U.S. patent application number 15/818710 was filed with the patent office on 2018-10-11 for therapeutic uses of genome editing with crispr/cas systems.
The applicant listed for this patent is The Children's Medical Center Corporation, President and Fellows of Harvard College. Invention is credited to Chad A. Cowan, Kiran Musunuru, Derrick J. Rossi.
Application Number | 20180291383 15/818710 |
Document ID | / |
Family ID | 51659363 |
Filed Date | 2018-10-11 |
United States Patent
Application |
20180291383 |
Kind Code |
A1 |
Musunuru; Kiran ; et
al. |
October 11, 2018 |
THERAPEUTIC USES OF GENOME EDITING WITH CRISPR/Cas SYSTEMS
Abstract
Disclosed herein are methods, compositions, and kits for high
efficiency, site-specific genomic editing of cells.
Inventors: |
Musunuru; Kiran; (Cambridge,
MA) ; Cowan; Chad A.; (Boston, MA) ; Rossi;
Derrick J.; (Roslindale, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
President and Fellows of Harvard College
The Children's Medical Center Corporation |
Cambridge
Boston |
MA
MA |
US
US |
|
|
Family ID: |
51659363 |
Appl. No.: |
15/818710 |
Filed: |
November 20, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14485288 |
Sep 12, 2014 |
9822370 |
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15818710 |
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PCT/US2014/033082 |
Apr 4, 2014 |
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14485288 |
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61808594 |
Apr 4, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/63 20130101;
A61P 43/00 20180101; A61P 31/00 20180101; A61P 31/18 20180101; A61P
35/00 20180101; C12N 15/907 20130101; A61K 48/00 20130101 |
International
Class: |
C12N 15/63 20060101
C12N015/63; C12N 15/90 20060101 C12N015/90 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under
HL107440, R01-HL118744, R00-HL098364 and R01-DK095384 awarded by
the National Institutes of Health. The government has certain
rights in the invention.
Claims
1. A composition comprising or encoding at least one guide
ribonucleic acid sequence consisting of (a) a nucleotide sequence
complementary to or identical to a target nucleotide sequence
selected from SEQ ID NOs: 1-139 and 304-333, or (b) a nucleotide
sequence with a single nucleotide mismatch to the target nucleotide
sequence selected from SEQ ID NOs: 1-139 and 304-333.
2. The composition of claim 1, comprising or encoding two guide
ribonucleic acid sequences.
3. The composition of claim 1, wherein at least one of the
ribonucleic acids of the guide ribonucleic acid is a modified
ribonucleic acid.
4. The composition of claim 3, wherein the modified ribonucleic
acid is selected from the group consisting of pseudouridine,
5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate,
4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate,
and 5-azauridine-5'-triphosphate.
5. The composition of claim 1, wherein the target nucleotide
sequence is selected from the group consisting of SEQ ID NOs:
304-333.
6. The composition of claim 1, wherein the target nucleotide
sequence is selected from the group consisting of SEQ ID NOs: 309,
314, 315, 316, 317, 331, and 332.
7. The composition of claim 1, wherein the target nucleotide
sequence is selected from the group consisting of SEQ ID NOs: 1, 4,
13, 14, 18, 21, 22, 26, 27, 32, 33, 37, 58, 64, 66, 72, 73, 79, 82,
83, 84, 85, 86, 87, 89, 93, 94, 112, 113, 114, 117, 120, 305, 308,
and 331.
8. The composition of claim 1, further comprising a nucleic acid
encoding a Cas protein.
9. The composition of claim 8, further comprising a promoter
operably linked to the nucleic acid encoding a Cas protein.
10. The composition of claim 9, wherein the promoter is optimized
for increased expression in human stem cells.
11. The composition of claim 10, wherein the promoter is selected
from the group consisting of a Cytomegalovirus (CMV) early enhancer
element and a chicken beta-actin promoter, a chicken beta-actin
promoter, an elongation factor-1 alpha promoter, and a ubiquitin
promoter.
12. The composition of claim 1, wherein the composition further
comprises a nucleic acid encoding a fluorescent protein.
13. The composition of claim 1, wherein the composition comprises a
plasmid encoding the at least one guide ribonucleic acid
sequence.
14. A vector comprising a nucleic acid sequence encoding at least
one guide ribonucleic acid sequence consisting of (a) a nucleotide
sequence complementary to or identical to a target nucleotide
sequence selected from SEQ ID NOs: 1-139 and 304-333, or (b) a
nucleotide sequence with a single nucleotide mismatch to the target
nucleotide sequence selected from SEQ ID NOs: 1-139 and
304-333.
15. A cell comprising one or two guide ribonucleic acid sequences
consisting of (a) a nucleotide sequence complementary to or
identical to a target nucleotide sequence selected from SEQ ID NOs:
1-139 and 304-333, or (b) a nucleotide sequence with a single
nucleotide mismatch to the target nucleotide sequence selected from
SEQ ID NOs: 1-139 and 304-333.
16. The cell of claim 15, further comprising a Cas protein.
Description
RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 14/485,288, filed on Sep. 12, 2014, which is a
continuation of PCT Application No. PCT/US2014/033082, filed Apr.
4, 2014, which claims the benefit of U.S. Provisional Application
Ser. No. 61/808,594, filed Apr. 4, 2013, the teachings of which are
incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0003] Clustered regularly interspaced short palindromic repeats
(CRISPR)/CRISPR-associated (Cas) systems are a new class of
genome-editing tools that target desired genomic sites in mammalian
cells. Recently published type II CRISPR/Cas systems use Cas9
nuclease that is targeted to a genomic site by complexing with a
synthetic guide RNA that hybridizes to a 20-nucleotide DNA sequence
and immediately preceding an NGG motif recognized by Cas9 (thus, a
(N).sub.20NGG target DNA sequence). This results in a double-strand
break three nucleotides upstream of the NGG motif. The double
strand break instigates either non-homologous end-joining, which is
error-prone and conducive to frameshift mutations that knock out
gene alleles, or homology-directed repair, which can be exploited
with the use of an exogenously introduced double-strand or
single-strand DNA repair template to knock in or correct a mutation
in the genome. Thus, CRISPR/Cas systems could be useful tools for
therapeutic applications, but unfortunately prior published reports
have demonstrated an efficiency of allele targeting of only 2%-4%
in human stem cells (Mali et al., Science 339:823-826 (2013)).
SUMMARY OF THE INVENTION
[0004] Work described herein demonstrates methods of allele
targeting using CRISPR/Cas systems resulting in mutant cells with
efficiencies of up to 80%. In particular, work described herein
surprisingly and unexpectedly demonstrates that a multiple guide
strategy (e.g., using two or more ribonucleic acids which guide Cas
protein to and hybridize to a target polynucleotide sequence)
efficiently and effectively deletes target polynucleotide sequences
(e.g., B2M, HPRT. CCR5 and/or CXCR4) in primary somatic cells
(e.g., human blood cells, e.g., CD34+ and T cells), in contrast to
a single guide strategy which has been demonstrated by the
inventors to efficiently delete target polynucleotide sequences in
cell lines (e.g., 293T) but not in primary somatic cells. These
vastly improved methods permit CRISPR/Cas systems to be utilized
effectively for the first time for therapeutic purposes. Methods of
delivery of CRISPR/Cas systems to human stem cells are provided. In
addition, methods of specifically identifying useful RNA guide
sequences are provided, along with particular guide sequences
useful in targeting specific genes (e.g., B2M. HPRT, CCR5 and/or
CXCR4). Moreover, methods of treatment (e.g., methods of treating
HIV infection) utilizing the compositions and methods disclosed
herein are provided.
[0005] In some aspects, the present invention provides a method for
altering a target polynucleotide sequence in a cell comprising
contacting the polynucleotide sequence with a clustered regularly
interspaced short palindromic repeats-associated (Cas) protein and
from one to two ribonucleic acids, wherein the ribonucleic acids
direct Cas protein to and hybridize to a target motif of the target
polynucleotide sequence, wherein the target polynucleotide sequence
is cleaved, and wherein the efficiency of alteration of cells that
express Cas protein is from about 50% to about 80%.
[0006] In some aspects, the present invention provides a method for
treating or preventing a disorder associated with expression of a
polynucleotide sequence in a subject, the method comprising (a)
altering a target polynucleotide sequence in a cell ex vivo by
contacting the polynucleotide sequence with a clustered regularly
interspaced short palindromic repeats-associated (Cas) protein and
from one to two ribonucleic acids, wherein the ribonucleic acids
direct Cas protein to and hybridize to a target motif of the target
polynucleotide sequence, wherein the target polynucleotide sequence
is cleaved, and wherein the efficiency of alteration of cells that
express Cas protein is from about 50% to about 80%, and (b)
introducing the cell into the subject, thereby treating or
preventing a disorder associated with expression of the
polynucleotide sequence.
[0007] In some aspects, the present invention provides a method for
simultaneously altering multiple target polynucleotide sequences in
a cell comprising contacting the polynucleotide sequences with a
clustered regularly interspaced short palindromic
repeats-associated (Cas) protein and multiple ribonucleic acids,
wherein the ribonucleic acids direct Cas protein to and hybridize
to target motifs of the target polynucleotide sequences, wherein
the target polynucleotide sequences are cleaved, and wherein the
efficiency of alteration of cells that express Cas protein is from
about 50% to about 80%.
[0008] In some aspects, the present invention provides a method for
treating or preventing a disorder associated with expression of
polynucleotide sequences in a subject, the method comprising (a)
altering target polynucleotide sequences in a cell ex vivo by
contacting the polynucleotide sequences with a clustered regularly
interspaced short palindromic repeats-associated (Cas) protein and
multiple ribonucleic acids, wherein the ribonucleic acids direct
Cas protein to and hybridize to target motifs of the target
polynucleotide sequences, wherein the target polynucleotide
sequences are cleaved, and wherein the efficiency of alteration of
cells that express Cas protein is from about 50% to about 80%, and
(b) introducing the cell into the subject, thereby treating or
preventing a disorder associated with expression of the
polynucleotide sequences.
[0009] In some embodiments, the Cas protein is Streptococcus
pyogenes Cas9 protein or a functional portion thereof. In some
embodiments, the functional portion comprises a combination of
operably linked Cas9 protein functional domains selected from the
group consisting of a DNA binding domain, at least one RNA binding
domain, a helicase domain, and an endonuclease domain. In some
embodiments, the functional domains form a complex.
[0010] In some embodiments, the Cas protein is Cas9 protein from
any bacterial species or functional portion thereof. In some
embodiments, the functional portion comprises a combination of
operably linked Cas9 protein functional domains selected from the
group consisting of a DNA binding domain, at least one RNA binding
domain, a helicase domain, and an endonuclease domain. In some
embodiments, the functional domains form a complex.
[0011] In some embodiments, the Cas protein is complexed with the
one to two ribonucleic acids. In some embodiments, the Cas protein
is complexed with the multiple ribonucleic acids.
[0012] In some embodiments, the target motif is a 20-nucleotide DNA
sequence. In some embodiments, each target motif is a 20-nucleotide
DNA sequence. In some embodiments, the target motif is a
20-nucleotide DNA sequence beginning with G and immediately
precedes an NGG motif recognized by the Cas protein. In some
embodiments, each target motif is a 20-nucleotide DNA sequence
beginning with G and immediately precedes an NGG motif recognized
by the Cas protein. In some embodiments, the target motif is a
20-nucleotide DNA sequence and immediately precedes an NGG motif
recognized by the Cas protein. In some embodiments, each target
motif is a 20-nucleotide DNA sequence and immediately precedes an
NGG motif recognized by the Cas protein. In some embodiments, the
target motif is G(N).sub.19NGG. In some embodiments, each target
motif is G(N).sub.19NGG. In some embodiments, the target motif is
(N).sub.20NGG. In some embodiments, each target motif is
(N).sub.20NGG.
[0013] In some embodiments, the target polynucleotide sequence is
cleaved such that a double-strand break results. In some
embodiments, each target polynucleotide sequence is cleaved such
that a double-strand break results. In some embodiments, the target
polynucleotide sequence is cleaved such that a single-strand break
results. In some embodiments, each target polynucleotide sequence
is cleaved such that a single-strand break results.
[0014] In some embodiments, the alteration is an indel. In some
embodiments, the alteration results in reduced expression of the
target polynucleotide sequence. In some embodiments, the alteration
results in reduced expression of the target polynucleotide
sequences. In some embodiments, the alteration results in a knock
out of the target polynucleotide sequence.
[0015] In some embodiments, the alteration results in a knock out
of the target polynucleotide sequences. In some embodiments, the
alteration results in correction of the target polynucleotide
sequence from an undesired sequence to a desired sequence. In some
embodiments, the alteration results in correction of the target
polynucleotide sequences from undesired sequences to desired
sequences. In some embodiments, the alteration is a homozygous
alteration. In some embodiments, each alteration is a homozygous
alteration.
[0016] In some embodiments, subsequent to cleavage of the target
polynucleotide sequence, homology-directed repair occurs. In some
embodiments, homology-directed repair is performed using an
exogenously introduced DNA repair template. In some embodiments,
the exogenously introduced DNA repair template is single-stranded.
In some embodiments, the exogenously introduced DNA repair template
is double-stranded.
[0017] In some embodiments, subsequent to cleavage of the target
polynucleotide sequences, homology-directed repair occurs. In some
embodiments, homology-directed repair is performed using an
exogenously introduced DNA repair template. In some embodiments,
the exogenously introduced DNA repair template is single-stranded.
In some embodiments, the exogenously introduced DNA repair template
is double-stranded.
[0018] In some embodiments, the cell is a peripheral blood cell. In
some embodiments, the cell is a stem cell or a pluripotent cell. In
some embodiments, the cell is a hematopoietic stem cell. In some
embodiments, the cell is a CD34.sup.+ cell. In some embodiments,
the cell is a CD34.sup.+ mobilized peripheral blood cell. In some
embodiments, the cell is a CD34.sup.+ cord blood cell. In some
embodiments, the cell is a CD34.sup.+ bone marrow cell. In some
embodiments, the cell is a
CD34.sup.+CD38-Lineage-CD90.sup.+CD45RA.sup.- cell. In some
embodiments, the cell is a hepatocyte.
[0019] In some embodiments, the target polynucleotide sequence is
CCR5. In some embodiments, at least one of the one to two
ribonucleic acids comprises a sequence selected from the group
consisting of the ribonucleic acid sequences of FIG. 1. In some
embodiments, at least one of the one to two ribonucleic acids
comprises a sequence with a single nucleotide mismatch to a
sequence selected from the group consisting of the ribonucleic acid
sequences of FIG. 1.
[0020] In some embodiments, the target polynucleotide sequence is
CXCR4. In some embodiments, at least one of the one to two
ribonucleic acids comprises a sequence selected from the group
consisting of the ribonucleic acid sequences of FIG. 2. In some
embodiments, at least one of the one to two ribonucleic acids
comprises a sequence with a single nucleotide mismatch to a
sequence selected from the group consisting of the ribonucleic acid
sequences of FIG. 2.
[0021] In some embodiments, the target polynucleotide sequences
comprise multiple different portions of CCR5. In some embodiments,
each of the multiple ribonucleic acids comprises a different
sequence selected from the group consisting of the ribonucleic acid
sequences of FIG. 1. In some embodiments, each of the multiple
ribonucleic acids comprises a sequence with a single nucleotide
mismatch to a different sequence selected from the group consisting
of the ribonucleic acid sequences of FIG. 1.
[0022] In some embodiments, the target polynucleotide sequences
comprise multiple different portions of CXCR4. In some embodiments,
each of the multiple ribonucleic acids comprises a different
sequence selected from the group consisting of the ribonucleic acid
sequences of FIG. 2. In some embodiments, each of the multiple
ribonucleic acids comprises a sequence with a single nucleotide
mismatch to a different sequence selected from the group consisting
of the ribonucleic acid sequences of FIG. 2.
[0023] In some embodiments, the target polynucleotide sequences
comprise at least a portion of CCR5 and at least a portion of
CXCR4. In some embodiments, each of the multiple ribonucleic acids
comprises a different sequence selected from the group consisting
of the ribonucleic acid sequences of FIG. 1 and the ribonucleic
acid sequences of FIG. 2. In some embodiments, each of the multiple
ribonucleic acids comprises a sequence with a single nucleotide
mismatch to a different sequence selected from the group consisting
of the ribonucleic acid sequences of FIG. 1 and the ribonucleic
acid sequences of FIG. 2.
[0024] In some embodiments, the disorder is a genetic disorder. In
some embodiments, the disorder is a monogenic disorder. In some
embodiments, the disorder is human immunodeficiency virus (HIV)
infection. In some embodiments, the disorder is acquired
immunodeficiency syndrome (AIDS).
[0025] In some embodiments, the one to two ribonucleic acids are
designed to hybridize to a target motif immediately adjacent to a
deoxyribonucleic acid motif recognized by the Cas protein. In some
embodiments, each of the one to two ribonucleic acids are designed
to hybridize to target motifs immediately adjacent to
deoxyribonucleic acid motifs recognized by the Cas protein which
flank a mutant allele located between the target motifs. In some
embodiments, the multiple ribonucleic acids are designed to
hybridize to target motifs immediately adjacent to deoxyribonucleic
acid motifs recognized by the Cas protein. In some embodiments, the
multiple ribonucleic acids are designed to hybridize to target
motifs immediately adjacent to deoxyribonucleic acid motifs
recognized by the Cas protein which flank mutant alleles located
between the target motifs. In some embodiments, the one to two
ribonucleic acids are selected to minimize hybridization with
nucleic acid sequences other than the target polynucleotide
sequence. In some embodiments, the multiple ribonucleic acids are
selected to minimize hybridization with nucleic acid sequences
other than the target polynucleotide sequence.
[0026] In some embodiments, the target motif is selected such that
it contains at least two mismatches when compared with all other
genomic nucleotide sequences in the cell. In some embodiments, each
target motif is selected such that it contains at least two
mismatches when compared with all other genomic nucleotide
sequences in the cell. In some embodiments, the target motif is
selected such that it contains at least one mismatch when compared
with all other genomic nucleotide sequences in the cell.
[0027] In some embodiments, the target motif is selected such that
it contains at least one mismatch when compared with all other
genomic nucleotide sequences in the cell. In some embodiments, the
one to two ribonucleic acids hybridize to a target motif that it
contains at least two mismatches when compared with all other
genomic nucleotide sequences in the cell.
[0028] In some embodiments, each of the multiple ribonucleic acids
hybridize to target motifs that contain at least two mismatches
when compared with all other genomic nucleotide sequences in the
cell. In some embodiments, the one to two ribonucleic acids
hybridize to a target motif that contains at least one mismatch
when compared with all other genomic nucleotide sequences in the
cell. In some embodiments, each of the multiple ribonucleic acids
hybridize to target motifs that contain at least one mismatch when
compared with all other genomic nucleotide sequences in the
cell.
[0029] In some embodiments, the efficiency of alteration at each
loci is from about 50% to about 80%. In some embodiments, the
efficiency of alteration is at least about 5%. In some embodiments,
the efficiency of alteration is at least about 10%. In some
embodiments, the efficiency of alteration is from about 50% to
about 80%.
[0030] In some embodiments, the Cas protein is encoded by a
modified nucleic acid. In some embodiments, the modified nucleic
acid comprises a ribonucleic acid containing at least one modified
nucleotide selected from the group consisting of pseudouridine,
5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate,
4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate,
and 5-azauridine-5'-triphosphate. In some embodiments, at least one
of the ribonucleic acids is a modified ribonucleic acid comprising
one to two modified nucleotides selected from the group consisting
of pseudouridine, 5-methylcytodine, 2-thio-uridine,
5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate,
5,6-dihydrouridine-5'-triphosphate, and
5-azauridine-5'-triphosphate.
[0031] In some embodiments, any of the Cas protein or the
ribonucleic acids are expressed from a plasmid.
[0032] In some embodiments, any of the Cas protein or the
ribonucleic acids are expressed using a promoter optimized for
increased expression in stem cells. In some embodiments, the
promoter is selected from the group consisting of a Cytomegalovirus
(CMV) early enhancer element and a chicken beta-actin promoter, a
chicken beta-actin promoter, an elongation factor-1 alpha promoter,
and a ubiquitin promoter.
[0033] In some embodiments, the method further comprises selecting
cells that express the Cas protein. In some embodiments, selecting
cells comprises FACS. In some embodiments, FACs is used to select
cells which co-express Cas and a fluorescent protein selected from
the group consisting of green fluorescent protein and red
fluorescent protein.
[0034] In some aspects, the present invention provides a method for
altering a target polynucleotide sequence in a cell comprising
contacting the polynucleotide sequence in a cell selected from the
group consisting of a human pluripotent cell, a primary human cell,
and a non-transformed human cell, with a clustered regularly
interspaced short palindromic repeats-associated (Cas) protein and
from one to two ribonucleic acids, wherein the ribonucleic acids
direct Cas protein to and hybridize to a target motif of the target
polynucleotide sequence, wherein the target polynucleotide sequence
is cleaved, and wherein the efficiency of alteration of cells that
express Cas protein is from about 8% to about 80%.
[0035] In some aspects, the present invention provides a method for
treating or preventing a disorder associated with expression of a
polynucleotide sequence in a subject, the method comprising (a)
altering a target polynucleotide sequence in a cell ex vivo by
contacting the polynucleotide sequence in a cell selected from the
group consisting of a human pluripotent cell, a primary human cell,
and a non-transformed human cell, with a clustered regularly
interspaced short palindromic repeats-associated (Cas) protein and
from one to two ribonucleic acids, wherein the ribonucleic acids
direct Cas protein to and hybridize to a target motif of the target
polynucleotide sequence, wherein the target polynucleotide sequence
is cleaved, and wherein the efficiency of alteration is from about
8% to about 80%, and (b) introducing the cell into the subject,
thereby treating or preventing a disorder associated with
expression of the polynucleotide sequence.
[0036] In some aspects, the present invention provides a method for
simultaneously altering multiple target polynucleotide sequences in
a cell comprising contacting the polynucleotide sequences in a cell
selected from the group consisting of a human pluripotent cell, a
primary human cell, and a non-transformed human cell, with a
clustered regularly interspaced short palindromic
repeats-associated (Cas) protein and multiple ribonucleic acids,
wherein the ribonucleic acids direct Cas protein to and hybridize
to target motifs of the target polynucleotide sequences, wherein
the target polynucleotide sequences are cleaved, and wherein the
efficiency of alteration of cells that express Cas protein is from
about 8% to about 80%.
[0037] In some aspects, the present invention provides a method for
treating or preventing a disorder associated with expression of
polynucleotide sequences in a subject, the method comprising (a)
altering target polynucleotide sequences in a cell ex vivo by
contacting the polynucleotide sequences in a cell selected from the
group consisting of a human pluripotent cell, a primary human cell,
and a non-transformed human cell, with a clustered regularly
interspaced short palindromic repeats-associated (Cas) protein and
multiple ribonucleic acids, wherein the ribonucleic acids direct
Cas protein to and hybridize to target motifs of the target
polynucleotide sequences, wherein the target polynucleotide
sequences are cleaved, and wherein the efficiency of alteration of
cells that express Cas protein is from about 8% to about 80%, and
(b) introducing the cell into the subject, thereby treating or
preventing a disorder associated with expression of the
polynucleotide sequences.
[0038] In some aspects, the present invention provides a
composition comprising at least one ribonucleic acid having a
sequence selected from the group consisting of the ribonucleic acid
sequences of FIG. 1.
[0039] In some aspects, the present invention provides a
composition comprising at least one ribonucleic acid comprising a
sequence with a single nucleotide mismatch to a sequence selected
from the group consisting of the ribonucleic acid sequences of FIG.
1.
[0040] In some aspects, the present invention provides a
composition comprising at least one ribonucleic acid having a
sequence selected from the group consisting of the ribonucleic acid
sequences of FIG. 2.
[0041] In some aspects, the present invention provides a
composition comprising at least one ribonucleic acid comprising a
sequence with a single nucleotide mismatch to a sequence selected
from the group consisting of the ribonucleic acid sequences of FIG.
2.
[0042] In some aspects, the present invention provides a
composition comprising at least one ribonucleic acid having a
sequence selected from the group consisting of the ribonucleic acid
sequences of FIG. 1, the ribonucleic acid sequences of FIG. 2, a
sequence with a single nucleotide mismatch to a ribonucleic acid
sequence of FIG. 1, and a sequence with a single nucleotide
mismatch to a ribonucleic acid sequences of FIG. 2.
[0043] In some embodiments, the composition further comprises a
nucleic acid sequence encoding a Cas protein. In some embodiments,
the composition further comprises a nucleic acid sequence encoding
a Cas9 protein or a functional portion thereof. In some
embodiments, the nucleic acid comprises a modified ribonucleic acid
comprising at least one modified nucleotide selected from the group
consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine,
5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate,
5,6-dihydrouridine-5'-triphosphate, and
5-azauridine-5'-triphosphate.
[0044] In some aspects, the present invention provides a
composition comprising a chimeric nucleic acid comprising a
ribonucleic acid encoding a Cas protein and at least one additional
ribonucleic acid having a sequence selected from the group
consisting of the ribonucleic acid sequences of FIG. 1.
[0045] In some aspects, the present invention provides a
composition comprising a chimeric nucleic acid comprising a
ribonucleic acid encoding a Cas protein and at least one additional
ribonucleic acid having a sequence selected from the group
consisting of the ribonucleic acid sequences of FIG. 2.
[0046] In some aspects, the present invention provides a
composition comprising a chimeric nucleic acid comprising a
ribonucleic acid encoding a Cas protein and at least one additional
ribonucleic acid sequence comprising a sequence with a single
nucleotide mismatch to a sequence selected from the group
consisting of the ribonucleic acid sequences of FIG. 1.
[0047] In some aspects, the present invention provides a
composition comprising a chimeric nucleic acid comprising a
ribonucleic acid encoding a Cas protein and at least one additional
ribonucleic acid sequence comprising a sequence with a single
nucleotide mismatch to a sequence selected from the group
consisting of the ribonucleic acid sequences of FIG. 2.
[0048] In some aspects, the present invention provides a
composition comprising a chimeric nucleic acid comprising a
ribonucleic acid encoding a Cas protein and at least one additional
ribonucleic acid having a sequence selected from the group
consisting of the ribonucleic acid sequences of FIG. 1, the
ribonucleic acid sequences of FIG. 2, a sequence with a single
nucleotide mismatch to a ribonucleic acid sequence of FIG. 1, and a
sequence with a single nucleotide mismatch to a ribonucleic acid
sequences of FIG. 2.
[0049] In some embodiments, the composition further comprises a
nucleic acid sequence encoding a fluorescent protein selected from
the group consisting of green fluorescent protein and red
fluorescent protein.
[0050] In some embodiments, the composition further comprises a
promoter operably linked to the chimeric nucleic acid. In some
embodiments, the promoter is optimized for increased expression in
human stem cells. In some embodiments, the promoter is selected
from the group consisting of a Cytomegalovirus (CMV) early enhancer
element and a chicken beta-actin promoter, a chicken beta-actin
promoter, an elongation factor-1 alpha promoter, and a ubiquitin
promoter.
[0051] In some embodiments, the Cas protein comprises a Cas9
protein or a functional portion thereof.
[0052] In some aspects, the present invention provides a kit for
altering a target polynucleotide sequence in a cell comprising a
Cas9 protein or a nucleic acid encoding the Cas9 protein, and at
least one ribonucleic acid sequence selected from the group
consisting of the ribonucleic acid sequences of FIG. 1, the
ribonucleic acid sequences of FIG. 2, a sequence with a single
nucleotide mismatch to a ribonucleic acid sequence of FIG. 1, and a
sequence with a single nucleotide mismatch to a ribonucleic acid
sequences of FIG. 2. In some embodiments, the kit further comprises
one or more cell lines, cultures, or populations selected from the
group consisting of human pluripotent cells, primary human cells,
and non-transformed cells. In some embodiments, the kit further
comprises a DNA repair template.
[0053] In some embodiments, the cell comprises a primary cell. In
some embodiments, the cell comprises a primary somatic cell. In
some embodiments, the cell comprises an autologous primary somatic
cell. In some embodiments, the cell comprises an allogeneic primary
somatic cell. In some embodiments, the target polynucleotide
sequence is B2M. In some embodiments, at least one of the one to
two ribonucleic acids comprises a sequence optimized to target the
B2M gene. In some embodiments, at least one of the one to two
ribonucleic acids comprises a sequence with a single nucleotide
mismatch to a sequence optimized to target the B2M gene. In some
embodiments, the target polynucleotide sequences comprises multiple
different portions of B2M. In some embodiments, each of the
multiple ribonucleic acids comprises a different sequence optimized
to target the B2M gene. In some embodiments, each of the multiple
ribonucleic acids comprises a sequence with a single nucleotide
mismatch to a different sequence optimized to target the B2M gene.
In some embodiments, the one to two ribonucleic acids comprise two
guide ribonucleic acid sequences.
[0054] In some embodiments, the one to two ribonucleic acids
comprise two guide ribonucleic acid sequences. In some embodiments,
the target polynucleotide sequence comprises CCR5. In some
embodiments, the cell comprises a primary CD34+ hematopoietic
progenitor cell. In some embodiments, the two guide ribonucleic
acid sequences comprise any combination of two guide ribonucleic
acid sequences which are complementary to a different sequence
selected from the group consisting of SEQ ID NOs: 1-139. In some
embodiments, the two guide ribonucleic acid sequences comprise any
combination of two guide ribonucleic acid sequences which are
complementary to offset sequences selected from the group
consisting of SEQ ID NOs: 1-139. In some embodiments, the two guide
ribonucleic acid sequences comprise any combination of two guide
ribonucleic acid sequences which hybridize to and target Cas
protein to offset target sites in CCR5 selected from the group
consisting of SEQ ID NOs: 1-139. In some embodiments, the two guide
ribonucleic acid sequences comprise any combination of two guide
ribonucleic acid sequences from SEQ ID NOs: 298-303. In some
embodiments, the two guide ribonucleic acid sequences comprise a
pair of guide ribonucleic acids selected from the group consisting
of SEQ ID NOs: 299 and 303, SEQ ID NOs: 298 and 300, SEQ ID NOs:
299 and 300, SEQ ID NOs: 298 and 303, SEQ ID NOs: 299 and 301, SEQ
ID NOs: 298 and 299, SEQ ID NOs: 301 and 303, SEQ ID NOs: 298 and
302, and SEQ ID NOs: 298 and 301. In some embodiments, the two
guide ribonucleic acid sequences comprise any combination of two
guide ribonucleic acid sequences which are complementary to a
different sequence selected from the group consisting of SEQ ID
NOs: 304-333. In some embodiments, the two guide ribonucleic acid
sequences comprise any combination of two guide ribonucleic acid
sequences which are complementary to offset sequences selected from
the group consisting of SEQ ID NOs: 304-333. In some embodiments,
the two guide ribonucleic acid sequences comprise any combination
of two guide ribonucleic acid sequences which hybridize to and
target Cas protein to offset target sites in CCR5 selected from the
group consisting of SEQ ID NOs: 304-333. In some embodiments, the
target polynucleotide sequence comprises CXCR4. In some
embodiments, the cell comprises a primary CD34+ hematopoietic
progenitor cell. In some embodiments, the two guide ribonucleic
acid sequences comprise any combination of two guide ribonucleic
acid sequences which are complementary to a different sequence
selected from the group consisting of SEQ ID NOs: 140-297. In some
embodiments, the two guide ribonucleic acid sequences comprise any
combination of two guide ribonucleic acid sequences which are
complementary to offset sequences selected from the group
consisting of SEQ ID NOs: 140-297. In some embodiments, the two
guide ribonucleic acid sequences comprise any combination of two
guide ribonucleic acid sequences which hybridize to and target Cas
protein to offset target sites in CXCR4 selected from the group
consisting of SEQ ID NOs: 140-297. In some embodiments, the target
polynucleotide sequence comprises B2M. In some embodiments, the
cell comprises a primary cell. In some embodiments, the two guide
ribonucleic acid sequences comprise any combination of two guide
ribonucleic acid sequences which are complementary to different
sequences in the B2M gene. In some embodiments, the two guide
ribonucleic acid sequences comprise any combination of two guide
ribonucleic acid sequences which are complementary to offset
sequences in the B2M gene. In some embodiments, the two guide
ribonucleic acid sequences comprise any combination of two guide
ribonucleic acid sequences which hybridize to and target Cas
protein to offset target sites in B2M.
[0055] In some aspects, the invention provides a method for
altering a target polynucleotide sequence in a primary cell
comprising contacting the polynucleotide sequence with a clustered
regularly interspaced short palindromic repeats-associated (Cas)
protein and at least two ribonucleic acids, wherein the at least
two ribonucleic acids comprise guide ribonucleic acids which direct
Cas protein to and hybridize to a target motif of the target
polynucleotide sequence, wherein the target polynucleotide sequence
is cleaved. In some embodiments, the efficiency of alteration of
cells that express Cas protein is from about 50% to about 80%.
[0056] In some aspects, the invention provides a method for
altering a target polynucleotide sequence in a primary cell
comprising contacting the polynucleotide sequence with a clustered
regularly interspaced short palindromic repeats-associated (Cas)
protein and at least two ribonucleic acids, wherein the at least
two ribonucleic acids comprise guide ribonucleic acids which direct
Cas protein to and hybridize to a target motif of the target
polynucleotide sequence, wherein the target polynucleotide sequence
is cleaved, and wherein the efficiency of alteration of cells that
express Cas protein is from about 50% to about 80%.
[0057] In some aspects, the invention provides a method for
treating or preventing a disorder associated with expression of a
polynucleotide sequence in a subject, the method comprising (a)
altering a target polynucleotide sequence in a primary cell ex vivo
by contacting the polynucleotide sequence with a clustered
regularly interspaced short palindromic repeats-associated (Cas)
protein and at least two ribonucleic acids, wherein the at least
two ribonucleic acids comprise guide ribonucleic acids which direct
Cas protein to and hybridize to a target motif of the target
polynucleotide sequence, wherein the target polynucleotide sequence
is cleaved, and (b) introducing the cell into the subject, thereby
treating or preventing a disorder associated with expression of the
polynucleotide sequence. In some embodiments, the efficiency of
alteration of cells that express Cas protein is from about 50% to
about 80%.
[0058] In some aspects, the invention provides a method for
treating or preventing a disorder associated with expression of a
polynucleotide sequence in a subject, the method comprising (a)
altering a target polynucleotide sequence in a primary cell ex vivo
by contacting the polynucleotide sequence with a clustered
regularly interspaced short palindromic repeats-associated (Cas)
protein and at least two ribonucleic acids, wherein the at least
two ribonucleic acids comprise guide ribonucleic acids which direct
Cas protein to and hybridize to a target motif of the target
polynucleotide sequence, wherein the target polynucleotide sequence
is cleaved, and wherein the efficiency of alteration of cells that
express Cas protein is from about 50% to about 80%, and (b)
introducing the cell into the subject, thereby treating or
preventing a disorder associated with expression of the
polynucleotide sequence.
[0059] In some aspects, the invention provides, a method for
simultaneously altering multiple target polynucleotide sequences in
a primary cell comprising contacting the polynucleotide sequences
with a clustered regularly interspaced short palindromic
repeats-associated (Cas) protein and multiple ribonucleic acids,
wherein the ribonucleic acids comprise guide ribonucleic acids
which direct Cas protein to and hybridize to target motifs of the
target polynucleotide sequences, wherein the target polynucleotide
sequences are cleaved. In some embodiments, the efficiency of
alteration of cells that express Cas protein is from about 50% to
about 80%.
[0060] In some aspects, the invention provides, a method for
simultaneously altering multiple target polynucleotide sequences in
a primary cell comprising contacting the polynucleotide sequences
with a clustered regularly interspaced short palindromic
repeats-associated (Cas) protein and multiple ribonucleic acids,
wherein the ribonucleic acids comprise guide ribonucleic acids
which direct Cas protein to and hybridize to target motifs of the
target polynucleotide sequences, wherein the target polynucleotide
sequences are cleaved, and wherein the efficiency of alteration of
cells that express Cas protein is from about 50% to about 80%.
[0061] In some aspects, the disclosure provides a method for
treating or preventing a disorder associated with expression of
polynucleotide sequences in a subject, the method comprising (a)
altering target polynucleotide sequences in a primary cell ex vivo
by contacting the polynucleotide sequences with a clustered
regularly interspaced short palindromic repeats-associated (Cas)
protein and multiple ribonucleic acids, wherein the ribonucleic
acids comprise guide ribonucleic acids which direct Cas protein to
and hybridize to target motifs of the target polynucleotide
sequences, wherein the target polynucleotide sequences are cleaved,
and (b) introducing the cell into the subject, thereby treating or
preventing a disorder associated with expression of the
polynucleotide sequences. In some embodiments, the efficiency of
alteration of cells that express Cas protein is from about 50% to
about 80%.
[0062] In some aspects, the disclosure provides a method for
treating or preventing a disorder associated with expression of
polynucleotide sequences in a subject, the method comprising (a)
altering target polynucleotide sequences in a primary cell ex vivo
by contacting the polynucleotide sequences with a clustered
regularly interspaced short palindromic repeats-associated (Cas)
protein and multiple ribonucleic acids, wherein the ribonucleic
acids comprise guide ribonucleic acids which direct Cas protein to
and hybridize to target motifs of the target polynucleotide
sequences, wherein the target polynucleotide sequences are cleaved,
and wherein the efficiency of alteration of cells that express Cas
protein is from about 50% to about 80%, and (b) introducing the
cell into the subject, thereby treating or preventing a disorder
associated with expression of the polynucleotide sequences.
[0063] In some embodiments, the Cas protein is Streptococcus
pyogenes Cas9 protein or a functional portion thereof. In some
embodiments, the functional portion comprises a combination of
operably linked Cas9 protein functional domains selected from the
group consisting of a DNA binding domain, at least one RNA binding
domain, a helicase domain, and an endonuclease domain. In some
embodiments, the functional domains form a complex. In some
embodiments, the Cas protein is Cas9 protein from any bacterial
species or functional portion thereof. In some embodiments, the
functional portion comprises a combination of operably linked Cas9
protein functional domains selected from the group consisting of a
DNA binding domain, at least one RNA binding domain, a helicase
domain, and an endonuclease domain. In some embodiments, the
functional domains form a complex. In some embodiments, the Cas
protein is complexed with the one to two ribonucleic acids. In some
embodiments, the Cas protein is complexed with the multiple
ribonucleic acids.
[0064] In some embodiments, the target motif is a 20-nucleotide DNA
sequence. In some embodiments, each target motif is a 20-nucleotide
DNA sequence. In some embodiments, the target motif is a
20-nucleotide DNA sequence beginning with G and immediately
precedes an NGG motif recognized by the Cas protein. In some
embodiments, each target motif is a 20-nucleotide DNA sequence
beginning with G and immediately precedes an NGG motif recognized
by the Cas protein. In some embodiments, the target motif is a
20-nucleotide DNA sequence and immediately precedes an NGG motif
recognized by the Cas protein. In some embodiments, each target
motif is a 20-nucleotide DNA sequence and immediately precedes an
NGG motif recognized by the Cas protein. In some embodiments, the
target motif is G(N).sub.19NGG. In some embodiments, each target
motif is G(N).sub.19NGG. In some embodiments, the target motif is
(N).sub.20NGG. In some embodiments, each target motif is
(N).sub.20NGG. In some embodiments, the target motif comprises a
sequence selected from the group consisting of SEQ ID NOs: 1-297 or
304-333. In some embodiments, the target motif comprises a sequence
selected from the group consisting of SEQ ID NOs: 1-297 or 304-333.
In some embodiments, the target polynucleotide sequence is cleaved
such that a double-strand break results. In some embodiments, each
target polynucleotide sequence is cleaved such that a double-strand
break results. In some embodiments, the target polynucleotide
sequence is cleaved such that a single-strand break results. In
some embodiments, each target polynucleotide sequence is cleaved
such that a single-strand break results. In some embodiments, the
alteration is an indel. In some embodiments, the alteration results
in reduced expression of the target polynucleotide sequence. In
some embodiments, the alteration results in reduced expression of
the target polynucleotide sequences. In some embodiments, the
alteration results in a knock out of the target polynucleotide
sequence. In some embodiments, the alteration results in a knock
out of the target polynucleotide sequences. In some embodiments,
the alteration results in correction of the target polynucleotide
sequence from an undesired sequence to a desired sequence. In some
embodiments, the alteration results in correction of the target
polynucleotide sequences from undesired sequences to desired
sequences. In some embodiments, the alteration is a homozygous
alteration.
[0065] In some embodiments, each alteration is a homozygous
alteration. In some embodiments, subsequent to cleavage of the
target polynucleotide sequence, homology-directed repair occurs. In
some embodiments, homology-directed repair is performed using an
exogenously introduced DNA repair template. In some embodiments,
the exogenously introduced DNA repair template is single-stranded.
In some embodiments, the exogenously introduced DNA repair template
is double-stranded. In some embodiments, subsequent to cleavage of
the target polynucleotide sequences, homology-directed repair
occurs. In some embodiments, homology-directed repair is performed
using an exogenously introduced DNA repair template. In some
embodiments, the exogenously introduced DNA repair template is
single-stranded. In some embodiments, the exogenously introduced
DNA repair template is double-stranded. In some embodiments, the
cell is a peripheral blood cell. In some embodiments, the cell is a
stem cell or a pluripotent cell. In some embodiments, the cell is a
hematopoietic stem cell. In some embodiments, the cell is a
CD34.sup.+ cell. In some embodiments, the cell is a CD34.sup.+
mobilized peripheral blood cell. In some embodiments, the cell is a
CD34.sup.+ cord blood cell. In some embodiments, the cell is a
CD34.sup.+ bone marrow cell. In some embodiments, the cell is a
CD34.sup.+CD38-Lineage-CD90.sup.+CD45RA.sup.- cell. In some
embodiments, the cell is a hepatocyte. In some embodiments, the
cell is a primary cell. In some embodiments, the target
polynucleotide sequence is CCR5.
[0066] In some embodiments, the two ribonucleic acids comprise a
different sequence selected from the group consisting of SEQ ID
NOs: 298-303. In some embodiments, the two guide ribonucleic acid
sequences comprise a pair of guide ribonucleic acids selected from
the group consisting of SEQ ID NOs: 299 and 303, SEQ ID NOs: 298
and 300, SEQ ID NOs: 299 and 300, SEQ ID NOs: 298 and 303. SEQ ID
NOs: 299 and 301, SEQ ID NOs: 298 and 299, SEQ ID NOs: 301 and 303,
SEQ ID NOs: 298 and 302, and SEQ ID NOs: 298 and 301. In some
embodiments, the two ribonucleic acids comprise sequences which are
complementary to and/or hybridize to different sequences selected
from the group consisting of SEQ ID NOs: 1-139 and 304-333. In some
embodiments, the two ribonucleic acids comprise sequences which are
complementary to and/or hybridize to different sequences with a
single nucleotide mismatch to a sequence selected from the group
consisting of SEQ ID NOs: 1-139 and 304-333. In some embodiments,
the two ribonucleic acids comprise sequences which are
complementary to and/or hybridizes to offset sequences selected
from the group consisting of SEQ ID NOs: 1-139 and 304-333. In some
embodiments, the two ribonucleic acids comprise sequences which are
complementary to and/or hybridize to offsets sequences with a
single nucleotide mismatch to a sequence selected from the group
consisting of SEQ ID NOs: 1-139 and 304-333. In some embodiments,
the target polynucleotide sequence is CXCR4. In some embodiments,
the two ribonucleic acids comprise sequences which are
complementary to and/or hybridize to different sequences selected
from the group consisting of SEQ ID NO: 140-297. In some
embodiments, the two ribonucleic acids comprise sequences which are
complementary to and/or hybridize to different sequences with a
single nucleotide mismatch to a sequence selected from the group
consisting of SEQ ID NO: 140-297. In some embodiments, the two
ribonucleic acids comprise sequences which are complementary to
and/or hybridize to offset sequences selected from the group
consisting of SEQ ID NO: 140-297. In some embodiments, the two
ribonucleic acids comprise sequences which are complementary to
and/or hybridize to offset sequences with a single nucleotide
mismatch to a sequence selected from the group consisting of SEQ ID
NO: 140-297. In some embodiments, the target polynucleotide
sequences comprise multiple different portions of CCR5. In some
embodiments, each of the multiple ribonucleic acids comprises a
sequence which is complementary to and/or hybridizes to a different
sequence selected from the group consisting of SEQ ID NOs: 1-139
and 304-333. In some embodiments, each of the multiple ribonucleic
acids comprises a sequence which is complementary to and/or
hybridizes to an offset sequence selected from the group consisting
of SEQ ID NOs: 1-139 and 304-333. In some embodiments, each of the
multiple ribonucleic acids comprises a sequence which is
complementary to and/or hybridizes to a sequence with a single
nucleotide mismatch to a different sequence selected from the group
consisting of SEQ ID NOs: 1-139 and 304-333. In some embodiments,
each of the multiple ribonucleic acids comprises a sequence which
is complementary to and/or hybridizes to a sequence with a single
nucleotide mismatch to an offset sequence selected from the group
consisting of SEQ ID NOs: 1-139 and 304-333.
[0067] In some embodiments, the target polynucleotide sequences
comprise multiple different portions of CXCR4. In some embodiments,
each of the multiple ribonucleic acids comprises a sequence which
is complementary to and/or hybridizes to a different sequence
selected from the group consisting of SEQ ID NOs: 140-297 and
304-333. In some embodiments, each of the multiple ribonucleic
acids comprises a sequence which is complementary to and/or
hybridizes to an offset sequence selected from the group consisting
of SEQ ID NOs: 140-297 and 304-333. In some embodiments, each of
the multiple ribonucleic acids comprises a sequence which is
complementary to and/or hybridizes to a sequence with a single
nucleotide mismatch to a different sequence selected from the group
consisting of SEQ ID NOs: 140-297 and 304-333. In some embodiments,
each of the multiple ribonucleic acids comprises a sequence which
is complementary to and/or hybridizes to a sequence with a single
nucleotide mismatch to an offset sequence selected from the group
consisting of SEQ ID NOs: 140-297 and 304-333.
[0068] In some embodiments, the target polynucleotide sequences
comprise at least a portion of CCR5 and at least a portion of
CXCR4. In some embodiments, each of the multiple ribonucleic acids
comprises a sequence which is complementary to and/or hybridizes to
a different sequence selected from the group consisting of SEQ ID
NOs: 1-297 and 304-333. In some embodiments, each of the multiple
ribonucleic acids comprises a sequence which is complementary to
and/or hybridizes to an offset sequence selected from the group
consisting of SEQ ID NOs: 1-297 and 304-333. In some embodiments,
each of the multiple ribonucleic acids comprises a sequence which
is complementary to and/or hybridizes to a sequence with a single
nucleotide mismatch to a different sequence selected from the group
consisting of SEQ ID NOs: 1-297 and 304-333. In some embodiments,
each of the multiple ribonucleic acids comprises a sequence which
is complementary to and/or hybridizes to a sequence with a single
nucleotide mismatch to an offset sequence selected from the group
consisting of SEQ ID NOs: 1-297 and 304-333. In some embodiments,
the multiple ribonucleic acids comprise at least two ribonucleic
acid sequences which are complementary to and/or hybridize to
offset sequences selected from the group consisting of SEQ ID NOs:
1-139 and 304-333, and at least two ribonucleic acid sequences
which are complementary to and/or hybridize to offset sequences
selected from the group consisting of SEQ ID NOs: 140-297. In some
embodiments, the multiple ribonucleic acids comprises at least two
ribonucleic acid sequences which are complementary to and/or
hybridize to different sequences with a single nucleotide mismatch
to an offset sequence selected from the group consisting of SEQ ID
NOs: 1-139 and 304-333, and at least two ribonucleic acid sequences
which are complementary to and/or hybridize to different sequences
with a single nucleotide mismatch to an offset sequence selected
from the group consisting of SEQ ID NOs: 140-297.
[0069] In some embodiments, the disorder is a genetic disorder. In
some embodiments, the disorder is a monogenic disorder. In some
embodiments, the disorder is human immunodeficiency virus (HIV)
infection. In some embodiments, the disorder is acquired
immunodeficiency syndrome (AIDS). In some embodiments, the two
ribonucleic acids are designed to hybridize to a target motif
immediately adjacent to a deoxyribonucleic acid motif recognized by
the Cas protein. In some embodiments, each of the two ribonucleic
acids are designed to hybridize to target motifs immediately
adjacent to deoxyribonucleic acid motifs recognized by the Cas
protein which flank a mutant allele located between the target
motifs. In some embodiments, the multiple ribonucleic acids are
designed to hybridize to target motifs immediately adjacent to
deoxyribonucleic acid motifs recognized by the Cas protein. In some
embodiments, the multiple ribonucleic acids are designed to
hybridize to target motifs immediately adjacent to deoxyribonucleic
acid motifs recognized by the Cas protein which flank mutant
alleles located between the target motifs. In some embodiments, the
two ribonucleic acids are selected to minimize hybridization with
nucleic acid sequences other than the target polynucleotide
sequence. In some embodiments, the multiple ribonucleic acids are
selected to minimize hybridization with nucleic acid sequences
other than the target polynucleotide sequence. In some embodiments,
the target motif is selected such that it contains at least two
mismatches when compared with all other genomic nucleotide
sequences in the cell. In some embodiments, each target motif is
selected such that it contains at least two mismatches when
compared with all other genomic nucleotide sequences in the cell.
In some embodiments, the target motif is selected such that it
contains at least one mismatch when compared with all other genomic
nucleotide sequences in the cell. In some embodiments, the target
motif is selected such that it contains at least one mismatch when
compared with all other genomic nucleotide sequences in the cell.
In some embodiments, the two ribonucleic acids hybridize to a
target motif that contains at least two mismatches when compared
with all other genomic nucleotide sequences in the cell. In some
embodiments, each of the multiple ribonucleic acids hybridize to
target motifs that contain at least two mismatches when compared
with all other genomic nucleotide sequences in the cell.
[0070] In some embodiments, the two ribonucleic acids hybridize to
a target motif that contains at least one mismatch when compared
with all other genomic nucleotide sequences in the cell. In some
embodiments, each of the multiple ribonucleic acids hybridize to
target motifs that contain at least one mismatch when compared with
all other genomic nucleotide sequences in the cell. In some
embodiments, the efficiency of alteration at each loci is from
about 50% to about 80%.
[0071] In some embodiments, the Cas protein is encoded by a
modified nucleic acid. In some embodiments, the modified nucleic
acid comprises a ribonucleic acid containing at least one modified
nucleotide selected from the group consisting of pseudouridine,
5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate,
4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate,
and 5-azauridine-5'-triphosphate. In some embodiments, at least one
of the two ribonucleic acids is a modified ribonucleic acid
comprising one to two modified nucleotides selected from the group
consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine,
5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate,
5,6-dihydrouridine-5'-triphosphate, and
5-azauridine-5'-triphosphate. In some embodiments, the two
ribonucleic acids comprise modified ribonucleic acids comprising
one to two modified nucleotides selected from the group consisting
of pseudouridine, 5-methylcytodine, 2-thio-uridine,
5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate,
5,6-dihydrouridine-5'-triphosphate, and
5-azauridine-5'-triphosphate.
[0072] In some embodiments, any of the Cas protein or the
ribonucleic acids are expressed from a plasmid. In some
embodiments, any of the Cas protein or the ribonucleic acids are
expressed using a promoter optimized for increased expression in
stem cells. In some embodiments, the promoter is selected from the
group consisting of a Cytomegalovirus (CMV) early enhancer element
and a chicken beta-actin promoter, a chicken beta-actin promoter,
an elongation factor-1 alpha promoter, and a ubiquitin promoter. In
some embodiments, the method comprises selecting cells that express
the Cas protein. In some embodiments, selecting cells comprises
FACS. In some embodiments, FACs is used to select cells which
co-express Cas and a fluorescent protein.
[0073] In some aspects, the invention provides a method for
altering a target polynucleotide sequence in a cell comprising
contacting the polynucleotide sequence in a cell selected from the
group consisting of a human pluripotent cell, a primary human cell,
and a non-transformed human cell, with a clustered regularly
interspaced short palindromic repeats-associated (Cas) protein and
two ribonucleic acids, wherein the ribonucleic acids direct Cas
protein to and hybridize to a target motif of the target
polynucleotide sequence, wherein the target polynucleotide sequence
is cleaved. In some embodiments, the efficiency of alteration of
cells that express Cas protein is from about 8% to about 80%.
[0074] In some aspects, the invention provides a method for
altering a target polynucleotide sequence in a cell comprising
contacting the polynucleotide sequence in a cell selected from the
group consisting of a human pluripotent cell, a primary human cell,
and a non-transformed human cell, with a clustered regularly
interspaced short palindromic repeats-associated (Cas) protein and
two ribonucleic acids, wherein the ribonucleic acids direct Cas
protein to and hybridize to a target motif of the target
polynucleotide sequence, wherein the target polynucleotide sequence
is cleaved, and wherein the efficiency of alteration of cells that
express Cas protein is from about 8% to about 80%.
[0075] In some aspects, the invention provides a method for
treating or preventing a disorder associated with expression of a
polynucleotide sequence in a subject, the method comprising (a)
altering a target polynucleotide sequence in a cell ex vivo by
contacting the polynucleotide sequence in a cell selected from the
group consisting of a human pluripotent cell, a primary human cell,
and a non-transformed human cell, with a clustered regularly
interspaced short palindromic repeats-associated (Cas) protein and
two ribonucleic acids, wherein the ribonucleic acids direct Cas
protein to and hybridize to a target motif of the target
polynucleotide sequence, wherein the target polynucleotide sequence
is cleaved, and (b) introducing the cell into the subject, thereby
treating or preventing a disorder associated with expression of the
polynucleotide sequence. In some embodiments, the efficiency of
alteration is from about 8% to about 80%.
[0076] In some aspects, the invention provides a method for
treating or preventing a disorder associated with expression of a
polynucleotide sequence in a subject, the method comprising (a)
altering a target polynucleotide sequence in a cell ex vivo by
contacting the polynucleotide sequence in a cell selected from the
group consisting of a human pluripotent cell, a primary human cell,
and a non-transformed human cell, with a clustered regularly
interspaced short palindromic repeats-associated (Cas) protein and
two ribonucleic acids, wherein the ribonucleic acids direct Cas
protein to and hybridize to a target motif of the target
polynucleotide sequence, wherein the target polynucleotide sequence
is cleaved, and wherein the efficiency of alteration is from about
8% to about 80%, and (b) introducing the cell into the subject,
thereby treating or preventing a disorder associated with
expression of the polynucleotide sequence.
[0077] In some aspects, the invention provides a method for
simultaneously altering multiple target polynucleotide sequences in
a cell comprising contacting the polynucleotide sequences in a cell
selected from the group consisting of a human pluripotent cell, a
primary human cell, and a non-transformed human cell, with a
clustered regularly interspaced short palindromic
repeats-associated (Cas) protein and multiple ribonucleic acids,
wherein the ribonucleic acids direct Cas protein to and hybridize
to target motifs of the target polynucleotide sequences, wherein
the target polynucleotide sequences are cleaved. In some
embodiments, the efficiency of alteration of cells that express Cas
protein is from about 8% to about 80%.
[0078] In some aspects, the invention provides a method for
simultaneously altering multiple target polynucleotide sequences in
a cell comprising contacting the polynucleotide sequences in a cell
selected from the group consisting of a human pluripotent cell, a
primary human cell, and a non-transformed human cell, with a
clustered regularly interspaced short palindromic
repeats-associated (Cas) protein and multiple ribonucleic acids,
wherein the ribonucleic acids direct Cas protein to and hybridize
to target motifs of the target polynucleotide sequences, wherein
the target polynucleotide sequences are cleaved, and wherein the
efficiency of alteration of cells that express Cas protein is from
about 8% to about 80%.
[0079] In some aspects, the invention provides a method for
treating or preventing a disorder associated with expression of
polynucleotide sequences in a subject, the method comprising (a)
altering target polynucleotide sequences in a cell ex vivo by
contacting the polynucleotide sequences in a cell selected from the
group consisting of a human pluripotent cell, a primary human cell,
and a non-transformed human cell, with a clustered regularly
interspaced short palindromic repeats-associated (Cas) protein and
multiple ribonucleic acids, wherein the ribonucleic acids direct
Cas protein to and hybridize to target motifs of the target
polynucleotide sequences, wherein the target polynucleotide
sequences are cleaved, and (b) introducing the cell into the
subject, thereby treating or preventing a disorder associated with
expression of the polynucleotide sequences. In some embodiments,
the efficiency of alteration of cells that express Cas protein is
from about 8% to about 80%.
[0080] In some aspects, the invention provides a method for
treating or preventing a disorder associated with expression of
polynucleotide sequences in a subject, the method comprising (a)
altering target polynucleotide sequences in a cell ex vivo by
contacting the polynucleotide sequences in a cell selected from the
group consisting of a human pluripotent cell, a primary human cell,
and a non-transformed human cell, with a clustered regularly
interspaced short palindromic repeats-associated (Cas) protein and
multiple ribonucleic acids, wherein the ribonucleic acids direct
Cas protein to and hybridize to target motifs of the target
polynucleotide sequences, wherein the target polynucleotide
sequences are cleaved, and wherein the efficiency of alteration of
cells that express Cas protein is from about 8% to about 80%, and
(b) introducing the cell into the subject, thereby treating or
preventing a disorder associated with expression of the
polynucleotide sequences.
[0081] In some aspects, the disclosure provides a composition
comprising at least two ribonucleic acids each comprising a
different sequence selected from the group consisting of SEQ ID
NOs: 298-303.
[0082] In some aspects, the disclosure provides a composition
comprising at least two ribonucleic acids each comprising a
sequence which is complementary to and/or hybridizes to a different
sequence selected from the group consisting of SEQ ID NOs: 1-139
and 304-333.
[0083] In some aspects, the disclosure provides a composition
comprising at least two ribonucleic acids each comprising a
sequence which is complementary to and/or hybridizes to a sequence
with a single nucleotide mismatch to a sequence selected from the
group consisting of SEQ ID NOs: 1-139 and 304-333.
[0084] In some aspects, the disclosure provides a composition
comprising at least two ribonucleic acids each comprising a
sequence which is complementary to and/or hybridizes to a different
sequence selected from the group consisting of SEQ ID NOs:
140-297.
[0085] In some aspects, the disclosure provides a composition
comprising at least two ribonucleic acids each comprising a
sequence which is complementary to and/or hybridizes to a sequence
with a single nucleotide mismatch to a sequence selected from the
group consisting of SEQ ID NOs: 140-297.
[0086] In some embodiments, at least one of the two ribonucleic
acids is a modified ribonucleic acid comprising one to two modified
nucleotides selected from the group consisting of pseudouridine,
5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate,
4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate,
and 5-azauridine-5'-triphosphate.
[0087] In some embodiments, the two ribonucleic acids comprise
modified ribonucleic acids comprising one to two modified
nucleotides selected from the group consisting of pseudouridine,
5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate,
4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate,
and 5-azauridine-5'-triphosphate. In some embodiments, the
composition includes a nucleic acid sequence encoding a Cas
protein. In some embodiments, the composition includes a nucleic
acid sequence encoding a Cas9 protein or a functional portion
thereof. In some embodiments, the nucleic acid comprises a modified
ribonucleic acid comprising at least one modified nucleotide
selected from the group consisting of pseudouridine,
5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate,
4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate,
and 5-azauridine-5'-triphosphate.
[0088] In some aspects, the invention provides a composition
comprising a chimeric nucleic acid comprising a ribonucleic acid
encoding a Cas protein and at least two additional ribonucleic
acids each having a sequence selected from the group consisting of
the ribonucleic acid sequences of SEQ ID NOs: 298-303.
[0089] In some aspects, the invention provides a chimeric nucleic
acid comprising a ribonucleic acid encoding a Cas protein and at
least two additional ribonucleic acids each having a sequence which
is complementary to and/or hybridizes to a different sequence
selected from the group consisting of SEQ ID NOs: 1-139 and
304-333.
[0090] In some aspects, the invention provides a composition
comprising a chimeric nucleic acid comprising a ribonucleic acid
encoding a Cas protein and at least two additional ribonucleic
acids each having a sequence which is complementary to and/or
hybridizes to a different sequence selected from the group
consisting of SEQ ID NOs: 140-297.
[0091] In some aspects, the invention provides a composition
comprising a chimeric nucleic acid comprising a ribonucleic acid
encoding a Cas protein and at least two additional ribonucleic acid
sequences each comprising a sequence which is complementary to
and/or hybridizes to a sequence with a single nucleotide mismatch
to a sequence selected from the group consisting of SEQ ID NOs:
1-139 and 304-333.
[0092] In some aspects, the invention provides a composition
comprising a chimeric nucleic acid comprising a ribonucleic acid
encoding a Cas protein and at least two additional ribonucleic acid
sequences each comprising a sequence which is complementary to
and/or hybridizes to a sequence with a single nucleotide mismatch
to a sequence selected from the group consisting of SEQ ID NOs:
140-297.
[0093] In some embodiments, the composition includes a nucleic acid
sequence encoding a detectable marker. In some embodiments, the
composition includes a nucleic acid sequence encoding a fluorescent
protein. In some embodiments, the composition includes a promoter
operably linked to the chimeric nucleic acid. In some embodiments,
the promoter is optimized for increased expression in human stem
cells. In some embodiments, the promoter is selected from the group
consisting of a Cytomegalovirus (CMV) early enhancer element and a
chicken beta-actin promoter, a chicken beta-actin promoter, an
elongation factor-1 alpha promoter, and a ubiquitin promoter. In
some embodiments, the chimeric nucleic acid comprises at least one
modified nucleotide selected from the group consisting of
pseudouridine, 5-methylcytodine, 2-thio-uridine,
5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate,
5,6-dihydrouridine-5'-triphosphate, and
5-azauridine-5'-triphosphate. In some embodiments, the Cas protein
comprises a Cas9 protein or a functional portion thereof.
[0094] In some aspects, the invention provides a kit for altering a
target polynucleotide sequence in a cell comprising a Cas9 protein
or a nucleic acid encoding the Cas9 protein, and at least two
ribonucleic acids each comprising a different sequence selected
from the group consisting of the ribonucleic acid sequences of SEQ
ID NOs: 298-303.
[0095] In some aspects, the invention provides a kit for altering a
target polynucleotide sequence in a cell comprising a Cas9 protein
or a nucleic acid encoding the Cas9 protein, and at least two
ribonucleic acids each comprising a sequence which is complementary
to and/or hybridizes to a different sequence selected from the
group consisting of SEQ ID NOs: 1-139 and 304-333.
[0096] In some aspects, the invention provides a kit for altering a
target polynucleotide sequence in a cell comprising a Cas9 protein
or a nucleic acid encoding the Cas9 protein, and at least two
ribonucleic acids each comprising a sequence which is complementary
to and/or hybridizes to a different sequence selected from the
group consisting of SEQ ID NOs: 140-297.
[0097] In some embodiments, the kit includes one or more cell
lines, cultures, or populations selected from the group consisting
of human pluripotent cells, primary human cells, and
non-transformed cells. In some aspects, the kit includes a DNA
repair template.
[0098] In some aspects, the invention provides a method of
administering cells to a subject in need of such cells, the method
comprising: (a) contacting a cell or population of cells ex vive
with a Cas protein and two ribonucleic acids which direct Cas
protein to and hybridize to a target polynucleotide sequence
encoding B2M in the cell or population of cells, wherein the target
polynucleotide sequence is cleaved; and (b) administering the
resulting cells from (a) to a subject in need of such cells.
[0099] In some aspects, the invention provides a method of
administering cells to a subject in need of such cells, the method
comprising: (a) contacting a cell or population of cells ex vivo
with (i) a Cas protein. (ii) at least two ribonucleic acids which
direct Cas protein to and hybridize to a target polynucleotide
sequence encoding B2M in the cell or population of cells, and (iii)
at least two additional ribonucleic acids which direct Cas protein
to and hybridize to a target polynucleotide sequence in the cell or
population of cells, wherein the target polynucleotide sequences
are cleaved; and (b) administering the resulting cell or cells from
(a) to a subject in need of such cells.
[0100] In some embodiments, cleavage of the target polynucleotide
sequence encoding B2M in the cell or population of cells reduces
the likelihood that the resulting cell or cells will trigger a host
immune response when the cells are administered to the subject. In
some aspects, the target polynucleotide sequence comprises CCR5. In
some embodiments, the at least two ribonucleic acids comprise two
different sequences selected from the group consisting of SEQ ID
NOs: 298-303. In some embodiments, the at least two ribonucleic
acids each comprise sequences which are complementary to and/or
hybridize to a different sequence selected from the group
consisting of SEQ ID NOs: 1-139 and 304-333. In some embodiments,
the at least two ribonucleic acids each comprise sequences which
are complementary to and/or hybridize to sequences comprising at
least one nucleotide mismatch to different sequences selected from
the group consisting of SEQ ID NOs: 1-139 and 304-333. In some
embodiments, the target polynucleotide sequence comprises CXCR4. In
some embodiments, the at least two ribonucleic acids each comprise
sequences which are complementary to and/or hybridize to a
different sequence selected from the group consisting of SEQ ID
NOs: 140-297. In some embodiments, the at least two ribonucleic
acids each comprise sequences which are complementary to and/or
hybridize to sequences comprising at least one nucleotide mismatch
to different sequences selected from the group consisting of SEQ ID
NOs: 140-297. In some embodiments, the cell or population of cells
comprises primary cells. In some embodiments, the subject in need
of administration of cells is suffering from a disorder. In some
embodiments, the disorder comprises a genetic disorder. In some
embodiments, the disorder comprises an infection. In some
embodiments, the disorder comprises HIV or AIDs. In some
embodiments, the disorder comprises cancer.
[0101] In some aspects, the invention provides a method of reducing
the likelihood that cells administered to a subject will trigger a
host immune response in the subject, the method comprising: (a)
contacting a cell or population of cells ex vivo with a Cas protein
and two ribonucleic acids which direct Cas protein to and hybridize
to a target polynucleotide sequence encoding B2M in the cell or
population of cells, wherein the target polynucleotide sequence
encoding B2M is cleaved, thereby reducing the likelihood that cells
administered to the subject will trigger a host immune response in
the subject; and (b) administering the resulting cells from (a) to
a subject in need of such cells.
[0102] In some aspects, the invention provides a method of reducing
the likelihood that cells administered to a subject will trigger a
host immune response in the subject, the method comprising: (a)
contacting a cell or population of cells ex vivo with (i) a Cas
protein, (ii) at least two ribonucleic acids which direct Cas
protein to and hybridize to a target polynucleotide sequence
encoding B2M in the cell or population of cells, wherein the target
polynucleotide sequence encoding B2M in the cell or population of
cells is cleaved, thereby reducing the likelihood that the cell or
population of cells will trigger a host immune response in the
subject, and (iii) at least two additional ribonucleic acids which
direct Cas protein to and hybridize to a target polynucleotide
sequence in the cell or population of cells, wherein the target
polynucleotide sequence is cleaved; and (b) administering the
resulting cell or cells from (a) to a subject in need of such
cells.
[0103] In some embodiments, the target polynucleotide sequence
comprises CCR5. In some embodiments, the at least two ribonucleic
acids comprise two different sequences selected from the group
consisting of SEQ ID NOs: 298-303. In some embodiments, the at
least two ribonucleic acids each comprise sequences which are
complementary to and/or hybridize to a different sequence selected
from the group consisting of SEQ ID NOs: 1-139 and 304-333. In some
embodiments, the at least two ribonucleic acids each comprise
sequences which are complementary to and/or hybridize to sequences
comprising at least one nucleotide mismatch to different sequences
selected from the group consisting of SEQ ID NOs: 1-139 and
304-333. In some embodiments, the target polynucleotide sequence
comprises CXCR4. In some embodiments, the at least two ribonucleic
acids each comprise sequences which are complementary to and/or
hybridize to a different sequence selected from the group
consisting of SEQ ID NOs: 140-297. In some embodiments, the at
least two ribonucleic acids each comprise sequences which are
complementary to and/or hybridize to sequences comprising at least
one nucleotide mismatch to different sequences selected from the
group consisting of SEQ ID NOs: 140-297. In some embodiments, the
cell or population of cells comprises primary cells. In some
embodiments, the subject in need of administration of cells is
suffering from a disorder. In some embodiments, the disorder
comprises a genetic disorder. In some embodiments, the disorder
comprises an infection. In some embodiments, the disorder comprises
HIV or AIDs. In some embodiments, the disorder comprises
cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0104] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0105] FIG. 1 shows exemplary guide RNA sequences useful when the
target polynucleotide sequence is human CCR5.
[0106] FIG. 2 shows exemplary guide RNA sequences useful when the
target polynucleotide sequence is human CXCR4.
[0107] FIG. 3 shows an exemplary amino acid sequence of a Cas
protein. Yellow highlights indicate Ruv-C-like domain. Underlining
indicates HNH nuclease domain.
[0108] FIGS. 4A, 4B, 4C, 4D and 4E demonstrate that a single guide
strategy achieves high efficiency genome editing in cell lines, but
not in clinically relevant primary somatic cells. FIG. 4A is a
table showing CRISPR-targeting sites in the CCR5 locus (single
guides), which were found by scanning the human chemokine receptor
CCR5 gene for optimized guide RNA sequences using a CRISPR design
program (available on the world wide web at http://CRISPR.mit.edu)
(left panel). A total of 11 guide RNAs having a score greater than
50 was tested for editing efficiency in a K562 cell line. FIG. 4A
(right panel) shows the editing efficiency of 7 of selected guides
(% indels) was measured by a CEL surveyor assay. FIG. 4B shows a
comparative analysis of genome-editing efficiency in cell lines
293T, K562 (left two panels) and primary human CD34+ HSPCs (right
two panels) illustrating inefficient genome editing efficiency in
primary CD34+ cells. Cells were transfected with Cas9 (lane 1)
together with guide RNA (lane 2) or expression plasmids (lane 3).
FIG. 4C is a schematic illustrating CRISPR-targeting sites in the
CCR5 locus (single guides). FIG. 4D shows the results of targeting
the B2M locus with single guide RNAs in 293T cells. FIG. 4E shows
the results of flow cytometry analysis using a single guide
strategy targeting B2M in 293T cell, which demonstrate that B2M
CRISPRs ablate B2M surface expression with high efficiency.
[0109] FIGS. 5A. 5B and 5C demonstrate that a double guide strategy
achieves genome editing with high efficacy in clinically relevant
cells. FIG. 5A shows that as compared to single guide (A or B),
2-guide combination (A+B) showed robust editing efficiency in
targeting CCR5 in K562 cell line. FIG. 5B shows various guide
combinations and spacing between each guide pair with orientation
(upper panel). The PCR results (bottom left panel) and CEL assay
(bottom right) show robust genome editing for tested guide pairs.
FIG. 5C shows the results of PCR analysis indicating that with
2-guide combination wild-type Cas9 effectively deleted the DNA
sequence between the two guides, in contrast to Nickase (D10A)
which did not effectively delete the DNA sequence between the two
guides. FIG. 5D is a schematic showing double B2M CRISPR
combinations.
[0110] FIGS. 6A and 6B demonstrate effective genome-editing in
human CD34+ HSPC using a two-guide approach. FIG. 6A is a
representative gel picture showing efficient clonal deletion
frequency using two guides. Clonal deletion efficiency was
determined by PCR carried on individual colony grown on methyl
cellulose. FIG. 6B is a Table showing data obtained from two
independent clonal deletion experiments, which suggests efficacious
genome-editing in primary human CD34+ cells using a two-guide
approach.
[0111] FIGS. 7A, 7B and 7C demonstrate that in contrast to primary
cells, the double guide strategy does not improve B2M editing
efficiency in 293T cells. FIG. 7A shows the gating strategy for
flow cytometry analysis of 293T cells electroporated with 1 .mu.g
Cas9 plus either 0.5 .mu.g gRNA or 0.25 .mu.g+0.25 .mu.g gRNA
targeting B2M 72 hours post-transfection in a 6-well format. FIG.
7B shows the results of a SURVEYOR assay with B2M CRISPR gRNAs in
293T cells (72 h). FIG. 7C shows that the double guide strategy
does not improve B2M cutting efficiency in 293T cells, in contrast
to the double guide strategy which significantly improves B2M
cutting efficiency in primary cells (FIG. 5).
[0112] FIGS. 8A, 8B, 8C and 8D demonstrate ablation of B2M surface
expression in somatic cells (e.g., primary CD4+ T cells) using a
double guide strategy. FIG. 8A shows the results of a flow
cytometry analysis demonstrating B2M knock-out efficiency in CD4+ T
cells (total live cells). FIG. 8B shows the results of a flow
cytometry analysis demonstrating B2M knock-out efficiency in CD4+ T
cells (gated on GFP+ cells). FIG. 8C shows a Table quantifying the
results of a flow cytometry analysis demonstrating B2M knock-out
efficiency in CD4+ T cells. FIG. 8D shows the results of a flow
cytometry analysis of cells gated on live/7AAD neg/GFP+ cells,
demonstrating that the double guide strategy results in ablation of
B2M surface expression.
DETAILED DESCRIPTION OF THE INVENTION
[0113] Work described herein demonstrates methods of allele
targeting using CRISPR/Cas systems resulting in mutant cells with
efficiencies of up to 80%. In particular, work described herein
surprisingly and unexpectedly demonstrates that a multiple guide
strategy (e.g., using two or more ribonucleic acids which guide Cas
protein to and hybridize to a target polynucleotide sequence)
efficiently and effectively deletes target polynucleotide sequences
(e.g., B2M, HPRT, CCR5 and/or CXCR4) in primary somatic cells
(e.g., human blood cells, e.g., CD34+ and T cells), in contrast to
a single guide strategy which has been demonstrated by the
inventors to efficiently delete target polynucleotide sequences in
cell lines (e.g., 293T) but not in primary somatic cells. These
vastly improved methods permit CRISPR/Cas systems to be utilized
effectively for the first time for therapeutic purposes. Methods of
delivery of CRISPR/Cas systems to human stem cells are provided. In
addition, methods of specifically identifying useful RNA guide
sequences are provided, along with particular guide sequences
useful in targeting specific genes (e.g., B2M, HPRT, CCR5 and/or
CXCR4). Moreover, methods of treatment (e.g., methods of treating
HIV infection) utilizing the compositions and methods disclosed
herein are provided. Moreover, methods of administering cells
(e.g., methods of administering a cell that has a reduced
likelihood of triggering a host immune response) utilizing the
compositions and methods disclosed herein are provided.
[0114] In one aspect, the present invention provides a method for
altering a target polynucleotide sequence in a cell.
[0115] An exemplary method for altering a target polynucleotide
sequence in a cell comprises contacting the polynucleotide sequence
with a clustered regularly interspaced short palindromic
repeats-associated (Cas) protein and from one to two ribonucleic
acids, wherein the ribonucleic acids direct Cas protein to and
hybridize to a target motif of the target polynucleotide sequence,
wherein the target polynucleotide sequence is cleaved, and wherein
the efficiency of alteration of cells that express Cas protein is
from about 50% to about 80%.
[0116] As used herein, the term "contacting" (i.e., contacting a
polynucleotide sequence with a clustered regularly interspaced
short palindromic repeats-associated (Cas) protein and/or
ribonucleic acids) is intended to include incubating the Cas
protein and/or the ribonucleic acids in the cell together in vitro
(e.g., adding the Cas protein or nucleic acid encoding the Cas
protein to cells in culture) or contacting a cell ex vivo. The step
of contacting a target polynucleotide sequence with a Cas protein
and/or ribonucleic acids as disclosed herein can be conducted in
any suitable manner. For example, the cells may be treated in
adherent culture, or in suspension culture. It is understood that
the cells contacted with a Cas protein and/or ribonucleic acids as
disclosed herein can also be simultaneously or subsequently
contacted with another agent, such as a growth factor or other
differentiation agent or environments to stabilize the cells, or to
differentiate the cells further.
[0117] In another aspect, the present invention provides a method
for treating or preventing a disorder associated with expression of
a polynucleotide sequence in a subject.
[0118] The terms "treat", "treating", "treatment", etc., as applied
to an isolated cell, include subjecting the cell to any kind of
process or condition or performing any kind of manipulation or
procedure on the cell. As applied to a subject, the terms refer to
providing a cell in which a target polynucleotide sequence has been
altered ex vivo according to the methods described herein to an
individual. The individual is usually ill or injured, or at
increased risk of becoming ill relative to an average member of the
population and in need of such attention, care, or management.
[0119] As used herein, the term "treating" and "treatment" refers
to administering to a subject an effective amount of cells with
target polynucleotide sequences altered ex vivo according to the
methods described herein so that the subject has a reduction in at
least one symptom of the disease or an improvement in the disease,
for example, beneficial or desired clinical results. For purposes
of this invention, beneficial or desired clinical results include,
but are not limited to, alleviation of one or more symptoms,
diminishment of extent of disease, stabilized (i.e., not worsening)
state of disease, delay or slowing of disease progression,
amelioration or palliation of the disease state, and remission
(whether partial or total), whether detectable or undetectable.
Treating can refer to prolonging survival as compared to expected
survival if not receiving treatment. Thus, one of skill in the art
realizes that a treatment may improve the disease condition, but
may not be a complete cure for the disease. As used herein, the
term "treatment" includes prophylaxis. Alternatively, treatment is
"effective" if the progression of a disease is reduced or halted.
"Treatment" can also mean prolonging survival as compared to
expected survival if not receiving treatment. Those in need of
treatment include those already diagnosed with a disorder
associated with expression of a polynucleotide sequence, as well as
those likely to develop such a disorder due to genetic
susceptibility or other factors.
[0120] By "treatment." "prevention" or "amelioration" of a disease
or disorder is meant delaying or preventing the onset of such a
disease or disorder, reversing, alleviating, ameliorating,
inhibiting, slowing down or stopping the progression, aggravation
or deterioration the progression or severity of a condition
associated with such a disease or disorder. In one embodiment, the
symptoms of a disease or disorder are alleviated by at least 5%, at
least 10%, at least 20%, at least 30%, at least 40%, or at least
50%.
[0121] An exemplary method for treating or preventing a disorder
associated with expression of a polynucleotide sequence in a
subject comprises (a) altering a target polynucleotide sequence in
a cell ex vivo by contacting the polynucleotide sequence with a
clustered regularly interspaced short palindromic
repeats-associated (Cas) protein and from one to two ribonucleic
acids, wherein the ribonucleic acids direct Cas protein to and
hybridize to a target motif of the target polynucleotide sequence,
wherein the target polynucleotide sequence is cleaved, and wherein
the efficiency of alteration of cells that express Cas protein is
from about 50% to about 80%, and (b) introducing the cell into the
subject, thereby treating or preventing a disorder associated with
expression of the polynucleotide sequence.
[0122] The present invention contemplates altering target
polynucleotide sequences in any manner which is available to the
skilled artisan utilizing a CRISPR/Cas system of the present
invention. Any CRISPR/Cas system that is capable of altering a
target polynucleotide sequence in a cell can be used. Such
CRISPR-Cas systems can employ a variety of Cas proteins (Haft et
al. PLoS Comput Biol. 2005; 1(6)e60). The molecular machinery of
such Cas proteins that allows the CRISPR/Cas system to alter target
polynucleotide sequences in cells include RNA binding proteins,
endo- and exo-nucleases, helicases, and polymerases. In some
embodiments, the CRISPR/Cas system is a CRISPR type I system. In
some embodiments, the CRISPR/Cas system is a CRISPR type II
system.
[0123] The CRISPR/Cas systems of the present invention can be used
to alter a target polynucleotide sequence in a cell. The present
invention contemplates altering target polynucleotide sequences in
a cell for any purpose. In some embodiments, the target
polynucleotide sequence in a cell is altered to produce a mutant
cell. As used herein, a "mutant cell" refers to a cell with a
resulting genotype that differs from its original genotype. In some
instances, a "mutant cell" exhibits a mutant phenotype, for example
when a normally functioning gene is altered using the CRISPR/Cas
systems of the present invention. In other instances, a "mutant
cell" exhibits a wild-type phenotype, for example when a CRISPR/Cas
system of the present invention is used to correct a mutant
genotype. In some embodiments, the target polynucleotide sequence
in a cell is altered to correct or repair a genetic mutation (e.g.,
to restore a normal phenotype to the cell). In some embodiments,
the target polynucleotide sequence in a cell is altered to induce a
genetic mutation (e.g., to disrupt the function of a gene or
genomic element).
[0124] In some embodiments, the alteration is an indel. As used
herein, "indel" refers to a mutation resulting from an insertion,
deletion, or a combination thereof. As will be appreciated by those
skilled in the art, an indel in a coding region of a genomic
sequence will result in a frameshift mutation, unless the length of
the indel is a multiple of three. In some embodiments, the
alteration is a point mutation. As used herein, "point mutation"
refers to a substitution that replaces one of the nucleotides. A
CRISPR/Cas system of the present invention can be used to induce an
indel of any length or a point mutation in a target polynucleotide
sequence.
[0125] In some embodiments, the alteration results in a knock out
of the target polynucleotide sequence or a portion thereof.
Knocking out a target polynucleotide sequence or a portion thereof
using a CRISPR/Cas system of the present invention can be useful
for a variety of applications. For example, knocking out a target
polynucleotide sequence in a cell can be performed in vitro for
research purposes. For ex vivo purposes, knocking out a target
polynucleotide sequence in a cell can be useful for treating or
preventing a disorder associated with expression of the target
polynucleotide sequence (e.g., by knocking out a mutant allele in a
cell ex vivo and introducing those cells comprising the knocked out
mutant allele into a subject).
[0126] As used herein, "knock out" includes deleting all or a
portion of the target polynucleotide sequence in a way that
interferes with the function of the target polynucleotide sequence.
For example, a knock out can be achieved by altering a target
polynucleotide sequence by inducing an indel in the target
polynucleotide sequence in a functional domain of the target
polynucleotide sequence (e.g., a DNA binding domain). Those skilled
in the art will readily appreciate how to use the CRISPR/Cas
systems of the present invention to knock out a target
polynucleotide sequence or a portion thereof based upon the details
described herein.
[0127] In some embodiments, the alteration results in reduced
expression of the target polynucleotide sequence. The terms
"decrease," "reduced," "reduction," and "decrease" are all used
herein generally to mean a decrease by a statistically significant
amount. However, for avoidance of doubt, "decrease," "reduced,"
"reduction," "decrease" means a decrease by at least 10% as
compared to a reference level, for example a decrease by at least
about 20%, or at least about 30%, or at least about 40%, or at
least about 50%, or at least about 60%, or at least about 70%, or
at least about 80%, or at least about 90% or up to and including a
100% decrease (i.e. absent level as compared to a reference
sample), or any decrease between 10-100% as compared to a reference
level.
[0128] The terms "increased", "increase" or "enhance" or "activate"
are all used herein to generally mean an increase by a statically
significant amount; for the avoidance of any doubt, the terms
"increased", "increase" or "enhance" or "activate" means an
increase of at least 10% as compared to a reference level, for
example an increase of at least about 20%, or at least about 30%,
or at least about 40%, or at least about 50%, or at least about
60%, or at least about 70%, or at least about 80%, or at least
about 90% or up to and including a 100% increase or any increase
between 10-100% as compared to a reference level, or at least about
a 2-fold, or at least about a 3-fold, or at least about a 4-fold,
or at least about a 5-fold or at least about a 10-fold increase, or
any increase between 2-fold and 10-fold or greater as compared to a
reference level.
[0129] The term "statistically significant" or "significantly"
refers to statistical significance and generally means a two
standard deviation (2SD) below normal, or lower, concentration of
the marker. The term refers to statistical evidence that there is a
difference. It is defined as the probability of making a decision
to reject the null hypothesis when the null hypothesis is actually
true. The decision is often made using the p-value.
[0130] In some embodiments, the alteration is a homozygous
alteration. In some embodiments, the alteration is a heterozygous
alteration.
[0131] In some embodiments, the alteration results in correction of
the target polynucleotide sequence from an undesired sequence to a
desired sequence. The CRISPR/Cas systems of the present invention
can be used to correct any type of mutation or error in a target
polynucleotide sequence. For example, the CRISPR/Cas systems of the
present invention can be used to insert a nucleotide sequence that
is missing from a target polynucleotide sequence due to a deletion.
The CRISPR/Cas systems of the present invention can also be used to
delete or excise a nucleotide sequence from a target polynucleotide
sequence due to an insertion mutation. In some instances, the
CRISPR/Cas systems of the present invention can be used to replace
an incorrect nucleotide sequence with a correct nucleotide sequence
(e.g., to restore function to a target polynucleotide sequence that
is impaired due to a loss of function mutation, i.e., a SNP).
[0132] The CRISPR/Cas systems of the present invention can alter
target polynucleotides with surprisingly high efficiency as
compared to conventional CRISPR/Cas systems. In certain
embodiments, the efficiency of alteration is at least about 5%. In
certain embodiments, the efficiency of alteration is at least about
10%. In certain embodiments, the efficiency of alteration is from
about 10% to about 80%. In certain embodiments, the efficiency of
alteration is from about 30% to about 80%. In certain embodiments,
the efficiency of alteration is from about 50% to about 80%. In
some embodiments, the efficiency of alteration is greater than or
equal to about 80%.
[0133] The CRISPR/Cas systems of the present invention can be used
to alter any target polynucleotide sequence in a cell. Those
skilled in the art will readily appreciate that desirable target
polynucleotide sequences to be altered in any particular cell may
correspond to any genomic sequence for which expression of the
genomic sequence is associated with a disorder or otherwise
facilitates entry of a pathogen into the cell. For example, a
desirable target polynucleotide sequence to alter in a cell may be
a polynucleotide sequence corresponding to a genomic sequence which
contains a disease associated single polynucleotide polymorphism.
In such example, the CRISPR/Cas systems of the present invention
can be used to correct the disease associated SNP in a cell by
replacing it with a wild-type allele. As another example, a
polynucleotide sequence of a target gene which is responsible for
entry or proliferation of a pathogen into a cell may be a suitable
target for deletion or insertion to disrupt the function of the
target gene to prevent the pathogen from entering the cell or
proliferating inside the cell.
[0134] In some embodiments, the target polynucleotide sequence is a
genomic sequence. In some embodiments, the target polynucleotide
sequence is a human genomic sequence. In some embodiments, the
target polynucleotide sequence is a mammalian genomic sequence. In
some embodiments, the target polynucleotide sequence is a
vertebrate genomic sequence.
[0135] In some embodiments, a target polynucleotide sequence is a
pathogenic genomic sequence. Exemplary pathogenic genomic sequences
include, but are not limited to a viral genomic sequence, a
bacterial genomic sequence, a fungal genomic sequence, a toxin
genomic sequence, or a parasitic genomic sequence. In such
embodiments, the CRISPR/Cas systems of the present invention can be
used to disrupt the function of a pathogen (e.g., to treat or
prevent an infection by the pathogen) by cleaving a genomic
sequence of the pathogen (e.g., a genomic sequence that is critical
for entry into a cell, or responsible for multiplication, growth or
survival once the pathogen is inside a cell).
[0136] In some embodiments, the target polynucleotide sequence is
beta-2-microglobulin (B2M; Gene ID: 567). The B2M polynucleotide
sequence encodes a serum protein associated with the heavy chain of
the major histocompatibility complex (MHC) class I molecules which
are expressed on the surface of virtually all nucleated cells. B2M
protein comprises a beta-pleated sheet structure that has been
found to form amyloid fibrils in certain pathological conditions.
The B2M gene has 4 exons which span approximately 8 kb. B2M has
been observed in the serum of normal individuals and in elevated
amounts in urine from patients having Wilson disease, cadmium
poisoning, and various conditions leading to renal tubular
dysfunction. Other pathological conditions known to be associated
with the B2M include, without limitation, a homozygous mutation
(e.g., ala11pro) in the B2M gene has been reported in individuals
having familial hypercatabolic hypoproteinemia, a heterozygous
mutation (e.g., asp76asn) in the B2M gene has been reported in
individuals having familial visceral amyloidosis
[0137] In some embodiments, the target polynucleotide sequence is a
variant of B2M. In some embodiments, the target polynucleotide
sequence is a homolog of B2M. In some embodiments, the target
polynucleotide sequence is an ortholog of B2M.
[0138] In some embodiments, the target polynucleotide sequence is
hypoxanthine phosphoribosyltransferase 1 (HPRT1; Gene ID:
3251).
[0139] In some embodiments, the target polynucleotide sequence is
CCR5 (Gene ID: 1234, also known as CC-CKR-5, CCCKR5, CCR-5, CD195,
CKR-5, CKR5, CMKBR5, and IDDM22). In some embodiments, the target
polynucleotide sequence is a variant of CCR5. In some embodiments,
the target polynucleotide sequence is a homolog of CCR5. In some
embodiments, the target polynucleotide sequence is an ortholog of
CCR5.
[0140] In some embodiments, the target polynucleotide sequence is
CXCR4 (Gene ID: 7852, also known as FB22; HM89; LAP3; LCR1; NPYR;
WHIM; CD184; LESTR; NPY3R; NPYRL; HSY3RR; NPYY3R; and D2S201E). In
some embodiments, the target polynucleotide sequence is a variant
of CXCR4. In some embodiments, the target polynucleotide sequence
is a homolog of CXCR4. In some embodiments, the target
polynucleotide sequence is an ortholog of CXCR4. It should be
appreciated that the CRISPR/Cas systems of the present invention
can cleave target polynucleotide sequences in a variety of ways. In
some embodiments, the target polynucleotide sequence is cleaved
such that a double-strand break results. In some embodiments, the
target polynucleotide sequence is cleaved such that a single-strand
break results.
[0141] The methods of the present invention can be used to alter
any target polynucleotide sequence in a cell, as long as the target
polynucleotide sequence in the cell contains a suitable target
motif that allows at least one ribonucleic acid of the CRISPR/Cas
system to direct the Cas protein to and hybridize to the target
motif. Those skilled in the art will appreciate that the target
motif for targeting a particular polynucleotide depends on the
CRISPR/Cas system being used, and the sequence of the
polynucleotide to be targeted.
[0142] In some embodiments, the target motif is at least 20 bp in
length. In some embodiments, the target motif is a 20-nucleotide
DNA sequence. In some embodiments, the target motif is a
20-nucleotide DNA sequence beginning with G and immediately
precedes an NGG motif recognized by the Cas protein. In some
embodiments, the target motif is G(N).sub.19NGG. In some
embodiments, the target motif is a 20-nucleotide DNA sequence and
immediately precedes an NGG motif recognized by the Cas protein. In
some embodiments, the target motif is (N).sub.20NGG.
[0143] The target motifs of the present invention can be selected
to minimize off-target effects of the CRISPR/Cas systems of the
present invention. In some embodiments, the target motif is
selected such that it contains at least two mismatches when
compared with all other genomic nucleotide sequences in the cell.
In some embodiments, the target motif is selected such that it
contains at least one mismatch when compared with all other genomic
nucleotide sequences in the cell. Those skilled in the art will
appreciate that a variety of techniques can be used to select
suitable target motifs for minimizing off-target effects (e.g.,
bioinformatics analyses).
[0144] In some embodiments, the target motif comprises a DNA
sequence selected from the group consisting of SEQ ID NOs: 1-139.
In some embodiments, the target motif comprises a DNA sequence
comprising at least one nucleotide mismatch compared to a DNA
sequence selected from the group consisting of SEQ ID NOs: 1-139.
In some embodiments, the target motif comprises a DNA sequence
comprising at least two nucleotide mismatches compared to a DNA
sequence selected from the group consisting of SEQ ID NOs: 1-139.
In some embodiments, the target motif comprises a DNA sequence
selected from the group consisting of SEQ ID NOs: 140-297. In some
embodiments, the target motif comprises a DNA sequence comprising
at least one nucleotide mismatch compared to a DNA sequence
selected from the group consisting of SEQ ID NOs: 140-297. In some
embodiments, the target motif comprises a DNA sequence comprising
at least two nucleotide mismatches compared to a DNA sequence
selected from the group consisting of SEQ ID NOs: 140-297. In some
embodiments, the target motif comprises a DNA sequence selected
from the group consisting of SEQ ID NOs: 304-333. In some
embodiments, the target motif comprises a DNA sequence comprising
at least one nucleotide mismatch compared to a DNA sequence
selected from the group consisting of SEQ ID NOs: 304-333. In some
embodiments, the target motif comprises a DNA sequence comprising
at least two nucleotide mismatches compared to a DNA sequence
selected from the group consisting of SEQ ID NOs: 304-333. In some
embodiments, the CRISPR/Cas systems of the present invention
utilize homology-directed repair to correct target polynucleotide
sequences. In some embodiments, subsequent to cleavage of the
target polynucleotide sequence, homology-directed repair occurs. In
some embodiments, homology-directed repair is performed using an
exogenously introduced DNA repair template. The exogenously
introduced DNA repair template can be single-stranded or
double-stranded. The DNA repair template can be of any length.
Those skilled in the art will appreciate that the length of any
particular DNA repair template will depend on the target
polynucleotide sequence that is to be corrected. The DNA repair
template can be designed to repair or replace any target
polynucleotide sequence, particularly target polynucleotide
sequences comprising disease associated polymorphisms (e.g., SNPs).
For example, homology-directed repair of a mutant allele comprising
such SNPs can be achieved with a CRISPR/Cas system by selecting two
target motifs which flank the mutant allele, and an designing a DNA
repair template to match the wild-type allele.
[0145] In some embodiments, a CRISPR/Cas system of the present
invention includes a Cas protein and at least one to two one
ribonucleic acids that are capable of directing the Cas protein to
and hybridizing to a target motif of a target polynucleotide
sequence.
[0146] As used herein, "protein" and "polypeptide" are used
interchangeably to refer to a series of amino acid residues joined
by peptide bonds (i.e., a polymer of amino acids) and include
modified amino acids (e.g., phosphorylated, glycated, glycosolated,
etc.) and amino acid analogs. Exemplary polypeptides or proteins
include gene products, naturally occurring proteins, homologs,
paralogs, fragments and other equivalents, variants, and analogs of
the above.
[0147] In some embodiments, a Cas protein comprises one or more
amino acid substitutions or modifications. In some embodiments, the
one or more amino acid substitutions comprises a conservative amino
acid substitution. In some instances, substitutions and/or
modifications can prevent or reduce proteolytic degradation and/or
extend the half-life of the polypeptide in a cell. In some
embodiments, the Cas protein can comprise a peptide bond
replacement (e.g., urea, thiourea, carbamate, sulfonyl urea, etc.).
In some embodiments, the Cas protein can comprise a naturally
occurring amino acid. In some embodiments, the Cas protein can
comprise an alternative amino acid (e.g., D-amino acids, beta-amino
acids, homocysteine, phosphoserine, etc.). In some embodiments, a
Cas protein can comprise a modification to include a moiety (e.g.,
PEGylation, glycosylation, lipidation, acetylation, end-capping,
etc.).
[0148] In some embodiments, a Cas protein comprises a core Cas
protein. Exemplary Cas core proteins include, but are not limited
to Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8 and Cas9. In some
embodiments, a Cas protein comprises a Cas protein of an E. coli
subtype (also known as CASS2). Exemplary Cas proteins of the E.
Coli subtype include, but are not limited to Cse1. Cse2, Cse3,
Cse4, and Cas5e. In some embodiments, a Cas protein comprises a Cas
protein of the Ypest subtype (also known as CASS3). Exemplary Cas
proteins of the Ypest subtype include, but are not limited to Csy1,
Csy2. Csv3, and Csy4. In some embodiments, a Cas protein comprises
a Cas protein of the Nmeni subtype (also known as CASS4). Exemplary
Cas proteins of the Nmeni subtype include, but are not limited to
Csn1 and Csn2. In some embodiments, a Cas protein comprises a Cas
protein of the Dvulg subtype (also known as CASS1). Exemplary Cas
proteins of the Dvulg subtype include Csd1, Csd2, and Cas5d. In
some embodiments, a Cas protein comprises a Cas protein of the
Tneap subtype (also known as CASS7). Exemplary Cas proteins of the
Tneap subtype include, but are not limited to, Cst1, Cst2, Cas5t.
In some embodiments, a Cas protein comprises a Cas protein of the
Hmari subtype. Exemplary Cas proteins of the Hmari subtype include,
but are not limited to Csh1, Csh2, and Cas5h. In some embodiments,
a Cas protein comprises a Cas protein of the Apern subtype (also
known as CASS5). Exemplary Cas proteins of the Apern subtype
include, but are not limited to Csa1, Csa2, Csa3, Csa4, Csa5, and
Cas5a. In some embodiments, a Cas protein comprises a Cas protein
of the Mtube subtype (also known as CASS6). Exemplary Cas proteins
of the Mtube subtype include, but are not limited to Csm1, Csm2,
Csm3, Csm4, and Csm5. In some embodiments, a Cas protein comprises
a RAMP module Cas protein. Exemplary RAMP module Cas proteins
include, but are not limited to, Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and
Cmr6.
[0149] In some embodiments, the Cas protein is a Streptococcus
pyogenes Cas9 protein or a functional portion thereof. In some
embodiments, the Cas protein is Cas9 protein from any bacterial
species or functional portion thereof. Cas9 protein is a member of
the type II CRISPR systems which typically include a trans-coded
small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas
protein. Cas 9 protein (also known as CRISPR-associated
endonuclease Cas9/Csn1) is a polypeptide comprising 1368 amino
acids. An exemplary amino acid sequence of a Cas9 protein (SEQ ID
NO: 298) is shown in FIG. 3. Cas 9 contains 2 endonuclease domains,
including an RuvC-like domain (residues 7-22, 759-766 and 982-989)
which cleaves target DNA that is noncomplementary to crRNA, and an
HNH nuclease domain (residues 810-872) which cleave target DNA
complementary to crRNA. In FIG. 3, the RuvC-like domain is
highlighted in yellow and the HNH nuclease domain is
underlined.
[0150] As used herein, "functional portion" refers to a portion of
a peptide which retains its ability to complex with at least one
ribonucleic acid (e.g., guide RNA (gRNA)) and cleave a target
polynucleotide sequence. In some embodiments, the functional
portion comprises a combination of operably linked Cas9 protein
functional domains selected from the group consisting of a DNA
binding domain, at least one RNA binding domain, a helicase domain,
and an endonuclease domain. In some embodiments, the functional
domains form a complex.
[0151] In some embodiments, a functional portion of the Cas9
protein comprises a functional portion of a RuvC-like domain. In
some embodiments, a functional portion of the Cas9 protein
comprises a functional portion of the HNH nuclease domain.
[0152] It should be appreciated that the present invention
contemplates various of ways of contacting a target polynucleotide
sequence with a Cas protein (e.g., Cas9). In some embodiments,
exogenous Cas protein can be introduced into the cell in
polypeptide form. In certain embodiments, Cas proteins can be
conjugated to or fused to a cell-penetrating polypeptide or
cell-penetrating peptide. As used herein, "cell-penetrating
polypeptide" and "cell-penetrating peptide" refers to a polypeptide
or peptide, respectively, which facilitates the uptake of molecule
into a cell. The cell-penetrating polypeptides can contain a
detectable label.
[0153] In certain embodiments, Cas proteins can be conjugated to or
fused to a charged protein (e.g., that carries a positive, negative
or overall neutral electric charge). Such linkage may be covalent.
In some embodiments, the Cas protein can be fused to a
superpositively charged GFP to significantly increase the ability
of the Cas protein to penetrate a cell (Cronican et al. ACS Chem
Biol. 2010; 5(8):747-52).
[0154] In certain embodiments, the Cas protein can be fused to a
protein transduction domain (PTD) to facilitate its entry into a
cell. Exemplary PTDs include Tat, oligoarginine, and
penetratin.
[0155] In some embodiments, the Cas9 protein comprises a Cas9
polypeptide fused to a cell-penetrating peptide. In some
embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to
a PTD. In some embodiments, the Cas9 protein comprises a Cas9
polypeptide fused to a tat domain. In some embodiments, the Cas9
protein comprises a Cas9 polypeptide fused to an oligoarginine
domain. In some embodiments, the Cas9 protein comprises a Cas9
polypeptide fused to a penetratin domain. In some embodiments, the
Cas9 protein comprises a Cas9 polypeptide fused to a
superpositively charged GFP.
[0156] In some embodiments, the Cas protein can be introduced into
a cell containing the target polynucleotide sequence in the form of
a nucleic acid encoding the Cas protein (e.g., Cas9). The process
of introducing the nucleic acids into cells can be achieved by any
suitable technique. Suitable techniques include calcium phosphate
or lipid-mediated transfection, electroporation, and transduction
or infection using a viral vector. In some embodiments, the nucleic
acid comprises DNA. In some embodiments, the nucleic acid comprises
a modified DNA, as described herein. In some embodiments, the
nucleic acid comprises mRNA. In some embodiments, the nucleic acid
comprises a modified mRNA, as described herein (e.g., a synthetic,
modified mRNA).
[0157] In some embodiments, the Cas protein is complexed with the
one to two ribonucleic acids. In some embodiments, the Cas protein
is complexed with two ribonucleic acids. In some embodiments, the
Cas protein is encoded by a modified nucleic acid, as described
herein (e.g., a synthetic, modified mRNA).
[0158] The methods of the present invention contemplate the use of
any ribonucleic acid that is capable of directing a Cas protein to
and hybridizing to a target motif of a target polynucleotide
sequence. In some embodiments, at least one of the ribonucleic
acids comprises tracrRNA. In some embodiments, at least one of the
ribonucleic acids comprises CRISPR RNA (crRNA). In some
embodiments, at least one of the ribonucleic acids comprises a
guide RNA that directs the Cas protein to and hybridizes to a
target motif of the target polynucleotide sequence in a cell. In
some embodiments, both of the one to two ribonucleic acids comprise
a guide RNA that directs the Cas protein to and hybridizes to a
target motif of the target polynucleotide sequence in a cell. The
ribonucleic acids of the present invention can be selected to
hybridize to a variety of different target motifs, depending on the
particular CRISPR/Cas system employed, and the sequence of the
target polynucleotide, as will be appreciated by those skilled in
the art. The one to two ribonucleic acids can also be selected to
minimize hybridization with nucleic acid sequences other than the
target polynucleotide sequence. In some embodiments, the one to two
ribonucleic acids hybridize to a target motif that contains at
least two mismatches when compared with all other genomic
nucleotide sequences in the cell. In some embodiments, the one to
two ribonucleic acids hybridize to a target motif that contains at
least one mismatch when compared with all other genomic nucleotide
sequences in the cell. In some embodiments, the one to two
ribonucleic acids are designed to hybridize to a target motif
immediately adjacent to a deoxyribonucleic acid motif recognized by
the Cas protein. In some embodiments, each of the one to two
ribonucleic acids are designed to hybridize to target motifs
immediately adjacent to deoxyribonucleic acid motifs recognized by
the Cas protein which flank a mutant allele located between the
target motifs.
[0159] In some embodiments, at least one of the one to two
ribonucleic acids comprises a sequence selected from the group
consisting of the ribonucleic acid sequences of FIG. 1. In some
embodiments, at least one of the one to two ribonucleic acids
comprises a sequence with a single nucleotide mismatch to a
sequence selected from the group consisting of the ribonucleic acid
sequences of FIG. 1.
[0160] In some embodiments, at least one of the one to two
ribonucleic acids comprises a sequence selected from the group
consisting of the ribonucleic acid sequences of FIG. 2. In some
embodiments, at least one of the one to two ribonucleic acids
comprises a sequence with a single nucleotide mismatch to a
sequence selected from the group consisting of the ribonucleic acid
sequences of FIG. 2.
[0161] In some embodiments, each of the one to two ribonucleic
acids comprises guide RNAs that directs the Cas protein to and
hybridizes to a target motif of the target polynucleotide sequence
in a cell. In some embodiments, the two guide RNA sequences
comprise any combination of two guide ribonucleic acid sequences
selected from the group consisting of SEQ ID NOs: 1-139. In some
embodiments, the two guide RNA sequences comprise any combination
of two guide ribonucleic acid sequences comprising RNA sequences
which are complementary to two different sequences selected from
the group consisting of SEQ ID NOs: 1-139. In some embodiments, the
two guide RNA sequences comprise any combination of two guide
ribonucleic acid sequences comprising RNA sequences which are
complementary to two different offset sequences selected from the
group consisting of SEQ ID NOs: 1-139. In some embodiments, the two
guide RNA sequences comprise any combination of two guide
ribonucleic acid sequences comprising RNA sequences which are
complementary to two different sequences comprising at least one
nucleotide mismatch compared to a sequence selected from the group
consisting of SEQ ID NOs: 1-139. In some embodiments, the two guide
RNA sequences comprise any combination of two guide ribonucleic
acid sequences comprising RNA sequences which are complementary to
two different offset sequences comprising at least one nucleotide
mismatch compared to a sequence selected from the group consisting
of SEQ ID NOs: 1-139. In some embodiments, the two guide RNA
sequences comprise any combination of two guide ribonucleic acid
sequences comprising RNA sequences which hybridize to two different
sequences selected from the group consisting of SEQ ID NOs: 1-139.
In some embodiments, the two guide RNA sequences comprise any
combination of two guide ribonucleic acid sequences comprising RNA
sequences which hybridize to two different offset sequences
selected from the group consisting of SEQ ID NOs: 1-139. In some
embodiments, the two guide RNA sequences comprise any combination
of two guide ribonucleic acid sequences comprising RNA sequences
which hybridize to two different sequences comprising at least one
nucleotide mismatch compared to a sequence selected from the group
consisting of SEQ ID NOs: 1-139. In some embodiments, the two guide
RNA sequences comprise any combination of two guide ribonucleic
acid sequences comprising RNA sequences which hybridize to two
different offset sequences comprising at least one nucleotide
mismatch compared to a sequence selected from the group consisting
of SEQ ID NOs: 1-139.
[0162] In some embodiments, the two guide RNA sequences comprise
any combination of two guide ribonucleic acid sequences selected
from the group consisting of SEQ ID NOs: 140-297. In some
embodiments, the two guide RNA sequences comprise any combination
of two guide ribonucleic acid sequences comprising RNA sequences
which are complementary to two different sequences selected from
the group consisting of SEQ ID NOs: 140-297. In some embodiments,
the two guide RNA sequences comprise any combination of two guide
ribonucleic acid sequences comprising RNA sequences which are
complementary to two different offset sequences selected from the
group consisting of SEQ ID NOs: 140-297. In some embodiments, the
two guide RNA sequences comprise any combination of two guide
ribonucleic acid sequences comprising RNA sequences which are
complementary to two different sequences comprising at least one
nucleotide mismatch compared to a sequence selected from the group
consisting of SEQ ID NOs: 140-297. In some embodiments, the two
guide RNA sequences comprise any combination of two guide
ribonucleic acid sequences comprising RNA sequences which are
complementary to two different offset sequences comprising at least
one nucleotide mismatch compared to a sequence selected from the
group consisting of SEQ ID NOs: 140-297. In some embodiments, the
two guide RNA sequences comprise any combination of two guide
ribonucleic acid sequences comprising RNA sequences which hybridize
to two different sequences selected from the group consisting of
SEQ ID NOs: 140-297. In some embodiments, the two guide RNA
sequences comprise any combination of two guide ribonucleic acid
sequences comprising RNA sequences which hybridize to two different
offset sequences selected from the group consisting of SEQ ID NOs:
140-297. In some embodiments, the two guide RNA sequences comprise
any combination of two guide ribonucleic acid sequences comprising
RNA sequences which hybridize to two different sequences comprising
at least one nucleotide mismatch compared to a sequence selected
from the group consisting of SEQ ID NOs: 140-297. In some
embodiments, the two guide RNA sequences comprise any combination
of two guide ribonucleic acid sequences comprising RNA sequences
which hybridize to two different offset sequences comprising at
least one nucleotide mismatch compared to a sequence selected from
the group consisting of SEQ ID NOs: 140-297.
[0163] In some embodiments, the two guide RNA sequences comprise
any combination of two guide ribonucleic acid sequences selected
from the group consisting of SEQ ID NOs: 298-303. In some
embodiments, the two guide ribonucleic acid sequences comprise a
pair of guide ribonucleic acids selected from the group consisting
of SEQ ID NOs: 299 and 303, SEQ ID NOs: 298 and 300, SEQ ID NOs:
299 and 300, SEQ ID NOs: 298 and 303. SEQ ID NOs: 299 and 301, SEQ
ID NOs: 298 and 299, SEQ ID NOs: 301 and 303. SEQ ID NOs: 298 and
302, and SEQ ID NOs: 298 and 301. In some embodiments, the two
guide RNA sequences comprise any combination of two guide
ribonucleic acid sequences comprising RNA sequences which are
complementary to two different sequences selected from the group
consisting of SEQ ID NOs: 304-333. In some embodiments, the two
guide RNA sequences comprise any combination of two guide
ribonucleic acid sequences comprising RNA sequences which are
complementary to two different offset sequences selected from the
group consisting of SEQ ID NOs: 304-333. In some embodiments, the
two guide RNA sequences comprise any combination of two guide
ribonucleic acid sequences comprising RNA sequences which are
complementary to two different sequences comprising at least one
nucleotide mismatch compared to a sequence selected from the group
consisting of SEQ ID NOs: 304-333. In some embodiments, the two
guide RNA sequences comprise any combination of two guide
ribonucleic acid sequences comprising RNA sequences which are
complementary to two different offset sequences comprising at least
one nucleotide mismatch compared to a sequence selected from the
group consisting of SEQ ID NOs: 304-333. In some embodiments, the
two guide RNA sequences comprise any combination of two guide
ribonucleic acid sequences comprising RNA sequences which hybridize
to two different sequences selected from the group consisting of
SEQ ID NOs: 304-333. In some embodiments, the two guide RNA
sequences comprise any combination of two guide ribonucleic acid
sequences comprising RNA sequences which hybridize to two different
offset sequences selected from the group consisting of SEQ ID NOs:
304-333. In some embodiments, the two guide RNA sequences comprise
any combination of two guide ribonucleic acid sequences comprising
RNA sequences which hybridize to two different sequences comprising
at least one nucleotide mismatch compared to a sequence selected
from the group consisting of SEQ ID NOs: 304-333. In some
embodiments, the two guide RNA sequences comprise any combination
of two guide ribonucleic acid sequences comprising RNA sequences
which hybridize to two different offset sequences comprising at
least one nucleotide mismatch compared to a sequence selected from
the group consisting of SEQ ID NOs: 304-333.
[0164] In some embodiments, the two ribonucleic acids (e.g., guide
RNAs) are complementary to and/or hybridize to sequences on the
same strand of a target polynucleotide sequence. In some
embodiments, the two ribonucleic acids (e.g., guide RNAs) are
complementary to and/or hybridize to sequences on the opposite
strands of a target polynucleotide sequence. In some embodiments,
the two ribonucleic acids (e.g., guide RNAs) are not complementary
to and/or do not hybrize to sequences on the opposite strands of a
target polynucleotide sequence. In some embodiments, the two
ribonucleic acids (e.g., guide RNAs) are complementary to and/or
hybridize to overlapping target motifs of a target polynucleotide
sequence. In some embodiments, the two ribonucleic acids (e.g.,
guide RNAs) are complementary to and/or hybridize to offset target
motifs of a target polynucleotide sequence.
[0165] The present invention also contemplates multiplex genomic
editing. Those skilled in the art will appreciate that the
description above with respect to genomic editing of a single gene
is equally applicable to the multiplex genomic editing embodiments
described below.
[0166] In another aspect, the present invention provides a method
for simultaneously altering multiple target polynucleotide
sequences in a cell.
[0167] An exemplary method for simultaneously altering multiple
target polynucleotide sequences in a cell comprises contacting the
polynucleotide sequences with a clustered regularly interspaced
short palindromic repeats-associated (Cas) protein and multiple
ribonucleic acids, wherein the ribonucleic acids direct Cas protein
to and hybridize to target motifs of the target polynucleotide
sequences, wherein the target polynucleotide sequences are cleaved,
and wherein the efficiency of alteration of cells that express Cas
protein is from about 50% to about 80%.
[0168] In yet another aspect, the present invention provides a
method for treating or preventing a disorder associated with
expression of polynucleotide sequences in a subject.
[0169] An exemplary method for treating or preventing a disorder
associated with expression of polynucleotide sequences in a subject
comprises (a) altering target polynucleotide sequences in a cell ex
vivo by contacting the polynucleotide sequences with a clustered
regularly interspaced short palindromic repeats-associated (Cas)
protein and multiple ribonucleic acids, wherein the ribonucleic
acids direct Cas protein to and hybridize to target motifs of the
target polynucleotide sequences, wherein the target polynucleotide
sequences are cleaved, and wherein the efficiency of alteration of
cells that express Cas protein is from about 50% to about 80%, and
(b) introducing the cell into the subject, thereby treating or
preventing a disorder associated with expression of the
polynucleotide sequences.
[0170] As used herein, the terms "administering," "introducing" and
"transplanting" are used interchangeably in the context of the
placement of cells, e.g. cells described herein comprising a target
polynucleotide sequence altered according to the methods of the
invention into a subject, by a method or route which results in at
least partial localization of the introduced cells at a desired
site. The cells can be implanted directly to the desired site, or
alternatively be administered by any appropriate route which
results in delivery to a desired location in the subject where at
least a portion of the implanted cells or components of the cells
remain viable. The period of viability of the cells after
administration to a subject can be as short as a few hours, e. g.
twenty-four hours, to a few days, to as long as several years. In
some instances, the cells can also be administered a location other
than the desired site, such as in the liver or subcutaneously, for
example, in a capsule to maintain the implanted cells at the
implant location and avoid migration of the implanted cells.
[0171] For ex vivo methods, cells can include autologous cells,
i.e., a cell or cells taken from a subject who is in need of
altering a target polynucleotide sequence in the cell or cells
(i.e., the donor and recipient are the same individual). Autologous
cells have the advantage of avoiding any immunologically-based
rejection of the cells. Alternatively, the cells can be
heterologous, e.g., taken from a donor. The second subject can be
of the same or different species. Typically, when the cells come
from a donor, they will be from a donor who is sufficiently
immunologically compatible with the recipient, i.e., will not be
subject to transplant rejection, to lessen or remove the need for
immunosuppression. In some embodiments, the cells are taken from a
xenogeneic source, i.e., a non-human mammal that has been
genetically engineered to be sufficiently immunologically
compatible with the recipient, or the recipient's species. Methods
for determining immunological compatibility are known in the art,
and include tissue typing to assess donor-recipient compatibility
for HLA and ABO determinants. See, e.g., Transplantation
Immunology, Bach and Auchincloss, Eds. (Wiley, John & Sons,
Incorporated 1994).
[0172] Any suitable cell culture media can be used for ex vivo
methods of the invention.
[0173] The terms "subject" and "individual" are used
interchangeably herein, and refer to an animal, for example, a
human from whom cells can be obtained and/or to whom treatment,
including prophylactic treatment, with the cells as described
herein, is provided. For treatment of those infections, conditions
or disease states which are specific for a specific animal such as
a human subject, the term subject refers to that specific animal.
The "non-human animals" and "non-human mammals" as used
interchangeably herein, includes mammals such as rats, mice,
rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The
term "subject" also encompasses any vertebrate including but not
limited to mammals, reptiles, amphibians and fish. However,
advantageously, the subject is a mammal such as a human, or other
mammals such as a domesticated mammal. e.g. dog, cat, horse, and
the like, or production mammal, e.g. cow, sheep, pig, and the
like.
[0174] In some embodiments, the alteration results in reduced
expression of the target polynucleotide sequences. In some
embodiments, the alteration results in a knock out of the target
polynucleotide sequences. In some embodiments, the alteration
results in correction of the target polynucleotide sequences from
undesired sequences to desired sequences. In some embodiments, each
alteration is a homozygous alteration. In some embodiments, the
efficiency of alteration at each loci is from about 5% to about
80%. In some embodiments, the efficiency of alteration at each loci
is from about 10% to about 80%. In some embodiments, the efficiency
of alteration at each loci is from about 30% to about 80%. In some
embodiments, the efficiency of alteration at each loci is from
about 50% to about 80%. In some embodiments, the efficiency of
alteration at each loci is from greater than or equal to about
80%.
[0175] In some embodiments, each target polynucleotide sequence is
cleaved such that a double-strand break results. In some
embodiments, each target polynucleotide sequence is cleaved such
that a single-strand break results.
[0176] In some embodiments, the target polynucleotide sequences
comprise multiple different portions of B2M. In some embodiments,
the target polynucleotide sequences comprise multiple different
portions of CCR5. In some embodiments, the target polynucleotide
sequences comprise multiple different portions of CXCR4. In some
embodiments, the target polynucleotide sequences comprise at least
a portion of CCR5 and at least a portion of CXCR4.
[0177] In some embodiments, each target motif is a 20-nucleotide
DNA sequence. In some embodiments, each target motif is a
20-nucleotide DNA sequence beginning with G and immediately
precedes an NGG motif recognized by the Cas protein. In some
embodiments, each target motif is a 20-nucleotide DNA sequence and
immediately precedes an NGG motif recognized by the Cas protein. In
some embodiments, each target motif is G(N)19NGG. In some
embodiments, each target motif is (N)20NGG. In some embodiments,
each target motif is selected such that it contains at least two
mismatches when compared with all other genomic nucleotide
sequences in the cell. In some embodiments, each target motif is
selected such that it contains at least two mismatches when
compared with all other genomic nucleotide sequences in the
cell.
[0178] In some embodiments, each target motif comprises a different
DNA sequence selected from the group consisting of SEQ ID NOs:
1-139. In some embodiments, each target motif comprises a different
DNA sequence comprising at least one nucleotide mismatch compared
to a DNA sequence selected from the group consisting of SEQ ID NOs:
1-139. In some embodiments, each target motif comprises a DNA
sequence comprising at least two nucleotide mismatches compared to
a DNA sequence selected from the group consisting of SEQ ID NOs:
1-139. In some embodiments, each target motif comprises a different
DNA sequence selected from the group consisting of SEQ ID NOs:
140-297. In some embodiments, each target motif comprises a
different DNA sequence comprising at least one nucleotide mismatch
compared to a DNA sequence selected from the group consisting of
SEQ ID NOs: 140-297. In some embodiments, each target motif
comprises a different DNA sequence comprising at least two
nucleotide mismatches compared to a DNA sequence selected from the
group consisting of SEQ ID NOs: 140-297. In some embodiments, each
target motif comprises a different DNA sequence selected from the
group consisting of SEQ ID NOs: 304-333. In some embodiments, each
target motif comprises a different DNA sequence comprising at least
one nucleotide mismatch compared to a DNA sequence selected from
the group consisting of SEQ ID NOs: 304-333. In some embodiments,
each target motif comprises a different DNA sequence comprising at
least two nucleotide mismatches compared to a DNA sequence selected
from the group consisting of SEQ ID NOs: 304-333.
[0179] In some embodiments, subsequent to cleavage of the target
polynucleotide sequences, homology-directed repair occurs. In some
embodiments, homology-directed repair is performed using an
exogenously introduced DNA repair template. In some embodiments,
exogenously introduced DNA repair template is single-stranded. In
some embodiments, exogenously introduced DNA repair template is
double-stranded.
[0180] In some embodiments, the Cas protein (e.g., Cas9) is
complexed with the multiple ribonucleic acids. In some embodiments,
the multiple ribonucleic acids are selected to minimize
hybridization with nucleic acid sequences other than the target
polynucleotide sequence (e.g., multiple alterations of a single
target polynucleotide sequence). In some embodiments, the multiple
ribonucleic acids are selected to minimize hybridization with
nucleic acid sequences other than the target polynucleotide
sequences (e.g., one or more alterations of multiple target
polynucleotide sequences). In some embodiments, each of the
multiple ribonucleic acids hybridize to target motifs that contain
at least two mismatches when compared with all other genomic
nucleotide sequences in the cell. In some embodiments, each of the
multiple ribonucleic acids hybridize to target motifs that contain
at least one mismatch when compared with all other genomic
nucleotide sequences in the cell. In some embodiments, each of the
multiple ribonucleic acids are designed to hybridize to target
motifs immediately adjacent to deoxyribonucleic acid motifs
recognized by the Cas protein. In some embodiments, each of the
multiple ribonucleic acids are designed to hybridize to target
motifs immediately adjacent to deoxyribonucleic acid motifs
recognized by the Cas protein which flank mutant alleles located
between the target motifs.
[0181] In some embodiments, each of the multiple ribonucleic acids
comprises a different sequence selected from the group consisting
of the ribonucleic acid sequences of FIG. 1. In some embodiments,
each of the multiple ribonucleic acids comprises a sequence with a
single nucleotide mismatch to a different sequence selected from
the group consisting of the ribonucleic acid sequences of FIG. 1.
In some embodiments, each of the multiple ribonucleic acids
comprises a different sequence selected from the group consisting
of the ribonucleic acid sequences of FIG. 2. In some embodiments,
each of the multiple ribonucleic acids comprises a sequence with a
single nucleotide mismatch to a different sequence selected from
the group consisting of the ribonucleic acid sequences of FIG. 2.
In some embodiments, each of the multiple ribonucleic acids
comprises a different sequence selected from the group consisting
of the ribonucleic acid sequences of FIG. 1 and the ribonucleic
acid sequences of FIG. 2. In some embodiments, each of the multiple
ribonucleic acids comprises a sequence with a single nucleotide
mismatch to a different sequence selected from the group consisting
of the ribonucleic acid sequences of FIG. 1 and the ribonucleic
acid sequences of FIG. 2.
[0182] In some embodiments, each of the multiple ribonucleic acids
comprises a different ribonucleic acid sequence which is
complementary to and/or hybridizes to a sequence selected from the
group consisting of SEQ ID NOs: 1-139 (FIG. 1). In some
embodiments, each of the multiple ribonucleic acids comprises a
different ribonucleic acid sequence which is complementary to
and/or hybridizes to a sequence with a single nucleotide mismatch
to a sequence selected from the group consisting of SEQ ID NOs:
1-139 (FIG. 1). In some embodiments, each of the multiple
ribonucleic acids comprises a different ribonucleic acid sequence
which is complementary to and/or hybridizes to a sequence selected
from the group consisting of SEQ ID NOs: 140-297 (FIG. 2). In some
embodiments, each of the multiple ribonucleic acids comprises a
different ribonucleic acid sequence which is complementary to
and/or hybridizes to a sequence with a single nucleotide mismatch
to a sequence selected from the group consisting of SEQ ID NOs:
140-297 (FIG. 2).
[0183] In some embodiments, each of the multiple ribonucleic acids
comprises a different sequence selected from the group consisting
of the ribonucleic acid sequences of SEQ ID NOs: 298-303. In some
embodiments, each of the multiple ribonucleic acids comprises a
different ribonucleic acid sequence which is complementary to
and/or hybridizes to a sequence selected from the group consisting
of SEQ ID NOs: 304-333. In some embodiments, each of the multiple
ribonucleic acids comprises a different ribonucleic acid sequence
which is complementary to and/or hybridizes to a sequence with a
single nucleotide mismatch to a sequence selected from the group
consisting of SEQ ID NOs: 304-333.
[0184] It should be appreciated that any of the Cas protein or the
ribonucleic acids can be expressed from a plasmid. In some
embodiments, any of the Cas protein or the ribonucleic acids are
expressed using a promoter optimized for increased expression in
stem cells (e.g., human stem cells). In some embodiments, the
promoter is selected from the group consisting of a Cytomegalovirus
(CMV) early enhancer element and a chicken beta-actin promoter, a
chicken beta-actin promoter, an elongation factor-1 alpha promoter,
and a ubiquitin promoter.
[0185] In some embodiments, the methods of the present invention
further comprise selecting cells that express the Cas protein. The
present invention contemplates any suitable method for selecting
cells. In some embodiments, selecting cells comprises FACS. In some
embodiments, FACs is used to select cells which co-express Cas and
a fluorescent protein selected from the group consisting of green
fluorescent protein and red fluorescent protein.
[0186] The present invention contemplates treating and/or
preventing a variety of disorders which are associated with
expression of a target polynucleotide sequences. It should be
appreciated that the methods and compositions described herein can
be used to treat or prevent disorders associated with increased
expression of a target polynucleotide sequence, as well as
decreased expression of a target polynucleotide sequence in a cell.
Increased and decreased expression of a target polynucleotide
sequence includes circumstances where the expression levels of the
target polynucleotide sequence are increased or decreased,
respectively, as well as circumstances in which the function and/or
level of activity of an expression product of the target
polynucleotide sequence increases or decreases, respectively,
compared to normal expression and/or activity levels. Those skilled
in the art will appreciate that treating or preventing a disorder
associated with increased expression of a target polynucleotide
sequence can be assessed by determining whether the levels and/or
activity of the target polynucleotide sequence (or an expression
product thereof) are decreased in a relevant cell after contacting
a cell with a composition described herein. The skilled artisan
will also appreciate that treating or preventing a disorder
associated with decreased expression of a target polynucleotide
sequence can be assessed by determining whether the levels and/or
activity of the target polynucleotide sequence (or an expression
product thereof) are increased in the relevant cell after
contacting a cell with a composition described herein.
[0187] In some embodiments, the disorder is a genetic disorder. In
some embodiments, the disorder is a monogenic disorder. In some
embodiments, the disorder is a multigenic disorder. In some
embodiments, the disorder is a disorder associated with one or more
SNPs. Exemplary disorders associated with one or more SNPs include
a complex disease described in U.S. Pat. No. 7,627,436, Alzheimer's
disease as described in PCT International Application Publication
No. WO/2009/112882, inflammatory diseases as described in U.S.
Patent Application Publication No. 2011/0039918, polycystic ovary
syndrome as described in U.S. Patent Application Publication No.
2012/0309642, cardiovascular disease as described in U.S. Pat. No.
7,732,139, Huntington's disease as described in U.S. Patent
Application Publication No. 2012/0136039, thromboembolic disease as
described in European Patent Application Publication No. EP2535424,
neurovascular diseases as described in PCT International
Application Publication No. WO/2012/001613, psychosis as described
in U.S. Patent Application Publication No. 2010/0292211, multiple
sclerosis as described in U.S. Patent Application Publication No.
2011/0319288, schizophrenia, schizoaffective disorder, and bipolar
disorder as described in PCT International Application Publication
No. WO/2006/023719A2, bipolar disorder and other ailments as
described in U.S. Patent Application Publication No. U.S.
2011/0104674, colorectal cancer as described in PCT International
Application Publication No. WO/2006/104370A1, a disorder associated
with a SNP adjacent to the AKT1 gene locus as described in U.S.
Patent Application Publication No. U.S. 2006/0204969, an eating
disorder as described in PCT International Application Publication
No. WO/2003/012143A1, autoimmune disease as described in U.S.
Patent Application Publication No. U.S. 2007/0269827,
fibrostenosing disease in patients with Crohn's disease as
described in U.S. Pat. No. 7,790,370, and Parkinson's disease as
described in U.S. Pat. No. 8,187,811, each of which is incorporated
herein by reference in its entirety. Other disorders associated
with one or more SNPs which can be treated or prevented according
to the methods of the present invention will be apparent to the
skilled artisan.
[0188] In some embodiments, the disorder is human immunodeficiency
virus (HIV) infection. In some embodiments, the disorder is
acquired immunodeficiency syndrome (AIDS).
[0189] The methods of the present invention are capable of altering
target polynucleotide sequences in a variety of different cells. In
some embodiments, the methods of the present invention are used to
alter target polynucleotide sequences in cells ex vivo for
subsequent introduction into a subject. In some embodiments, the
cell is a peripheral blood cell. In some embodiments, the cell is a
stem cell or a pluripotent cell. In some embodiments, the cell is a
hematopoietic stem cell. In some embodiments, the cell is a CD34+
cell. In some embodiments, the cell is a CD34+ mobilized peripheral
blood cell. In some embodiments, the cell is a CD34+ cord blood
cell. In some embodiments, the cell is a CD34+ bone marrow cell. In
some embodiments, the cell is a CD34+CD38-Lineage-CD90+CD45RA-
cell. In some embodiments, the cell is a CD4+ cell. In some
embodiments, the cell is a CD4+ T cell. In some embodiments, the
cell is a hepatocyte. In some embodiments, the cell is a human
pluripotent cell. In some embodiments, the cell is a primary human
cell. In some embodiments, the cell is a primary CD34+ cell. In
some embodiments, the cell is a primary CD34+ hematopoietic
progenitor cell (HPC). In some embodiments, the cell is a primary
CD4+ cell. In some embodiments, the cell is a primary CD4+ T cell.
In some embodiments, the cell is an autologous primary cell. In
some embodiments, the cell is an autologous primary somatic cell.
In some embodiments, the cell is an allogeneic primary cell. In
some embodiments, the cell is an allogeneic primary somatic cell.
In some embodiments, the cell is a nucleated cell. In some
embodiments, the cell is a non-transformed cell. In some
embodiments, the cell is not a cancer cell. In some embodiments,
the cell is not a tumor cell. In some embodiments, the cell is not
a transformed cell.
[0190] In some aspects, the present invention provides a method for
altering a target polynucleotide sequence in a cell comprising
contacting the polynucleotide sequence in a cell selected from the
group consisting of a human pluripotent cell, a primary human cell,
and a non-transformed human cell, with a clustered regularly
interspaced short palindromic repeats-associated (Cas) protein and
from one to two ribonucleic acids, wherein the ribonucleic acids
direct Cas protein to and hybridize to a target motif of the target
polynucleotide sequence, wherein the target polynucleotide sequence
is cleaved, and wherein the efficiency of alteration of cells that
express Cas protein is from about 8% to about 80%.
[0191] In some aspects, the present invention provides a method for
treating or preventing a disorder associated with expression of a
polynucleotide sequence in a subject, the method comprising (a)
altering a target polynucleotide sequence in a cell ex vivo by
contacting the polynucleotide sequence in a cell selected from the
group consisting of a human pluripotent cell, a primary human cell,
and a non-transformed human cell, with a clustered regularly
interspaced short palindromic repeats-associated (Cas) protein and
from one to two ribonucleic acids, wherein the ribonucleic acids
direct Cas protein to and hybridize to a target motif of the target
polynucleotide sequence, wherein the target polynucleotide sequence
is cleaved, and wherein the efficiency of alteration is from about
8% to about 80%, and (b) introducing the cell into the subject,
thereby treating or preventing a disorder associated with
expression of the polynucleotide sequence.
[0192] In some aspects, the present invention provides a method for
simultaneously altering multiple target polynucleotide sequences in
a cell comprising contacting the polynucleotide sequences in a cell
selected from the group consisting of a human pluripotent cell, a
primary human cell, and a non-transformed human cell, with a
clustered regularly interspaced short palindromic
repeats-associated (Cas) protein and multiple ribonucleic acids,
wherein the ribonucleic acids direct Cas protein to and hybridize
to target motifs of the target polynucleotide sequences, wherein
the target polynucleotide sequences are cleaved, and wherein the
efficiency of alteration of cells that express Cas protein is from
about 8% to about 80%.
[0193] In some aspects, the present invention provides a method for
treating or preventing a disorder associated with expression of
polynucleotide sequences in a subject, the method comprising (a)
altering target polynucleotide sequences in a cell ex vivo by
contacting the polynucleotide sequences in a cell selected from the
group consisting of a human pluripotent cell, a primary human cell,
and a non-transformed human cell, with a clustered regularly
interspaced short palindromic repeats-associated (Cas) protein and
multiple ribonucleic acids, wherein the ribonucleic acids direct
Cas protein to and hybridize to target motifs of the target
polynucleotide sequences, wherein the target polynucleotide
sequences are cleaved, and wherein the efficiency of alteration of
cells that express Cas protein is from about 8% to about 80%, and
(b) introducing the cell into the subject, thereby treating or
preventing a disorder associated with expression of the
polynucleotide sequences.
[0194] The present invention also provides compositions comprising
Cas proteins of the present invention or functional portions
thereof, nucleic acids encoding the Cas proteins or functional
portions thereof, and ribonucleic acid sequences which direct Cas
proteins to and hybridize to target motifs of target
polynucleotides in a cell.
[0195] In some aspects, the present invention provides a
composition comprising at least one ribonucleic acid having a
sequence selected from the group consisting of the ribonucleic acid
sequences of FIG. 1. In some aspects, the present invention
provides a composition comprising at least one ribonucleic acid
having a sequence which is complementary to and/or hybridizes to a
sequence selected from the group consisting of SEQ ID NOs: 1-139
(FIG. 1).
[0196] In some aspects, the present invention provides a
composition comprising at least one ribonucleic acid comprising a
sequence with a single nucleotide mismatch to a sequence selected
from the group consisting of the ribonucleic acid sequences of FIG.
1. In some aspects, the present invention provides a composition
comprising at least one ribonucleic acid having a sequence which is
complementary to and/or hybridizes to a sequence with a single
nucleotide mismatch to a sequence selected from the group
consisting of SEQ ID NOs: 1-139 (FIG. 1).
[0197] In some aspects, the present invention provides a
composition comprising at least one ribonucleic acid having a
sequence selected from the group consisting of the ribonucleic acid
sequences of FIG. 2. In some aspects, the present invention
provides a composition comprising at least one ribonucleic acid
having a sequence which is complementary to and/or hybridizes to a
sequence selected from the group consisting of SEQ ID NOs: 140-297
(FIG. 2).
[0198] In some aspects, the present invention provides a
composition comprising at least one ribonucleic acid comprising a
sequence with a single nucleotide mismatch to a sequence selected
from the group consisting of the ribonucleic acid sequences of FIG.
2. In some aspects, the present invention provides a composition
comprising at least one ribonucleic acid having a sequence which is
complementary to and/or hybridizes to a sequence with a single
nucleotide mismatch to a sequence selected from the group
consisting of SEQ ID NOs: 140-297 (FIG. 2).
[0199] In some aspects, the present invention provides a
composition comprising at least one ribonucleic acid having a
sequence selected from the group consisting of the ribonucleic acid
sequences of SEQ ID NOs: 298-303. In some aspects, the present
invention provides a composition comprising at least one
ribonucleic acid having a sequence which is complementary to and/or
hybridizes to a sequence selected from the group consisting of SEQ
ID NOs: 304-333. In some aspects, the present invention provides a
composition comprising at least one ribonucleic acid having a
sequence which is complementary to and/or hybridizes to a sequence
comprising at least one nucleotide mismatch compared to a sequence
selected from the group consisting of SEQ ID NOs: 304-333. In some
aspects, the present invention provides a composition comprising at
least one ribonucleic acid having a sequence selected from the
group consisting of the ribonucleic acid sequences of FIG. 1, the
ribonucleic acid sequences of FIG. 2, a sequence with a single
nucleotide mismatch to a ribonucleic acid sequence of FIG. 1, and a
sequence with a single nucleotide mismatch to a ribonucleic acid
sequences of FIG. 2.
[0200] In some embodiments, at least one of the ribonucleic acids
in the composition is a modified ribonucleic acid as described
herein (e.g., a synthetic, modified ribonucleic acid, e.g.,
comprising one to two modified nucleotides selected from the group
consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine,
5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate,
5,6-dihydrouridine-5'-triphosphate, and
5-azauridine-5'-triphosphate, or any other modified nucleotides or
modifications described herein).
[0201] In some embodiments, a composition of the present invention
comprises a nucleic acid sequence encoding a Cas protein. In some
embodiments, a composition of the present invention comprises
nucleic acid sequence encoding Cas9 protein or a functional portion
thereof.
[0202] In some embodiments, the nucleic acid encoding the Cas
protein (e.g., Cas9) comprises a modified ribonucleic acid as
described herein (e.g., a synthetic, modified mRNA described
herein, e.g., comprising at least one modified nucleotide selected
from the group consisting of pseudouridine, 5-methylcytodine,
2-thio-uridine, 5-methyluridine-5'-triphosphate,
4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate,
and 5-azauridine-5'-triphosphate or any other modified nucleotides
or modifications described herein).
[0203] In some aspects, the present invention provides a
composition comprising a chimeric nucleic acid comprising a
ribonucleic acid encoding a Cas protein and at least one additional
ribonucleic acid having a sequence selected from the group
consisting of the ribonucleic acid sequences of FIG. 1. In some
aspects, the present invention provides a composition comprising a
chimeric nucleic acid comprising a ribonucleic acid encoding a Cas
protein and at least one additional ribonucleic acid having a
sequence which is complementary to and/or hybridizes to a sequence
selected from the group consisting of SEQ ID NOs: 1-139 (FIG. 1).
In some aspects, the present invention provides a composition
comprising a chimeric nucleic acid comprising a ribonucleic acid
encoding a Cas protein and two additional ribonucleic acid each
having a sequence which is complementary to and/or hybridizes to a
different sequence selected from the group consisting of SEQ ID
NOs: 1-139 (FIG. 1). In some aspects, the present invention
provides a composition comprising a chimeric nucleic acid
comprising a ribonucleic acid encoding a Cas protein and two
additional ribonucleic acid each having a sequence which is
complementary to and/or hybridizes to an offset sequence selected
from the group consisting of SEQ ID NOs: 1-139 (FIG. 1). In some
aspects, the present invention provides a composition comprising a
chimeric nucleic acid comprising a ribonucleic acid encoding a Cas
protein and at least one additional ribonucleic acid having a
sequence selected from the group consisting of the ribonucleic acid
sequences of FIG. 2. In some aspects, the present invention
provides a composition comprising a chimeric nucleic acid
comprising a ribonucleic acid encoding a Cas protein and at least
one additional ribonucleic acid having a sequence which is
complementary to and/or hybridizes to a sequence selected from the
group consisting of SEQ ID NOs: 140-297 (FIG. 2). In some aspects,
the present invention provides a composition comprising a chimeric
nucleic acid comprising a ribonucleic acid encoding a Cas protein
and two additional ribonucleic acids each having a sequence which
is complementary to and/or hybridizes to a different sequence
selected from the group consisting of SEQ ID NOs: 140-297 (FIG. 2).
In some aspects, the present invention provides a composition
comprising a chimeric nucleic acid comprising a ribonucleic acid
encoding a Cas protein and two additional ribonucleic acids each
having a sequence which is complementary to and/or hybridizes to an
offset sequence selected from the group consisting of SEQ ID NOs:
140-297 (FIG. 2).
[0204] In some aspects, the present invention provides a
composition comprising a chimeric nucleic acid comprising a
ribonucleic acid encoding a Cas protein and at least one additional
ribonucleic acid sequence comprising a sequence with a single
nucleotide mismatch to a sequence selected from the group
consisting of the ribonucleic acid sequences of FIG. 1. In some
aspects, the present invention provides a composition comprising a
chimeric nucleic acid comprising a ribonucleic acid encoding a Cas
protein and at least one additional ribonucleic acid sequence which
is complementary to and/or hybridizes to a sequence with a single
nucleotide mismatch to a sequence selected from the group
consisting of SEQ ID NOs: 1-139 (FIG. 1). In some aspects, the
present invention provides a composition comprising a chimeric
nucleic acid comprising a ribonucleic acid encoding a Cas protein
and two additional ribonucleic acid sequences each of which are
complementary to and/or hybridize to different sequences with
single nucleotide mismatches to a sequence selected from the group
consisting of SEQ ID NOs: 1-139 (FIG. 1). In some aspects, the
present invention provides a composition comprising a chimeric
nucleic acid comprising a ribonucleic acid encoding a Cas protein
and two additional ribonucleic acid sequences each of which are
complementary to and/or hybridize to offset sequences with single
nucleotide mismatches to a sequence selected from the group
consisting of SEQ ID NOs: 1-139 (FIG. 1).
[0205] In some aspects, the present invention provides a
composition comprising a chimeric nucleic acid comprising a
ribonucleic acid encoding a Cas protein and at least one additional
ribonucleic acid sequence comprising a sequence with a single
nucleotide mismatch to a sequence selected from the group
consisting of the ribonucleic acid sequences of FIG. 2. In some
aspects, the present invention provides a composition comprising a
chimeric nucleic acid comprising a ribonucleic acid encoding a Cas
protein and at least one additional ribonucleic acid sequence which
is complementary to and/or hybridizes to a sequence with a single
nucleotide mismatch to a sequence selected from the group
consisting of SEQ ID NOs: 140-297 (FIG. 1). In some aspects, the
present invention provides a composition comprising a chimeric
nucleic acid comprising a ribonucleic acid encoding a Cas protein
and two additional ribonucleic acid sequences each of which is
complementary to and/or hybridizes to a different sequence with a
single nucleotide mismatch to a sequence selected from the group
consisting of SEQ ID NOs: 140-297 (FIG. 1). In some aspects, the
present invention provides a composition comprising a chimeric
nucleic acid comprising a ribonucleic acid encoding a Cas protein
and two additional ribonucleic acid sequences each of which is
complementary to and/or hybridizes to an offset sequence with a
single nucleotide mismatch to a sequence selected from the group
consisting of SEQ ID NOs: 140-297 (FIG. 1).
[0206] In some aspects, the present invention provides a
composition comprising a chimeric nucleic acid comprising a
ribonucleic acid encoding a Cas protein and at least one additional
ribonucleic acid having a sequence selected from the group
consisting of the ribonucleic acid sequences of SEQ ID NOs:
298-303.
[0207] In some aspects, the present invention provides a
composition comprising a chimeric nucleic acid comprising a
ribonucleic acid encoding a Cas protein and at least two additional
ribonucleic acid having a sequence selected from the group
consisting of the ribonucleic acid sequences of SEQ ID NOs:
298-303.
[0208] In some aspects, the present invention provides a
composition comprising a chimeric nucleic acid comprising a
ribonucleic acid encoding a Cas protein and two guide ribonucleic
acids comprising SEQ ID NOs: 299 and 303. In some aspects, the
present invention provides a composition comprising a chimeric
nucleic acid comprising a ribonucleic acid encoding a Cas protein
and two guide ribonucleic acids comprising SEQ ID NOs: 298 and 300.
In some aspects, the present invention provides a composition
comprising a chimeric nucleic acid comprising a ribonucleic acid
encoding a Cas protein and two guide ribonucleic acids comprising
SEQ ID NOs: 299 and 300. In some aspects, the present invention
provides a composition comprising a chimeric nucleic acid
comprising a ribonucleic acid encoding a Cas protein and two guide
ribonucleic acids comprising SEQ ID NOs: 298 and 303. In some
aspects, the present invention provides a composition comprising a
chimeric nucleic acid comprising a ribonucleic acid encoding a Cas
protein and two guide ribonucleic acids comprising SEQ ID NOs: 299
and 301. In some aspects, the present invention provides a
composition comprising a chimeric nucleic acid comprising a
ribonucleic acid encoding a Cas protein and two guide ribonucleic
acids comprising SEQ ID NOs: 298 and 299. In some aspects, the
present invention provides a composition comprising a chimeric
nucleic acid comprising a ribonucleic acid encoding a Cas protein
and two guide ribonucleic acids comprising SEQ ID NOs: 301 and 303.
In some aspects, the present invention provides a composition
comprising a chimeric nucleic acid comprising a ribonucleic acid
encoding a Cas protein and two guide ribonucleic acids comprising
SEQ ID NOs: 298 and 302. In some aspects, the present invention
provides a composition comprising a chimeric nucleic acid
comprising a ribonucleic acid encoding a Cas protein and two guide
ribonucleic acids comprising SEQ ID NOs: 298 and 301.
[0209] In some aspects, the present invention provides a
composition comprising at least one ribonucleic acid having a
sequence which is complementary to and/or hybridizes to a sequence
selected from the group consisting of SEQ ID NOs: 304-333. In some
aspects, the present invention provides a composition comprising at
least two ribonucleic acids each having a sequence which is
complementary to and/or hybridizes to a different sequence selected
from the group consisting of SEQ ID NOs: 304-333. In some aspects,
the present invention provides a composition comprising at least
two ribonucleic acids each having a sequence which is complementary
to and/or hybridizes to an offset sequence selected from the group
consisting of SEQ ID NOs: 304-333. In some aspects, the present
invention provides a composition comprising at least one
ribonucleic acid having a sequence which is complementary to and/or
hybridizes to a sequence comprising at least one nucleotide
mismatch compared to a sequence selected from the group consisting
of SEQ ID NOs: 304-333. In some aspects, the present invention
provides a composition comprising at least two ribonucleic acids
each having a sequence which is complementary to and/or hybridizes
to a different sequence comprising at least one nucleotide mismatch
compared to a sequence selected from the group consisting of SEQ ID
NOs: 304-333. In some aspects, the present invention provides a
composition comprising at least two ribonucleic acids each having a
sequence which is complementary to and/or hybridizes to an offset
sequence comprising at least one nucleotide mismatch compared to a
sequence selected from the group consisting of SEQ ID NOs:
304-333.
[0210] In some aspects, the present invention provides a
composition comprising a chimeric nucleic acid comprising a
ribonucleic acid encoding a Cas protein and at least one additional
ribonucleic acid having a sequence selected from the group
consisting of the ribonucleic acid sequences of FIG. 1, the
ribonucleic acid sequences of FIG. 2, a sequence with a single
nucleotide mismatch to a ribonucleic acid sequence of FIG. 1, and a
sequence with a single nucleotide mismatch to a ribonucleic acid
sequences of FIG. 2.
[0211] In some embodiments, a composition of the present invention
comprises a nucleic acid sequence encoding a fluorescent protein
selected from the group consisting of green fluorescent protein and
red fluorescent protein. In some embodiments, a composition of the
present invention comprises a promoter operably linked to the
chimeric nucleic acid. In some embodiments, the promoter is
optimized for increased expression in human stem cells. In some
embodiments, the promoter is optimized for increased expression in
primary human cells. In some embodiments, the promoter is selected
from the group consisting of a Cytomegalovirus (CMV) early enhancer
element and a chicken beta-actin promoter, a chicken beta-actin
promoter, an elongation factor-1 alpha promoter, and a ubiquitin
promoter.
[0212] In some embodiments, the Cas protein comprises a Cas9
protein or a functional portion thereof.
[0213] The present invention also provides kits for practicing any
of the methods of the present invention, as well as kits comprising
the compositions of the present invention, and instructions for
using the kits for altering target polynucleotide sequences in a
cell.
[0214] In some aspects, the present invention comprises a kit for
altering a target polynucleotide sequence in a cell comprising a
Cas9 protein or a nucleic acid encoding the Cas9 protein, and at
least one ribonucleic acid sequence selected from the group
consisting of the ribonucleic acid sequences of FIG. 1, the
ribonucleic acid sequences of FIG. 2, a sequence with a single
nucleotide mismatch to a ribonucleic acid sequence of FIG. 1, and a
sequence with a single nucleotide mismatch to a ribonucleic acid
sequences of FIG. 2.
[0215] In some aspects, the present invention comprises a kit for
altering a target polynucleotide sequence in a cell comprising a
Cas9 protein or a nucleic acid encoding the Cas9 protein, and at
least one ribonucleic acid sequence selected from the group
consisting of the ribonucleic acid sequences of SEQ ID NO: 1-139
(FIG. 1).
[0216] In some aspects, the present invention comprises a kit for
altering a target polynucleotide sequence in a cell comprising a
Cas9 protein or a nucleic acid encoding the Cas9 protein, and at
least one ribonucleic acid sequence which is complementary to
and/or hybridizes to a sequence selected from the group consisting
of SEQ ID NO: 1-139 (FIG. 1).
[0217] In some aspects, the present invention comprises a kit for
altering a target polynucleotide sequence in a cell comprising a
Cas9 protein or a nucleic acid encoding the Cas9 protein, and at
least one ribonucleic acid sequence which is complementary to
and/or hybridizes to a sequence comprising at least one nucleotide
mismatch to a sequence selected from the group consisting of SEQ ID
NO: 1-139 (FIG. 1).
[0218] In some aspects, the present invention comprises a kit for
altering a target polynucleotide sequence in a cell comprising a
Cas9 protein or a nucleic acid encoding the Cas9 protein, and at
least two ribonucleic acid sequences selected from the group
consisting of the ribonucleic acid sequences of SEQ ID NO: 1-139
(FIG. 1).
[0219] In some aspects, the present invention comprises a kit for
altering a target polynucleotide sequence in a cell comprising a
Cas9 protein or a nucleic acid encoding the Cas9 protein, and at
least two ribonucleic acid sequences each of which is complementary
to and/or hybridizes to a different sequence selected from the
group consisting of SEQ ID NO: 1-139 (FIG. 1).
[0220] In some aspects, the present invention comprises a kit for
altering a target polynucleotide sequence in a cell comprising a
Cas9 protein or a nucleic acid encoding the Cas9 protein, and at
least two ribonucleic acid sequences each of which is complementary
to and/or hybridizes to an offset sequence selected from the group
consisting of SEQ ID NO: 1-139 (FIG. 1).
[0221] In some aspects, the present invention comprises a kit for
altering a target polynucleotide sequence in a cell comprising a
Cas9 protein or a nucleic acid encoding the Cas9 protein, and at
least two ribonucleic acid sequences each of which is complementary
to and/or hybridizes to a different sequence comprising at least
one nucleotide mismatch to a sequence selected from the group
consisting of SEQ ID NO: 1-139 (FIG. 1).
[0222] In some aspects, the present invention comprises a kit for
altering a target polynucleotide sequence in a cell comprising a
Cas9 protein or a nucleic acid encoding the Cas9 protein, and at
least two ribonucleic acid sequences each of which is complementary
to and/or hybridizes to an offset sequence comprising at least one
nucleotide mismatch to a sequence selected from the group
consisting of SEQ ID NO: 1-139 (FIG. 1).
[0223] In some aspects, the present invention comprises a kit for
altering a target polynucleotide sequence in a cell comprising a
Cas9 protein or a nucleic acid encoding the Cas9 protein, and at
least one ribonucleic acid sequence selected from the group
consisting of the ribonucleic acid sequences of SEQ ID NO: 140-297
(FIG. 2).
[0224] In some aspects, the present invention comprises a kit for
altering a target polynucleotide sequence in a cell comprising a
Cas9 protein or a nucleic acid encoding the Cas9 protein, and at
least one ribonucleic acid sequence which is complementary to
and/or hybridizes to a sequence selected from the group consisting
of SEQ ID NO: 140-297 (FIG. 2).
[0225] In some aspects, the present invention comprises a kit for
altering a target polynucleotide sequence in a cell comprising a
Cas9 protein or a nucleic acid encoding the Cas9 protein, and at
least one ribonucleic acid sequence which is complementary to
and/or hybridizes to a sequence comprising at least one nucleotide
mismatch to a sequence selected from the group consisting of SEQ ID
NO: 140-297 (FIG. 2).
[0226] In some aspects, the present invention comprises a kit for
altering a target polynucleotide sequence in a cell comprising a
Cas9 protein or a nucleic acid encoding the Cas9 protein, and at
least two ribonucleic acid sequences selected from the group
consisting of the ribonucleic acid sequences of SEQ ID NO: 140-297
(FIG. 2).
[0227] In some aspects, the present invention comprises a kit for
altering a target polynucleotide sequence in a cell comprising a
Cas9 protein or a nucleic acid encoding the Cas9 protein, and at
least two ribonucleic acid sequences each of which is complementary
to and/or hybridizes to a different sequence selected from the
group consisting of SEQ ID NO: 140-297 (FIG. 2).
[0228] In some aspects, the present invention comprises a kit for
altering a target polynucleotide sequence in a cell comprising a
Cas9 protein or a nucleic acid encoding the Cas9 protein, and at
least two ribonucleic acid sequences each of which is complementary
to and/or hybridizes to an offset sequence selected from the group
consisting of SEQ ID NO: 140-297 (FIG. 2).
[0229] In some aspects, the present invention comprises a kit for
altering a target polynucleotide sequence in a cell comprising a
Cas9 protein or a nucleic acid encoding the Cas9 protein, and at
least two ribonucleic acid sequences each of which is complementary
to and/or hybridizes to a different sequence comprising at least
one nucleotide mismatch to a sequence selected from the group
consisting of SEQ ID NO: 140-297 (FIG. 2).
[0230] In some aspects, the present invention comprises a kit for
altering a target polynucleotide sequence in a cell comprising a
Cas9 protein or a nucleic acid encoding the Cas9 protein, and at
least two ribonucleic acid sequences each of which is complementary
to and/or hybridizes to an offset sequence comprising at least one
nucleotide mismatch to a sequence selected from the group
consisting of SEQ ID NO: 140-297 (FIG. 2).
[0231] In some aspects, the present invention comprises a kit for
altering a target polynucleotide sequence in a cell comprising a
Cas9 protein or a nucleic acid encoding the Cas9 protein, and at
least two ribonucleic acid sequences selected from the group
consisting of the ribonucleic acid sequences of SEQ ID NO: 298-303.
In some embodiments, the at least two ribonucleic acid sequences of
SEQ ID NO: 298-303 are complementary to and/or hybridize to offset
target sequences.
[0232] In some aspects, the present invention comprises a kit for
altering a target polynucleotide sequence in a cell comprising a
Cas9 protein or a nucleic acid encoding the Cas9 protein, and at
least two ribonucleic acid sequences each of which is complementary
to and/or hybridizes to a different sequence selected from the
group consisting of SEQ ID NO: 304-333.
[0233] In some aspects, the present invention comprises a kit for
altering a target polynucleotide sequence in a cell comprising a
Cas9 protein or a nucleic acid encoding the Cas9 protein, and at
least two ribonucleic acid sequences each of which is complementary
to and/or hybridizes to an offset sequence selected from the group
consisting of SEQ ID NO: 304-333.
[0234] In some aspects, the present invention comprises a kit for
altering a target polynucleotide sequence in a cell comprising a
Cas9 protein or a nucleic acid encoding the Cas9 protein, and at
least two ribonucleic acid sequences each of which is complementary
to and/or hybridizes to a different sequence comprising at least
one nucleotide mismatch to a sequence selected from the group
consisting of SEQ ID NO: 304-333.
[0235] In some aspects, the present invention comprises a kit for
altering a target polynucleotide sequence in a cell comprising a
Cas9 protein or a nucleic acid encoding the Cas9 protein, and at
least two ribonucleic acid sequences each of which is complementary
to and/or hybridizes to an offset sequence comprising at least one
nucleotide mismatch to a sequence selected from the group
consisting of SEQ ID NO: 304-333.
[0236] In some embodiments, the kit comprises one or more cell
lines, cultures, or populations selected from the group consisting
of human pluripotent cells, primary human cells, and
non-transformed cells. In some embodiments, the kit comprises a DNA
repair template.
[0237] In some aspects, the invention provides a method of
administering cells to a subject in need of such cells, the method
comprising: (a) contacting a cell or population of cells ex vivo
with a Cas protein and two ribonucleic acids which direct Cas
protein to and hybridize to a target polynucleotide sequence
encoding B2M in the cell or population of cells, wherein the target
polynucleotide sequence is cleaved, and (b) administering the
resulting cells from (a) to a subject in need of such cells.
[0238] In some aspects, the invention provides a method of
administering cells to a subject in need of such cells, the method
comprising: (a) contacting a cell or population of cells ex vivo
with (i) a Cas protein, (ii) at least two ribonucleic acids which
direct Cas protein to and hybridize to a target polynucleotide
sequence encoding B2M in the cell or population of cells, and (iii)
at least two additional ribonucleic acids which direct Cas protein
to and hybridize to a target polynucleotide sequence in the cell or
population of cells, wherein the target polynucleotide sequences
are cleaved, and (b) administering the resulting cell or cells from
(a) to a subject in need of such cells.
[0239] B2M is an accessory chain of the MHC class I proteins which
is necessary for the expression of MHC class I proteins on the
surface of cells. It is believed that engineering cells (e.g.,
mutant cells) devoid of surface MHC class I may reduce the
likelihood that the engineered cells will be detected by cytotoxic
T cells when the engineered cells are administered to a host.
Accordingly, in some embodiments, cleavage of the target
polynucleotide sequence encoding B2M in the cell or population of
cells reduces the likelihood that the resulting cell or cells will
trigger a host immune response when the cells are administered to
the subject.
[0240] In some aspects, the invention provides a method of reducing
the likelihood that cells administered to a subject will trigger a
host immune response in the subject, the method comprising: (a)
contacting a cell or population of cells ex vivo with a Cas protein
and two ribonucleic acids which direct Cas protein to and hybridize
to a target polynucleotide sequence encoding B2M in the cell or
population of cells, wherein the target polynucleotide sequence
encoding B2M is cleaved, thereby reducing the likelihood that cells
administered to the subject will trigger a host immune response in
the subject; and (b) administering the resulting cells from (a) to
a subject in need of such cells.
[0241] In some aspects, the invention provides a method of reducing
the likelihood that cells administered to a subject will trigger a
host immune response in the subject, the method comprising: (a)
contacting a cell or population of cells ex vivo with (i) a Cas
protein. (ii) at least two ribonucleic acids which direct Cas
protein to and hybridize to a target polynucleotide sequence
encoding B2M in the cell or population of cells, wherein the target
polynucleotide sequence encoding B2M in the cell or population of
cells is cleaved, thereby reducing the likelihood that the cell or
population of cells will trigger a host immune response in the
subject, and (iii) at least two additional ribonucleic acids which
direct Cas protein to and hybridize to a target polynucleotide
sequence in the cell or population of cells, wherein the target
polynucleotide sequence is cleaved; and (b) administering the
resulting cell or cells from (a) to a subject in need of such
cells.
[0242] It is contemplated that the methods of administering cells
can be adapted for any purpose in which administering such cells is
desirable. In some embodiments, the subject in need of
administration of cells is suffering from a disorder. For example,
the subject may be suffering from a disorder in which the
particular cells are decreased in function or number, and it may be
desirable to administer functional cells obtained from a healthy or
normal individual in which the particular cells are functioning
properly and to administer an adequate number of those healthy
cells to the individual to restore the function provided by those
cells (e.g., hormone producing cells which have decreased in cell
number or function, immune cells which have decreased in cell
number or function, etc.). In such instances, the healthy cells can
be engineered to decrease the likelihood of host rejection of the
healthy cells. In some embodiments, the disorder comprises a
genetic disorder. In some embodiments, the disorder comprises an
infection. In some embodiments, the disorder comprises HIV or AIDs.
In some embodiments, the disorder comprises cancer.
[0243] In some aspects, the invention provides a method of reducing
the likelihood that cells administered to a subject will trigger a
host immune response in the subject, the method comprising: (a)
contacting a cell or population of cells ex vivo with a Cas protein
and two ribonucleic acids which direct Cas protein to and hybridize
to a target polynucleotide sequence encoding B2M in the cell or
population of cells, wherein the target polynucleotide sequence
encoding B2M is cleaved, thereby reducing the likelihood that cells
administered to the subject will trigger a host immune response in
the subject; and (b) administering the resulting cells from (a) to
a subject in need of such cells.
[0244] In some aspects, the invention provides a method of reducing
the likelihood that cells administered to a subject will trigger a
host immune response in the subject, the method comprising: (a)
contacting a cell or population of cells ex vivo with (i) a Cas
protein, (ii) at least two ribonucleic acids which direct Cas
protein to and hybridize to a target polynucleotide sequence
encoding B2M in the cell or population of cells, wherein the target
polynucleotide sequence encoding B2M in the cell or population of
cells is cleaved, thereby reducing the likelihood that the cell or
population of cells will trigger a host immune response in the
subject, and (iii) at least two additional ribonucleic acids which
direct Cas protein to and hybridize to a target polynucleotide
sequence in the cell or population of cells, wherein the target
polynucleotide sequence is cleaved; and (b) administering the
resulting cell or cells from (a) to a subject in need of such
cells. As used herein "nucleic acid," in its broadest sense,
includes any compound and/or substance that comprise a polymer of
nucleotides linked via a phosphodiester bond. Exemplary nucleic
acids include ribonucleic acids (RNAs), deoxyribonucleic acids
(DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs),
peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or
hybrids thereof. They may also include RNAi-inducing agents, RNAi
agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes,
catalytic DNA, tRNA, RNAs that induce triple helix formation,
aptamers, vectors, etc. In some embodiments, the nucleic acid
encoding the Cas protein is an mRNA. In some embodiments, the Cas
protein is encoded by a modified nucleic acid (e.g., a synthetic,
modified mRNA described herein).
[0245] The present invention contemplates the use of any nucleic
acid modification available to the skilled artisan. The nucleic
acids of the present invention can include any number of
modifications. In some embodiments, the nucleic acid comprises one
or more modifications selected from the group consisting of
pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine,
2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine,
5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine,
1-carboxymethyl-pseudouridine, 5-propynyl-uridine,
1-propynyl-pseudouridine, 5-taurinomethyluridine,
1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,
1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,
1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,
2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,
2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,
dihydropseudouridine, 2-thio-dihydrouridine,
2-thio-dihydropseudouridine, 2-methoxyuridine,
2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,
4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine,
3-methyl-cytidine, N4-acctylcytidine, 5-formylcytidine,
N4-methylcytidine, 5-hydroxymethylcytidine,
1-methyl-pseudoisocytidine, pyrrolo-cytidine,
pyrrolo-pseudoisocytidine, 2-thio-cytidine,
2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,
4-thio-1-methyl-pseudoisocytidine,
4-thio-1-methyl-1-deaza-pseudoisocytidine,
1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,
5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,
2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,
4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,
2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine,
7-deaza-8-aza-adenine, 7-deaza-2-aminopurine,
7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine,
7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine,
N6-methyladenosine, N6-isopentenyladenosine,
N6-(cis-hydroxyisopentenyl)adenosine,
2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine,
N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,
2-methylthio-N6-threonyl carbamoyladenosine,
N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and
2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine,
7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine,
6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine,
7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine,
6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine,
N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine,
1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and
N2,N2-dimethyl-6-thio-guanosine, and combinations thereof.
[0246] Preparation of modified nucleosides and nucleotides used in
the manufacture or synthesis of modified RNAs of the present
invention can involve the protection and deprotection of various
chemical groups. The need for protection and deprotection, and the
selection of appropriate protecting groups can be readily
determined by one skilled in the art.
[0247] The chemistry of protecting groups can be found, for
example, in Greene, et al., Protective Groups in Organic Synthesis,
2d. Ed., Wiley & Sons, 1991, which is incorporated herein by
reference in its entirety.
[0248] Modified nucleosides and nucleotides can be prepared
according to the synthetic methods described in Ogata et al.
Journal of Organic Chemistry 74:2585-2588, 2009; Purmal et al.
Nucleic Acids Research 22(1): 72-78, 1994; Fukuhara et al.
Biochemistry 1(4): 563-568, 1962; and Xu et al. Tetrahedron 48(9):
1729-1740, 1992, each of which are incorporated by reference in
their entirety.
[0249] Modified nucleic acids (e.g., ribonucleic acids) need not be
uniformly modified along the entire length of the molecule.
Different nucleotide modifications and/or backbone structures may
exist at various positions in the nucleic acid. One of ordinary
skill in the art will appreciate that the nucleotide analogs or
other modification(s) may be located at any position(s) of a
nucleic acid such that the function of the nucleic acid is not
substantially decreased. A modification may also be a 5' or 3'
terminal modification. The nucleic acids may contain at a minimum
one and at maximum 100% modified nucleotides, or any intervening
percentage, such as at least 50% modified nucleotides, at least 80%
modified nucleotides, or at least 90% modified nucleotides.
[0250] In some embodiments, at least one of the one to two
ribonucleic acids is a modified ribonucleic acid. In some
embodiments, each of the one to two ribonucleic acids is a modified
ribonucleic acid. In some embodiments, at least one of the multiple
ribonucleic acids is a modified ribonucleic acid. In some
embodiments, a plurality of the multiple ribonucleic acids are
modified. In some embodiments, each of the multiple ribonucleic
acids are modified. Those skilled in the art will appreciate that
the modified ribonucleic acids can include one or more of the
nucleic acid modification described herein.
[0251] In some aspects, provided herein are synthetic, modified RNA
molecules encoding polypeptides, where the synthetic, modified RNA
molecules comprise one or more modifications, such that introducing
the synthetic, modified RNA molecules to a cell results in a
reduced innate immune response relative to a cell contacted with
synthetic RNA molecules encoding the polypeptides not comprising
the one or more modifications. In some embodiments, the Cas protein
comprises a synthetic, modified RNA molecule encoding a Cas
protein. In some embodiments, the Cas protein comprises a
synthetic, modified RNA molecule encoding a Cas9 protein.
[0252] The synthetic, modified RNAs described herein include
modifications to prevent rapid degradation by endo- and
exo-nucleases and to avoid or reduce the cell's innate immune or
interferon response to the RNA. Modifications include, but are not
limited to, for example, (a) end modifications, e.g., 5' end
modifications (phosphorylation dephosphorylation, conjugation,
inverted linkages, etc.), 3' end modifications (conjugation, DNA
nucleotides, inverted linkages, etc.), (b) base modifications,
e.g., replacement with modified bases, stabilizing bases,
destabilizing bases, or bases that base pair with an expanded
repertoire of partners, or conjugated bases, (c) sugar
modifications (e.g., at the 2' position or 4' position) or
replacement of the sugar, as well as (d) internucleoside linkage
modifications, including modification or replacement of the
phosphodiester linkages. To the extent that such modifications
interfere with translation (i.e., results in a reduction of 50% or
more in translation relative to the lack of the modification--e.g.,
in a rabbit reticulocyte in vitro translation assay), the
modification is not suitable for the methods and compositions
described herein. Specific examples of synthetic, modified RNA
compositions useful with the methods described herein include, but
are not limited to, RNA molecules containing modified or
non-natural internucleoside linkages. Synthetic, modified RNAs
having modified internucleoside linkages include, among others,
those that do not have a phosphorus atom in the internucleoside
linkage. In other embodiments, the synthetic, modified RNA has a
phosphorus atom in its internucleoside linkage(s).
[0253] Non-limiting examples of modified internucleoside linkages
include phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates and chiral phosphonates, phosphinates,
phosphoramidates including 3'-amino phosphoramidate and
aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs
of these, and those) having inverted polarity wherein the adjacent
pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to
5'-2'. Various salts, mixed salts and free acid forms are also
included.
[0254] Representative U.S. patents that teach the preparation of
the above phosphorus-containing linkages include, but are not
limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;
5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302;
5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;
5,466,677, 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111;
5,563,253, 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445;
6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199;
6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167;
6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933;
7,321,029; and U.S. Pat. RE39464, each of which is herein
incorporated by reference in its entirety.
[0255] Modified internucleoside linkages that do not include a
phosphorus atom therein have internucleoside linkages that are
formed by short chain alkyl or cycloalkyl internucleoside linkages,
mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages,
or one or more short chain heteroatomic or heterocyclic
internucleoside linkages. These include those having morpholino
linkages (formed in part from the sugar portion of a nucleoside);
siloxane backbones; sulfide, sulfoxide and sulfone backbones;
formacetyl and thioformacetyl backbones; methylene formacetyl and
thioformacetyl backbones; alkene containing backbones; sulfamate
backbones; methyleneimino and methylenehydrazino backbones;
sulfonate and sulfonamide backbones; amide backbones; and others
having mixed N, O, S and CH2 component parts.
[0256] Representative U.S. patents that teach the preparation of
modified oligonucleosides include, but are not limited to, U.S.
Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141;
5,235,033; 564,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677;
5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240;
5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360;
5,677,437; and 5,677,439, each of which is herein incorporated by
reference in its entirety.
[0257] Some embodiments of the synthetic, modified RNAs described
herein include nucleic acids with phosphorothioate internucleoside
linkages and oligonucleosides with heteroatom internucleoside
linkage, and in particular --CH2-NH--CH2-,
--CH2-N(CH3)-O--CH2-[known as a methylene (methylimino) or MMI],
--CH2-O--N(CH3)-CH2-, --CH2-N(CH3)-N(CH3)-CH2- and
--N(CH3)-CH2-CH2- [wherein the native phosphodiester
internucleoside linkage is represented as --O--P--O--CH2-] of the
above-referenced U.S. Pat. No. 5,489,677, and the amide backbones
of the above-referenced U.S. Pat. No. 5,602,240, both of which are
herein incorporated by reference in their entirety. In some
embodiments, the nucleic acid sequences featured herein have
morpholino backbone structures of the above-referenced U.S. Pat.
No. 5,034,506, herein incorporated by reference in its
entirety.
[0258] Synthetic, modified RNAs described herein can also contain
one or more substituted sugar moieties. The nucleic acids featured
herein can include one of the following at the 2' position: H
(deoxyribose); OH (ribose); F; O-, S-, or N-alkyl; O-, S-, or
N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the
alkyl, alkenyl and alkynyl can be substituted or unsubstituted C1
to C10 alkyl or C2 to C10 alkenyl and alkynyl. Exemplary
modifications include O[(CH2)nO]mCH3, O(CH2).nOCH3, O(CH2)nNH2.
O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m
are from 1 to about 10. In some embodiments, synthetic, modified
RNAs include one of the following at the 2' position: C1 to C10
lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl
or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3,
ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl,
aminoalkylamino, polyalkylamino, substituted silyl, a reporter
group, an intercalator, a group for improving the pharmacokinetic
properties of an RNA, or a group for improving the pharmacodynamic
properties of a synthetic, modified RNA, and other substituents
having similar properties. In some embodiments, the modification
includes a 2' methoxyethoxy (2'-O--CH2CH2OCH3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta,
1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary
modification is 2'-dimethylaminooxyethoxy, i.e., a O(CH2)20N(CH3)2
group, also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy
(also known in the art as 2'-O-dimethylaminoethoxyethyl or
2'-DMAEOE), i.e., 2'-O--CH2-O--CH2-N(CH2)2.
[0259] Other modifications include 2'-methoxy (2'-OCH3),
2'-aminopropoxy (2'-OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar
modifications can also be made at other positions on the nucleic
acid sequence, particularly the 3' position of the sugar on the 3'
terminal nucleotide or in 2'-5' linked nucleotides and the 5'
position of 5' terminal nucleotide. A synthetic, modified RNA can
also have sugar mimetics such as cyclobutyl moieties in place of
the pentofuranosyl sugar. Representative U.S. patents that teach
the preparation of such modified sugar structures include, but are
not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080;
5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134;
5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053;
5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain
of which are commonly owned with the instant application, and each
of which is herein incorporated by reference in its entirety.
[0260] As non-limiting examples, synthetic, modified RNAs described
herein can include at least one modified nucleoside including a
2'-O-methyl modified nucleoside, a nucleoside comprising a 5'
phosphorothioate group, a 2'-amino-modified nucleoside,
2'-alkyl-modified nucleoside, morpholino nucleoside, a
phosphoramidate or a non-natural base comprising nucleoside, or any
combination thereof.
[0261] In some embodiments of this aspect and all other such
aspects described herein, the at least one modified nucleoside is
selected from the group consisting of 5-methylcytidine (5mC),
N6-methyladenosine (m6A), 3,2'-O-dimethyluridine (m4U),
2-thiouridine (s2U), 2' fluorouridine, pseudouridine,
2'-O-methyluridine (Um), 2' deoxyuridine (2' dU), 4-thiouridine
(s4U), 5-methyluridine (m5U), 2'-O-methyladenosine (m6A),
N6,2'-O-dimethyladenosine (m6Am), N6,N6,2'-O-trimethyladenosine
(m62Am), 2'-O-methylcytidine (Cm), 7-methylguanosine (m7G),
2'-O-methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G).
N2,N2,7-trimethylguanosine (m2,2,7G), and inosine (I).
[0262] Alternatively, a synthetic, modified RNA can comprise at
least two modified nucleosides, at least 3, at least 4, at least 5,
at least 6, at least 7, at least 8, at least 9, at least 10, at
least 15, at least 20 or more, up to the entire length of the
nucleotide. At a minimum, a synthetic, modified RNA molecule
comprising at least one modified nucleoside comprises a single
nucleoside with a modification as described herein. It is not
necessary for all positions in a given synthetic, modified RNA to
be uniformly modified, and in fact more than one of the
aforementioned modifications can be incorporated in a single
synthetic, modified RNA or even at a single nucleoside within a
synthetic, modified RNA. However, it is preferred, but not
absolutely necessary, that each occurrence of a given nucleoside in
a molecule is modified (e.g., each cytosine is a modified cytosine
e.g., 5mC). However, it is also contemplated that different
occurrences of the same nucleoside can be modified in a different
way in a given synthetic, modified RNA molecule (e.g., some
cytosines modified as 5mC, others modified as 2'-O-methylcytidine
or other cytosine analog). The modifications need not be the same
for each of a plurality of modified nucleosides in a synthetic,
modified RNA. Furthermore, in some embodiments of the aspects
described herein, a synthetic, modified RNA comprises at least two
different modified nucleosides. In some such preferred embodiments
of the aspects described herein, the at least two different
modified nucleosides are 5-methylcytidine and pseudouridine. A
synthetic, modified RNA can also contain a mixture of both modified
and unmodified nucleosides.
[0263] As used herein, "unmodified" or "natural" nucleosides or
nucleobases include the purine bases adenine (A) and guanine (G),
and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
In some embodiments, a synthetic, modified RNA comprises at least
one nucleoside ("base") modification or substitution. Modified
nucleosides include other synthetic and natural nucleobases such as
inosine, xanthine, hypoxanthine, nubularine, isoguanisine,
tubercidine, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine,
2 (amino)adenine, 2-(aminoalkyl)adenine, 2 (aminopropyl)adenine, 2
(methylthio) N6 (isopentenyl)adenine, 6 (alkyl)adenine, 6
(methyl)adenine, 7 (deaza)adenine, 8 (alkenyl)adenine,
8-(alkyl)adenine, 8 (alkynyl)adenine, 8 (amino)adenine,
8-(halo)adenine, 8-(hydroxyl)adenine, 8 (thioalkyl)adenine,
8-(thiol)adenine, N6-(isopentyl)adenine, N6 (methyl)adenine, N6, N6
(dimethyl)adenine, 2-(alkyl)guanine, 2 (propyl)guanine,
6-(alkyl)guanine, 6 (methyl)guanine, 7 (alkyl)guanine, 7
(methyl)guanine, 7 (deaza)guanine, 8 (alkyl)guanine,
8-(alkenyl)guanine, 8 (alkyl)guanine, 8-(amino)guanine, 8
(halo)guanine, 8-(hydroxyl)guanine, 8 (thioalkyl)guanine,
8-(thiol)guanine, N (methyl)guanine, 2-(thio)cytosine, 3 (deaza) 5
(aza)cytosine, 3-(alkyl)cytosine, 3 (methyl)cytosine,
5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5 (halo)cytosine, 5
(methyl)cytosine, 5 (propynyl)cytosine, 5 (propynyl)cytosine, 5
(trifluoromethyl)cytosine, 6-(azo)cytosine, N4 (acetyl)cytosine, 3
(3 amino-3 carboxypropyl)uracil, 2-(thio)uracil, 5 (methyl) 2
(thio)uracil, 5 (methylaminomethyl)-2 (thio)uracil, 4-(thio)uracil,
5 (methyl) 4 (thio)uracil, 5 (methylaminomethyl)-4 (thio)uracil, 5
(methyl) 2.4 (dithio)uracil, 5 (methylaminomethyl)-2,4
(dithio)uracil, 5 (2-aminopropyl)uracil, 5-(alkyl)uracil,
5-(alkynyl)uracil, 5-(allylamino)uracil, 5 (aminoallyl)uracil, 5
(aminoalkyl)uracil, 5 (guanidiniumalkyl)uracil, 5
(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil,
5-(dialkylaminoalkyl)uracil, 5 (dimethylaminoalkyl)uracil,
5-(halo)uracil, 5-(methoxy)uracil, uracil-5 oxyacetic acid, 5
(methoxycarbonylmethyl)-2-(thio)uracil, 5
(methoxycarbonyl-methyl)uracil, 5 (propynyl)uracil, 5
(propynyl)uracil, 5 (trifluoromethyl)uracil, 6 (azo)uracil,
dihydrouracil, N3 (methyl)uracil, 5-uracil (i.e., pseudouracil), 2
(thio)pseudouracil, 4 (thio)pseudouracil, 2,4-(dithio)psuedouracil,
5-(alkyl)pseudouracil, 5-(methyl)pseudouracil,
5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil,
5-(alkyl)-4 (thio)pseudouracil, 5-(methyl)-4 (thio)pseudouracil,
5-(alkyl)-2,4 (dithio)pseudouracil, 5-(methyl)-2,4
(dithio)pseudouracil, 1 substituted pseudouracil, 1 substituted
2(thio)-pseudouracil, 1 substituted 4 (thio)pseudouracil, 1
substituted 2,4-(dithio)pseudouracil, 1
(aminocarbonylethylenyl)-pseudouracil, 1
(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1
(aminocarbonylethylenyl)-4 (thio)pseudouracil, 1
(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1
(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1
(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil, 1
(aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil, 1
(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil,
1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,
1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,
1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,
1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted
1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted
1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted
1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted
1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,
7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,
7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,
7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,
7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,
7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,
7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1l-yl,
7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,
7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1I-yl,
1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine,
hypoxanthine, nubularine, tubercidine, isoguanisine, inosinyl,
2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl,
nitrobenzimidazolyl, nitroindazolyl, aminoindolyl,
pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl,
5-(methyl)isocarbostyrilyl,
3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl,
6-(methyl)-7-(aza)indolyl, imidizopyridinyl,
9-(methyl)-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl,
7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl,
2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl,
phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl,
stilbenzyl, tetracenyl, pentacenyl, difluorotolyl,
4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole,
6-(azo)thymine, 2-pyridinone, 5 nitroindole, 3 nitropyrrole,
6-(aza)pyrimidine, 2 (amino)purine, 2,6-(diamino)purine, 5
substituted pyrimidines, N2-substituted purines. N6-substituted
purines, 06-substituted purines, substituted 1,2,4-triazoles,
pyrrolo-pyridin-2-on-3-yl, 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,
para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,
ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,
bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,
para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,
ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,
bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,
pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl,
2-oxo-pyridopyrimidine-3-yl, or any O-alkylated or N-alkylated
derivatives thereof. Modified nucleosides also include natural
bases that comprise conjugated moieties, e.g. a ligand. As
discussed herein above, the RNA containing the modified nucleosides
must be translatable in a host cell (i.e., does not prevent
translation of the polypeptide encoded by the modified RNA). For
example, transcripts containing s2U and m6A are translated poorly
in rabbit reticulocyte lysates, while pseudouridine, m5U, and m5C
are compatible with efficient translation. In addition, it is known
in the art that 2'-fluoro-modified bases useful for increasing
nuclease resistance of a transcript, leads to very inefficient
translation. Translation can be assayed by one of ordinary skill in
the art using e.g., a rabbit reticulocyte lysate translation
assay.
[0264] Further modified nucleobases include those disclosed in U.S.
Pat. No. 3,687,808, those disclosed in Modified Nucleosides in
Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed.
Wiley-VCH, 2008; those disclosed in Int. Appl. No.
PCT/US09/038,425, filed Mar. 26, 2009; those disclosed in The
Concise Encyclopedia Of Polymer Science And Engineering, pages
858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, and
those disclosed by Englisch et al., Angewandte Chemie,
International Edition, 1991, 30, 613.
[0265] Representative U.S. patents that teach the preparation of
certain of the above noted modified nucleobases as well as other
modified nucleobases include, but are not limited to, the above
noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205;
5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187;
5,457,191; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;
5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 6,015,886;
6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640;
6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of
which is herein incorporated by reference in its entirety, and U.S.
Pat. No. 5,750,692, also herein incorporated by reference in its
entirety.
[0266] Another modification for use with the synthetic, modified
RNAs described herein involves chemically linking to the RNA one or
more ligands, moieties or conjugates that enhance the activity,
cellular distribution or cellular uptake of the RNA. The synthetic,
modified RNAs described herein can further comprise a 5' cap. In
some embodiments of the aspects described herein, the synthetic,
modified RNAs comprise a 5' cap comprising a modified guanine
nucleotide that is linked to the 5' end of an RNA molecule using a
5'-5' triphosphate linkage. As used herein, the term "5' cap" is
also intended to encompass other 5' cap analogs including, e.g., 5'
diguanosine cap, tetraphosphate cap analogs having a
methylene-bis(phosphonate) moiety (see e.g., Rydzik, A M et al.,
(2009) Org Biomol Chem 7(22):4763-76), dinucleotide cap analogs
having a phosphorothioate modification (see e.g., Kowalska, J. et
al., (2008) RNA 14(6): 1119-1131), cap analogs having a sulfur
substitution for a non-bridging oxygen (see e.g.,
Grudzien-Nogalska, E. et al., (2007) RNA 13(10): 1745-1755),
N7-benzylated dinucleoside tetraphosphate analogs (see e.g.,
Grudzien, E. et al., (2004) RNA 10(9):1479-1487), or anti-reverse
cap analogs (see e.g., Jemielity, J. et al., (2003) RNA 9(9):
1108-1122 and Stepinski, J. et al., (2001) RNA 7(10): 1486-1495).
In one such embodiment, the 5' cap analog is a 5' diguanosine cap.
In some embodiments, the synthetic, modified RNA does not comprise
a 5' triphosphate.
[0267] The 5' cap is important for recognition and attachment of an
mRNA to a ribosome to initiate translation. The 5' cap also
protects the synthetic, modified RNA from 5' exonuclease mediated
degradation. It is not an absolute requirement that a synthetic,
modified RNA comprise a 5' cap, and thus in other embodiments the
synthetic, modified RNAs lack a 5' cap. However, due to the longer
half-life of synthetic, modified RNAs comprising a 5' cap and the
increased efficiency of translation, synthetic, modified RNAs
comprising a 5' cap are preferred herein.
[0268] The synthetic, modified RNAs described herein can further
comprise a 5' and/or 3' untranslated region (UTR). Untranslated
regions are regions of the RNA before the start codon (5') and
after the stop codon (3'), and are therefore not translated by the
translation machinery. Modification of an RNA molecule with one or
more untranslated regions can improve the stability of an mRNA,
since the untranslated regions can interfere with ribonucleases and
other proteins involved in RNA degradation. In addition,
modification of an RNA with a 5' and/or 3' untranslated region can
enhance translational efficiency by binding proteins that alter
ribosome binding to an mRNA. Modification of an RNA with a 3' UTR
can be used to maintain a cytoplasmic localization of the RNA,
permitting translation to occur in the cytoplasm of the cell. In
one embodiment, the synthetic, modified RNAs described herein do
not comprise a 5' or 3' UTR. In another embodiment, the synthetic,
modified RNAs comprise either a 5' or 3' UTR. In another
embodiment, the synthetic, modified RNAs described herein comprise
both a 5' and a 3' UTR. In one embodiment, the 5' and/or 3' UTR is
selected from an mRNA known to have high stability in the cell
(e.g., a murine alpha-globin 3' UTR). In some embodiments, the 5'
UTR, the 3' UTR, or both comprise one or more modified
nucleosides.
[0269] In some embodiments, the synthetic, modified RNAs described
herein further comprise a Kozak sequence. The "Kozak sequence"
refers to a sequence on eukaryotic mRNA having the consensus
(gcc)gccRccAUGG, where R is a purine (adenine or guanine) three
bases upstream of the start codon (AUG), which is followed by
another `G`. The Kozak consensus sequence is recognized by the
ribosome to initiate translation of a polypeptide. Typically,
initiation occurs at the first AUG codon encountered by the
translation machinery that is proximal to the 5' end of the
transcript. However, in some cases, this AUG codon can be bypassed
in a process called leaky scanning. The presence of a Kozak
sequence near the AUG codon will strengthen that codon as the
initiating site of translation, such that translation of the
correct polypeptide occurs. Furthermore, addition of a Kozak
sequence to a synthetic, modified RNA will promote more efficient
translation, even if there is no ambiguity regarding the start
codon. Thus, in some embodiments, the synthetic, modified RNAs
described herein further comprise a Kozak consensus sequence at the
desired site for initiation of translation to produce the correct
length polypeptide. In some such embodiments, the Kozak sequence
comprises one or more modified nucleosides.
[0270] In some embodiments, the synthetic, modified RNAs described
herein further comprise a "poly (A) tail", which refers to a 3'
homopolymeric tail of adenine nucleotides, which can vary in length
(e.g., at least 5 adenine nucleotides) and can be up to several
hundred adenine nucleotides). The inclusion of a 3' poly(A) tail
can protect the synthetic, modified RNA from degradation in the
cell, and also facilitates extra-nuclear localization to enhance
translation efficiency. In some embodiments, the poly(A) tail
comprises between 1 and 500 adenine nucleotides; in other
embodiments the poly(A) tail comprises at least 5, at least 10, at
least 20, at least 30, at least 40, at least 50, at least 60, at
least 70, at least 80, at least 90, at least 100, at least 110, at
least 120, at least 130, at least 140, at least 150, at least 160,
at least 170, at least 180, at least 190, at least 200, at least
225, at least 250, at least 275, at least 300, at least 325, at
least 350, at least 375, at least 400, at least 425, at least 450,
at least 475, at least 500 adenine nucleotides or more. In one
embodiment, the poly(A) tail comprises between 1 and 150 adenine
nucleotides. In another embodiment, the poly(A) tail comprises
between 90 and 120 adenine nucleotides. In some such embodiments,
the poly(A) tail comprises one or more modified nucleosides.
[0271] It is contemplated that one or more modifications to the
synthetic, modified RNAs described herein permit greater stability
of the synthetic, modified RNA in a cell. To the extent that such
modifications permit translation and either reduce or do not
exacerbate a cell's innate immune or interferon response to the
synthetic, modified RNA with the modification, such modifications
are specifically contemplated for use herein. Generally, the
greater the stability of a synthetic, modified RNA, the more
protein can be produced from that synthetic, modified RNA.
Typically, the presence of AU-rich regions in mammalian mRNAs tend
to destabilize transcripts, as cellular proteins are recruited to
AU-rich regions to stimulate removal of the poly(A) tail of the
transcript. Loss of a poly(A) tail of a synthetic, modified RNA can
result in increased RNA degradation. Thus, in one embodiment, a
synthetic, modified RNA as described herein does not comprise an
AU-rich region. In particular, it is preferred that the 3' UTR
substantially lacks AUUUA sequence elements.
[0272] In one embodiment, a ligand alters the cellular uptake,
intracellular targeting or half-life of a synthetic, modified RNA
into which it is incorporated. In some embodiments a ligand
provides an enhanced affinity for a selected target, e.g.,
molecule, cell or cell type, intracellular compartment. e.g.,
mitochondria, cytoplasm, peroxisome, lysosome, as, e.g., compared
to a composition absent such a ligand. Preferred ligands do not
interfere with expression of a polypeptide from the synthetic,
modified RNA.
[0273] The ligand can be a substance, e.g., a drug, which can
increase the uptake of the synthetic, modified RNA or a composition
thereof into the cell, for example, by disrupting the cell's
cytoskeleton, e.g., by disrupting the cell's microtubules,
microfilaments, and/or intermediate filaments. The drug can be, for
example, taxol, vincristine, vinblastine, cytochalasin, nocodazole,
japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine,
or myoservin.
[0274] In another aspect, the ligand is a moiety, e.g., a vitamin,
which is taken up by a host cell. Exemplary vitamins include
vitamin A, E, and K. Other exemplary vitamins include B vitamin,
e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other
vitamins or nutrients taken up, for example, by cancer cells. Also
included are HSA and low density lipoprotein (LDL).
[0275] In another aspect, the ligand is a cell-permeation agent,
preferably a helical cell-permeation agent. Preferably, the agent
is amphipathic. An exemplary agent is a peptide such as tat or
antennopedia. If the agent is a peptide, it can be modified,
including a peptidylmimetic, invertomers, non-peptide or
pseudo-peptide linkages, and use of D-amino acids. The helical
agent is preferably an alpha-helical agent, which preferably has a
lipophilic and a lipophobic phase.
[0276] A "cell permeation peptide" is capable of permeating a cell,
e.g., a microbial cell, such as a bacterial or fungal cell, or a
mammalian cell, such as a human cell. A microbial cell-permeating
peptide can be, for example, an .alpha.-helical linear peptide
(e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide
(e.g., .alpha.-defensin, .beta.-defensin or bactenecin), or a
peptide containing only one or two dominating amino acids (e.g.,
PR-39 or indolicidin). For example, a cell permeation peptide can
be a bipartite amphipathic peptide, such as MPG, which is derived
from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40
large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724,
2003).
[0277] The synthetic, modified RNAs described herein can be
synthesized and/or modified by methods well established in the art,
such as those described in "Current Protocols in Nucleic Acid
Chemistry," Beaucage, S. L. et al. (Edrs.), John Wiley & Sons,
Inc., New York, N.Y., USA, which is hereby incorporated herein by
reference in its entirety. Transcription methods are described
further herein in the Examples.
[0278] In one embodiment of the aspects described herein, a
template for a synthetic, modified RNA is synthesized using
"splint-mediated ligation," which allows for the rapid synthesis of
DNA constructs by controlled concatenation of long oligos and/or
dsDNA PCR products and without the need to introduce restriction
sites at the joining regions. It can be used to add generic
untranslated regions (UTRs) to the coding sequences of genes during
T7 template generation. Splint mediated ligation can also be used
to add nuclear localization sequences to an open reading frame, and
to make dominant-negative constructs with point mutations starting
from a wild-type open reading frame. Briefly, single-stranded
and/or denatured dsDNA components are annealed to splint oligos
which bring the desired ends into conjunction, the ends are ligated
by a thermostable DNA ligase and the desired constructs amplified
by PCR. A synthetic, modified RNA is then synthesized from the
template using an RNA polymerase in vitro. After synthesis of a
synthetic, modified RNA is complete, the DNA template is removed
from the transcription reaction prior to use with the methods
described herein.
[0279] In some embodiments of these aspects, the synthetic,
modified RNAs are further treated with an alkaline phosphatase.
[0280] One skilled in the art readily appreciates that the present
invention is well adapted to carry out the objects and obtain the
ends and advantages mentioned, as well as those inherent therein.
The details of the description and the examples herein are
representative of certain embodiments, are exemplary, and are not
intended as limitations on the scope of the invention.
Modifications therein and other uses will occur to those skilled in
the art. These modifications are encompassed within the spirit of
the invention. It will be readily apparent to a person skilled in
the art that varying substitutions and modifications may be made to
the invention disclosed herein without departing from the scope and
spirit of the invention.
[0281] The articles "a" and "an" as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to include the plural referents.
Claims or descriptions that include "or" between one or more
members of a group are considered satisfied if one, more than one,
or all of the group members are present in, employed in, or
otherwise relevant to a given product or process unless indicated
to the contrary or otherwise evident from the context. The
invention includes embodiments in which exactly one member of the
group is present in, employed in, or otherwise relevant to a given
product or process. The invention also includes embodiments in
which more than one, or all of the group members are present in,
employed in, or otherwise relevant to a given product or process.
Furthermore, it is to be understood that the invention provides all
variations, combinations, and permutations in which one or more
limitations, elements, clauses, descriptive terms, etc., from one
or more of the listed claims is introduced into another claim
dependent on the same base claim (or, as relevant, any other claim)
unless otherwise indicated or unless it would be evident to one of
ordinary skill in the art that a contradiction or inconsistency
would arise. It is contemplated that all embodiments described
herein are applicable to all different aspects of the invention
where appropriate. It is also contemplated that any of the
embodiments or aspects can be freely combined with one or more
other such embodiments or aspects whenever appropriate. Where
elements are presented as lists, e.g., in Markush group or similar
format, it is to be understood that each subgroup of the elements
is also disclosed, and any element(s) can be removed from the
group. It should be understood that, in general, where the
invention, or aspects of the invention, is/are referred to as
comprising particular elements, features, etc., certain embodiments
of the invention or aspects of the invention consist, or consist
essentially of, such elements, features, etc. For purposes of
simplicity those embodiments have not in every case been
specifically set forth in so many words herein. It should also be
understood that any embodiment or aspect of the invention can be
explicitly excluded from the claims, regardless of whether the
specific exclusion is recited in the specification. For example,
any one or more active agents, additives, ingredients, optional
agents, types of organism, disorders, subjects, or combinations
thereof, can be excluded.
[0282] Where the claims or description relate to a composition of
matter, it is to be understood that methods of making or using the
composition of matter according to any of the methods disclosed
herein, and methods of using the composition of matter for any of
the purposes disclosed herein are aspects of the invention, unless
otherwise indicated or unless it would be evident to one of
ordinary skill in the art that a contradiction or inconsistency
would arise. Where the claims or description relate to a method,
e.g., it is to be understood that methods of making compositions
useful for performing the method, and products produced according
to the method, are aspects of the invention, unless otherwise
indicated or unless it would be evident to one of ordinary skill in
the art that a contradiction or inconsistency would arise.
[0283] Where ranges are given herein, the invention includes
embodiments in which the endpoints are included, embodiments in
which both endpoints are excluded, and embodiments in which one
endpoint is included and the other is excluded. It should be
assumed that both endpoints are included unless indicated
otherwise. Furthermore, it is to be understood that unless
otherwise indicated or otherwise evident from the context and
understanding of one of ordinary skill in the art, values that are
expressed as ranges can assume any specific value or subrange
within the stated ranges in different embodiments of the invention,
to the tenth of the unit of the lower limit of the range, unless
the context clearly dictates otherwise. It is also understood that
where a series of numerical values is stated herein, the invention
includes embodiments that relate analogously to any intervening
value or range defined by any two values in the series, and that
the lowest value may be taken as a minimum and the greatest value
may be taken as a maximum. Numerical values, as used herein,
include values expressed as percentages. For any embodiment of the
invention in which a numerical value is prefaced by "about" or
"approximately", the invention includes an embodiment in which the
exact value is recited. For any embodiment of the invention in
which a numerical value is not prefaced by "about" or
"approximately", the invention includes an embodiment in which the
value is prefaced by "about" or "approximately".
[0284] As used herein "A and/or B", where A and B are different
claim terms, generally means at least one of A, B, or both A and B.
For example, one sequence which is complementary to and/or
hybridizes to another sequence includes (i) one sequence which is
complementary to the other sequence even though the one sequence
may not necessarily hybridize to the other sequence under all
conditions, (ii) one sequence which hybridizes to the other
sequence even if the one sequence is not perfectly complementary to
the other sequence, and (iii) sequences which are both
complementary to and hybridize to the other sequence.
[0285] "Approximately" or "about" generally includes numbers that
fall within a range of 1% or in some embodiments within a range of
5% of a number or in some embodiments within a range of 10% of a
number in either direction (greater than or less than the number)
unless otherwise stated or otherwise evident from the context
(except where such number would impermissibly exceed 100% of a
possible value). It should be understood that, unless clearly
indicated to the contrary, in any methods claimed herein that
include more than one act, the order of the acts of the method is
not necessarily limited to the order in which the acts of the
method are recited, but the invention includes embodiments in which
the order is so limited. It should also be understood that unless
otherwise indicated or evident from the context, any product or
composition described herein may be considered "isolated".
[0286] As used herein the term "comprising" or "comprises" is used
in reference to compositions, methods, and respective component(s)
thereof, that are essential to the invention, yet open to the
inclusion of unspecified elements, whether essential or not.
[0287] As used herein the term "consisting essentially of" refers
to those elements required for a given embodiment. The term permits
the presence of additional elements that do not materially affect
the basic and novel or functional characteristic(s) of that
embodiment of the invention.
[0288] The term "consisting of" refers to compositions, methods,
and respective components thereof as described herein, which are
exclusive of any element not recited in that description of the
embodiment.
EXAMPLES
Example 1
[0289] Transcription activator-like effector nucleases (TALENs)
bind as a pair around a genomic site, in which a double-strand
break (DSB) is introduced by a dimer of FokI nuclease domains. The
use of a TALEN genome-editing system to rapidly and efficiently
generate mutant alleles of 15 different genes in human pluripotent
stem cells (hPSCs) as a means of performing rigorous disease
modeling was recently reported (Ding et al., Cell Stem Cell
12:238-251 (2013)); the proportions of clones bearing at least one
mutant allele ranged from 2%-34%.
[0290] As described below, the relative efficacies of CRISPRs and
TALENs targeting the same genomic sites in the same hPSC lines was
assessed with the use of the same delivery platform described
previously (Ding et al., Cell Stem Cell 12:238-251 (2013)). In the
TALEN genome-editing system, the CAG promoter was used to
co-translate (via a viral 2A peptide) each TALEN with green
fluorescent protein (GFP) or red fluorescent protein (RFP). For
CRISPRs, a human codon-optimized Cas9 gene was subcloned with a
C-terminal nuclear localization signal (Mali et al., Science
339:823-826 (2013)) into the same CAG expression plasmid with GFP,
and the guide RNA (gRNA) was separately expressed from a plasmid
with the human U6 polymerase III promoter (Mali et al., Science
339:823-826 (2013)). The 20-nucleotide protospacer sequence for
each gRNA was introduced using polymerase chain reaction
(PCR)-based methods. Whether using TALENs or CRISPRs, equal amounts
of the two plasmids were co-electroporated into hPSCs (either 25
.mu.g of each plasmid, or 12.5 .mu.g of each plasmid along with 25
.mu.g of a DNA repair template if attempting knock-in) followed by
fluorescence-activated cell sorting (FACS) after 24-48 hours,
clonal expansion of single cells, and screening for mutations at
the genomic target site via PCR.
[0291] gRNAs were designed matching G(N)19NGG sequences in seven
loci in six genes (AKT2, CELSR2, CIITA, GLUT4, LINC00116, and
SORT1) previously successfully targeted with TALENs (Ding et al.,
Cell Stem Cell 12:238-251 (2013)) and one additional locus in LDLR.
In this system, CRISPRs consistently and substantially outperformed
TALENs across loci and hPSC lines (see Table S1). The TALENs
yielded clones with at least one mutant allele at efficiencies of
0%-34%, but matched CRISPRs yielded mutant clones at efficiencies
of 51%-79% (Table S1). Just as with TALENs, CRISPRs produced a
variety of indels of sizes ranging from one nucleotide to several
dozen nucleotides in size, centered on the predicted cleavage
sites, suggesting that non-homologous end-joining mutagenesis
occurs in the same way regardless of whether CRISPRs or TALENs are
used. Moreover, CRISPRs readily generated homozygous mutant clones
(7%-25% of all clones; Table S1) as discerned by sequencing.
[0292] Knock-in of E17K mutations into AKT2 was also attempted
using a 67-nucleotide single-stranded DNA oligonucleotide as
previously described (Ding et al., Cell Stem Cell 12:238-251
(2013)). Although the predicted CRISPR cleavage site lay 11 and 13
nucleotides from the point mutations, respectively, the CRISPR
yielded knock-in clones at a rate of 11%, whereas TALENs yielded
only 1.6% (Table S1).
TABLE-US-00001 TABLE S1 Targeting Efficiency of CRISPRs Versus
TALENs in Human Pluripotent Stem Cells Chromo- CRISPRs some: TALENs
Effi- Position Efficiency Efficiency ciency (Start of (Mutants/
(Mutants/ of Homo- Target Cell Clones Clones zygous Gene Sequence)
Target Sequence.sup.a Line.sup.b Screened).sup.c Screened).sup.c
Mutants AKT2 chr19: TCCCTTCCTGCCTCATTTCAGGTGAATACAT HUES 8.9% (SEQ
ID 40762982 CAAGACCTGGAGGCCA 9 (17/192) NO: 335) AKT2 chr19:
TCCCTTCCTGCC|TCATTTCAGGTGAATAC HUES (SEQ ID 60.6% 12.7% 40762982
ATCAAGACCTGGAGGCCA 9 NO: 336) (86/142) (18/142) CELSR2 chr1:
TGCTGGCTCGGCTGCCCTGAGGTTGCTCAAT HUES 3.5% (SEQ ID 109817568
CAAGCACAGGTTTCAA 1 (18/506) NO: 337) CELSR2 chr1:
TGCTGGCTCGGCTGCCCT HUES (SEQ ID 66.2% 7.4% 109817568
GAGGTTGCTCAATCAAG|CACAGGTTTCAA 1 NO: 338) (45/68) (5/68) CIITA
chr16: TAACAGCGATGCTGACCCCCTGTGCCTCTAC BJ- 12.7% (SEQ ID 10989200
CACTTCTATGACCAGA RiPS (37/292) NO: 339) CIITA chr16:
CGATGCTGACCCCCTGTGCCTCTACCA BJ- (SEQ ID 78.7% 11.5% 10989206
CTT|CTATGACCAGATGGACC RiPS NO: 340) (96/122) (14/122) GLUT4 chr17:
TGGTCCTTGCTGTGTTCTCTGCGGTGCTTGGCT HUES 33.5% (SEQ ID 7186601
CCCTGCAGTTTGGGTA 9 (52/155) NO: 341) GLUT4 chr17:
TGGTCCTTGCTGTGTTCT|CTG HUES (SEQ ID 66.5% 24.9% 7186601
CGGTGCTTGGCTCCCTGCAGTTTGGGTA 9 NO: 342) (123/185) (46/185) LDLR
chr19: TGGGCGACAGATGCGAAAGAAACGAGTTCCA HUES 0% (SEQ ID 11210899
GTGCCAAGACGGGAAA 9 (0/568) NO: 343) LDLR chr19:
GAAACGAGTTCCAGTGCCAAGACG HUES (SEQ ID 51.1% 8.0% 11210917
GGAAATGCATCTCCTAC|AAGTGG 9 NO: 344) (90/176) (14/176) LINC00116
chr2: TCAGAGAGGACACTGCAGTTGTCCGTGCTAGT HUES 29.5% (SEQ ID 110970090
AGCCTTCGCTTCTGGA 9 (26/88) NO: 345) LINC00116 chr2:
TCAGAGAGGACACTGCAGTTGTCC HUES (SEQ ID 57.4% 8.6% 110970090
GTGCTAGTAGCCTTCGC|TTCTGGA 9 NO: 346) (93/162) (14/162) SORT1 chr1:
TGATGATCTCAGAGGCTCAGTATCCTTGTCC HUES 22.2% (SEQ ID exon 2 109912203
TGGGTTGGAGATAGCA 1 (128/576) NO: 347) SORT1 chr1:
TGATGATCTCAGAGGCTCAGTATCCTTG|TCC HUES (SEQ ID 68.5% 13.0% exon 2
109912203 TGGGTTGGAGATAGCA 1 NO: 348) (100/146) (19/146) SORT1
chr1: TGGTAATTATGACTTTTGGACAGTCCAAGCTAT HUES 10.9% (SEQ ID exon 3
109910069 ATCGAAGGTGAGATCA 9 (21/192) NO: 349) SORT1 chr1:
TGGTAATTATGACTTTTG HUES (SEQ ID 75.9% 10.3% exon 3 109910069
GACAGTCCAAGCTATAT|CGAAGGTGAGATCA 9 NO: 350) (148/195) (20/195) AKT2
chr19: TCCCTTCCTGCCTCATTTCAGGT A HUES 1.6% (SEQ ID E17K 40762982
TACATCAAGACCTGGAGGCCA 9 (3/192).sup.d NO: 351) AKT2 chr19:
TCCCTTCCT HUES (SEQ ID 10.6% 1.1% E17K 40762982 ATCAAGACCTGGAGGCCA
9 NO: 352) (10/94).sup.d (1/94).sup.d AKT2 off- chr5: CTATGCCCT
HUES (SEQ ID 0% 0% target 22683972 GAAATCCCTGGAGCTTGG 9 NO: 353)
(0/142) (0/142) .sup.aFor TALENs, the binding sites are indicated
with underlines, with the cleavage site predicted to be midway
between the binding sites; for CRISPRs, the protospacer is
underlined, the NGG motif is in bold (may be on the antisense
strand), and the predicted cleavage site is indicated with "|"; for
the AKT2 E17K target sequence, the sites of the knock-in mutations
are indicated in bold/italics; for the AKT2 off-target site, the
two mismatches in the protospacer are indicated in bold/italics
.sup.bHUES 1 and HUES 9 are human embryonic stem cell lines;
BJ-RiPS is an induced pluripotent stem cell line .sup.cMutants
include single heterozygotes, compound heterozygotes, and
homozygous mutants; TALEN data is from Table 1 of Ding et al.
(2013), with the exception of LDLR .sup.dSuccessfully inserted E17K
knock-in mutations into an AKT2 allele(s) using single-stranded DNA
oligonucleotide (refer to FIG. 3 of Ding et al., 2013)
[0293] It is worth noting that the requirement for a G(N) 19NGG
target sequence somewhat limits site selection. Because either DNA
strand can be targeted, a target sequence occurs on average every
32 basepairs. This is no barrier for gene knockout, where any
coding sequence can be targeted, but it may present difficulties
when trying to knock in or correct a mutation at a specific
location. However, the requirement for a G at the start of the
protospacer is dictated by the use of the U6 promoter to express
the gRNA, and alternative CRISPR/Cas systems can relieve this
requirement (Cong et al., Science 339:819-823 (2013)). This allows
for the use of (N)20NGG target sequences, which are found on
average every 8 basepairs.
[0294] In addition, the extent of CRISPR off-target effects remains
to be defined and is highly sequence-dependent. Previous analyses
have suggested that one-nucleotide mismatches in the first half of
the protospacer are better tolerated than mismatches in second half
(Jinek et al., Science 337:816-821 (2012); Cong et al., Science
339:819-823 (2013)). For the AKT2 sequence, there is a two-mismatch
sequence differing at nucleotides 1 and 3, in the more "tolerant"
half of the protospacer. Zero clones were obtained with mutations
at this potential off-target site, as compared to 61% at the
on-target site (Table S1). For one of the SORT1 sequences, use of a
different human pluripotent stem cell line in which a single
nucleotide polymorphism results in a one-nucleotide mismatch at the
target site yielded mutant clones at an efficiency of 42%, compared
to 66% in the original cell line. Thus, judicious selection of
target sites is necessary to minimize systematic off-target
effects; target sites with perfect-match or
single-nucleotide-mismatch sequences elsewhere in the genome should
be avoided.
[0295] From a practical standpoint, CRISPRs are easier to implement
than TALENs. Each TALEN pair must be constructed de novo, whereas
for CRISPRs the Cas9 component is fixed and the gRNA requires only
swapping of the 20-nucleotide protospacer. Given this consideration
and the demonstration herein of substantially increased efficiency
as a result of replacing TALENs with CRISPRs in an otherwise
identical system, CRISPRs appear to be a very powerful and broadly
applicable tool for genome editing, particularly in a therapeutic
context.
Example 2: Efficient Targeting of Clinically Relevant Genes in
Primary Somatic Cells
[0296] Work described herein shows for the first time that the
CRISPR/Cas9 system can be used to edit the genome of somatic cells
(e.g., primary) with high efficiency by using a double guide
strategy. The inventors posit that this work will help bring genome
editing in clinically relevant primary cells into reality.
[0297] The advent of genome editing tools that allow one to target
any desired genomic site has greatly advanced the investigation of
human biology and disease. In particular, the CRISPR/Cas9 system
has become the gold standard in targeted genome editing technology,
due to its flexibility and high efficacy. This system is
constituted by the Cas9 nuclease from the microbial type II
CRISPR/Cas system, which is targeted to specific genomic loci by a
20-nucleotide region in a synthetic guide RNA molecule. Similar to
other targeted nucleases (ZFNs and TALENs), Cas9 induces double
strand breaks (DSBs) that are repaired mainly by error-prone
non-homologous end joining (NHEJ) (Cong et al., 2013; Jinek et al.,
2013; Mali et al., 2013).
[0298] Implementation of the CRISPR/Cas9 system has made it
possible to achieve unprecedentedly high targeting efficiencies in
immortalized cell lines (Cong et al., 2013; Jinek et al., 2013;
Mali et al., 2013), human pluripotent stem cells (Ding et al.,
2013) and even zygotes of mice (Wang et al., 2013), rats (Li et
al., 2013) and, most recently, monkeys (Niu et al., 2014), leading
to the generation of knock-out or knock-in animals in very short
periods of time when compared to classical strategies.
[0299] However, it remains to be proven whether CRISPR/Cas9
technology can be used to edit the genome of clinically relevant
primary somatic cells with high efficiency, an essential step for
the full realization of the promise of genome editing for
regenerative medicine and transplantation therapies.
[0300] The inventors sought to test the amenability of the
CRISPR/Cas9 system to edit clinically relevant genes in primary
somatic cells. For this purpose the inventors chose to target two
therapy-related genes: CCR5, a co-receptor for HIV, in CD34+
hematopoietic progenitor cells (HPCs), and B2M, the accessory chain
of MHC class I molecules, in CD4+ T cells. The inventors found that
a single guide strategy yielded very low to undetectable mutational
rates in HPCs and T cells, despite high efficiencies in
immortalized cell lines such 293T and K562. In contrast,
surprisingly and unexpectedly a double guide strategy with a pair
of gRNAs with different offsets targeting the locus of interest
resulted in up to 40% homozygous deletion efficiency in HPCs and T
cells. These results establish a novel approach through which the
CRISPR/Cas9 system can be used to edit the genome in clinically
relevant somatic cells with high efficiency.
[0301] Results
[0302] Efficient and Rapid Genome Editing Using the CRISPR/Cas9
System in Cell Lines
[0303] The inventors transfected HEK293T cells with Cas9 and a
series of CRISPR guide RNAs targeting the B2M locus and measured
cutting efficiency based on SURVEYOR assays (FIG. 4), as well as
flow cytometry, taking advantage of the fact that B2M is a surface
antigen. These experiments were performed only 72h
post-transfection, in order to account for the half-life of B2M on
the cell membrane. Of note, B2M surface expression was abrogated in
up to 60% of transfected HEK293T cells (FIG. 4). In addition, the
inventors observed a wide variation of efficiency between
individual guide RNAs, even if targeting the same exon. For
instance, variation between single guide cutting efficiencies was
several-fold amongst the seven guide RNAs binding within the 67 bp
long protein coding portion of the first exon of B2M (FIG. 1X),
strongly suggesting that CRISPR cutting efficiency is primarily
guide sequence-dependent.
[0304] Primary Somatic Cells are Refractory to CRISPR/Cas9
Targeting
[0305] Next, the inventors tested the CRISPR/Cas9 system in primary
cells. We chose two clinically relevant immune cell types: primary
CD34.sup.+ hematopoietic progenitor cells (HPCs) and primary
CD4.sup.+ T cells isolated from peripheral blood. Surprisingly, the
same guide RNAs that resulted in up to 60% cutting efficiency in a
cell line (B2M in 293T cells, FIG. 4) revealed ineffective in
somatic cells (FIG. 4). The inventors speculate that such dramatic
drop in targeting efficiency in primary cells is due to either a
lower expression level of Cas9 nuclease in nucleofected cells,
enhanced DNA repair mechanisms, or a combination of both.
[0306] Double Guide Strategy Dramatically Increases Targeting
Efficiency in Primary Cells
[0307] The inventors sought to determine whether genome editing
efficacy in clinically relevant primary cells using the CRISPR/Cas9
system could be improved, hoping to achieve targeting efficiencies
high enough to be potentially used in therapy. The inventors
devised a double guide strategy, where two CRISPR guide RNAs
targeting the same locus were delivered to cells
simultaneously.
[0308] Addition of another guide RNA targeting the HPRT locus
almost invariably resulted in increased mutation efficiency
compared with the first guide RNA alone. Cells deficient in HPRT
were selected by resistance to 6-thioguanine (6-TG). The use of
additional gRNAs invariably resulted in increased HPRT mutant
frequency. In an embodiment, the target polynucleotide sequence
comprises a HPRT gene sequence.
[0309] Different guide RNA pairs were tested for each locus, and
the most active one was used for further studies with primary
cells. FIGS. 4A-4E demonstrate that the single guide strategy
achieves high efficiency genome editing in cell lines, but not in
clinically relevant primary somatic cells. In the two systems we
used, the double guide strategy consistently and substantially
outperformed the traditional single guide strategy in primary
somatic cells. These results are demonstrated in FIGS. 5A-5E, which
show that the double guide strategy achieves genome editing with
high efficiency in clinically relevant cells.
DISCUSSION
[0310] One of the major focuses in the field of CRISPR/Cas9 genome
editing field is the search for parameters that modulate cutting
efficiency by Cas9. The data described herein suggest that this
phenomenon appears to be mostly determined by gRNA sequence, as
gRNAs matching very close or even partially overlapping sequences
within the same exon result in significantly different targeting
efficiencies (FIG. 4).
[0311] In a previous report, an approach combining a Cas9 nickase
mutant with paired guide RNAs to introduce targeted double-strand
breaks has been used to drastically reduce CRISPR off-target
effects without sacrificing on-target efficiency (Ran et al.,
2013). In our hands, however, this strategy did not yield a
significant mutation rate (Max & Pankaj). We thus combined WT
Cas9 with pairs of gRNAs to increase cutting efficiency in cell
types refractory to targeting--primary somatic cells.
[0312] B2M is an accessory chain of the MHC class I proteins, being
necessary for their expression on the cells surface. Engineering
cells devoid of surface MHC class I, hence invisible to cytotoxic T
cells, is of utmost importance in transplantation and adoptive cell
therapy.
[0313] Altogether, our data shows that the CRISPR/Cas9 system can
be used to edit the genome of clinically relevant primary somatic
cells with significant efficiencies by using a double guide
strategy. We predict that this strategy has the potential to be a
general approach to target genes in somatic cells with a high
enough efficiency that it becomes relevant for potential
translation into therapeutics.
[0314] Experimental Procedures
[0315] Flow Cytometry.
[0316] Cells were stained with mouse monoclonal anti-B2M antibody
2M2 (Biolegend).
[0317] Primary Blood Cell Electroporation.
[0318] Primary CD4+ T cells were isolated from lecukopacs (MGH)
using RosetteSep CD4 T cell enrichment cocktail (Stem Cell
Technologies) and electroporated with endotoxin-free DNA using
Amaxa T cell nucleofection kit (Lonza).
[0319] 6-TG Selection for HPRT Deficiency.
[0320] 5*10 6 cells were used per electroporation, with 25ugCas9
and 12.5 ug of each gRNA. For the Cas9 control a non-cutting gRNA
was used to keep the total DNA amount the same. FACS sorting ended
up being relatively similar at 5-8% GFP 48 hours after EP. Cells
were plated out at 40,000 per 10 cm plate per sample, and grown
until colonies could clearly be seen. 30 uM 6-Thioguanine (6-TG) in
mTESR (e.g., at a concentration of 30 .mu.m) and was used as
selection medium for 8-9 days and colonies were counted again. The
results are shown in Table 1 below.
TABLE-US-00002 TABLE 1 Starting Percentage - gRNA colonies Final
colonies Percentage Cas9 background Cas9 105 17 0.161904762 0.00 1
121 55 0.454545455 0.29 3 118 67 0.56779661 0.41 5 124 76
0.612903226 0.45 7 125 27 0.216 0.05 9 131 29 0.221374046 0.06 11
93 63 0.677419355 0.52 1 + 5 64 43 0.671875 0.51 1 + 3 77 45
0.584415584 0.42 1 + 7 55 19 0.345454545 0.18 1 + 9 60 26
0.433333333 0.27 1 + 11 52 32 0.615384615 0.45 3 + 5 69 46
0.666666667 0.50 3 + 7 55 33 0.6 0.44 3 + 11 38 30 0.789473684 0.63
7 + 11 72 41 0.569444444 0.41
[0321] Table 2 below shows the results from Table 1 above ranked
according to editing efficiency.
TABLE-US-00003 TABLE 2 gRNA Percentage 3 + 11 0.63 11 0.52 1 + 5
0.51 3 + 5 0.50 1 + 11 0.45 5 0.45 3 + 7 0.44 1 + 3 0.42 7 + 11
0.41 3 0.41 1 0.29 1 + 9 0.27 1 + 7 0.18 9 0.06 7 0.05 Cas9
0.00
[0322] gRNAs used in the experiments are shown below:
TABLE-US-00004 1- (SEQ ID NO: 298) gtcttgctcgagatgtgatg 3- (SEQ ID
NO: 299) taaattctttgctgacctgc 5- (SEQ ID NO: 300)
tagatccattcctatgactg 7- (SEQ ID NO: 301) cttcagtctgataaaatcta 9-
(SEQ ID NO: 302) tttgatgtaatccagcaggt 11- (SEQ ID NO: 303)
cacagagggctacaatgtga
REFERENCES
[0323] 1. Cong, L., et al., 2013. Multiplex genome engineering
using CRISPR/Cas systems. Science. 339, 819-23. [0324] 2. Ding, Q.,
et al., 2013. Enhanced efficiency of human pluripotent stem cell
genome editing through replacing TALENs with CRISPRs. Cell Stem
Cell. 12, 393-4. [0325] 3. Jinek, M., et al., 2013. RNA-programmed
genome editing in human cells. Elife. 2, e00471. [0326] 4. Li, D.,
et al., 2013. Heritable gene targeting in the mouse and rat using a
CRISPR-Cas system. Nat Biotechnol. 31, 681-3. [0327] 5. Malli, P.,
et al., 2013. RNA-guided human genome engineering via Cas9.
Science. 339, 823-6. [0328] 6. Niu, Y., et al., 2014. Generation of
Gene-Modified Cynomolgus Monkey via Cas9/RNA-Mediated Gene
Targeting in One-Cell Embryos. Cell. 156, 836-43. [0329] 7. Ran, F.
A., et al., 2013. Double nicking by RNA-guided CRISPR Cas9 for
enhanced genome editing specificity. Cell. 154, 1380-9. [0330] 8.
Wang, H., et al., 2013. One-step generation of mice carrying
mutations in multiple genes by CRISPR/Cas-mediated genome
engineering. Cell. 153, 910-8.
Sequence CWU 1
1
353123DNAArtificialCCR5 gRNA target site sequence 1tgacatcaat
tattatacat cgg 23223DNAArtificialCCR5 gRNA target site sequence
2cctgcctccg ctctactcac tgg 23323DNAArtificialCCR5 gRNA target site
sequence 3tactcactgg tgttcatctt tgg 23423DNAArtificialCCR5 gRNA
target site sequence 4ggtgttcatc tttggttttg tgg
23523DNAArtificialCCR5 gRNA target site sequence 5gtgttcatct
ttggttttgt ggg 23623DNAArtificialCCR5 gRNA target site sequence
6tggttttgtg ggcaacatgc tgg 23723DNAArtificialCCR5 gRNA target site
sequence 7tcatcctgat aaactgcaaa agg 23823DNAArtificialCCR5 gRNA
target site sequence 8tgacatctac ctgctcaacc tgg
23923DNAArtificialCCR5 gRNA target site sequence 9tccttcttac
tgtccccttc tgg 231023DNAArtificialCCR5 gRNA target site sequence
10ccttcttact gtccccttct ggg 231123DNAArtificialCCR5 gRNA target
site sequence 11ctcactatgc tgccgcccag tgg 231223DNAArtificialCCR5
gRNA target site sequence 12tcactatgct gccgcccagt ggg
231323DNAArtificialCCR5 gRNA target site sequence 13gctgccgccc
agtgggactt tgg 231423DNAArtificialCCR5 gRNA target site sequence
14acaatgtgtc aactcttgac agg 231523DNAArtificialCCR5 gRNA target
site sequence 15caatgtgtca actcttgaca ggg 231623DNAArtificialCCR5
gRNA target site sequence 16ttgacagggc tctattttat agg
231723DNAArtificialCCR5 gRNA target site sequence 17tattttatag
gcttcttctc tgg 231823DNAArtificialCCR5 gRNA target site sequence
18tcatcctcct gacaatcgat agg 231923DNAArtificialCCR5 gRNA target
site sequence 19cctgacaatc gataggtacc tgg 232023DNAArtificialCCR5
gRNA target site sequence 20ctgtgtttgc tttaaaagcc agg
232123DNAArtificialCCR5 gRNA target site sequence 21gtttgcttta
aaagccagga cgg 232223DNAArtificialCCR5 gRNA target site sequence
22aaagccagga cggtcacctt tgg 232323DNAArtificialCCR5 gRNA target
site sequence 23aagccaggac ggtcaccttt ggg 232423DNAArtificialCCR5
gRNA target site sequence 24agccaggacg gtcacctttg ggg
232523DNAArtificialCCR5 gRNA target site sequence 25caggacggtc
acctttgggg tgg 232623DNAArtificialCCR5 gRNA target site sequence
26tggtgacaag tgtgatcact tgg 232723DNAArtificialCCR5 gRNA target
site sequence 27ggtgacaagt gtgatcactt ggg 232823DNAArtificialCCR5
gRNA target site sequence 28gacaagtgtg atcacttggg tgg
232923DNAArtificialCCR5 gRNA target site sequence 29aagtgtgatc
acttgggtgg tgg 233023DNAArtificialCCR5 gRNA target site sequence
30gctgtgtttg cgtctctccc agg 233123DNAArtificialCCR5 gRNA target
site sequence 31tttaccagat ctcaaaaaga agg 233223DNAArtificialCCR5
gRNA target site sequence 32catacagtca gtatcaattc tgg
233323DNAArtificialCCR5 gRNA target site sequence 33gacattaaag
atagtcatct tgg 233423DNAArtificialCCR5 gRNA target site sequence
34acattaaaga tagtcatctt ggg 233523DNAArtificialCCR5 gRNA target
site sequence 35cattaaagat agtcatcttg ggg 233623DNAArtificialCCR5
gRNA target site sequence 36aaagatagtc atcttggggc tgg
233723DNAArtificialCCR5 gRNA target site sequence 37ggtcctgccg
ctgcttgtca tgg 233823DNAArtificialCCR5 gRNA target site sequence
38tgtcatggtc atctgctact cgg 233923DNAArtificialCCR5 gRNA target
site sequence 39gtcatggtca tctgctactc ggg 234023DNAArtificialCCR5
gRNA target site sequence 40gaatcctaaa aactctgctt cgg
234123DNAArtificialCCR5 gRNA target site sequence 41ggtgtcgaaa
tgagaagaag agg 234223DNAArtificialCCR5 gRNA target site sequence
42gaaatgagaa gaagaggcac agg 234323DNAArtificialCCR5 gRNA target
site sequence 43aaatgagaag aagaggcaca ggg 234423DNAArtificialCCR5
gRNA target site sequence 44agaagaggca cagggctgtg agg
234523DNAArtificialCCR5 gRNA target site sequence 45tgattgttta
ttttctcttc tgg 234623DNAArtificialCCR5 gRNA target site sequence
46gattgtttat tttctcttct ggg 234723DNAArtificialCCR5 gRNA target
site sequence 47ccttctcctg aacaccttcc agg 234823DNAArtificialCCR5
gRNA target site sequence 48aacaccttcc aggaattctt tgg
234923DNAArtificialCCR5 gRNA target site sequence 49ataattgcag
tagctctaac agg 235023DNAArtificialCCR5 gRNA target site sequence
50ttgcagtagc tctaacaggt tgg 235123DNAArtificialCCR5 gRNA target
site sequence 51caggttggac caagctatgc agg 235223DNAArtificialCCR5
gRNA target site sequence 52atgcaggtga cagagactct tgg
235323DNAArtificialCCR5 gRNA target site sequence 53tgcaggtgac
agagactctt ggg 235423DNAArtificialCCR5 gRNA target site sequence
54cccatcatct atgcctttgt cgg 235523DNAArtificialCCR5 gRNA target
site sequence 55ccatcatcta tgcctttgtc ggg 235623DNAArtificialCCR5
gRNA target site sequence 56catcatctat gcctttgtcg ggg
235723DNAArtificialCCR5 gRNA target site sequence 57ctgttctatt
ttccagcaag agg 235823DNAArtificialCCR5 gRNA target site sequence
58tcagtttaca cccgatccac tgg 235923DNAArtificialCCR5 gRNA target
site sequence 59cagtttacac ccgatccact ggg 236023DNAArtificialCCR5
gRNA target site sequence 60agtttacacc cgatccactg ggg
236123DNAArtificialCCR5 gRNA target site sequence 61cacccgatcc
actggggagc agg 236223DNAArtificialCCR5 gRNA target site sequence
62tggggagcag gaaatatctg tgg 236323DNAArtificialCCR5 gRNA target
site sequence 63ggggagcagg aaatatctgt ggg 236423DNAArtificialCCR5
gRNA target site sequence 64taataattga tgtcatagat tgg
236523DNAArtificialCCR5 gRNA target site sequence 65ttcacattga
ttttttggca ggg 236623DNAArtificialCCR5 gRNA target site sequence
66cttcacattg attttttggc agg 236723DNAArtificialCCR5 gRNA target
site sequence 67tttgcttcac attgattttt tgg 236823DNAArtificialCCR5
gRNA target site sequence 68gtagagcgga ggcaggaggc ggg
236923DNAArtificialCCR5 gRNA target site sequence 69agtagagcgg
aggcaggagg cgg 237023DNAArtificialCCR5 gRNA target site sequence
70gtgagtagag cggaggcagg agg 237123DNAArtificialCCR5 gRNA target
site sequence 71ccagtgagta gagcggaggc agg 237223DNAArtificialCCR5
gRNA target site sequence 72aacaccagtg agtagagcgg agg
237323DNAArtificialCCR5 gRNA target site sequence 73atgaacacca
gtgagtagag cgg 237423DNAArtificialCCR5 gRNA target site sequence
74ttttgcagtt tatcaggatg agg 237523DNAArtificialCCR5 gRNA target
site sequence 75tcagcctttt gcagtttatc agg 237623DNAArtificialCXCR5
gRNA target site sequence 76cagagatggc caggttgagc agg
237723DNAArtificialCCR5 gRNA target site sequence 77aaaacaggtc
agagatggcc agg 237823DNAArtificialCCR5 gRNA target site sequence
78aaggaaaaac aggtcagaga tgg 237923DNAArtificialCCR5 gRNA target
site sequence 79ggacagtaag aaggaaaaac agg 238023DNAArtificialCCR5
gRNA target site sequence 80cccagaaggg gacagtaaga agg
238123DNAArtificialCCR5 gRNA target site sequence 81cagcatagtg
agcccagaag ggg 238223DNAArtificialCCR5 gRNA target site sequence
82gcagcatagt gagcccagaa ggg 238323DNAArtificialCCR5 gRNA target
site sequence 83ggcagcatag tgagcccaga agg 238423DNAArtificialCCR5
gRNA target site sequence 84atttccaaag tcccactggg cgg
238523DNAArtificialCCR5 gRNA target site sequence 85tgtatttcca
aagtcccact ggg 238623DNAArtificialCCR5 gRNA target site sequence
86ttgtatttcc aaagtcccac tgg 238723DNAArtificialCCR5 gRNA target
site sequence 87ggtacctatc gattgtcagg agg 238823DNAArtificialCCR5
gRNA target site sequence 88ccaggtacct atcgattgtc agg
238923DNAArtificialCCR5 gRNA target site sequence 89acacagcatg
gacgacagcc agg 239023DNAArtificialCCR5 gRNA target site sequence
90cttttaaagc aaacacagca tgg 239123DNAArtificialCCR5 gRNA target
site sequence 91caccccaaag gtgaccgtcc tgg 239223DNAArtificialCCR5
gRNA target site sequence 92cacacttgtc accaccccaa agg
239323DNAArtificialCCR5 gRNA target site sequence 93atctggtaaa
gatgattcct ggg 239423DNAArtificialCCR5 gRNA target site sequence
94gatctggtaa agatgattcc tgg 239523DNAArtificialCCR5 gRNA target
site sequence 95aagaccttct ttttgagatc tgg 239623DNAArtificialCCR5
gRNA target site sequence 96gtatggaaaa tgagagctgc agg
239723DNAArtificialCCR5 gRNA target site sequence 97cagaattgat
actgactgta tgg 239823DNAArtificialCCR5 gRNA target site sequence
98agatgactat ctttaatgtc tgg 239923DNAArtificialCCR5 gRNA target
site sequence 99tgaccatgac aagcagcggc agg 2310023DNAArtificialCCR5
gRNA target site sequence 100cagatgacca tgacaagcag cgg
2310123DNAArtificialCCR5 gRNA target site sequence 101gacaccgaag
cagagttttt agg 2310223DNAArtificialCCR5 gRNA target site sequence
102gagaaaataa acaatcatga tgg 2310323DNAArtificialCCR5 gRNA target
site sequence 103aggagaagga caatgttgta ggg 2310423DNAArtificialCCR5
gRNA target site sequence 104caggagaagg acaatgttgt agg
2310523DNAArtificialCCR5 gRNA target site sequence 105cctggaaggt
gttcaggaga agg 2310623DNAArtificialCCR5 gRNA target site sequence
106agaattcctg gaaggtgttc agg 2310723DNAArtificialCCR5 gRNA target
site sequence 107caggccaaag aattcctgga agg 2310823DNAArtificialCCR5
gRNA target site sequence 108tattcaggcc aaagaattcc tgg
2310923DNAArtificialCCR5 gRNA target site sequence 109tagagctact
gcaattattc agg 2311023DNAArtificialCCR5 gRNA target site sequence
110tctctgtcac ctgcatagct tgg 2311123DNAArtificialCCR5 gRNA target
site sequence 111cgacaaaggc atagatgatg ggg 2311223DNAArtificialCCR5
gRNA target site sequence 112ccgacaaagg catagatgat ggg
2311323DNAArtificialCCR5 gRNA target site sequence 113cccgacaaag
gcatagatga tgg 2311423DNAArtificialCCR5 gRNA target site sequence
114tctgaacttc tccccgacaa agg 2311523DNAArtificialCCR5 gRNA target
site sequence 115gcttttggaa gaagactaag agg 2311623DNAArtificialCCR5
gRNA target site sequence 116agcgtttggc aatgtgcttt tgg
2311723DNAArtificialCCR5 gRNA target site sequence 117acagcatttg
cagaagcgtt tgg 2311823DNAArtificialCCR5 gRNA target site sequence
118ctcgctcggg agcctcttgc tgg 2311923DNAArtificialCCR5 gRNA target
site sequence 119taaactgagc ttgctcgctc ggg 2312023DNAArtificialCCR5
gRNA target site sequence 120gtaaactgag cttgctcgct cgg
2312123DNAArtificialCCR5 gRNA target site sequence 121ttcctgctcc
ccagtggatc ggg 2312223DNAArtificialCCR5 gRNA target site sequence
122tttcctgctc cccagtggat cgg 2312323DNAArtificialCCR5 gRNA target
site sequence 123agatatttcc tgctccccag tgg 2312428DNAArtificialCCR5
gRNA target site sequence 124atcatcttta ccagatctca aaaagaag
2812528DNAArtificialCCR5 gRNA target site sequence 125aactctgctt
cggtgtcgaa atgagaag 2812628DNAArtificialCCR5 gRNA target site
sequence 126tctgcttcgg tgtcgaaatg agaagaag 2812728DNAArtificialCCR5
gRNA target site sequence 127catcatctat gcctttgtcg gggagaag
2812828DNAArtificialCCR5 gRNA target site sequence 128atgcctttgt
cggggagaag ttcagaaa 2812928DNAArtificialCCR5 gRNA target site
sequence 129agtgagccca gaaggggaca gtaagaag 2813028DNAArtificialCCR5
gRNA target site sequence 130ctgggcggca gcatagtgag cccagaag
2813128DNAArtificialCCR5 gRNA target site sequence 131gatgatgaag
aagattccag agaagaag 2813228DNAArtificialCCR5 gRNA target site
sequence 132gaggatgatg aagaagattc cagagaag 2813328DNAArtificialCCR5
gRNA target site sequence 133atcgattgtc aggaggatga tgaagaag
2813428DNAArtificialCCR5 gRNA target site sequence 134caatgttgta
gggagcccag aagagaaa 2813528DNAArtificialCCR5 gRNA target site
sequence 135aaggacaatg ttgtagggag cccagaag 2813628DNAArtificialCCR5
gRNA target site sequence 136agaattcctg gaaggtgttc aggagaag
2813728DNAArtificialCCR5 gRNA target site sequence 137gcgtttggca
atgtgctttt ggaagaag 2813828DNAArtificialCCR5 gRNA target site
sequence 138ctggaaaata gaacagcatt tgcagaag 2813928DNAArtificialCCR5
gRNA target site sequence 139tcgggagcct cttgctggaa aatagaac
2814023DNAArtificialCXCR4 gRNA target site sequence 140acttgaagac
tcagactcag tgg 2314123DNAArtificialCXCR4 gRNA target site sequence
141atgtccacct cgctttcctt tgg 2314223DNAArtificialCXCR4 gRNA target
site sequence 142cacctcgctt tcctttggag agg
2314323DNAArtificialCXCR4 gRNA target site sequence 143ttcctttgga
gaggatcttg agg 2314423DNAArtificialCXCR4 gRNA target site sequence
144tttggagagg atcttgaggc tgg 2314523DNAArtificialCXCR4 gRNA target
site sequence 145gctggaccct ctgctcacag agg
2314623DNAArtificialCXCR4 gRNA target site sequence 146tcacagaggt
gagtgcgtgc tgg 2314723DNAArtificialCXCR4 gRNA target site sequence
147cacagaggtg agtgcgtgct ggg 2314823DNAArtificialCXCR4 gRNA target
site sequence 148ggtgagtgcg tgctgggcag agg
2314923DNAArtificialCXCR4 gRNA target site sequence 149ctgggcagag
gttttaaatt tgg 2315023DNAArtificialCXCR4 gRNA target site sequence
150aggttttaaa tttggctcca agg 2315123DNAArtificialCXCR4 gRNA target
site sequence 151tggctccaag gaaagcatag agg
2315223DNAArtificialCXCR4 gRNA target site sequence 152tccaaggaaa
gcatagagga tgg 2315323DNAArtificialCXCR4 gRNA target site sequence
153ccaaggaaag catagaggat ggg 2315423DNAArtificialCXCR4 gRNA target
site sequence 154caaggaaagc atagaggatg ggg
2315523DNAArtificialCXCR4 gRNA target site sequence 155ggatggggtt
cagacaacag tgg 2315623DNAArtificialCXCR4 gRNA target site sequence
156gacaacagtg gaagaaagct agg 2315723DNAArtificialCXCR4 gRNA target
site sequence 157acaacagtgg aagaaagcta ggg
2315823DNAArtificialCXCR4 gRNA target site sequence 158gtggaagaaa
gctagggcct cgg 2315923DNAArtificialCXCR4 gRNA target site sequence
159gaaagctagg gcctcggtga tgg 2316023DNAArtificialCXCR4 gRNA target
site sequence 160acccttgctt gatgatttcc agg
2316123DNAArtificialCXCR4 gRNA target site sequence 161cttgcttgat
gatttccagg agg 2316223DNAArtificialCXCR4 gRNA target site sequence
162gatgatttcc aggaggatga agg 2316323DNAArtificialCXCR4 gRNA target
site sequence 163atgctgatcc caatgtagta agg
2316423DNAArtificialCXCR4 gRNA target site sequence 164aatgtagtaa
ggcagccaac agg 2316523DNAArtificialCXCR4 gRNA target site sequence
165gccaacaggc gaagaaagcc agg 2316623DNAArtificialCXCR4 gRNA target
site sequence 166aggcgaagaa agccaggatg agg
2316723DNAArtificialCXCR4 gRNA target site sequence 167agccaggatg
aggatgactg tgg 2316823DNAArtificialCXCR4 gRNA target site sequence
168tgaggatgac tgtggtcttg agg 2316923DNAArtificialCXCR4 gRNA target
site sequence 169gaggatgact gtggtcttga ggg
2317023DNAArtificialCXCR4 gRNA target site sequence 170tcttgagggc
cttgcgcttc tgg 2317123DNAArtificialCXCR4 gRNA target site sequence
171tgagggcctt gcgcttctgg tgg 2317223DNAArtificialCXCR4 gRNA target
site sequence 172cttgcgcttc tggtggccct tgg
2317323DNAArtificialCXCR4 gRNA target site sequence 173gcccttggag
tgtgacagct tgg 2317423DNAArtificialCXCR4 gRNA target site sequence
174ggagatgata atgcaatagc agg 2317523DNAArtificialCXCR4 gRNA target
site sequence 175tgataatgca atagcaggac agg
2317623DNAArtificialCXCR4 gRNA target site sequence 176caggacagga
tgacaatacc agg 2317723DNAArtificialCXCR4 gRNA target site sequence
177acaggatgac aataccaggc agg 2317823DNAArtificialCXCR4 gRNA target
site sequence 178tgacaatacc aggcaggata agg
2317923DNAArtificialCXCR4 gRNA target site sequence 179caaccatgat
gtgctgaaac tgg 2318023DNAArtificialCXCR4 gRNA target site sequence
180cacaaccacc cacaagtcat tgg 2318123DNAArtificialCXCR4 gRNA target
site sequence 181acaaccaccc acaagtcatt ggg
2318223DNAArtificialCXCR4 gRNA target site sequence 182caaccaccca
caagtcattg ggg 2318323DNAArtificialCXCR4 gRNA target site sequence
183acaagtcatt ggggtagaag cgg 2318423DNAArtificialCXCR4 gRNA target
site sequence 184gtcatctgcc tcactgacgt tgg
2318523DNAArtificialCXCR4 gRNA target site sequence 185gacgttggca
aagatgaagt cgg 2318623DNAArtificialCXCR4 gRNA target site sequence
186acgttggcaa agatgaagtc ggg 2318723DNAArtificialCXCR4 gRNA target
site sequence 187tgaagtcggg aatagtcagc agg
2318823DNAArtificialCXCR4 gRNA target site sequence 188agtcgggaat
agtcagcagg agg 2318923DNAArtificialCXCR4 gRNA target site sequence
189gtcgggaata gtcagcagga ggg 2319023DNAArtificialCXCR4 gRNA target
site sequence 190ggaatagtca gcaggagggc agg
2319123DNAArtificialCXCR4 gRNA target site sequence 191gaatagtcag
caggagggca ggg 2319223DNAArtificialCXCR4 gRNA target site sequence
192ttttcagcca acagcttcct tgg 2319323DNAArtificialCXCR4 gRNA target
site sequence 193cttccttggc ctctgactgt tgg
2319423DNAArtificialCXCR4 gRNA target site sequence 194ccttggcctc
tgactgttgg tgg 2319523DNAArtificialCXCR4 gRNA target site sequence
195gcctctgact gttggtggcg tgg 2319623DNAArtificialCXCR4 gRNA target
site sequence 196actgttggtg gcgtggacga tgg
2319723DNAArtificialCXCR4 gRNA target site sequence 197tggtggcgtg
gacgatggcc agg 2319823DNAArtificialCXCR4 gRNA target site sequence
198cgtggacgat ggccaggtag cgg 2319923DNAArtificialCXCR4 gRNA target
site sequence 199gtagcggtcc agactgatga agg
2320023DNAArtificialCXCR4 gRNA target site sequence 200ggtccagact
gatgaaggcc agg 2320123DNAArtificialCXCR4 gRNA target site sequence
201gactgatgaa ggccaggatg agg 2320223DNAArtificialCXCR4 gRNA target
site sequence 202ggatgaggac actgctgtag agg
2320323DNAArtificialCXCR4 gRNA target site sequence 203ggttgactgt
gtagatgaca tgg 2320423DNAArtificialCXCR4 gRNA target site sequence
204tgacatggac tgccttgcat agg 2320523DNAArtificialCXCR4 gRNA target
site sequence 205cccaaagtac cagtttgcca cgg
2320623DNAArtificialCXCR4 gRNA target site sequence 206cacggcatca
actgcccaga agg 2320723DNAArtificialCXCR4 gRNA target site sequence
207acggcatcaa ctgcccagaa ggg 2320823DNAArtificialCXCR4 gRNA target
site sequence 208agggaagcgt gatgacaaag agg
2320923DNAArtificialCXCR4 gRNA target site sequence 209gaagcgtgat
gacaaagagg agg 2321023DNAArtificialCXCR4 gRNA target site sequence
210cgtgatgaca aagaggaggt cgg 2321123DNAArtificialCXCR4 gRNA target
site sequence 211agaggaggtc ggccactgac agg
2321223DNAArtificialCXCR4 gRNA target site sequence 212tcatgcttct
cagtttcttc tgg 2321323DNAArtificialCXCR4 gRNA target site sequence
213tcttctggta acccatgacc agg 2321423DNAArtificialCXCR4 gRNA target
site sequence 214aatgccagtt aagaagatga tgg
2321523DNAArtificialCXCR4 gRNA target site sequence 215taagaagatg
atggagtaga tgg 2321623DNAArtificialCXCR4 gRNA target site sequence
216gaagatgatg gagtagatgg tgg 2321723DNAArtificialCXCR4 gRNA target
site sequence 217aagatgatgg agtagatggt ggg
2321823DNAArtificialCXCR4 gRNA target site sequence 218tgatggagta
gatggtgggc agg 2321923DNAArtificialCXCR4 gRNA target site sequence
219tgaaattagc attttcttca cgg 2322023DNAArtificialCXCR4 gRNA target
site sequence 220agcattttct tcacggaaac agg
2322123DNAArtificialCXCR4 gRNA target site sequence 221gcattttctt
cacggaaaca ggg 2322223DNAArtificialCXCR4 gRNA target site sequence
222acggaaacag ggttccttca tgg 2322323DNAArtificialCXCR4 gRNA target
site sequence 223gtcccctgag cccatttcct cgg
2322423DNAArtificialCXCR4 gRNA target site sequence 224gaagtgtata
tctgcaaaag agg 2322523DNAArtificialCXCR4 gRNA target site sequence
225tatatctgca aaagaggcaa agg 2322623DNAArtificialCXCR4 gRNA target
site sequence 226ctgcaaaaga ggcaaaggaa tgg
2322723DNAArtificialCXCR4 gRNA target site sequence 227ctctccaaag
gaaagcgagg tgg 2322823DNAArtificialCXCR4 gRNA target site sequence
228atcctctcca aaggaaagcg agg 2322923DNAArtificialCXCR4 gRNA target
site sequence 229agcctcaaga tcctctccaa agg
2323023DNAArtificialCXCR4 gRNA target site sequence 230cactcacctc
tgtgagcaga ggg 2323123DNAArtificialCXCR4 gRNA target site sequence
231gcactcacct ctgtgagcag agg 2323223DNAArtificialCXCR4 gRNA target
site sequence 232cccatcctct atgctttcct tgg
2323323DNAArtificialCXCR4 gRNA target site sequence 233caagtggatt
tccatcaccg agg 2323423DNAArtificialCXCR4 gRNA target site sequence
234ttgagaacac tgtgcacaag tgg 2323523DNAArtificialCXCR4 gRNA target
site sequence 235tcctggaaat catcaagcaa ggg
2323623DNAArtificialCXCR4 gRNA target site sequence 236ctcctggaaa
tcatcaagca agg 2323723DNAArtificialCXCR4 gRNA target site sequence
237catcgactcc ttcatcctcc tgg 2323823DNAArtificialCXCR4 gRNA target
site sequence 238gttggctgcc ttactacatt ggg
2323923DNAArtificialCXCR4 gRNA target site sequence 239tgttggctgc
cttactacat tgg 2324023DNAArtificialCXCR4 gRNA target site sequence
240tcctggcttt cttcgcctgt tgg 2324123DNAArtificialCXCR4 gRNA target
site sequence 241gaccacagtc atcctcatcc tgg
2324223DNAArtificialCXCR4 gRNA target site sequence 242caagggccac
cagaagcgca agg 2324323DNAArtificialCXCR4 gRNA target site sequence
243tccaagctgt cacactccaa ggg 2324423DNAArtificialCXCR4 gRNA target
site sequence 244ctccaagctg tcacactcca agg
2324523DNAArtificialCXCR4 gRNA target site sequence 245atggttggcc
ttatcctgcc tgg 2324623DNAArtificialCXCR4 gRNA target site sequence
246cagtttcagc acatcatggt tgg 2324723DNAArtificialCXCR4 gRNA target
site sequence 247gttccagttt cagcacatca tgg
2324823DNAArtificialCXCR4 gRNA target site sequence 248ctaccccaat
gacttgtggg tgg 2324923DNAArtificialCXCR4 gRNA target site sequence
249cttctacccc aatgacttgt ggg 2325023DNAArtificialCXCR4 gRNA target
site sequence 250gcttctaccc caatgacttg tgg
2325123DNAArtificialCXCR4 gRNA target site sequence 251catctttgcc
aacgtcagtg agg 2325223DNAArtificialCXCR4 gRNA target site sequence
252aggtggtcta tgttggcgtc tgg 2325323DNAArtificialCXCR4 gRNA target
site sequence 253gctgaaaagg tggtctatgt tgg
2325423DNAArtificialCXCR4 gRNA target site sequence 254gaagctgttg
gctgaaaagg tgg 2325523DNAArtificialCXCR4 gRNA target site sequence
255aaggaagctg ttggctgaaa agg 2325623DNAArtificialCXCR4 gRNA target
site sequence 256tcagaggcca aggaagctgt tgg
2325723DNAArtificialCXCR4 gRNA target site sequence 257ccaccaacag
tcagaggcca agg 2325823DNAArtificialCXCR4 gRNA target site sequence
258tccacgccac caacagtcag agg 2325923DNAArtificialCXCR4 gRNA target
site sequence 259catcagtctg gaccgctacc tgg
2326023DNAArtificialCXCR4 gRNA target site sequence 260catcctggcc
ttcatcagtc tgg 2326123DNAArtificialCXCR4 gRNA target site sequence
261ctacagcagt gtcctcatcc tgg 2326223DNAArtificialCXCR4 gRNA target
site sequence 262ctttgggaac ttcctatgca agg
2326323DNAArtificialCXCR4 gRNA target site sequence 263ccgtggcaaa
ctggtacttt ggg 2326423DNAArtificialCXCR4 gRNA target site sequence
264gccgtggcaa actggtactt tgg 2326523DNAArtificialCXCR4 gRNA target
site sequence 265cagttgatgc cgtggcaaac tgg
2326623DNAArtificialCXCR4 gRNA target site sequence 266cttctgggca
gttgatgccg tgg 2326723DNAArtificialCXCR4 gRNA target site sequence
267tgtcatcacg cttcccttct ggg 2326823DNAArtificialCXCR4 gRNA target
site sequence 268ttgtcatcac
gcttcccttc tgg 2326923DNAArtificialCXCR4 gRNA target site sequence
269gtacaggctg cacctgtcag tgg 2327023DNAArtificialCXCR4 gRNA target
site sequence 270gaagcatgac ggacaagtac agg
2327123DNAArtificialCXCR4 gRNA target site sequence 271gaagaaactg
agaagcatga cgg 2327223DNAArtificialCXCR4 gRNA target site sequence
272ggattggtca tcctggtcat ggg 2327323DNAArtificialCXCR4 gRNA target
site sequence 273tggattggtc atcctggtca tgg
2327423DNAArtificialCXCR4 gRNA target site sequence 274gggcaatgga
ttggtcatcc tgg 2327523DNAArtificialCXCR4 gRNA target site sequence
275tggcattgtg ggcaatggat tgg 2327623DNAArtificialCXCR4 gRNA target
site sequence 276ttaactggca ttgtgggcaa tgg
2327723DNAArtificialCXCR4 gRNA target site sequence 277atcttcttaa
ctggcattgt ggg 2327823DNAArtificialCXCR4 gRNA target site sequence
278catcttctta actggcattg tgg 2327923DNAArtificialCXCR4 gRNA target
site sequence 279tactccatca tcttcttaac tgg
2328023DNAArtificialCXCR4 gRNA target site sequence 280aggggactat
gactccatga agg 2328123DNAArtificialCXCR4 gRNA target site sequence
281caccgaggaa atgggctcag ggg 2328223DNAArtificialCXCR4 gRNA target
site sequence 282acaccgagga aatgggctca ggg
2328323DNAArtificialCXCR4 gRNA target site sequence 283tacaccgagg
aaatgggctc agg 2328423DNAArtificialCXCR4 gRNA target site sequence
284gataactaca ccgaggaaat ggg 2328523DNAArtificialCXCR4 gRNA target
site sequence 285agataactac accgaggaaa tgg
2328623DNAArtificialCXCR4 gRNA target site sequence 286cacttcagat
aactacaccg agg 2328728DNAArtificialCXCR4 gRNA target site sequence
287gatggggttc agacaacagt ggaagaaa 2828828DNAArtificialCXCR4 gRNA
target site sequence 288gtagtaaggc agccaacagg cgaagaaa
2828928DNAArtificialCXCR4 gRNA target site sequence 289aaccacccac
aagtcattgg ggtagaag 2829028DNAArtificialCXCR4 gRNA target site
sequence 290gtttgccacg gcatcaactg cccagaag
2829128DNAArtificialCXCR4 gRNA target site sequence 291tccattgccc
acaatgccag ttaagaag 2829228DNAArtificialCXCR4 gRNA target site
sequence 292catcaagcaa gggtgtgagt ttgagaac
2829328DNAArtificialCXCR4 gRNA target site sequence 293gctgtcacac
tccaagggcc accagaag 2829428DNAArtificialCXCR4 gRNA target site
sequence 294tcatgggtta ccagaagaaa ctgagaag
2829528DNAArtificialCXCR4 gRNA target site sequence 295catcctggtc
atgggttacc agaagaaa 2829628DNAArtificialCXCR4 gRNA target site
sequence 296ggtcatcctg gtcatgggtt accagaag
2829728DNAArtificialCXCR4 gRNA target site sequence 297atgaaggaac
cctgtttccg tgaagaaa 282981368PRTUnknownCAS protein 298Met Asp Lys
Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 1 5 10 15 Gly
Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25
30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr
Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys
Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met
Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser
Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile
Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr
Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr
Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155
160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln
Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser
Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys
Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu
Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser
Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala
Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp
Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280
285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser
Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp
Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu
Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr
Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr
Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu
Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405
410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro
Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr
Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn
Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile
Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala
Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys
Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu
Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530
535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val
Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile
Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg
Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile
Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp
Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp
Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His
Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650
655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp
Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp
Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser
Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu
Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val
Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys
Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr
Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775
780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu
Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu
Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile
Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys
Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn
Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900
905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr
Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val
Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys
Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His
His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala
Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015
1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe
1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr
Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu
Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly
Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro
Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly
Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser
Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Lys
Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135
1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys
1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg
Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala
Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys
Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg
Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly
Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu
Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255
1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys
1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu
Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu
Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn
Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr
Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu
Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr
Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365
29920DNAArtificialgRNA 299taaattcttt gctgacctgc
2030020DNAArtificialgRNA 300tagatccatt cctatgactg
2030120DNAArtificialgRNA 301cttcagtctg ataaaatcta
2030220DNAArtificialgRNA 302tttgatgtaa tccagcaggt
2030320DNAArtificialgRNA 303cacagagggc tacaatgtga
2030423DNAArtificialTarget site sequence with NGG 304gtagagcgga
ggcaggaggc ggg 2330523DNAArtificialTarget site sequence with NGG
305gtgagtagag cggaggcagg agg 2330623DNAArtificialTarget site
sequence with NGG 306ggtgttcatc tttggttttg tgg
2330723DNAArtificialTarget site sequence with NGG 307gtgttcatct
ttggttttgt ggg 2330823DNAArtificialTarget site sequence with NGG
308ggacagtaag aaggaaaaac agg 2330923DNAArtificialTarget site
sequence with NGG 309gctgccgccc agtgggactt tgg
2331023DNAArtificialTarget site sequence with NGG 310gcagcatagt
gagcccagaa ggg 2331123DNAArtificialTarget site sequence with NGG
311ggcagcatag tgagcccaga agg 2331223DNAArtificialTarget site
sequence with NGG 312ggtacctatc gattgtcagg agg
2331323DNAArtificialTarget site sequence with NGG 313gtttgcttta
aaagccagga cgg 2331423DNAArtificialTarget site sequence with NGG
314ggtgacaagt gtgatcactt ggg 2331523DNAArtificialTarget site
sequence with NGG 315gacaagtgtg atcacttggg tgg
2331623DNAArtificialTarget site sequence with NGG 316gctgtgtttg
cgtctctccc agg 2331723DNAArtificialTarget site sequence with NGG
317gatctggtaa agatgattcc tgg 2331823DNAArtificialTarget site
sequence with NGG 318gtatggaaaa tgagagctgc agg
2331923DNAArtificialTarget site sequence with NGG 319gacattaaag
atagtcatct tgg 2332023DNAArtificialTarget site sequence with NGG
320ggtcctgccg ctgcttgtca tgg
2332123DNAArtificialTarget site sequence with NGG 321gtcatggtca
tctgctactc ggg 2332223DNAArtificialTarget site sequence with NGG
322gaatcctaaa aactctgctt cgg 2332323DNAArtificialTarget site
sequence with NGG 323ggtgtcgaaa tgagaagaag agg
2332423DNAArtificialTarget site sequence with NGG 324gacaccgaag
cagagttttt agg 2332523DNAArtificialTarget site sequence with NGG
325gaaatgagaa gaagaggcac agg 2332623DNAArtificialTarget site
sequence with NGG 326gattgtttat tttctcttct ggg
2332723DNAArtificialTarget site sequence with NGG 327gagaaaataa
acaatcatga tgg 2332823DNAArtificialTarget site sequence with NGG
328gcttttggaa gaagactaag agg 2332923DNAArtificialTarget site
sequence with NGG 329gtaaactgag cttgctcgct cgg
2333023DNAArtificialTarget site sequence with NGG 330ggggagcagg
aaatatctgt ggg 2333123DNAArtificialTarget site sequence with NGG
331acaatgtgtc aactcttgac agg 2333223DNAArtificialTarget site
sequence with NGG 332tcactatgct gccgcccagt ggg
2333323DNAArtificialTarget site sequence with NGG 333ggtacctatc
gattgtcagg agg 2333420DNAArtificialgRNA 334gtcttgctcg agatgtgatg
2033547DNAArtificialtarget sequence 335tcccttcctg cctcatttca
ggtgaataca tcaagacctg gaggcca 4733647DNAArtificialtarget sequence
336tcccttcctg cctcatttca ggtgaataca tcaagacctg gaggcca
4733747DNAArtificialtarget sequence 337tgctggctcg gctgccctga
ggttgctcaa tcaagcacag gtttcaa 4733847DNAArtificialtarget sequence
338tgctggctcg gctgccctga ggttgctcaa tcaagcacag gtttcaa
4733947DNAArtificialtarget sequence 339taacagcgat gctgaccccc
tgtgcctcta ccacttctat gaccaga 4734047DNAArtificialtarget sequence
340cgatgctgac cccctgtgcc tctaccactt ctatgaccag atggacc
4734149DNAArtificialtarget sequence 341tggtccttgc tgtgttctct
gcggtgcttg gctccctgca gtttgggta 4934249DNAArtificialtarget sequence
342tggtccttgc tgtgttctct gcggtgcttg gctccctgca gtttgggta
4934347DNAArtificialtarget sequence 343tgggcgacag atgcgaaaga
aacgagttcc agtgccaaga cgggaaa 4734447DNAArtificialtarget sequence
344gaaacgagtt ccagtgccaa gacgggaaat gcatctccta caagtgg
4734548DNAArtificialtarget sequence 345tcagagagga cactgcagtt
gtccgtgcta gtagccttcg cttctgga 4834648DNAArtificialtarget sequence
346tcagagagga cactgcagtt gtccgtgcta gtagccttcg cttctgga
4834747DNAArtificialtarget sequence 347tgatgatctc agaggctcag
tatccttgtc ctgggttgga gatagca 4734847DNAArtificialtarget sequence
348tgatgatctc agaggctcag tatccttgtc ctgggttgga gatagca
4734949DNAArtificialtarget sequence 349tggtaattat gacttttgga
cagtccaagc tatatcgaag gtgagatca 4935049DNAArtificialtarget sequence
350tggtaattat gacttttgga cagtccaagc tatatcgaag gtgagatca
4935147DNAArtificialtarget sequence 351tcccttcctg cctcatttca
ggtgaataca tcaagacctg gaggcca 4735247DNAArtificialtarget sequence
352tcccttcctg cctcatttca ggtgaataca tcaagacctg gaggcca
4735347DNAArtificialtarget sequence 353ctatgccctg cctcatttca
ggtgaagatg aaatccctgg agcttgg 47
* * * * *
References