U.S. patent application number 15/531751 was filed with the patent office on 2017-09-21 for rna-guided systems for in vivo gene editing.
The applicant listed for this patent is President and Fellows of Harvard College. Invention is credited to Wei Leong Chew, George M. Church, Mohammadsharif Tabebordbar, Amy J. Wagers.
Application Number | 20170266320 15/531751 |
Document ID | / |
Family ID | 56092326 |
Filed Date | 2017-09-21 |
United States Patent
Application |
20170266320 |
Kind Code |
A1 |
Wagers; Amy J. ; et
al. |
September 21, 2017 |
RNA-Guided Systems for In Vivo Gene Editing
Abstract
Methods of editing target nucleic acids are provided using a
guide RNA and a Cas9 protein to excise exons in a target gene and
where the edited gene is expressed to produce a truncated
polypeptide.
Inventors: |
Wagers; Amy J.; (Cambridge,
MA) ; Tabebordbar; Mohammadsharif; (Cambridge,
MA) ; Chew; Wei Leong; (Boston, MA) ; Church;
George M.; (Brookline, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
President and Fellows of Harvard College |
Cambridge |
MA |
US |
|
|
Family ID: |
56092326 |
Appl. No.: |
15/531751 |
Filed: |
December 1, 2015 |
PCT Filed: |
December 1, 2015 |
PCT NO: |
PCT/US15/63181 |
371 Date: |
May 31, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62085785 |
Dec 1, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/86 20130101;
C12N 15/907 20130101; C12N 9/22 20130101; C12N 2310/20 20170501;
C12N 15/11 20130101; A61K 48/005 20130101; C12N 15/102 20130101;
C12N 2750/14143 20130101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 9/22 20060101 C12N009/22; C12N 15/11 20060101
C12N015/11; C12N 15/90 20060101 C12N015/90; C12N 15/86 20060101
C12N015/86 |
Goverment Interests
STATEMENT OF GOVERNMENT INTERESTS
[0002] This invention was made with government support under
U01HL100402, DP2 OD004345, 5P50HG005550-04S1 and 5 PN2 Ey018244
awarded by National Institutes of Health. The government has
certain rights in the invention.
Claims
1. A method of producing an altered gene product in a eukaryotic
cell comprising providing to the cell two or more guide RNAs and a
Cas9 protein, wherein the two or more guide RNAs are complementary
to two or more target genomic DNA sequences flanking a target
excision sequence including one or more exons in a target gene
encoding a biologically functional polypeptide, wherein the two or
more guide RNAs bind to the two or more complementary target
genomic DNA sequences and the Cas9 protein cleaves the two or more
target genomic DNA sequences thereby removing the one or more exons
from the target gene to produce an altered target gene and wherein
the altered target gene recombines, and wherein the eukaryotic cell
expresses the altered target gene to produce an altered
biologically functional polypeptide.
2. The method of claim 1 wherein the altered biologically
functional polypeptide lacks a polypeptide sequence corresponding
to the one or more removed exons.
3. The method of claim 1 wherein the two or more guide RNAs and the
Cas9 protein are foreign to the eukaryotic cell.
4. The method of claim 1 wherein the two or more guide RNAs and the
Cas9 protein are foreign to each other.
5. The method of claim 1 wherein the two or more guide RNAs and the
Cas9 protein are non-naturally occurring.
6. The method of claim 1 wherein the two or more guide RNAs are
provided to the cell by electroporation of the two or more guide
RNAs into the cell.
7. The method of claim 1 wherein the Cas9 protein is provided to
the cell by electroporation of the Cas9 protein into the cell.
8. The method of claim 1 wherein the two or more guide RNAs are
provided to the cell by introducing into the cell a first foreign
nucleic acid sequence encoding the two or more guide RNAs.
9. The method of claim 1 wherein the two or more guide RNAs are
provided to the cell by introducing into the cell a first foreign
nucleic acid sequence encoding the two or more guide RNAs present
in a plasmid or vector.
10. The method of claim 1 wherein the Cas 9 protein is provided to
the cell by introducing into the cell a second foreign nucleic acid
sequence encoding the Cas 9 protein.
11. The method of claim 1 wherein the Cas 9 protein is provided to
the cell by introducing into the cell a second foreign nucleic acid
sequence encoding the Cas 9 protein present in a plasmid or
vector.
12. The method of claim 1 wherein the eukaryotic cell is a yeast
cell, a plant cell, a vertebrate cell, a mammalian cell or a human
cell.
13. The method of claim 1 wherein the eukaryotic cell is within a
mammal
14. The method of claim 1 wherein the eukaryotic cell is a skeletal
muscle cell.
15. The method of claim 1 wherein the target excision sequence is
greater than 45 kb.
16. The method of claim 1 wherein the target gene encodes
dystrophin protein.
17. The method of claim 1 wherein the target gene encodes
dystrophin protein and the one or more exons is exon 23.
18. The method of claim 1 wherein the target gene encodes
dystrophin protein and the one or more exons is exon 52 and exon
53.
19. The method of claim 1 wherein the RNA includes between about 10
to about 250 nucleotides.
20. The method of claim 1 wherein the RNA includes between about 20
to about 100 nucleotides.
21. The method of claim 1 wherein the guide RNA includes a guide
sequence fused to a trans-activating cr (tracr) sequence.
22. The method of claim 1 wherein the ratio of plasmid encoding the
Cas9 protein to the plasmid encoding the guide RNA is between 1:5
and 2:1.
23. The method of claim 1 wherein the plasmid encoding the guide
RNA is modified to increase the expression of the RNA by removing a
potential premature transcription termination site.
24. The method of claim 1 wherein the one or more exons includes a
mutation.
25. The method of claim 1 wherein the Cas9 protein is provided to
the cell by electroporation of the Cas9 mRNA into the cell.
26. The method of claim 1 wherein the guide RNA and the Cas9
protein co-localize to the target genomic DNA sequence to form a
complex.
27. The method of claim 1 wherein the target nucleic acid is
chromosomal DNA.
28. The method of claim 1 wherein the Cas9 protein is wild type
Cas9, Cas9 nickase or a nuclease null Cas9 including a
nuclease.
29. The method of claim 1 wherein the guide RNA and the Cas9
protein are combined and then contacted with the target gene.
30. The method of claim 1 wherein the guide RNA and the Cas9
protein are combined and then contacted with the target gene within
a cell.
31. The method of claim 1 comprising providing to the cell a
plurality of guide RNAs with each having a portion complementary to
a target genomic DNA sequence.
32. The method of claim 1 wherein the cell is a transplantable
cell.
33. The method of claim 1 wherein the cell is a progenitor
cell.
34. The method of claim 1 wherein the cell is a stem cell.
35. The method of claim 1 wherein the cell is a muscle stem
cell.
36. A skeletal muscle cell including a Cas9 protein and two or more
guide RNAs complementary to two or more target genomic DNA
sequences flanking a target excision sequence including one or more
exons in a target gene encoding dystrophin protein.
37. The skeletal muscle cell of claim 36 wherein the one or more
exons are in the exon 45-55 region.
38. The skeletal muscle cell of claim 36 wherein the one or more
exons include exon 23, exon 52 or exon 53.
39. A skeletal muscle cell including a first nucleic acid encoding
two or more guide RNAs complementary to two or more target genomic
DNA sequences flanking a target excision sequence including one or
more exons in a target gene encoding dystrophin protein and a
second nucleic acid encoding a Cas9 protein.
40. The skeletal muscle cell of claim 39 wherein the one or more
exons are in the exon 45-55 region.
41. The skeletal muscle cell of claim 39 wherein the one or more
exons include exon 23, exon 52 or exon 53.
42. The skeletal muscle cell of claim 39 wherein the first nucleic
acid is within a plasmid or vector.
43. The skeletal muscle cell of claim 39 wherein the second nucleic
acid is within a plasmid or vector.
44. The skeletal muscle cell of claim 39 wherein the second nucleic
acid is within a viral vector.
45. The skeletal muscle cell of claim 39 wherein the second nucleic
acid is within a viral vector selected from the group consisting of
lentivirus, adenovirus, adeno-associated virus, retrovirus, herpes
simplex virus, or sendai virus.
46. A skeletal muscle cell including a Cas9 protein and two or more
guide RNAs complementary to two or more target genomic DNA
sequences flanking a target excision sequence including one or more
exons in a target gene encoding dystrophin protein.
47. The skeletal muscle cell of claim 46 wherein the one or more
exons are in the exon 45-55 region.
48. The skeletal muscle cell of claim 46 wherein the one or more
exons include exon 23, exon 52 or exon 53.
49. A muscle stem cell including a first nucleic acid encoding two
or more guide RNAs complementary to two or more target genomic DNA
sequences flanking a target excision sequence including one or more
exons in a target gene encoding dystrophin protein and a second
nucleic acid encoding a Cas9 protein.
50. The muscle stem cell of claim 49 wherein the one or more exons
are in the exon 45-55 region.
51. The muscle stem cell of claim 49 wherein the one or more exons
include exon 23, exon 52 or exon 53.
52. The muscle stem cell of claim 49 wherein the first nucleic acid
is within a plasmid or vector.
53. The muscle stem cell of claim 49 wherein the second nucleic
acid is within a plasmid or vector.
54. The muscle stem cell of claim 49 wherein the second nucleic
acid is within a viral vector.
55. The muscle stem cell of claim 49 wherein the second nucleic
acid is within a viral vector selected from the group consisting of
lentivirus, adenovirus, adeno-associated virus, retrovirus, herpes
simplex virus, or sendai virus.
56. A genetically modified skeletal muscle cell including a first
nucleic acid encoding two or more guide RNAs complementary to two
or more target genomic DNA sequences flanking a target excision
sequence including one or more exons in a target gene encoding
dystrophin protein and a second nucleic acid encoding a Cas9
protein and wherein the target gene encoding dystrophin protein
lacks one or more of exon 23, exon 52 or exon 53.
57. A genetically modified muscle stem cell including a first
nucleic acid encoding two or more guide RNAs complementary to two
or more target genomic DNA sequences flanking a target excision
sequence including one or more exons in a target gene encoding
dystrophin protein and a second nucleic acid encoding a Cas9
protein and wherein the target gene encoding dystrophin protein
lacks one or more of exon 23, exon 52 or exon 53.
58. A method of producing an altered gene product in a eukaryotic
cell within a mammal comprising injecting two plasmids into the
mammal, wherein the two plasmids include a first nucleic acid
encoding two or more guide RNAs complementary to two or more target
genomic DNA sequences flanking a target excision sequence including
one or more exons in a target gene encoding dystrophin protein and
a second nucleic acid encoding a Cas9 protein, wherein the two or
more guide RNAs bind to the two or more complementary target
genomic DNA sequences and the Cas9 protein cleaves the two or more
target genomic DNA sequences thereby removing the one or more exons
from the target gene to produce an altered target gene and wherein
the altered target gene recombines, and wherein the eukaryotic cell
expresses the altered target gene to produce an altered
biologically functional polypeptide.
59. The method of claim 58 wherein the eukaryotic cell is a
skeletal muscle cell.
60. The method of claim 58 wherein the eukaryotic cell is a muscle
stem cell.
61. The method of claim 58 wherein the eukaryotic cell is a member
of the group consisting of a skeletal muscle cell, a muscle stem
cell, a progenitor cell and a stem cell.
62. The method of claim 58 wherein the one or more exons is exon
23, exon 52 or exon 53.
63. A method of removing one or more mutations from a target gene
encoding a dystrophin protein in a eukaryotic cell comprising
providing to the cell two or more guide RNAs and a Cas9 protein,
wherein the two or more guide RNAs are complementary to two or more
target genomic DNA sequences flanking a target excision sequence
including one or more exons having one or more mutations in the
target gene, wherein the two or more guide RNAs bind to the two or
more complementary target genomic DNA sequences and the Cas9
protein cleaves the two or more target genomic DNA sequences
thereby removing the one or more exons having one or more mutations
from the target gene to produce an altered target gene and wherein
the altered target gene recombines, and wherein the eukaryotic cell
expresses the altered target gene to produce a functional truncated
dystrophin protein.
64. The method of claim 63 wherein the eukaryotic cell is a
skeletal muscle cell.
65. The method of claim 63 wherein the eukaryotic cell is a muscle
stem cell.
66. The method of claim 63 wherein the eukaryotic cell is a member
of the group consisting of a skeletal muscle cell, a muscle stem
cell, a progenitor cell and a stem cell.
67. The method of claim 63 wherein the one or more exons is exon
23, exon 52 or exon 53.
68. The method of claim 63 wherein the eukaryotic cell is within a
mammal
69. The method of claim 63 wherein the one or more exons are in the
exon 45-55 region.
70. The method of claim 1 wherein the one or more exons are in the
exon 45-55 region.
71. The method of claim 11 wherein the second nucleic acid is
within a viral vector.
72. The method of claim 11 wherein the second nucleic acid is
within a viral vector selected from the group consisting of
lentivirus, adenovirus, adeno-associated virus, retrovirus, herpes
simplex virus, or sendai virus.
Description
RELATED APPLICATION DATA
[0001] This application claims priority to U.S. provisional
application no. 62/085,785, filed Dec. 1, 2014 which is hereby
incorporated herein by reference in its entirety for all
purposes.
BACKGROUND
[0003] Bacterial and archaeal CRISPR-Cas systems rely on short
guide RNAs (gRNA) in complex with Cas proteins to direct
degradation of complementary sequences present within invading
foreign nucleic acid. See Deltcheva, E. et al. CRISPR RNA
maturation by trans-encoded small RNA and host factor RNase III.
Nature 471, 602-607 (2011); Gasiunas, G., Barrangou, R., Horvath,
P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates
specific DNA cleavage for adaptive immunity in bacteria.
Proceedings of the National Academy of Sciences of the United
States of America 109, E2579-2586 (2012); Jinek, M. et al. A
programmable dual-RNA-guided DNA endonuclease in adaptive bacterial
immunity. Science 337, 816-821 (2012); Sapranauskas, R. et al. The
Streptococcus thermophilus CRISPR/Cas system provides immunity in
Escherichia coli. Nucleic acids research 39, 9275-9282 (2011); and
Bhaya, D., Davison, M. & Barrangou, R. CRISPR-Cas systems in
bacteria and archaea: versatile small RNAs for adaptive defense and
regulation. Annual review of genetics 45, 273-297 (2011). A recent
in vitro reconstitution of the S. pyogenes type II CRISPR system
demonstrated that crRNA ("CRISPR RNA") fused to a normally
trans-encoded tracrRNA ("trans-activating CRISPR RNA") is
sufficient to direct Cas9 protein to sequence-specifically cleave
target DNA sequences matching the crRNA. Expressing a gRNA with a
`spacer` homologous to a target site results in Cas9 recruitment
and endonucleolytic cleavage of the target DNA protospacer. See H.
Deveau et al., Phage response to CRISPR-encoded resistance in
Streptococcus thermophilus. Journal of Bacteriology 190, 1390 (Feb,
2008). Various uses of CRISPR/Cas9 systems are known. See
WO2014/099744, WO2013176772, U.S. Pat. No. 8,697,359 and Sternberg
et al., Nature, Vol. 507, pp. 62-67 (2014).
[0004] Duchenne muscular dystrophy (DMD) is one of the most common
X-linked genetic disorders in humans, and arises from mutations in
the Dystrophin gene that cause loss of protein expression (see A.
H. Burghes, C. Logan, X. Hu, B. Belfall, R. G. Worton, P. N. Ray, A
cDNA clone from the Duchenne/Becker muscular dystrophy gene. Nature
328, 434-437 (1987)). Dystrophin is an essential structural protein
in muscle fibers, J. M. Ervasti, K. P. Campbell, Membrane
organization of the dystrophin-glycoprotein complex. Cell 66,
1121-1131 (1991), and its absence destabilizes the muscle fiber
membrane, increasing the susceptibility of muscle fibers to
contraction-induced injury, K. P. Campbell, S. D. Kahl, Association
of dystrophin and an integral membrane glycoprotein. Nature 338,
259-262 (1989), which eventually leads to loss of mobility and
premature death in patients.
SUMMARY
[0005] Aspects of the present disclosure are directed to a method
of altering the sequence of a full length biologically active
polypeptide to produce an altered biologically active polypeptide.
The gene encoding the full length biologically active polypeptide
is altered to remove sequences encoding for portions of the
biologically active polypeptide that do not prevent the biological
activity of the polypeptide, or otherwise allow the altered or
modified polypeptide to have an activity similar to the full length
or unmodified polypeptide. According to one aspect, the gene is
altered using a DNA binding protein, such as a Cas9 protein.
According to one aspect, the gene is altered using a CRISPR/Cas9
system. According to one aspect, the gene is altered using a
CRISPR/Cas9 system in vivo. According to one aspect, a CRISPR/Cas9
system is delivered into a cell where the CRISPR/Cas9 system cuts
the target gene to remove sequences encoding for portions of the
biologically active polypeptide that do not prevent the biological
activity of the polypeptide. In this manner, the altered or
modified or truncated polypeptide has an activity the same as or
similar to that of the full length, unaltered, unmodified or
non-truncated polypeptide. According to one aspect, the cell may be
a cell in a cell culture, an embryonic cell or a cell in an animal
that is post embryonic stage, such as a mature animal. Accordingly,
aspects of the present disclose include carrying out the methods
described herein in vivo in an animal. According to one aspect, the
altered gene is expressed to produce an altered biologically active
polypeptide which may be referred to herein as a truncated
biologically active polypeptide. The biologically active
polypeptide is truncated to the extent that it lacks one or more
sequences found in the full length polypeptide. The polypeptide is
"truncated" to the extent that the truncated polypeptide excludes
amino acid sequences that are present in the full length or
non-truncated polypeptide. A truncated biologically active
polypeptide lacks one or more sequences found in the full length
polypeptide yet retains the same or substantially similar or
similar biological activity of the full length polypeptide. In this
aspect, the biological activity of the full length polypeptide is
retained in the truncated biologically active polypeptide despite
having one or more portions of the polypeptide removed. In another
aspect, the biological activity may be altered by the modification
of one or more sequences in the truncated biologically active
polypeptide. For example, the modification may result in a
partially active protein or a dominant negative protein.
[0006] Aspects of the present disclosure are directed to a method
of genetically altering a nucleic acid sequence, such as a target
gene, to excise one or more nucleic acid portions or sections or
sequences of the gene encoding a biologically functional
polypeptide using a CRISPR/Cas9 system. According to certain
aspects, the CRISPR Cas9 system includes a Cas9 protein and two or
more guide RNA. The two or more guide RNA are complementary to
corresponding target nucleic acid sequences on either side of, i.e.
flanking, the nucleic acid portion or section or sequence of the
target gene to be excised or removed. The guide RNA bind to the
corresponding target nucleic acid sequences and the Cas9 protein
cuts the double stranded nucleic acid at two or more cut sites
flanking the one or more nucleic acid portions or sections or
sequences thereby excising or removing the one or more nucleic acid
portions or sections or sequences. The gene having two or more cut
sites recombines to form an altered gene with the one or more
nucleic acid portions or sections or sequences being absent. The
gene is referred to herein as an altered target gene, which may
also be referred to as a modified target gene or truncated target
gene. The altered target gene is then expressed to produce an
altered biologically functional polypeptide, which may retain full
or partial biological activity, or may exhibit loss of function or
dominant inhibitory activity.
[0007] According to one aspect, the method is carried out within a
cell, i.e. in vivo. The cell can be a eukaryotic cell. According to
one aspect, the nucleic acid portion is a target excision sequence
and the two or more guide RNAs are complementary to flanking
sequences on either side of the target excision sequence. According
to one aspect, the target excision sequence includes one or more
exons in the target gene encoding a biologically functional
polypeptide. According to one aspect, the two or more guide RNAs
bind to complementary target genomic DNA sequences and the Cas9
protein cleaves the two or more target genomic DNA sequences
thereby removing the one or more exons from the target gene to
produce an altered target gene. According to one aspect, the
altered target gene recombines. According to one aspect, the
altered target gene recombines at a reading frame in the target
gene. The cell expresses the altered target gene which has
recombined to produce a biologically functional polypeptide which
is truncated compared to the full length polypeptide. According to
one aspect, the cell expresses the altered and recombined target
gene to produce an altered biologically functional polypeptide.
According to one aspect, the altered biologically functional
polypeptide is truncated relative to the full length biologically
functional polypeptide.
[0008] According to methods described herein, a complex is formed
including a guide RNA, a DNA binding protein, such as a Cas9
protein, and a double stranded DNA target sequence. According to
certain aspects, DNA binding proteins within the scope of the
present disclosure include a protein that forms a complex with the
guide RNA and with the guide RNA and or the complex binding to a
double stranded DNA sequence. This aspect of the present disclosure
may be referred to as co-localization of the RNA and DNA binding
protein to or with the double stranded DNA. In this manner, a DNA
binding protein-guide RNA complex may be used to cut DNA at a
specific target DNA sequence.
[0009] According to certain aspects, the term "guide RNA" in the
context of a CRISPR Cas9 system is known to those of skill in the
art and includes a portion, such as a 20 nucleotide spacer portion,
that is complementary to a target nucleic acid protospacer. Methods
of designing guide RNA are well known to those of skill in the art.
Methods described herein include contacting the target nucleic acid
sequence with a plurality of guide RNA sequences, each having a
portion complementary to the target nucleic acid sequence.
According to one aspect, the target nucleic acid is a double
stranded nucleic acid.
[0010] According to one aspect, the target nucleic acid is double
stranded genomic DNA. According to one aspect, the target nucleic
acid is chromosomal DNA. According to one aspect, the target
nucleic acid is RNA.
[0011] According to one aspect, the Cas9 protein is wild type Cas9,
a Cas9 nickase or a nuclease null Cas9, as known to those of skill
in the art. According to one aspect, the nuclease null Cas9
excludes one or more nucleases. Methods of isolating wild type Cas9
are known to those of skill in the art. Methods of making a Cas9
nickase are known to those of skill in the art. Methods of making a
nuclease null Cas9 are known to those of skill in the art.
[0012] According to one aspect, the cell is a eukaryotic cell.
According to one aspect, the cell is a yeast cell, a plant cell, a
vertebrate cell, or an animal cell, such as an adult animal cell.
According to one aspect, the cell is a mammalian cell. According to
one aspect, the cell is a human cell.
[0013] According to one aspect, the guide RNA is between about 10
to about 500 nucleotides. According to one aspect, the guide RNA is
between about 20 to about 100 nucleotides. According to one aspect,
a scaffold for the guide RNA is between about 80 to about 95
nucleotides and is fused to a variable spacer sequence between
about 16 to about 40 nucleotides. According to one aspect, the
guide RNA is a tracrRNA-crRNA fusion.
[0014] According to one aspect, a method of producing an altered
gene product in a eukaryotic cell includes the steps of providing
to the cell two or more guide RNAs and a Cas9 protein, wherein the
two or more guide RNAs are complementary to two or more target
genomic DNA sequences flanking a target excision sequence including
one or more exons in a target gene encoding a biologically
functional polypeptide, wherein the two or more guide RNAs bind to
the two or more complementary target genomic DNA sequences and the
Cas9 protein cleaves the two or more target genomic DNA sequences
thereby removing the one or more exons from the target gene to
produce an altered target gene, and wherein the altered target gene
recombines in the target gene, and wherein the eukaryotic cell
expresses the altered target gene to produce an altered
biologically functional polypeptide.
[0015] According to one aspect, the altered target gene recombines
at a reading frame in the target gene. According to one aspect, the
altered target gene recombines at a shifted reading frame in the
target gene, such as a frameshift downstream of the excision.
[0016] According to one aspect, an altered biologically functional
polypeptide lacks a polypeptide sequence corresponding to the one
or more exons removed from the target gene.
[0017] According to one aspect, the two or more guide RNAs and the
Cas9 protein are foreign to the eukaryotic cell. According to one
aspect, the two or more guide RNAs and the Cas9 protein are foreign
to each other. According to one aspect, the two or more guide RNAs
and the Cas9 protein are non-naturally occurring.
[0018] According to one aspect, the two or more guide RNAs and the
Cas9 protein are provided to a cell using methods known to those of
skill in the art. According to one aspect, the two or more guide
RNAs are provided to the cell by electroporation of the two or more
guide RNAs into the cell. According to one aspect, the Cas9 protein
is provided to the cell by electroporation of the Cas9 protein into
the cell. According to one aspect, the Cas9 protein is provided to
the cell by liposomal encapsulation, such as by lipofection.
According to one aspect, the Cas9 protein is provided to the cell
by viral delivery, such as by lentivirus, adenovirus,
adeno-associated virus, retrovirus, herpes simplex virus, or sendai
virus.
[0019] According to one aspect, the two or more guide RNAs are
provided to the cell by introducing into the cell a first foreign
nucleic acid sequence encoding the two or more guide RNAs, wherein
the first foreign nucleic acid is expressed. According to one
aspect, the two or more guide RNAs are provided to the cell by
introducing into the cell a first foreign nucleic acid sequence
encoding the two or more guide RNAs present in a vector wherein the
first foreign nucleic acid is expressed.
[0020] According to one aspect, the Cas 9 protein is provided to
the cell by introducing into the cell a second foreign nucleic acid
sequence encoding the Cas 9 protein wherein the second foreign
nucleic acid sequence is expressed.
[0021] According to one aspect, the Cas 9 protein is provided to
the cell by introducing into the cell a second foreign nucleic acid
sequence encoding the Cas 9 protein present in a vector wherein the
second foreign nucleic acid sequence is expressed.
[0022] According to one aspect, the eukaryotic cell is a yeast
cell, a plant cell, a vertebrate cell, a mammalian cell (e.g., a
human cell), or a non-mammalian cell (e.g., a fish cell, bird
cell). According to one aspect, the eukaryotic cell is within a
mammal According to one aspect, the eukaryotic cell is a skeletal
muscle cell or a cardiac muscle cell.
[0023] According to one aspect, the target excision sequence is
greater than 45 kb. According to one aspect, the target gene
encodes dystrophin protein. According to one aspect, the target
gene encodes dystrophin protein, and the one or more exons is exon
23. According to one aspect, the target gene encodes dystrophin
protein, and the one or more exons is exon 52 and exon 53.
According to one aspect, the one or more exons are in the exon
45-55 region.
[0024] According to one aspect, the guide RNA includes between
about 10 to about 250 nucleotides. According to one aspect, the
guide RNA includes between about 20 to about 100 nucleotides.
According to one aspect, the guide RNA includes a guide sequence
fused to a trans-activating cr (tracr) sequence.
[0025] According to one aspect, the ratio range of plasmid encoding
the Cas9 protein to the plasmid encoding the guide RNA is between
1:10 and 10:1. An exemplary ratio range of plasmid encoding the
Cas9 protein to the plasmid encoding the guide RNA is between 1:5
and 2:1. One of skill in the art would readily understand based on
the present disclosure that any particular ratio depends on at
least cell type. According to one aspect, the plasmid encoding the
guide RNA is modified to increase the expression of the RNA by
removing a potential premature transcription termination site.
[0026] According to one aspect, the one or more exons include a
mutation. According to one aspect, the Cas9 protein is provided to
the cell by electroporation of the Cas9 mRNA into the cell, by
liposomal encapsulation, or by viral delivery. According to one
aspect, the guide RNA and the Cas9 protein co-localize to the
target genomic DNA sequence to form a complex.
[0027] According to one aspect, the guide RNA and the Cas9 protein
are combined and then contacted with the target gene. According to
one aspect, the guide RNA and the Cas9 protein are combined and
then contacted with the target gene within a cell.
[0028] According to one aspect, a plurality of guide RNAs with each
having a portion complementary to a target genomic DNA sequence are
provided to the cell.
[0029] Further features and advantages of certain embodiments of
the present invention will become more fully apparent in the
following description of embodiments and drawings thereof, and from
the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee. The foregoing and
other features and advantages of the present invention will be more
fully understood from the following detailed description of
illustrative embodiments taken in conjunction with the accompanying
drawings in which:
[0031] FIG. 1 is a schematic diagram of the Dystrophin pre-mRNA of
a Duchenne Patient with exon 50 deletion. Skipping exon 51 at the
mRNA level leads to restoration of reading frame and production of
a truncated but partially functional Dystrophin protein. Exon
skipping can be caused by antisense oligonucleotides at the RNA
splicing stage, which needs repeated administration of high doses
of the oligonucleotide, or by a one-time deletion of exon 51 from
the genomic DNA using a CRISPR Cas9 system.
[0032] FIG. 2A is a schematic diagram of the mdx mouse genomic DNA
at the mutated exon 23 locus before (top) and after (bottom)
cutting the DNA with two gRNAs targeting 5' and 3' of exon 23.
[0033] FIG. 2B is an image illustrating PCR products amplified by a
primer pair spanning exon 23 of mouse Dystrophin gene from genomic
DNA of C2C12 myoblasts transfected with Cas9 only or Cas9 and two
gRNAs targeting 5' and 3' of exon 23. Cutting both sides of exon 23
with two gRNAs leads to excision of the exon from the DNA and
amplification of a smaller PCR product corresponding to the deleted
locus.
[0034] FIG. 2C is a graph illustrating results from deep sequencing
of exon 23 genomic DNA amplicons from C2C12 cells transfected with
Cas9 and two gRNAs targeting 5' and 3' of exon 23.
[0035] FIG. 3A is a schematic diagram of the mdx mouse mRNA at the
mutated exon 23 locus before (top) and after (bottom) cutting the
DNA with two gRNAs targeting 5' and 3' of exon 23.
[0036] Removal of exon 23 from the mRNA restores the reading frame
and leads to expression of a truncated but partially functional
Dystrophin protein in dystrophic mouse muscle (Lu et. al,
Functional amounts of dystrophin produced by skipping the mutated
exon in the mdx dystrophic mouse, see Nature Medicine 9, 1009-1014
(2003).
[0037] FIG. 3B is an image illustrating RT-PCR products amplified
by a primer pair spanning exon 23 of mouse Dystrophin mRNA from
cDNA of C2C12 myoblasts transfected with Cas9 only or Cas9 and two
gRNAs targeting 5' and 3' of exon 23. Cutting both sides of
Dystrophin exon 23 with two gRNAs leads to excision of the exon
from the mRNA and amplification of a smaller PCR product
corresponding to the transcript lacking exon 23.
[0038] FIG. 4A is a diagram illustrating brightfield and
epiflourescent images of a multinucleated single fiber.
[0039] FIG. 4B is a graph illustrating results from deep sequencing
of exon 23 genomic DNA amplicons from a mdx single fiber
electroporated with Cas9 and two gRNAs targeting 5' and 3' of exon
23. The red signal shows the percentage of the deletion in the
amplicons and the dashed lines indicate the gRNA targeting
sites.
[0040] FIG. 5A is a schematic diagram of the mdx4cv mouse genomic
DNA at the mutated exon 53 locus before (top) and after (bottom)
cutting the DNA with two gRNAs targeting 5' of exon 52 and 3' of
exon 53. Deletion of exons 52 and 53 from the DNA and skipping
these exons in the mRNA restores the reading frames and leads to
expression of a truncated but functional protein in mdx4cv mouse
muscle (see Mitrpant et. al, By-passing the nonsense mutation in
the 4 CV mouse model of muscular dystrophy by induced exon
skipping, J Gene Med. 2009 Jan;11(1):46-56).
[0041] FIG. 5B is an image illustrating PCR product amplified by a
primer pair spanning exons 52 and 53 of Dystrophin gene from
genomic DNA of C2C12 myoblasts transfected with Cas9 only or Cas9
and two gRNAs targeting 5' of exon 52 and 3' of exon 53.
Amplification only occurs when 45 kb of genomic DNA containing
exons 52 and 53 between the two gRNA target sites is excised.
[0042] FIG. 6A is a schematic diagram of an in vivo CRISPR activity
reporter system in mouse muscle, wherein the mouse strain was
obtained from the Jackson Library (stock number 007905, world wide
website jaxmice.jax.org/strain/007905.html). The Ai9 reporter
construct integrated into the mouse Rosa26 locus contains a 3xSTOP
cassette between the promoter and the tdtomato coding sequence,
which upon excision of the 3xSTOP cassette by cutting with two
gRNAs at 5' and 3' sites of the cassette (or by Cre-mediated
excision), expresses tdTomato under the control of a CAG
promoter.
[0043] FIG. 6B illustrates pictures of Ai9 muscle sections
electroporated with GFP and Cas9 only (top), or GFP, Cas9 and two
gRNAs targeting 5' and 3' of the STOP cassette (bottom).
[0044] FIG. 7A is a schematic diagram of a multiplex-able
CRISPR-dependent fluorescent reporter, derived from Brainbowl.0 `L`
(see Livit et al., Nature, 450(7166):56-62 (2007)). Excision of the
dTomato cassette by guide RNAs CB12 or CB10 with guide RNA CB13
leads to mCerulean expression, while excision of tdTomato and
mCerulean cassette by guide RNAs CB12 or CB10 with guide RNA CB15,
or by guide RNA CB8 only, leads to EYFP expression.
[0045] FIG. 7B illustrates results from transfection of CRISPR in
HEK293 cells expressing the Brainbowl1.0 `L` construct results in
CRISPR-mediated fluorescent activation.
[0046] FIG. 7C illustrates results from linkage of guide RNA CB8 to
guide RNAs targeting endogenous genes of interest (GOI) which
selectively enriches for GOI mutants in the EYFP-positive
subpopulations. C2C12 were co-lipofected with plasmids encoding
Brainbowl1.0 `L`, Cas9, and a single plasmid encoding guide RNA CB8
together with 2 guide RNAs targeting GOI. Cells were sorted based
on dTomato and EYFP fluorescence intensities. Subpopulations were
then deep sequenced for the GOI loci. EYFP-positive subpopulations
(P5-P9) harbor GOI mutations>10-fold more than non-fluorescent
subpopulation (P4).
[0047] FIG. 8 is a diagram illustrating muscle hypertrophy induced
by a non-functional myostatin-activin receptor pathway.
[0048] FIG. 9 is a schematic diagram of guide RNAs targeting the
mouse activin receptor JIB (mActRIIB), activin receptor IIA
(mActRIIA), and myostatin (mMstn) genes.
[0049] FIG. 10A illustrates results of mutation frequencies in
unselected C2C12 cells lipofected with either single guide RNAs or
paired guide RNAs, using four forms of Cas9-expressing constructs
(pSMVP: plasmid with an SV40enhancer-CMV-chimeric intron promoter;
MC-SMVP: minicircle with the same promoter; with or without
P2A-turboGFP for co-translational expression of yurboGFP).
[0050] FIG. 10B illustrates deletion sizes generated by single or
paired guide RNAs.
[0051] FIG. 10C is a diagram illustrating results from examining
breakpoint junctions. The results reveal that genomic loci with GGG
PAM exhibit cut-site wobble, where the CRISPR-induced double-strand
break is 3 bp or 4 bp upstream of the PAM.
[0052] FIG. 11A illustrates results from CRISPR-mediated gene
editing in adult muscle fibers. 2-10% of genomes in multi-nucleated
muscle fibers were mutated after electroporation of CRISPR into
adult mouse TA muscle. Using paired guide RNAs allows precise
deletion of intervening genomic sequences.
[0053] FIG. 11B illustrates results from deep sequencing alignments
of a single fiber for each gene locus.
[0054] FIG. 12 is a graph illustrating inter-fiber mutational
variability within the same animal. Each black dot depicts the
mutational frequency of a single fiber. Bars denote the mean
frequency of mutational subtypes for each animal. Guide RNAs used
were mActRIIB1 and mActRIIB3. n=1 mouse for each condition
tested.
[0055] FIG. 13 illustrates immune response towards Cas9 and GFP, as
examined with DAPI nuclear stain, CD45 antibody stain, and immune
cell FACS profiling. T-cell infiltration into the
transgene-expressing tissue can be alleviated with FK506
immuno-suppression.
[0056] FIG. 14A is a schematic of the Ai9 allele used for tdTomato
fluorescent reporting of gene editing in CRISPR transduced cells.
Precise excision of the 3XSTOP cassette induced by paired CRISPR
targeting enables tdTomato expression from the ubiquitous CAGGs
promoter.
[0057] FIG. 14B is a schematic of CRISPR-mediated excision of DMD
exon 23, which in mdx mice bears a nonsense mutation (E23*) that
results in destabilization of DMD mRNA and absence of Dystrophin
expression in muscle. Targeted excision of E23* creates a hybrid
intron (122/23), and subsequent transcription and splicing
generates an exon "skipped" mRNA in which exon 22 (E22) is fused
directly to exon 24 (E24), restoring Dystrophin reading frame and
producing a truncated but still functional Dystrophin protein.
[0058] FIG. 14C is a schematic of the Ai9 targeting gRNA
constructs.
[0059] FIG. 14D is a schematic of the coupled Ai9-DMD23 gRNA
constructs.
[0060] FIG. 14E depicts detection of permanent exon skipping by
genomic PCR using primers spanning DMD exon 23. DNA was isolated
from myotubes differentiated from sorted tdTomato+cells derived
from satellite cells previously transfected with Sp hCas9 and Ai9
gRNAs (left lanes) or coupled Ai9-DMD23 gRNAs (right lanes).
Unedited genomic product, 1572bp; gene-edited product, 1189bp.
Sanger sequencing trace confirms precise deletion of exon 23 from
the genome. (SEQ ID NO:1)
[0061] FIG. 14F illustrates FACS plots from mdx;Ai9 satellite cells
transfected with plasmids encoding for Sp hCas9 and gRNAs targeting
Ai9 locus (middle panel), Sp hCas9 and coupled gRNAs targeting Ai9
and DMD23 loci (right panel) or no plasmids (left panel).
[0062] FIG. 14G depicts detection of exon 23-skipped mRNA using
RT-PCR. M, molecular weight ladder, Unedited RT-PCR product 738bp;
exon skipped product 525bp. Analysis of four representative
cultures is shown for each set of gRNAs. Sanger sequencing traces
confirms precise deletion of exon 23 from the mRNA. (SEQ ID
NO:2-3)
[0063] FIG. 14H graphically depicts the quantification of percent
exon skipping in targeted satellite cell-derived myotubes by
Taqman-based qPCR.
[0064] FIG. 141 depicts the Western blot for Dystrophin and GAPDH
(loading control) in lysates of myotubes derived from gene-edited
satellite cells. Analysis of four representative cultures is shown
for each set of gRNAs, compared to lysates from cultures containing
100% unedited mdx myotubes or 80% mdx +20% wild-type myotubes.
Signal intensity is quantified by densitometry at the bottom. A.U.:
Arbitrary Unit normalized to GAPDH.
[0065] FIG. 14J illustrates dystrophin immunofluorescence in mdx
muscles transplanted with satellite cells targeted with Sp hCas9
+Ai9 gRNAs (top) or Sp hCas9 +Ai9-DMD23 coupled gRNAs (bottom).
Green, dystrophin; red, tdTomato; blue: DAPI. Scale bar: 100
um.
[0066] FIG. 15A is representative immunofluorescence analysis of
muscles from adult mdx;Ai9 mice injected intramuscularly with
vehicle (left) or dual AAVs encoding for SaCas9 and Ai9 gRNAs
(right). The dual AAV-CRISPR system efficiently targets the Ai9
locus in adult dystrophic muscle, resulting in tdTomato expression
(red). Individual muscle fibers are marked by laminin (green), and
nuclei by DAPI (blue). Scale bar: 500 um.
[0067] FIG. 15B depicts the detection of permanent exon skipping by
genomic PCR using primers spanning DMD exon 23 (left). DNA was
isolated from tibialis anterior muscle harvested from mdx;Ai9 mice
injected intramuscularly with AAV-CRISPR targeting the Ai9 or DMD23
locus. Exon 23 excision in DNA is detected only in muscles
receiving AAV-DMD23 gRNAs. Sanger sequencing trace confirms precise
deletion of exon 23 from the genome (right). (SEQ ID NO:4)
[0068] FIG. 15C depicts the detection of exon skipping in the mRNA
by RT-PCR (left). Sequencing result from unedited and exon skipped
mRNA, confirms skipping of exon 23 in the mRNA (right). (SEQ ID
NO:5-6)
[0069] FIG. 15D graphically depicts the quantification of exon
skipping in injected muscles by Taqman-based real time PCR. Data
plotted for individual mice (n=5 receiving DMD23 gRNAs (blue) and
n=3 receiving Ai9 gRNAs (red). Overlay indicates mean .+-.SD for Sa
Cas9+DMD gRNA group.
[0070] FIG. 15E illustrates the Western blot for Dystrophin and
GAPDH (loading control) in muscles injected with AAV-CRISPR using
Ai9 (left) or DMD (right) gRNAs, quantified by densitometry at the
bottom. A.U.: Arbitrary Unit normalized to GAPDH.
[0071] FIG. 15F displays images of immunofluorescence staining for
Dystrophin (green) in mdx;Ai9 muscles injected with CRISPR AAVs
targeting Ai9 (Left) or DMD23 (Right).
[0072] FIG. 15G graphically depicts the analysis of muscle specific
force, and FIG. 15H graphically depicts the decrease in force after
eccentric damage. n=6 for wild type mice injected with vehicle, n=9
for mdx;Ai9 mice injected with AAV-DMD CRISPR in the right leg and
vehicle in the left leg and n=8 for mdx;Ai9 mice injected with
AAV-Ai9 CRISPR in the right leg and vehicle in the left leg.
P-values calculated by paired student t-test for comparing
contralateral legs and unpaired student t-test for comparing
muscles from different mice.
[0073] FIG. 16A illustrates representative immunofluorescence
analysis of different muscles from 3 weeks old mdx;Ai9 mice
injected systemically with vehicle or dual AAVs encoding for
[0074] SaCas9 and Ai9 gRNAs on P3. The dual AAV-CRISPR system
efficiently targets the Ai9 locus in neonatal dystrophic muscle
after systemic injection, resulting in tdTomato expression (red).
Individual muscle fibers are marked by laminin (green), and nuclei
by DAPI (blue). Scale bar: 200 um.
[0075] FIG. 16B depicts the detection of exon skipping by RT-PCR
using primers spanning DMD exon 23. RNA was isolated from the
indicated tissues of mdx;Ai9 mice injected intraperitoneally with
AAV-SaCas9+AAV-Ai9 (left) or AAV-DMD23 (right) gRNAs. Exon
23-skipped mRNA is detected only in muscles receiving AAV-DMD23
gRNAs.
[0076] FIG. 16C graphically depicts the quantification of exon
skipping in injected muscles by Taqman assay.
[0077] FIG. 16D depicts the detection of Dystrophin and GAPDH
(loading control) by Western blot in the indicated muscles of
mdx;Ai9 mice receiving systemic AAV-CRISPR. Right lanes correspond
to muscles from 7 different mice injected systemically with AAV-DMD
CRISPR. Signal intensity is quantified by densitometry at the
bottom. A.U.: Arbitrary Unit normalized to GAPDH.
[0078] FIG. 16E displays the representative images of
immunofluorescence staining for Dystrophin (green) in mdx;Ai9
muscles injected with CRISPR AAVs targeting Ai9 or DMD23. Scale
bar: 200 um.
[0079] FIG. 17A depicts the experimental design schematic.
Pax7-ZsGreen;Mdx;Ai9 mice were injected intramuscularly or
intraperitoneally with Cre or CRISPR AAV targeting Ai9 or
DMD23.
[0080] Two weeks later, Pax7+satellite cells were isolated by FACS,
expanded in culture, differentiated to myotubes and analyzed for
gene editing.
[0081] FIG. 17B is representative of FACS plots of tdTomato
expression among Zsgreen+satellite cells isolated from mice
injected intramuscularly with vehicle (left), AAV9-Cre (middle) or
AAV9-Ai9 CRISPR (right). FACS plots were previously gated for
Pax7-ZsGreen positive, Scal, CD45, Ter119 and Mac1 negative, live
mononuclear cells, and numbers indicate percent tdTomato+cells for
each plot.
[0082] FIG. 17C graphically depicts the quantification of
tdTomato+cells among ZsGreen+satellite cells isolated from mice
injected intramuscularly with vehicle, AAV9-Cre or AAV9-Ai9
CRISPR.
[0083] FIG. 17D depicts the representative immunofluorescence
images of myotubes differentiated from FACSorted Pax7-ZsGreen+cells
from vehicle (top), AAV9-Cre (middle) and AAV9-Ai9 CRISPR (bottom)
injected muscles. Myosin heavy chain (MHC, green); tdTomato (red).
Scale bar: 200 um.
[0084] FIG. 17E is representative of FACS plots of tdTomato
expression among ZsGreen+satellite cells isolated from mice
injected systemically with vehicle (left), AAV9-Cre (middle) or
AAV9-Ai9 CRISPR (right). FACS plots were previously gated for
Pax7-ZsGreen positive, Scal, CD45, Ter119 and Macl negative, live
mononuclear cells, and numbers indicate percent tdTomato+cells for
each plot.
[0085] FIG. 17F graphically depicts the quantification of
tdTomato+cells among ZsGreen+satellite cells isolated from mice
injected systemically with vehicle, AAV9-Cre or AAV9-Ai9
CRISPR.
[0086] FIG. 17G is representative immunofluorescence images of
myotubes differentiated from FACSorted Pax7-ZsGreen+cells from mice
injected systemically with vehicle (top), AAV9-Cre (middle) and
AAV9-Ai9 CRISPR (bottom). Myosin heavy chain (MHC, green); tdTomato
(red). Scale bar: 200 um.
[0087] FIG. 17H illustrates RT-PCR with exon23 spanning primers
indicates expression of exon 23-skipped DMD mRNA in myotubes
differentiated in vitro from satellite cells isolated from adult
mice receiving AAV-DMD CRISPR (right lanes), but not those from
muscles injected with AAV-Ai9 CRISPR (left lanes) after
intramuscular delivery of AAVs. Sequencing result from unedited and
exon skipped mRNA, confirms skipping of exon 23 in the mRNA. (SEQ
ID NO:7-8)
[0088] FIG. 17I graphically depicts quantification of exon skipping
in myotubes derived from satellite cells isolated from
intramuscularly injected muscles by Taqman assay.
[0089] FIG. 17J illustrates RT-PCR with exon23 spanning primers
indicates expression of exon 23-skipped DMD mRNA in myotubes
differentiated in vitro from satellite cells isolated from 3 weeks
old mice receiving AAV-DMD CRISPR (right lanes), but not those from
muscles injected with AAV-Ai9 CRISPR (left lanes) after systemic
delivery of AAVs on P3. Sequencing result from unedited and exon
skipped mRNA, confirms skipping of exon 23 in the mRNA. (SEQ ID
NO:9-10)
[0090] FIG. 17K graphically depicts quantification of exon skipping
in myotubes derived from satellite cells isolated from systemically
injected mice by Taqman assay.
[0091] FIG. 18A schematically depicts the sequence of the original
(left) and optimized (left) Sa gRNA scaffold. (SEQ ID NO:11-12)
[0092] FIG. 18B illustrates the representative FACS plots from Ai9
mouse tail tip fibroblasts transfected with no plasmids (left
panel), plasmids encoding SaCas9 and Ai9 gRNAs with the original
scaffold (middle panel) or the optimized scaffold (right
panel).
[0093] FIG. 18C graphically depicts quantification of percent
tdTomato+targeted cells in transfected Ai9 tail tip
fibroblasts.
[0094] FIG. 18D is an image illustrating the result of the
screening for different pairs of Sa DMD23 gRNAs by genomic PCR
using primers spanning exon 23. Intensity of the cut band was
quantified by densitometry. A.U.: Arbitrary Unit normalized to the
wild-type band.
[0095] FIG. 19A depicts the schematics of AAV-SaCas9 (top)
AAV-DMD23 gRNAs (middle) and AAV-Ai9 gRNAs (bottom) constructs used
for dual CRISPR AAV experiments.
[0096] FIG. 19B depicts the schematic of 173CMV_SaCas9_DMD23 gRNAs
and EFS_SaCas9_DMD23 gRNAs single AAV constructs.
[0097] FIG. 19C is an image of the detection of exon skipping by
RT-PCR using primers spanning DMD exon 23. RNA was isolated from
mdx;Ai9 in vitro differentiated myotubes, transduced with AAV DJ
encoding the indicated constructs. Exon 23-skipped mRNA is detected
only in myotubes receiving AAV-DMD CRISPR. Sequencing result from
unedited and exon skipped mRNA, confirms skipping of exon 23 in the
mRNA. (SEQ ID NO:13)
[0098] FIG. 19D graphically depicts Taqman-based quantification of
exon skipping in myotubes transduced with AAV DJ encoding dual or
single DMD23 CRISPR constructs.
[0099] FIG. 19E graphically depicts Taqman-based quantification of
exon skipping in muscles injected locally with dual AAVs encoding
SaCas9+DMD23 gRNAs or a single AAV encoding EFS_SaCas9_DMD23 gRNAs.
*: P<0.05.
[0100] FIG. 19F graphically depicts Taqman-based quantification of
exon skipping in different muscles of mice systemically injected
with dual AAVs encoding SaCas9+DMD23 gRNAs or a single AAV encoding
EFS_SaCas9_DMD23 gRNAs. *: P<0.05.
[0101] FIG. 20A is an image of the detection of Dystrophin and
GAPDH (loading control) by Western blot in the abdominal (top),
heart (middle) and diaphragm (bottom) muscles of mdx;Ai9 mice
receiving systemic AAV-CRISPR. Right lanes correspond to muscles
from 7 different mice injected systemically with AAV-DMD CRISPR.
Signal intensity is quantified by densitometry at the bottom. A.U.:
Arbitrary Unit normalized to GAPDH.
[0102] FIG. 20B includes Brightfield (left) and chemiluminescent
(right) images of uncropped Dystrophin and GAPDH Western blots from
gastrocnemius (top) and TA (bottom) muscles of mice systemically
injected with AAV CRISPR.
DETAILED DESCRIPTION
[0103] Embodiments of the present disclosure are based on the use
of DNA binding proteins and guide RNA to co-localize at or complex
at a target nucleic acid and then cut or cleave the target nucleic
acid in a manner to remove a nucleic acid sequence, which may be
referred to herein as an excision sequence. Such DNA binding
proteins include RNA-guided DNA binding proteins readily known to
those of skill in the art to bind to DNA for various purposes. Such
DNA binding proteins may be naturally occurring. DNA binding
proteins included within the scope of the present disclosure
include those which may be guided by RNA, referred to herein as
guide RNA. According to this aspect, the guide RNA and the RNA
guided DNA binding protein form a co-localization complex at the
DNA. According to certain aspects, the DNA binding protein may be a
nuclease-null DNA binding protein which otherwise may have one or
more nucleases attached thereto. According to this aspect, the
nuclease-null DNA binding protein may result from the alteration or
modification of a DNA binding protein having nuclease activity.
Such DNA binding proteins having nuclease activity are known to
those of skill in the art, and include naturally occurring DNA
binding proteins having nuclease activity, such as Cas9 proteins
present, for example, in Type II CRISPR systems. Such Cas9 proteins
and Type II CRISPR systems are well documented in the art. See
Makarova et al., Nature Reviews, Microbiology, Vol. 9, June 2011,
pp. 467-477 including all supplementary information hereby
incorporated by reference in its entirety.
[0104] In general, a CRISPR locus is characterized by an array of
repetitive sequences (direct repeats) interspaced by short
stretches of non-repetitive sequences (spacers). The non-coding
CRISPR array is transcribed and cleaved within direct repeats into
short crRNAs containing individual spacer sequences, which direct
Cas nucleases to the target site (protospacer). The Type
[0105] II CRISPR system carries out targeted DNA double-strand
break in four sequential steps. First, two non-coding RNA, the
pre-crRNA array and tracrRNA, are transcribed from the CRISPR
locus. Second, tracrRNA hybridizes to the repeat regions of the
pre-crRNA and mediates the processing of pre-crRNA into mature
crRNAs containing individual spacer sequences. Third, the mature
crRNA:tracrRNA complex directs Cas9 to the target DNA via
Watson-Crick base-pairing between the spacer on the crRNA and the
protospacer on the target DNA next to the protospacer adjacent
motif (PAM), an additional requirement for target recognition.
Finally, Cas9 mediates cleavage of target DNA to create a
double-stranded break within the protospacer.
[0106] Exemplary DNA binding proteins having nuclease activity
function to nick or cut double stranded DNA. Such nuclease activity
may result from the DNA binding protein having one or more
polypeptide sequences exhibiting nuclease activity. Such exemplary
DNA binding proteins may have two separate nuclease domains with
each domain responsible for cutting or nicking a particular strand
of the double stranded DNA. Exemplary polypeptide sequences having
nuclease activity known to those of skill in the art include the
McrA-HNH nuclease related domain and the RuvC-like nuclease domain.
Accordingly, exemplary DNA binding proteins are those that in
nature contain one or more of the McrA-HNH nuclease related domain
and the RuvC-like nuclease domain. According to certain aspects,
the DNA binding protein is altered or otherwise modified to
inactivate the nuclease activity. Such alteration or modification
includes altering one or more amino acids to inactivate the
nuclease activity or the nuclease domain. Such modification
includes removing the polypeptide sequence or polypeptide sequences
exhibiting nuclease activity, i.e. the nuclease domain, such that
the polypeptide sequence or polypeptide sequences exhibiting
nuclease activity, i.e. nuclease domain, are absent from the DNA
binding protein. Other modifications to inactivate nuclease
activity will be readily apparent to one of skill in the art based
on the present disclosure. Accordingly, a nuclease-null DNA binding
protein includes polypeptide sequences modified to inactivate
nuclease activity or removal of a polypeptide sequence or sequences
to inactivate nuclease activity. The nuclease-null DNA binding
protein retains the ability to bind to DNA even though the nuclease
activity has been inactivated. When a nuclease null DNA binding
protein is used, the nuclease null DNA binding protein has been
modified to include one or more DNA nucleases, which may be more
specific, such as Fok1, in cutting DNA than the nucleases
associated with wild type Cas9. Accordingly, the DNA binding
protein includes the polypeptide sequence or sequences required for
DNA binding but may lack the one or more or all of the nuclease
sequences exhibiting nuclease activity. Accordingly, the DNA
binding protein includes the polypeptide sequence or sequences
required for DNA binding but may have one or more or all of the
nuclease sequences exhibiting nuclease activity inactivated.
[0107] According to one aspect, a DNA binding protein having two or
more nuclease domains may be modified or altered to inactivate all
but one of the nuclease domains. Such a modified or altered DNA
binding protein is referred to as a DNA binding protein nickase, to
the extent that the DNA binding protein cuts or nicks only one
strand of double stranded DNA. When guided by RNA to DNA, the DNA
binding protein nickase is referred to as an RNA guided DNA binding
protein nickase. Accordingly, useful Cas9 proteins may be a wild
type Cas9, a Cas9 nickase or a nuclease null Cas9 and homologs and
orthologs thereof. See Jinek et al., Science 337, 816-821 (2012)
hereby incorporated by reference in its entirety.
[0108] In S. pyogenes, Cas9 generates a blunt-ended double-stranded
break 3bp or 4bp upstream of the protospacer-adjacent motif (PAM)
via a process mediated by two catalytic domains in the protein: an
HNH domain that cleaves the complementary strand of the DNA and a
RuvC-like domain that cleaves the non-complementary strand. See
Jinek et al., Science 337, 816-821 (2012) hereby incorporated by
reference in its entirety. Cas9 proteins are known to exist in many
Type II CRISPR systems including the following as identified in the
supplementary information to Makarova et al., Nature Reviews,
Microbiology, Vol. 9, June 2011, pp. 467-477: Methanococcus
Maripaludis C7; Corynebacterium diphtheriae; Corynebacterium
efficiens YS-314; Corynebacterium glutamicum ATCC 13032 Kitasato;
Corynebacterium glutamicum ATCC 13032 Bielefeld; Corynebacterium
glutamicum R; Corynebacterium kroppenstedtii DSM 44385;
Mycobacterium abscessus ATCC 19977; Nocardia farcinica IFM10152;
Rhodococcus erythropolis PR4; Rhodococcus jostii RHA1; Rhodococcus
opacus B4 uid36573; Acidothermus cellulolyticus 11B; Arthrobacter
chlorophenolicus A6; Kribbella flavida DSM 17836 uid43465;
Thermomonospora curvata DSM 43183; Bifidobacterium dentium Bd1;
Bifidobacterium longum DJO10A; Slackia heliotrinireducens DSM
20476; Persephonella marina EX H1; Bacteroides fragilis NCTC 9434;
Capnocytophaga ochracea DSM 7271; Flavobacterium psychrophilum
JIP02 86; Akkermansia muciniphila ATCC BAA 835; Roseiflexus
castenholzii DSM 13941; Roseiflexus RS1; Synechocystis PCC6803;
Elusimicrobium minutum Pei191; uncultured Termite group 1 bacterium
phylotype Rs D17; Fibrobacter succinogenes S85; Bacillus cereus
ATCC 10987; Listeria innocua;Lactobacillus casei; Lactobacillus
rhamnosus GG; Lactobacillus salivarius UCC118; Streptococcus
agalactiae A909; Streptococcus agalactiae NEM316; Streptococcus
agalactiae 2603; Streptococcus dysgalactiae equisimilis GGS 124;
Streptococcus equi zooepidemicus MGCS10565; Streptococcus
gallolyticus UCN34 uid46061; Streptococcus gordonii Challis subst
CH1; Streptococcus mutans NN2025 uid46353; Streptococcus mutans;
Streptococcus pyogenes M1 GAS; Streptococcus pyogenes MGAS5005;
Streptococcus pyogenes MGAS2096; Streptococcus pyogenes MGAS9429;
Streptococcus pyogenes MGAS10270; Streptococcus pyogenes MGAS6180;
Streptococcus pyogenes MGAS315; Streptococcus pyogenes SSI-1;
Streptococcus pyogenes MGAS10750; Streptococcus pyogenes NZ131;
Streptococcus thermophiles CNRZ1066; Streptococcus thermophiles
LMD-9; Streptococcus thermophiles LMG 18311; Clostridium botulinum
A3 Loch Maree; Clostridium botulinum B Eklund 17B; Clostridium
botulinum Ba4 657; Clostridium botulinum F Langeland; Clostridium
cellulolyticum H10; Finegoldia magna ATCC 29328; Eubacterium
rectale ATCC 33656; Mycoplasma gallisepticum; Mycoplasma mobile
163K; Mycoplasma penetrans; Mycoplasma synoviae 53; Streptobacillus
moniliformis DSM 12112; Bradyrhizobium BTAi1; Nitrobacter
hamburgensis X14; Rhodopseudomonas palustris BisB18;
Rhodopseudomonas palustris BisB5; Parvibaculum lavamentivorans
DS-1; Dinoroseobacter shibae DFL 12; Gluconacetobacter
diazotrophicus Pal 5 FAPERJ; Gluconacetobacter diazotrophicus Pal 5
JGI; Azospirillum B510 uid46085; Rhodospirillum rubrum ATCC 11170;
Diaphorobacter TPSY uid29975; Verminephrobacter eiseniae EF01-2;
Neisseria meningitides 053442; Neisseria meningitides alphal4;
Neisseria meningitides Z2491; Desulfovibrio salexigens DSM 2638;
Campylobacter jejuni doylei 269 97; Campylobacter jejuni 81116;
Campylobacter jejuni; Campylobacter lari RM2100; Helicobacter
hepaticus; Wolinella succinogenes; Tolumonas auensis DSM 9187;
Pseudoalteromonas atlantica T6c; Shewanella pealeana ATCC 700345;
Legionella pneumophila Paris; Actinobacillus succinogenes 130Z;
Pasteurella multocida; Francisella tularensis novicida U112;
Francisella tularensis holarctica; Francisella tularensis FSC 198;
Francisella tularensis tularensis; Francisella tularensis
WY96-3418; and Treponema denticola ATCC 35405. Accordingly, aspects
of the present disclosure are directed to a Cas9 protein present in
a Type II CRISPR system, which has been rendered nuclease null or
which has been rendered a nickase as described herein.
[0109] The Cas9 protein may be referred by one of skill in the art
in the literature as Csnl. The S. pyogenes Cas9 protein sequence is
shown below. See Deltcheva et al., Nature 471, 602-607 (2011)
hereby incorporated by reference in its entirety. There may be
protein-level modifications that are made to the S. pyogenes Cas9
protein sequence for activity in eukaryotic cells. One example may
be nuclear-localization signals, including 2 or 3 in the N or C
termini of S. pyogenes Cas9.
TABLE-US-00001 (SEQ ID NO: 14) ##STR00001## ##STR00002##
[0110] Target nucleic acids include any nucleic acid sequence to
which a co-localization complex as described herein can be useful
to cut. Target nucleic acids include genes. The target nucleic acid
may be within DNA extracted from a single cell. The target nucleic
acid may be DNA extracted from a single chromosome. The target
nucleic acid may be within DNA within a single cell. For purposes
of the present disclosure, DNA, such as double stranded DNA, can
include the target nucleic acid and a co-localization complex can
bind to or otherwise co-localize with the DNA at or adjacent or
near the target nucleic acid and in a manner to cut the target
nucleic acid.
[0111] Such target nucleic acids can include endogenous (or
naturally occurring) nucleic acids and exogenous (or foreign)
nucleic acids.
[0112] As used herein, the term "chromosome" refers to the support
for the genes carrying heredity in a living cell, including DNA,
protein, RNA and other associated factors. The conventional
international system for identifying and numbering the chromosomes
of the human genome is used herein. The size of an individual
chromosome may vary within a multi-chromosomal genome and from one
genome to another. A chromosome can be obtained from any species. A
chromosome can be obtained from an adult subject, a juvenile
subject, an infant subject, from an unborn subject (e.g., from a
fetus, e.g., via prenatal test such as amniocentesis, chorionic
villus sampling, and the like or directly from the fetus, e.g.,
during a fetal surgery) from a biological sample (e.g., a
biological tissue, fluid or cells (e.g., sputum, blood, blood
cells, tissue or fine needle biopsy samples, urine, cerebrospinal
fluid, peritoneal fluid, and pleural fluid, or cells therefrom) or
from a cell culture sample (e.g., primary cells, immortalized
cells, partially immortalized cells or the like). In certain
exemplary embodiments, one or more chromosomes can be obtained from
one or more genera including, but not limited to, Homo, Drosophila,
Caenorhabiditis, Danio, Cyprinus, Equus, Canis, Ovis, Ocorynchus,
Salmo, Bos, Sus, Gallus, Solanum, Triticum, Oryza, Zea, Hordeum,
Musa, Avena, Populus, Brassica, Saccharum and the like.
[0113] According to certain aspects, a guide RNA corresponding to
the target double stranded nucleic acid sequence of interest, i.e.
the guide RNA may bind to the target double stranded nucleic acid
sequence of interest and also complex with Cas9, is designed using
methods known to those of skill in the art, preincubated with Cas9
and then added to a sample, which may be a single cell or
collection of cells, containing the target DNA. The guide RNA and
Cas9 will then co-localize to and form a complex with the target
DNA. One of skill based on the present disclosure will readily be
able to identify or design guide RNAs and Cas9 proteins which
co-localize to a DNA including a target nucleic acid. The guide
RNAs can be made by direct solid-phase synthesis of RNA (available
from vendors such as IDT) or by in vitro transcription of
solid-phase synthesized DNA oligos. The gRNA can be synthesized
from array-synthesized oligos, and amplified.
[0114] The following examples are set forth as being representative
of the present disclosure. These examples are not to be construed
as limiting the scope of the present disclosure as these and other
equivalent embodiments will be apparent in view of the present
disclosure, figures and accompanying claims.
EXAMPLE I
In Vivo Gene Editing in Cells
[0115] According to one aspect, methods are described herein for
site-specific exon removal (i.e., skipping) in a target gene in a
cell using a CRISPR/Cas9 system. The edited gene is then expressed
to produce a truncated polypeptide. According to one aspect, the
truncated polypeptide has an activity similar to the full length
polypeptide. According to one aspect, the cell can be a somatic
cell, such as a tissue cell, such as a mammalian tissue cell. The
cell can be a skeletal muscle cell, liver cell, blood cell, heart
cell, skin cell, brain cell, peripheral neuronal cell, gut cell,
lung cell, kidney cell, bladder cell, bone cell, fat cell, blood
vessel cell or stem cell. According to one aspect, the site
specific exon skipping is permanent to the extent that the target
exon is removed from the target gene and the target gene is then
expressed, such as by the cell, to form a protein lacking the
polypeptide sequence corresponding to the removed exon. According
to one aspect, the exon to be removed from the target gene includes
a mutation which negatively affects the biological activity of the
polypeptide. When the exon with the mutation is removed from the
gene, expression of the altered gene produces an altered
polypeptide which exhibits the biological activity of the full
length polypeptide without the mutation.
[0116] One exemplary embodiment of the present disclosure is a
method of exon removal in the Dystrophin gene in skeletal muscle
using a CRISPR/Cas9 system. Duchenne Muscular Dystrophy (DMD) is an
X-linked skeletal muscle disorder that afflicts 1 in every 3000 to
4000 male births and is characterized by progressive muscle wasting
and weakness. DMD is caused by mutations in the Dystrophin locus,
including large deletions and point mutations. Many DMD mutations
cause frameshifts in the coding sequence of Dystrophin, resulting
in degradation of the Dystrophin mRNA via nonsense mediated decay
and absence of Dystrophin protein from the surface of muscle
fibers. Loss of Dystrophin destabilizes the muscle fiber membrane,
leading to contraction-mediated muscle damage and progressive loss
of functional muscle. The Dystrophin protein is large and includes
a central "rod" domain formed from repeating spectrin-like coils
that appear largely dispensable, as reducing the number of repeats
from 24 to 8 allows for relatively intact function.
[0117] According to one aspect, methods are provided herein for
removing exons within the rod domain that introduce frameshifting
mutations using a CRISPR/Cas9 system. Such an altered gene when
expressed can produce a truncated and partially functional protein
that can counter loss of functional or biologically active
Dystrophin. Accordingly, aspects of the present disclosure are
directed to removing one or more mutations from the Dystrophin gene
using a CRISPR/Cas9 system with two or more guide RNAs to excise
the one or more mutations resulting in an edited Dystrophin gene.
The edited Dystrophin gene is then expressed to produce a
functional truncated Dystrophin protein.
[0118] According to one aspect, guide RNA are designed to target
sequences to direct Cas9-mediated cleavage of DNA at specific
genetic sequences within the Dystrophin gene flanking a particular
target mutation, such as a point mutation. Cleavage at both
flanking sites excises the intervening DNA to remove the mutation
and to introduce a precise deletion that restores the proper
reading frame of the protein.
[0119] One of ordinary skill in the art will readily identify other
target genes for exon excision where the exon includes a mutation
and where excision of the exon produces an altered polypeptide
having biological activity similar to the full length polypeptide
without the mutation. Additional exemplary target genes include
genes known to be associate with cancer, such as oncogenes, genes
associated with amyotrophic lateral sclerosis, genes associated
with trinucleotide repeat disorders including polyglutamine
diseases such as Huntington's disease, prior-related diseases such
as Creutzfeldt-Jakob disease and Fatal Familial Insomnia and genes
associated with Marfan syndrome and Dysferlin-associated
dystrophies (Aartsma-Rus et. al, Therapeutic exon skipping for
dysferlinopathies?, Eur J Hum Genet. 2010 Aug; 18(8):889-94.)
EXAMPLE II
Construction and Screening of Guide RNA Plasmids
[0120] Candidate guide RNAs (gRNAs) were designed by searching the
sequences flanking exon 23 and exons 52 and 53 of the mouse
Dystrophin gene for appropriate sequence features (e.g., presence
of PAM sequence). The sequences for the DNA binding portion of the
guide RNAs were determined and are listed below:
TABLE-US-00002 Exon 23 5' (left) guide RNA: (SEQ ID NO: 15)
GAATAATTTCTATTATATTACA Exon 23 3' (right) guide RNA: (SEQ ID NO:
16) TTCGAAAATTTCAGGTAAGCCG Exon 52 5' (left) guide RNA: (SEQ ID NO:
17) TCATTTCTAAAAGTCTTTTGCC Exon 53 3' (right) guide RNA: (SEQ ID
NO: 18) TTTGAGACACAGTATAGGTTAT
[0121] Full length sequences are provided below.
TABLE-US-00003 Exon 23 5' (left) guide RNA: (SEQ ID NO: 19)
GAATAATTTCTATTATATTACAGTTTaAGAGCTAtgctgGAAAcagcaTA
GCAAGTTtAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA GTCGGTGCTTTTTTT
Exon 23 3' (right) guide RNA: (SEQ ID NO: 20)
TTCGAAAATTTCAGGTAAGCCGGTTTaAGAGCTAtgctgGAAAcagcaTA
GCAAGTTtAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA GTCGGTGCTTTTTTT
Exon 52 5' (left) guide RNA: (SEQ ID NO: 21)
TCATTTCTAAAAGTCTTTTGCCGTTTaAGAGCTAtgctgGAAAcagcaTA
GCAAGTTtAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA GTCGGTGCTTTTTTT
Exon 53 3' (right) guide RNA: (SEQ ID NO: 22)
TTTGAGACACAGTATAGGTTATGTTTaAGAGCTAtgctgGAAAcagcaTA
GCAAGTTtAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
GTCGGTGCTTTTTTT
[0122] Lack of predicted off target sites in other coding regions
of the mouse genome was confirmed for candidate gRNAs by aligning
the protospacers to the mouse genome using Blast online tool.
Confirmed protospacers were cloned into gRNA cloning vector (world
wide website addgene.org/41824/) using overlap extension PCR.
Different pairs of guide RNA plasmids flanking exon 23 or exons 52
and 53 were co-transfected with Sp hCas9_p2A GFP encoding plasmid
into mouse C2C12 myoblast cells using lipofectamine 2000 Reagent
(Invitrogen) according to the manufacturer's instructions. After
three to four days, the transfected cells were sorted out based on
GFP expression, and genomic DNA was extracted using the
quickextract DNA extraction solution (Epicentre). Primers flanking
the gRNA target sites were used to amplify exon 23 and also exons
52 and 53 by PCR. The PCR products were purified and sequenced by
Sanger and deep sequencing. Guide RNA pairs with the highest
efficiency for removing the targeted Dystrophin exons were used for
the in vivo experiments.
EXAMPLE III
In Vivo Electroporation
[0123] Animals were anesthetized using Isoflurane and injected with
50 ul of 2 mg/ml hyaluronidase in the tibialis anterior muscles.
After 1 hour, two plasmids encoding the two gRNAs, hCas9 encoding
plasmid or hCas9 mRNA (Trilink), and GFP encoding plasmid were
co-injected in the hyaluronidase-injected muscles. hCas9 is
humanized codon optimized S. pyogenes Cas9, and the gene encoding
hCas9 was obtained from addgene (world wide website
addgene.org/41815.) Muscles were electroporated by applying 10
pulses of 20 ms at 200 V/cm with 100 ms intervals using an ECM 830
electro square porator (BTX Harvard apparatus) and a two needle
array.
EXAMPLE IV
Single Fiber Isolation and Sequencing
[0124] Animals were sacrificed and injected muscles were harvested
10 days after electroporation and digested with 0.2% collagenase
type II in DMEM for 50 min in a 37.degree. C. water bath. Muscles
were triturated with a fire polished Pasteur pipette and GFP+
transduced single fibers were isolated using a fluorescent
dissection microscope. DNA was isolated from single fibers by
quickextract DNA extraction solution (Epicentre). Targeted exons
were PCR amplified using primers flanking the gRNA target sites.
Deep sequencing libraries were prepared by adding adaptors to the
PCR products using PCR, and libraries were sequenced by Illumina
Miseq.
EXAMPLE V
Excision of Target Exon Using a CRISPR Cas9 System
[0125] Cas9 with addition of 3xNLSs and multiplexed guide RNAs
flanking exon 23 (mutated in the mdx mouse model of DMD) were
delivered directly into skeletal muscle fibers in vivo (see FIG. 4)
and into cultured mouse C2C12 myoblasts (See FIGS. 2 and 3).
[0126] The sequence of the Cas9 protein (with the addition of
3xNLSs in italics) that was used is shown below:
TABLE-US-00004 (SEQ ID NO: 23)
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY
LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIV
DEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS
GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS
LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQ
YADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDE
HHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGA
SQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG
SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY
VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSF
IERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVT
EGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKK
IECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENE
DILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR
RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKK
GILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQ
KNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYY
LQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK
VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ
RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQIL
DSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV
REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYK
VYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANG
EIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVK KTEVQTGGF
SKESILPKRNSDKLIARKKDWDPKKYGGFDS
PTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN
PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG
ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLF
VEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRD
KPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKE
VLDATLIHQSITGLYETRIDLSQLGGDSRADPKKKRKVDPKK KRKVDPKKKRKV-
[0127] PCR amplification and DNA sequencing of the PCR product from
individual muscle fibers harvested from electroporated muscle at 10
days after electroporation demonstrated that electroporation into
skeletal muscle of the combination of Cas9 and exon 23 guide RNAs
resulted in double cutting of the genomic DNA and deletion of the
intervening sequence to restore transcript reading frame (See FIG.
4).
[0128] Cas9 and multiplexed guide RNAs flanking exons 52 and 53
(mutated in the mdx4cv mouse model of DMD) are delivered directly
into cultured mouse C2C12 myoblasts. C2C12 transfection of the
combination of Cas9 and guide RNAs flanking exons 52 and 53 results
in double cutting of the genomic DNA and deletion of the
intervening sequence to restore transcript reading frame (See FIG.
5). According to this aspect, the CRISPR/ Cas9 system can be used
to excise large nucleic acid segments from a target gene, for
example, so as to produce a biologically functional expressed
protein.
EXAMPLE VI
In Vivo Gene Editing of Loci Corresponding to Myostatin and Its
Receptors ActRIIA and ActRIIB
[0129] Paired Sp gRNAs were used to generate two double-strand
breaks (DSBs) flanking the desired genomic excision, promoting
end-joining of the genomic sequences. Target sites were chosen to
disrupt the myostatin-activin receptor pathway, thereby
derepressing the myostatin-mediated inhibition of muscle growth and
leading to musclehypertrophy (see Lee et al., Proc. Natl. Acad.
Sci. USA, 2001;98(16):9306-11). For the myostatin gene, the
physiological RSRR cleavage site between the propeptide and activin
receptor-binding domains was targeted, to create deletions or
mis-sense mutations that result in an uncleaved myostatin protein,
and nonsense mutations that result in a truncated myostatin, both
dominant negative forms of myostatin. Accordingly, aspects of the
present disclosure are directed to producing dominant negative
forms of a protein using the Cas9 mediated methods derscribed
herein. For TGF.beta.-family activin receptors IIB and HA, the
region between the trans-membrane and kinase domains was targeted,
excising the ATP-binding domains, which likewise results in
dominant-negative activin ligand traps that bind cognate ligands
but lack functional kinase activity.
[0130] FIGS. 10A and 10B depict CRISPR lipofection in unselected
C2C12 cell culture. FIG. 10A shows the panel of guide RNAs (single
or paired) co-delivered with four forms of Cas9-expressing DNA
constructs. For singly-transfected gRNAs, the majority of mutants
harbor deletions, with the deletion window concentrated at <7bp,
but large deletions that span the entire amplicon (444-829bp) are
also observed at low frequencies (FIG. 10B). Transfection of paired
gRNAs dramatically shifts the deletion profile towards intervening
excision. Deep sequencing reveals that the significant proportion
of breakpoint junctions are formed by end-joining of the flanking
genomic sequences, corresponding to end-ligation between the SpCas9
cut-sites 3bp 5' from the PAM NGG. Shifting the gRNA by 1bp
(compare mActRIIB1 with mActRIIB2 or mActRIIB3) shifts the excision
product by 1bp. Precise excisions can be generated through all
combinations of PAMs-orientation of paired gRNAs, such as same
direction, `inwards pointing`, or `outwards pointing` PAMs. For
some gRNAs pairs, wobble was observed in the breakpoints, where the
putative cut-sites were shifted 1bp from the expected sites (i.e.
4bp 5' of PAM sequence, such that co-delivery of mActRIIB1 and
mActRIIB3 gives rise to both 124bp and 125bp deletion sizes).
Sequence consensus reveals that cut-site wobble manifests in the
context of a GGG PAM (FIG. 10C).
[0131] Cas9 and multiplexed guide RNAs flanking sp 3r.sup.d ActRIIA
and sp 4.sup.th ActRIIA were delivered directly into skeletal
muscle fibers in vivo. The spacer sequences for the Sp gRNAs are
listed below:
TABLE-US-00005 1st ActRIIB (SEQ ID NO: 24)
GGGCCATGTGGACATCCATGAGGTGAGACAGTGCCAGCGT 2nd ActRIIB (SEQ ID NO:
25) GGCCTGAAGCCACTACAGCTGCTGGAGATCAAGGCTC 3rd ActRIIB (SEQ ID NO:
26) GGCCTGAAGCCACTACAGCTGCTGGAGATCAAGGCTCG 1st ActRIIA (SEQ ID NO:
27) GCCATTGCAGCTGTTAGAAGTGAAAGCAAG 3rd ActRIIA (SEQ ID NO: 28)
GGCCCTAGCATCTAAGTTCTCGCAGGC 4th ActRIIA (SEQ ID NO: 29)
GGTCATTCCATCTCAGCTGTGACAGCAGCGCAGAA 1st Mstn (SEQ ID NO: 30)
GGAAGTCAAGGTGACAGACACACCCAAGAGGTCC 2nd Mstn (SEQ ID NO: 31)
GGACACACCCAAGAGGTCCCGGAGAGACTTT 3rd Mstn (SEQ ID NO: 32)
GTCAAGCCCAAAGTCTCTCCGGGACCTCTT 4th Mstn (SEQ ID NO: 33)
GGAATCCCGGTGCTGCCGCTACCCCCTCA
[0132] All guide RNAs use the scaffold sequence shown below of Mali
et al., Science, 2013;339(6121):836-6 hereby incorporated by
reference in its entirety for all purposes.
TABLE-US-00006 (SEQ ID NO: 34)
GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC
TTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT
[0133] Deep sequencing of the PCR product from individual muscle
fibers harvested from electroporated muscle at 10 days after
electroporation demonstrated that electroporation into skeletal
muscle of the combination of Cas9 and guide RNAs sp 3.sup.rd
ActRIIA and sp 4.sup.th ActRIIA resulted in double cutting of the
genomic DNA and deletion of the intervening sequence to generate a
dominant negative ActRIIA coding gene. According to certain
aspects, the protein is truncated specifically after the upstream
guide RNA. Paired guide RNAs were chosen to delete critical domains
of the proteins, while allowing precise endpoints for the
deletions, such that the N-terminal portions remain expressed, at
least up to the location of the upstream guide RNA or a created
premature stop codon. In the case of mActRIIB and mActRIIA, these
truncated proteins bind to the cognate ligands but do not transduce
the relevant signals because they lack the C-terminal kinase
domains. In the case of mMstn, the RSRR cleavage signal is deleted,
such that with or without a frameshift, the N-terminal propeptide
serves as a dominant negative protein. It is possible to retain the
reading frame, if desired, by choosing guide RNAs that have their
cut sites in phase of 3n within the protein-coding sequence.
[0134] Cas9 and multiplexed guide RNAs sp 1st ActRIIB and sp
3.sup.rd ActRIIB were delivered directly into skeletal muscle
fibers in vivo. Deep sequencing of the PCR product from individual
muscle fibers harvested from electroporated muscle at 10 days after
electroporation demonstrated that electroporation into skeletal
muscle of the combination of Cas9 and guide RNAs sp 1st ActRIIB and
sp 3r.sup.d ActRIIB resulted in double cutting of the genomic DNA
and deletion of the intervening sequence to generate a dominant
negative ActRIIB coding gene.
[0135] Cas9 and multiplexed guide RNAs sp 3.sup.rd myostatin (Mstn)
and sp 4.sup.th myostatin were delivered directly into skeletal
muscle fibers in vivo. Deep sequencing of the PCR product from
individual muscle fibers harvested from electroporated muscle at 10
days after electroporation demonstrated that electroporation into
skeletal muscle of the combination of Cas9 and guide RNAs sp
3.sup.rd myostatin and sp 4.sup.th myostatin resulted in double
cutting of the genomic DNA and deletion of the intervening sequence
to generate a dominant negative myostatin coding gene.
[0136] Precise cutting of genomic sequence by multiplexed guide
RNAs was confirmed at the above loci as shown in FIGS. 10 and 11
indicating that the methods of in vivo gene editing can be extended
to any desired gene target.
EXAMPLE VII
Ratio of Cas9 to Guide RNA
[0137] According to certain aspect, the ratio of Cas9 to guide RNA
can affect the rate of genome modification in vivo. As shown in
FIG. 12, an exemplary ratio of plasmid encoding the Cas9 protein to
the plasmids encoding the guide RNA is 1:1.5 to 1:2. For the
results of FIG. 12, C57B1/6 mouse tibialis anterior (TA) muscles
were electroporated with 15 ug GFP, 30ug Cas9 and 0, 15, 30, 45,
60, 75, 90 or 105 ug of sp 1st ActRIIB and sp 3rd ActRIIB gRNA
plasmids. PCR amplification and DNA sequencing of the PCR product
from individual muscle fibers harvested from electroporated muscle
at 10 days after electroporation demonstrated that electroporation
into skeletal muscle of the combination of Cas9 and guide is most
efficient when the ratio of plasmid coding Cas9 to the plasmids
coding the gRNAs is 1:1.5 to 1:2.
EXAMPLE VIII
In Vivo Gene Editing in C2C12 Myoblasts
[0138] As shown in FIGS. 2, 3 and 10, the methods described herein
can be applied to cultured cells, such as progenitor cells, for
transplantation into an animal, such as a human. FIG. 2A is a
schematic diagram of the mdx mouse genomic DNA at the mutated exon
23 locus before (top) and after (bottom) cutting the DNA with two
gRNAs targeting 5' and 3' of exon 23. FIG. 2B is an image
illustrating PCR products amplified by a primer pair spanning exon
23 of mouse Dystrophin gene from genomic DNA of C2C12 myoblasts
transfected with Cas9 only or Cas9 and two gRNAs targeting 5' and
3' of exon 23. Cutting both sides of exon 23 with two gRNAs leads
to excision of the exon from the DNA and amplification of a smaller
PCR product corresponding to the deleted locus. FIG. 2C is a graph
illustrating results from deep sequencing of exon 23 genomic DNA
amplicons from C2C12 cells transfected with Cas9 and two gRNAs
targeting 5' and 3' of exon 23.
[0139] FIG. 3A is a schematic diagram of the mdx mouse mRNA at the
mutated exon 23 locus before (top) and after (bottom) cutting the
DNA with two gRNAs targeting 5' and 3' of exon 23. Removal of exon
23 from the mRNA restores the reading frame and leads to expression
of a truncated but partially functional Dystrophin protein in
dystrophic mouse muscle (Lu et. al, Functional amounts of
dystrophin produced by skipping the mutated exon in the mdx
dystrophic mouse, Nature Medicine 9, 1009-1014 (2003). FIG. 3B is
an image illustrating RT-PCR products amplified by a primer pair
spanning exon 23 of mouse Dystrophin mRNA from cDNA of C2C12
myoblasts transfected with Cas9 only or Cas9 and two gRNAs
targeting 5' and 3' of exon 23. Cutting both sides of Dystrophin
exon 23 with two gRNAs leads to excision of the exon from the mRNA
and amplification of a smaller PCR product corresponding to the
transcript lacking exon 23.
[0140] FIG. 10A illustrates results of mutation frequencies in
unselected C2C12 cells lipofected with either single guide RNAs or
paired guide RNAs, using four forms of Cas9-expressing constructs
(pSMVP: plasmid with an SV40enhancer-CMV-chimeric intron promoter;
MC-SMVP: minicircle with the same promoter; with or without P2A-GFP
for co-translational expression of GFP). FIG. 10B illustrates
deletion sizes generated by single or paired guide RNAs. FIG. 10C
is a diagram illustrating results from examining breakpoint
junctions. The results reveal that genomic loci with GGG PAM
exhibit cut-site wobble, where the CRISPR-induced double-strand
break is 3bp or 4bp upstream of the PAM.
[0141] According to this aspect, precisely edited, autologous,
transplantable cells, such as progenitor cells, can be created and
implanted into an animal, such as a human. Exemplary cells include
stem cells such as muscle stem cells.
EXAMPLE IX
Developing a Reporter System for CRISPR in Vivo Activity
[0142] Ai9 mice (Madisen et. al A robust and high-throughput Cre
reporting and characterization system for the whole mouse brain.
Nat Neurosci, 2010 13(1):133-40) that harbor an inducible tdtomato
reporter construct in their Rosa26 locus were used as a reporter
for CRISPR in vivo double cut activity. The reporter construct
includes a CAG promoter followed by a 3xSTOP cassette (3x SV40
polyA translational terminators) and tdtomato coding sequence.
Therefore tdtomato is only expressed when the 3xSTOP cassette is
removed (See FIG. 6). Guide RNAs were designed to target 5' and 3'
sites of the 3xSTOP cassette, and Ai9 TA muscles were
electroporated with GFP and Cas9 only or GFP, Cas9 and two gRNAs
targeting 5' and 3' of the 3xSTOP cassette. Targeting both sides of
the 3xSTOP cassette leads to excision of the cassette from the
genome and expression of tdtomato (See FIG. 6). This system can be
used as a reporter for in vivo CRISPR activity in different
tissues. Guide RNAs targeting different genes can also be
co-delivered with the gRNAs targeting the 3xSTOP cassette, and the
targeted tissues can be detected and isolated based on tdtomato
expression. The 3xSTOP cassette is commonly used in LoxP mouse
strains, and the 3xSTOP gRNAs used are compatible with the large
mouse collection publicly available.
EXAMPLE X
Immune Response to Cas9
[0143] In the course of in vivo experiments, enrichment of
DAPI-stained nuclei was observed in the vicinity of
transgene-expressing fibers, reminiscent of immune cell
infiltration. In FIG. 13A, the green represents the DAPI nuclear
stain intensity around GFP-positive muscle fibers, the blue
represents the DAPI stain intensity around GFP-negative muscle
fibers adjacent to GFP-positive fibers (i.e., 1 degree neighbor),
the yellow represents 2 degree neighbor, and the red represents 3
degree or more neighbor. Both Cas9 and GFP are exogenous transgenes
that could elicit an immune response, which is a potential concern
that needs to be resolved, especially in anticipation of clinical
adoption of CRISPR-mediated gene therapy. The immunological
cell-types present within injected muscles were profiled by
antibody-staining and FACS (FIG. 13C). Similar to the
[0144] GFP-only control, CRISPR induced immune cell infiltration
throughout the injected muscle, enriched around
transgene-expressing fibers, was visualized by CD45 antibody
staining (FIGS. 13B, 13D). To alleviate the T-cell response, mice
were treated with the immunosuppressant FK506, which abolishes
T-cell infiltration in the injection muscle to mock-injection
levels (FIGS. 13B, 13C, 13D).
EXAMPLE XI
Therapeutic Gene Editing
[0145] According to certain aspects, methods and materials are
provided for editing a target gene in a cell to remove one or more
exons from the target gene by administering a Cas9 protein, such as
a Cas9 enzyme, and guide RNAs (CRISPR system) as described herein
to an individual in need of such target gene editing, such as for
treating a genetic disorder. Methods and materials are provided for
therapeutic treatment of an individual suffering from Duchenne
muscular dystrophy by in vivo genome editing to correct frame
shifting mutations in the Dmd gene using a Cas9 enzyme and guide
RNA delivered by AAV. The CRISPR system may be delivered directly
to post-mitotic skeletal muscle fibers and cardiomyocytes, as well
as proliferative muscle satellite cells, where the CRISPR system
targets the Dmd gene for permanent exon deletion, restores
Dystrophin protein expression and partially or totally recovers
functional deficiencies of dystrophic muscle. The individual may be
a human or a non-human animal. The Cas9 enzyme or guide RNAs may be
delivered intramuscularly or systemically. Systemic administration
may include intravenous administration or injection,
intraperitoneal administration or injection, intramuscular
administration or injection, intracranial administration or
injection, intraocular administration or injection, or subcutaneous
administration or injection. The Cas9 enzyme or guide RNAs may be
delivered in one or more viral vectors, such as an adeno-associated
virus. According to one aspect, the target gene may be the
dystrophin gene and the individual may be suffering from Duchenne
muscular dystrophy. According to one aspect, the target gene is in
an endogenous muscle cell. According to one aspect, the target gene
is in an endogenous muscle stem cell which may be referred to
herein as a satellite cell. According to one aspect, a Cas9 enzyme
and guide RNAs are administered to an individual to edit a target
gene in a muscle cell and a target gene in a muscle stem cell.
According to certain aspects, methods are provided using
CRISPR/Cas9 gene editing for therapeutic modification of
disease-specific alleles in muscle cells and primary muscle stem
cells.
[0146] Certain exemplary aspects of the present disclosure include
a permanent gene editing approach that restores Dystrophin
expression in skeletal muscles, cardiac muscle, as well as muscle
stem cells after intramuscular or systemic injection of
adeno-associated viruses (AAVs) encoding the components of
CRISPR/Cas9 gene editing system. According to certain aspects,
methods described herein permanently target DMD mutations in
endogenous muscle stem cells which then produce muscle cells
retaining the edit to the target gene providing for long-term
repair of dystrophic fibers with corrected muscle precursor
cells.
[0147] Vectors are contemplated for use with the methods and
constructs described herein. The term "vector" includes a nucleic
acid molecule capable of transporting another nucleic acid to which
it has been linked. Vectors used to deliver the nucleic acids to
cells as described herein include vectors known to those of skill
in the art and used for such purposes. Certain exemplary vectors
may be plasmids, lentiviruses or adeno-associated viruses known to
those of skill in the art. Vectors include, but are not limited to,
nucleic acid molecules that are single-stranded, doublestranded, or
partially double-stranded; nucleic acid molecules that comprise one
or more free ends, no free ends (e.g. circular); nucleic acid
molecules that comprise DNA, RNA, or both; and other varieties of
polynucleotides known in the art. One type of vector is a
"plasmid," which refers to a circular double stranded DNA loop into
which additional DNA segments can be inserted, such as by standard
molecular cloning techniques. Another type of vector is a viral
vector, wherein virally-derived DNA or RNA sequences are present in
the vector for packaging into a virus (e.g. retroviruses,
lentiviruses, replication defective retroviruses, adenoviruses,
replication defective adenoviruses, and adeno-associated viruses).
Viral vectors also include polynucleotides carried by a virus for
transfection into a host cell. Certain vectors are capable of
autonomous replication in a host cell into which they are
introduced (e.g. bacterial vectors having a bacterial origin of
replication and episomal mammalian vectors). Other vectors (e.g.,
non-episomal mammalian vectors) are integrated into the genome of a
host cell upon introduction into the host cell, and thereby are
replicated along with the host genome. Moreover, certain vectors
are capable of directing the expression of genes to which they are
operatively linked. Such vectors are referred to herein as
"expression vectors." Common expression vectors of utility in
recombinant DNA techniques are often in the form of plasmids.
Recombinant expression vectors can comprise a nucleic acid of the
invention in a form suitable for expression of the nucleic acid in
a host cell, which means that the recombinant expression vectors
include one or more regulatory elements, which may be selected on
the basis of the host cells to be used for expression, that is
operatively-linked to the nucleic acid sequence to be expressed.
Within a recombinant expression vector, "operably linked" is
intended to mean that the nucleotide sequence of interest is linked
to the regulatory element(s) in a manner that allows for expression
of the nucleotide sequence (e.g. in an in vitro
transcription/translation system or in a host cell when the vector
is introduced into the host cell). Representative AAV viruses
include those serotypes known in the art and referred to as AAV1,
AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11
and as described in the literature.
[0148] Methods of non-viral delivery of nucleic acids or native DNA
binding protein, native guide RNA or other native species include
lipofection, microinjection, biolistics, virosomes, liposomes,
immunoliposomes, polycation or lipid:nucleic acid conjugates, naked
DNA, artificial virions, and agent-enhanced uptake of DNA.
Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386,
4,946,787; and 4,897,355) and lipofection reagents are sold
commercially (e.g., Transfectam.TM. and Lipofectin.TM.). Cationic
and neutral lipids that are suitable for efficient
receptor-recognition lipofection of polynucleotides include those
of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells
(e.g. in vitro or ex vivo administration) or target tissues (e.g.
in vivo administration). The term native includes the protein,
enzyme or guide RNA species itself and not the nucleic acid
encoding the species.
[0149] Regulatory elements are contemplated for use with the
methods and constructs described herein. The term "regulatory
element" is intended to include promoters, enhancers, internal
ribosomal entry sites (IRES), and other expression control elements
(e.g. transcription termination signals, such as polyadenylation
signals and poly-U sequences). Such regulatory elements are
described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:
METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.
(1990). Regulatory elements include those that direct constitutive
expression of a nucleotide sequence in many types of host cell and
those that direct expression of the nucleotide sequence only in
certain host cells (e.g., tissue-specific regulatory sequences). A
tissue-specific promoter may direct expression primarily in a
desired tissue of interest, such as muscle, neuron, bone, skin,
blood, specific organs (e.g. liver, pancreas), or particular cell
types (e.g. lymphocytes). Regulatory elements may also direct
expression in a temporal-dependent manner, such as in a cell-cycle
dependent or developmental stage-dependent manner, which may or may
not also be tissue or cell-type specific. In some embodiments, a
vector may comprise one or more pol III promoter (e.g. 1, 2, 3, 4,
5, or more pol III promoters), one or more pol II promoters (e.g.
1, 2, 3, 4, 5, or more pol II promoters), one or more pol I
promoters (e.g. 1, 2, 3, 4, 5, or more pol I promoters), or
combinations thereof. Examples of pol III promoters include, but
are not limited to, U6 and H1 promoters. Examples of pol II
promoters include, but are not limited to, the retroviral Rous
sarcoma virus (RSV) LTR promoter (optionally with the RSV
enhancer), the cytomegalovirus (CMV) promoter (optionally with the
CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)],
the SV40 promoter, the dihydrofolate reductase promoter, the
13-actin promoter, the phosphoglycerol kinase (PGK) promoter, and
the EFla promoter and Pol II promoters described herein. Also
encompassed by the term "regulatory element" are enhancer elements,
such as WPRE; CMV enhancers; the R-U5' segment in LTR of HTLV-I
(Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; and
the intron sequence between exons 2 and 3 of rabbit .beta.-globin
(Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). It
will be appreciated by those skilled in the art that the design of
the expression vector can depend on such factors as the choice of
the host cell to be transformed, the level of expression desired,
etc. A vector can be introduced into host cells to thereby produce
transcripts, proteins, or peptides, including fusion proteins or
peptides, encoded by nucleic acids as described herein (e.g.,
clustered regularly interspersed short palindromic repeats (CRISPR)
transcripts, proteins, enzymes, mutant forms thereof, fusion
proteins thereof, etc.).
[0150] Aspects of the methods described herein may make use of
terminator sequences. A terminator sequence includes a section of
nucleic acid sequence that marks the end of a gene or operon in
genomic DNA during transcription. This sequence mediates
transcriptional termination by providing signals in the newly
synthesized mRNA that trigger processes which release the mRNA from
the transcriptional complex. These processes include the direct
interaction of the mRNA secondary structure with the complex and/or
the indirect activities of recruited termination factors. Release
of the transcriptional complex frees RNA polymerase and related
transcriptional machinery to begin transcription of new mRNAs.
Terminator sequences include those known in the art and identified
and described herein.
EXAMPLE XII
Materials and Methods
[0151] Methods described in Example XI use the following general
methods.
[0152] Genomic PCR: Genomic DNA were extracted from tissues and in
vitro cultured cells using Quick Extract solution (Epicenter)
according to manufacturer's instruction. DNA samples in Quick
Extract of volumes equal to 10% of the final PCR reaction were
used. Nested PCR was performed with 20 cycles of first round
amplification followed by 25 cycles of second round amplification
with a 1:10 dilution between the two rounds. PCR products were used
for electrophoresis on 1.5% agarose gel for visualization and on
0.6% agarose gel for gel extraction. Wild-type and exon-excised
bands were gel extracted and cloned into TOPO plasmids using
ZeroBlunt TOPO kit (LifeTech) and subsequently transformed TOP10
competent cells. Individual colonies were sent for Sanger
sequencing to confirm the correct excision of sequence flanked by
two guide RNAs.
[0153] RT-PCR and Taqman-based Real Time PCR: Total RNA was
isolated from tissues using TRIzol reagent (LifeTech) per
manufacturer's instructions. For tissues harvested from animals,
lug of RNA was used for cDNA synthesis with SuperScript III
MasterMix (LifeTech) in 20 uL reactions. For in vitro cultured
samples, 400 ng of RNA was used for cDNA synthesis with SuperScript
VILO MasterMix (LifeTech) in 20 uL reactions. RT-PCR was performed
using 1 uL or abovementioned cDNA with Q5 HotStart MasterMix (New
England Biolabs) with DMD RT forward and reverse primer. Both
wild-type and exon-skipped bands were gel extracted and cloned into
TOPO vectors using ZeroBlunt TOPO kit (LifeTech) and subsequently
transformed into TOP10 competent cells. Individual colonies were
sent for Sanger sequencing. Alignment to genomic sequence was
performed using Geneious software. Taqman quantitative RealTime-PCR
was performed as previously described. Taqman assays against exon
4-5, for quantification of total DMD transcripts, and against exon
22-24, for quantification of exon-23-skipped transcripts were used
in additional to 18s assay as endogenous control. Assays were
carried out in triplicates of 10 uL reactions for each probe and
with 20 ng of cDNA input. Taqman Fast Advanced master mix
(LifeTech) was used with fast cycling conditions recommended by the
manual. Delta-Ct values between exon 4-5 and exon 22-24 were used
to quantify the percentage of exon-skipped transcripts in
comparison to total DMD transcripts.
[0154] Satellite Cell Isolation and Culture: Satellite cell
isolation was performed as previously described. For in vitro
expansion experiments, CD45-Sca-1-Mac-1-CXCR4+b1 -integrin+cells
were seeded on collagen/laminin-coated plates in F10 (GIBCO)
containing 20% horse serum (Atlanta Biologics), 1%
penicillin-streptomycin (Invitrogen), and 1% glutamax (Invitrogen).
5 ng/ml bFGF (Sigma) was added to the medium daily. Medium was
changed for fresh medium every other day. After 7 days, satellite
cells were harvested, cell numbers were counted and cells were
re-plated in multiple wells of a 96 well plate for differentiation.
The next day, medium was changed to DMEM (GIBCO) containing 2%
horse serum (Atlanta Biologics), 1% penicillin-streptomycin
(Invitrogen). Cells were fixed after 60 or 72 hr in differentiation
medium.
[0155] AAV Production: AAVs were generated through the Gene
Transfer Vector Core (GTVC) at the Grousbeck Gene Therapy Center at
the Schepens Eye Research Institute and Massachusetts Eye and Ear
Infirmary (SERI/MEEI).
[0156] Western Blotting: Protein was extracted from tissues and
cultured cells using RIPA buffer (Cell Signaling). Tissues were
homogenized using GentleMACS M-tubes (Miltenyi Biotech) with
protein 1.1 program. Protein was concentrated using Amicon Ultra
10k centrifugal filter units. Protein concentration was determined
by BCA assay (Pierce). 25 ug, 25 ug and 50 ug of total protein per
lane were used for myotubes, IM injected TA muscle and IP injected
tissues, respectively. Different percentages of wild-type muscle
proteins were diluted in mdx proteins from the same muscle so that
the total protein of that lane was kept the same. Samples were
denatured at 99.degree. C. for 5 minutes before being loaded on to
4-20% Tris-HC1 precast Criterion gels (Bio-Rad). Dystrophin and
GAPDH (loading control) were detected by primary antibodies
NCL_DYS1 (1:100,
[0157] Novocastra) and sc-32233 (1:25,000, Santa-Cruz
Biotechnology) followed by horse anti-mouse IgG HRP-linked
(1:1,000, Cell Signaling Technology 7076P2). ChemiDoc imaging
system (Bio-Rad) was used to detect chemiluminescence after using
SuperDura ECL kit. Intensity of Dystrophin and GAPDH bands were
quantified using ImageJ gel analysis function. Different exposures
were used for some membranes for Dystrophin and GAPDH
quantification to avoid overexposed bands. Relative abundance of
Dystrophin in total protein was computed by the ratio of Dystrophin
signal and GAPDH and presented in Arbitrary Unit (AU).
[0158] Histology: Mouse skeletal and heart muscles were dissected.
Samples used for Dystrophin immunofluorescence were embedded in
O.C.T compound (Tissue-Tek) right after dissection and frozen in
liquid-nitrogen-cold isopentane. Sample used for tdTomato
immunofluorescence were fixed in 4% PFA for 1 h at room temperature
and immersed in 30% sucrose until submersion, before embedding in
O.C.T. and freezing. Subsequent cryosectioning was performed using
a Microm HM550 (Thermo Scientific); skeletal muscles were sectioned
to a thickness of 12 .mu.m and heart samples were sectioned at 30
.mu.m.
[0159] Immunofluorescence: Fixed cryosections were blocked with 5%
NGS, 2% BSA, 2% protein concentrate (M.O.M. Kit, Vector
Laboratories, BMK-2202), and 0.1% tween-20 at room temp. for 1 h,
followed by 3 washes with 1.times.DPBS for 5 min. each. Sections
were subsequently stained with rabbit polyclonal anti-dystrophin
(1:50, Abcam, ab15277) or anti-laminin (1:200, Abcam, ab11575) at
4.degree. C. overnight, followed by 3 washes with lx DPBS for 5
min. each. Slides were then incubated with secondary
goat-anti-rabbit IgG Alexa Fluor 488 (1:250, Life Technologies) at
room temp. for 1 h, followed by 3 washes with 1.times. DPBS for 5
min. each. Slides were then mounted with mounting media containing
DAPI (Vector Laboratories).
[0160] Muscle Force Analysis: Mice were anesthetized with sodium
pentobarbital (80-100 mg/kg body mass). Supplemental doses were
provided as necessary during the experiment. Small incisions were
made to expose the right tibialis anterior (TA) tendon and right
patellar tendon. The mouse was placed on the temperature controlled
platform (38 .degree. C.) of an in situ test stand (Aurora
[0161] Scientific model 809B, Aurora, Ontario, Canada). Silk suture
(4-0) was used to attach the severed TA tendon to the lever arm of
a dual mode muscle lever system (Aurora Scientific model 305C-LR).
The lower right limb was stabilized by using suture attached to the
patellar tendon to secure the knee to a horizontal support.
Supramaximal 200 .mu.Ls square-wave pulses, output by a high
current muscle stimulator (Aurora Scientific, model 701A), were
delivered to platinum electrodes inserted behind the knee to
depolarize the peroneal nerve. The lever system was interfaced to a
PC using a multi-function data acquisition board (National
Instruments model USB-6229, Austin, Tex.). Custom software written
in LabVIEW (National Instruments) was used to configure and trigger
stimulation, control lever arm position, and record data to disk.
After the right leg was studied, the animal was removed from the
test stand and the left leg prepared and studied in an identical
manner. All contractile measurements were initiated at the
empirically determined optimal length (L.sub.0) for tetanic tension
(200 Hz stimulation). Fiber length (FL) was calculated as 0.60
L.sub.0. Susceptibility to mechanical strain was evaluated by
subjecting the muscle to 5 lengthening (eccentric) trials. During
each lengthening trial the muscle was tetanically stimulated at
L.sub.0 for 100 ms and then lengthened to 1.20 FL at a velocity of
+1.5 FL/s. Stimulation ceased at the conclusion of the lengthening
ramp. The muscle was held for 200 ms before being returned to its
L.sub.0 at a velocity of -1.5 FL/s. The series of lengthening
contractions was bracketed by fixed-end tetanic contractions, which
were used to evaluate the overall change in force due to the
lengthening contractions. One minute separated all contractions.
Specific force was calculated as active tetanic force divided by
physiological cross-sectional area (pCSA). The pCSA of the TA was
calculated as muscle mass divided by the product of FL and muscle
density. Muscle density was taken as 1.06 mg/mm.sup.3.
EXAMPLE XIII
CRISPR Activity Reporter System
[0162] A CRISPR activity reporter system and related data is
depicted in FIGS. 14A-14J. The CRISPER activity reporter system is
a programmable system for fluorescent detection and enrichment of
gene-edited cells in vitro and in vivo.
[0163] According to certain aspects, a mouse reporter allele, Ai9,
encodes the fluorescent tdTomato protein downstream of a ubiquitous
CAGGS promoter and "floxed" STOP cassette (See
[0164] FIG. 14A). Exposure to Cas9, together with paired gRNAs
targeting near the 5' and 3' of loxP sites of the Ai9 allele
(hereafter Ai9 gRNAs), results in precise excision of the
intervening DNA and expression of the downstream tdTomato gene.
This Ai9 CRISPR system thus provides sensitive, fluorescence-based
detection of CRISPR activity with single cell resolution and the
capacity to prospectively detect and isolate gene-edited cells by
fluorescence activated cell sorting (FACS). A pair of gRNAs
directed at 5' and 3' sequences flanking exon 23 of the mouse Dmd
gene (hereafter Dmd23 gRNAs) were designed and tested and which
enabled efficient excision of the intervening DNA. Mdx mice, which
provide a genetic model of human DMD, carry a nonsense mutation in
exon 23 of the Dmd gene, resulting in loss of Dystrophin protein
and destabilization of DMD mRNA. According to methods described
herein, skipping of exon 23 restores Dystrophin reading frame and
results in production of an internally truncated, but still highly
functional protein that can complement dystrophin-deficiency in
dystrophic muscle. To facilitate detection of DMD gene-edited
cells, the Dmd23 gRNAs were coupled to Ai9 gRNAs using a two
plasmid system in which the 3' gRNAs for Ai9 and Dmd were encoded
in one vector and the 5' gRNAs in another vector. This hybrid
vector system effectively links expression of the CRISPR activity
reporter (tdTomato) to genome editing events at the Dmd locus,
because in order to express tdTomato after co-transfection with
these gRNA vectors and Sp humanized Cas9 (Sp hCas9), the target
cell must have received both the 5' and 3' Ai9 gRNAs, and therefore
also must have received both of the linked Dmd23 gRNAs. In vitro
transfection of primary satellite cells from mdx mice carrying the
Ai9 allele (hereafter referred to as mdx;Ai9 mice) with Sp hCas9
+Ai9-Dmd23 coupled gRNAs induced gene editing at both the Ai9 locus
(demonstrated by tdTomato expression, FIG. 14F, right panel) and
Dmd locus (detected by genomic PCR using primers flanking exon 23
and sequencing of amplions, which indicated precise excision and
generation of a hybrid intron fusing introns 22 and 23, FIG. 14E).
DMD gene editing was not seen in mdx;Ai9 cells receiving Ai9 gRNAs
alone (FIG. 14E), although tdTomato expression was equivalently
induced (FIG. 14F, middle panel), confirming that locus specificity
in this system is determined by the genomic complementarity of the
gRNAs used for programming Cas9.
[0165] To confirm that CRISPR-mediated editing of the DMD locus
results in permanent genomic modification and production of exon
skipped mRNA and protein, co-transfected primary satellite cells
were FACSorted based on tdTomato expression, expanded in vitro, and
differentiated to myotubes. RNA and protein were then harvested for
standard RT-PCR and sanger sequencing analysis, which demonstrated
the presence of the exon 23 skipped Dmd mRNA in cells receiving Sp
hCas9 and coupled Ai9-Dmd23 gRNAs, but not in cells receiving Sp
hCas9 and Ai9 gRNAs only (FIG. 14G). Sequencing of the exon skipped
amplicon confirmed the production of an in-frame transcript that
juxtaposes exons 22 and 24. Levels of exon skipped transcripts were
quantified using Taqman analysis, and represented 24-47% of total
DMD mRNA in cells receiving Ai9-Dmd23 coupled gRNAs (FIG. 14H). In
contrast, exon skipping was undetectable in cells receiving Sp
hCas9 with only Ai9 gRNAs (FIG. 14H). Dystrophin protein expression
was also restored in CRISPR-modified mdx;Ai9 cells, and was
detectable by Western blot (FIG. 141) in in vitro differentiated
myotubes and immunofluescence in in vivo engrafted muscle fibers
derived from gene edited satellite cells (FIG. 14J).
EXAMPLE XIV
Delivery of a CRISPR System Using AAV
[0166] Aspects of the present disclosure are directed to methods of
delivering a Cas9 protein and guide RNA to a cell, including a cell
within an individual, using a viral vector, such as an
adeno-associated virus. AAVs are currently in use in human clinical
trials and provide the opportunity for both local and systemic
delivery of virally encoded gene editing complexes. Given the
limited packaging capacity (4.8 kb) of AAVs, the orthologous Cas9
protein from Streptococcus aureus (Sa), which is .about.1 kb
smaller than Sp Cas9, and can target any desired locus in the
genome containing a "NNGRRT" PAM sequence was used. To also employ
tdTomato expression as a reporter of in vivo CRISPR activity,
paired Sa gRNAs targeting sequences flanking the STOP cassette of
the Ai9 allele were generated. Using the Ai9 CRISPR activity
reporter, the SaCas9 gRNA scaffold was optimized by incorporating
base modifications that have been reported to remove a putative RNA
polymerase III transcription terminator and enhance the assembly of
gRNA and catalytically inactive orthologous Sp Cas9. The same base
modifications in the gRNA scaffold that increase the efficiency of
Sp CRISPR complex formation also enhance gene targeting by SaCas9
(See FIG. 18A-18C). The optimized Sa gRNA scaffold was used to
generate Dmd23 Sa gRNAs. 16 pairs of Dmd23 Sa gRNAs were screened
and the pair with highest efficiency for precise DNA excision at
exon 23 (see FIG. 18D) was identified. AAV constructs were produced
encoding SaCas9 and Ai9 Sa gRNAs or Dmd23 Sa gRNAs in two different
vectors (FIG. 19A) or a single vector (FIG. 19B). Two different
small promoters (173CMV or elongation factor la short (EFS)) were
used to drive expression of SaCas9 in the single vector CRISPR
constructs, while SaCas9 was expressed from the SV40 enhancer and
CMV promoter in the dual vector system. Dual or single CRISPR AAV
constructs targeting Dmd23 (AAV-Dmd CRISPR) were used to generate
AAV serotype DJ and transduce myotubes derived from mdx primary
satellite cells in order to compare the efficiency of different
constructs for inducing exon skipping. Quantification of exon
skipping in transduced mdx myotubes showed that dual AAV constructs
induce exon skipping more efficiently than the single vector
constructs and that EFS-driven SaCas9 is more efficient than
173CMV-driven SaCas9 (FIG. 19C). Though both the single vector
system and the two vector system produced exon skipping, the dual
vector system was selected for in vivo Dmd targeting according to
the methods described herein. Accordingly, methods described herein
are directed to a first AAV constructs encoding SaCas9 and Ai9 Sa
gRNAs and a second AAV construct encoding SaCas9 and Dmd23 Sa
gRNAs. The first and second AAV constructs are introduced to a
cell.
EXAMPLE XV
In Vivo Delivery of a CRISPR System Using AAV
[0167] Aspects of the present disclosure are directed to methods of
gene editing by injecting a Cas9/guide RNA system encoded into one
or more AAV vectors into muscle of an animal. For in vivo
injections, dual AAVs were pseudotyped to serotype 9, which
exhibits robust transduction of mouse skeletal and cardiac muscle.
The tibialis anterior (TA) muscles of mdx;Ai9 mice were injected
with AAV9-SaCas9 +AAV9-Ai9 gRNAs (7.5E+11 vg each) or vehicle to
test the potential for in vivo targeting of an endogenous gene in
multinucleated muscle fibers. Four weeks later, muscles were
harvested for immunofluorescence to assess genomic editing events.
TdTomato fluorescence was detected in muscles injected with AAV-Ai9
CRISPR, but not in muscles injected with vehicle alone (FIG. 15A)
demonstrating precise genome editing in multinucleated skeletal
muscle fibers after in vivo delivery of CRISPR AAV.
[0168] Similar to targeting at the Ai9 locus, co-delivery of
AAV9-SaCas9 +AAV9-Dmd23 gRNAs resulted in robust and specific
modification of the Dmd locus in skeletal muscles in vivo. Genomic
PCR and sanger sequencing demonstrated precise excision of exon 23
in muscles of mice injected with AAV9-SaCas9 +AAV9-Dmd23 gRNAs, but
not AAV9-Sa Cas9 +AAV9-Ai9 gRNAs (FIG. 15B). Consistent with
genomic data, RT-PCR and sanger sequencing analysis demonstrated
the presence of exon skipped DMD mRNA specifically in muscles
receiving AAV9-SaCas9+AAV9-Dmd23 gRNAs (FIG. 15C). Quantification
of exon skipping efficiency by Taqman indicated an average
targeting rate of 39% (.+-.8.3%) (FIG. 15D). As seen for targeting
of primary satellite cells in culture, local, in vivo CRISPR-based
editing of skeletal muscle restored expression of Dystrophin
protein, which was detected by Western blot (FIG. 15E) and present
at the surface of muscle fibers of mdx;Ai9 mouse muscle for at
least four weeks after transduction with AAV-Dmd CRISPR (FIG. 15F).
In contrast, Dystrophin expression was undetectable by Western blot
(FIG. 15E) and present only on rare revertant fibers (FIG. 15F) in
mdx;Ai9 mice receiving control Ai9 gRNAs.
EXAMPLE XVI
In Situ Muscle Force Assessment
[0169] To evaluate the functional consequences of CRISPR-mediated
induction of exon-skipped DMD mRNA in mdx muscle, a subset of mice
injected intramuscularly with AAV-Dmd23 CRISPR were subjected to in
situ muscle force assessment. Muscles injected with AAV9-SaCas9
+AAV9-Dmd23 showed significantly increased specific force (FIG.
15G), and attenuated force drop after eccentric damage (FIG. 15H)
compared to the contralateral vehicle injected muscle,
demonstrating a therapeutic benefit of permanent exon skipping in
the mdx model. In contrast, differences in specific force (FIG.
15G) and force drop (FIG. 15H) for AAV9-SaCas9+AAV9-Ai9 gRNAs
injected mice were not statistically significant between the
virus-injected and vehicle-injected muscles. According to certain
aspects, CRISPR/Cas9 gene editing systems and methods described
herein are effective for in vivo genomic modification, including
the introduction of therapeutic gene deletions, even in highly
multinucleated, post-mitotic cell types such as muscle fibers. The
CRISPR-Cas9 system enables permanent modification of the targeted
loci, providing enduring production of the modified gene product
for as long as the targeted cell/nucleus survives.
EXAMPLE XVII
Systemic Delivery of Gene Editing Complex
[0170] Methods described herein restore Dystrophin expression after
intramuscular delivery of AAV-Dmd CRISPR in mdx mice. Aspects of
the present disclosure also include systemic administration of a
Cas9/guide RNA system using an AAV. According to this aspect, dual
AAV9 vectors (1.5E+12 vg each) were co-injected intraperitoneally
into mdx;Ai9 mice at postnatal day 3 (P3). 3 weeks later, muscles
were harvested and analyzed for locus-specific gene targeting.
RT-PCR and sanger sequencing demonstrated detectable exon 23
skipping in multiple skeletal muscles and cardiac muscle of mice
receiving systemic AAV9-SaCas9 +AAV9-Dmd23 gRNAs. In contrast, no
exon skipping was apparent in dystrophin mRNA in animals receiving
Ai9 gRNAs instead (FIG. 16B). Quantification of exon skipped
transcripts as a percentage of total DMD mRNA confirmed widespread
targeting in animals receiving systemic AAV9-SaCas9 +AAV9-Dmd23
gRNAs, with levels varying from 3-18% in different muscle groups
(FIG. 16C). Finally, Western blot (FIG. 16D and FIG. 20) and
immunofluorescence analysis (FIG. 16E) of Dystrophin protein
expression, which is normally lacking in mdx mice and absent from
cardiac and skeletal muscles of mdx;Ai9 mice receiving
AAV9-SaCas9+AAV9-Ai9 gRNAs, showed restoration of Dystrophin in
mice receiving AAV9-SaCas9 +AAV9-Dmd23 gRNAs in all muscle groups
examined. Levels of Dystrophin protein in dual AAV-Dmd CRIPSR
treated mice varied among individual mice and muscle groups, with
amounts as high as 5% and as low as <0.1% of wild-type. Local
and systemic injection of the single vector AAV-Dmd CRISPR, in
which SaCas9 is expressed under the control of the EFS promoter,
yielded lower exon skipping efficiencies compared to the dual
AAV-Dmd CRISPR system (FIG. 19E and 19F).
EXAMPLE XVIII
Targeting of Satellite Cells In Vivo
[0171] According to certain aspects, a Cas9/guide RNA system
encoded into one or more AAV vectors is targeted to stem cells
(satellite cells) where the target gene is edited in the stem cell
(satellite cell). According to certain aspects, dystrophic
pathology and other acute and chronic muscle injuries activate
satellite cells, leading to muscle regenerative responses that add
new nuclei to damaged fibers. In the context of gene therapy in
muscle as described herein, the target gene in the satellite cells
is permanently edited/corrected to avoid the addition of new,
non-targeted nuclei reducing the fraction of nuclei in muscle
fibers producing therapeutic exon skipped mRNAs. According to one
aspect, the satellite cells are targeted in vivo for target gene
modification or alteration so as to provide continual replenishment
of gene-edited myonuclei through normal muscle damage and repair
mechanisms.
[0172] AAV9-CRISPR gene editing in satellite cells in vivo was
monitored using the sensitive Ai9 fluorescent reporter system
described herein. To facilitate the discrimination of satellite
cells, the mdx;Ai9 mice were crossed with previously described
Pax7-ZsGreen animals, in which satellite cells are specifically
marked by green fluorescence. Pax7-ZsGreen.sup..+-.;Mdx;Ai9 mice
were injected intramuscularly (FIG. 17B-17D) or intraperitoneally
(FIG. 17E-17G,) with AAV9 encoding Cre recombinase or Ai9 CRISPR
components, and skeletal muscles were harvested 2 weeks later
for
[0173] FACS isolation of ZsGreen+muscle satellite cells (FIG. 17A).
Flow cytometric analysis demonstrated that about 36% (.+-.1.9%) of
Pax7-ZsGreen+cells expressed tdTomato when isolated from muscles
injected locally with AAV9-Cre (6E+11 vg), suggesting significant
transduction by AAV of endogenous satellite cells in these mice
(FIG. 17B, 17C). Systemic delivery of AAV9-Cre (3E+11 vg) also
resulted in transduction of muscle satellite cells, albeit at lower
frequencies (9.6% .+-.2.6%) (FIG. 17E, 17F). Myogenic
differentiation of ZsGreen+satellite cells isolated from mice
receiving either intramuscular or systemic AAV9-Cre produced
tdTomato+myotubes, demonstrating that permanent recombination at
the Ai9 locus was induced in these muscle progenitors by AAV9-Cre.
(FIG. 17D and 17G).
[0174] TdTomato expression was also detected in
Pax7-ZsGreen+satellite cells harvested from mice receiving
AAV9-SaCas9 +AAV9-Ai9 gRNAs intramuscularly (FIG. 17B, 17C) or
intraperitoneally (FIG. 17E, 17F) with efficiency of .about.3%.
These CRISPR-targeted satellite cells also differentiated to
produce tdTomato+myotubes (FIG. 17D and 17G), again consistent with
stable modification of the Ai9 allele following in vivo exposure to
Ai9-CRISPR.
EXAMPLE XVIII
Permanent Genetic Modification of Cells Derived from Satellite
Cells
[0175] Methods described herein are directed to in vivo
administration of a Cas9/guide RNA system as described herein to an
animal sufficient to introduce the Cas9/guide RNA system to in vivo
produced satellite cells and wherein the Cas9/guide RNA system
edits the target gene in the satellite cell and wherein the
genetically modified satellite cell differentiates into a muscle
cell that has retained the genetic modification. According to
certain aspects, methods described herein use a Cas9/guide RNA
system to transduce and genomically modify endogenous satellite
cells. The genomically modify endogenous satellite cells
differentiate into muscle cells with the genetic modification.
According to certain aspects, methods described herein use a
Cas9/guide RNA system targeting Dmd to transduce and genomically
modify endogenous satellite cells which are precursor cells in
dystrophic muscles, and wherein the transformed satellite cells
diferentiate into muscle cells with the genetically altered Dmd.
According to certain aspects, methods are provided for in situ gene
editing in muscle satellite cells as well as terminally
differentiated multinucleated fibers.
[0176] Myosatellite cells or satellite cells are small mononuclear
multipotent cells with virtually no cytoplasm found in mature
muscle. Satellite cells are precursors to skeletal muscle cells,
able to give rise to satellite cells or differentiated skeletal
muscle cells. They have the potential to provide additional
myonuclei to their parent muscle fiber, or return to a quiescent
state. More specifically, upon activation, satellite cells can
re-enter the cell cycle to proliferate and differentiate into
myoblasts.
[0177] Myosatellite cells are located between the basement membrane
and the sarcolemma of muscle fibers, and can lie in grooves either
parallel or transversely to the longitudinal axis of the fibre.
Their distribution across the fibre can vary significantly.
Non-proliferative, quiescent myosatellite cells, which adjoin
resting skeletal muscles, can be identified by their distinct
location between sarcolemma and basal lamina, a high
nuclear-to-cytoplasmic volume ratio, few organelles (e.g.
ribosomes, endoplasmic reticulum, mitochondria, golgi complexes),
small nuclear size, and a large quantity of nuclear heterochromatin
relative to myonuclei. On the other hand, activated satellite cells
have an increased number of caveolae, cytoplasmic organelles, and
decreased levels of heterochromatin. Satellite cells are able to
differentiate and fuse to augment existing muscle fibers and to
form new fibers. These cells represent the oldest known adult stem
cell niche, and are involved in the normal growth of muscle, as
well as regeneration following injury or disease. In undamaged
muscle, the majority of satellite cells are quiescent; they neither
differentiate nor undergo cell division. In response to mechanical
strain, satellite cells become activated. Activated satellite cells
initially proliferate as skeletal myoblasts before undergoing
myogenic differentiation.
[0178] Pax7-ZsGreen+satellite cells from Pax7-ZsGreen.+-.;Mdx;Ai9
mice injected intramuscularly or systemically with AAV9-SaCas9
+AAV9-Dmd gRNAs or AAV9-SaCas9 +AAV9-Ai9 gRNAs were isolated and
expanded and differentiated in vitro. RT-PCR analysis of mRNA
isolated from satellite cell-derived myotubes demonstrated the
presence of a truncated transcript of the expected size for
gene-edited Dmd in many of the AAV-Dmd CRISPR injected muscles, but
not AAV-Ai9 CRISPR injected ones. In addition, sequencing of this
shorter transcript confirmed site directed excision of exon 23 and
production of an exon-skipped mRNA in which exon 22 is fused to
exon 24 (FIG. 17H and 17J). Levels of exon-skipped transcripts in
the differentiated myotubes were also quantified using Taqman-based
real time PCR (FIGS. 171 and 17K).
EXAMPLE XIX
Embodiments
[0179] Aspects of the present disclosure include a method of
producing an altered gene product in a eukaryotic cell including
providing to the cell two or more guide RNAs and a Cas9 protein,
wherein the two or more guide RNAs are complementary to two or more
target genomic DNA sequences flanking a target excision sequence
including one or more exons in a target gene encoding a
biologically functional polypeptide, wherein the two or more guide
RNAs bind to the two or more complementary target genomic DNA
sequences and the Cas9 protein cleaves the two or more target
genomic DNA sequences thereby removing the one or more exons from
the target gene to produce an altered target gene and wherein the
altered target gene recombines, and wherein the eukaryotic cell
expresses the altered target gene to produce an altered
biologically functional polypeptide. According to one aspect, the
altered biologically functional polypeptide lacks a polypeptide
sequence corresponding to the one or more removed exons. According
to one aspect, the two or more guide RNAs and the Cas9 protein are
foreign to the eukaryotic cell. According to one aspect, the two or
more guide RNAs and the Cas9 protein are foreign to each other.
According to one aspect, the two or more guide RNAs and the Cas9
protein are non-naturally occurring.
[0180] According to one aspect, the two or more guide RNAs are
provided to the cell by electroporation of the two or more guide
RNAs into the cell. According to one aspect, the Cas9 protein is
provided to the cell by electroporation of the Cas9 protein into
the cell. According to one aspect, the two or more guide RNAs are
provided to the cell by introducing into the cell a first foreign
nucleic acid sequence encoding the two or more guide RNAs.
According to one aspect, the two or more guide
[0181] RNAs are provided to the cell by introducing into the cell a
first foreign nucleic acid sequence encoding the two or more guide
RNAs present in a plasmid or vector. According to one aspect, the
Cas 9 protein is provided to the cell by introducing into the cell
a second foreign nucleic acid sequence encoding the Cas 9 protein.
According to one aspect, the Cas 9 protein is provided to the cell
by introducing into the cell a second foreign nucleic acid sequence
encoding the Cas 9 protein present in a plasmid or vector.
According to one aspect, the eukaryotic cell is a yeast cell, a
plant cell, a vertebrate cell, a mammalian cell or a human cell.
According to one aspect, the eukaryotic cell is within a mammal
According to one aspect, the eukaryotic cell is a skeletal muscle
cell. According to one aspect, the eukaryotic cell is a cardiac
muscle cell. According to one aspect, the target excision sequence
is greater than 45 kb. According to one aspect, the target gene
encodes dystrophin protein. According to one aspect, the target
gene encodes dystrophin protein and the one or more exons is exon
23. According to one aspect, the target gene encodes dystrophin
protein and the one or more exons is exon 52 and exon 53. According
to one aspect, the RNA includes between about 10 to about 250
nucleotides. According to one aspect, the RNA includes between
about 20 to about 100 nucleotides. According to one aspect, the
guide RNA includes a guide sequence fused to a trans-activating cr
(tracr) sequence. According to one aspect, the ratio of plasmid
encoding the Cas9 protein to the plasmid encoding the guide RNA is
between 1:5 and 2:1. According to one aspect, the plasmid encoding
the guide RNA is modified to increase the expression of the RNA by
removing a potential premature transcription termination site.
According to one aspect, the one or more exons includes a mutation.
According to one aspect, the
[0182] Cas9 protein is provided to the cell by electroporation of
the Cas9 mRNA into the cell. According to one aspect, the guide RNA
and the Cas9 protein co-localize to the target genomic DNA sequence
to form a complex. According to one aspect, the target nucleic acid
is chromosomal DNA. According to one aspect, the Cas9 protein is
wild type Cas9, Cas9 nickase or a nuclease null Cas9 including a
nuclease. According to one aspect, the guide RNA and the Cas9
protein are combined and then contacted with the target gene.
According to one aspect, the guide RNA and the Cas9 protein are
combined and then contacted with the target gene within a cell.
According to one aspect, the method includes providing to the cell
a plurality of guide RNAs with each having a portion complementary
to a target genomic DNA sequence. According to one aspect, the cell
is a transplantable cell. According to one aspect, the cell is a
progenitor cell. According to one aspect, the cell is a stem cell.
According to one aspect, the cell is a muscle stem cell.
[0183] Aspects of the present disclosure are directed to a skeletal
muscle cell including a Cas9 protein and two or more guide RNAs
complementary to two or more target genomic DNA sequences flanking
a target excision sequence including one or more exons in a target
gene encoding dystrophin protein. According to one aspect, the one
or more exons are in the exon 45-55 region. According to one
aspect, the one or more exons include exon 23, exon 52 or exon
53.
[0184] Aspects of the present disclosure are directed to according
to a skeletal muscle cell including a first nucleic acid encoding
two or more guide RNAs complementary to two or more target genomic
DNA sequences flanking a target excision sequence including one or
more exons in a target gene encoding dystrophin protein and a
second nucleic acid encoding a Cas9 protein. According to one
aspect, the one or more exons are in the exon 45-55 region.
According to one aspect, the one or more exons include exon 23,
exon 52 or exon 53. According to one aspect, the first nucleic acid
is within a plasmid or vector. According to one aspect, the second
nucleic acid is within a plasmid or vector. According to one
aspect, the second nucleic acid is within a viral vector. According
to one aspect, the second nucleic acid is within a viral vector
selected from the group consisting of lentivirus, adenovirus,
adeno-associated virus, retrovirus, herpes simplex virus, or sendai
virus.
[0185] Aspects of the present disclosure are directed to a skeletal
muscle cell including a Cas9 protein and two or more guide RNAs
complementary to two or more target genomic DNA sequences flanking
a target excision sequence including one or more exons in a target
gene encoding dystrophin protein. According to one aspect, the one
or more exons are in the exon 45-55 region. According to one
aspect, the one or more exons include exon 23, exon 52 or exon
53.
[0186] Aspects of the present disclosure are directed to a muscle
stem cell including a first nucleic acid encoding two or more guide
RNAs complementary to two or more target genomic DNA sequences
flanking a target excision sequence including one or more exons in
a target gene encoding dystrophin protein and a second nucleic acid
encoding a Cas9 protein. According to one aspect, the one or more
exons are in the exon 45-55 region. According to one aspect, the
one or more exons include exon 23, exon 52 or exon 53. According to
one aspect, the first nucleic acid is within a plasmid or vector.
According to one aspect, the second nucleic acid is within a
plasmid or vector. According to one aspect, the second nucleic acid
is within a viral vector. According to one aspect, the second
nucleic acid is within a viral vector selected from the group
consisting of lentivirus, adenovirus, adeno-associated virus,
retrovirus, herpes simplex virus, or sendai virus.
[0187] Aspects of the present disclosure are directed to a
genetically modified skeletal muscle cell including a first nucleic
acid encoding two or more guide RNAs complementary to two or more
target genomic DNA sequences flanking a target excision sequence
including one or more exons in a target gene encoding dystrophin
protein and a second nucleic acid encoding a Cas9 protein and
wherein the target gene encoding dystrophin protein lacks one or
more of exon 23, exon 52 or exon 53.
[0188] Aspects of the present disclosure are directed to a
genetically modified muscle stem cell including a first nucleic
acid encoding two or more guide RNAs complementary to two or more
target genomic DNA sequences flanking a target excision sequence
including one or more exons in a target gene encoding dystrophin
protein and a second nucleic acid encoding a Cas9 protein and
wherein the target gene encoding dystrophin protein lacks one or
more of exon 23, exon 52 or exon 53.
[0189] Aspects of the present disclosure are directed to a method
of producing an altered gene product in a eukaryotic cell within a
mammal comprising injecting two plasmids into the mammal, wherein
the two plasmids include a first nucleic acid encoding two or more
guide RNAs complementary to two or more target genomic DNA
sequences flanking a target excision sequence including one or more
exons in a target gene encoding dystrophin protein and a second
nucleic acid encoding a Cas9 protein, wherein the two or more guide
RNAs bind to the two or more complementary target genomic DNA
sequences and the Cas9 protein cleaves the two or more target
genomic DNA sequences thereby removing the one or more exons from
the target gene to produce an altered target gene and wherein the
altered target gene recombines, and wherein the eukaryotic cell
expresses the altered target gene to produce an altered
biologically functional polypeptide. According to one aspect, the
eukaryotic cell is a skeletal muscle cell oir cardiac cell.
According to one aspect, the eukaryotic cell is a muscle stem cell
or cardiac stem cell. According to one aspect, the eukaryotic cell
is a member of the group consisting of a skeletal muscle cell, a
muscle stem cell, a progenitor cell and a stem cell. According to
one aspect, the one or more exons is exon 23, exon 52 or exon
53.
[0190] Aspects of the present disclosure are directed to a method
of removing one or more mutations from a target gene encoding a
dystrophin protein in a eukaryotic cell inclouding providing to the
cell two or more guide RNAs and a Cas9 protein, wherein the two or
more guide RNAs are complementary to two or more target genomic DNA
sequences flanking a target excision sequence including one or more
exons having one or more mutations in the target gene, wherein the
two or more guide RNAs bind to the two or more complementary target
genomic DNA sequences and the Cas9 protein cleaves the two or more
target genomic DNA sequences thereby removing the one or more exons
having one or more mutations from the target gene to produce an
altered target gene and wherein the altered target gene recombines,
and wherein the eukaryotic cell expresses the altered target gene
to produce a functional truncated dystrophin protein. According to
one aspect, the eukaryotic cell is a skeletal muscle cell or
cardiac cell. According to one aspect, the eukaryotic cell is a
muscle stem cell or cardiac stem cell. According to one aspect, the
eukaryotic cell is a member of the group consisting of a skeletal
muscle cell, a muscle stem cell, a progenitor cell and a stem cell.
According to one aspect, the one or more exons is exon 23, exon 52
or exon 53. According to one aspect, the eukaryotic cell is within
a mammal According to one aspect, the one or more exons are in the
exon 45-55 region.
Sequence CWU 1
1
34137DNAArtificialFigure 14E intron 22 and intron 23 1aaatataata
tgccctgtcc gaggtttggc ctttaaa 37237DNAArtificialFigure 14G exon 23
and exon 24 2taaacttcga aaatttcaga atcacataaa aacctta
37337DNAArtificialFigure 14G exon 22 and exon 24 3gagactcggg
aaattacaga atcacataaa aacctta 37437DNAArtificialFigure 15B Exon 23
excision in DNA 4taatataata gaaattattt tcttggattg tctgtat
37536DNAArtificialFigure 15C Exon 23 excision in DNA 5taaacttcga
aaatttcaga atcacataaa aacctt 36636DNAArtificialFigure 15C Exon 23
excision in DNA 6agactcggga aattacagaa tcacataaaa acctaa
36737DNAArtificialFigure 17H skipping of exon 23 in the mRNA
7taaacttcga aaatttcaga atcacataaa aacctta 37838DNAArtificialFigure
17H skipping of exon 23 in the mRNA 8gagactcggg aaattacaga
atcacataaa aaccttac 38938DNAArtificialFigure 17J exon 23 and 24
9taaacttcga aaatttcaga atcacataaa aaccttac
381037DNAArtificialFigure 17J exon 22 and 24 10agactcggga
aattacagaa tcacataaaa accttac 3711103DNAArtificialFigure 18A gRNA
scaffoldmisc_feature(1)..(20)n is a, c, g, t or u 11nnnnnnnnnn
nnnnnnnnnn guuuuaguac ucuggaaaca gaaucuacua aaacaaggca 60aaaugccgug
uuuaucucgu caacuuguug gcgagauuuu uuu 10312114DNAArtificialFigure
18A gRNA scaffoldmisc_feature(1)..(20)n is a, c, g, t or u
12nnnnnnnnnn nnnnnnnnnn guuuuaagua cucugugcug gaaacagcac agaaucuacu
60uaaacaaggc aaaaugccgu guuuaucucg ucaacuuguu ggcgagauuu uuuu
1141337DNAArtificialFigure 19C exon 22 and 24 13gagactcggg
aaattacaga atcacataaa aacctta 37141368PRTStreptococcus pyogenes
14Met 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 1522DNAArtificialgRNA 15gaataatttc tattatatta ca
221622DNAArtificialgRNA 16ttcgaaaatt tcaggtaagc cg
221722DNAArtificialgRNA 17tcatttctaa aagtcttttg cc
221822DNAArtificialgRNA 18tttgagacac agtataggtt at
2219115DNAArtificialgRNA 19gaataatttc tattatatta cagtttaaga
gctatgctgg aaacagcata gcaagtttaa 60ataaggctag tccgttatca acttgaaaaa
gtggcaccga gtcggtgctt ttttt 11520115DNAArtificialgRNA 20ttcgaaaatt
tcaggtaagc cggtttaaga gctatgctgg aaacagcata gcaagtttaa 60ataaggctag
tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttt
11521115DNAArtificialgRNA 21tcatttctaa aagtcttttg ccgtttaaga
gctatgctgg aaacagcata gcaagtttaa 60ataaggctag tccgttatca acttgaaaaa
gtggcaccga gtcggtgctt ttttt 11522115DNAArtificialgRNA 22tttgagacac
agtataggtt atgtttaaga gctatgctgg aaacagcata gcaagtttaa 60ataaggctag
tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttt
115231395PRTArtificialCas9 protein with NLS 23Met 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 Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val Asp
Pro Lys Lys 1370 1375 1380 Lys Arg Lys Val Asp Pro Lys Lys Lys Arg
Lys Val 1385 1390 1395 2440DNAArtificialSpacer sequence
24gggccatgtg gacatccatg aggtgagaca gtgccagcgt
402537DNAArtificialSpacer sequence 25ggcctgaagc cactacagct
gctggagatc aaggctc 372638DNAArtificialSpacer sequence 26ggcctgaagc
cactacagct gctggagatc aaggctcg 382730DNAArtificialSpacer sequence
27gccattgcag ctgttagaag tgaaagcaag 302827DNAArtificialSpacer
sequence 28ggccctagca tctaagttct cgcaggc 272935DNAArtificialSpacer
sequence 29ggtcattcca tctcagctgt gacagcagcg cagaa
353034DNAArtificialSpacer sequence 30ggaagtcaag gtgacagaca
cacccaagag gtcc 343131DNAArtificialSpacer sequence 31ggacacaccc
aagaggtccc ggagagactt t 313230DNAArtificialSpacer sequence
32gtcaagccca aagtctctcc gggacctctt 303329DNAArtificialSpacer
sequence 33ggaatcccgg tgctgccgct accccctca
293483DNAArtificialScaffold sequence 34gttttagagc tagaaatagc
aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60ggcaccgagt cggtgctttt
ttt 83
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