U.S. patent application number 14/318933 was filed with the patent office on 2014-11-20 for rna-guided human genome engineering.
The applicant listed for this patent is President and Fellows of Harvard College. Invention is credited to George M. CHURCH, Prashant G. MALI, Luhan Yang.
Application Number | 20140342456 14/318933 |
Document ID | / |
Family ID | 50979072 |
Filed Date | 2014-11-20 |
United States Patent
Application |
20140342456 |
Kind Code |
A1 |
MALI; Prashant G. ; et
al. |
November 20, 2014 |
RNA-Guided Human Genome Engineering
Abstract
A method of altering a eukaryotic cell is provided including
transfecting the eukaryotic cell with a nucleic acid encoding RNA
complementary to genomic DNA of the eukaryotic cell, transfecting
the eukaryotic cell with a nucleic acid encoding an enzyme that
interacts with the RNA and cleaves the genomic DNA in a site
specific manner, wherein the cell expresses the RNA and the enzyme,
the RNA binds to complementary genomic DNA and the enzyme cleaves
the genomic DNA in a site specific manner.
Inventors: |
MALI; Prashant G.;
(Somerville, MA) ; CHURCH; George M.; (Brookline,
MA) ; Yang; Luhan; (Somerville, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
President and Fellows of Harvard College |
Cambridge |
MA |
US |
|
|
Family ID: |
50979072 |
Appl. No.: |
14/318933 |
Filed: |
June 30, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2013/075317 |
Dec 16, 2013 |
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14318933 |
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61738355 |
Dec 17, 2012 |
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61799169 |
Mar 15, 2013 |
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Current U.S.
Class: |
435/441 ;
435/254.2; 435/325; 435/366; 435/419; 435/455; 435/468;
435/471 |
Current CPC
Class: |
C12N 2310/20 20170501;
C12N 15/01 20130101; C12N 15/85 20130101; C12N 15/87 20130101; C12N
2810/55 20130101; C12N 15/1024 20130101; C12N 15/102 20130101; C12N
15/10 20130101; C12N 15/8201 20130101; C12N 15/90 20130101; C12N
9/22 20130101; C12N 2800/80 20130101; C12N 15/63 20130101; C12Y
301/00 20130101; C12N 15/907 20130101; C12N 15/81 20130101 |
Class at
Publication: |
435/441 ;
435/471; 435/254.2; 435/468; 435/419; 435/455; 435/325;
435/366 |
International
Class: |
C12N 15/01 20060101
C12N015/01; C12N 15/82 20060101 C12N015/82; C12N 15/85 20060101
C12N015/85; C12N 15/81 20060101 C12N015/81 |
Goverment Interests
STATEMENT OF GOVERNMENT INTERESTS
[0002] This invention was made with government support under P50
HG005550 awarded by National Institutes of Health. The government
has certain rights in the invention.
Claims
1. A method of altering a eukaryotic cell comprising providing to
the eukaryotic cell a guide RNA sequence complementary to a target
nucleic acid sequence, providing to the eukaryotic cell a Cas9
enzyme that interacts with the guide RNA sequence and cleaves the
target nucleic acid sequence in a site specific manner, wherein the
guide RNA sequence binds to the complementary target nucleic acid
sequence and the Cas9 enzyme cleaves the target nucleic acid
sequence in a site specific manner; wherein the guide RNA sequence
includes a guide sequence of GN.sub.19 complementary to the target
nucleic acid sequence and a scaffold sequence and wherein the guide
RNA sequence is between about 100 to about 250 nucleotides.
2. The method of claim 1 wherein the guide RNA is about 250
nucleotides.
3. The method of claim 1 wherein the guide RNA is about 100
nucleotides.
4. The method of claim 1 wherein the eukaryotic cell is a yeast
cell, a plant cell or a mammalian cell.
5. The method or claim 1 wherein the eukaryotic cell is a human
cell.
6. The method of claim 1 wherein a plurality of guide RNAs are
provided to the eukaryotic cell that are complementary to different
target nucleic acid sequences and the Cas9 enzyme cleaves the
different target nucleic acid sequences in a site specific
manner
7. The method of claim 1 wherein the guide RNA is provided to the
eukaryotic cell by introducing to the eukaryotic cell a nucleic
acid encoding the guide RNA, wherein the Cas9 enzyme is provided to
the eukaryotic cell by introducing to the eukaryotic cell a nucleic
acid encoding the Cas9 enzyme, wherein the eukaryotic cell
expresses the guide RNA and the Cas9 enzyme, the guide RNA binds to
complementary target nucleic acid and the Cas9 enzyme cleaves the
target nucleic acid in a site specific manner.
8. A eukaryotic cell including a nucleic acid encoding a guide RNA
sequence complementary to a target nucleic acid sequence, a nucleic
acid encoding a Cas9 enzyme that interacts with the guide RNA
sequence and cleaves the target nucleic acid sequence in a site
specific manner, wherein the eukaryotic cell expresses the guide
RNA and the Cas9 enzyme, the guide RNA binds to the complementary
target nucleic acid and the Cas9 enzyme cleaves the target nucleic
acid in a site specific manner, wherein the guide RNA sequence
includes a guide sequence of GN.sub.19 complementary to the target
nucleic acid sequence and a scaffold sequence and wherein the guide
RNA sequence is between about 100 to about 250 nucleotides.
9. The eukaryotic cell of claim 8 wherein the guide RNA is about
250 nucleotides.
10. The eukaryotic cell of claim 8 wherein the guide RNA is about
100 nucleotides.
11. The eukaryotic cell of claim 8 wherein the eukaryotic cell is a
yeast cell, a plant cell or a mammalian cell.
12. The eukaryotic cell of claim 8 wherein the eukaryotic cell is a
human cell.
13. The eukaryotic cell of claim 8 further including a plurality of
nucleic acids encoding a plurality of guide RNA sequences
complementary to different target nucleic acid sequences.
14. A method of altering a eukaryotic cell comprising providing to
the eukaryotic cell a guide RNA complementary to a target nucleic
acid sequence, providing to the eukaryotic cell a Cas9 enzyme that
interacts with the guide RNA and cleaves the target nucleic acid
sequence in a site specific manner, wherein the guide RNA binds to
the complementary target nucleic acid sequence and the Cas9 enzyme
cleaves the target nucleic acid sequence in a site specific manner;
wherein the guide RNA includes a guide sequence complementary to
the target nucleic acid sequence and a scaffold sequence connected
to the guide sequence and having the following nucleic acid
sequence and structure: ##STR00001##
15. The method of claim 14 wherein the guide RNA is provided to the
eukaryotic cell by introducing to the eukaryotic cell a nucleic
acid encoding the guide RNA, wherein the Cas9 enzyme is provided to
the eukaryotic cell by introducing to the eukaryotic cell a nucleic
acid encoding the Cas9 enzyme, wherein the eukaryotic cell
expresses the guide RNA and the Cas9 enzyme, the guide RNA binds to
complementary target nucleic acid and the Cas9 enzyme cleaves the
target nucleic acid in a site specific manner.
16. The method of claim 14 wherein the eukaryotic cell is a yeast
cell, a plant cell or a mammalian cell.
17. The method or claim 14 wherein the eukaryotic cell is a human
cell.
18. The method of claim 14 wherein a plurality of guide RNAs are
provided to the eukaryotic cell that are complementary to different
target nucleic acid sequences and the Cas9 enzyme cleaves the
different target nucleic acid sequences in a site specific
manner.
19. A method of altering expression of a target nucleic acid
sequence in a eukaryotic cell comprising providing to the
eukaryotic cell a guide RNA sequence complementary to the target
nucleic acid sequence, providing to the eukaryotic cell a Cas9
enzyme that interacts with the guide RNA sequence and cleaves the
target nucleic acid sequence in a site specific manner, wherein the
guide RNA sequence binds to the complementary target nucleic acid
sequence and the Cas9 enzyme cleaves the target nucleic acid
sequence in a site specific manner whereby expression of the target
nucleic acid sequence is altered; wherein the guide RNA sequence
includes a guide sequence of GN.sub.19 complementary to the target
nucleic acid sequence and a scaffold sequence and wherein the guide
RNA sequence is between about 100 to about 250 nucleotides.
20. The method of claim 19 wherein the guide RNA is about 250
nucleotides.
21. The method of claim 19 wherein the guide RNA is about 100
nucleotides.
22. The method of claim 19 wherein the eukaryotic cell is a yeast
cell, a plant cell or a mammalian cell.
23. The method or claim 19 wherein the eukaryotic cell is a human
cell.
24. The method of claim 19 wherein a plurality of guide RNAs are
provided to the eukaryotic cell that are complementary to different
target nucleic acid sequences and the Cas9 enzyme cleaves the
different target nucleic acid sequences in a site specific
manner.
25. The method of claim 19 wherein the guide RNA is provided to the
eukaryotic cell by introducing to the eukaryotic cell a nucleic
acid encoding the guide RNA, wherein the Cas9 enzyme is provided to
the eukaryotic cell by introducing to the eukaryotic cell a nucleic
acid encoding the Cas9 enzyme, wherein the eukaryotic cell
expresses the guide RNA and the Cas9 enzyme, the guide RNA binds to
complementary target nucleic acid and the Cas9 enzyme cleaves the
target nucleic acid in a site specific manner.
Description
RELATED APPLICATION DATA
[0001] This application is a continuation of PCT application no.
PCT/US2013/075317, designating the United States and filed Dec. 16,
2013; which claims the benefit U.S. Provisional Patent Application
No. 61/779,169, filed on Mar. 13, 2013 and U.S. Provisional
Application No. 61/738,355, filed on Dec. 17, 2012; each of which
are hereby incorporated by reference in their entireties.
BACKGROUND
[0003] Bacterial and archaeal CRISPR systems rely on crRNAs in
complex with Cas proteins to direct degradation of complementary
sequences present within invading viral and plasmid DNA (1-3). A
recent in vitro reconstitution of the S. pyogenes type II CRISPR
system demonstrated that crRNA fused to a normally trans-encoded
tracrRNA is sufficient to direct Cas9 protein to
sequence-specifically cleave target DNA sequences matching the
crRNA (4).
SUMMARY
[0004] The present disclosure references documents numerically
which are listed at the end of the present disclosure. The document
corresponding to the number is incorporated by reference into the
specification as a supporting reference corresponding to the number
as if fully cited.
[0005] According to one aspect of the present disclosure, a
eukaryotic cell is transfected with a two component system
including RNA complementary to genomic DNA and an enzyme that
interacts with the RNA. The RNA and the enzyme are expressed by the
cell. The RNA of the RNA/enzyme complex then binds to complementary
genomic DNA. The enzyme then performs a function, such as cleavage
of the genomic DNA. The RNA includes between about 10 nucleotides
to about 250 nucleotides. The RNA includes between about 20
nucleotides to about 100 nucleotides. According to certain aspects,
the enzyme may perform any desired function in a site specific
manner for which the enzyme has been engineered. According to one
aspect, the eukaryotic cell is a yeast cell, plant cell or
mammalian cell. According to one aspect, the enzyme cleaves genomic
sequences targeted by RNA sequences (see references (4-6)), thereby
creating a genomically altered eukaryotic cell.
[0006] According to one aspect, the present disclosure provides a
method of genetically altering a human cell by including a nucleic
acid encoding an RNA complementary to genomic DNA into the genome
of the cell and a nucleic acid encoding an enzyme that performs a
desired function on genomic DNA into the genome of the cell.
According to one aspect, the RNA and the enzyme are expressed,
According to one aspect, the RNA hybridizes with complementary
genomic DNA. According to one aspect, the enzyme is activated to
perform a desired function, such as cleavage, in a site specific
manner when the RNA is hybridized to the complementary genomic DNA.
According to one aspect, the RNA and the enzyme are components of a
bacterial Type II CRISPR system.
[0007] According to one aspect, a method of altering a eukaryotic
cell is providing including transfecting the eukaryotic cell with a
nucleic acid encoding RNA complementary to genomic DNA of the
eukaryotic cell, transfecting the eukaryotic cell with a nucleic
acid encoding an enzyme that interacts with the RNA and cleaves the
genomic DNA in a site specific manner, wherein the cell expresses
the RNA and the enzyme, the RNA binds to complementary genomic DNA
and the enzyme cleaves the genomic DNA in a site specific manner.
According to one aspect, the enzyme is Cas9 or modified Cas9 or a
homolog of Cas9. According to one aspect, the eukaryotic cell is a
yeast cell, a plant cell or a mammalian cell. 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.
[0008] According to one aspect, a method of altering a human cell
is provided including transfecting the human cell with a nucleic
acid encoding RNA complementary to genomic DNA of the eukaryotic
cell, transfecting the human cell with a nucleic acid encoding an
enzyme that interacts with the RNA and cleaves the genomic DNA in a
site specific manner, wherein the human cell expresses the RNA and
the enzyme, the RNA binds to complementary genomic DNA and the
enzyme cleaves the genomic DNA in a site specific manner. According
to one aspect, the enzyme is Cas9 or modified Cas9 or a homolog of
Cas9. 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.
[0009] According to one aspect, a method of altering a eukaryotic
cell at a plurality of genomic DNA sites is provided including
transfecting the eukaryotic cell with a plurality of nucleic acids
encoding RNAs complementary to different sites on genomic DNA of
the eukaryotic cell, transfecting the eukaryotic cell with a
nucleic acid encoding an enzyme that interacts with the RNA and
cleaves the genomic DNA in a site specific manner, wherein the cell
expresses the RNAs and the enzyme, the RNAs bind to complementary
genomic DNA and the enzyme cleaves the genomic DNA in a site
specific manner. According to one aspect, the enzyme is Cas9.
According to one aspect, the eukaryotic cell is a yeast cell, a
plant cell or a mammalian cell. 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIGS. 1A-1C depict genome editing in human cells using an
engineered type II CRISPR system. (A) sets forth SEQ ID NO:17; (B)
sets forth SEQ ID NO:18.
[0011] FIGS. 2A-2F depict RNA-guided genome editing of the native
AAVS1 locus in multiple cell types. (A) sets forth SEQ ID NO:19;
(E) sets forth SEQ ID NOs:20 and 21.
[0012] FIGS. 3A-3C depict a process mediated by two catalytic
domains in the Cas9 protein. (A) sets forth SEQ ID NO:22; (B) sets
forth SEQ ID NO:23; (C) sets forth SEQ ID NOs:24-31.
[0013] FIG. 4 depicts that all possible combinations of the repair
DNA donor, Cas9 protein, and gRNA were tested for their ability to
effect successful HR in 293Ts.
[0014] FIGS. 5A-5B depict the analysis of gRNA and Cas9 mediated
genome editing. (B) sets forth SEQ ID NO:19.
[0015] FIGS. 6A-6B depict 293T stable lines each bearing a distinct
GFP reporter construct. (A) depicts sequences set forth as SEQ ID
NOs;32-34.
[0016] FIG. 7 depicts gRNAs targeting the flanking GFP sequences of
the reporter described in FIG. 1B (in 293Ts).
[0017] FIGS. 8A-8B depict 293T stable lines each bearing a distinct
GFP reporter construct. (A) depicts sequences set forth as SEQ ID
NOs;35-36.
[0018] FIGS. 9A-9C depict human iPS cells (PGP1) that were
nucleofected with constructs. (A) sets forth SEQ ID NO:19.
[0019] FIGS. 10A-10B depict RNA-guided NHEJ in K562 cells. (A) sets
forth SEQ ID NO:19.
[0020] FIGS. 11A-11B depict RNA-guided NHEJ in 293T cells. (A) sets
forth SEQ ID NO:19.
[0021] FIGS. 12A-12C depict HR at the endogenous AAVS1 locus using
either a dsDNA donor or a short oligonucleotide donor. (C) sets
forth SEQ ID NOs:37-38.
[0022] FIGS. 13A-13B depict the methodology for multiplex
synthesis, retrieval and U6 expression vector cloning of guide RNAs
targeting genes in the human genome. (A) sets forth SEQ ID
NOs:39-41.
[0023] FIGS. 14A-14D depict CRISPR mediated RNA-guided
transcriptional activation. (A) sets forth SEQ ID NOs:42-43.
[0024] FIGS. 15A-15B depict gRNA sequence flexibility and
applications thereof. (A) sets forth SEQ ID NO:44.
DETAILED DESCRIPTION
[0025] According to one aspect, a human codon-optimized version of
the Cas9 protein bearing a C-terminus SV40 nuclear localization
signal is synthetized and cloned into a mammalian expression system
(FIG. 1A and FIG. 3A). Accordingly, FIG. 1 is directed to genome
editing in human cells using an engineered type II CRISPR system.
As shown in FIG. 1A, RNA-guided gene targeting in human cells
involves co-expression of the Cas9 protein bearing a C-terminus
SV40 nuclear localization signal with one or more guide RNAs
(gRNAs) expressed from the human U6 polymerase III promoter. Cas9
unwinds the DNA duplex and cleaves both strands upon recognition of
a target sequence by the gRNA, but only if the correct
protospacer-adjacent motif (PAM) is present at the 3' end. Any
genomic sequence of the form GN.sub.20GG can in principle be
targeted. As shown in FIG. 1B, a genomically integrated GFP coding
sequence is disrupted by the insertion of a stop codon and a 68 bp
genomic fragment from the AAVS1 locus. Restoration of the GFP
sequence by homologous recombination (HR) with an appropriate donor
sequence results in GFP.sup.+ cells that can be quantitated by
FACS. T1 and T2 gRNAs target sequences within the AAVS1 fragment.
Binding sites for the two halves of the TAL effector nuclease
heterodimer (TALEN) are underlined. As shown in FIG. 1C, bar graph
depict HR efficiencies induced by T1, T2, and TALEN-mediated
nuclease activity at the target locus, as measured by FACS.
Representative FACS plots and microscopy images of the targeted
cells are depicted below (scale bar is 100 microns). Data is
mean+/-SEM (N=3).
[0026] According to one aspect, to direct Cas9 to cleave sequences
of interest, crRNA-tracrRNA fusion transcripts are expressed,
hereafter referred to as guide RNAs (gRNAs), from the human U6
polymerase III promoter. According to one aspect, gRNAs are
directly transcribed by the cell. This aspect advantageously avoids
reconstituting the RNA processing machinery employed by bacterial
CRISPR systems (FIG. 1A and FIG. 3B) (see references (4, 7-9)).
According to one aspect, a method is provided for altering genomic
DNA using a U6 transcription initiating with G and a PAM
(protospacer-adjacent motif) sequence -NGG following the 20 by
crRNA target. According to this aspect, the target genomic site is
in the form of GN.sub.20GG (See FIG. 3C).
[0027] According to one aspect, a GFP reporter assay (FIG. 1B) in
293T cells was developed similar to one previously described (see
reference (10)) to test the functionality of the genome engineering
methods described herein. According to one aspect, a stable cell
line was established bearing a genomically integrated GFP coding
sequence disrupted by the insertion of a stop codon and a 68 bp
genomic fragment from the AAVS1 locus that renders the expressed
protein fragment non-fluorescent. Homologous recombination (HR)
using an appropriate repair donor can restore the normal GFP
sequence, which allows one to quantify the resulting GFP cells by
flow activated cell sorting (FACS).
[0028] According to one aspect, a method is provided of homologous
recombination (HR). Two gRNAs are constructed, T1 and T2, that
target the intervening AAVS1 fragment (FIG. 1b). Their activity to
that of a previously described TAL effector nuclease heterodimer
(TALEN) targeting the same region (see reference (11)) was
compared. Successful HR events were observed using all three
targeting reagents, with gene correction rates using the T1 and T2
gRNAs approaching 3% and 8% respectively (FIG. 1C). This
RNA-mediated editing process was notably rapid, with the first
detectable GFP.sup.+ cells appearing .about.20 hours post
transfection compared to .about.40 hours for the AAVS1 TALENs. HR
was observed only upon simultaneous introduction of the repair
donor, Cas9 protein, and gRNA, confirming that all components are
required for genome editing (FIG. 4). While no apparent toxicity
associated with Cas9/crRNA expression was noted, work with ZFNs and
TALENs has shown that nicking only one strand further reduces
toxicity. Accordingly, a Cas9D10A mutant was tested that is known
to function as a nickase in vitro, which yielded similar HR but
lower non-homologous end joining (NHEJ) rates (FIG. 5) (see
references (4, 5)). Consistent with (4) where a related Cas9
protein is shown to cut both strands 6 by upstream of the PAM, NHEJ
data confirmed that most deletions or insertions occurred at the 3'
end of the target sequence (FIG. 5B). Also confirmed was that
mutating the target genomic site prevents the gRNA from effecting
HR at that locus, demonstrating that CRISPR-mediated genome editing
is sequence specific (FIG. 6). It was showed that two gRNAs
targeting sites in the GFP gene, and also three additional gRNAs
targeting fragments from homologous regions of the DNA methyl
transferase 3a (DNMT3a) and DNMT3b genes could sequence
specifically induce significant HR in the engineered reporter cell
lines (FIG. 7, 8). Together these results confirm that RNA-guided
genome targeting in human cells induces robust HR across multiple
target sites.
[0029] According to certain aspects, a native locus was modified.
gRNAs were used to target the AAVS1 locus located in the PPP1R12C
gene on chromosome 19, which is ubiquitously expressed across most
tissues (FIG. 2A) in 293Ts, K562s, and PGP1 human iPS cells (see
reference (12)) and analyzed the results by next-generation
sequencing of the targeted locus. Accordingly, FIG. 2 is directed
to RNA-guided genome editing of the native AAVS1 locus in multiple
cell types. As shown in FIG. 2A, T1 (red) and T2 (green) gRNAs
target sequences in an intron of the PPP1R12C gene within the
chromosome 19 AAVS1 locus. As shown in FIG. 2B, total count and
location of deletions caused by NHEJ in 293Ts, K562s, and PGP1 iPS
cells following expression of Cas9 and either T1 or T2 gRNAs as
quantified by next-generation sequencing is provided. Red and green
dash lines demarcate the boundaries of the T1 and T2 gRNA targeting
sites. NHEJ frequencies for T1 and T2 gRNAs were 10% and 25% in
293T, 13% and 38% in K562, and 2% and 4% in PGP1 iPS cells,
respectively. As shown in FIG. 2C, DNA donor architecture for HR at
the AAVS1 locus, and the locations of sequencing primers (arrows)
for detecting successful targeted events, are depicted. As shown in
FIG. 2D, PCR assay three days post transfection demonstrates that
only cells expressing the donor, Cas9 and T2 gRNA exhibit
successful HR events. As shown in FIG. 2E, successful HR was
confirmed by Sanger sequencing of the PCR amplicon showing that the
expected DNA bases at both the genome-donor and donor-insert
boundaries are present. As shown in FIG. 2F, successfully targeted
clones of 293T cells were selected with puromycin for 2 weeks.
Microscope images of two representative GFP+ clones is shown (scale
bar is 100 microns).
[0030] Consistent with results for the GFP reporter assay, high
numbers of NHEJ events were observed at the endogenous locus for
all three cell types. The two gRNAs T1 and T2 achieved NHEJ rates
of 10 and 25% in 293Ts, 13 and 38% in K562s, and 2 and 4% in
PGP1-iPS cells, respectively (FIG. 2B). No overt toxicity was
observed from the Cas9 and crRNA expression required to induce NHEJ
in any of these cell types (FIG. 9). As expected, NHEJ-mediated
deletions for T1 and T2 were centered around the target site
positions, further validating the sequence specificity of this
targeting process (FIG. 9, 10, 11). Simultaneous introduction of
both T1 and T2 gRNAs resulted in high efficiency deletion of the
intervening 19 bp fragment (FIG. 10), demonstrating that
multiplexed editing of genomic loci is feasible using this
approach.
[0031] According to one aspect, HR is used to integrate either a
dsDNA donor construct (see reference (H)) or an oligo donor into
the native AAVS1 locus (FIG. 2C, FIG. 12). HR-mediated integration
was confirmed using both approaches by PCR (FIG. 2D, FIG. 12) and
Sanger sequencing (FIG. 2E). 293T or iPS clones were readily
derived from the pool of modified cells using puromycin selection
over two weeks (FIG. 2F, FIG. 12). These results demonstrate that
Cas9 is capable of efficiently integrating foreign DNA at
endogenous loci in human cells. Accordingly, one aspect of the
present disclosure includes a method of integrating foreign DNA
into the genome of a cell using homologous recombination and
Cas9.
[0032] According to one aspect, an RNA-guided genome editing system
is provided which can readily be adapted to modify other genomic
sites by simply modifying the sequence of the gRNA expression
vector to match a compatible sequence in the locus of interest.
According to this aspect, 190,000 specifically gRNA-targetable
sequences targeting about 40.5% exons of genes in the human genome
were generated. These target sequences were incorporated into a 200
bp format compatible with multiplex synthesis on DNA arrays (see
reference (14)) (FIG. 13). According to this aspect, a ready
genome-wide reference of potential target sites in the human genome
and a methodology for multiplex gRNA synthesis is provided.
[0033] According to one aspect, methods are provided for
multiplexing genomic alterations in a cell by using one or more or
a plurality of RNA/enzyme systems described herein to alter the
genome of a cell at a plurality of locations. According to one
aspect, target sites perfectly match the PAM sequence NGG and the
8-12 base "seed sequence" at the 3' end of the gRNA. According to
certain aspects, perfect match is not required of the remaining
8-12 bases. According to certain aspects, Cas9 will function with
single mismatches at the 5' end. According to certain aspects, the
target locus's underlying chromatin structure and epigenetic state
may affect efficiency of Cas9 function. According to certain
aspects, Cas9 homologs having higher specificity are included as
useful enzymes. One of skill in the art will be able to identify or
engineer suitable Cas9 homologs. According to one aspect,
CRISPR-targetable sequences include those having different PAM
requirements (see reference (9)), or directed evolution. According
to one aspect, inactivating one of the Cas9 nuclease domains
increases the ratio of HR to NHEJ and may reduce toxicity (FIG. 3A,
FIG. 5) (4, 5), while inactivating both domains may enable Cas9 to
function as a retargetable DNA binding protein. Embodiments of the
present disclosure have broad utility in synthetic biology (see
references (21, 22)), the direct and multiplexed perturbation of
gene networks (see references (13, 23)), and targeted ex vivo (see
references (24-26)) and in vivo gene therapy (see reference
(27)).
[0034] According to certain aspects, a "re-engineerable organism"
is provided as a model system for biological discovery and in vivo
screening. According to one aspect, a "re-engineerable mouse"
bearing an inducible Cas9 transgene is provided, and localized
delivery (using adeno-associated viruses, for example) of libraries
of gRNAs targeting multiple genes or regulatory elements allow one
to screen for mutations that result in the onset of tumors in the
target tissue type. Use of Cas9 homologs or nuclease-null variants
bearing effector domains (such as activators) allow one to
multiplex activate or repress genes in vivo. According to this
aspect, one could screen for factors that enable phenotypes such
as: tissue-regeneration, trans-differentiation etc. According to
certain aspects, (a) use of DNA-arrays enables multiplex synthesis
of defined gRNA libraries (refer FIG. 13); and (b) gRNAs being
small in size (refer FIG. 3b) are packaged and delivered using a
multitude of non-viral or viral delivery methods.
[0035] According to one aspect, the lower toxicities observed with
"nickases" for genome engineering applications is achieved by
inactivating one of the Cas9 nuclease domains, either the nicking
of the DNA strand base-paired with the RNA or nicking its
complement. Inactivating both domains allows Cas9 to function as a
retargetable DNA binding protein. According to one aspect, the Cas9
retargetable DNA binding protein is attached [0036] (a) to
transcriptional activation or repression domains for modulating
target gene expression, including but not limited to chromatin
remodeling, histone modification, silencing, insulation, direct
interactions with the transcriptional machinery; [0037] (b) to
nuclease domains such as FokI to enable `highly specific` genome
editing contingent upon dimerization of adjacent gRNA-Cas9
complexes; [0038] (c) to fluorescent proteins for visualizing
genomic loci and chromosome dynamics; or [0039] (d) to other
fluorescent molecules such as protein or nucleic acid bound organic
fluorophores, quantum dots, molecular beacons and echo probes or
molecular beacon replacements; [0040] (e) to multivalent
ligand-binding protein domains that enable programmable
manipulation of genome-wide 3D architecture.
[0041] According to one aspect, the transcriptional activation and
repression components can employ CRISPR systems naturally or
synthetically orthogonal, such that the gRNAs only bind to the
activator or repressor class of Cas. This allows a large set of
gRNAs to tune multiple targets.
[0042] According to certain aspects, the use of gRNAs provide the
ability to multiplex than mRNAs in part due to the smaller
size--100 vs. 2000 nucleotide lengths respectively. This is
particularly valuable when nucleic acid delivery is size limited,
as in viral packaging. This enables multiple instances of cleavage,
nicking, activation, or repression--or combinations thereof. The
ability to easily target multiple regulatory targets allows the
coarse-or-fine-tuning or regulatory networks without being
constrained to the natural regulatory circuits downstream of
specific regulatory factors (e.g. the 4 mRNAs used in reprogramming
fibroblasts into IPSCs). Examples of multiplexing applications
include: [0043] 1. Establishing (major and minor)
histocompatibility alleles, haplotypes, and genotypes for human (or
animal) tissue/organ transplantation. This aspect results e.g. in
HLA homozygous cell lines or humanized animal breeds--or--a set of
gRNAs capable of superimposing such HLA alleles onto an otherwise
desirable cell lines or breeds. [0044] 2. Multiplex cis-regulatory
element (CRE=signals for transcription, splicing, translation, RNA
and protein folding, degradation, etc.) mutations in a single cell
(or a collection of cells) can be used for efficiently studying the
complex sets of regulatory interaction that can occur in normal
development or pathological, synthetic or pharmaceutical scenarios.
According to one aspect, the CREs are (or can be made) somewhat
orthogonal (i.e. low cross talk) so that many can be tested in one
setting--e.g. in an expensive animal embryo time series. One
exemplary application is with RNA fluorescent in situ sequencing
(FISSeq). [0045] 3. Multiplex combinations of CRE mutations and/or
epigenetic activation or repression of CREs can be used to alter or
reprogram iPSCs or ESCs or other stem cells or non-stem cells to
any cell type or combination of cell types for use in
organs-on-chips or other cell and organ cultures for purposes of
testing pharmaceuticals (small molecules, proteins, RNAs, cells,
animal, plant or microbial cells, aerosols and other delivery
methods), transplantation strategies, personalization strategies,
etc. [0046] 4. Making multiplex mutant human cells for use in
diagnostic testing (and/or DNA sequencing) for medical genetics. To
the extent that the chromosomal location and context of a human
genome allele (or epigenetic mark) can influence the accuracy of a
clinical genetic diagnosis, it is important to have alleles present
in the correct location in a reference genome--rather than in an
ectopic (aka transgenic) location or in a separate piece of
synthetic DNA. One embodiment is a series of independent cell lines
one per each diagnostic human SNP, or structural variant.
Alternatively, one embodiment includes multiplex sets of alleles in
the same cell. In some cases multiplex changes in one gene (or
multiple genes) will be desirable under the assumption of
independent testing. In other cases, particular haplotype
combinations of alleles allows testing of sequencing (genotyping)
methods which accurately establish haplotype phase (i.e. whether
one or both copies of a gene are affected in an individual person
or somatic cell type. [0047] 5. Repetitive elements or endogenous
viral elements can be targeted with engineered Cas+gRNA systems in
microbes, plants, animals, or human cells to reduce deleterious
transposition or to aid in sequencing or other analytic
genomic/transcriptomic/proteomic/diagnostic tools (in which nearly
identical copies can be problematic).
[0048] The following references identified by number in the
foregoing section are hereby incorporated by reference in their
entireties. [0049] 1. B. Wiedenheft, S. H. Sternberg, J. A. Doudna,
Nature 482, 331 (Feb. 16, 2012). [0050] 2. D. Bhaya, M. Davison, R.
Barrangou, Annual review of genetics 45, 273 (2011). [0051] 3. M.
P. Terns, R. M. Terns, Current opinion in microbiology 14, 321
(June, 2011). [0052] 4. M. Jinek et al., Science 337, 816 (Aug. 17,
2012). [0053] 5. G. Gasiunas, R. Barrangou, P. Horvath, V. Siksnys,
Proceedings of the National Academy of Sciences of the United
States of America 109, E2579 (Sep. 25, 2012). [0054] 6. R.
Sapranauskas et al., Nucleic acids research 39, 9275 (November,
2011). [0055] 7. T. R. Brummelkamp, R. Bernards, R. Agami, Science
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biotechnology 20, 497 (May, 2002). [0057] 9. E. Deltcheva et al.,
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Huang, S. N. Dowey, L. Cheng, Blood 118, 4599 (Oct. 27, 2011).
[0059] 11. N. E. Sanjana et al., Nature protocols 7, 171 (January,
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(November, 2009). [0061] 13. D. Hockemeyer et al., Nature
biotechnology 27, 851 (September, 2009). [0062] 14. S. Kosuri et
al., Nature biotechnology 28, 1295 (December, 2010). [0063] 15. V.
Pattanayak, C. L. Ramirez, J. K. Joung, D. R. Liu, Nature methods
8, 765 (September, 2011). [0064] 16. N. M. King, O.
Cohen-Haguenauer, Molecular therapy: the journal of the American
Society of Gene Therapy 16, 432 (March, 2008). [0065] 17. Y. G.
Kim, J. Cha, S. Chandrasegaran, Proceedings of the National Academy
of Sciences of the United States of America 93, 1156 (Feb. 6,
1996). [0066] 18. E. J. Rebar, C. O. Pabo, Science 263, 671 (Feb.
4, 1994). [0067] 19. J. Boch et al., Science 326, 1509 (Dec. 11,
2009). [0068] 20. M. J. Moscou, A. J. Bogdanove, Science 326, 1501
(Dec. 11, 2009). [0069] 21. A. S. Khalil, J. J. Collins, Nature
reviews. Genetics 11, 367 (May, 2010). [0070] 22. P. E. Purnick, R.
Weiss, Nature reviews. Molecular cell biology 10, 410 (June, 2009).
[0071] 23. J. Zou et al., Cell stem cell 5, 97 (Jul. 2, 2009).
[0072] 24. N. Holt et al., Nature biotechnology 28, 839 (August,
2010). [0073] 25. F. D. Urnov et al., Nature 435, 646 (Jun. 2,
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14, 2011).
[0076] 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
The Type II CRISPR-Cas System
[0077] According to one aspect, embodiments of the present
disclosure utilize short RNA to identify foreign nucleic acids for
activity by a nuclease in a eukaryotic cell. According to a certain
aspect of the present disclosure, a eukaryotic cell is altered to
include within its genome nucleic acids encoding one or more short
RNA and one or more nucleases which are activated by the binding of
a short RNA to a target DNA sequence. According to certain aspects,
exemplary short RNA/enzyme systems may be identified within
bacteria or archaea, such as (CRISPR)/CRISPR-associated (Cas)
systems that use short RNA to direct degradation of foreign nucleic
acids. CRISPR ("clustered regularly interspaced short palindromic
repeats") defense involves acquisition and integration of new
targeting "spacers" from invading virus or plasmid DNA into the
CRISPR locus, expression and processing of short guiding CRISPR
RNAs (crRNAs) consisting of spacer-repeat units, and cleavage of
nucleic acids (most commonly DNA) complementary to the spacer.
[0078] Three classes of CRISPR systems are generally known and are
referred to as Type I, Type II or Type III). According to one
aspect, a particular useful enzyme according to the present
disclosure to cleave dsDNA is the single effector enzyme, Cas9,
common to Type II. (See reference (1)). Within bacteria, the Type
II effector system consists of a long pre-crRNA transcribed from
the spacer-containing CRISPR locus, the multifunctional Cas9
protein, and a tracrRNA important for gRNA processing. The
tracrRNAs hybridize to the repeat regions separating the spacers of
the pre-crRNA, initiating dsRNA cleavage by endogenous RNase III,
which is followed by a second cleavage event within each spacer by
Cas9, producing mature crRNAs that remain associated with the
tracrRNA and Cas9. According to one aspect, eukaryotic cells of the
present disclosure are engineered to avoid use of RNase III and the
crRNA processing in general. See reference (2).
[0079] According to one aspect, the enzyme of the present
disclosure, such as Cas9 unwinds the DNA duplex and searches for
sequences matching the crRNA to cleave. Target recognition occurs
upon detection of complementarity between a "protospacer" sequence
in the target DNA and the remaining spacer sequence in the crRNA.
Importantly, Cas9 cuts the DNA only if a correct
protospacer-adjacent motif (PAM) is also present at the 3' end.
According to certain aspects, different protospacer-adjacent motif
can be utilized. For example, the S. pyogenes system requires an
NGG sequence, where N can be any nucleotide. S. thermophilus Type
II systems require NGGNG (see reference (3)) and NNAGAAW (see
reference (4)), respectively, while different S. mutans systems
tolerate NGG or NAAR (see reference (5)). Bioinformatic analyses
have generated extensive databases of CRISPR loci in a variety of
bacteria that may serve to identify additional useful PAMs and
expand the set of CRISPR-targetable sequences (see references (6,
7)). In S. thermophilus, Cas9 generates a blunt-ended
double-stranded break 3 bp prior to the 3' end of the protospacer
(see reference (8)), a process mediated by two catalytic domains in
the Cas9 protein: an HNH domain that cleaves the complementary
strand of the DNA and a RuvC-like domain that cleaves the
non-complementary strand (See FIG. 1A and FIG. 3). While the S.
pyogenes system has not been characterized to the same level of
precision, DSB formation also occurs towards the 3' end of the
protospacer. If one of the two nuclease domains is inactivated,
Cas9 will function as a nickase in vitro (see reference (2)) and in
human cells (see FIG. 5).
[0080] According to one aspect, the specificity of gRNA-directed
Cas9 cleavage is used as a mechanism for genome engineering in a
eukaryotic cell. According to one aspect, hybridization of the gRNA
need not be 100 percent in order for the enzyme to recognize the
gRNA/DNA hybrid and affect cleavage. Some off-target activity could
occur. For example, the S. pyogenes system tolerates mismatches in
the first 6 bases out of the 20 bp mature spacer sequence in vitro.
According to one aspect, greater stringency may be beneficial in
vivo when potential off-target sites matching (last 14 bp) NGG
exist within the human reference genome for the gRNAs. The effect
of mismatches and enzyme activity in general are described in
references (9), (2), (10), and (4).
[0081] According to certain aspects, specificity may be improved.
When interference is sensitive to the melting temperature of the
gRNA-DNA hybrid, AT-rich target sequences may have fewer off-target
sites. Carefully choosing target sites to avoid pseudo-sites with
at least 14 bp matching sequences elsewhere in the genome may
improve specificity. The use of a Cas9 variant requiring a longer
PAM sequence may reduce the frequency of off-target sites. Directed
evolution may improve Cas9 specificity to a level sufficient to
completely preclude off-target activity, ideally requiring a
perfect 20 bp gRNA match with a minimal PAM. Accordingly,
modification to the Cas9 protein is a representative embodiment of
the present disclosure. As such, novel methods permitting many
rounds of evolution in a short timeframe (see reference (11) and
envisioned. CRISPR systems useful in the present disclosure are
described in references (12, 13).
EXAMPLE II
Plasmid Construction
[0082] The Cas9 gene sequence was human codon optimized and
assembled by hierarchical fusion PCR assembly of 9 500 bp gBlocks
ordered from IDT. FIG. 3A for the engineered type II CRISPR system
for human cells shows the expression format and full sequence of
the cas9 gene insert. The RuvC-like and HNH motifs, and the
C-terminus SV40 NLS are respectively highlighted by blue, brown and
orange colors. Cas9_D10A was similarly constructed. The resulting
full-length products were cloned into the pcDNA3.3-TOPO vector
(Invitrogen). The target gRNA expression constructs were directly
ordered as individual 455 bp gBlocks from IDT and either cloned
into the pCR-BluntII-TOPO vector (Invitrogen) or per amplified.
FIG. 3B shows the U6 promoter based expression scheme for the guide
RNAs and predicted RNA transcript secondary structure. The use of
the U6 promoter constrains the 1.sup.st position in the RNA
transcript to be a `G` and thus all genomic sites of the form
GN.sub.20GG can be targeted using this approach. FIG. 3C shows the
7 gRNAs used.
[0083] The vectors for the HR reporter assay involving a broken GFP
were constructed by fusion PCR assembly of the GFP sequence bearing
the stop codon and 68 bp AAVS1 fragment (or mutants thereof; see
FIG. 6), or 58 bp fragments from the DNMT3a and DNMT3b genomic loci
(see FIG. 8) assembled into the EGIP lentivector from Addgene
(plasmid #26777). These lentivectors were then used to establish
the GFP reporter stable lines. TALENs used in this study were
constructed using the protocols described in (14). All DNA reagents
developed in this study are available at Addgene.
EXAMPLE III
Cell Culture
[0084] PGP1 iPS cells were maintained on Matrigel (BD
Biosciences)-coated plates in mTeSR1 (Stemcell Technologies).
Cultures were passaged every 5-7 d with TrypLE Express
(Invitrogen). K562 cells were grown and maintained in RPMI
(Invitrogen) containing 15% FBS. HEK 293T cells were cultured in
Dulbecco's modified Eagle's medium (DMEM, Invitrogen) high glucose
supplemented with 10% fetal bovine serum (FBS, Invitrogen),
penicillin/streptomycin (pen/strep, Invitrogen), and non-essential
amino acids (NEAA, Invitrogen). All cells were maintained at
37.degree. C. and 5% CO.sub.2 in a humidified incubator.
EXAMPLE IV
Gene Targeting of PGP1 iPS, K562 and 293Ts
[0085] PGP1 iPS cells were cultured in Rho kinase (ROCK) inhibitor
(Calbiochem) 2 h before nucleofection. Cells were harvest using
TrypLE Express (Invitrogen) and 2.times.10.sup.6 cells were
resuspended in P3 reagent (Lonza) with 1 .mu.g Cas9 plasmid, 1
.mu.g gRNA and/or 1 .mu.g DNA donor plasmid, and nucleofected
according to manufacturer's instruction (Lonza). Cells were
subsequently plated on an mTeSR1-coated plate in mTeSR1 medium
supplemented with ROCK inhibitor for the first 24 h. For K562s,
2.times.10.sup.6 cells were resuspended in SF reagent (Lonza) with
1 .mu.g Cas9 plasmid, 1 .mu.g gRNA and/or 1 .mu.g DNA donor
plasmid, and nucleofected according to manufacturer's instruction
(Lonza). For 293Ts, 0.1.times.10.sup.6 cells were transfected with
1 .mu.g Cas9 plasmid, 1 .mu.g gRNA and/or 1 .mu.g DNA donor plasmid
using Lipofectamine 2000 as per the manufacturer's protocols. The
DNA donors used for endogenous AAVS1 targeting were either a dsDNA
donor (FIG. 2C) or a 90 mer oligonucleotide. The former has
flanking short homology arms and a SA-2A-puromycin-CaGGS-eGFP
cassette to enrich for successfully targeted cells.
[0086] The targeting efficiency was assessed as follows. Cells were
harvested 3 days after nucleofection and the genomic DNA of
.about.1.times.10.sup.6 cells was extracted using prepGEM (ZyGEM).
PCR was conducted to amplify the targeting region with genomic DNA
derived from the cells and amplicons were deep sequenced by MiSeq
Personal Sequencer (Illumina) with coverage >200,000 reads. The
sequencing data was analyzed to estimate NHEJ efficiencies. The
reference AAVS1 sequence analyzed is:
TABLE-US-00001 (SEQ ID NO: 1)
CACTTCAGGACAGCATGTTTGCTGCCTCCAGGGATCCTGTGTCCCCGAGCTGGGACCACCTT
ATATTCCCAGGGCCGGTTAATGTGGCTCTGGTTCTGGGTACTTTTATCTGTCCCCTCCACCCC
ACAGTGGGGCCACTAGGGACAGGATTGGTGACAGAAAAGCCCCATCCTTAGGCCTCCTCCTT
CCTAGTCTCCTGATATTGGGTCTAACCCCCACCTCCTGTTAGGCAGATTCCTTATCTGGTGAC
ACACCCCCATTTCCTGGA
The PCR primers for amplifying the targeting regions in the human
genome are:
TABLE-US-00002 (SEQ ID NO: 2) AAVS1-R
CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTacaggaggtgggggttagac (SEQ ID NO:
3) AAVS1-F.1
ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATtatattcccagggccggtta (SEQ ID
NO: 4) AAVS1-F.2
ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGtatattcccagggccggtta (SEQ ID
NO: 5) AAVS1-F.3
ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCTAAtatattcccagggccggtta (SEQ ID
NO: 6) AAVS1-F.4
ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGTCAtatattcccagggccggtta (SEQ ID
NO: 7) AAVS1-F.5
ACACTCTTTCCCTACACGACGCTCTTCCGATCTCACTGTtatattcccagggccggtta (SEQ ID
NO: 8) AAVS1-F.6
ACACTCTTTCCCTACACGACGCTCTTCCGATCTATTGGCtatattcccagggccggtta (SEQ ID
NO: 9) AAVS1-F.7
ACACTCTTTCCCTACACGACGCTCTTCCGATCTGATCTGtatattcccagggccggtta (SEQ ID
NO: 10) AAVS1-F.8
ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCAAGTtatattcccagggccggtta (SEQ ID
NO: 11) AAVS1-F.9
ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGATCtatattcccagggccggtta (SEQ ID
NO: 12) AAVS1-F.10
ACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGCTAtatattcccagggccggtta (SEQ ID
NO: 13) AAVS1-F.11
ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTAGCCtatattcccagggccggtta (SEQ ID
NO: 14) AAVS1-F.12
ACACTCTTTCCCTACACGACGCTCTTCCGATCTTACAAGtatattcccagggccggtta
[0087] To analyze the HR events using the DNA donor in FIG. 2C, the
primers used were:
TABLE-US-00003 (SEQ ID NO: 15) HR_AAVS1-F CTGCCGTCTCTCTCCTGAGT (SEQ
ID NO: 16) HR_Puro-R GTGGGCTTGTACTCGGTCAT
EXAMPLE V
Bioinformatics Approach for Computing Human Exon CRISPR Targets and
Methodology for their Multiplexed Synthesis
[0088] A set of gRNA gene sequences that maximally target specific
locations in human exons but minimally target other locations in
the genome were determined as follows. According to one aspect,
maximally efficient targeting by a gRNA is achieved by 23 nt
sequences, the 5'-most 20 nt of which exactly complement a desired
location, while the three 3'-most bases must be of the form NGG.
Additionally, the 5'-most nt must be a G to establish a pol-III
transcription start site. However, according to (2), mispairing of
the six 5'-most nt of a 20 bp gRNA against its genomic target does
not abrogate Cas9-mediated cleavage so long as the last 14 nt pairs
properly, but mispairing of the eight 5'-most nt along with pairing
of the last 12 nt does, while the case of the seven 5-most nt
mispairs and 13 3' pairs was not tested. To be conservative
regarding off-target effects, one condition was that the case of
the seven 5'-most mispairs is, like the case of six, permissive of
cleavage, so that pairing of the 3'-most 13 nt is sufficient for
cleavage. To identify CRISPR target sites within human exons that
should be cleavable without off-target cuts, all 23 bp sequences of
the form 5'-GBBBB BBBBB BBBBB BBBBB NGG-3' (form 1) were examined,
where the B's represent the bases at the exon location, for which
no sequence of the form 5'-NNNNN NNBBB BBBBB BBBBB NGG-3' (form 2)
existed at any other location in the human genome. Specifically,
(i) a BED file of locations of coding regions of all RefSeq genes
the GRCh37/hg19 human genome from the UCSC Genome Browser (15-17)
was downloaded. Coding exon locations in this BED file comprised a
set of 346089 mappings of RefSeq mRNA accessions to the hg19
genome. However, some RefSeq mRNA accessions mapped to multiple
genomic locations (probable gene duplications), and many accessions
mapped to subsets of the same set of exon locations (multiple
isoforms of the same genes). To distinguish apparently duplicated
gene instances and consolidate multiple references to the same
genomic exon instance by multiple RefSeq isoform accessions, (ii)
unique numerical suffixes to 705 RefSeq accession numbers that had
multiple genomic locations were added, and (iii) the mergeBed
function of BEDToo1s (18) (v2.16.2-zip-87e3926) was used to
consolidate overlapping exon locations into merged exon regions.
These steps reduced the initial set of 346089 RefSeq exon locations
to 192783 distinct genomic regions. The hg19 sequence for all
merged exon regions were downloaded using the UCSC Table Browser,
adding 20 bp of padding on each end. (iv) Using custom perl code,
1657793 instances of form 1 were identified within this exonic
sequence. (v) These sequences were then filtered for the existence
of off-target occurrences of form 2: For each merged exon form 1
target, the 3'-most 13 bp specific (B) "core" sequences were
extracted and, for each core generated the four 16 bp sequences
5'-BBB BBBBB BBBBB NGG-3' (N=A, C, G, and T), and searched the
entire hg19 genome for exact matches to these 6631172 sequences
using Bowtie version 0.12.8 (19) using the parameters -1 16 -v 0 -k
2. Any exon target site for which there was more than a single
match was rejected. Note that because any specific 13 bp core
sequence followed by the sequence NGG confers only 15 bp of
specificity, there should be on average .about.5.6 matches to an
extended core sequence in a random .about.3Gb sequence (both
strands). Therefore, most of the 1657793 initially identified
targets were rejected; however 189864 sequences passed this filter.
These comprise the set of CRISPR-targetable exonic locations in the
human genome. The 189864 sequences target locations in 78028 merged
exonic regions (.about.40.5% of the total of 192783 merged human
exon regions) at a multiplicity of .about.2.4 sites per targeted
exonic region. To assess targeting at a gene level, RefSeq mRNA
mappings were clustered so that any two RefSeq accessions
(including the gene duplicates distinguished in (ii)) that overlap
a merged exon region are counted as a single gene cluster, the
189864 exonic specific CRISPR sites target 17104 out of 18872 gene
clusters (.about.90.6% of all gene clusters) at a multiplicity of
.about.11.1 per targeted gene cluster. (Note that while these gene
clusters collapse RefSeq mRNA accessions that represent multiple
isoforms of a single transcribed gene into a single entity, they
will also collapse overlapping distinct genes as well as genes with
antisense transcripts.) At the level of original RefSeq accessions,
the 189864 sequences targeted exonic regions in 30563 out of a
total of 43726 (.about.69.9%) mapped RefSeq accessions (including
distinguished gene duplicates) at a multiplicity of .about.6.2
sites per targeted mapped RefSeq accession.
[0089] According to one aspect, the database can be refined by
correlating performance with factors, such as base composition and
secondary structure of both gRNAs and genomic targets (20, 21), and
the epigenetic state of these targets in human cell lines for which
this information is available (22).
EXAMPLE VI
Multiplex Synthesis
[0090] The target sequences were incorporated into a 200 bp format
that is compatible for multiplex synthesis on DNA arrays (23, 24).
According to one aspect the method allows for targeted retrieval of
a specific or pools of gRNA sequences from the DNA array based
oligonucleotide pool and its rapid cloning into a common expression
vector (FIG. 13A). Specifically, a 12 k oligonucleotide pool from
CustomArray Inc. was synthesized. Furthermore, gRNAs of choice from
this library (FIG. 13B) were successfully retrieved. We observed an
error rate of .about.4 mutations per 1000 bp of synthesized
DNA.
EXAMPLE VII
RNA-Guided Genome Editing Requires both Cas9 and Guide RNA for
Successful Targeting
[0091] Using the GFP reporter assay described in FIG. 1B, all
possible combinations of the repair DNA donor, Cas9 protein, and
gRNA were tested for their ability to effect successful HR (in
293Ts). As shown in FIG. 4, GFP+ cells were observed only when all
the 3 components were present, validating that these CRISPR
components are essential for RNA-guided genome editing. Data is
mean+/-SEM (N=3).
EXAMPLE VIII
Analysis of gRNA and Cas9 Mediated Genome Editing
[0092] The CRISPR mediated genome editing process was examined
using either (A) a GFP reporter assay as described earlier results
of which are shown in FIGS. 5A, and (B) deep sequencing of the
targeted loci (in 293Ts), results of which are shown in FIG. 5B. As
comparison, a D10A mutant for Cas9 was tested that has been shown
in earlier reports to function as a nickase in in vitro assays. As
shown in FIG. 5, both Cas9 and Cas9D10A can effect successful HR at
nearly similar rates. Deep sequencing however confirms that while
Cas9 shows robust NHEJ at the targeted loci, the D10A mutant has
significantly diminished NHEJ rates (as would be expected from its
putative ability to only nick DNA). Also, consistent with the known
biochemistry of the Cas9 protein, NHEJ data confirms that most
base-pair deletions or insertions occurred near the 3' end of the
target sequence: the peak is .about.3-4 bases upstream of the PAM
site, with a median deletion frequency of .about.9-10 bp. Data is
mean+/-SEM (N=3).
EXAMPLE IX
RNA-Guided Genome Editing is Target Sequence Specific
[0093] Similar to the GFP reporter assay described in FIG. 1B, 3
293T stable lines each bearing a distinct GFP reporter construct
were developed. These are distinguished by the sequence of the
AAVS1 fragment insert (as indicated in the FIG. 6). One line
harbored the wild-type fragment while the two other lines were
mutated at 6 bp (highlighted in red). Each of the lines was then
targeted by one of the following 4 reagents: a GFP-ZFN pair that
can target all cell types since its targeted sequence was in the
flanking GFP fragments and hence present in along cell lines; a
AAVS1 TALEN that could potentially target only the wt-AAVS1
fragment since the mutations in the other two lines should render
the left TALEN unable to bind their sites; the T1 gRNA which can
also potentially target only the wt-AAVS1 fragment, since its
target site is also disrupted in the two mutant lines; and finally
the T2 gRNA which should be able to target all 3 cell lines since,
unlike the T1 gRNA, its target site is unaltered among the 3 lines.
ZFN modified all 3 cell types, the AAVS1 TALENs and the T1 gRNA
only targeted the wt-AAVS1 cell type, and the T2 gRNA successfully
targets all 3 cell types. These results together confirm that the
guide RNA mediated editing is target sequence specific. Data is
mean+/-SEM (N=3).
EXAMPLE X
Guide RNAs Targeted to the GFP Sequence Enable Robust Genome
Editing
[0094] In addition to the 2 gRNAs targeting the AAVS1 insert, two
additional gRNAs targeting the flanking GFP sequences of the
reporter described in FIG. 1B (in 293Ts) were tested. As shown in
FIG. 7, these gRNAs were also able to effect robust HR at this
engineered locus. Data is mean+/-SEM (N=3).
EXAMPLE XI
RNA-Guided Genome Editing is Target Sequence Specific, and
Demonstrates Similar Targeting Efficiencies as ZFNs or TALENs
[0095] Similar to the GFP reporter assay described in FIG. 1B, two
293T stable lines each bearing a distinct GFP reporter construct
were developed. These are distinguished by the sequence of the
fragment insert (as indicated in FIG. 8). One line harbored a 58 bp
fragment from the DNMT3a gene while the other line bore a
homologous 58 bp fragment from the DNMT3b gene. The sequence
differences are highlighted in red. Each of the lines was then
targeted by one of the following 6 reagents: a GFP-ZFN pair that
can target all cell types since its targeted sequence was in the
flanking GFP fragments and hence present in along cell lines; a
pair of TALENs that potentially target either DNMT3a or DNMT3b
fragments; a pair of gRNAs that can potentially target only the
DNMT3a fragment; and finally a gRNA that should potentially only
target the DNMT3b fragment. As indicated in FIG. 8, the ZFN
modified all 3 cell types, and the TALENs and gRNAs only their
respective targets. Furthermore the efficiencies of targeting were
comparable across the 6 targeting reagents. These results together
confirm that RNA-guided editing is target sequence specific and
demonstrates similar targeting efficiencies as ZFNs or TALENs. Data
is mean+/-SEM (N=3).
EXAMPLE XII
RNA-Guided NHEJ in Human iPS Cells
[0096] Human iPS cells (PGP1) were nucleofected with constructs
indicated in the left panel of FIG. 9. 4 days after nucleofection,
NHEJ rate was measured by assessing genomic deletion and insertion
rate at double-strand breaks (DSBs) by deep sequencing. Panel 1:
Deletion rate detected at targeting region. Red dash lines:
boundary of T1 RNA targeting site; green dash lines: boundary of T2
RNA targeting site. The deletion incidence at each nucleotide
position was plotted in black lines and the deletion rate as the
percentage of reads carrying deletions was calculated. Panel 2:
Insertion rate detected at targeting region. Red dash lines:
boundary of T1 RNA targeting site; green dash lines: boundary of T2
RNA targeting site. The incidence of insertion at the genomic
location where the first insertion junction was detected was
plotted in black lines and the insertion rate as the percentage of
reads carrying insertions was calculated. Panel 3: Deletion size
distribution. The frequencies of different size deletions among the
whole NHEJ population was plotted. Panel 4: insertion size
distribution. The frequencies of different sizes insertions among
the whole NHEJ population was plotted. iPS targeting by both gRNAs
is efficient (2-4%), sequence specific (as shown by the shift in
position of the NHEJ deletion distributions), and reaffirming the
results of FIG. 4, the NGS-based analysis also shows that both the
Cas9 protein and the gRNA are essential for NHEJ events at the
target locus.
EXAMPLE XIII
RNA-Guided NHEJ in K562 Cells
[0097] K562 cells were nucleated with constructs indicated in the
left panel of FIG. 10. 4 days after nucleofection, NHEJ rate was
measured by assessing genomic deletion and insertion rate at DSBs
by deep sequencing. Panel 1: Deletion rate detected at targeting
region. Red dash lines: boundary of T1 RNA targeting site; green
dash lines: boundary of T2 RNA targeting site. The deletion
incidence at each nucleotide position was plotted in black lines
and the deletion rate as the percentage of reads carrying deletions
was calculated. Panel 2: Insertion rate detected at targeting
region. Red dash lines: boundary of T1 RNA targeting site; green
dash lines: boundary of T2 RNA targeting site. The incidence of
insertion at the genomic location where the first insertion
junction was detected was plotted in black lines and the insertion
rate as the percentage of reads carrying insertions was calculated.
Panel 3: Deletion size distribution. The frequencies of different
size deletions among the whole NHEJ population was plotted. Panel
4: insertion size distribution. The frequencies of different sizes
insertions among the whole NHEJ population was plotted. K562
targeting by both gRNAs is efficient (13-38%) and sequence specific
(as shown by the shift in position of the NHEJ deletion
distributions). Importantly, as evidenced by the peaks in the
histogram of observed frequencies of deletion sizes, simultaneous
introduction of both T1 and T2 guide RNAs resulted in high
efficiency deletion of the intervening 19 bp fragment,
demonstrating that multiplexed editing of genomic loci is also
feasible using this approach.
EXAMPLE XIV
RNA-Guided NHEJ in 293T Cells
[0098] 293T cells were transfected with constructs indicated in the
left panel of FIG. 11. 4 days after nucleofection, NHEJ rate was
measured by assessing genomic deletion and insertion rate at DSBs
by deep sequencing. Panel 1: Deletion rate detected at targeting
region. Red dash lines: boundary of T1 RNA targeting site; green
dash lines: boundary of T2 RNA targeting site. The deletion
incidence at each nucleotide position was plotted in black lines
and the deletion rate as the percentage of reads carrying deletions
was calculated. Panel 2: Insertion rate detected at targeting
region. Red dash lines: boundary of T1 RNA targeting site; green
dash lines: boundary of T2 RNA targeting site. The incidence of
insertion at the genomic location where the first insertion
junction was detected was plotted in black lines and the insertion
rate as the percentage of reads carrying insertions was calculated.
Panel 3: Deletion size distribution. The frequencies of different
size deletions among the whole NHEJ population was plotted. Panel
4: insertion size distribution. The frequencies of different sizes
insertions among the whole NHEJ population was plotted. 293T
targeting by both gRNAs is efficient (10-24%) and sequence specific
(as shown by the shift in position of the NHEJ deletion
distributions).
EXAMPLE XV
HR at the Endogenous AAVS1 Locus Using either a dsDNA Donor or a
Short Oligonucleotide Donor
[0099] As shown in FIG. 12A, PCR screen (with reference to FIG. 2C)
confirmed that 21/24 randomly picked 293T clones were successfully
targeted. As shown in FIG. 12B, similar PCR screen confirmed 3/7
randomly picked PGP1-iPS clones were also successfully targeted. As
shown in FIG. 12C, short 90 mer oligos could also effect robust
targeting at the endogenous AAVS1 locus (shown here for K562
cells).
EXAMPLE XVI
Methodology for Multiplex Synthesis, Retrieval and U6 Expression
Vector Cloning of Guide RNAs Targeting Genes in the Human
Genome
[0100] A resource of about 190 k bioinformatically computed unique
gRNA sites targeting .about.40.5% of all exons of genes in the
human genome was generated. As shown in FIG. 13A, the gRNA target
sites were incorporated into a 200 bp format that is compatible for
multiplex synthesis on DNA arrays. Specifically, the design allows
for (i) targeted retrieval of a specific or pools of gRNA targets
from the DNA array oligonucleotide pool (through 3 sequential
rounds of nested PCR as indicated in the figure schematic); and
(ii) rapid cloning into a common expression vector which upon
linearization using an AflII site serves as a recipient for Gibson
assembly mediated incorporation of the gRNA insert fragment. As
shown in FIG. 13B, the method was used to accomplish targeted
retrieval of 10 unique gRNAs from a 12 k oligonucleotide pool
synthesized by CustomArray Inc.
EXAMPLE XVII
CRISPR Mediated RNA-Guided Transcriptional Activation
[0101] The CRISPR-Cas system has an adaptive immune defense system
in bacteria and functions to `cleave` invading nucleic acids.
According to one aspect, the CRISPR-CAS system is engineered to
function in human cells, and to `cleave` genomic DNA. This is
achieved by a short guide RNA directing a Cas9 protein (which has
nuclease function) to a target sequence complementary to the spacer
in the guide RNA. The ability to `cleave` DNA enables a host of
applications related to genome editing, and also targeted genome
regulation. Towards this, the Cas9 protein was mutated to make it
nuclease-null by introducing mutations that are predicted to
abrogate coupling to Mg2+ (known to be important for the nuclease
functions of the RuvC-like and HNH-like domains): specifically,
combinations of D10A, D839A, H840A and N863A mutations were
introduced. The thus generated Cas9 nuclease-null protein (as
confirmed by its ability to not cut DNA by sequencing analysis) and
hereafter referred to as Cas9R-H-, was then coupled to a
transcriptional activation domain, here VP64, enabling the
CRISPR-cas system to function as a RNA guided transcription factor
(see FIG. 14). The Cas9R-H-+VP64 fusion enables RNA-guided
transcriptional activation at the two reporters shown.
Specifically, both FACS analysis and immunofluorescence imaging
demonstrates that the protein enables gRNA sequence specific
targeting of the corresponding reporters, and furthermore, the
resulting transcription activation as assayed by expression of a
dTomato fluorescent protein was at levels similar to those induced
by a convention TALE-VP64 fusion protein.
EXAMPLE XVIII
gRNA Sequence Flexibility and Applications Thereof
[0102] Flexibility of the gRNA scaffold sequence to designer
sequence insertions was determined by systematically assaying for a
range of the random sequence insertions on the 5', middle and 3'
portions of the gRNA: specifically, 1 bp, 5 bp, 10 bp, 20 bp, and
40 bp inserts were made in the gRNA sequence at the 5', middle, and
3' ends of the gRNA (the exact positions of the insertion are
highlighted in `red` in FIG. 15). This gRNA was then tested for
functionality by its ability to induce HR in a GFP reporter assay
(as described herein). It is evident that gRNAs are flexible to
sequence insertions on the 5' and 3' ends (as measured by retained
HR inducing activity). Accordingly, aspects of the present
disclosure are directed to tagging of small-molecule responsive RNA
aptamers that may trigger onset of gRNA activity, or gRNA
visualization. Additionally, aspects of the present disclosure are
directed to tethering of ssDNA donors to gRNAs via hybridization,
thus enabling coupling of genomic target cutting and immediate
physical localization of repair template which can promote
homologous recombination rates over error-prone non-homologous
end-joining
[0103] The following references identified in the Examples section
by number are hereby incorporated by reference in their entireties
for all purposes.
REFERENCES
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the CRISPR-Cas systems. Nature reviews. Microbiology 9, 467 (June,
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DNA endonuclease in adaptive bacterial immunity Science 337, 816
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the immune system of bacteria and archaea. Science 327, 167 (Jan.
8, 2010). [0107] 4. H. Deveau et al., Phage response to
CRISPR-encoded resistance in Streptococcus thermophilus. Journal of
bacteriology 190, 1390 (February, 2008). [0108] 5. J. R. van der
Ploeg, Analysis of CRISPR in Streptococcus mutans suggests frequent
occurrence of acquired immunity against infection by M102-like
bacteriophages. Microbiology 155, 1966 (June, 2009). [0109] 6. M.
Rho, Y. W. Wu, H. Tang, T. G. Doak, Y. Ye, Diverse CRISPRs evolving
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Sequence CWU 1
1
441268DNAHomo sapiens 1cacttcagga cagcatgttt gctgcctcca gggatcctgt
gtccccgagc tgggaccacc 60ttatattccc agggccggtt aatgtggctc tggttctggg
tacttttatc tgtcccctcc 120accccacagt ggggccacta gggacaggat
tggtgacaga aaagccccat ccttaggcct 180cctccttcct agtctcctga
tattgggtct aacccccacc tcctgttagg cagattcctt 240atctggtgac
acacccccat ttcctgga 268253DNAArtificialPCR primer 2ctcggcattc
ctgctgaacc gctcttccga tctacaggag gtgggggtta gac
53359DNAArtificialPCR primer 3acactctttc cctacacgac gctcttccga
tctcgtgatt atattcccag ggccggtta 59459DNAArtificialPCR primer
4acactctttc cctacacgac gctcttccga tctacatcgt atattcccag ggccggtta
59559DNAArtificialPCR primer 5acactctttc cctacacgac gctcttccga
tctgcctaat atattcccag ggccggtta 59659DNAArtificialPCR primer
6acactctttc cctacacgac gctcttccga tcttggtcat atattcccag ggccggtta
59759DNAArtificialPCR primer 7acactctttc cctacacgac gctcttccga
tctcactgtt atattcccag ggccggtta 59859DNAArtificialPCR primer
8acactctttc cctacacgac gctcttccga tctattggct atattcccag ggccggtta
59959DNAArtificialPCR primer 9acactctttc cctacacgac gctcttccga
tctgatctgt atattcccag ggccggtta 591059DNAArtificialPCR primer
10acactctttc cctacacgac gctcttccga tcttcaagtt atattcccag ggccggtta
591159DNAArtificialPCR primer 11acactctttc cctacacgac gctcttccga
tctctgatct atattcccag ggccggtta 591259DNAArtificialPCR primer
12acactctttc cctacacgac gctcttccga tctaagctat atattcccag ggccggtta
591359DNAArtificialPCR primer 13acactctttc cctacacgac gctcttccga
tctgtagcct atattcccag ggccggtta 591459DNAArtificialPCR primer
14acactctttc cctacacgac gctcttccga tcttacaagt atattcccag ggccggtta
591520DNAArtificialPCR primer 15ctgccgtctc tctcctgagt
201620DNAArtificialPCR primer 16gtgggcttgt actcggtcat
201780RNAArtificialguide RNA 17guuuuagagc uagaaauagc aaguuaaaau
aaggcuaguc cguuaucaac uugaaaaagu 60ggcaccgagu cggugcuuuu
801871DNAHomo sapiens 18taatactttt atcttgtccc ctccacccca cagtggggcc
actagggaca ggattggtga 60cagaaagccc c 711952DNAHomo sapiens
19ttatctgtcc cctccacccc acagtggggc cactagggaa caggattggt ga
522022DNAHomo sapiens 20caggcaggtc ctgctttctc tg 222122DNAHomo
sapiens 21cacagtgggg caagcttctg ac 22224146DNAHomo sapiens
22gccaccatgg acaagaagta ctccattggg ctcgatatcg gcacaaacag cgtcggctgg
60gccgtcatta cggacgagta caaggtgccg agcaaaaaat tcaaagttct gggcaatacc
120gatcgccaca gcataaagaa gaacctcatt ggcgccctcc tgttcgactc
cggggagacg 180gccgaagcca cgcggctcaa aagaacagca cggcgcagat
atacccgcag aaagaatcgg 240atctgctacc tgcaggagat ctttagtaat
gagatggcta aggtggatga ctctttcttc 300cataggctgg aggagtcctt
tttggtggag gaggataaaa agcacgagcg ccacccaatc 360tttggcaata
tcgtggacga ggtggcgtac catgaaaagt acccaaccat atatcatctg
420aggaagaagc ttgtagacag tactgataag gctgacttgc ggttgatcta
tctcgcgctg 480gcgcatatga tcaaatttcg gggacacttc ctcatcgagg
gggacctgaa cccagacaac 540agcgatgtcg acaaactctt tatccaactg
gttcagactt acaatcagct tttcgaagag 600aacccgatca acgcatccgg
agttgacgcc aaagcaatcc tgagcgctag gctgtccaaa 660tcccggcggc
tcgaaaacct catcgcacag ctccctgggg agaagaagaa cggcctgttt
720ggtaatctta tcgccctgtc actcgggctg acccccaact ttaaatctaa
cttcgacctg 780gccgaagatg ccaagcttca actgagcaaa gacacctacg
atgatgatct cgacaatctg 840ctggcccaga tcggcgacca gtacgcagac
ctttttttgg cggcaaagaa cctgtcagac 900gccattctgc tgagtgatat
tctgcgagtg aacacggaga tcaccaaagc tccgctgagc 960gctagtatga
tcaagcgcta tgatgagcac caccaagact tgactttgct gaaggccctt
1020gtcagacagc aactgcctga gaagtacaag gaaattttct tcgatcagtc
taaaaatggc 1080tacgccggat acattgacgg cggagcaagc caggaggaat
tttacaaatt tattaagccc 1140atcttggaaa aaatggacgg caccgaggag
ctgctggtaa agcttaacag agaagatctg 1200ttgcgcaaac agcgcacttt
cgacaatgga agcatccccc accagattca cctgggcgaa 1260ctgcacgcta
tcctcaggcg gcaagaggat ttctacccct ttttgaaaga taacagggaa
1320aagattgaga aaatcctcac atttcggata ccctactatg taggccccct
cgcccgggga 1380aattccagat tcgcgtggat gactcgcaaa tcagaagaga
ccatcactcc ctggaacttc 1440gaggaagtcg tggataaggg ggcctctgcc
cagtccttca tcgaaaggat gactaacttt 1500gataaaaatc tgcctaacga
aaaggtgctt cctaaacact ctctgctgta cgagtacttc 1560acagtttata
acgagctcac caaggtcaaa tacgtcacag aagggatgag aaagccagca
1620ttcctgtctg gagagcagaa gaaagctatc gtggacctcc tcttcaagac
gaaccggaaa 1680gttaccgtga aacagctcaa agaagactat ttcaaaaaga
ttgaatgttt cgactctgtt 1740gaaatcagcg gagtggagga tcgcttcaac
gcatccctgg gaacgtatca cgatctcctg 1800aaaatcatta aagacaagga
cttcctggac aatgaggaga acgaggacat tcttgaggac 1860attgtcctca
cccttacgtt gtttgaagat agggagatga ttgaagaacg cttgaaaact
1920tacgctcatc tcttcgacga caaagtcatg aaacagctca agaggcgccg
atatacagga 1980tgggggcggc tgtcaagaaa actgatcaat gggatccgag
acaagcagag tggaaagaca 2040atcctggatt ttcttaagtc cgatggattt
gccaaccgga acttcatgca gttgatccat 2100gatgactctc tcacctttaa
ggaggacatc cagaaagcac aagtttctgg ccagggggac 2160agtcttcacg
agcacatcgc taatcttgca ggtagcccag ctatcaaaaa gggaatactg
2220cagaccgtta aggtcgtgga tgaactcgtc aaagtaatgg gaaggcataa
gcccgagaat 2280atcgttatcg agatggcccg agagaaccaa actacccaga
agggacagaa gaacagtagg 2340gaaaggatga agaggattga agagggtata
aaagaactgg ggtcccaaat ccttaaggaa 2400cacccagttg aaaacaccca
gcttcagaat gagaagctct acctgtacta cctgcagaac 2460ggcagggaca
tgtacgtgga tcaggaactg gacatcaatc ggctctccga ctacgacgtg
2520gatcatatcg tgccccagtc ttttctcaaa gatgattcta ttgataataa
agtgttgaca 2580agatccgata aaaatagagg gaagagtgat aacgtcccct
cagaagaagt tgtcaagaaa 2640atgaaaaatt attggcggca gctgctgaac
gccaaactga tcacacaacg gaagttcgat 2700aatctgacta aggctgaacg
aggtggcctg tctgagttgg ataaagccgg cttcatcaaa 2760aggcagcttg
ttgagacacg ccagatcacc aagcacgtgg cccaaattct cgattcacgc
2820atgaacacca agtacgatga aaatgacaaa ctgattcgag aggtgaaagt
tattactctg 2880aagtctaagc tggtctcaga tttcagaaag gactttcagt
tttataaggt gagagagatc 2940aacaattacc accatgcgca tgatgcctac
ctgaatgcag tggtaggcac tgcacttatc 3000aaaaaatatc ccaagcttga
atctgaattt gtttacggag actataaagt gtacgatgtt 3060aggaaaatga
tcgcaaagtc tgagcaggaa ataggcaagg ccaccgctaa gtacttcttt
3120tacagcaata ttatgaattt tttcaagacc gagattacac tggccaatgg
agagattcgg 3180aagcgaccac ttatcgaaac aaacggagaa acaggagaaa
tcgtgtggga caagggtagg 3240gatttcgcga cagtccggaa ggtcctgtcc
atgccgcagg tgaacatcgt taaaaagacc 3300gaagtacaga ccggaggctt
ctccaaggaa agtatcctcc cgaaaaggaa cagcgacaag 3360ctgatcgcac
gcaaaaaaga ttgggacccc aagaaatacg gcggattcga ttctcctaca
3420gtcgcttaca gtgtactggt tgtggccaaa gtggagaaag ggaagtctaa
aaaactcaaa 3480agcgtcaagg aactgctggg catcacaatc atggagcgat
caagcttcga aaaaaacccc 3540atcgactttc tcgaggcgaa aggatataaa
gaggtcaaaa aagacctcat cattaagctt 3600cccaagtact ctctctttga
gcttgaaaac ggccggaaac gaatgctcgc tagtgcgggc 3660gagctgcaga
aaggtaacga gctggcactg ccctctaaat acgttaattt cttgtatctg
3720gccagccact atgaaaagct caaagggtct cccgaagata atgagcagaa
gcagctgttc 3780gtggaacaac acaaacacta ccttgatgag atcatcgagc
aaataagcga attctccaaa 3840agagtgatcc tcgccgacgc taacctcgat
aaggtgcttt ctgcttacaa taagcacagg 3900gataagccca tcagggagca
ggcagaaaac attatccact tgtttactct gaccaacttg 3960ggcgcgcctg
cagccttcaa gtacttcgac accaccatag acagaaagcg gtacacctct
4020acaaaggagg tcctggacgc cacactgatt catcagtcaa ttacggggct
ctatgaaaca 4080agaatcgacc tctctcagct cggtggagac agcagggctg
accccaagaa gaagaggaag 4140gtgtga 414623455DNAHomo
sapiensmisc_feature(320)..(338)n is a, c, g, or t 23tgtacaaaaa
agcaggcttt aaaggaacca attcagtcga ctggatccgg taccaaggtc 60gggcaggaag
agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
120gttagagaga taattagaat taatttgact gtaaacacaa agatattagt
acaaaatacg 180tgacgtagaa agtaataatt tcttgggtag tttgcagttt
taaaattatg ttttaaaatg 240gactatcata tgcttaccgt aacttgaaag
tatttcgatt tcttggcttt atatatcttg 300tggaaaggac gaaacaccgn
nnnnnnnnnn nnnnnnnngt tttagagcta gaaatagcaa 360gttaaaataa
ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
420tctagaccca gctttcttgt acaaagttgg catta 4552499RNAArtificialguide
RNA 24gnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc aaguuaaaag
aaggguagug 60gguuaucaac uugaaaaagc ggcaccgagu caauacuuu
992523DNAHomo sapiens 25gtgaaccgca tcgagctgaa ggg 232623DNAHomo
sapiens 26ggagcgcacc atcttcttca agg 232723DNAHomo sapiens
27gtcccctcca ccccacagtg ggg 232823DNAHomo sapiens 28ggggccacta
gggacaggat tgg 232923DNAHomo sapiens 29gcatgatgcg cggcccaagg agg
233023DNAHomo sapiens 30gagatgatcg ccccttcttc tgg 233123DNAHomo
sapiens 31gaattactca cgccccaagg agg 233272DNAHomo sapiens
32taatactttt atcttgtccc ctccacccca cagtggggcc actagggaca ggattggtga
60cagaaaagcc cc 723370DNAHomo sapiens 33taatactttt atctgtcaaa
aaaaccccac agtggggcca ctaggacagg attggtgaca 60gaaaagcccc
703471DNAHomo sapiens 34taatactttt atctgtcggg gggaccccac agtggggcca
ctagggacag gattggtgac 60agaaaagccc c 713561DNAHomo sapiens
35taatgcatga tgcgcggccc aaggagggag atgatcgccc cttcttctgg ctctttgaga
60a 613661DNAHomo sapiens 36taatgaatta ctcacgcccc aaggagggtg
atgaccggcc gttcttctgg atgtttgaga 60a 6137110DNAHomo sapiens
37ggtactttta tctgtcccct ccaccccaca gtgggccact cgggacagga ttggtgacag
60aaaagcccca tccttaggcc tcctccttcc tagtctcctg atattgggtc
1103890DNAHomo sapiens 38ttatctgtcc cctccacccc acagtggggc
cactagggac aggaaaggtg acagaaaagc 60cccatcctta ggcctcctcc ttcctagtct
903923DNAHomo sapiensmisc_feature(2)..(21)wherein N is G, A, T or C
39gnnnnnnnnn nnnnnnnnnn ngg 2340201DNAHomo
sapiensmisc_feature(27)..(51)wherein N is G, A, T or C 40tatgaggacg
aatctccccg cttatannnn nnnnnnnnnn nnnnnnnnnn nttcttggct 60ttatatatct
tgtggaaagg acgaaaacac cgnnnnnnnn nnnnnnnnnn ngttttagag
120ctagaaatag caagttaaaa taaggctagt cnnnnnnnnn nnnnnnnnnn
nnnnnngtac 180aagcacacgt ttgtcaagac c 20141415DNAHomo sapiens
41attcgccctt tgtacaaaaa agcaggcttt aaaggaacca attcagtcga ctggatccgg
60taccaaggtc gggcaggaag agggcctatt tcccatgatt ccttcatatt tcatatacga
120tacaaggctg ttagagagat aataagaatt aatttgactg taaacacaaa
gatattagta 180caaaatacgt gacgtagaaa gtaataattt cttgggtagt
ttgcagtttt aaaattatgt 240tttaaaatgg actatcatat gcttaccgta
acttgaaagt atttcgattt cttggcttta 300tatatcttaa gttaaaataa
ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg 360gtgctttttt
tctagaccca gctttcttgt acaaagttgg cattaaaggg cgaat
4154223DNAArtificialreporter oligonucleotide 42gtcccctcca
ccccacagtg ggg 234323DNAArtificialreporter oligonucleotide
43ggggccacta gggacaggat tgg 2344100DNAArtificialguide RNA
44ggggccacta gggacaccat guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc
60cguuaucaac uucaaaaaca cccacccaga cggugcuuuu 100
* * * * *