017 TABLE 17 Overlaping Primers for Generation of
sn-casRNA.sup.EX3-Cys Components Type of sn-casRNA.sup.EX3-Cys
Component Oligonucleotides* sn1-casRNA.sup.Ex3Csy-Csy-AAVS1 A, AK,
AL, AM sn2-casRNA.sup.EX3Csy-Csy A, AN, AO, AC
sn1-casRNA.sup.EX3Csy-lnkCsy-AAVS1 A, AK, AP, AQ
sn2-casRNA.sup.EX3Csy-lnkCsy A, AR, AS, AC *DNA primer sequences
are shown in FIG. 13
[0394] Generation of double-stranded DNA template for RNA
transcription was performed as described in Example 1. Transcribed
sn-casRNA.sup.EX3-Cys sequences are shown in Table 18.
TABLE-US-00018 TABLE 18 sn-casRNA Sequences Type of Cas9 RNA
Component RNA sequence SEQ ID NO sn1-casRNAs.sup.EX3Csy-Csy-AAVS1
5'-GGGGCCACUA GGGACAGGAU GUCUCAGAGC SEQ ID NO: 85 UAUGCAGUCC
UGGAAACAGG ACUGCAUAGC AAGUUGAGAU AAGGCUACUG CCGUAUAGGC AG-3'
sn2-casRNAs.sup.EX3Csy-Csy 5'-CUGCCGUAUA GGCAGGUCCG UUAUCAACUU SEQ
ID NO: 86 GAAAAAGUGG CACCGAGUCG GUGCUU-3'
sn1-casRNAs.sup.EX3Csy-InkCsy-AAVS1 5'-GGGGCCACUA GGGACAGGAU
GUCUCAGAGC SEQ ID NO: 87 UAUGCAGUCC UGGAAACAGG ACUGCAUAGC
AAGUUGAGAU AAGGCUAGAC ACUGCCCGUAU AGGCAG-3'
sn2-casRNAs.sup.EX3Csy-InkCsy 5'-CUGCCGUAUA GGCAGAGACA GUCCGUUAUC
SEQ ID NO: 88 AACUUGAAAA AGUGGCACCG AGUCGGUGCUU-3'
[0395] Target double-stranded DNA for use in the in vitro Cas9
cleavage assays was produced using PCR amplification as described
in Example 2. The forward and reverse primers used for
amplification from gDNA were as follows: AAVS-1, oligonucleotides H
and I (FIG. 13). The amplified double-stranded DNA target for
AAVS-1 was 495 bp. In vitro cleavage was performed as described in
Example 3.
[0396] FIG. 11 presents the results of the Cas9 cleavage assay
using the sn-casRNAs described above. In the figure, the cleavage
activity is shown at the bottom of each lane (except for lanes 1
and 10, which are molecular weight standards). For lanes indicated
as LOD, any cleavage activity was below the limit of detection. The
systems used in each of the Cas9 cleavage assay reactions were as
shown in Table 6 (see Brief Description of the Figures, FIG.
11).
[0397] As can be seen from the data presented in FIG. 11, both
sn1-casRNA.sup.EX3Csy-Csy-AAVS1 and sn2-casRNA.sup.EX3Csy-Csy (FIG.
11, lanes 2 and 3) or sn1-casRNA.sup.EX3Csy-lnkCsy-AAVS1 and
sn2-casRNA.sup.EX3Csy-lnkCsy are necessary for detectable cleavage
activity (FIG. 11, lanes 6 and 7). Furthermore, enhanced cleavage
was detectable when a linker element nucleotide sequence was
introduced between the split nexus element (FIG. 11, lane 8
compared to lane 4). Additionally, when Csy4* protein is introduced
enhanced cleavage is observed with
sn1-casRNA.sup.EX3Csy-lnkCsy-AAVS1 and sn2-casRNA.sup.EX3Csy-lnkCsy
(FIG. 11 lane 9 compared to lane 8), but not in the absence of the
linker sequences (sn1-casRNA.sup.EX3Csy-Csy-AAVS1 and
sn2-casRNA.sup.EX3Csy-Csy; FIG. 11 lane 5 compared to lane 4).
[0398] The data presented in FIG. 11 demonstrate that the
sn1-casRNA and sn2-casRNA constructs as described herein facilitate
the in vitro Cas9 mediated site-specific cleavage of a
double-stranded DNA target. These data support that the split-nexus
Cas9-associated polynucleotide systems of the present invention
facilitate in vivo Cas9-mediated site-specific cleavage of genomic
loci.
[0399] Following the guidance of the present specification and
examples, the Cas9 cleavage assay described in this example can be
practiced by one of ordinary skill in the art with other Type II
CRISPR Cas9 proteins including, but not limited to, Cas9 and Cas9
fusions combined with their cognate polynucleotide components
modified as described herein to comprise a split nexus element.
EXAMPLE 8
Identification and Screening of Trans-Activating CRISPR RNA
[0400] This example illustrates the method through which
trans-activating CRISPR RNAs (tracrRNAs) of species having
CRISPR-Cas9 Type II system may be identified. The method presented
here is adapted from Chylinski, et. al. ("The tracrRNA and Cas9
families of type II CRISPR-Cas immunity systems," RNA Biol. 2013
May; 10(5):726-37.). Not all of the following steps are required
for screening nor must the order of the steps be as presepted.
[0401] A. Identify a Species Containing a CRISPR-Cas9 Type-II
System
[0402] Using the Basic Local Alignment Search Tool (BLAST,
blast.ncbi.nlm.nih.gov/Blast.cgi), a search of various species'
genomes is conducted to identify Cas9 or Cas9-like proteins.
CRISPR-Cas9 system exhibit a high diversity in sequence across
species, however Cas9 orthologs exhibit conserved domain
architecture of central HNH endonuclease domain and a split
RuvC/RNase H domain. Primary BLAST results are filtered for
identified domains; incomplete or truncated sequences are discarded
and Cas9 orthologs identified.
[0403] When a Cas9 ortholog is identified in a species, sequences
adjacent to the Cas9 ortholog coding sequence are probed for other
Cas proteins and an associated repeat-spacer array to identify all
sequences belonging to the CRISPR-Cas9 locus. This may be done by
alignment to other CRISPR-Cas9 Type-II loci already known in the
public domain, with the knowledge that closely related species
exhibit similar CRISPR-Cas9 locus architecture (i.e., Cas protein
composition, size, orientation, location of array, location of
tracrRNA, etc.). The tracrRNA element is typically contained within
the CRISPR-Cas9 Type-II locus and is readily identified by its
sequence complementarity to the repeat elements in the
repeat-spacer array (tracr anti-repeat sequence).
[0404] Once the sequence of the CRISPR-Cas9 locus for the Cas9
ortholog is identified for the species, in silico predictive
screening is used to extract the anti-repeat sequence to identify
the associated tracrRNA. Putative anti-repeats are screened, for
example, as follows.
[0405] If the repeat sequence is from a known species, it is
identified in and retrieved from the CRISPRdb database
(crispr.u-psud.fr/crispr/). If the repeat sequence is not known to
be associated with a species, repeat sequences are predicted using
CRISPRfinder software (crispr.u-psud.fr/Server/) using the
CRISPR-Cas9 Type-II locus for the species as described above.
[0406] The identified repeat sequence for the species is used to
probe the CRISPR-Cas9 locus for the anti-repeat sequence (e.g.,
using the BLASTp algorithm or the like). The search is typically
restricted to intronic regions of the CRISPR-Cas9 locus.
[0407] An identified anti-repeat region is validated for
complementarity to the identified repeat sequence.
[0408] A putative anti-repeat region is probed both 5' and 3' of
the putative anti-repeat for a Rho-independent transcriptional
terminator (TransTerm HP, transterm.cbcb.umd.edu/).
[0409] Thus, the identified sequence comprising the anti-repeat
element and the Rho-independent transcriptional terminator is
determined to be the putative tracrRNA of the given species.
[0410] B. Preparation of RNA-Seq Library
[0411] The putative tracrRNA that was identified in silico is
further validated using RNA sequencing (RNAseq).
[0412] Cells from species from which the putative tracrRNA was
identified are procured from a commercial repository (e.g., ATCC,
Manassas Va.; DSMZ, Braunschweig, Germany).
[0413] Cells are grown to mid-log phase and total RNA prepped using
Trizol reagent (Sigma-Aldrich, St. Louis, Mo.) and treated with
DNaseI (Fermentas, Vilnius, Lithuania).
[0414] 10 ug of the total RNA is treated with Ribo-Zero rRNA
Removal Kit (Illumina, San Diego, Calif.) and the remaining RNA
purified using RNA Clean and Concentrators (Zymo Research, Irvine,
Calif.).
[0415] A library is then prepared using TruSeq Small RNA Library
Preparation Kit (Illumina, San Diego, Calif.) following the
manufacturer's instructions, which results in the presence of
adapter sequences associated with the cDNA.
[0416] The resulting cDNA library is sequenced using MiScq
Sequencer (Illumina, San Diego, Calif.).
[0417] C. Processing of Sequencing Data
[0418] Sequencing reads of the cDNA library are processed using the
following method.
[0419] Adapter sequences are removed using cutadapt 1.1
(pypi.python.org/pypi/cutadapt/1.1) and 15 nt are trimmed from the
3'end of the read to improve read quality.
[0420] Reads are aligned back to respective species' genome (from
which the putative tracrRNA was identified) with a mismatch
allowance of 2 nucleotides.
[0421] Read coverage is calculated using BedTools
(bedtools.readthedocs.org/en/latest/).
[0422] Integrative Genomics Viewer (TGV,
www.broadinstitute.org/igv/) is used to map the starting (5') and
ending (3') position of reads. Total reads retrieved for the
putative tracrRNA are calculated from the SAM file of
alignments.
[0423] The RNA-seq data is used to validate that a putative
tracrRNA element is actively transcribed in vivo. Confirmed hits
from the composite of the in silico and RNA-scq screens are
validated for functional ability of the identified tracrRNA
sequence and its cognate crRNA to support Cas9 mediated cleavage of
a double-stranded DNA target using methods outline herein (see
Examples 1, 2, and 3).
[0424] Following the guidance of the present specification and the
examples herein, the identification of novel tracrRNA sequences can
be practiced by one of ordinary skill in the art.
EXAMPLE 9
T7E1 Assay for Detection of Target Modifications in Eukaryotic
Cells
[0425] This example illustrates the use of T7E1 assays to evaluate
and compare the percent cleavage in vivo of sn-casPNs/Cas9 systems
relative to selected double-stranded DNA target sequences.
[0426] A. Cell Transfections Using Cas Polynucleotide
Components
[0427] sn-casPNs are transfected into HEK293 cells constitutively
expressing SpyCas9-GFP fusion (HEK293-Cas9-GFP), using the
Nucicofector.RTM. 96-well Shuttle System (Lonza, Allendale, N.J.)
and the following protocol. Equal molar amounts of Cas
polynucleotide components are prepared in an annealing buffer (1.25
mM HEPES, 0.625 mM MgCl.sub.2, 9.375 mM KCl at pH 7.5), are
incubated for 2 minutes at 95.degree. C., are removed from
thermocycler, allowed to equilibrate to room temperature, and
dispensed in a 10 .mu.L final volume in a 96-well plate. Culture
medium is aspirated from HEK293-Cas9-GFP cells, and the cells are
washed once with calcium and magnesium-free PBS then are
trypsinized by the addition of TrypLE (Life Technologies, Grand
Island, N.Y.) followed by incubation at 37.degree. C. for 3-5
minutes. Trypsinized cells are gently pipetted up and down to form
a single cell suspension and added to DMEM complete culture medium
composed of DMEM culture medium (Life Technologies, Grand Island,
N.Y.) containing 10% FBS (Fisher Scientific, Pittsburgh, Pa.) and
supplemented with penicillin and streptomycin (Life Technologies,
Grand Island, N.Y.).
[0428] The cells are then pelleted by centrifugation for 3 minutes
at 200.times.g, the culture medium aspirated and cells are
resuspended in PBS. The cells are counted using the Countess.RTM. H
Automated Cell Counter (Life Technologies, Grand Island, N.Y.).
2.2.times.10.sup.7 cells are transferred to a 50 ml tube and
pelleted. The PBS is aspirated and the cells are resuspended in
Nucleofector.TM. SF (Lonza, Allendale, N.J.) solution to a density
of 1.times.10.sup.7 cells/mL. 20 .mu.L of the cell suspension are
then added to individual wells containing 10 uL of Cas
polynucleotide components and the entire volume is transferred to
the wells of a 96-well Nucleocuvette.TM. Plate (Lonza, Allendale,
N.J.). The plate is loaded onto the Nucleofector.TM. 96-well
Shuttle.TM. (Lonza, Allendale, N.J.) and cells are nucleofected
using the 96-CM-130 Nucleofector.TM. program (Lonza, Allendale,
N.J.). Post-nucleofection, 70 .mu.L DMEM complete culture medium is
added to each well and 50 .mu.L of the cell suspension are
transferred to a collagen coated 96-well cell culture plate
containing 150 .mu.L pre-warmed DMEM complete culture medium. The
plate is then transferred to a tissue culture incubator and
maintained at 37.degree. C. in 5% CO.sub.2 for 48 hours.
[0429] B. Target Double-Stranded DNA Generation for T7E1 Assay
[0430] gDNA is isolated from HEK-293-SpyCas9 cells 48 hours after
Cas polynucleotide component transfection using 50 .mu.L
QuickExtract DNA Extraction solution (Epicentre, Madison, Wis.) per
well followed by incubation at 37.degree. C. for 10 minutes,
65.degree. C. for 6 minutes and 95.degree. C. for 3 minutes to stop
the reaction. gDNA is then diluted with 150 .mu.L water and samples
are stored at -80.degree. C.
[0431] DNA for T7E1 is generated by PCR amplification of a target
double-stranded DNA sequence (e.g., AAVS-1) from isolated gDNA. PCR
reactions are set up using 8 mL gDNA as template with KAPA HiFi Hot
Start polymerase and containing 0.5 U of polymerase, 1.times.
reaction buffer, 0.4 mM dNTPs and 300 nM forward and reverse
primers directed to the target double-stranded DNA (e.g., AAVS-1,
oligonucleotides K and L (FIG. 4)) in a total volume of 25 mL.
Target DNA is amplified using the following conditions: 95.degree.
C. for 5 minutes, 4 cycles of 20 s at 98.degree. C., 20 s at
70.degree. C., minus 2.degree. C/cycle, 30 s at 72.degree. C.,
followed by 30 cycles of 15 s at 98.degree. C., 20 s at 62.degree.
C., 20 s at 72.degree. C., and a final extension at 72.degree. C.
for 1 minute.
[0432] C. T7E1 Assay
[0433] PCR amplified target double-stranded DNA for T7E1 assays is
denatured at 95.degree. C. for 10 minutes and then allowed to
re-anneal by cooling to 25.degree. C. at -0.5.degree. C/s in a
thermal cycler. The re-annealed DNA is incubated with 0.5 mL T7
Endonuclease 1 in 1.times. NEBuffer 2 buffer (New England Biolabs,
Ipswich, Mass.) in a total volume of 15 mL for 25 minutes at
37.degree. C. T7E1 reactions are analyzed using the Fragment
Analyzer.TM. System (Advanced Analytical Technologies, Inc., Ames,
Iowa) and the DNF-910 Double-stranded DNA Reagent Kit (Advanced
Analytical Technologies, Inc., Ames, Iowa). The Fragment
Analyzer.TM. System provides the concentration of each cleavage
fragment and of the target double-stranded DNA that remains after
cleavage.
[0434] Cleavage percentages of the target double-stranded DNA are
calculated from the concentration of each cleavage fragment and the
target double-stranded DNA, which remains after cleavage has take
place, using the following formula:
% cleavage = ( 1 - ( 1 - ( frag 1 + frag 2 ) ( frag 1 + frag 2 +
parent ) ) ) EQUATION 1 ##EQU00001##
[0435] In Equation 1, "frag1" and "frag2" concentrations correspond
to the concentration of Cas9 cleavage fragments of the
double-stranded DNA target and "parent" corresponds to the target
double-stranded DNA that remains after cleavage has take place.
[0436] The T7E1 assay for detection of target modifications in
eukaryotic cells provides data to demonstrate that the
sn-casPNs/Cas9 systems as described herein facilitate Cas9-mediated
site-specific in vivo cleavage of target double-stranded DNA. sgRNA
and/or tracrRNA/crRNA polynucleotides having the same DNA target
binding sequence as the sn-casPNs can also be included in the assay
to compare the Cas9-mediated site-specific cleavage percentages
between the constructs.
[0437] Following the guidance of the present specification and
examples, the T7E1 assay described in this example can be practiced
by one of ordinary skill in the art with other Type II CRISPR Cas9
proteins including, but not limited to, Cas9 and Cas9 fusions
combined with their cognate polynucleotide components modified as
described herein to comprise a split nexus element.
EXAMPLE 10
Split Nexus Testing of Identified tracrRNAs
[0438] This example describes the generation and testing of split
nexus modifications in tracrRNAs, for example, based on
crRNA/tracrRNAs know in the art or identified by methods described
in Example 8.
[0439] A tracrRNA sequence and its cognate crRNA sequences are
joined, with the crRNA sequence placed 5' of the tracrRNA sequence
maintaining 5' to 3' polarity, with a linker sequence to generate a
sgRNA. A suitable linker sequence is 5'-GAAA-3'.
[0440] The sgRNA is analyzed for secondary structural motifs using
publically available RNA folding software. One such software is
RNAstnicture
(rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1/Predict1.html).
[0441] The secondary structures of the sgRNA are analyzed for
secondary structure similar to known sgRNA that support Cas9
directed cleavage activity, traditionally comprising, in a 5' to 3'
direction, a first stem element, a hairpin element that comprises a
second stem clement (herein referred to as a nexus clement), and
zero, one, or two, hairpin elements 3' of the nexus element.
[0442] The sgRNA is then split at the nexus element into at least
two polynucleotides: a first polynucleotide (e.g., a sn1-casPN,
FIG. 3B) comprising in a 5' to 3' direction a selected DNA
targeting binding sequence, the first stem element, and first
portion of the nexus (i.e., a split nexus stem element nucleotide
sequence I); and a second polynucleotide (e.g., a sn2-casPN, FIG.
3B) comprising in a 5' to 3' direction a second portion of the
nexus (i.e., a split nexus stem element nucleotide sequence II),
and the zero, one, or two 3' hairpins.
[0443] A library of first polynucleotide sequences and second
polynucleotide sequences is constructed, using method describe in
Example 1 of the present specification, wherein a split in the
nexus of the sgRNA is made at each nucleotide position of the
sequence comprising the native nexus.
[0444] The library is then tested for the ability of each split
nexus first polynucleotide sequence and its cognate split nexus
second polynucleotide sequence to support Cas9 mediated cleavage of
a selected double-stranded DNA target following the methods
described in Example 2 through 4 of the present specification.
[0445] Putative split nexus arrangements of known tracrRNA
sequences from various species are shown in FIG. 12. In the figure,
the first column is an identifying number for the bacterial species
(see Table 7, Brief Description of the Figures), the second column
is the sequence of the sn1-casRNA/sn2-casRNA. A split nexus of a S.
pyogenes sn1-casRNA/sn2-casRNA of the present invention is shown
for reference (FIG. 12, row 1).
[0446] It is known that a single species can have more than one
CRISPR locus of the same Type, or more than one CRISPR locus of
different Types (e.g., Type-I and Type-II). Typically repeat
elements of one CRISPR locus are only usable to identify the
anti-repeat element (and therefore the tracrRNA sequences)
contained within the same CRISPR locus.
[0447] Following the guidance of the present specification and
examples, the testing described in this example can be practiced by
one of ordinary skill in the art with other Type II CRISPR Cas9
proteins including, but not limited to, Cas9 and Cas9 fusions
combined with their cognate polynucleotide components modified as
described herein to comprise a split nexus clement.
EXAMPLE 11
Screening of Multiple sn-casRNAs Comprising DNA Target-Binding
Sequences
[0448] This example illustrates the use of sn-casRNAs of the
present invention to modify targets present in human genomic DNA
and measure the level of cleavage activity at those sites. Target
sites are first selected from genomic DNA and then sn-casRNAs are
designed to target those selected sequences. Measurements are then
carried out to determine the level of target cleavage that has
taken place. Not all of the following steps are required for every
screening nor must the order of the steps be as presented, and the
screening can be coupled to other experiments, or form part of a
larger experiment.
[0449] A. Select a DNA Target Region from Genomic DNA
[0450] Identify all PAM sequences (e.g. `NGG`) within the selected
genomic region.
[0451] Identify and select one or more 20 nucleotide sequence long
sequences (target DNA sequence) that is 5' adjacent to PAM
sequences.
[0452] Selection criteria can include but are not limited to:
homology to other regions in the genome; percent G-C content;
melting temperature; presences of homopolymer within the spacer;
and other criteria known to one skilled in the art.
[0453] Append an appropriate sn-casRNA sequence (e.g., an
sn1-casRNA, as illustrated in FIG. 3B, with the spacer sequence
removed) to the 3' end of the identified target DNA sequence
(sn-casRNA-DNAtbs (DNA target binding sequence)). A
sn-casRNA-DNAtbs construct is typically synthesized by a commercial
manufacturer or produced as described in Example 1 by in vitro
transcription.
[0454] A sn-casRNA-DNAtbs as described herein is used with cognate
sn-casRNA(s) to complete a sn-casRNA system (e.g., a
sn1-casRNA-DNAtbs/sn2-casRNA two polynucleotide split nexus system)
for use with a cognate Cas protein.
[0455] B. Determination of Cleavage Percentages and Specificity
[0456] In vitro cleavage percentages and specificity associated
with a sn-casRNA-DNAtbs/sn-casRNA(s) system are compared, for
example, using the Cas9 cleavage assays of Example 3, as
follows:
[0457] (a) If only a single target DNA sequence is identified or
selected, the cleavage percentage and specificity for the DNA
target region is determined. If so desired, cleavage percentage
and/or specificity are altered in further experiments using methods
of the present invention including but not limited to modifying the
RNA, introducing effector proteins/effector protein-binding
sequences or ligand/ligand binding moieties.
[0458] (b) The percentage cleavage data and site-specificity data
obtained from the cleavage assays is compared between different
DNAs comprising the target binding sequence to identify the target
DNA sequences having the best cleavage percentage and highest
specificity. Cleavage percentage data and specificity data provide
criteria on which to base choices for a variety of applications.
For example, in some situations the activity of the sn-casRNA may
be the most important factor. In other situations, the specificity
of the cleavage site may be relatively more important than the
cleavage percentage. If so desired, cleavage percentage and/or
specificity are altered in further experiments using methods of the
present invention including but not limited to modifying the RNA,
introducing effector proteins/effector protein-binding sequences or
ligand/ligand binding moieties.
[0459] Optionally, or instead of, the in vitro analysis, in vivo
cleavage percentages and specificity associated with a
sn-casRNA-DNAtbs/sn-casRNA(s) system are compared, for example,
using Deep Sequencing Analysis for Detection of Target
Modifications in Eukaryotic Cells of Example 5, as follows:
[0460] (a) If only a target DNA sequence is identified the cleavage
percentage and specificity for the DNA target region is determined.
If so desired, cleavage percentage and/or specificity are altered
in further experiments using methods of the present invention
including but not limited to modifying the RNA, introducing
effector proteins/effector protein-binding sequences or
ligand/ligand binding moieties.
[0461] (b) The percentage cleavage data and site specificity data
obtained from the cleavage assays is compared between different
target DNAs to identify the sn-casRNA sequences that resulting the
highest percentage cleavage or target DNA and the highest
specificity for the target DNA. Cleavage percentage data and
specificity data provide criteria on which to base choices for a
variety of applications. For example, in some situations the
activity of the sn-casRNA may be the most important factor. In
other situations, the specificity of the cleavage site may be
relatively more important than the cleavage percentage. If so
desired, cleavage percentage and/or specificity are altered in
further experiments using methods of the present invention
including but not limited to modifying the RNA, introducing
effector proteins/effector protein-binding sequences or
ligand/ligand binding moieties.
[0462] Following the guidance of the present specification and
examples, the screening described in this example can be practiced
by one of ordinary skill in the art with other Type II CRISPR Cas9
proteins including, but not limited to, Cas9 and Cas9 fusions
combined with their cognate polynucleotide components modified as
described herein to comprise a split nexus element.
EXAMPLE 12
Functional Genomics Screening
[0463] This example describes use of the split-nexus
Cas9-associated polynucleotides (sn-casPNs) of the present
invention for identification of the functional role of genes
utilizing a functional screening method and sequence data.
[0464] A two-part sn-casRNA (sn1-casRNA and sn2-casRNA) system
(see, e.g., FIG. 3B) is used in a modification of the methods
described in Shalem et al. ("Genome-scale CRISPR-Cas9 knockout
screening in human cells," Science. 2014 Jan. 3; 343(6166):84-7.)
and Zhou et al. ("High-throughput screening of a CRISPR/Cas9
library for functional genomics in human cells", Nature 509, 2014
May 22, 487-491) which used a single guide RNA having a continuous
sequence. The screen described herein is designed around the
vulnerability of the A375 melanoma cell line to the drug
vemerafenib; when treated with vemurafenib, cells growth is
arrested. A375 cells are transduced with a library of sn1-casRNA,
and these cells are subsequently treated with vemerafenib.
sn1-casRNA knockout of genes important for A375 sensitivity to
vemerafenib will be enriched in the surviving cell population and
can be sequenced and identified.
[0465] Examples of suitable vectors, media, culture conditions,
etc. are described. Modifications of these components and
conditions will be understood by one of ordinary skill in the art
in view of the teachings of the present specification.
[0466] A. Lentiviral Library and Cas9 Constructs
[0467] A viral library of sn1-casRNAs is generated by synthesizing
oligonucleotides containing the designed spacer sequences appended
to universal tag sequences for cloning into a transfer plasmid for
lentivirus production (e.g. pD2107-CMV-DNA 2.0, Menlo Park,
Calif.). Oligonucleotide libraries are synthesized on programmable
microarrays and cleaved from the microarray by the array
manufacturer (e.g. Agilent technologies, Santa Clara, Calif.).
Full-length oligonucleotides are amplified by PCR using Q5
polymerase (NEB) and primers designed to amplify DNA containing the
universal tag sequences. Cloning into the transfer vector is
carried out using standard techniques known to one skilled in the
art. One example includes digesting the vector with a Type II
restriction enzyme (e.g. BsbI) to reveal single-stranded overhangs,
treating with alkaline phosphatase (Fermentas) and purifying the
cut vector from uncut by gel purification. Oligonucleotide
libraries are digested with a restriction enzyme to reveal
compatible ends, and ligated into the vector using DNA ligase
(Fermentas).
[0468] The transfer vector can include a human codon optimized S.
pyogenes Cas9 gene N-terminally and C-terminally tagged with a SV40
nuclear localization signal under the control of the elongation
factor-1.alpha. short promoter (EFS) promoter. This NLS-Cas9-NLS
sequence is joined to a 2A self-cleaving peptide and a selection
maker suitable for mammalian cells (i.e. puromycin).
[0469] Alternatively, Cas9 can be delivered to the cells in a
separate viral vector, or stable cell-lines can be generated that
express Cas9 constitutively. Viral vector-expressed sn1-casRNA
libraries can then be used to transduce the Cas9-expressing cell
lines.
[0470] B. Lentivirus Production and Purification
[0471] HEK293T cells are seeded at approximately 40% confluence 24
hours before transfection in DMEM (Life Technologies, Grand Island,
N.Y.) supplemented with 10% fetal bovine serum (FBS). Cells are
transferred into reduced serum OptiMEM (Life Technologies, Grand
Island, N.Y.) and transfected using Lipofectamine 2000 and Plus
reagent according to manufacturer's instructions. For transfection,
the lentiviral transfer vector is combined with plasmids for
lentiviral packaging such as the LentiX.TM. HTX Packaging System
(Takara Clontech, Mountain View, Calif.) according to
manufacturer's instructions.
[0472] After 60 hours, media is removed and centrifuged at 3000 rpm
to remove cell debris. Supernatant is filtered through a 0.45 um
low protein binding membrane (e.g. Millipore Steriflip HV/PVDF).
The pooled library can be concentrated by ultracentrifugation and
then resuspended in DMEM supplemented with 10% FBS and 1% BSA
(Sigma-Aldrich, St. Louis, Mo.).
[0473] C. Cell Culture
[0474] A375 (ATCC CRL-1619) cells are obtained from ATCC (Manassas,
Va.) and cultured in R8758 medium (Sigma-Aldrich, St. Louis, Mo.),
supplemented with 10% FBS (Life Technologies, Grand Island, N.Y.),
1% Penicillin-Streptomycin (Sigma-Aldrich, St. Louis, Mo.), 20 mM
HEPES (Sigma-Aldrich, St. Louis, Mo.).
[0475] D. Lentiviral Transduction
[0476] Multiplicity of Infection (MOI) for the viral vector library
is determined using standard methods based upon transduction of
cells with predetermined virus volumes. Approximately
3.times.10.sup.6 A375 cells, are plated per well on a 12 well plate
in appropriate media supplemented with 8 mg/ml polybrene
(Sigma-Aldrich, St. Louis, Mo.). Cells are mixed with the
predetermined virus volume to identify a multiplicity of infection
(MOI) of between 0.3-0.5. Plated cells are centrifuged at 2,000 rpm
for 2 hours at 37.degree. C., after which the media is aspirated
and fresh media for each cell type is added, without polybrene.
Cells are incubated for 24 hours at 37.degree. C., 5% CO2. A
non-transduced control is included.
[0477] After 24 hours, cells arc detached and counted,
approximately 2.5.times.10.sup.6 cells are re-plated into both a
`selection well` and a `non-selection well`. Selection wells are
put under selection specific to the lentiviral library construct
(i.e. puromycin, Sigma-Aldrich, St. Louis, Mo.). Non-selection
wells are not treated with puromycin. Cells are incubated until no
surviving cells placed under selection in the non-transduced
control remained. Cells are counted, and the number of cells in
`selection wells` divided by the number of cells in the
corresponding `non-selection wells` multiplied by 100 yields the
MOI, with a MOI close to 0.4 being the ideal value.
[0478] E. Drug Resistance Screen
[0479] Cells are plated into wells of 2.times.10.sup.6 cells per
well for each condition to be tested. The cells in each well are
transduced with 1 Oul of the library to reach a transduction
efficiency of 30% (minimum of 3-400 cells per clone in the
library). Puromycin is added to the wells 24 hours post
transduction and the cells are maintained for 7 days. Cells are
split into drug conditions in duplicate with a minimum of
2.times.10.sup.7 cells per replicate well. One well is supplemented
with 2 uM drug compound (e.g. PLX4032, Thermo Fisher Scientific,
South San Francisco, Calif.) and the other with DMSO (Thermo Fisher
Scientific, South San Francisco, Calif.). Cells are incubated at
37.degree. C., 5% CO2 for 14 days, and passaged every 2-3 days in
to fresh media, supplemented with either PLX4032 or DMSO as
appropriate. After 14 days, genomic DNA (gDNA) is prepared from
cells using the QuickExtract DNA extraction solution (Illumina, San
Diego, Calif.) as per manufacturer instructions.
[0480] F. gDNA Sequencing
[0481] PCR primers are designed to amplify lentiviral sn1-casPN
target sequences from genomic DNA. Using isolated gDNA, a first PCR
is performed using Herculase II Fusion DNA Polymerase (Agilent,
Santa Clara, Calif.) with primers comprising an adapter sequences
and a sequence specific to the lentiviral sn1-casPN cassette. A
second PCR is performed using the amplicons of the first round as
template at 1/20.sup.th the volume of the second PCR reaction
volume. The second PCR uses a second set of primers comprising:
sequence complementary to the universal adapter sequence of the
first primer pair, a barcode index sequence unique to each sample,
and a flow cell adapter sequence. PCR reactions are pooled to
ensure a 300.times. sequencing coverage of each transduced sample.
Pooled PCR reactions are analyzed on a 2% TBE gel, bands of
expected amplicon sizes are gel purified using the QIAEX II Gel
Extraction Kit (Qiagen, Venlo, Limburg). The concentrations of
purified amplicons are evaluated using the Double-strand DNA BR
Assay Kit and Qubit System (Life Technologies, Grand Island, N.Y.)
and library quality determined using the Agilent DNA100Chip and
Agilent Bioanalyzer 2100 System (Agilent, Santa Clara, Calif.).
Pooled library are sequenced on a MiSeq 2500 (Illumina, San Diego,
Calif.).
[0482] G. Processing and Analysis of Sequencing Data
[0483] Raw sequencing reads are processed to only contain the
sn1-casPN cassette sequence. sn1-casPN reads are aligned to the
target sequences contained within the lentiviral screening library
and the number of reads for each unique target sequence are
counted. Counted reads per target sequence are normalized by
dividing the reads per target by total aligned reads for all
targets in the sample and multiplying by 10.sup.6 and adding 1.
[0484] Normalized target reads identified in drug-treated samples
are compared to normalize targets reads identified in the DMSO
control treated samples. Targets with high read count present in
the drug treated sample that are absent or reduced in the DMSO
control treated samples can be further evaluated as candidate
genes, important in resistance to drug treatment.
[0485] Other functional genomic screens using a sn1-casRNA library
and the method of screening outlined here can be used to identify
candidate genes important in to those screens.
[0486] This procedure provides data to verify that the Cas9
sn1-casRNA/sn2-casRNA system of the present invention can be used
in functional screening to interrogate gene-function on a
genome-wide scale.
[0487] Following the guidance of the present specification and
examples, the screening described in this example can be practiced
by one of ordinary skill in the art with other Type II CRISPR Cas9
proteins including, but not limited to, Cas9 and Cas9 fusions
combined with their cognate polynucleotide components modified as
described herein to comprise a split nexus element.
EXAMPLE 13
Repression/Activation
[0488] This example describes use of the split-nexus
Cas9-associated polynucleotides (sn-casPNs) of the present
invention for the repression or activation of endogenous genes in
human cells.
[0489] A two-part sn-casRNA (sn1-casRNA and sn2-casRNA) system
(see, e.g., FIG. 3) is used in a modification of the methods
described in Gilbert et al. (CRISPR-Mediated Modular RNA-Guided
Regulation of Transcription in Eukaryotes," Cell. 2013 Jul. 18;
154(2):442-51. doi: 10.1016/j.cell.2013.06.044.) which used a
single guide RNA having a continuous sequence.
[0490] Examples of suitable vectors, media, culture conditions,
etc. are described. Modifications of these components and
conditions will be understood by one of ordinary skill in the art
in view of the teachings of the present specification.
[0491] A. dCas9 Activator and Repressor Constructs
[0492] The nuclease deficient S. pyogenes Cas9 (dCas9) with
mutation D10A and H840A is codon optimized for expression in
mammalian cells and C-terminally tagged with a SV40 nuclear
localization signal and either the Kruppel associated box (KRAB)
repression domain (dCas9-KRAB) or four copies of the
transcriptional activator VP16 (dCas9-VP64). Both the dCas9-KRAB
and dCas9-VP64 are inserted into a vector adjacent to a suitable
mammalian promoter, such as the cytomegalovirus (CMV) promoter. One
such vector, pJ607-03 (DNA2.0, Menlo Park, Calif.), is commercially
available.
[0493] B. sn-casPN Construction
[0494] The sn1-casRNA-CD71 sequence comprises a 20 nucleotide
spacer sequence targeted toward the upstream untranslated region
(UTR) of the of the transferrin receptor CD71. The sn1-casRNA-CD71
sequence is assembled into a suitable vector also comprising the
independent sn2-casRNA sequence. Each sequence is under independent
control by a human U6 promoter that directs transcription by RNA
polymerase III. Once suitable vector backbone for the expression of
sn 1-casRNA and sn2-casRNA sequences is the pRSFDuet-1 vector
(Novagen, Merck, Darmstadt, Germany).
[0495] C. Cell Culture
[0496] HeLa (ATCC CCL-2) cells are obtained from ATCC (Manassas,
Va.) and cultured in Dulbecco's modified Eagle medium (DMEM, Life
Technologies, Grand Island, N.Y.), supplemented with 10% FBS (Life
Technologies, Grand Island, N.Y.), 1% Penicillin-Streptomycin
(Sigma-Aldrich, St. Louis, Mo.), 2 mM glutamine (Life Technologies,
Grand Island, N.Y.) and cultured at 37.degree. C., 5% CO2.
[0497] D. Transfection and FACS Sorting
[0498] HeLa cells are transiently transfected with equal weight
Cas9-containing plasmid and sn1-casRNA-CD71 vector using
TransIT-LT1 (Mirus, Madison, Wis.). A non-transfected control is
included. 72 hours after transfection cells are trypsinized (Life
Technologies, South San Francisco, Calif.) and dissociated with 10
nM EDTA-PBS (Lonza, Basel, Switzerland). Cells are incubated in the
presents of an anti-human CD71-specific antibody conjugated to a
FITC fluorophore (eBiosceince, San Diego, Calif.) in Flow Cytometry
Staining Buffer (eBiosceince, San Diego, Calif.).
Fluorescence-activated cell sorting (FACS) of transfected cells is
preformed using the using blue laser (excitation 488 nm) and the
LSR II flow cytometer (BD Biosciences, Franklin Lakes, N.J.) for
detection of the CD71-FITC antibody.
[0499] Activation of CD71 expression in dCas9-VP64 transfected
samples is measured by the increase in detected fluorescence (a.u.
Log.sub.10) compared to the measured fluorescence of a
non-transfected control population of HeLa cells as detected by
FACS sorting.
[0500] Repression of CD71 expression in dCas9-KRAB transfected
samples is measured by the decrease in detected fluorescence (a.u.
Log.sub.10) compared to the measured fluorescence of a
non-transfected control population of HeLa cells as detected by
FACS sorting.
[0501] Other genes are similarly activated or repressed using the
sn-casPN of the present invention and the methods outlined here. As
apparent to one skilled in the art, other activation and repression
domain can be fused to a dCas9 to achieve a similar result to the
methods describe here.
[0502] This procedure provides data to verify that the Cas9
sn1-casRNA/sn2-casRNA system of the present invention can be used
in the activation of repression of endogenous genes.
[0503] Following the guidance of the present specification and
examples, the repression/activation assays described in this
example can be practiced by one of ordinary skill in the art with
other Type II CRISPR Cas9 proteins including, but not limited to,
Cas9 and Cas9 fusions combined with their cognate polynucleotide
components modified as described herein to comprise a split nexus
element.
EXAMPLE 14
Modification of CHO Cells for Industrial Application
[0504] This example describes use of the split-nexus
Cas9-associated polynucleotides (sn-casPNs) of the present
invention for modifying the genome of a Chinese Hamster Ovary cell
(CHO cell). Also contained in this example is an outline for the
sequence validation and selection of sn-casPN modified cells for
future uses in industrial applications (i.e. production of
antibodies).
[0505] A two-part sn-casRNA (sn1-casRNA and sn2-casRNA) system
(see, e.g., FIG. 3) is used in a modification of the method
described in Ronda et al. ("Accelerating genome editing in CHO
cells using CRISPR Cas9 and CRISPy, a web-based target finding
tool," Biotechnology and Bioengineering. Volume 111, Issue 8, pages
1604-1616, August 2014), which used a single guide RNA having a
continuous sequence.
[0506] Examples of suitable vectors, media, culture conditions,
etc. are described. Modifications of these components and
conditions will be understood by one of ordinary skill in the art
in view of the teachings of the present specification.
[0507] A. Plasmid Construction
[0508] The S. pyogenes Cas9 sequence is codon optimized for
expression in CHO cells and C-terminally tagged with a SV40 nuclear
localization signal and inserted into a vector adjacent to a
suitable mammalian promoter, such as the cytomegalovirus (CMV)
promoter. One such vector, pJ607-03 (DNA2.0, Menlo Park, Calif.),
is commercially available.
[0509] The sn1-casRNA-FUT8 sequence comprises a 20 nucleotide FUT8
spacer sequence. The sn1-casRNA-FUT8 sequence is assembled into a
suitable vector also comprising the independent sn2-casRNA
sequence. Each sequence is under independent control by a U6
promoter that directs transcription by RNA polymerase III. One
suitable vector backbone for the expression of sn1-casRNA and
sn2-casRNA sequences is the pRSFDuet-1 vector (Novagen, Merck,
Darmstadt, Germany).
[0510] B. Cell Culture
[0511] CHO-Ki cells are obtained from ATCC (Manassas, Virginia) and
cultured in CHO-Ki F-12K medium (ATCC, Manassas, Va.), 10% FBS
(Life Technologies, Grand Island, N.Y.) and 1%
Penicillin-Streptomycin (Sigma-Aldrich, St. Louis, Mo.). CHO-Ki
cells are transfected with equal weight Cas9 containing plasmid and
sn1-casRNA-FUT8/sn2-casRNA comprising vector using the Nucleofector
2b Device (Lonza, Basel, Switzerland) and the Amaxa Cell line
Nucleofector Kit V (Lonza, Basel, Switzerland) as per the
manufacturers recommendations. Cells are incubated at 30.degree. C.
in 5% CO2 for the first 24 hours and then moved to 37.degree. C.,
5% CO2 for another 24 hour periods.
[0512] C. Selection of FUT8 Knockout Cells
[0513] FUT8 knockout cells are selected by the addition of 50
.mu.g/mL Lens culinaris agglutinin (LCA, Vector Laboratories,
Burlingame, Calif.) five days after transfection of Cas9 vector and
the sn1-casRNA-FUT8. Cells are subject to 7 days of selection on
LCA, cells are passaged and fresh medium added, with LCA, every 2-3
days or as necessary. Only cells that have disruptions in the Fut8
gene, caused by the Cas9 sn-casPN system, will have resistance to
the LCA.
[0514] To confirm FUT8 knockout, selected cells are re-seeded into
complete media without LCA, and incubated for 48 hours. After
re-seeding, genomic DNA (gDNA) is prepped using the QuickExtract
DNA extraction solution (Illumina, San Diego, Calif.) as per
manufacturer instructions.
[0515] D. Sequence Validation of Cas9 Modification & Myseq
Library Construction
[0516] Sequencing amplicons of between 150 bp-200 bp are designed
to span the sn1-casRNA-FUT8 target site. Using previously isolated
gDNA, a first PCR is performed using Herculase II Fusion DNA
Polymerase (Agilent, Santa Clara, Calif.) with primers comprising
an adapter sequences and a sequence specific to the region flanking
the FUT8 target site. A second PCR is performed using the amplicons
of the first round of PCR as template at 1/20.sup.th the volume of
the PCR reaction volume. The second PCR uses a second set of
primers comprising: sequence complementary to the adapter sequence
of the first primer pair, a barcode index sequence unique to a each
sample, and a flow cell adapter sequence. Amplicons are pooled and
analyzed on a 2% TBE gel, bands of expected amplicon sizes are gel
purified using the QIAEX II Gel Extraction Kit (Qiagen, Venlo,
Netherlands). The concentrations of purified amplicons are
evaluated using the Double-strand DNA BR Assay Kit and Qubit System
(Life Technologies, Grand Island, N.Y.) and library quality
determined using the Agilent DNA100Chip and Agilent Bioanalyzer
2100 System (Agilent, Santa Clara, Calif.). After validation of
library quality, the library is sequenced on a MiSeq Benchtop
Sequencer (Illumina, San Diego, Calif.) with the MiSeq Reagent Kit
v2 (300 cycles, Illumina, San Diego, Calif.) per manufacturer
instructions for 151 bp paired end reads.
[0517] E. Deep Sequencing Data Analysis
[0518] The identity of products in the sequencing data is analyzed
based upon the index barcode sequence adapted onto the amplicon in
the second round of PCR. A computational script is used to process
the MiSeq data by executing the following tasks:
[0519] 1. Joining of paired end reads with the aid of fastq-join
(Aronesty 2011: code.google.com/p/ea-utils)
[0520] 2. Validation of the sequence reads for appropriate primer
sequences being present at both 5' and 3' ends of the read sequence
using fastx_barcode_splitter
(hannonlab.cshl.edu/fastx_toolkit/index.html). Reads lacking
correct primer sequences at both ends arc discarded.
[0521] 3. Compare Read sequences to expected wild type FUT8
sequence, identical read sequences are classified as having the
same indel modification.
[0522] Other chromosomal loci within CHO cells are similarly
modified by selection of appropriate spacer sequence for the
sn1-casRNA. Selection is specific to a specific gene target and the
procedure outlined in this example is readily modifiable by one of
ordinary skill in the art for other gene targets.
[0523] This procedure provides data to verify the Cas9
sn1-casRNA/sn2-casRNA system of the present invention provides
sequence specific RNA-directed endonuclease activity at targeted
loci in CHO cell and outlines the methods for selection of said
modified CHO cells for continued use.
[0524] Following the guidance of the present specification and
examples, the assay described in this example can be practiced by
one of ordinary skill in the art with other Type II CRISPR Cas9
proteins including, but not limited to, Cas9 and Cas9 fusions
combined with their cognate polynucleotide components modified as
described herein to comprise a split nexus element.
EXAMPLE 15
Genome Engineering in Saccharomyces cerevisiae
[0525] This example describes use of the split-nexus
Cas9-associated polynucleotides (sn-casPNs) of the present
invention for modifying the genome of the yeast S. cerevisiae.
[0526] A two-part sn-casRNA (sn1-casRNA and sn2-casRNA) system
(see, e.g., FIG. 3) is used in a modification of the method of
DiCarlo, et al. ("Genome engineering in Saccharomyces cerevisiae
using CRISPR-Cas systems," Nucleic Acids Res. 2013 April; 41(7):
4336-4343), which used a single guide RNA having a continuous
sequence.
[0527] Examples of suitable vectors, media, culture conditions,
etc. are described. Modifications of these components and
conditions will be understood by one of ordinary skill in the art
in view of the teachings of the present specification.
[0528] A. Site-Specific Genomic Mutations
[0529] A Streptococcus pyogenes Cas9 gene, codon-optimized for
expression in yeast cells, is C-terminally tagged with a SV40
nuclear localization signal and inserted into a low copy number
vector adjacent an inducible promoter, for example, GalL promoter
sequences. The vector also contains a selectable marker, such as a
URA3 selectable marker. One such vector, p415-GalL-Cas9-CYC1t
(Addgene, Cambridge, Mass.), is commercially available. Expression
of the Cas9 gene is under the inducible control of the GalL
promoter.
[0530] The sn1-casRNA-CAN1.Y sequence comprises a 20 nucleotide
CAN1.Y spacer sequence. The sn1-casRNA-CAN1.Z comprises a 20
nucleotide CAN1.Z spacer sequence. Expression cassettes are
assembled comprising each sn1-casRNA, the SNR52 promoter, and SUF*4
30 flanking sequence. Each expression cassette is assembled into a
vector comprising a 2 micron replication origin and a selectable
marker, for example, p426. DNA sequences encoding sn1-casRNA
expression cassette are inserted into a vector containing a HIS3
selectable marker. DNA sequences encoding sn2-casRNA expression
cassette are inserted into a vector containing a LEU2 selectable
marker. One suitable backbone vector for the sn-casRNA encoding
sequences is p426 GPD (American Type Culture Collection, Manassas,
Va.), wherein the URA3 coding sequences are mutated or deleted and
the appropriate selectable marker is inserted. Expression of the
sn-casRNA sequences is under the constitutive control of the SNR52
promoter that directs transcription by RNA polymerase III.
[0531] The Cas9 vector and each sn1-casRNA/sn2-casRNA vector pair
are transformed using standard methods into ATCC 200895 (MATa
his3delta200 leu2delta0 met15delta0 trp1delta63 ura3delta0)
(American Type Culture Collection, Manassas, Va.) and presence of
the vectors is selected for using SC dropout media without uracil,
histidine or leucine. Negative control yeast strains are also
constructed by transformation of the individual vectors comprising
sn1-casRNA-CANI.Y, sn1-casRNA-CAN1.Z, sn2-casRNA, and Cas9 into
ATCC 200895. Appropriate selection media are used for each
vector.
[0532] Cells comprising Cas9 and sn1-casRNA/sn2-casRNA are cultured
in liquid SC dropout media without uracil, histidine and leucine,
and containing 2% galactose and 1% raffinose. Cells are grown for
approximately 16 hours, pelleted and plated on YPAD,
SC-uracil-histidine-leucine plates containing 60 mg/ml L-canavanine
(Sigma-Aldrich, St. Louis, Mo.), and SC-lysine containing 100 mg/ml
thialysine (S-2-aminoethyl-1-cysteine, Sigma-Aldrich, St. Louis,
Mo.). Approximately 10.sup.7-10.sup.8 cells are plated on
canavanine and thialysine containing media, and cells arc diluted
appropriately for plating on rich media.
[0533] The ratio of the colony count on canavanine or thialysine
plates divided by the colony count on rich media (YPAD) plates for
each culture is used as a measure of mutation frequency. Negative
control strains are similarly cultured and plated.
[0534] To control for a potential genome-wide mutator phenotype,
the mutation frequency of the non-targeted endogenous LYP1 gene, a
lysine permease, is monitored by selecting for lyp1 mutants using a
toxic lysine analogue, thialysine.
[0535] LYP1 and CAN1 genes are on separate chromosomes.
Accordingly, local mutation frequency in each locus should be
independent in the absence of a genome-wide mutator.
[0536] The sn1-casRNA-CAN1.Y/sn2-casRNA directs Cas9 endonuclease
activity to a target site located 207bp downstream of the start
codon of the CAN1 gene. The sn1-casRNA-CAN1.Z/sn2-casRNA directs
Cas9 endonuclease activity to a target site located 58 bp
downstream of the ATG start codon of the CAN1 gene.
[0537] When expression of Cas9 is induced by galactose, a decrease
in cell viability on SC-uracil-histidine-leucine plates containing
60 mg/ml L-canavanine versus YPAD media indicates a higher mutation
frequency in the CAN1 gene. The mutation rate in the LYP1 gene
provides an indication of the background mutation rate. When the
LYP1 gene mutation rate remains constant across all strains it
suggests that the sn1-casRNA/sn2-casRNA and Cas9 system does not
induce random mutations genome-wide. To further validate that
mutations are caused by the sn1-casRNA/sn2-casRNA and Cas9 system,
the CAN1 gene can be isolated and sequenced from canavanine
resistant populations. The sequences are then aligned to identify
the location and types of mutations in the CAN1 gene relative to
the target binding sequence (i.e., the spacer sequence) present in
the sn1-casRNA.
[0538] Other chromosomal loci in S. cerevisiae are similarly
targeted for modification by selection of appropriate spacer
sequences for sn1-casRNA.
[0539] This analysis provides data to verify that the Cas9 and
sn1-casRNA/sn2-casRNA systems of the present invention provide
specific RNA-directed endonuclease activity at targeted endogenous
genomic loci in yeast.
[0540] B. Site-Specific Homologous Recombination with Donor DNA
[0541] A KanMX oligonucleotide sequence is PCR amplified with 50 bp
homology arms to the CAN1 locus from the pFA6a-KanMX6 plasmid,
which is commonly used for creation of gene knockouts in yeast. The
KanMX is used as a donor DNA. The KanMX oligonucleotide confers
G418 resistance and is designed to disrupt the CAN1.Y associated
PAM sequence. Upon integration, this donor DNA results in
canavanine resistance and G418 resistance.
[0542] Cells containing the sn1-casRNA-CAN1.Y, sn2-casRNA, Cas9
expression vectors are grown to saturation in SC dropout media
without uracil, histidine and leucine. This culture is used to
inoculated liquid SC media without uracil, histidine and leucine
and the culture is grown to approximately OD600 of 1.8. Cells are
collected via centrifugation and donor oligonucleotides are
transformed into the cells by electroporation. Electroporated cells
are transferred into SC-ura-his-leu media containing 2% galactose
and 1% raffinose and grown for approximately 12 hours. Negative
control strains are similarly treated but no donor oligonucleotide
is provided.
[0543] Approximately 10.sup.6-10.sup.7 cells are plated on
selective media, and cells are diluted appropriately on rich media.
Negative control strains are similarly cultured and plated.
[0544] Colonies containing the plasmids are replica plated to
canavanine media as well as rich media with G418 to select for the
KanMX integration event. The ratio of colony count on selective
plates (i.e., colonies that are both canavanine and G418 resistant)
over colony count on rich plates is used as a measure of correction
frequency which suggests homologous recombination of the KanMX
sequences at the site of sn1-casRNA/sn2-casRNA directed cleavage.
To further validate that the integration events are directed by the
sn1-casRNA/sn2-casRNA and Cas9 system, the CAN1 gene including the
integrated KanMX sequences can be isolated and sequenced from
canavanine/G418 resistant populations. The sequences are then
aligned to identify the location and types of insertions in the
CAN1 gene relative to the target binding sequence (i.e., the spacer
sequence) present in the sn1-casRNA.
[0545] Other chromosomal loci in S. cerevisiae are similarly
targeted for modification by selection of appropriate spacer
sequences for sn1-casRNA and donor oligonucleotides. Functional
genes can be introduced into the S. cerevisiae genome without
disruption of endogenous genes. Also, introduction of selectable
markers into endogenous target genes can be used to provide
selectable knock-out mutations of the target genes.
[0546] This analysis provides data to verify that the Cas9 and
sn1-casRNA/sn2-casRNA systems of the present invention provide
specific RNA-directed endonuclease activity at targeted endogenous
genomic loci in yeast and can stimulate homologous recombination
events at such loci using donor DNA.
[0547] Following the guidance of the present specification and
examples, the methods described in this example can be practiced by
one of ordinary skill in the art with other Type II CRISPR Cas9
proteins including, but not limited to, Cas9 and Cas9 fusions
combined with their cognate polynucleotide components modified as
described herein to comprise a split nexus element.
EXAMPLE 16
Targeted Mutagenesis in Zea mays
[0548] This example describes use of the split-nexus
Cas9-associated polynucleotides (sn-casPNs) of the present
invention for creating genomic modifications in plants. Although a
two component sn-casRNA polynucleotide system is described, other
embodiments of the present invention can be used as well (e.g., a
three component sn-casRNA polynucleotide system).
[0549] A three-part sn-casRNA (sn1-casRNA, sn-2-casRNA and
sn3-casRNA) system (see, e.g., FIG. 3A) is used in a modification
of the method of Cigan, A. M., et al., "Genome modification using
guide polynucicotidc/cas endonuclease systems and methods of use,"
U.S. Patent Publication No. 20150059010, published Feb. 26, 2015,
which used guide RNAs each having a continuous sequence.
[0550] Examples of suitable vectors, media, culture conditions,
etc. are described. Modifications of these components and
conditions will be understood by one of ordinary skill in the art
in view of the teachings of the present specification.
[0551] A. Expression Cassettes
[0552] The Cas9 gene from Streptococcus pyogenes M1 GAS (SF370) is
maize codon optimized per standard techniques known in the art. The
potato ST-LS1 intron is introduced in order to eliminate its
expression in E. coli and Agrobacterium. Nuclear localization of
the Cas9 protein in maize cells is facilitated by simian virus 40
(SV40) monopartite and Agrobacterium tumejaciens bipartite VirD2
T-DNA border endonuclease nuclear localization signals incorporated
at the amino and carboxyl-termini of the Cas9 open reading frame,
respectively. The Cas9 gene was operably linked to a maize
constitutive (e.g. a plant Ubiquitin promoter) or regulated
promoter by standard molecular biological techniques.
[0553] Expression cassettes for the expression of the sn1-casRNA,
sn2-casRNA, and sn3-casRNA utilize the maize U6 polymerase HI
promoter (5' of each sn-casRNA coding sequence) and maize U6
polymerase III terminator (3' of each sn-casRNA coding sequence)
operably linked to sn-casRNA DNA coding sequences using standard
molecular biology techniques to create sn-casRNA expression
cassettes. As shown in FIG. 3A, sn3-casRNA comprises a 20 spacer
region complementarity to the DNA target (VT domain). A target
region upstream of a PAM sequence is selected for target site
recognition and cleavage.
[0554] The expression cassettes for the Cas9 protein and sn-casRNAs
can be placed in suitable backbone vectors (e.g., as described by
Belhaj, K., et al., (2013) "Plant genome editing made easy:
targeted mutagenesis in model and crop plants using the CRISPR/Cas
system," Plant Methods. 9(1):39; Weber E., et al., (2011) "A
Modular Cloning System for Standardized Assembly of Multigene
Constructs," PLoS ONE 6(2): el6765) using standard molecular
biology techniques.
[0555] B. Generating Mutations
[0556] Three different maize genomic target sequences are targeted
for cleavage using the sn1-casRNA/sn2-casRNA/sn3-casRNA/Cas9
system. The three target sequences are located at the LIG locus
(approximately 600 bp upstream of the Liguleless 1 gene start
codon) and examined by deep sequencing for the presence of
mutations. Spacer sequences for each target site (LIGCas-1,
LIGCas-2, and LIGCas3) are as described in U.S. Patent Publication
No. 20150059010 (see, VT domains complementary to the antisense
strand of the maize genomic target sequences listed in Table 1 of
U.S. Patent Publication No. 20150059010). The resulting sn-casRNAs
are as follows: sn1-casRNA/sn2-casRNA/sn3-casRNA-LIGCas-1;
sn1-casRNA/sn2-casRNA/sn3-casRNA-LIGCas-2; and
sn1-casRNA/sn2-casRNA/sn3-casRNA-LIGCas-3.
[0557] Expression cassettes comprising the three component
sn-casRNA systems and Cas expression cassettes are codelivered to
60-90 Hi-II immature maize embryos by particle-mediated delivery.
Hi-II maize embryos are transformed with the Cas9 and long guide
RNA expression cassettes (as described in U.S. Published Patent
Application 20150082478, published Mar. 19, 2015) targeting the
L1GCas-3 genomic target site for cleavage to provide a positive
control. Hi-II maize embryos transformed with only the Cas9
expression cassette provides a negative control.
[0558] Maize cars are husked and surface sterilized and rinsed two
times with sterile water. The immature embryos are isolated and
placed embryo axis side down (scutellum side up), 25 embryos per
plate, on 560Y medium for 4 hours and then aligned within the
2.5-cm target zone in preparation for bombardment.
[0559] Vectors comprising the sn1-casRNA/sn2-casRNA1sn3-casRNA/Cas9
systems are co-bombarded with vectors containing the developmental
genes ODP2 (Ovule development protein 2, an AP2 domain
transcription factor; see, e.g., U.S. Published Patent Application
No. 20090328252, published Dec. 31, 2009) and Wushel (U.S.
Published Patent Application No. 20110167516, published Jul. 7,
2011).
[0560] For each sn1-casRNA/sn2-casRNA/sn3-casRNA/Cas9 system, the
corresponding vectors are precipitated onto 0.6 .sub.I-LM (average
diameter) gold pellets using a water-soluble cationic lipid
transfection reagent. DNA solution is prepared on ice using
sn1-casRNA/sn2-casRNA/sn3-casRNA/Cas9 vectors and plasmids
containing the developmental genes ODP2 and Wushel. Prepared gold
particles are added to the pre-mixed DNA. The water-soluble
cationic lipid transfection reagent is added in water and mixed
carefully. Gold particles are pelleted in a microfuge the
supernatant is removed. The resulting pellet is carefully rinsed
with ethanol (EtOH) without resuspending the pellet and the EtOH
rinse is carefully removed. 100% EtOH is added and the particles
are resuspended by brief sonication. Then, the mixture is spotted
onto the center of each macrocarrier and allowed to dry about 2
minutes before bombardment (Kildcert J. R., et al., (2005) "Stable
transformation of plant cells by particle bombardment/biolistics,"
Methods Mol Biol. 286:61-78).
[0561] Plates with the embryos are bombarded at level #4 with a
Helios.RTM. Gene Gun System (Biorad, Hercules Calif.). All samples
receive a single shot at 450 PSI of prepared particles/DNA.
Following bombardment, the embryos are incubated on maintenance
medium for 12 to 48 hours at temperatures ranging from 26.degree.
C. to 37.degree. C., and are then placed at 26.degree. C.
[0562] After 7 days, approximately 30 of the most uniformly
transformed embryos from each treatment are pooled and total
genomic DNA is extracted. The region surrounding the intended
target site is PCR amplified with Phusion.RTM. HighFidelity PCR
Master Mix (New England Biolabs, Ipswich, Mass.). The PCR
amplification is also used to add amplicon-specific barcodes and
Illumnia sequencing primers (Illumina, Madison Wis.). The resulting
PCR amplification products arc purified with a PCR purification
spin column (Qiagen, Valencia Calif.), concentration measured with
a Hoechst dye-based fluorometric assay, combined in an equimolar
ratio, and single-read 100 nucleotide-length deep sequencing was
performed on MiSeq Personal Sequencer (Illumina, Madison Wis.).
[0563] The frequencies of NHEJ mutations recovered by deep
sequencing for the sn1-casRNA/sn2-casRNA/sn3-casRNA/Cas9 systems
targeting the three LIGCas targets compared to the single long
guide RNA/Cas9 endonuclease system targeting the corresponding
locus are determined. These data are to demonstrate that the
sn1-casRNA/sn2-casRNA/sn3-casRNA/Cas9 systems as described herein
cleaves maize chromosomal DNA and generates NHEJ-mediated
mutations.
[0564] Following the guidance of the present specification and
examples, the methods described in this example can be practiced by
one of ordinary skill in the art with other Type II CRISPR Cas9
proteins including, but not limited to, Cas9 and Cas9 fusions
combined with their cognate polynucleotide components modified as
described herein to comprise a split nexus clement.
EXAMPLE 17
Generation of Transgenic Mice
[0565] This example describes use of the split-nexus
Cas9-associated polynucleotides (sn-casPNs) of the present
invention for creating genomic modifications in animals.
[0566] A two-part sn-casRNA (sn1-casRNA and sn2-casRNA) system
(see, e.g., FIG. 3) is used in a modification of the method of
Wang, et al. ("One-step generation of mice carrying mutations in
multiple genes by CRISPR/Cas-mediated genome engineering," Cell
(2013) 153(4):910-918), which used a single guide RNAs each having
a continuous sequence.
[0567] Examples of suitable vectors, media, culture conditions,
etc. are described. Modifications of these components and
conditions will be understood by one of ordinary skill in the art
in view of the teachings of the present specification.
[0568] A. Production of Cas9 mRNA and sn1-casRNA/sn2-casRNA
[0569] A T7 promoter is added to Cas9 coding region optimized for
mammalian expression (e.g., the Cas9 coding sequence can be PCR
amplified from pX330-U6-Chimeric_BB-CBh-hSpCas9; Addgene,
Cambridge, Mass.). The T7 promoter is added by PCR amplification.
The T7-Cas9 PCR product is gel purified and used as the template
for in vitro transcription using mMESSAGE mMACHINE T7 ULTRA Kit
(Life Technologies, Grand Island, N.Y.). The Cas9-mRNA is purified
using MEGAclear Kit (Life Technologies, Grand Island, N.Y.) and
eluted in RNase-free water.
[0570] DNA sequences encoding the sn1-casRNAs and sn2-casRNAs (see,
e.g., FIG. 3B) are chemically synthesized. The 20 nucleotide spacer
sequences for the sn1-casRNAs are as follows: sn1-casRNA-Tet1,
GGCTGCTGTC AGGGAGCTCA (SEQ ID NO:89); and sn1-casRNA-Tet 2,
GAAAGTGCCA ACAGATATCC (SEQ ID NO:90) (see, FIG. 1A of Wang, et al.,
Cell (2013) 153(4):910-918)). The T7 promoter is added to each of
the sn1-casRNA and sn2-casRNA templates by PCR amplification. The
T7-sn-casRNA PCR products are gel purified and used as the template
for in vitro transcription using MEGAshortscript T7 Kit (Life
Technologies, Grand Island, N.Y.). The sn-casRNAs are purified
using MEGAclear Kit (Life Technologies, Grand Island, N.Y.) and
eluted in RNase-free water.
[0571] B. One-Cell Embryo Injection
[0572] All animal procedures are performed according to NIH
guidelines. B6D2F1 (C57BL/6.times. DBA2) female mice are used as
embryo donors. ICR mouse strains are used as foster mothers.
Superovulated, seven to eight week old female B6D2F1 mice arc mated
to B6D2F1 males. Fertilized embryos are collected from oviducts.
Cas9 mRNAs (administered to individual embryos over a range of
approximately 20 ng/ml to approximately 200 ng/ml),
sn1-casRNA/sn2-casRNA (administered to individual embryos over a
range of from 20 ng/ml to 50 ng/ml) are injected into the cytoplasm
of fertilizedembryos (having well recognized pronuclei) in M2
medium (Sigma-Aldrich, St. Louis, Mo.).
[0573] When a donor oligonucleotide is also being injected the
concentration of the split-nexus Cas9-associated
polynucleotides/Cas9 protein system components are as follows: Cas9
mRNA (approximately 100 ng/ml), sn1-casRNA/sn2-casRNA (50 ng/ml);
and donor oligonucleotide (100 ng/ml). The components are mixed and
injected into zygotes at the pronuclei stage. Injected zygotes are
cultured in PrimeQ.TM. KSOM Embryo Culture Medium, w/Amino Acids
and Phenol Red (MTI-GlobalStem, Gaithersburg, Md.) at 37.degree. C.
under 5% CO.sub.2 in air for about 3.5 days (until blastocyst
stage). 15-25 blastocysts are transferred into the uteri of
pseudopregnant ICR females at approximately 2.5 days
postcoitum.
[0574] C. Double-Gene Mutant Mice
[0575] sn1-casRNA-Tet1/sn2-casRNA and sn1-casRNA-Tet2/sn2-casRNA
are coinjected as described above into zygotes. The genomic DNA of
pups is evaluated by RFLP (restriction fragment length polymorphism
analysis), Southern blot analysis, and sequencing analysis to
identify mice carrying targeted mutations at all four alleles of
the Tet1 and Tet2 genes. The results of these analyses are to
demonstrate that postnatal mice carrying bi-allelic mutations in
two different genes (i.e., the Tea and Tet2 genes) can be
efficiently generated.
[0576] In vivo off-target effects arc also evaluated. Previous work
in vitro, in bacteria, and in cultured human cells suggests that
the protospacer-adjacent motif sequence NGG and the 8 to 12 base
"seed sequence" of the spacer sequence is important for determining
the DNA cleavage specificity (Cong, L., et al., (2013) "Multiplex
genome engineering using CRISPR/Cas systems," Science 339:819-82;
Jiang, W., et al., (2013) "RNA-guided editing of bacterial genomes
using CRISPR-Cas systems," Nat. Biotechnol. 31:233-239; and Jinek,
M., et al., (2012) "A programmable dual-RNA-guided DNA endonuclease
in adaptive bacterial immunity," Science 337:816-821). Using this
rule, Wang, et al., identified that only three Tet1 and four Tet2
potential off-target sites exist in the mouse genome. Off-target
effects are evaluated using the Surveyor Assay (Guschin, D. Y., et
al., (2010) "A rapid and general assay for monitoring endogenous
gene modification," Methods Mol. Biol. 649: 247-256). The number of
off-target effects provides an estimate of in vivo targeting
accuracy of the sn1-casRNA/sn-2-casRNA/Cas9 protein complex.
[0577] D. In Vivo Gene Repair Modification
[0578] To evaluate in vivo gene repair using the sn1-casRNA and
sn2-casRNA system, a donor oligonucleotide is used to target Tet1
to change two base pairs of a SacI restriction site to create an
EcoRI site (Tet1 oligonucleotide; 126 bp, for sequence see FIG. 3A
of Wang, et al.). A second donor oligonucleotide is used to target
Tet2 to change two base pairs of an EcoRV site into an EcoRI site
(Tet2 oligonucleotide; 126 bp, for sequence see FIG. 3A of Wang, et
al.). Blastocysts are derived from zygotes'injected with Cas9 mRNA,
sn1-casRNA-Tet1/sn2-casRNA, and Tet1 oligonucleotide, Cas9 mRNA,
sn1-casRNA-Tet2/sn2-casRNA, and Tet2 oligonucleotide, and Cas9
mRNA, sn1-casRNA-Tet1/sn2-casRNA, Tet1 oligonucleotide,
sn1-casRNA-Tet2/sn2-casRNA, and Tet2 oligonucleotide.
[0579] DNA is isolated from the Cas9 mRNA,
sn1-casRNA-Tet1/sn2-casRNA, and Tet1 oligonucleotide blastocysts,
amplified, and digested with EcoRI to detect
oligonucleotide-mediated gene repair events. DNA is isolated from
the Cas9 mRNA, sn1-casRNA-Tet2/sn2-casRNA, and Tet2 oligonucleotide
blastocysts, amplified, and digested with EcoRI to detect
oligonucleotide-mediated gene repair events. DNA is isolated from
the Cas9 mRNA, sn1-casRNA-Tet1/sn2-casRNA, Tet1 oligonucleotide,
sn1-casRNA-Tet2/sn2-casRNA, and Tet2 oligonucleotide blastocysts,
amplified, and digested with EcoRI to detect
oligonucleotide-mediated gene repair events. The genomic DNA from
the blastocysts is evaluated by RFLP, Southern blot analysis, and
sequencing analysis to identify blastocysts carrying modified
restriction sites of the Tet1 and Tet2 genes. The results of these
analyses are to demonstrate that in vivo repair of mouse genes
(i.e., the Tet1 and Tet2 genes) can be efficiently carried out.
[0580] RFLP analyses using Sad and EcoRV cleavage to evaluate the
Tet1 and Tet2 loci, respectively, are used to demonstrate that
alleles not targeted by a selected Cas9 mRNA, sn1-casRNA-Tet1 or
Tet2/sn2-casRNA, and oligonucleotide (in each of the above listed
combinations) are not affected.
[0581] Furthermore, blastocysts with double oligonucleotide
injections are implanted into foster mothers. The genomic DNA from
resulting pups is evaluated by RFLP, Southern blot analysis, and
sequencing analysis to identify blastocysts carrying modified
restriction sites of the Tet1 and Tet2 genes. The results of these
analyses are to demonstrate that mice with genomic repair
modifications in multiple genes can be generated.
[0582] Following the guidance of the present specification and
examples, the methods described in this example can be practiced by
one of ordinary skill in the art with other Type II CRISPR Cas9
proteins including, but not limited to, Cas9 and Cas9 fusions
combined with their cognate polynucleotide components modified as
described herein to comprise a split nexus element.
EXAMPLE 18
sn-casRNAs/Cas9 Complexes in Delivery Vectors Comprising Cationic
Molecules
[0583] A. Production of Cas9 mRNA and
sn1-casRNA/sn2-casRNA/sn3-casRNA
[0584] A T7 promoter is added to Cas9 coding region optimized for
mammalian expression and tagged at the C-terminal with two nuclear
localization sequences (NLS). The T7 promoter is added by PCR
amplification. The T7-Cas9 PCR product is gel purified and cloned
into a vector for cell free protein expression (e.g., pT7CFE1-NFtag
Vector for Mammalian Cell-Free Protein Expression, Life
Technologies, Grand Island N.Y.). Cas9 protein is expressed and
isolated using a cell free protein expression system (e.g., 1-Step
CHO High-Yield IVT Kit, Life Technologies, Grand Island N.Y.) and
suspended in RNase-free water.
[0585] DNA sequences encoding the sn1-casRNA, sn2-casRNA, and
sn3-casRNA-AAVS-1 are prepared as described in Example 1. The
T7-sn-casRNA PCR products are gel purified and used as the template
for in vitro transcription using T7 High Yield RNA Synthesis Kit
(New England Biolabs, Ipswich, Mass.). The sn-casRNAs are purified
using GeneJet RNA Cleanup and Concentration Kit (Life Technologies,
Grand Island, N.Y.) and eluted in RNase-free water.
[0586] B. Formation of Ribonucleoprotein Complexes
[0587] Ribonucleoprotein (RNP) complexes are prepared at two
concentrations, 50 pmol Cas9:150pmols sn-casRNAs and 200 pmols
Cas9:600 pmols sn-casRNAs. All three sn-casRNA components in
equimolar amounts are mixed in an annealing buffer (1.25 mM HEPES,
0.625 mM MgCl2, 9.375 mM KCl at pH7.5) to desired concentration
(150 pmols or 600 pmols) in a final volume of 5 .mu.L, are
incubated for 2 minutes at 95.degree. C., are removed from
thermocycler and are allowed to equilibrate to room temperature.
Cas9 protein is diluted to appropriate concentration in binding
buffer (20 mM HEPES, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, and 5%
glycerol at pH 7.4) in a final volume of 5 .mu.L and is mixed with
the 5 .mu.L of heat-denatured sn-casRNAs followed by incubation at
37.degree. C. for 30 minutes to form the sn-casRNAs/Cas9
complexes.
[0588] C. Preparation of SC12CDClickpropylamine Vector
sn-casRNAs/Cas9 Complexes
[0589] SC12CDClickpropylamine (a cationic betacyclodextrin;
O'Mahony A. M., et al., (2013) "Cationic and PEGylated Amphiphilic
Cyclodextrins: Co-Formulation Opportunities for Neuronal Sirna
Delivery," PLoS ONE 8(6): e66413) is weighed out and dissolved in
chloroform (approximately 1 mg/ml), then mixed together in
appropriate volumes to provide molar ratios of cationic to
PEGylated-Cyclodextrin (U.S. Patent Application Publication No.
20140079770, published Mar. 20, 2014, "Vector for Pulmonary
Delivery, Inducing Agent, and Uses"). The solvent is removed under
a stream of nitrogen to provide a dry cyclodextrin (CD)
composition.
[0590] The CD composition is rehydrated with binding buffer (final
concentration approximately 1 mg/ml) and is sonicated for one hour
at room temperature followed by the immediate addition of the
sn-casRNAs/Cas9 complexes in binding buffer. The sn-casRNAs/Cas9
complexes in binding buffer are added in an equal volume. The
solution is mixed and is incubated for 20-30 minutes at room
temperature to produce a CD composition comprising the
sn-casRNAs/Cas9 complexes (CD-sn-casRNAs/Cas9).
[0591] D. Preparation of Liposomal Entrapped sn-casRNAs/Cas9
Complexes
[0592] Liposomes are formed without the casRNAs/Cas9 complexes to
provide negative controls (empty liposomes).
[0593] In a suitably sized round bottom flask the liposome
components are added and solubilized in a suitable solvent or
solvent mixture. Example liposome components are as follows:
[0594] Liposome 1: EPC (EtOH solution) and Cholesterol (EtOH
solution) are prepared in a molar ratio of 70/30.
[0595] Liposome 1-PEG: Stearylated PEG2000 (EtOH solution) is added
to be 5 mol % with respect to the total lipid amount of liposome 1
(EPC+Cholesterol).
[0596] Liposome 2: DOTMA (EtOH solution), Cholesterol (EtOH
solution), and EPC (EtOH solution) are prepared in a molar ratio of
30/40/30.
[0597] Liposome 2-PEG: Stearylated PEG2000 (EtOH solution) is added
to be 5 mol % with respect to the total lipid amount of liposome 2
(DOTMA+Cholesterol+EPC).
[0598] Liposome 3: DODAP (EtOH solution), Cholesterol (EtOH
solution), and EPC (EtOH solution) were added at a molar ratio of
30/40/30.
[0599] Liposome 3-PEG: Stearylated PEG2000 (EtOH solution) is added
to be 5 mol % with respect to the total lipid amount of liposome 3
(DODAP+Cholesterol+EPC).
[0600] An amount of EtOH is added to solubilize all components. The
flask is attached to a rotary evaporator spinning at 50-100 rpm and
immersed in a water bath set above the highest gel-liquid crystal
phase transition (Tc) temperature of the lipids used. The flask is
allowed to rotate in the water bath for approximately 1 minute to
equilibrate. A slow vacuum is pulled, to as low as <10 Torr, to
obtain a thin dry film on the walls of the flask without
precipitation. To remove any residual solvent, the flask is
subjected to high vacuum at room temperature for a few hours or
overnight.
[0601] A solution of either sn-casRNAs/Cas9 complexes or
CD-sn-casRNAs/Cas9 is added to obtain a final lipid concentration
of between about 2 mM to about 0.5 mM. Lipid rehydration is
conducted at room temperature for 15 minutes or longer. Liposomes
are prepared by ultrasonication for approximately 1 minute.
[0602] The above methods produce the particle compositions and
liposome compositions shown in Table 19.
TABLE-US-00019 TABLE 19 Particle and Liposome Compositions Particle
sn-casRNAs/Cas9 complex Particle CD-sn-casRNAs/Cas9 complex
Liposome 1 sn-casRNAs/Cas9 complex Liposome 1 CD-sn-casRNAs/Cas9
complex Liposome 1-PEG sn-casRNAs/Cas9 complex Liposome 1-PEG
CD-sn-casRNAs/Cas9 complex Liposome 2 sn-casRNAs/Cas9 complex
Liposome 2 CD-sn-casRNAs/Cas9 complex Liposome 2-PEG
sn-casRNAs/Cas9 complex Liposome 2-PEG CD-sn-casRNAs/Cas9 complex
Liposome 3 sn-casRNAs/Cas9 complex Liposome 3 CD-sn-casRNAs/Cas9
complex Liposome 3-PEG sn-casRNAs/Cas9 complex Liposome 3-PEG
CD-sn-casRNAs/Cas9 complex
[0603] E. Characterization of Particle Compositions and Liposomes
Compositions
[0604] The particle compositions and liposome compositions
described above are characterized (see e.g., Laouini, A., et al.,
(2012) "Preparation, Characterization and Applications of
Liposomes: State of the Art," Journal of Colloid Science and
Biotechnology Vol. 1, 147-168, 2012) using standard methods as
follows.
[0605] (i) Size Analysis
[0606] The sizes of the particles and liposomes are evaluated by
standard techniques. Several techniques are available for assessing
liposome size and size distribution including microscopy
techniques, size-exclusion chromatography (SEC), field-flow
fractionation and static or dynamic light scattering. Furthermore,
particle sizes can be evaluated using non-denaturing agarose gels
(e.g., 1.5% agarose gels, SYBRO Safe, Life Technologies, Grand
Island, N.Y.). Different sizes of particles and liposomes are
useful for different applications, for example, cell transfection
in culture or therapeutic administration to an animal.
[0607] (ii) Charge Measurements
[0608] The average size and charge the particles and liposomes are
measured with a Zetasizer Nano ZS (Malvern, Westborough Mass.)
using dynamic light scattering (DLS). If all the particles in a
suspension have a large negative or large positive zeta potential
they tend to repel each other. Thus reducing or eliminating the
tendency to aggregation. However, particles with low zeta potential
value have no force to prevent the particles flocculating.
[0609] (iii) Morphology
[0610] The morphologies of the compositions are evaluated using
transmission electron microscopy (TEM), for example, a JEOL 2000
FXII transmission electron microscope (Jeol Ltd., Tokyo, Japan).
Generally compositions having uniform particle or liposome
morphology is most desirable.
[0611] (iv) Aggregation Studies
[0612] The effects of salt-containing medium and serum on the
aggregation of the particles and liposomes is evaluated by
incubating complexes in either Opti-MEM.RTM. transfection media
(Life Technologies, Grand Island, N.Y.) or fetal bovine serum for
24 hours at 37.degree. C. Size measurements are carried out using
the Zetasizer Nano ZS. The absence of aggregation is often a
desirable quality. However, in some applications (for example,
transfection experiments) some aggregation may be desirable.
[0613] (v) Encapsulation Efficiency
[0614] The liposome preparations are a mixture of encapsulated and
unencapsulated CD-sn-casRNAs/Cas9 or sn-casRNAs/Cas9 fractions.
Several techniques are know for the determination of the
encapsulation efficiency including HPLC and field-flow
fractionation. Typically, the encapsulation percent is expressed as
the ratio of the unencapsulated peak area to that of a reference
standard at the same initial concentration. This method can be
applied if the liposomes do not undergo any purification following
preparation. General a high degree of encapsulation efficiency is
an important parameter for liposomes in therapeutic applications.
Low encapsulation efficiency necessitates the incorporation of a
post encapsulation separation step (such as dialysis, size
exclusion chromatography or ultrafiltration) to remove
unencapsulated complexes. Typically an encapsulation efficiency of
greater than 45%, more preferably greater than 80%, and most
preferably greater than 95% is desirable.
[0615] (v) In Vivo Activity
[0616] The particle compositions and liposome compositions
comprising the sn1-casRNA, sn2-casRNA, and sn3-casRNA-AAVS-1 are
evaluated for their relative abilities to deliver the
sn-casRNAs/Cas9 complexes into cells for gene repair. For this
experiment a donor DNA molecule is included in the preparation of
each of the particle compositions and liposome compositions. The
donor DNA is an EGFP fragment for use with the AAVS1 Positive
Control EGIP 293T Reporter Cell Line (System Biosciences, Mountain
View, Calif.). Other reporter systems are suitable for use in this
analysis. Liposome compositions are concentrated as necessary using
standard techniques.
[0617] The particle compositions and liposome compositions are
transfected into cells. One day before transfection, the cells are
plated in growth medium without antibiotics. The cells should be at
confluence at the time of transfection. The particle compositions
and liposome compositions are diluted into Opti-MEM.RTM. I Medium
(Life Technologies, Grand Island N.Y.) without serum to provide a
range of concentration for the sn-cas1RNAs/Cas9 complexes.
Typically a volume of about 200 .mu.l of these suspensions is
applied to cells in multiwell plates. The cells and suspensions are
gently mixed by rocking the plates back and forth. The cells are
incubated at 37.degree. C. in a CO.sub.2 incubator for 5-24 hours.
The following day, complete growth medium is added to the cells.
The cells are incubated cells at 37.degree. C. in a CO.sub.2
incubator for 24-48 hours prior to testing.
[0618] Gene repair is evaluated using the AAVS1 Positive Control
EGIP 293T Reporter Cell Line for monitoring HDR efficiency of EGFP
donor DNA. The sn3-casRNA-AAVS1 RNA sequence directs the
sn-casRNA/Cas9 complex to target and cleave a 53 bp AAVS1 sequence
integrated in a Enhanced Green Fluorescent Inhibited Protein (EGIP)
reporter cell line. The EGIP comprises a stop codon in the middle
to inactivate expression of the Enhanced Green Fluorescent Protein
(EGFP). In the presence of an active sn-casRNA/Cas9 complex, the
EGFP donor DNA recombines at the target site and restores the open
reading of the EGFP gene by homologous recombination. Cleavage
efficiency of the sn-casRNAs/Cas99 complex targeting the AAVS1
locus is measured using the Surveyor Assay. Efficiency of
restoration of EGFP expression is monitored using fluorescence
microscopy. Results are expressed as a percent EGFP gene expression
relative to particle composition (sn-casRNAs/Cas9 and
CD-sn-casRNA/Cas9) and the empty liposome controls. The results of
the in vivo activity studies provide guidance for the selection of
optimal particle and/or liposome components and compositions.
[0619] Taken together, these data provide information that allows
establishing criteria for selecting optimal liposomal compositions
for encapsulation of sn-casRNAs/Cas9 complexes of the present
invention according to their advantages and limitations.
[0620] Following the guidance of the present specification and
examples, the methods described in this example can be practiced by
one of ordinary skill in the art with other Type II CRISPR Cas9
proteins including, but not limited to, Cas9 and Cas9 fusions
combined with their cognate polynucleotide components modified as
described herein to comprise a split nexus element.
[0621] As is apparent to one of skill in the art, various
modification and variations of the above embodiments can be made
without departing from the spirit and scope of this invention. Such
modifications and variations are within the scope of this
invention.
Sequence CWU 1
1
90131RNAArtificial SequenceDerived from Streptococcus pyogenes
1caaaacagca uagcaaguua aaauaaggcu a 31246RNAArtificial
SequenceDerived from Streptococcus pyogenes 2guccguuauc aacuugaaaa
aguggcaccg agucggugcu uuuuuu 46334RNAArtificial SequenceDerived
from Streptococcus thermophilus CRISPR-I 3uaaaucuugc agaagcuaca
acgauaaggc uuca 34453RNAArtificial SequenceDerived from
Streptococcus thermophilus CRISPR-I 4ugccgaaauc aacacccugu
cauuuuaugg caggguguuu ucguuauuuu uuu 53532RNAArtificial
SequenceDerived from Listeria innocua 5caaaauaaca uagcaaguua
aaauaaggcu uu 32648RNAArtificial SequenceDerived from Listeria
innocua 6guccguuauc aacuuuuaau uaaguagcgc uguuucggcg cuuuuuuu
48745RNAArtificial SequenceDerived from Neisseria meningitidis
7cugcgaaaug agaaccguug cuacaauaag gccgucugaa aagau
45835RNAArtificial SequenceDerived from Neisseria meningitidis
8gugccgcaac gcucugcccc uuaaagcuuc ugcuu 35943RNAArtificial
SequenceDerived from Streptococcus gallolyticus 9uuggagcuau
ucgaaacaac acagcgaguu aaaauaaggc uuu 431049RNAArtificial
SequenceDerived from Streptococcus gallolyticus 10guccguacac
aacuuguaaa aguggcaccc gauucgggug cguuuuuuu 491146RNAArtificial
SequenceDerived from Staphylococcus aureus 11auuguacuua uaccuaaaau
uacagaaucu acuaaaacaa ggcaaa 461239RNAArtificial SequenceDerived
from Staphylococcus aureus 12augccguguu uaucucguca acuuguuggc
gagauuuuu 391348RNAArtificial SequenceDerived from Corynebacterium
diphtheriae 13agucacuaac uuaauuaaau agaacugaac cucaguaagc auuggcuc
481458RNAArtificial SequenceDerived from Corynebacterium
diphtheriae 14guuuccaaug uugauugcuc cgccggugcu ccuuauuuuu
aagggcgccg gcuuucuu 581546RNAArtificial SequenceDerived from
Parvibaculum lavamentivorans 15uagcaaaucg agaggcgguc gcuuuucgca
agcaaauuga ccccuu 461677RNAArtificial SequenceDerived from
Parvibaculum lavamentivorans 16gugcgggcuc ggcaucccaa ggucagcugc
cgguuauuau cgaaaaggcc caccgcaagc 60agcgcguggg ccuuuuu
771751RNAArtificial SequenceDerived from Campylobacter lari
17aauucuugcu aaagaaauuu aaaaagagac uaaaauaagu gguuuuuggu c
511873RNAArtificial SequenceDerived from Campylobacter lari
18auccacgcag gguuacaauc ccuuuaaaac cauuaaaauu caaauaaacu agguuguauc
60aacuuaguuu uuu 731954RNAArtificial SequenceDerived from Neisseria
cinerea 19auugucgcac ugcgaaauga gaaccguugc uacaauaagg ccgucugaaa
agau 542085RNAArtificial SequenceDerived from Neisseria cinerea
20gugccgcaac gcucugcccc uuaaagcuuc ugcuuuaagg ggcaucguuu auuucgguua
60aaaaugccgu cugaaaccgg uuuuu 852147RNAArtificial SequenceDerived
from Streptococcus pasteurianus 21cuugcacggu uacuuaaauc uugcugagcc
uacaaagaua aggcuuu 472243RNAArtificial SequenceDerived from
Streptococcus pasteurianus 22augccgaauu caagcacccc auguuuugac
augaggugcu uuu 432323DNAArtificial SequenceOligonucleotide Primer
23agtaataata cgactcacta tag 232424DNAArtificial
SequenceOligonucleotide Primer 24aagcaccgac tcggtgccac tttt
242558DNAArtificial SequenceOligonucleotide Primer 25taatacgact
cactatagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctt
582649DNAArtificial SequenceOligonucleotide Primer 26taatacgact
cactatagca ggacagcata gcaagttgag ataaggcta 492749DNAArtificial
SequenceOligonucleotide Primer 27tagccttatc tcaacttgct atgctgtcct
gctatagtga gtcgtatta 492855DNAArtificial SequenceOligonucleotide
Primer 28taatacgact cactataggg gccactaggg acaggatgtc tcagagctat
gctgt 552955DNAArtificial SequenceOligonucleotide Primer
29acagcatagc tctgagacat cctgtcccta gtggccccta tagtgagtcg tatta
553020DNAArtificial SequenceOligonucleotide Primer 30ccccgttctc
ctgtggattc 203120DNAArtificial SequenceOligonucleotide Primer
31atcctctctg gctccatcgt 203252DNAArtificial SequenceOligonucleotide
Primer 32cactctttcc ctacacgacg ctcttccgat cttctggcaa ggagagagat gg
523348DNAArtificial SequenceOligonucleotide Primer 33ggagttcaga
cgtgtgctct tccgatctta tattcccagg gccggtta 483457DNAArtificial
SequenceOligonucleotide Primer 34caagcagaag acggcatacg agattacgtg
atgtgactgg agttcagacg tgtgctc 573558DNAArtificial
SequenceOligonucleotide Primer 35aatgatacgg cgaccaccga gatctacacc
gtctaataca ctctttccct acacgacg 583658DNAArtificial
SequenceOligonucleotide Primer 36aatgatacgg cgaccaccga gatctacact
ctctccgaca ctctttccct acacgacg 583758DNAArtificial
SequenceOligonucleotide Primer 37aatgatacgg cgaccaccga gatctacact
cgactagaca ctctttccct acacgacg 583858DNAArtificial
SequenceOligonucleotide Primer 38aatgatacgg cgaccaccga gatctacact
tctagctaca ctctttccct acacgacg 583958DNAArtificial
SequenceOligonucleotide Primer 39aatgatacgg cgaccaccga gatctacacc
ctagagtaca ctctttccct acacgacg 584058DNAArtificial
SequenceOligonucleotide Primer 40aatgatacgg cgaccaccga gatctacacc
tattaagaca ctctttccct acacgacg 584162DNAArtificial
SequenceOligonucleotide Primer 41ggcagtagcc ttatctcaac ttgctatgct
gtcctgtttc caggacagca tagctctgag 60ac 624220DNAArtificial
SequenceOligonucleotide Primer 42ggcagtagcc ttatctcaac
204363DNAArtificial SequenceOligonucleotide Primer 43taatacgact
cactataggc aggtccgtta tcaacttgaa aaagtggcac cgagtcggtg 60ctt
634467DNAArtificial SequenceOligonucleotide Primer 44ggcagtgaac
tagccttatc tcaacttgct atgctgtcct gtttccagga cagcatagct 60ctgagac
674520DNAArtificial SequenceOligonucleotide Primer 45ggcagtgaac
tagccttatc 204668DNAArtificial SequenceOligonucleotide Primer
46taatacgact cactataggc agctaaggtc cgttatcaac ttgaaaaagt ggcaccgagt
60cggtgctt 684756DNAArtificial SequenceOligonucleotide Primer
47taatacgact cactataggg gccactaggg acaggatgtc tcagagctat gctgtc
564856DNAArtificial SequenceOligonucleotide Primer 48taatacgact
cactatagtt tgtgtttcca taaactggtc tcagagctat gctgtc
564956DNAArtificial SequenceOligonucleotide Primer 49taatacgact
cactatagcc cgccaccacc aggatgtgtc tcagagctat gctgtc
565056DNAArtificial SequenceOligonucleotide Primer 50taatacgact
cactataggc agccagcatg atgagacgtc tcagagctat gctgtc
565120DNAArtificial SequenceOligonucleotide Primer 51aagcaccgac
tcggtgccac 205252DNAArtificial SequenceOligonucleotide Primer
52cactctttcc ctacacgacg ctcttccgat ctacatgcac acccatgttt tg
525348DNAArtificial SequenceOligonucleotide Primer 53ggagttcaga
cgtgtgctct tccgatctaa catttccagg tgacaggc 485450DNAArtificial
SequenceOligonucleotide Primer 54cactctttcc ctacacgacg ctcttccgat
ctgttccgac gctccttgaa 505548DNAArtificial SequenceOligonucleotide
Primer 55ggagttcaga cgtgtgctct tccgatctca gatgcgatga cctttgtg
485652DNAArtificial SequenceOligonucleotide Primer 56cactctttcc
ctacacgacg ctcttccgat ctaagaaagg caagaagcct gg 525748DNAArtificial
SequenceOligonucleotide Primer 57ggagttcaga cgtgtgctct tccgatctgc
tggcctgaga cattccta 485857DNAArtificial SequenceOligonucleotide
Primer 58tagccttatc tcaacttgct atgctgtcct gtttccagga cagcatagct
ctgagac 575957DNAArtificial SequenceOligonucleotide Primer
59taatacgact cactataggg gccactaggg acaggatgtc tcagagctat gcagtcc
576060DNAArtificial SequenceOligonucleotide Primer 60cagtagcctt
atctcaactt gctatgcagt cctgtttcca ggactgcata gctctgagac
606140DNAArtificial SequenceOligonucleotide Primer 61ctgcctatac
ggcagtagcc ttatctcaac ttgctatgca 406257DNAArtificial
SequenceOligonucleotide Primer 62taatacgact cactatagct gccgtatagg
caggtccgtt atcaacttga aaaagtg 576343DNAArtificial
SequenceOligonucleotide Primer 63aagcaccgac tcggtgccac tttttcaagt
tgataacgga cct 436460DNAArtificial SequenceOligonucleotide Primer
64gtctagcctt atctcaactt gctatgcagt cctgtttcca ggactgcata gctctgagac
606540DNAArtificial SequenceOligonucleotide Primer 65ctgcctatac
ggcagtgtct agccttatct caacttgcta 406657DNAArtificial
SequenceOligonucleotide Primer 66taatacgact cactatagct gccgtatagg
cagagacagt ccgttatcaa cttgaaa 576748DNAArtificial
SequenceOligonucleotide Primer 67aagcaccgac tcggtgccac tttttcaagt
tgataacgga ctgtctct 486841RNAArtificial SequenceSplit-nexus
Cas9-associated RNA 68guccguuauc aacuugaaaa aguggcaccg agucggugcu u
416932RNAArtificial SequenceSplit-nexus Cas9-associated RNA
69gcaggacagc auagcaaguu gagauaaggc ua 327038RNAArtificial
SequenceSplit-nexus Cas9-associated RNA 70ggggccacua gggacaggau
gucucagagc uaugcugu 387182RNAArtificial SequenceSplit-nexus
Cas9-associated RNA 71ggggccacua gggacaggau gucucagagc uaugcugucc
uggaaacagg acagcauagc 60aaguugagau aaggcuacug cc
827282RNAArtificial SequenceSplit-nexus Cas9-associated RNA
72guuuguguuu ccauaaacug gucucagagc uaugcugucc uggaaacagg acagcauagc
60aaguugagau aaggcuacug cc 827382RNAArtificial SequenceSplit-nexus
Cas9-associated RNA 73gcccgccacc accaggaugu gucucagagc uaugcugucc
uggaaacagg acagcauagc 60aaguugagau aaggcuacug cc
827482RNAArtificial SequenceSplit-nexus Cas9-associated RNA
74ggcagccagc augaugagac gucucagagc uaugcugucc uggaaacagg acagcauagc
60aaguugagau aaggcuacug cc 827546RNAArtificial SequenceSplit-nexus
Cas9-associated RNA 75ggcagguccg uuaucaacuu gaaaaagugg caccgagucg
gugcuu 467687RNAArtificial SequenceSplit-nexus Cas9-associated RNA
76ggggccacua gggacaggau gucucagagc uaugcugucc uggaaacagg acagcauagc
60aaguugagau aaggcuaguu cacugcc 877787RNAArtificial
SequenceSplit-nexus Cas9-associated RNA 77guuuguguuu ccauaaacug
gucucagagc uaugcugucc uggaaacagg acagcauagc 60aaguugagau aaggcuaguu
cacugcc 877887RNAArtificial SequenceSplit-nexus Cas9-associated RNA
78gcccgccacc accaggaugu gucucagagc uaugcugucc uggaaacagg acagcauagc
60aaguugagau aaggcuaguu cacugcc 877987RNAArtificial
SequenceSplit-nexus Cas9-associated RNA 79ggcagccagc augaugagac
gucucagagc uaugcugucc uggaaacagg acagcauagc 60aaguugagau aaggcuaguu
cacugcc 878068RNAArtificial SequenceSplit-nexus Cas9-associated RNA
80gggcagugaa cuagccuuau cucaacuugc uaugcugucc uguuuccagg acagcauagc
60ucugagac 688177RNAArtificial SequenceSplit-nexus Cas9-associated
RNA 81ggggccacua gggacaggau gucucagagc uaugcugucc uggaaacagg
acagcauagc 60aaguugagau aaggcua 778277RNAArtificial
SequenceSplit-nexus Cas9-associated RNA 82gcccgccacc accaggaugu
gucucagagc uaugcugucc uggaaacagg acagcauagc 60aaguugagau aaggcua
778377RNAArtificial SequenceSplit-nexus Cas9-associated RNA
83ggcagccagc augaugagac gucucagagc uaugcugucc uggaaacagg acagcauagc
60aaguugagau aaggcua 778441RNAArtificial SequenceSplit-nexus
Cas9-associated RNA 84guccguuauc aacuugaaaa aguggcaccg agucggugcu u
418592RNAArtificial SequenceSplit-nexus Cas9-associated RNA
85ggggccacua gggacaggau gucucagagc uaugcagucc uggaaacagg acugcauagc
60aaguugagau aaggcuacug ccguauaggc ag 928656RNAArtificial
SequenceSplit-nexus Cas9-associated RNA 86cugccguaua ggcagguccg
uuaucaacuu gaaaaagugg caccgagucg gugcuu 568796RNAArtificial
SequenceSplit-nexus Cas9-associated RNA 87ggggccacua gggacaggau
gucucagagc uaugcagucc uggaaacagg acugcauagc 60aaguugagau aaggcuagac
acugccguau aggcag 968861RNAArtificial SequenceSplit-nexus
Cas9-associated RNA 88cugccguaua ggcagagaca guccguuauc aacuugaaaa
aguggcaccg agucggugcu 60u 618920DNAArtificial SequenceTet1-Spacer
89ggctgctgtc agggagctca 209020DNAArtificial SequenceTet2-Spacer
90gaaagtgcca acagatatcc 20
* * * * *
References