U.S. patent application number 16/938110 was filed with the patent office on 2020-11-12 for crispr-cas nickase systems, methods and compositions for sequence manipulation in eukaryotes.
This patent application is currently assigned to The Broad Institute, Inc.. The applicant listed for this patent is The Broad Institute, Inc., Massachusetts Institute of Technology, President and Fellows of Harvard College. Invention is credited to Le Cong, Fei Ran, Feng ZHANG.
Application Number | 20200354742 16/938110 |
Document ID | / |
Family ID | 1000004976608 |
Filed Date | 2020-11-12 |
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United States Patent
Application |
20200354742 |
Kind Code |
A1 |
ZHANG; Feng ; et
al. |
November 12, 2020 |
CRISPR-Cas Nickase Systems, Methods And Compositions For Sequence
Manipulation in Eukaryotes
Abstract
The invention provides for systems, methods, and compositions
for manipulation of sequences and/or activities of target
sequences. Provided are vectors and vector systems, some of which
encode one or more components of a CRISPR complex, as well as
methods for the design and use of such vectors. Also provided are
methods of directing CRISPR complex formation in eukaryotic cells
and methods for selecting specific cells by introducing precise
mutations utilizing the CRISPR/Cas system.
Inventors: |
ZHANG; Feng; (Cambridge,
MA) ; Cong; Le; (Cambridge, MA) ; Ran;
Fei; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Broad Institute, Inc.
Massachusetts Institute of Technology
President and Fellows of Harvard College |
Cambridge
Cambridge
Cambridge |
MA
MA
MA |
US
US
US |
|
|
Assignee: |
The Broad Institute, Inc.
Cambridge
MA
Massachusetts Institute of Technology
Cambridge
MA
President and Fellows of Harvard College
Cambridge
MA
|
Family ID: |
1000004976608 |
Appl. No.: |
16/938110 |
Filed: |
July 24, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15229702 |
Aug 5, 2016 |
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16938110 |
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14105031 |
Dec 12, 2013 |
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15229702 |
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61736527 |
Dec 12, 2012 |
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61748427 |
Jan 2, 2013 |
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61791409 |
Mar 15, 2013 |
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61802174 |
Mar 15, 2013 |
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61835931 |
Jun 17, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/907 20130101;
C12N 2310/531 20130101; C12N 9/22 20130101; C12N 15/113 20130101;
C12N 2310/3519 20130101; C12N 2310/20 20170501; C12N 15/1082
20130101; C12N 15/8509 20130101; C12N 15/8213 20130101; C12N 15/85
20130101; C12N 15/63 20130101; C12N 2310/10 20130101 |
International
Class: |
C12N 15/85 20060101
C12N015/85; C12N 15/113 20060101 C12N015/113; C12N 9/22 20060101
C12N009/22; C12N 15/63 20060101 C12N015/63; C12N 15/82 20060101
C12N015/82; C12N 15/90 20060101 C12N015/90 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0004] This invention was made with government support under Grant
No. MH100706 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. An engineered composition comprising a vector system comprising
one or more eukaryotic expression vectors comprising I. a
nucleotide sequence encoding a CRISPR-Cas complex chimeric RNA
(chiRNA) having two or more hairpins and comprising (a) a guide
sequence capable of hybridizing to a target eukaryotic genome DNA
sequence adjacent to a Protospacer Adjacent Motif (PAM) in a
eukaryotic cell (b) a tracr mate sequence, and (c) a tracr sequence
comprising 30 or more nucleotides in length, and II. a nucleotide
sequence encoding a Cas9 comprising one or more nuclear
localization signals (NLSs) in the proximity of a terminus of the
Cas9 and one or more mutations in a catalytic domain thereby
rendering it a nickase that cleaves a single DNA strand, wherein:
components I and II are located on the same or different vectors of
the system, and components I and II each are operably linked to a
regulatory element for transcription thereof in the eukaryotic cell
and there can be one or more regulatory elements for transcription
of components I and II.
2. A CRISPR composition for multiplexing, wherein the composition
comprises a vector system comprising one or more eukaryotic
expression vectors comprising I. a nucleotide sequence encoding
more than one CRISPR-Cas complex each having two or more hairpins
and comprising (a) a guide sequence capable of hybridizing to a
different target eukaryotic genome DNA sequence adjacent to a PAM
in a eukaryotic, (b) a tracr mate sequence, and (c) a tracr
sequence comprising 30 or more nucleotides in length, and II. a
nucleotide sequence encoding a Cas9 comprising one or more NLSs in
the proximity of a terminus of the Cas9 and one or more mutations
in a catalytic domain thereby rendering it a nickase that cleaves a
single DNA strand, wherein: each of components I and II are
operably linked to a regulatory element for transcription thereof
in the eukaryotic cell and there can be one or more regulatory
elements for transcription of components I and II.
3. The composition of claim 1, wherein the regulatory element
operably linked to component I comprises a polymerase III
promoter.
4. The composition of claim 1, wherein the regulatory element
operably linked to component II comprises a polymerase II
promoter.
5. The composition of claim 1, wherein the guide sequence comprises
about or more than about 10-75 nucleotides in length, and the tracr
sequence comprises about or more than about 30-50 nucleotides in
length, and the tracr sequence exhibits at least 50% sequence
complementarity along the length of the tracr mate.
6. The composition of claim 1, wherein the tracr sequence exhibits
at least 50% of sequence complementarity along the length of the
tracr mate sequence.
7. The composition of claim 1, wherein the nucleotide sequence
encoding the Cas9 is codon-optimized for expression in a eukaryotic
cell.
8. The composition of claim 1, wherein the guide sequence is at
least 15 nucleotides in length.
9. The composition of claim 1, wherein the chiRNA comprises two,
three, four or five hairpins.
10. The composition of claim 1, wherein the catalytic domain
comprises RuvCI, RuvCII, RuvCIII or HNH domain.
11. The composition of claim 1, wherein the Cas9 comprises a
mutation in a residue selected from the group consisting of D10,
E762, H840, N854, N863, or D986, with reference to S. pyogenes
Cas9.
12. The composition of claim 1, wherein the Cas9 comprises a
mutation comprising D10A, E762A, H840A, N854A, N863A or D986A, with
reference to S. pyogenes Cas9.
13. An engineered composition comprising a vector system comprising
one or more vectors comprising I. a nucleotide sequence encoding
(a) a guide sequence capable of hybridizing to a eukaryotic genome
DNA target sequence adjacent to a PAM in a eukaryotic cell, and (b)
a tracr mate sequence, II. a nucleotide sequence encoding a Cas9
comprising one or more NLSs in the proximity of a terminus of the
Cas9 and one or more mutations in a catalytic domain thereby
rendering it a nickase that cleaves a single DNA strand, and III. a
nucleotide sequence encoding a tracr sequence comprising 30 or more
nucleotides in length, wherein: components I, II and III are
located on the same or different vectors of the system, and
components I, II and III are each operably linked to a regulatory
element and there can be one or more regulatory elements for
transcription of components I, II and III.
14. A CRISPR composition for multiplexing, wherein the composition
comprises a vector system comprising one or more vectors comprising
I. (a) nucleotide sequences encoding more than one guide sequences
each capable of hybridizing to a different eukaryotic genome DNA
target sequence adjacent to a PAM in a eukaryotic cell, and (b) a
tracr mate sequence, II. a nucleotide sequence encoding a Cas9
comprising one or more NLSs in the proximity of a terminus of the
Cas9 and one or more mutations in a catalytic domain thereby
rendering it a nickase that cleaves a single DNA strand, and III. a
nucleotide sequence encoding a tracr sequence comprising 30 or more
nucleotides in length, wherein: components I, II and III are
located on the same or different vectors of the system, and each of
components I, II and III are operably linked to a regulatory
element for transcription thereof in the eukaryotic cell and there
can be one or more regulatory elements therefor.
15. The composition of claim 13, wherein the regulatory element
operably linked to component I comprises a polymerase III
promoter.
16. The composition of claim 13, wherein the regulatory element
operably linked to component II comprises a polymerase II
promoter.
17. The composition of claim 13, wherein the regulatory element
operably linked to component III comprises a polymerase III
promoter.
18. The composition of claim 13, wherein the guide sequence
comprises about or more than about 10-75 nucleotides in length, and
the tracr sequence comprises about or more than about 30-50
nucleotides in length, and the tracr sequence exhibits at least 50%
sequence complementarity along the length of the tracr mate.
19. The composition of claim 13, wherein the tracr sequence
exhibits at least 50% of sequence complementarity along the length
of the tracr mate sequence.
20. The composition of claim 13, wherein the nucleotide sequence
encoding the Cas9 is codon-optimized for expression in a eukaryotic
cell.
21. The composition of claim 13, wherein the guide sequence is at
least 15 nucleotides in length.
22. The composition of claim 13, wherein the catalytic domain
comprises RuvCI, RuvCII, RuvCIII or HNH domain.
23. The composition of claim 13, wherein the Cas9 comprises a
mutation in a residue selected from the group consisting of D10,
E762, H840, N854, N863, or D986, with reference to S. pyogenes
Cas9.
24. The composition of claim 13, wherein the Cas9 comprises a
mutation comprising D10A, E762A, H840A, N854A, N863A or D986A, with
reference to S. pyogenes Cas9.
25. An isolated eukaryotic host cell comprising the composition of
claim 1.
26. A non-human animal comprising the eukaryotic host cell of claim
25.
27. A non-human organism or plant comprising the eukaryotic host
cell of claim 25.
28. A kit comprising the composition of claim 1, and instructions
for using said kit.
29. A method of altering the expression of a genomic locus of
interest in a eukaryotic cell comprising contacting the eukaryotic
cell with the composition of claim 1 and thereby delivering the one
or more vectors and allowing a CRISPR-Cas complex to form and bind
to target and determining if the expression of the genomic locus
has been altered.
30. The composition of claim 1, wherein components I and II are on
the same vector.
31. An isolated eukaryotic cell or a eukaryotic cell of a non-human
organism comprising the composition of claim 30.
32. The composition of claim 13, wherein components I, II and III
are on the same vector.
33. An isolated eukaryotic cell or a eukaryotic cell of a non-human
organism comprising the composition of claim 32.
34. The composition of claim 1, wherein the Cas9 comprises one or
more heterologous effector domains.
35. The composition according to claim 34, wherein the one or more
effector domains comprises a transposase domain, integrase domain,
recombinase domain, resolvase domain, invertase domain, protease
domain, DNA methyltransferase domain, DNA demethylase domain,
histone acetylase domain, histone deacetylases domain, nuclease
domain, repressor domain, activator domain, transcription-protein
recruiting domain, cellular uptake activity associated domain,
nucleic acid binding domain or antibody presentation domain.
36. A method of modifying the target eukaryotic genome DNA sequence
adjacent to a PAM in the eukaryotic cell, the method comprising
contacting the eukaryotic cell with the composition of claim 1 and
thereby delivering the one or more vectors to the eukaryotic cell
and allowing a CRISPR complex to form and bind to the target to
effect DNA cleavage of said target thereby modifying the
target.
37. The method of claim 36, wherein said one or more vectors are
delivered to the eukaryotic cell in a subject.
38. The method of claim 36, wherein said modifying takes place in
said eukaryotic cell in a cell culture.
39. The method of claim 36, further comprising isolating said
eukaryotic cell from a subject prior to said modifying.
40. The method of claim 39, further comprising returning said
eukaryotic cell and/or progeny cell(s) therefrom to said subject
after said modifying.
41. An isolated eukaryotic cell or a eukaryotic cell of a non-human
organism from the method of claim 36.
42. A vector comprising a regulatory element operably linked to a
coding sequence encoding a Cas9 comprising one or more NLSs,
wherein said regulatory element is configured to drive
transcription of the Cas9 in a eukaryotic cell in an amount
effective to accumulate the Cas9 in the nucleus of the eukaryotic
cell; wherein the Cas9 comprises one or more mutations in a
catalytic domain thereby rendering the Cas9 a nickase that cleaves
a single DNA strand.
43. The vector of claim 42, wherein said regulatory element is a
polymerase II promoter.
44. An isolated eukaryotic cell or a eukaryotic cell of a non-human
organism comprising the vector of claim 42.
45. An isolated eukaryotic cell or a eukaryotic cell of a non-human
organism comprising a Cas9; wherein the Cas9 comprises one or more
mutations in a catalytic domain thereby rendering the Cas9 a
nickase that cleaves a single DNA strand.
46. An isolated eukaryotic cell culture from the cell of claim
31.
47. An isolated eukaryotic cell culture from the cell of claim
33.
48. An isolated eukaryotic cell culture from the cell of claim
41.
49. An isolated eukaryotic cell culture from the cell of claim
44.
50. The composition of claim 1 wherein component I is operably
linked to a first regulatory element and component II is operably
linked to a second regulatory element.
51. The composition of claim 13 wherein component I is operably
linked to a first regulatory element, component II is operably
linked to a second regulatory element, and component III is
operably linked to a third regulatory element.
52. The composition of claim 1 wherein the target eukaryotic genome
DNA sequence adjacent to a PAM in a eukaryotic cell is a target
mammalian genome DNA sequence adjacent to a PAM in a mammalian
cell.
53. A method of modifying the target mammalian genome DNA sequence
adjacent to a PAM in the mammalian cell, the method comprising
contacting the mammalian cell with the composition of claim 52 and
thereby delivering the vector to the mammalian cell and allowing a
CRISPR complex to form and bind to the target to effect DNA
cleavage of said target, thereby modifying the target.
54. The method of claim 53 wherein the modifying obtains increased
expression as to a product of the mammalian cell, in comparison
with a cell that has not been subjected to the method.
55. The method of claim 36 wherein the modifying obtains increased
expression as to a product of the cell, in comparison with a cell
that has not been subjected to the method.
56. An isolated cell having increased expression as to a product of
the cell, from the method of claim 54, wherein the increased
expression is in comparison with a cell that has not been subjected
to method modifying the target genome DNA sequence in the cell.
57. An isolated cell having increased expression as to a product of
the cell, from the method of claim 55, wherein the increased
expression is in comparison with a cell that has not been subjected
to method modifying the target genome DNA sequence in the cell.
58. An isolated cell line of the cell of claim 56.
59. An isolated cell line of the cell of claim 57.
Description
RELATED APPLICATIONS AND INCORPORATION BY REFERENCE
[0001] This application is a continuation of U.S. application Ser.
No. 15/229,702, filed on Aug. 5, 2016, which is a continuation of
U.S. application Ser. No. 14/105,031, filed Dec. 12, 2013, which
claims priority to U.S. provisional patent applications 61/736,527,
61/748,427, 61/791,409 and 61/835,931 all entitled SYSTEMS METHODS
AND COMPOSITIONS FOR SEQUENCE MANIPULATION, filed on Dec. 12, 2012,
Jan. 2, 2013, Mar. 15, 2013 and Jun. 17, 2013, respectively. This
application also claims priority to U.S. provisional patent
application 61/802,174 entitled ENGINEERING AND OPTIMIZATION OF
SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION, filed
on Mar. 15, 2013.
[0002] Reference is made to U.S. provisional patent applications
61/758,468; 61/769,046; 61/802,174; 61/806,375; 61/814,263;
61/819,803 and 61/828,130, each entitled ENGINEERING AND
OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE
MANIPULATION, filed on Jan. 30, 2013; Feb. 25, 2013; Mar. 15, 2013;
Mar. 28, 2013; Apr. 20, 2013; May 6, 2013 and May 28, 2013
respectively. Reference is also made to U.S. provisional patent
applications 61/835,936, 61/836,127, 61/836,101, 61/836,080 and
61/835,973 each filed Jun. 17, 2013. Reference is also made to U.S.
provisional patent application 61/842,322 and U.S. patent
application Ser. No. 14/054,414, entitled CRISPR-CAS SYSTEMS AND
METHODS FOR ALTERING EXPRESSION OF GENE PRODUCTS filed on Jul. 2,
2013 and Oct. 15, 2013 respectively.
[0003] The foregoing applications, and all documents cited therein
or during their prosecution ("appin cited documents") and all
documents cited or referenced in the appin cited documents, and all
documents cited or referenced herein ("herein cited documents"),
and all documents cited or referenced in herein cited documents,
together with any manufacturer's instructions, descriptions,
product specifications, and product sheets for any products
mentioned herein or in any document incorporated by reference
herein, are hereby incorporated herein by reference, and may be
employed in the practice of the invention. More specifically, all
referenced documents are incorporated by reference to the same
extent as if each individual document was specifically and
individually indicated to be incorporated by reference.
SEQUENCE LISTING
[0005] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Aug. 28, 2014, is named 44790.06.2003_SL.txt and is 321,104
bytes in size.
FIELD OF THE INVENTION
[0006] The present invention generally relates to systems, methods
and compositions used for the control of gene expression involving
sequence targeting, such as genome perturbation or gene-editing,
that may use vector systems related to Clustered Regularly
Interspaced Short Palindromic Repeats (CRISPR) and components
thereof.
BACKGROUND OF THE INVENTION
[0007] Recent advances in genome sequencing techniques and analysis
methods have significantly accelerated the ability to catalog and
map genetic factors associated with a diverse range of biological
functions and diseases. Precise genome targeting technologies are
needed to enable systematic reverse engineering of causal genetic
variations by allowing selective perturbation of individual genetic
elements, as well as to advance synthetic biology,
biotechnological, and medical applications. Although genome-editing
techniques such as designer zinc fingers, transcription
activator-like effectors (TALEs), or homing meganucleases are
available for producing targeted genome perturbations, there
remains a need for new genome engineering technologies that are
affordable, easy to set up, scalable, and amenable to targeting
multiple positions within the eukaryotic genome.
SUMMARY OF THE INVENTION
[0008] There exists a pressing need for alternative and robust
systems and techniques for sequence targeting with a wide array of
applications. This invention addresses this need and provides
related advantages. The CRISPR/Cas or the CRISPR-Cas system (both
terms are used interchangeably throughout this application) does
not require the generation of customized proteins to target
specific sequences but rather a single Cas enzyme can be programmed
by a short RNA molecule to recognize a specific DNA target, in
other words the Cas enzyme can be recruited to a specific DNA
target using said short RNA molecule. Adding the CRISPR-Cas system
to the repertoire of genome sequencing techniques and analysis
methods may significantly simplify the methodology and accelerate
the ability to catalog and map genetic factors associated with a
diverse range of biological functions and diseases. To utilize the
CRISPR-Cas system effectively for genome editing without
deleterious effects, it is critical to understand aspects of
engineering and optimization of these genome engineering tools,
which are aspects of the claimed invention.
[0009] In one aspect, the invention provides a vector system
comprising one or more vectors. In some embodiments, the system
comprises: (a) a first regulatory element operably linked to a
tracr mate sequence and one or more insertion sites for inserting
one or more guide sequences upstream of the tracr mate sequence,
wherein when expressed, the guide sequence directs
sequence-specific binding of a CRISPR complex to a target sequence
in a eukaryotic cell, wherein the CRISPR complex comprises a CRISPR
enzyme complexed with (1) the guide sequence that is hybridized to
the target sequence, and (2) the tracr mate sequence that is
hybridized to the tracr sequence; and (b) a second regulatory
element operably linked to an enzyme-coding sequence encoding said
CRISPR enzyme comprising a nuclear localization sequence; wherein
components (a) and (b) are located on the same or different vectors
of the system. In some embodiments, component (a) further comprises
the tracr sequence downstream of the tracr mate sequence under the
control of the first regulatory element. In some embodiments,
component (a) further comprises two or more guide sequences
operably linked to the first regulatory element, wherein when
expressed, each of the two or more guide sequences direct sequence
specific binding of a CRISPR complex to a different target sequence
in a eukaryotic cell. In some embodiments, the system comprises the
tracr sequence under the control of a third regulatory element,
such as a polymerase III promoter. In some embodiments, the tracr
sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of
sequence complementarity along the length of the tracr mate
sequence when optimally aligned. Determining optimal alignment is
within the purview of one of skill in the art. For example, there
are publically and commercially available alignment algorithms and
programs such as, but not limited to, ClustalW, Smith-Waterman in
matlab, Bowtie, Geneious, Biopython and SeqMan. In some
embodiments, the CRISPR complex comprises one or more nuclear
localization sequences of sufficient strength to drive accumulation
of said CRISPR complex in a detectable amount in the nucleus of a
eukaryotic cell. Without wishing to be bound by theory, it is
believed that a nuclear localization sequence is not necessary for
CRISPR complex activity in eukaryotes, but that including such
sequences enhances activity of the system, especially as to
targeting nucleic acid molecules in the nucleus. In some
embodiments, the CRISPR enzyme is a type II CRISPR system enzyme.
In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some
embodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes, or S.
thermophilus Cas9, and may include mutated Cas9 derived from these
organisms. The enzyme may be a Cas9 homolog or ortholog. In some
embodiments, the CRISPR enzyme is codon-optimized for expression in
a eukaryotic cell. In some embodiments, the CRISPR enzyme directs
cleavage of one or two strands at the location of the target
sequence. In some embodiments, the CRISPR enzyme lacks DNA strand
cleavage activity. In some embodiments, the first regulatory
element is a polymerase III promoter. In some embodiments, the
second regulatory element is a polymerase II promoter. In some
embodiments, the guide sequence is at least 15, 16, 17, 18, 19, 20,
25 nucleotides, or between 10-30, or between 15-25, or between
15-20 nucleotides in length. In general, and throughout this
specification, the term "vector" refers to a nucleic acid molecule
capable of transporting another nucleic acid to which it has been
linked. Vectors include, but are not limited to, nucleic acid
molecules that are single-stranded, double-stranded, 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,
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.
[0010] 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).
[0011] 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
comprises 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 .beta.-actin promoter, the phosphoglycerol
kinase (PGK) promoter, and the EF1.alpha. promoter. 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.).
[0012] Advantageous vectors include lentiviruses and
adeno-associated viruses, and types of such vectors can also be
selected for targeting particular types of cells.
[0013] In one aspect, the invention provides a vector comprising a
regulatory element operably linked to an enzyme-coding sequence
encoding a CRISPR enzyme comprising one or more nuclear
localization sequences. In some embodiments, said regulatory
element drives transcription of the CRISPR enzyme in a eukaryotic
cell such that said CRISPR enzyme accumulates in a detectable
amount in the nucleus of the eukaryotic cell. In some embodiments,
the regulatory element is a polymerase II promoter. In some
embodiments, the CRISPR enzyme is a type II CRISPR system enzyme.
In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some
embodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes or S.
thermophilus Cas9, and may include mutated Cas9 derived from these
organisms. In some embodiments, the CRISPR enzyme is
codon-optimized for expression in a eukaryotic cell. In some
embodiments, the CRISPR enzyme directs cleavage of one or two
strands at the location of the target sequence. In some
embodiments, the CRISPR enzyme lacks DNA strand cleavage
activity.
[0014] In one aspect, the invention provides a CRISPR enzyme
comprising one or more nuclear localization sequences of sufficient
strength to drive accumulation of said CRISPR enzyme in a
detectable amount in the nucleus of a eukaryotic cell. In some
embodiments, the CRISPR enzyme is a type II CRISPR system enzyme.
In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some
embodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes or S.
thermophilus Cas9, and may include mutated Cas9 derived from these
organisms. The enzyme may be a Cas9 homolog or ortholog. In some
embodiments, the CRISPR enzyme lacks the ability to cleave one or
more strands of a target sequence to which it binds.
[0015] In one aspect, the invention provides a eukaryotic host cell
comprising (a) a first regulatory element operably linked to a
tracr mate sequence and one or more insertion sites for inserting
one or more guide sequences upstream of the tracr mate sequence,
wherein when expressed, the guide sequence directs
sequence-specific binding of a CRISPR complex to a target sequence
in a eukaryotic cell, wherein the CRISPR complex comprises a CRISPR
enzyme complexed with (1) the guide sequence that is hybridized to
the target sequence, and (2) the tracr mate sequence that is
hybridized to the tracr sequence; and/or (b) a second regulatory
element operably linked to an enzyme-coding sequence encoding said
CRISPR enzyme comprising a nuclear localization sequence. In some
embodiments, the host cell comprises components (a) and (b). In
some embodiments, component (a), component (b), or components (a)
and (b) are stably integrated into a genome of the host eukaryotic
cell. In some embodiments, component (a) further comprises the
tracr sequence downstream of the tracr mate sequence under the
control of the first regulatory element. In some embodiments,
component (a) further comprises two or more guide sequences
operably linked to the first regulatory element, wherein when
expressed, each of the two or more guide sequences direct sequence
specific binding of a CRISPR complex to a different target sequence
in a eukaryotic cell. In some embodiments, the eukaryotic host cell
further comprises a third regulatory element, such as a polymerase
III promoter, operably linked to said tracr sequence. In some
embodiments, the tracr sequence exhibits at least 50%, 60%, 70%,
80%, 90%, 95%, or 99% of sequence complementarity along the length
of the tracr mate sequence when optimally aligned. In some
embodiments, the CRISPR enzyme comprises one or more nuclear
localization sequences of sufficient strength to drive accumulation
of said CRISPR enzyme in a detectable amount in the nucleus of a
eukaryotic cell. In some embodiments, the CRISPR enzyme is a type
II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is
a Cas9 enzyme. In some embodiments, the Cas9 enzyme is S.
pneumoniae, S. pyogenes or S. thermophilus Cas9, and may include
mutated Cas9 derived from these organisms. The enzyme may be a Cas9
homolog or ortholog. In some embodiments, the CRISPR enzyme is
codon-optimized for expression in a eukaryotic cell. In some
embodiments, the CRISPR enzyme directs cleavage of one or two
strands at the location of the target sequence. In some
embodiments, the CRISPR enzyme lacks DNA strand cleavage activity.
In some embodiments, the first regulatory element is a polymerase
III promoter. In some embodiments, the second regulatory element is
a polymerase II promoter. In some embodiments, the guide sequence
is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or between
10-30, or between 15-25, or between 15-20 nucleotides in length. In
an aspect, the invention provides a non-human eukaryotic organism;
preferably a multicellular eukaryotic organism, comprising a
eukaryotic host cell according to any of the described embodiments.
In other aspects, the invention provides a eukaryotic organism;
preferably a multicellular eukaryotic organism, comprising a
eukaryotic host cell according to any of the described embodiments.
The organism in some embodiments of these aspects may be an animal;
for example a mammal. Also, the organism may be an arthropod such
as an insect. The organism also may be a plant. Further, the
organism may be a fungus.
[0016] In one aspect, the invention provides a kit comprising one
or more of the components described herein. In some embodiments,
the kit comprises a vector system and instructions for using the
kit. In some embodiments, the vector system comprises (a) a first
regulatory element operably linked to a tracr mate sequence and one
or more insertion sites for inserting one or more guide sequences
upstream of the tracr mate sequence, wherein when expressed, the
guide sequence directs sequence-specific binding of a CRISPR
complex to a target sequence in a eukaryotic cell, wherein the
CRISPR complex comprises a CRISPR enzyme complexed with (1) the
guide sequence that is hybridized to the target sequence, and (2)
the tracr mate sequence that is hybridized to the tracr sequence;
and/or (b) a second regulatory element operably linked to an
enzyme-coding sequence encoding said CRISPR enzyme comprising a
nuclear localization sequence. In some embodiments, the kit
comprises components (a) and (b) located on the same or different
vectors of the system. In some embodiments, component (a) further
comprises the tracr sequence downstream of the tracr mate sequence
under the control of the first regulatory element. In some
embodiments, component (a) further comprises two or more guide
sequences operably linked to the first regulatory element, wherein
when expressed, each of the two or more guide sequences direct
sequence specific binding of a CRISPR complex to a different target
sequence in a eukaryotic cell. In some embodiments, the system
further comprises a third regulatory element, such as a polymerase
III promoter, operably linked to said tracr sequence. In some
embodiments, the tracr sequence exhibits at least 50%, 60%, 70%,
80%, 90%, 95%, or 99% of sequence complementarity along the length
of the tracr mate sequence when optimally aligned. In some
embodiments, the CRISPR enzyme comprises one or more nuclear
localization sequences of sufficient strength to drive accumulation
of said CRISPR enzyme in a detectable amount in the nucleus of a
eukaryotic cell. In some embodiments, the CRISPR enzyme is a type
II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is
a Cas9 enzyme. In some embodiments, the Cas9 enzyme is S.
pneumoniae, S. pyogenes or S. thermophilus Cas9, and may include
mutated Cas9 derived from these organisms. The enzyme may be a Cas9
homolog or ortholog. In some embodiments, the CRISPR enzyme is
codon-optimized for expression in a eukaryotic cell. In some
embodiments, the CRISPR enzyme directs cleavage of one or two
strands at the location of the target sequence. In some
embodiments, the CRISPR enzyme lacks DNA strand cleavage activity.
In some embodiments, the first regulatory element is a polymerase
III promoter. In some embodiments, the second regulatory element is
a polymerase II promoter. In some embodiments, the guide sequence
is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or between
10-30, or between 15-25, or between 15-20 nucleotides in
length.
[0017] In one aspect, the invention provides a method of modifying
a target polynucleotide in a eukaryotic cell. In some embodiments,
the method comprises allowing a CRISPR complex to bind to the
target polynucleotide to effect cleavage of said target
polynucleotide thereby modifying the target polynucleotide, wherein
the CRISPR complex comprises a CRISPR enzyme complexed with a guide
sequence hybridized to a target sequence within said target
polynucleotide, wherein said guide sequence is linked to a tracr
mate sequence which in turn hybridizes to a tracr sequence. In some
embodiments, said cleavage comprises cleaving one or two strands at
the location of the target sequence by said CRISPR enzyme. In some
embodiments, said cleavage results in decreased transcription of a
target gene. In some embodiments, the method further comprises
repairing said cleaved target polynucleotide by homologous
recombination with an exogenous template polynucleotide, wherein
said repair results in a mutation comprising an insertion,
deletion, or substitution of one or more nucleotides of said target
polynucleotide. In some embodiments, said mutation results in one
or more amino acid changes in a protein expressed from a gene
comprising the target sequence. In some embodiments, the method
further comprises delivering one or more vectors to said eukaryotic
cell, wherein the one or more vectors drive expression of one or
more of: the CRISPR enzyme, the guide sequence linked to the tracr
mate sequence, and the tracr sequence. In some embodiments, said
vectors are delivered to the eukaryotic cell in a subject. In some
embodiments, said modifying takes place in said eukaryotic cell in
a cell culture. In some embodiments, the method further comprises
isolating said eukaryotic cell from a subject prior to said
modifying. In some embodiments, the method further comprises
returning said eukaryotic cell and/or cells derived therefrom to
said subject.
[0018] In one aspect, the invention provides a method of modifying
expression of a polynucleotide in a eukaryotic cell. In some
embodiments, the method comprises allowing a CRISPR complex to bind
to the polynucleotide such that said binding results in increased
or decreased expression of said polynucleotide; wherein the CRISPR
complex comprises a CRISPR enzyme complexed with a guide sequence
hybridized to a target sequence within said polynucleotide, wherein
said guide sequence is linked to a tracr mate sequence which in
turn hybridizes to a tracr sequence. In some embodiments, the
method further comprises delivering one or more vectors to said
eukaryotic cells, wherein the one or more vectors drive expression
of one or more of: the CRISPR enzyme, the guide sequence linked to
the tracr mate sequence, and the tracr sequence.
[0019] In one aspect, the invention provides a method of generating
a model eukaryotic cell comprising a mutated disease gene. In some
embodiments, a disease gene is any gene associated an increase in
the risk of having or developing a disease. In some embodiments,
the method comprises (a) introducing one or more vectors into a
eukaryotic cell, wherein the one or more vectors drive expression
of one or more of: a CRISPR enzyme, a guide sequence linked to a
tracr mate sequence, and a tracr sequence; and (b) allowing a
CRISPR complex to bind to a target polynucleotide to effect
cleavage of the target polynucleotide within said disease gene,
wherein the CRISPR complex comprises the CRISPR enzyme complexed
with (1) the guide sequence that is hybridized to the target
sequence within the target polynucleotide, and (2) the tracr mate
sequence that is hybridized to the tracr sequence, thereby
generating a model eukaryotic cell comprising a mutated disease
gene. In some embodiments, said cleavage comprises cleaving one or
two strands at the location of the target sequence by said CRISPR
enzyme. In some embodiments, said cleavage results in decreased
transcription of a target gene. In some embodiments, the method
further comprises repairing said cleaved target polynucleotide by
homologous recombination with an exogenous template polynucleotide,
wherein said repair results in a mutation comprising an insertion,
deletion, or substitution of one or more nucleotides of said target
polynucleotide. In some embodiments, said mutation results in one
or more amino acid changes in a protein expression from a gene
comprising the target sequence.
[0020] In one aspect, the invention provides a method for
developing a biologically active agent that modulates a cell
signaling event associated with a disease gene. In some
embodiments, a disease gene is any gene associated an increase in
the risk of having or developing a disease. In some embodiments,
the method comprises (a) contacting a test compound with a model
cell of any one of the described embodiments; and (b) detecting a
change in a readout that is indicative of a reduction or an
augmentation of a cell signaling event associated with said
mutation in said disease gene, thereby developing said biologically
active agent that modulates said cell signaling event associated
with said disease gene.
[0021] In one aspect, the invention provides a recombinant
polynucleotide comprising a guide sequence upstream of a tracr mate
sequence, wherein the guide sequence when expressed directs
sequence-specific binding of a CRISPR complex to a corresponding
target sequence present in a eukaryotic cell. In some embodiments,
the target sequence is a viral sequence present in a eukaryotic
cell. In some embodiments, the target sequence is a proto-oncogene
or an oncogene.
[0022] In one aspect the invention provides for a method of
selecting one or more prokaryotic cell(s) by introducing one or
more mutations in a gene in the one or more prokaryotic cell (s),
the method comprising: introducing one or more vectors into the
prokaryotic cell (s), wherein the one or more vectors drive
expression of one or more of: a CRISPR enzyme, a guide sequence
linked to a tracr mate sequence, a tracr sequence, and a editing
template; wherein the editing template comprises the one or more
mutations that abolish CRISPR enzyme cleavage; allowing homologous
recombination of the editing template with the target
polynucleotide in the cell(s) to be selected; allowing a CRISPR
complex to bind to a target polynucleotide to effect cleavage of
the target polynucleotide within said gene, wherein the CRISPR
complex comprises the CRISPR enzyme complexed with (1) the guide
sequence that is hybridized to the target sequence within the
target polynucleotide, and (2) the tracr mate sequence that is
hybridized to the tracr sequence, wherein binding of the CRISPR
complex to the target polynucleotide induces cell death, thereby
allowing one or more prokaryotic cell(s) in which one or more
mutations have been introduced to be selected. In a preferred
embodiment, the CRISPR enzyme is Cas9. In another aspect of the
invention the cell to be selected may be a eukaryotic cell. Aspects
of the invention allow for selection of specific cells without
requiring a selection marker or a two-step process that may include
a counter-selection system.
[0023] Accordingly, it is an object of the invention not to
encompass within the invention any previously known product,
process of making the product, or method of using the product such
that Applicants reserve the right and hereby disclose a disclaimer
of any previously known product, process, or method. It is further
noted that the invention does not intend to encompass within the
scope of the invention any product, process, or making of the
product or method of using the product, which does not meet the
written description and enablement requirements of the USPTO (35
U.S.C. .sctn. 112, first paragraph) or the EPO (Article 83 of the
EPC), such that Applicants reserve the right and hereby disclose a
disclaimer of any previously described product, process of making
the product, or method of using the product.
[0024] It is noted that in this disclosure and particularly in the
claims and/or paragraphs, terms such as "comprises", "comprised",
"comprising" and the like can have the meaning attributed to it in
U.S. Patent law; e.g., they can mean "includes", "included",
"including", and the like; and that terms such as "consisting
essentially of" and "consists essentially of" have the meaning
ascribed to them in U.S. Patent law, e.g., they allow for elements
not explicitly recited, but exclude elements that are found in the
prior art or that affect a basic or novel characteristic of the
invention. These and other embodiments are disclosed or are obvious
from and encompassed by, the following Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0026] FIG. 1 shows a schematic model of the CRISPR system. The
Cas9 nuclease from Streptococcus pyogenes (yellow) is targeted to
genomic DNA by a synthetic guide RNA (sgRNA) consisting of a 20-nt
guide sequence (blue) and a scaffold (red). The guide sequence
base-pairs with the DNA target (blue), directly upstream of a
requisite 5'-NGG protospacer adjacent motif (PAM; magenta), and
Cas9 mediates a double-stranded break (DSB) .about.3 bp upstream of
the PAM (red triangle).
[0027] FIGS. 2A-2F show an exemplary CRISPR system, a possible
mechanism of action, an example adaptation for expression in
eukaryotic cells, and results of tests assessing nuclear
localization and CRISPR activity. FIG. 2A illustrates a schematic
showing the CRISPR locus 1 from Streptococcus pyogenes SF370 and a
proposed mechanism of CRISPR-mediated DNA cleavage by this system.
FIG. 2B illustrates engineering of S. pyogenes Cas9 (SpCas9) and
RNase III (SpRNase III) with nuclear localization signals (NLSs) to
enable import into the mammalian nucleus. FIG. 2C illustrates
mammalian expression of SpCas9 and SpRNase III driven by the
constitutive EF1a promoter and tracrRNA and pre-crRNA array
(DR-Spacer-DR) driven by the RNA Pol3 promoter U6 to promote
precise transcription initiation and termination. FIG. 2D
illustrates surveyor nuclease assay for SpCas9-mediated minor
insertions and deletions. SpCas9 was expressed with and without
SpRNase III, tracrRNA, and a pre-crRNA array carrying the
EMX1-target spacer. FIG. 2E illustrates a schematic representation
of base pairing between target locus and EMX1-targeting crRNA, as
well as an example chromatogram showing a micro deletion adjacent
to the SpCas9 cleavage site. FIG. 2F illustrates mutated alleles
identified from sequencing analysis of 43 clonal amplicons showing
a variety of micro insertions and deletions. FIG. 2C discloses SEQ
ID NOS 281-282, respectively, in order of appearance. FIG. 2E
discloses SEQ ID NOS 283-285, respectively, in order of appearance.
FIG. 2F discloses SEQ ID NOS 286-290, respectively, in order of
appearance.
[0028] FIG. 3A-3C shows an exemplary expression cassette for
expression of CRISPR system elements in eukaryotic cells, predicted
structures of example guide sequences, and CRISPR system activity
as measured in eukaryotic and prokaryotic cells (SEQ ID NOS
291-300, respectively, in order of appearance). FIG. 3A provides a
schematic of a bi-cistronic vector for driving the expression of a
synthetic crRNA-tracrRNA chimera (chimeric RNA) as well as SpCas9.
FIG. 3B provides a schematic showing guide sequences targeting the
human EMX1, PVALB, and mouse Th loci, as well as their predicted
secondary structures. FIG. 3C shows evaluation of crRNA targeting
efficiency.
[0029] FIGS. 4A-4D show results of an evaluation of SpCas9
specificity for an example target. FIG. 4A shows analysis of the
effect of single-nucleotide mutations in the guide sequence on
protospacer cleavage in the mammalian genome using a series of
EMX1-targeting chimeric crRNAs with single point mutations. FIG. 4A
discloses SEQ ID NOS 301, 284 and 302-312, respectively, in order
of appearance. FIG. 4B illustrates results of a Surveyor nuclease
assay comparing the cleavage efficiency of Cas9 when paired with
different mutant chimeric RNAs. FIG. 4C provides a schematic
showing the design of TALENs targeting EMX1. FIG. 4C discloses SEQ
ID NO: 301. FIG. 4D shows a Surveyor gel comparing the efficiency
of TALEN and Cas9 (n=3).
[0030] FIGS. 5A-5G show an exemplary vector system and results for
its use in directing homologous recombination in eukaryotic cells.
FIG. 5A discloses SpCas9n. FIG. 5B discloses co-expression of
EMX1-targeting chimeric crRNA with SpCas9 produced indels in the
target site, whereas co-expression with SpCas9n did not. FIG. 5C
provides a schematic illustration of the HR strategy. FIG. 5D shows
restriction fragment length polymorphism gel analysis. FIG. 5E
discloses SEQ ID NO: 313. FIG. 5F discloses SEQ ID NOS 314-315,
respectively, in order of appearance. FIG. 5G discloses SEQ ID NOS
316-320, respectively, in order of appearance.
[0031] FIG. 6 provides a table of protospacer sequences (SEQ ID NOS
33, 32, 31, 324-329, 35, 34 and 332-336, respectively, in order of
appearance) and summarizes modification efficiency results for
protospacer targets designed based on exemplary S. pyogenes and S.
thermophilus CRISPR systems with corresponding PAMs against loci in
human and mouse genomes. Cells were transfected with Cas9 and
either pre-crRNA/tracrRNA or chimeric RNA, and analyzed 72 hours
after transfection. Percent indels are calculated based on Surveyor
assay results from indicated cell lines (N=3 for all protospacer
targets, errors are S.E.M., N.D. indicates not detectable using the
Surveyor assay, and N.T. indicates not tested in this study).
[0032] FIGS. 7A-7C show a comparison of different tracrRNA
transcripts for Cas9-mediated gene targeting. FIG. 7A discloses SEQ
ID NOS 337-338, respectively, in order of appearance. FIG. 7A shows
expression constructs for tracrRNA. FIG. 7B shows results of the
Surveyor assay. FIG. 7C shows results of a Northern blot analysis
of total RNA extracted from 293FT cells transfected with U6
expression constructs carrying long or short tracrRNA, as well as
SpCas9 and DR-EMX1(1)-DR.
[0033] FIG. 8 shows a schematic of a surveyor nuclease assay for
detection of double strand break-induced micro-insertions and
-deletions.
[0034] FIGS. 9A-9B show exemplary bicistronic expression vectors
for expression of CRISPR system elements in eukaryotic cells. FIG.
9A discloses SEQ ID NOS 339-341, respectively, in order of
appearance. FIG. 9B discloses SEQ ID NOS 342-344, respectively, in
order of appearance.
[0035] FIG. 10A-10D shows a bacterial plasmid transformation
interference assay, expression cassettes and plasmids used therein,
and transformation efficiencies of cells used therein. FIG. 10A
shows pCRISPR plasmid comprising elements of the S. pyogenes CRISPR
locus 1 sufficient for CRISPR activity in E. coli. FIG. 10A
discloses SEQ ID NOS 345-347, respectively, in order of appearance.
FIG. 10B shows challenge plasmids used in the interference assay
were constructed by inserting the protospacer sequence along with
an adjacent CRISPR motif sequence (PAM) into pUC19. FIG. 10C
provides a schematic representation of the interference assay. FIG.
10D illustrates competence of each pCRISPR-carrying E. coli strain
used in assays illustrated in FIG. 4C.
[0036] FIGS. 11A-11C show histograms of distances between adjacent
S. pyogenes SF370 locus 1 PAM (NGG) (FIG. 11A) and S. thermophilus
LMD9 locus 2 PAM (NNAGAAW) (FIG. 11B) in the human genome; and
distances for each PAM by chromosome (Chr) (FIG. 11C).
[0037] FIGS. 12A-12C show an exemplary CRISPR system, an example
adaptation for expression in eukaryotic cells, and results of tests
assessing CRISPR activity. FIG. 12A provides a Schematic
illustration of CRISPR 1 from S. thermophilus LMD-9. FIG. 12B
illustrates the design of an expression system for the S.
thermophilus CRISPR system. FIG. 12B discloses SEQ ID NOS 348-349,
respectively, in order of appearance. FIG. 12C provides a schematic
showing guide sequences targeting the human EMX1 locus as well as
their predicted secondary structures. FIG. 12C discloses SEQ ID NO:
350.
[0038] FIGS. 13A-13C show exemplary manipulations of a CRISPR
system for targeting of genomic loci in mammalian cells. FIG. 13A
provides a schematic of the human EMX1 locus showing the location
of five protospacers, indicated by the underlined sequences. FIG.
13A discloses SEQ ID NO: 351. FIG. 13B provides a schematic of the
pre-crRNA/trcrRNA complex showing hybridization between the direct
repeat region of the pre-crRNA and tracrRNA (top), and a schematic
of a chimeric RNA design comprising a 20 bp guide sequence, and
tracr mate and tracr sequences consisting of partial direct repeat
and tracrRNA sequences hybridized in a hairpin structure (bottom).
FIG. 13B discloses SEQ ID NOS 352-354, respectively, in order of
appearance. FIG. 13C shows results of a Surveyor assay comparing
the efficacy of Cas9-mediated cleavage at five protospacers in the
human EMX1 locus.
[0039] FIGS. 14A-14B show the results of a Northern blot analysis
of crRNA processing in mammalian cells. FIG. 14A illustrates a
schematic showing the expression vector for a single spacer flanked
by two direct repeats. FIG. 14A discloses SEQ ID NO: 355. FIG. 14B
shows a Northern blot analysis of total RNA extracted from 293FT
cells transfected with U6 expression constructs carrying
DR-EMX1(1)-DR.
[0040] FIG. 15A-15B shows an exemplary selection of protospacers in
the human PVALB and mouse Th loci. FIG. 15A discloses SEQ ID NO:
356. FIG. 15B discloses SEQ ID NO: 357.
[0041] FIG. 16 shows example protospacer and corresponding PAM
sequence targets of the S. thermophilus CRISPR system in the human
EMX1 locus (SEQ ID NO: 350).
[0042] FIG. 17 provides a table of sequences for primers and probes
(SEQ ID NOS 36-39 and 358-365, respectively, in order of
appearance) used for Surveyor, RFLP, genomic sequencing, and
Northern blot assays.
[0043] FIGS. 18A-18C show exemplary manipulation of a CRISPR system
with chimeric RNAs and results of SURVEYOR assays for system
activity in eukaryotic cells. FIG. 18A illustrates a schematic of a
bicistronic expression vector for chimeric RNA and Cas9. FIG. 18A
discloses SEQ ID NO: 366, respectively, in order of appearance.
FIGS. 18B and 18C show results of SURVEYOR assays for Cas9-mediated
indels at the human EMX1 and PVALB loci, respectively.
[0044] FIGS. 19A-19B show a graphical representation of the results
of SURVEYOR assays for CRISPR system activity in eukaryotic cells.
FIG. 19A shows results from the EMX1 locus. FIG. 19B shows results
from the PVALB locus.
[0045] FIG. 20 shows an exemplary visualization of some S. pyogenes
Cas9 target sites in the human genome using the UCSC genome browser
(SEQ ID NOS 367-445, respectively, in order of appearance).
[0046] FIG. 21 shows predicted secondary structures for exemplary
chimeric RNAs comprising a guide sequence, tracr mate sequence, and
tracr sequence (SEQ ID NOS 446-465, respectively, in order of
appearance).
[0047] FIG. 22 shows exemplary bicistronic expression vectors for
expression of CRISPR system elements in eukaryotic cells (SEQ ID
NOS 466 and 343-344, respectively, in order of appearance).
[0048] FIG. 23A-23B shows that Cas9 nuclease activity against
endogenous targets may be exploited for genome editing. (a) Concept
of genome editing using the CRISPR system. The CRISPR targeting
construct directed cleavage of a chromosomal locus and was
co-transformed with an editing template that recombined with the
target to prevent cleavage. Kanamycin-resistant transformants that
survived CRISPR attack contained modifications introduced by the
editing template. tracr, trans-activating CRISPR RNA; aphA-3,
kanamycin resistance gene. (b) Transformation of crR6M DNA in
R6.sup.8232.5 cells with no editing template, the R6 wild-type srtA
or the R6370.1 editing templates. Recombination of either R6 srtA
or R6.sup.370.1 prevented cleavage by Cas9. Transformation
efficiency was calculated as colony forming units (cfu) per .mu.g
of crR6M DNA; the mean values with standard deviations from at
least three independent experiments are shown. PCR analysis was
performed on 8 clones in each transformation. "Un." indicates the
unedited srtA locus of strain R6.sup.8232.5; "Ed." shows the
editing template. R6.sup.8232.5 and R6.sup.370.1 targets are
distinguished by restriction with EaeI.
[0049] FIG. 24A-24C shows analysis of PAM and seed sequences that
eliminate Cas9 cleavage. (a) PCR products with randomized PAM
sequences or randomized seed sequences were transformed in crR6
cells (SEQ ID NOS 467-471, respectively, in order of appearance).
These cells expressed Cas9 loaded with a crRNA that targeted a
chromosomal region of R6.sup.8232.5 cells (highlighted in pink)
that is absent from the R6 genome. More than 2.times.105
chloramphenicol-resistant transformants, carrying inactive PAM or
seed sequences, were combined for amplification and deep sequencing
of the target region. (b) Relative proportion of number of reads
after transformation of the random PAM constructs in crR6 cells
(compared to number of reads in R6 transformants). The relative
abundance for each 3-nucleotide PAM sequence is shown. Severely
underrepresented sequences (NGG) are shown in red; partially
underrepresented one in orange (NAG) (c) Relative proportion of
number of reads after transformation of the random seed sequence
constructs in crR6 cells (compared to number of reads in R6
transformants). The relative abundance of each nucleotide for each
position of the first 20 nucleotides of the protospacer sequence is
shown (SEQ ID NO: 472). High abundance indicates lack of cleavage
by Cas9, i.e. a CRISPR inactivating mutation. The grey line shows
the level of the WT sequence. The dotted line represents the level
above which a mutation significantly disrupts cleavage (See section
"Analysis of deep sequencing data" in Example 5)
[0050] FIG. 25A-25F shows introduction of single and multiple
mutations using the CRISPR system in S. pneumoniae. (FIG. 25A)
Nucleotide and amino acid sequences of the wild-type and edited
(green nucleotides; underlined amino acid residues) bgaA. The
protospacer, PAM and restriction sites are shown (SEQ ID NOS
473-477 and 474, respectively, in order of appearance). (FIG. 25B)
Transformation efficiency of cells transformed with targeting
constructs in the presence of an editing template or control. (FIG.
25C) PCR analysis for 8 transformants of each editing experiment
followed by digestion with BtgZI (R.fwdarw.A) and TseI
(NE.fwdarw.AA). Deletion of bgaA was revealed as a smaller PCR
product. (FIG. 25D) Miller assay to measure the
.beta.-galactosidase activity of WT and edited strains. (FIG. 25E)
For a single-step, double deletion the targeting construct
contained two spacers (in this case matching srtA and bgaA) and was
co-transformed with two different editing templates (FIG. 25F) PCR
analysis for 8 transformants to detect deletions in srtA and bgaA
loci. 6/8 transformants contained deletions of both genes.
[0051] FIG. 26A-26D provides mechanisms underlying editing using
the CRISPR system. (FIG. 26A) A stop codon was introduced in the
erythromycin resistance gene ermAM to generate strain JEN53. The
wild-type sequence can be restored by targeting the stop codon with
the CRISPR::ermAM(stop) construct, and using the ermAM wild-type
sequence as an editing template. (FIG. 26B) Mutant and wild-type
ermAM sequences (SEQ ID NOS 478-481, respectively, in order of
appearance). (FIG. 26C) Fraction of erythromicyn-resistant
(erm.sup.R) cfu calculated from total or kanamycin-resistant cfu.
(FIG. 26D) Fraction of total cells that acquire both the CRISPR
construct and the editing template. Co-transformation of the CRISPR
targeting construct produced more transformants (t-test, p=0.011).
In all cases the values show the mean.+-.s.d. for three independent
experiments.
[0052] FIG. 27A-27D illustrates genome editing with the CRISPR
system in E. coli. (FIG. 27A) A kanamycin-resistant plasmid
carrying the CRISPR array (pCRISPR) targeting the gene to edit may
be transformed in the H1VIE63 recombineering strain containing a
chloramphenicol-resistant plasmid harboring cas9 and tracr (pCas9),
together with an oligonucleotide specifying the mutation. (FIG.
27B) A K42T mutation conferring streptomycin resistance was
introduced in the rpsL gene (SEQ ID NOS 482-485, respectively, in
order of appearance) (FIG. 27C) Fraction of streptomicyn-resistant
(strep.sup.R) cfu calculated from total or kanamycin-resistant
(kan.sup.R) cfu. (FIG. 27D) Fraction of total cells that acquire
both the pCRISPR plasmid and the editing oligonucleotide.
Co-transformation of the pCRISPR targeting plasmid produced more
transformants (t-test, p=0.004). In all cases the values showed the
mean.+-.s.d. for three independent experiments.
[0053] FIG. 28A-28B illustrates the transformation of crR6 genomic
DNA leads to editing of the targeted locus (FIG. 28A) The IS1167
element of S. pneumoniae R6 was replaced by the CRISPR01 locus of
S. pyogenes SF370 to generate crR6 strain. This locus encodes for
the Cas9 nuclease, a CRISPR array with six spacers, the tracrRNA
that is required for crRNA biogenesis and Cas1, Cas2 and Csn2,
proteins not necessary for targeting. Strain crR6M contains a
minimal functional CRISPR system without cas1, cast and csn2. The
aphA-3 gene encodes kanamycin resistance. Protospacers from the
streptococcal bacteriophages .PHI.8232.5 and .PHI.370.1 were fused
to a chloramphenicol resistance gene (cat) and integrated in the
srtA gene of strain R6 to generate strains R68232.5 and R6370.1.
(FIG. 28B) Left panel: Transformation of crR6 and crR6M genomic DNA
in R6.sup.8232.5 and R.sup.6370.1. As a control of cell competence
a streptomycin resistant gene was also transformed. Right panel:
PCR analysis of 8 R6.sup.8232.5 transformants with crR6 genomic
DNA. Primers that amplify the srtA locus were used for PCR. 7/8
genotyped colonies replaced the R68232.5 srtA locus by the WT locus
from the crR6 genomic DNA.
[0054] FIG. 29A-29F provides chromatograms of DNA sequences of
edited cells obtained in this study. In all cases the wild-type and
mutant protospacer and PAM sequences (or their reverse complement)
are indicated. When relevant, the amino acid sequence encoded by
the protospacer is provided. For each editing experiment, all
strains for which PCR and restriction analysis corroborated the
introduction of the desired modification were sequenced. A
representative chromatogram is shown. (FIG. 29A) Chromatogram for
the introduction of a PAM mutation into the R6.sup.8232.5 target
(FIG. 23D) (SEQ ID NOS 486-487, respectively, in order of
appearance). (FIG. 29B) Chromatograms for the introduction of the
R>A and NE>AA mutations into .beta.-galactosidase (bgaA)
(FIG. 25C) (SEQ ID NOS 473-477 and 474, respectively, in order of
appearance). (FIG. 29C) Chromatogram for the introduction of a 6664
bp deletion within bgaA ORF (FIGS. 25C and 25F). The dotted line
indicates the limits of the deletion (SEQ ID NOS 488-490,
respectively, in order of appearance). (FIG. 29D) Chromatogram for
the introduction of a 729 bp deletion within srtA ORF (FIG. 25F).
The dotted line indicates the limits of the deletion (SEQ ID NOS
491-493, respectively, in order of appearance). (FIG. 29E)
Chromatograms for the generation of a premature stop codon within
ermAM (FIG. 33) (SEQ ID NOS 494-497, respectively, in order of
appearance). (FIG. 29F) rpsL editing in E. coli (FIG. 27) (SEQ ID
NOS 482-485, respectively, in order of appearance).
[0055] FIG. 30A-30C illustrates CRISPR immunity against random S.
pneumoniae targets containing different PAMs. FIG. 30A shows the
position of the 10 random targets on the S. pneumoniae R6 genome.
The chosen targets have different PAMs and are on both strands.
FIG. 30B shows spacers corresponding to the targets were cloned in
a minimal CRISPR array on plasmid pLZ12 and transformed into strain
crR6Rc, which supplies the processing and targeting machinery in
trans. FIG. 30C shows transformation efficiency of the different
plasmids in strain R6 and crR6Rc. No colonies were recovered for
the transformation of pDB99-108 (T1-T10) in crR6Rc. The dashed line
represents limit of detection of the assay.
[0056] FIG. 31 provides a general scheme for targeted genome
editing. To facilitate targeted genome editing, crR6M was further
engineered to contain tracrRNA, Cas9 and only one repeat of the
CRISPR array followed by kanamycin resistance marker (aphA-3),
generating strain crR6Rk. DNA from this strain is used as a
template for PCR with primers designed to introduce a new spacer
(green box designated with N). The left and right PCRs are
assembled using the Gibson method to create the targeting
construct. Both the targeting and editing constructs are then
transformed into strain crR6Rc, which is a strain equivalent to
crR6Rk but has the kanamycin resistance marker replaced by a
chloramphenicol resistance marker (cat). About 90% of the
kanamycin-resistant transformants contain the desired mutation.
[0057] FIG. 32 illustrates the distribution of distances between
PAMs. NGG and CCN that are considered to be valid PAMs. Data is
shown for the S. pneumoniae R6 genome as well as for a random
sequence of the same length and with the same GC-content (39.7%).
The dotted line represents the average distance (12) between PAMs
in the R6 genome.
[0058] FIG. 33A-33D illustrates CRISPR-mediated editing of the
ermAM locus using genomic DNA as targeting construct. To use
genomic DNA as targeting construct it is necessary to avoid CRISPR
autoimmunity, and therefore a spacer against a sequence not present
in the chromosome must be used (in this case the ermAM erythromycin
resistance gene). FIG. 33A shows a nucleotide and amino acid
sequences of the wild-type and mutated (red letters) ermAM gene.
The protospacer and PAM sequences are shown (SEQ ID NOS 494-497,
respectively, in order of appearance). FIG. 33B shows a schematic
for CRISPR-mediated editing of the ermAM locus using genomic DNA. A
construct carrying an ermAM-targeting spacer (blue box) is made by
PCR and Gibson assembly, and transformed into strain crR6Rc,
generating strain JEN37. The genomic DNA of JEN37 was then used as
a targeting construct, and was co-transformed with the editing
template into JEN38, a strain in which the srtA gene was replaced
by a wild-type copy of ermAM. Kanamycin-resistant transformants
contain the edited genotype (JEN43). FIG. 33C shows a number of
kanamycin-resistant cells obtained after co-transformation of
targeting and editing or control templates. In the presence of the
control template 5.4.times.10.sup.3 cfu/ml were obtained, and
4.3.times.10.sup.5 cfu/ml when the editing template was used. This
difference indicates an editing efficiency of about 99%
[(4.3.times.10.sup.5-5.4.times.10.sup.3)/4.3.times.10.sup.5]. FIG.
33D illustrates a check for the presence of edited cells seven
kanamycin-resistant clones and JEN38 were streaked on agar plates
with (erm+) or without (erm-) erythromycin. Only the positive
control displayed resistance to erythromycin. The ermAM mut
genotype of one of these transformants was also verified by DNA
sequencing (FIG. 29E).
[0059] FIG. 34A-34D illustrates sequential introduction of
mutations by CRISPR-mediated genome editing. FIG. 34A is a
schematic for sequential introduction of mutations by
CRISPR-mediated genome editing. First, R6 is engineered to generate
crR6Rk. crR6Rk is co-transformed with a srtA-targeting construct
fused to cat for chloramphenicol selection of edited cells, along
with an editing construct for a .DELTA.srtA in-frame deletion.
Strain crR6 .DELTA.srtA is generated by selection on
chlramphenicol. Subsequently, the .DELTA.srtA strain is
co-transformed with a bgaA-targeting construct fused to aphA-3 for
kanamycin selection of edited cells, and an editing construct
containing a .DELTA.bgaA in-frame deletion. Finally, the engineered
CRISPR locus can be erased from the chromosome by first
co-transforming R6 DNA containing the wild-type IS1167 locus and a
plasmid carrying a bgaA protospacer (pDB97), and selection on
spectinomycin. FIG. 34B is a PCR analysis for 8 chloramphenicol
(Cam)-resistant transformants to detect the deletion in the srtA
locus. FIG. 34C shows .beta.-galactosidase activity as measured by
Miller assay. In S. pneumoniae, this enzyme is anchored to the cell
wall by sortase A. Deletion of the srtA gene results in the release
of .beta.-galactosidase into the supernatant. .DELTA.bgaA mutants
show no activity. FIG. 34D is a PCR analysis for 8 spectinomycin
(Spec)-resistant transformants to detect the replacement of the
CRISPR locus by wild-type IS1167.
[0060] FIG. 35A-35C illustrates the background mutation frequency
of CRISPR in S. pneumoniae. (FIG. 35A) Transformation of the
CRISPR::O or CRISPR::erm(stop) targeting constructs in JEN53, with
or without the ermAM editing template. The difference in kan.sup.R
CFU between CRISPR::O and CRISPR::erm(stop) indicates that Cas9
cleavage kills non-edited cells. Mutants that escape CRISPR
interference in the absence of editing template are observed at a
frequency of 3.times.10.sup.-3. (FIG. 35B) PCR analysis of the
CRISPR locus of escapers shows that 7/8 have a spacer deletion.
(FIG. 35C) Escaper #2 carries a point mutation in cas9 (SEQ ID NOS
498-501, respectively, in order of appearance).
[0061] FIG. 36 illustrates that the essential elements of the S.
pyogenes CRISPR locus 1 are reconstituted in E. coli using pCas9.
The plasmid contained tracrRNA, Cas9, as well as a leader sequence
driving the crRNA array. The pCRISPR plasmids contained the leader
and the array only. Spacers may be inserted into the crRNA array
between BsaI sites using annealed oligonucleotides (SEQ ID NOS 345,
502 and 127, respectively, in order of appearance). Oligonucleotide
design is shown at bottom. pCas9 carried chloramphenicol resistance
(CmR) and is based on the low-copy pACYC184 plasmid backbone.
pCRISPR is based on the high-copy number pZE21 plasmid. Two
plasmids were required because a pCRISPR plasmid containing a
spacer targeting the E. coli chromosome may not be constructed
using this organism as a cloning host if Cas9 is also present (it
will kill the host).
[0062] FIG. 37 illustrates CRISPR-directed editing in E. coli
MG1655. An oligonucleotide (W542) carrying a point mutation that
both confers streptomycin resistance and abolishes CRISPR immunity,
together with a plasmid targeting rpsL (pCRISPR::rpsL) or a control
plasmid (pCRISPR::O) were co-transformed into wild-type E. coli
strain MG1655 containing pCas9. Transformants were selected on
media containing either streptomycin or kanamycin. Dashed line
indicates limit of detection of the transformation assay.
[0063] FIG. 38A-38B illustrates the background mutation frequency
of CRISPR in E. coli HME63. (FIG. 38A) Transformation of the
pCRISPR::O or pCRISPR::rpsL plasmids into HME63 competent cells.
Mutants that escape CRISPR interference were observed at a
frequency of 2.6.times.10.sup.-4. (FIG. 38B) Amplification of the
CRISPR array of escapers showed that 8/8 have deleted the
spacer.
[0064] FIGS. 39A-39D show a circular depiction of the phylogenetic
analysis revealing five families of Cas9s, including three groups
of large Cas9s (.about.1400 amino acids) and two of small Cas9s
(.about.1100 amino acids).
[0065] FIGS. 40A-40F show the linear depiction of the phylogenetic
analysis revealing five families of Cas9s, including three groups
of large Cas9s (.about.1400 amino acids) and two of small Cas9s
(.about.1100 amino acids).
[0066] FIG. 41A-41M shows sequences where the mutation points are
located within the SpCas9 gene (SEQ ID NOS 503-504, respectively,
in order of appearance).
[0067] FIG. 42 shows a schematic construct in which the
transcriptional activation domain (VP64) is fused to Cas9 with two
mutations in the catalytic domains (D10 and H840).
[0068] FIG. 43A-43D shows genome editing via homologous
recombination. FIG. 43A is a schematic of SpCas9 nickase, with D10A
mutation in the RuvC I catalytic domain. FIG. 43B is a schematic
representing homologous recombination (HR) at the human EMX1 locus
using either sense or antisense single stranded oligonucleotides as
repair templates. Red arrow above indicates sgRNA cleavage site;
PCR primers for genotyping (Tables J and K) are indicated as arrows
in right panel. FIG. 43C shows the sequence of region modified by
HR. FIG. 43D shows a SURVEYOR assay for wildtype (wt) and nickase
(D10A) SpCas9-mediated indels at the EMX1 target 1 locus (n=3) (SEQ
ID NOS 505-507, 505, 508 and 507, respectively, in order of
appearance). Arrows indicate positions of expected fragment
sizes.
[0069] FIGS. 44A-44B show single vector designs for SpCas9. FIG.
44A illustrates a single expression vector incorporating a U6
promoter linked to an insertion site for a guide oligo, and a Cbh
promoter linked to SpCas9 coding sequence. FIG. 44A discloses SEQ
ID NOS 322-323 and 330, respectively, in order of appearance. FIG.
44B illustrates a vector that includes a tracrRNA coding sequence
linked to an HI promoter. FIG. 44B discloses SEQ ID NO: 331.
[0070] FIG. 45 shows quantification of cleavage of NLS-Csn1
constructs NLS-Csn1, Csn1, Csn1-NLS, NLS-Csn1-NLS, NLS-Csn1-GFP-NLS
and UnTFN.
[0071] FIG. 46 shows index frequency of NLS-Cas9, Cas9, Cas9-NLS
and NLS-Cas9-NLS.
[0072] FIG. 47 shows a gel demonstrating that SpCas9 with nickase
mutations (individually) do not induce double strand breaks.
[0073] FIG. 48A-48B shows a design of the oligo DNA used as
Homologous Recombination (HR) template in this experiment (FIG.
48A) and a comparison of HR efficiency induced by different
combinations of Cas9 protein and HR template (FIG. 48B).
[0074] FIG. 49A shows the Conditional Cas9, Rosa26 targeting vector
map.
[0075] FIG. 49B shows the Constitutive Cas9, Rosa26 targeting
vector map.
[0076] FIG. 50A-5011 show the sequences of each element present in
the vector maps of FIGS. 49A-B (SEQ ID NOS 509-518, respectively,
in order of appearance).
[0077] FIG. 51 shows a schematic of the important elements in the
Constitutive and Conditional Cas9 constructs.
[0078] FIG. 52 shows the functional validation of the expression of
Constitutive and Conditional Cas9 constructs.
[0079] FIG. 53 shows the validation of Cas9 nuclease activity by
Surveyor.
[0080] FIG. 54 shows the quantification of Cas9 nuclease
activity.
[0081] FIG. 55 shows construct design and homologous recombination
(HR) strategy.
[0082] FIG. 56 shows the genomic PCR genotyping results for the
constitutive (Right) and conditional (Left) constructs at two
different gel exposure times (top row for 3 min and bottom row for
1 min).
[0083] FIG. 57 shows Cas9 activation in mESCs.
[0084] FIG. 58 shows a schematic of the strategy used to mediate
gene knockout via NHEJ using a nickase version of Cas9 along with
two guide RNAs.
[0085] FIG. 59 shows how DNA double-strand break (DSB) repair
promotes gene editing. In the error-prone non-homologous end
joining (NHEJ) pathway, the ends of a DSB are processed by
endogenous DNA repair machineries and rejoined together, which can
result in random insertion/deletion (indel) mutations at the site
of junction. Indel mutations occurring within the coding region of
a gene can result in frame-shift and a premature stop codon,
leading to gene knockout. Alternatively, a repair template in the
form of a plasmid or single-stranded oligodeoxynucleotides (ssODN)
can be supplied to leverage the homology-directed repair (HDR)
pathway, which allows high fidelity and precise editing.
[0086] FIG. 60 shows the timeline and overview of experiments.
Steps for reagent design, construction, validation, and cell line
expansion. Custom sgRNAs (light blue bars) for each target, as well
as genotyping primers, are designed in silico via our online design
tool (available at the website genome-engineering.org/tools). sgRNA
expression vectors are then cloned into a plasmid containing Cas9
(PX330) and verified via DNA sequencing. Completed plasmids
(pCRISPRs), and optional repair templates for facilitating homology
directed repair, are then transfected into cells and assayed for
ability to mediate targeted cleavage. Finally, transfected cells
can be clonally expanded to derive isogenic cell lines with defined
mutations.
[0087] FIG. 61A-61C shows Target selection and reagent preparation.
FIG. 61A shows for S. pyogenes Cas9, 20-bp targets (highlighted in
blue) must be followed by 5'-NGG, which can occur in either strand
on genomic DNA. We recommend using the online tool described in
this protocol in aiding target selection
(www.genome-engineering.org/tools). FIG. 61B is a schematic for
co-transfection of Cas9 expression plasmid (PX165) and
PCR-amplified U6-driven sgRNA expression cassette. Using a U6
promoter-containing PCR template and a fixed forward primer (U6
Fwd), sgRNA-encoding DNA can appended onto the U6 reverse primer
(U6 Rev) and synthesized as an extended DNA oligo (Ultramer oligos
from IDT). Note the guide sequence (blue N's) in U6 Rev is the
reverse complement of the 5'-NGG flanking target sequence (SEQ ID
NOS 519 and 519-521, respectively, in order of appearance). FIG.
61C is a schematic for scarless cloning of the guide sequence
oligos into a plasmid containing Cas9 and sgRNA scaffold (PX330).
The guide oligos (blue N's) contain overhangs for ligation into the
pair of BbsI sites on PS330, with the top and bottom strand
orientations matching those of the genomic target (i.e. top oligo
is the 20-bp sequence preceding 5'-NGG in genomic DNA). Digestion
of PX330 with BbsI allows the replacement of the Type IIs
restriction sites (blue outline) with direct insertion of annealed
oligos. It is worth noting that an extra G was placed before the
first base of the guide sequence. Applicants have found that an
extra G in front of the guide sequence does not adversely affect
targeting efficiency. In cases when the 20-nt guide sequence of
choice does not begin with guanine, the extra guanine will ensure
the sgRNA is efficiently transcribed by the U6 promoter, which
prefers a guanine in the first base of the transcript (SEQ ID NOS
322-323 and 330, respectively, in order of appearance).
[0088] FIG. 62A-62D shows the anticipated results for multiplex
NHEJ. FIG. 62A shows the schematic of the SURVEYOR assay used to
determine indel percentage. First, genomic DNA from the
heterogeneous population of Cas9-targeted cells is amplified by
PCR. Amplicons are then reannealed slowly to generate
heteroduplexes. The reannealed heteroduplexes are cleaved by
SURVEYOR nuclease, whereas homoduplexes are left intact.
Cas9-mediated cleavage efficiency (% indel) is calculated based on
the fraction of cleaved DNA, as determined by integrated intensity
of gel bands. FIG. 62B shows the two sgRNAs (orange and blue bars)
are designed to target the human GRIN2B and DYRK1A loci. SURVEYOR
gel shows modification at both loci in transfected cells. Colored
arrows indicated expected fragment sizes for each locus. FIG. 62C
shows a pair of sgRNAs (light blue and green bars) are designed to
excise an exon (dark blue) in the human EMX1 locus. Target
sequences and PAMs (red) are shown in respective colors, and sites
of cleavage indicated by red triangle. Predicted junction is shown
below. Individual clones isolated from cell populations transfected
with sgRNA 3, 4, or both are assayed by PCR (OUT Fwd, OUT Rev),
reflecting a deletion of .about.270-bp. Representative clones with
no modification (12/23), mono-allelic (10/23), and bi-allelic
(1/23) modifications are shown. IN Fwd and IN Rev primers are used
to screen for inversion events (FIG. 6D) (SEQ ID NOS 522-524,
respectively, in order of appearance). FIG. 62D shows the
quantification of clonal lines with EMX1 exon deletions. Two pairs
of sgRNAs (3.1, 3.2 left-flanking sgRNAs; 4.1, 4.2, right flanking
sgRNAs) are used to mediate deletions of variable sizes around one
EMX1 exon. Transfected cells are clonally isolated and expanded for
genotyping analysis for deletions and inversion events. Of the 105
clones are screened, 51 (49%) and 11 (10%) carrying heterozygous
and homozygous deletions, respectively. Approximate deletion sizes
are given since junctions may be variable.
[0089] FIG. 63A-63C shows the application of ssODNs and targeting
vector to mediate HR with both wildtype and nickase mutant of Cas9
in HEK293FT and HUES9 cells with efficiencies ranging from 1.0-27%.
FIG. 63B discloses SEQ ID NOS 505-507, 505, 508 and 507,
respectively, in order of appearance.
[0090] FIG. 64 shows a schematic of a PCR-based method for rapid
and efficient CRISPR targeting in mammalian cells. A plasmid
containing the human RNA polymerase III promoter U6 is
PCR-amplified using a U6-specific forward primer and a reverse
primer carrying the reverse complement of part of the U6 promoter,
the sgRNA(+85) scaffold with guide sequence, and 7 T nucleotides
for transcriptional termination. The resulting PCR product is
purified and co-delivered with a plasmid carrying Cas9 driven by
the CBh promoter (SEQ ID NOS 519, 525, 520 and 526-527,
respectively, in order of appearance).
[0091] FIG. 65 shows SURVEYOR Mutation Detection Kit from
Transgenomics results for each gRNA and respective controls. A
positive SURVEYOR result is one large band corresponding to the
genomic PCR and two smaller bands that are the product of the
SURVEYOR nuclease making a double-strand break at the site of a
mutation. Each gRNA was validated in the mouse cell line,
Neuro-N2a, by liposomal transient co-transfection with hSpCas9. 72
hours post-transfection genomic DNA was purified using QuickExtract
DNA from Epicentre. PCR was performed to amplify the locus of
interest.
[0092] FIG. 66 shows Surveyor results for 38 live pups (lanes 1-38)
1 dead pup (lane 39) and 1 wild-type pup for comparison (lane 40).
Pups 1-19 were injected with gRNA Chd8.2 and pups 20-38 were
injected with gRNA Chd8.3. Of the 38 live pups, 13 were positive
for a mutation. The one dead pup also had a mutation. There was no
mutation detected in the wild-type sample. Genomic PCR sequencing
was consistent with the SURVEYOR assay findings (SEQ ID NOS
528-530, respectively, in order of appearance).
[0093] FIG. 67 shows a design of different Cas9 NLS constructs. All
Cas9 were the human-codon-optimized version of the Sp Cas9. NLS
sequences are linked to the cas9 gene at either N-terminus or
C-terminus. All Cas9 variants with different NLS designs were
cloned into a backbone vector containing so it is driven by EF1a
promoter. On the same vector there is a chimeric RNA targeting
human EMX1 locus driven by U6 promoter, together forming a
two-component system.
[0094] FIG. 68 shows the efficiency of genomic cleavage induced by
Cas9 variants bearing different NLS designs. The percentage
indicate the portion of human EMX1 genomic DNA that were cleaved by
each construct. All experiments are from 3 biological replicates.
n=3, error indicates S.E.M.
[0095] FIG. 69A shows a design of the CRISPR-TF (Transcription
Factor) with transcriptional activation activity. The chimeric RNA
is expressed by U6 promoter, while a human-codon-optimized,
double-mutant version of the Cas9 protein (hSpCas9m), operably
linked to triple NLS and a VP64 functional domain is expressed by a
EF1a promoter. The double mutations, D10A and H840A, renders the
cas9 protein unable to introduce any cleavage but maintained its
capacity to bind to target DNA when guided by the chimeric RNA.
[0096] FIG. 69B shows transcriptional activation of the human SOX2
gene with CRISPR-TF system (Chimeric RNA and the Cas9-NLS-VP64
fusion protein). 293FT cells were transfected with plasmids bearing
two components: (1) U6-driven different chimeric RNAs targeting
20-bp sequences within or around the human SOX2 genomic locus, and
(2) EF1a-driven hSpCas9m (double mutant)-NLS-VP64 fusion protein.
96 hours post transfection, 293FT cells were harvested and the
level of activation is measured by the induction of mRNA expression
using a qRT-PCR assay. All expression levels are normalized against
the control group (grey bar), which represents results from cells
transfected with the CRISPR-TF backbone plasmid without chimeric
RNA. The qRT-PCR probes used for detecting the SOX2 mRNA is Taqman
Human Gene Expression Assay (Life Technologies). All experiments
represents data from 3 biological replicates, n=3, error bars show
s.e.m.
[0097] FIG. 70 depicts NLS architecture optimization for
SpCas9.
[0098] FIG. 71 shows a QQ plot for NGGNN sequences.
[0099] FIG. 72 shows a histogram of the data density with fitted
normal distribution (black line) and 0.99 quantile (dotted
line).
[0100] FIG. 73A-73C shows RNA-guided repression of bgaA expression
by dgRNA::cas9**. FIG. 73A shows the Cas9 protein binds to the
tracrRNA, and to the precursor CRISPR RNA which is processed by
RNAseIII to form the crRNA. The crRNA directs binding of Cas9 to
the bgaA promoter and represses transcription. FIG. 73B shows the
targets used to direct Cas9** to the bgaA promoter are represented
(SEQ ID NO: 531). Putative -35, -10 as well as the bgaA start codon
are in bold. FIG. 73C shows the Betagalactosidase activity as
measure by Miller assay in the absence of targeting and for the
four different targets.
[0101] FIG. 74A-74E shows characterization of Cas9** mediated
repression. FIG. 74A shows the gffimut2 gene and its promoter,
including the -35 and -10 signals are represented together with the
position of the different target sites used the study. FIG. 74B
shows the relative fluorescence upon targeting of the coding
strand. FIG. 74C shows the relative fluorescence upon targeting of
the non-coding strand. FIG. 74D shows the northern blot with probes
B477 and B478 on RNA extracted from T5, T10, B10 or a control
strain without a target. FIG. 74E shows the effect of an increased
number of mutations in the 5' end of the crRNA of B1, T5 and
B10.
[0102] The figures herein are for illustrative purposes only and
are not necessarily drawn to scale.
DETAILED DESCRIPTION OF THE INVENTION
[0103] The terms "polynucleotide", "nucleotide", "nucleotide
sequence", "nucleic acid" and "oligonucleotide" are used
interchangeably. They refer to a polymeric form of nucleotides of
any length, either deoxyribonucleotides or ribonucleotides, or
analogs thereof. Polynucleotides may have any three dimensional
structure, and may perform any function, known or unknown. The
following are non-limiting examples of polynucleotides: coding or
non-coding regions of a gene or gene fragment, loci (locus) defined
from linkage analysis, exons, introns, messenger RNA (mRNA),
transfer RNA, ribosomal RNA, short interfering RNA (siRNA),
short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA,
recombinant polynucleotides, branched polynucleotides, plasmids,
vectors, isolated DNA of any sequence, isolated RNA of any
sequence, nucleic acid probes, and primers. A polynucleotide may
comprise one or more modified nucleotides, such as methylated
nucleotides and nucleotide analogs. If present, modifications to
the nucleotide structure may be imparted before or after assembly
of the polymer. The sequence of nucleotides may be interrupted by
non-nucleotide components. A polynucleotide may be further modified
after polymerization, such as by conjugation with a labeling
component.
[0104] In aspects of the invention the terms "chimeric RNA",
"chimeric guide RNA", "guide RNA", "single guide RNA" and
"synthetic guide RNA" are used interchangeably and refer to the
polynucleotide sequence comprising the guide sequence, the tracr
sequence and the tracr mate sequence. The term "guide sequence"
refers to the about 20 bp sequence within the guide RNA that
specifies the target site and may be used interchangeably with the
terms "guide" or "spacer". The term "tracr mate sequence" may also
be used interchangeably with the term "direct repeat(s)".
[0105] As used herein the term "wild type" is a term of the art
understood by skilled persons and means the typical form of an
organism, strain, gene or characteristic as it occurs in nature as
distinguished from mutant or variant forms.
[0106] As used herein the term "variant" should be taken to mean
the exhibition of qualities that have a pattern that deviates from
what occurs in nature.
[0107] The terms "non-naturally occurring" or "engineered" are used
interchangeably and indicate the involvement of the hand of man.
The terms, when referring to nucleic acid molecules or polypeptides
mean that the nucleic acid molecule or the polypeptide is at least
substantially free from at least one other component with which
they are naturally associated in nature and as found in nature.
[0108] "Complementarity" refers to the ability of a nucleic acid to
form hydrogen bond(s) with another nucleic acid sequence by either
traditional Watson-Crick base pairing or other non-traditional
types. A percent complementarity indicates the percentage of
residues in a nucleic acid molecule which can form hydrogen bonds
(e.g., Watson-Crick base pairing) with a second nucleic acid
sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%,
80%, 90%, and 100% complementary). "Perfectly complementary" means
that all the contiguous residues of a nucleic acid sequence will
hydrogen bond with the same number of contiguous residues in a
second nucleic acid sequence. "Substantially complementary" as used
herein refers to a degree of complementarity that is at least 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a
region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to
two nucleic acids that hybridize under stringent conditions.
[0109] As used herein, "stringent conditions" for hybridization
refer to conditions under which a nucleic acid having
complementarity to a target sequence predominantly hybridizes with
the target sequence, and substantially does not hybridize to
non-target sequences. Stringent conditions are generally
sequence-dependent, and vary depending on a number of factors. In
general, the longer the sequence, the higher the temperature at
which the sequence specifically hybridizes to its target sequence.
Non-limiting examples of stringent conditions are described in
detail in Tijssen (1993), Laboratory Techniques In Biochemistry And
Molecular Biology-Hybridization With Nucleic Acid Probes Part I,
Second Chapter "Overview of principles of hybridization and the
strategy of nucleic acid probe assay", Elsevier, N.Y.
[0110] "Hybridization" refers to a reaction in which one or more
polynucleotides react to form a complex that is stabilized via
hydrogen bonding between the bases of the nucleotide residues. The
hydrogen bonding may occur by Watson Crick base pairing, Hoogstein
binding, or in any other sequence specific manner. The complex may
comprise two strands forming a duplex structure, three or more
strands forming a multi stranded complex, a single self hybridizing
strand, or any combination of these. A hybridization reaction may
constitute a step in a more extensive process, such as the
initiation of PCR, or the cleavage of a polynucleotide by an
enzyme. A sequence capable of hybridizing with a given sequence is
referred to as the "complement" of the given sequence.
[0111] As used herein, "expression" refers to the process by which
a polynucleotide is transcribed from a DNA template (such as into
and mRNA or other RNA transcript) and/or the process by which a
transcribed mRNA is subsequently translated into peptides,
polypeptides, or proteins. Transcripts and encoded polypeptides may
be collectively referred to as "gene product." If the
polynucleotide is derived from genomic DNA, expression may include
splicing of the mRNA in a eukaryotic cell.
[0112] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to polymers of amino acids of any
length. The polymer may be linear or branched, it may comprise
modified amino acids, and it may be interrupted by non amino acids.
The terms also encompass an amino acid polymer that has been
modified; for example, disulfide bond formation, glycosylation,
lipidation, acetylation, phosphorylation, or any other
manipulation, such as conjugation with a labeling component. As
used herein the term "amino acid" includes natural and/or unnatural
or synthetic amino acids, including glycine and both the D or L
optical isomers, and amino acid analogs and peptidomimetics.
[0113] The terms "subject," "individual," and "patient" are used
interchangeably herein to refer to a vertebrate, preferably a
mammal, more preferably a human. Mammals include, but are not
limited to, murines, simians, humans, farm animals, sport animals,
and pets. Tissues, cells and their progeny of a biological entity
obtained in vivo or cultured in vitro are also encompassed.
[0114] The terms "therapeutic agent", "therapeutic capable agent"
or "treatment agent" are used interchangeably and refer to a
molecule or compound that confers some beneficial effect upon
administration to a subject. The beneficial effect includes
enablement of diagnostic determinations; amelioration of a disease,
symptom, disorder, or pathological condition; reducing or
preventing the onset of a disease, symptom, disorder or condition;
and generally counteracting a disease, symptom, disorder or
pathological condition.
[0115] As used herein, "treatment" or "treating," or "palliating"
or "ameliorating" are used interchangeably. These terms refer to an
approach for obtaining beneficial or desired results including but
not limited to a therapeutic benefit and/or a prophylactic benefit.
By therapeutic benefit is meant any therapeutically relevant
improvement in or effect on one or more diseases, conditions, or
symptoms under treatment. For prophylactic benefit, the
compositions may be administered to a subject at risk of developing
a particular disease, condition, or symptom, or to a subject
reporting one or more of the physiological symptoms of a disease,
even though the disease, condition, or symptom may not have yet
been manifested.
[0116] The term "effective amount" or "therapeutically effective
amount" refers to the amount of an agent that is sufficient to
effect beneficial or desired results. The therapeutically effective
amount may vary depending upon one or more of: the subject and
disease condition being treated, the weight and age of the subject,
the severity of the disease condition, the manner of administration
and the like, which can readily be determined by one of ordinary
skill in the art. The term also applies to a dose that will provide
an image for detection by any one of the imaging methods described
herein. The specific dose may vary depending on one or more of: the
particular agent chosen, the dosing regimen to be followed, whether
it is administered in combination with other compounds, timing of
administration, the tissue to be imaged, and the physical delivery
system in which it is carried.
[0117] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of immunology,
biochemistry, chemistry, molecular biology, microbiology, cell
biology, genomics and recombinant DNA, which are within the skill
of the art. See Sambrook, Fritsch and Maniatis, MOLECULAR CLONING:
A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); the series
METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL
APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds.
(1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY
MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).
[0118] Several aspects of the invention relate to vector systems
comprising one or more vectors, or vectors as such. Vectors can be
designed for expression of CRISPR transcripts (e.g. nucleic acid
transcripts, proteins, or enzymes) in prokaryotic or eukaryotic
cells. For example, CRISPR transcripts can be expressed in
bacterial cells such as Escherichia coli, insect cells (using
baculovirus expression vectors), yeast cells, or mammalian cells.
Suitable host cells are discussed further in Goeddel, GENE
EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press,
San Diego, Calif. (1990). Alternatively, the recombinant expression
vector can be transcribed and translated in vitro, for example
using T7 promoter regulatory sequences and T7 polymerase.
[0119] Vectors may be introduced and propagated in a prokaryote. In
some embodiments, a prokaryote is used to amplify copies of a
vector to be introduced into a eukaryotic cell or as an
intermediate vector in the production of a vector to be introduced
into a eukaryotic cell (e.g. amplifying a plasmid as part of a
viral vector packaging system). In some embodiments, a prokaryote
is used to amplify copies of a vector and express one or more
nucleic acids, such as to provide a source of one or more proteins
for delivery to a host cell or host organism. Expression of
proteins in prokaryotes is most often carried out in Escherichia
coli with vectors containing constitutive or inducible promoters
directing the expression of either fusion or non-fusion proteins.
Fusion vectors add a number of amino acids to a protein encoded
therein, such as to the amino terminus of the recombinant protein.
Such fusion vectors may serve one or more purposes, such as: (i) to
increase expression of recombinant protein; (ii) to increase the
solubility of the recombinant protein; and (iii) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, in fusion expression vectors, a
proteolytic cleavage site is introduced at the junction of the
fusion moiety and the recombinant protein to enable separation of
the recombinant protein from the fusion moiety subsequent to
purification of the fusion protein. Such enzymes, and their cognate
recognition sequences, include Factor Xa, thrombin and
enterokinase. Example fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40),
pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,
Piscataway, N.J.) that fuse glutathione S-transferase (GST),
maltose E binding protein, or protein A, respectively, to the
target recombinant protein. Examples of suitable inducible
non-fusion E. coli expression vectors include pTrc (Amrann et al.,
(1988) Gene 69:301-315) and pET 11d (Studier et al., GENE
EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press,
San Diego, Calif. (1990) 60-89).
[0120] In some embodiments, a vector is a yeast expression vector.
Examples of vectors for expression in yeast Saccharomyces cerivisae
include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa
(Kuijan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et
al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San
Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
[0121] In some embodiments, a vector drives protein expression in
insect cells using baculovirus expression vectors. Baculovirus
vectors available for expression of proteins in cultured insect
cells (e.g., SF9 cells) include the pAc series (Smith, et al.,
1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow
and Summers, 1989. Virology 170: 31-39).
[0122] In some embodiments, a vector is capable of driving
expression of one or more sequences in mammalian cells using a
mammalian expression vector. Examples of mammalian expression
vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC
(Kaufman, et al., 1987. EMBO J. 6: 187-195). When used in mammalian
cells, the expression vector's control functions are typically
provided by one or more regulatory elements. For example, commonly
used promoters are derived from polyoma, adenovirus 2,
cytomegalovirus, simian virus 40, and others disclosed herein and
known in the art. For other suitable expression systems for both
prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of
Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed.,
Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989.
[0123] In some embodiments, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes
Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton,
1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell
receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and
immunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen and
Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters
(e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc.
Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters
(Edlund, et al., 1985. Science 230: 912-916), and mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No.
4,873,316 and European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, e.g., the
murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379)
and the .alpha.-fetoprotein promoter (Campes and Tilghman, 1989.
Genes Dev. 3: 537-546).
[0124] In some embodiments, a regulatory element is operably linked
to one or more elements of a CRISPR system so as to drive
expression of the one or more elements of the CRISPR system. In
general, CRISPRs (Clustered Regularly Interspaced Short Palindromic
Repeats), also known as SPIDRs (SPacer Interspersed Direct
Repeats), constitute a family of DNA loci that are usually specific
to a particular bacterial species. The CRISPR locus comprises a
distinct class of interspersed short sequence repeats (SSRs) that
were recognized in E. coli (Ishino et al., J. Bacteriol.,
169:5429-5433 [1987]; and Nakata et al., J. Bacteriol.,
171:3553-3556 [1989]), and associated genes. Similar interspersed
SSRs have been identified in Haloferax mediterranei, Streptococcus
pyogenes, Anabaena, and Mycobacterium tuberculosis (See, Groenen et
al., Mol. Microbiol., 10:1057-1065 [1993]; Hoe et al., Emerg.
Infect. Dis., 5:254-263 [1999]; Masepohl et al., Biochim. Biophys.
Acta 1307:26-30 [1996]; and Mojica et al., Mol. Microbiol.,
17:85-93 [1995]). The CRISPR loci typically differ from other SSRs
by the structure of the repeats, which have been termed short
regularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ.
Biol., 6:23-33 [2002]; and Mojica et al., Mol. Microbiol.,
36:244-246 [2000]). In general, the repeats are short elements that
occur in clusters that are regularly spaced by unique intervening
sequences with a substantially constant length (Mojica et al.,
[2000], supra). Although the repeat sequences are highly conserved
between strains, the number of interspersed repeats and the
sequences of the spacer regions typically differ from strain to
strain (van Embden et al., J. Bacteriol., 182:2393-2401 [2000]).
CRISPR loci have been identified in more than 40 prokaryotes (See
e.g., Jansen et al., Mol. Microbiol., 43:1565-1575 [2002]; and
Mojica et al., [2005]) including, but not limited to Aeropyrum,
Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula,
Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus,
Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium,
Mycobacterium, Streptomyces, Aquifex, Porphyromonas, Chlorobium,
Thermus, Bacillus, Listeria, Staphylococcus, Clostridium,
Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus,
Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter,
Myxococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia,
Escherichia, Legionella, Methylococcus, Pasteurella,
Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema, and
Thermotoga.
[0125] In general, "CRISPR system" refers collectively to
transcripts and other elements involved in the expression of or
directing the activity of CRISPR-associated ("Cas") genes,
including sequences encoding a Cas gene, a tracr (trans-activating
CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a
tracr-mate sequence (encompassing a "direct repeat" and a
tracrRNA-processed partial direct repeat in the context of an
endogenous CRISPR system), a guide sequence (also referred to as a
"spacer" in the context of an endogenous CRISPR system), or other
sequences and transcripts from a CRISPR locus. In some embodiments,
one or more elements of a CRISPR system is derived from a type I,
type II, or type III CRISPR system. In some embodiments, one or
more elements of a CRISPR system is derived from a particular
organism comprising an endogenous CRISPR system, such as
Streptococcus pyogenes. In general, a CRISPR system is
characterized by elements that promote the formation of a CRISPR
complex at the site of a target sequence (also referred to as a
protospacer in the context of an endogenous CRISPR system). In the
context of formation of a CRISPR complex, "target sequence" refers
to a sequence to which a guide sequence is designed to have
complementarity, where hybridization between a target sequence and
a guide sequence promotes the formation of a CRISPR complex. Full
complementarity is not necessarily required, provided there is
sufficient complementarity to cause hybridization and promote
formation of a CRISPR complex. A target sequence may comprise any
polynucleotide, such as DNA or RNA polynucleotides. In some
embodiments, a target sequence is located in the nucleus or
cytoplasm of a cell. In some embodiments, the target sequence may
be within an organelle of a eukaryotic cell, for example,
mitochondrion or chloroplast. A sequence or template that may be
used for recombination into the targeted locus comprising the
target sequences is referred to as an "editing template" or
"editing polynucleotide" or "editing sequence". In aspects of the
invention, an exogenous template polynucleotide may be referred to
as an editing template. In an aspect of the invention the
recombination is homologous recombination.
[0126] Typically, in the context of an endogenous CRISPR system,
formation of a CRISPR complex (comprising a guide sequence
hybridized to a target sequence and complexed with one or more Cas
proteins) results in cleavage of one or both strands in or near
(e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base
pairs from) the target sequence. Without wishing to be bound by
theory, the tracr sequence, which may comprise or consist of all or
a portion of a wild-type tracr sequence (e.g. about or more than
about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a
wild-type tracr sequence), may also form part of a CRISPR complex,
such as by hybridization along at least a portion of the tracr
sequence to all or a portion of a tracr mate sequence that is
operably linked to the guide sequence. In some embodiments, the
tracr sequence has sufficient complementarity to a tracr mate
sequence to hybridize and participate in formation of a CRISPR
complex. As with the target sequence, it is believed that complete
complementarity is not needed, provided there is sufficient to be
functional. In some embodiments, the tracr sequence has at least
50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity
along the length of the tracr mate sequence when optimally aligned.
In some embodiments, one or more vectors driving expression of one
or more elements of a CRISPR system are introduced into a host cell
such that expression of the elements of the CRISPR system direct
formation of a CRISPR complex at one or more target sites. For
example, a Cas enzyme, a guide sequence linked to a tracr-mate
sequence, and a tracr sequence could each be operably linked to
separate regulatory elements on separate vectors. Alternatively,
two or more of the elements expressed from the same or different
regulatory elements, may be combined in a single vector, with one
or more additional vectors providing any components of the CRISPR
system not included in the first vector. CRISPR system elements
that are combined in a single vector may be arranged in any
suitable orientation, such as one element located 5' with respect
to ("upstream" of) or 3' with respect to ("downstream" of) a second
element. The coding sequence of one element may be located on the
same or opposite strand of the coding sequence of a second element,
and oriented in the same or opposite direction. In some
embodiments, a single promoter drives expression of a transcript
encoding a CRISPR enzyme and one or more of the guide sequence,
tracr mate sequence (optionally operably linked to the guide
sequence), and a tracr sequence embedded within one or more intron
sequences (e.g. each in a different intron, two or more in at least
one intron, or all in a single intron). In some embodiments, the
CRISPR enzyme, guide sequence, tracr mate sequence, and tracr
sequence are operably linked to and expressed from the same
promoter.
[0127] In some embodiments, a vector comprises one or more
insertion sites, such as a restriction endonuclease recognition
sequence (also referred to as a "cloning site"). In some
embodiments, one or more insertion sites (e.g. about or more than
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are
located upstream and/or downstream of one or more sequence elements
of one or more vectors. In some embodiments, a vector comprises an
insertion site upstream of a tracr mate sequence, and optionally
downstream of a regulatory element operably linked to the tracr
mate sequence, such that following insertion of a guide sequence
into the insertion site and upon expression the guide sequence
directs sequence-specific binding of a CRISPR complex to a target
sequence in a eukaryotic cell. In some embodiments, a vector
comprises two or more insertion sites, each insertion site being
located between two tracr mate sequences so as to allow insertion
of a guide sequence at each site. In such an arrangement, the two
or more guide sequences may comprise two or more copies of a single
guide sequence, two or more different guide sequences, or
combinations of these. When multiple different guide sequences are
used, a single expression construct may be used to target CRISPR
activity to multiple different, corresponding target sequences
within a cell. For example, a single vector may comprise about or
more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more
guide sequences. In some embodiments, about or more than about 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-sequence-containing
vectors may be provided, and optionally delivered to a cell.
[0128] In some embodiments, a vector comprises a regulatory element
operably linked to an enzyme-coding sequence encoding a CRISPR
enzyme, such as a Cas protein. Non-limiting examples of Cas
proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cash, Cas7,
Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3,
Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6,
Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14,
Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4,
homologs thereof, or modified versions thereof. These enzymes are
known; for example, the amino acid sequence of S. pyogenes Cas9
protein may be found in the SwissProt database under accession
number Q99ZW2. In some embodiments, the unmodified CRISPR enzyme
has DNA cleavage activity, such as Cas9. In some embodiments the
CRISPR enzyme is Cas9, and may be Cas9 from S. pyogenes or S.
pneumoniae. In some embodiments, the CRISPR enzyme directs cleavage
of one or both strands at the location of a target sequence, such
as within the target sequence and/or within the complement of the
target sequence. In some embodiments, the CRISPR enzyme directs
cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from
the first or last nucleotide of a target sequence. In some
embodiments, a vector encodes a CRISPR enzyme that is mutated to
with respect to a corresponding wild-type enzyme such that the
mutated CRISPR enzyme lacks the ability to cleave one or both
strands of a target polynucleotide containing a target sequence.
For example, an aspartate-to-alanine substitution (D10A) in the
RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from
a nuclease that cleaves both strands to a nickase (cleaves a single
strand). Other examples of mutations that render Cas9 a nickase
include, without limitation, H840A, N854A, and N863A. In some
embodiments, a Cas9 nickase may be used in combination with guide
sequenc(es), e.g., two guide sequences, which target respectively
sense and antisense strands of the DNA target. This combination
allows both strands to be nicked and used to induce NHEJ.
Applicants have demonstrated (data not shown) the efficacy of two
nickase targets (i.e., sgRNAs targeted at the same location but to
different strands of DNA) in inducing mutagenic NHEJ. A single
nickase (Cas9-D10A with a single sgRNA) is unable to induce NHEJ
and create indels but Applicants have shown that double nickase
(Cas9-D10A and two sgRNAs targeted to different strands at the same
location) can do so in human embryonic stem cells (hESCs). The
efficiency is about 50% of nuclease (i.e., regular Cas9 without D10
mutation) in hESCs.
[0129] As a further example, two or more catalytic domains of Cas9
(RuvC I, RuvC II, and RuvC III) may be mutated to produce a mutated
Cas9 substantially lacking all DNA cleavage activity. In some
embodiments, a D10A mutation is combined with one or more of H840A,
N854A, or N863A mutations to produce a Cas9 enzyme substantially
lacking all DNA cleavage activity. In some embodiments, a CRISPR
enzyme is considered to substantially lack all DNA cleavage
activity when the DNA cleavage activity of the mutated enzyme is
less than about 25%, 10%, 5%, 1%, 0.1%, 0.01%, or lower with
respect to its non-mutated form. Other mutations may be useful;
where the Cas9 or other CRISPR enzyme is from a species other than
S. pyogenes, mutations in corresponding amino acids may be made to
achieve similar effects.
[0130] In some embodiments, an enzyme coding sequence encoding a
CRISPR enzyme is codon optimized for expression in particular
cells, such as eukaryotic cells. The eukaryotic cells may be those
of or derived from a particular organism, such as a mammal,
including but not limited to human, mouse, rat, rabbit, dog, or
non-human primate. In general, codon optimization refers to a
process of modifying a nucleic acid sequence for enhanced
expression in the host cells of interest by replacing at least one
codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25,
50, or more codons) of the native sequence with codons that are
more frequently or most frequently used in the genes of that host
cell while maintaining the native amino acid sequence. Various
species exhibit particular bias for certain codons of a particular
amino acid. Codon bias (differences in codon usage between
organisms) often correlates with the efficiency of translation of
messenger RNA (mRNA), which is in turn believed to be dependent on,
among other things, the properties of the codons being translated
and the availability of particular transfer RNA (tRNA) molecules.
The predominance of selected tRNAs in a cell is generally a
reflection of the codons used most frequently in peptide synthesis.
Accordingly, genes can be tailored for optimal gene expression in a
given organism based on codon optimization. Codon usage tables are
readily available, for example, at the "Codon Usage Database", and
these tables can be adapted in a number of ways. See Nakamura, Y.,
et al. "Codon usage tabulated from the international DNA sequence
databases: status for the year 2000" Nucl. Acids Res. 28:292
(2000). Computer algorithms for codon optimizing a particular
sequence for expression in a particular host cell are also
available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also
available. In some embodiments, one or more codons (e.g. 1, 2, 3,
4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence
encoding a CRISPR enzyme correspond to the most frequently used
codon for a particular amino acid.
[0131] In some embodiments, a vector encodes a CRISPR enzyme
comprising one or more nuclear localization sequences (NLSs), such
as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
NLSs. In some embodiments, the CRISPR enzyme comprises about or
more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or
near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a
combination of these (e.g. one or more NLS at the amino-terminus
and one or more NLS at the carboxy terminus). When more than one
NLS is present, each may be selected independently of the others,
such that a single NLS may be present in more than one copy and/or
in combination with one or more other NLSs present in one or more
copies. In a preferred embodiment of the invention, the CRISPR
enzyme comprises at most 6 NLSs. In some embodiments, an NLS is
considered near the N- or C-terminus when the nearest amino acid of
the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50,
or more amino acids along the polypeptide chain from the N- or
C-terminus. Typically, an NLS consists of one or more short
sequences of positively charged lysines or arginines exposed on the
protein surface, but other types of NLS are known. Non-limiting
examples of NLSs include an NLS sequence derived from: the NLS of
the SV40 virus large T-antigen, having the amino acid sequence
PKKKRKV (SEQ ID NO: 1); the NLS from nucleoplasmin (e.g. the
nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ
ID NO: 2)); the c-myc NLS having the amino acid sequence PAAKRVKLD
(SEQ ID NO: 3) or RQRRNELKRSP (SEQ ID NO: 4); the hRNPA1 M9 NLS
having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID
NO: 5); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV
(SEQ ID NO: 6) of the IBB domain from importin-alpha; the sequences
VSRKRPRP (SEQ ID NO: 7) and PPKKARED (SEQ ID NO: 8) of the myoma T
protein; the sequence PQPKKKPL (SEQ ID NO: 9) of human p53; the
sequence SALIKKKKKMAP (SEQ ID NO: 10) of mouse c-abl IV; the
sequences DRLRR (SEQ ID NO: 11) and PKQKKRK (SEQ ID NO: 12) of the
influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 13) of the
Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO:
14) of the mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK
(SEQ ID NO: 15) of the human poly(ADP-ribose) polymerase; and the
sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 16) of the steroid hormone
receptors (human) glucocorticoid.
[0132] In general, the one or more NLSs are of sufficient strength
to drive accumulation of the CRISPR enzyme in a detectable amount
in the nucleus of a eukaryotic cell. In general, strength of
nuclear localization activity may derive from the number of NLSs in
the CRISPR enzyme, the particular NLS(s) used, or a combination of
these factors. Detection of accumulation in the nucleus may be
performed by any suitable technique. For example, a detectable
marker may be fused to the CRISPR enzyme, such that location within
a cell may be visualized, such as in combination with a means for
detecting the location of the nucleus (e.g. a stain specific for
the nucleus such as DAPI). Examples of detectable markers include
fluorescent proteins (such as Green fluorescent proteins, or GFP;
RFP; CFP), and epitope tags (HA tag, flag tag, SNAP tag). Cell
nuclei may also be isolated from cells, the contents of which may
then be analyzed by any suitable process for detecting protein,
such as immunohistochemistry, Western blot, or enzyme activity
assay. Accumulation in the nucleus may also be determined
indirectly, such as by an assay for the effect of CRISPR complex
formation (e.g. assay for DNA cleavage or mutation at the target
sequence, or assay for altered gene expression activity affected by
CRISPR complex formation and/or CRISPR enzyme activity), as
compared to a control no exposed to the CRISPR enzyme or complex,
or exposed to a CRISPR enzyme lacking the one or more NLSs.
[0133] In general, a guide sequence is any polynucleotide sequence
having sufficient complementarity with a target polynucleotide
sequence to hybridize with the target sequence and direct
sequence-specific binding of a CRISPR complex to the target
sequence. In some embodiments, the degree of complementarity
between a guide sequence and its corresponding target sequence,
when optimally aligned using a suitable alignment algorithm, is
about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%,
99%, or more. Optimal alignment may be determined with the use of
any suitable algorithm for aligning sequences, non-limiting example
of which include the Smith-Waterman algorithm, the Needleman-Wunsch
algorithm, algorithms based on the Burrows-Wheeler Transform (e.g.
the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign
(Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP
(available at soap.genomics.org.cn), and Maq (available at
maq.sourceforge.net). In some embodiments, a guide sequence is
about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or
more nucleotides in length. In some embodiments, a guide sequence
is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer
nucleotides in length. The ability of a guide sequence to direct
sequence-specific binding of a CRISPR complex to a target sequence
may be assessed by any suitable assay. For example, the components
of a CRISPR system sufficient to form a CRISPR complex, including
the guide sequence to be tested, may be provided to a host cell
having the corresponding target sequence, such as by transfection
with vectors encoding the components of the CRISPR sequence,
followed by an assessment of preferential cleavage within the
target sequence, such as by Surveyor assay as described herein.
Similarly, cleavage of a target polynucleotide sequence may be
evaluated in a test tube by providing the target sequence,
components of a CRISPR complex, including the guide sequence to be
tested and a control guide sequence different from the test guide
sequence, and comparing binding or rate of cleavage at the target
sequence between the test and control guide sequence reactions.
Other assays are possible, and will occur to those skilled in the
art.
[0134] A guide sequence may be selected to target any target
sequence. In some embodiments, the target sequence is a sequence
within a genome of a cell. Exemplary target sequences include those
that are unique in the target genome. For example, for the S.
pyogenes Cas9, a unique target sequence in a genome may include a
Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGG (SEQ ID NO:
534) where NNNNNNNNNNNNXGG (SEQ ID NO: 535) (N is A, G, T, or C;
and X can be anything) has a single occurrence in the genome. A
unique target sequence in a genome may include an S. pyogenes Cas9
target site of the form MMMMMMMMNNNNNNNNNNNXGG (SEQ ID NO: 536)
where NNNNNNNNNNNXGG (SEQ ID NO: 537) (N is A, G, T, or C; and X
can be anything) has a single occurrence in the genome. For the S.
thermophilus CRISPR1 Cas9, a unique target sequence in a genome may
include a Cas9 target site of the form MMMMMMMNNNNNNNNNNNNXXAGAAW
(SEQ ID NO: 17) where NNNNNNNNNNNNXXAGAAW (SEQ ID NO: 18) (N is A,
G, T, or C; X can be anything; and W is A or T) has a single
occurrence in the genome. A unique target sequence in a genome may
include an S. thermophilus CRISPR1 Cas9 target site of the form
MMMMMMMMMNNNNNNNNNNNXXAGAAW (SEQ ID NO: 19) where
NNNNNNNNNNNXXAGAAW (SEQ ID NO: 20) (N is A, G, T, or C; X can be
anything; and W is A or T) has a single occurrence in the genome.
For the S. pyogenes Cas9, a unique target sequence in a genome may
include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGGXG
(SEQ ID NO: 538) where NNNNNNNNNNNNXGGXG (SEQ ID NO: 539) (N is A,
G, T, or C; and X can be anything) has a single occurrence in the
genome. A unique target sequence in a genome may include an S.
pyogenes Cas9 target site of the form MMMMMMMMNNNNNNNNNNNXGGXG (SEQ
ID NO: 540) where NNNNNNNNNNNXGGXG (SEQ ID NO: 541) (N is A, G, T,
or C; and X can be anything) has a single occurrence in the genome.
In each of these sequences "M" may be A, G, T, or C, and need not
be considered in identifying a sequence as unique.
[0135] In some embodiments, a guide sequence is selected to reduce
the degree of secondary structure within the guide sequence.
Secondary structure may be determined by any suitable
polynucleotide folding algorithm. Some programs are based on
calculating the minimal Gibbs free energy. An example of one such
algorithm is mFold, as described by Zuker and Stiegler (Nucleic
Acids Res. 9 (1981), 133-148). Another example folding algorithm is
the online webserver RNAfold, developed at Institute for
Theoretical Chemistry at the University of Vienna, using the
centroid structure prediction algorithm (see e.g. A. R. Gruber et
al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009,
Nature Biotechnology 27(12): 1151-62). Further algorithms may be
found in U.S. application Ser. No. TBA (attorney docket
44790.11.2022; Broad Reference BI-2013/004A); incorporated herein
by reference.
[0136] In general, a tracr mate sequence includes any sequence that
has sufficient complementarity with a tracr sequence to promote one
or more of: (1) excision of a guide sequence flanked by tracr mate
sequences in a cell containing the corresponding tracr sequence;
and (2) formation of a CRISPR complex at a target sequence, wherein
the CRISPR complex comprises the tracr mate sequence hybridized to
the tracr sequence. In general, degree of complementarity is with
reference to the optimal alignment of the tracr mate sequence and
tracr sequence, along the length of the shorter of the two
sequences. Optimal alignment may be determined by any suitable
alignment algorithm, and may further account for secondary
structures, such as self-complementarity within either the tracr
sequence or tracr mate sequence. In some embodiments, the degree of
complementarity between the tracr sequence and tracr mate sequence
along the length of the shorter of the two when optimally aligned
is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, 97.5%, 99%, or higher. Example illustrations of optimal
alignment between a tracr sequence and a tracr mate sequence are
provided in FIGS. 12B and 13B. In some embodiments, the tracr
sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in
length. In some embodiments, the tracr sequence and tracr mate
sequence are contained within a single transcript, such that
hybridization between the two produces a transcript having a
secondary structure, such as a hairpin. Preferred loop forming
sequences for use in hairpin structures are four nucleotides in
length, and most preferably have the sequence GAAA. However, longer
or shorter loop sequences may be used, as may alternative
sequences. The sequences preferably include a nucleotide triplet
(for example, AAA), and an additional nucleotide (for example C or
G). Examples of loop forming sequences include CAAA and AAAG. In an
embodiment of the invention, the transcript or transcribed
polynucleotide sequence has at least two or more hairpins. In
preferred embodiments, the transcript has two, three, four or five
hairpins. In a further embodiment of the invention, the transcript
has at most five hairpins. In some embodiments, the single
transcript further includes a transcription termination sequence;
preferably this is a polyT sequence, for example six T nucleotides.
An example illustration of such a hairpin structure is provided in
the lower portion of FIG. 13B, where the portion of the sequence 5'
of the final "N" and upstream of the loop corresponds to the tracr
mate sequence, and the portion of the sequence 3' of the loop
corresponds to the tracr sequence. Further non-limiting examples of
single polynucleotides comprising a guide sequence, a tracr mate
sequence, and a tracr sequence are as follows (listed 5' to 3'),
where "N" represents a base of a guide sequence, the first block of
lower case letters represent the tracr mate sequence, and the
second block of lower case letters represent the tracr sequence,
and the final poly-T sequence represents the transcription
terminator: (1)
NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataa-
ggctt catgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT
(SEQ ID NO: 21); (2)
NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaa-
atca acaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO:
22); (3)
NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaa-
atca acaccctgtcattttatggcagggtgtTTTTTT (SEQ ID NO: 23); (4)
NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttg-
aaaa agtggcaccgagtcggtgcTTTTTT (SEQ ID NO: 24); (5)
NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaacttg-
aa aaagtgTTTTTTT (SEQ ID NO: 25); and (6)
NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTTTTT
TTT (SEQ ID NO: 26). In some embodiments, sequences (1) to (3) are
used in combination with Cas9 from S. thermophilus CRISPR1. In some
embodiments, sequences (4) to (6) are used in combination with Cas9
from S. pyogenes. In some embodiments, the tracr sequence is a
separate transcript from a transcript comprising the tracr mate
sequence (such as illustrated in the top portion of FIG. 13B).
[0137] In some embodiments, a recombination template is also
provided. A recombination template may be a component of another
vector as described herein, contained in a separate vector, or
provided as a separate polynucleotide. In some embodiments, a
recombination template is designed to serve as a template in
homologous recombination, such as within or near a target sequence
nicked or cleaved by a CRISPR enzyme as a part of a CRISPR complex.
A template polynucleotide may be of any suitable length, such as
about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200,
500, 1000, or more nucleotides in length. In some embodiments, the
template polynucleotide is complementary to a portion of a
polynucleotide comprising the target sequence. When optimally
aligned, a template polynucleotide might overlap with one or more
nucleotides of a target sequences (e.g. about or more than about 1,
5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more
nucleotides). In some embodiments, when a template sequence and a
polynucleotide comprising a target sequence are optimally aligned,
the nearest nucleotide of the template polynucleotide is within
about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000,
5000, 10000, or more nucleotides from the target sequence.
[0138] In some embodiments, the CRISPR enzyme is part of a fusion
protein comprising one or more heterologous protein domains (e.g.
about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
domains in addition to the CRISPR enzyme). A CRISPR enzyme fusion
protein may comprise any additional protein sequence, and
optionally a linker sequence between any two domains. Examples of
protein domains that may be fused to a CRISPR enzyme include,
without limitation, epitope tags, reporter gene sequences, and
protein domains having one or more of the following activities:
methylase activity, demethylase activity, transcription activation
activity, transcription repression activity, transcription release
factor activity, histone modification activity, RNA cleavage
activity and nucleic acid binding activity. Non-limiting examples
of epitope tags include histidine (His) tags, V5 tags, FLAG tags,
influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and
thioredoxin (Trx) tags. Examples of reporter genes include, but are
not limited to, glutathione-S-transferase (GST), horseradish
peroxidase (HRP), chloramphenicol acetyltransferase (CAT)
beta-galactosidase, beta-glucuronidase, luciferase, green
fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein
(CFP), yellow fluorescent protein (YFP), and autofluorescent
proteins including blue fluorescent protein (BFP). A CRISPR enzyme
may be fused to a gene sequence encoding a protein or a fragment of
a protein that bind DNA molecules or bind other cellular molecules,
including but not limited to maltose binding protein (MBP), S-tag,
Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain
fusions, and herpes simplex virus (HSV) BP16 protein fusions.
Additional domains that may form part of a fusion protein
comprising a CRISPR enzyme are described in US20110059502,
incorporated herein by reference. In some embodiments, a tagged
CRISPR enzyme is used to identify the location of a target
sequence.
[0139] In some aspects, the invention provides methods comprising
delivering one or more polynucleotides, such as or one or more
vectors as described herein, one or more transcripts thereof,
and/or one or proteins transcribed therefrom, to a host cell. In
some aspects, the invention further provides cells produced by such
methods, and organisms (such as animals, plants, or fungi)
comprising or produced from such cells. In some embodiments, a
CRISPR enzyme in combination with (and optionally complexed with) a
guide sequence is delivered to a cell. Conventional viral and
non-viral based gene transfer methods can be used to introduce
nucleic acids in mammalian cells or target tissues. Such methods
can be used to administer nucleic acids encoding components of a
CRISPR system to cells in culture, or in a host organism. Non-viral
vector delivery systems include DNA plasmids, RNA (e.g. a
transcript of a vector described herein), naked nucleic acid, and
nucleic acid complexed with a delivery vehicle, such as a liposome.
Viral vector delivery systems include DNA and RNA viruses, which
have either episomal or integrated genomes after delivery to the
cell. For a review of gene therapy procedures, see Anderson,
Science 256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217
(1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon,
TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van
Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative
Neurology and Neuroscience 8:35-36 (1995); Kremer &
Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada
et al., in Current Topics in Microbiology and Immunology, Doerfler
and Bohm (eds) (1995); and Yu et al., Gene Therapy 1:13-26
(1994).
[0140] Methods of non-viral delivery of nucleic acids include
lipofection, nucleofection, 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 Felgner, 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).
[0141] The preparation of lipid:nucleic acid complexes, including
targeted liposomes such as immunolipid complexes, is well known to
one of skill in the art (see, e.g., Crystal, Science 270:404-410
(1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et
al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate
Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995);
Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos.
4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728,
4,774,085, 4,837,028, and 4,946,787).
[0142] The use of RNA or DNA viral based systems for the delivery
of nucleic acids takes advantage of highly evolved processes for
targeting a virus to specific cells in the body and trafficking the
viral payload to the nucleus. Viral vectors can be administered
directly to patients (in vivo) or they can be used to treat cells
in vitro, and the modified cells may optionally be administered to
patients (ex vivo). Conventional viral based systems could include
retroviral, lentivirus, adenoviral, adeno-associated and herpes
simplex virus vectors for gene transfer. Integration in the host
genome is possible with the retrovirus, lentivirus, and
adeno-associated virus gene transfer methods, often resulting in
long term expression of the inserted transgene. Additionally, high
transduction efficiencies have been observed in many different cell
types and target tissues.
[0143] The tropism of a retrovirus can be altered by incorporating
foreign envelope proteins, expanding the potential target
population of target cells. Lentiviral vectors are retroviral
vectors that are able to transduce or infect non-dividing cells and
typically produce high viral titers. Selection of a retroviral gene
transfer system would therefore depend on the target tissue.
Retroviral vectors are comprised of cis-acting long terminal
repeats with packaging capacity for up to 6-10 kb of foreign
sequence. The minimum cis-acting LTRs are sufficient for
replication and packaging of the vectors, which are then used to
integrate the therapeutic gene into the target cell to provide
permanent transgene expression. Widely used retroviral vectors
include those based upon murine leukemia virus (MuLV), gibbon ape
leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human
immuno deficiency virus (HIV), and combinations thereof (see, e.g.,
Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J.
Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59
(1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et
al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700), In
applications where transient expression is preferred, adenoviral
based systems may be used. Adenoviral based vectors are capable of
very high transduction efficiency in many cell types and do not
require cell division. With such vectors, high titer and levels of
expression have been obtained. This vector can be produced in large
quantities in a relatively simple system. Adeno-associated virus
("AAV") vectors may also be used to transduce cells with target
nucleic acids, e.g., in the in vitro production of nucleic acids
and peptides, and for in vivo and ex vivo gene therapy procedures
(see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No.
4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994);
Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of
recombinant AAV vectors are described in a number of publications,
including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell.
Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol.
4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470
(1984); and Samulski et al., J. Virol. 63:03822-3828 (1989).
[0144] Packaging cells are typically used to form virus particles
that are capable of infecting a host cell. Such cells include 293
cells, which package adenovirus, and .psi.2 cells or PA317 cells,
which package retrovirus. Viral vectors used in gene therapy are
usually generated by producing a cell line that packages a nucleic
acid vector into a viral particle. The vectors typically contain
the minimal viral sequences required for packaging and subsequent
integration into a host, other viral sequences being replaced by an
expression cassette for the polynucleotide(s) to be expressed. The
missing viral functions are typically supplied in trans by the
packaging cell line. For example, AAV vectors used in gene therapy
typically only possess ITR sequences from the AAV genome which are
required for packaging and integration into the host genome. Viral
DNA is packaged in a cell line, which contains a helper plasmid
encoding the other AAV genes, namely rep and cap, but lacking ITR
sequences. The cell line may also be infected with adenovirus as a
helper. The helper virus promotes replication of the AAV vector and
expression of AAV genes from the helper plasmid. The helper plasmid
is not packaged in significant amounts due to a lack of ITR
sequences. Contamination with adenovirus can be reduced by, e.g.,
heat treatment to which adenovirus is more sensitive than AAV.
Additional methods for the delivery of nucleic acids to cells are
known to those skilled in the art. See, for example, US20030087817,
incorporated herein by reference.
[0145] In some embodiments, a host cell is transiently or
non-transiently transfected with one or more vectors described
herein. In some embodiments, a cell is transfected as it naturally
occurs in a subject. In some embodiments, a cell that is
transfected is taken from a subject. In some embodiments, the cell
is derived from cells taken from a subject, such as a cell line. A
wide variety of cell lines for tissue culture are known in the art.
Examples of cell lines include, but are not limited to, C8161,
CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC,
HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rath,
CV1, RPTE, A10, T24, J82, A375, ARH-77, Calul, SW480, SW620, SKOV3,
SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat,
J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E,
MRCS, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A,
BS-C-1 monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast,
3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse
fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172,
A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B,
bEnd.3, BHK-21, BR 293, BxPC3, C3H-10T1/2, C6/36, Cal-27, CHO,
CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr -/-, COR-L23,
COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7, COV-434, CML T1,
CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1,
EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa,
Hepalc1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812,
KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A,
MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR/0.2R,
MONO-MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20,
NCI-H69/LX4, NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT
2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D,
T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39,
WT-49, X63, YAC-1, YAR, and transgenic varieties thereof. Cell
lines are available from a variety of sources known to those with
skill in the art (see, e.g., the American Type Culture Collection
(ATCC) (Manassas, Va.)). In some embodiments, a cell transfected
with one or more vectors described herein is used to establish a
new cell line comprising one or more vector-derived sequences. In
some embodiments, a cell transiently transfected with the
components of a CRISPR system as described herein (such as by
transient transfection of one or more vectors, or transfection with
RNA), and modified through the activity of a CRISPR complex, is
used to establish a new cell line comprising cells containing the
modification but lacking any other exogenous sequence. In some
embodiments, cells transiently or non-transiently transfected with
one or more vectors described herein, or cell lines derived from
such cells are used in assessing one or more test compounds.
[0146] In some embodiments, one or more vectors described herein
are used to produce a non-human transgenic animal or transgenic
plant. In some embodiments, the transgenic animal is a mammal, such
as a mouse, rat, or rabbit. In certain embodiments, the organism or
subject is a plant. In certain embodiments, the organism or subject
or plant is algae. Methods for producing transgenic plants and
animals are known in the art, and generally begin with a method of
cell transfection, such as described herein.
[0147] In one aspect, the invention provides for methods of
modifying a target polynucleotide in a eukaryotic cell. In some
embodiments, the method comprises allowing a CRISPR complex to bind
to the target polynucleotide to effect cleavage of said target
polynucleotide thereby modifying the target polynucleotide, wherein
the CRISPR complex comprises a CRISPR enzyme complexed with a guide
sequence hybridized to a target sequence within said target
polynucleotide, wherein said guide sequence is linked to a tracr
mate sequence which in turn hybridizes to a tracr sequence.
[0148] In one aspect, the invention provides a method of modifying
expression of a polynucleotide in a eukaryotic cell. In some
embodiments, the method comprises allowing a CRISPR complex to bind
to the polynucleotide such that said binding results in increased
or decreased expression of said polynucleotide; wherein the CRISPR
complex comprises a CRISPR enzyme complexed with a guide sequence
hybridized to a target sequence within said polynucleotide, wherein
said guide sequence is linked to a tracr mate sequence which in
turn hybridizes to a tracr sequence.
[0149] With recent advances in crop genomics, the ability to use
CRISPR-Cas systems to perform efficient and cost effective gene
editing and manipulation will allow the rapid selection and
comparison of single and multiplexed genetic manipulations to
transform such genomes for improved production and enhanced traits.
In this regard reference is made to US patents and publications:
U.S. Pat. No. 6,603,061--Agrobacterium-Mediated Plant
Transformation Method; U.S. Pat. No. 7,868,149--Plant Genome
Sequences and Uses Thereof and US 2009/0100536--Transgenic Plants
with Enhanced Agronomic Traits, all the contents and disclosure of
each of which are herein incorporated by reference in their
entirety. In the practice of the invention, the contents and
disclosure of Morrell et al "Crop genomics:advances and
applications" Nat Rev Genet. 2011 Dec. 29; 13(2):85-96 are also
herein incorporated by reference in their entirety. In an
advantageous embodiment of the invention, the CRISPR/Cas9 system is
used to engineer microalgae (Example 15). Accordingly, reference
herein to animal cells may also apply, mutatis mutandis, to plant
cells unless otherwise apparent.
[0150] In one aspect, the invention provides for methods of
modifying a target polynucleotide in a eukaryotic cell, which may
be in vivo, ex vivo or in vitro. In some embodiments, the method
comprises sampling a cell or population of cells from a human or
non-human animal or plant (including micro-algae), and modifying
the cell or cells. Culturing may occur at any stage ex vivo. The
cell or cells may even be re-introduced into the non-human animal
or plant (including micro-algae).
[0151] In plants, pathogens are often host-specific. For example,
Fusarium oxysporum f. sp. lycopersici causes tomato wilt but
attacks only tomato, and F. oxysporum f. dianthii Puccinia graminis
f sp. tritici attacks only wheat. Plants have existing and induced
defenses to resist most pathogens. Mutations and recombination
events across plant generations lead to genetic variability that
gives rise to susceptibility, especially as pathogens reproduce
with more frequency than plants. In plants there can be non-host
resistance, e.g., the host and pathogen are incompatible. There can
also be Horizontal Resistance, e.g., partial resistance against all
races of a pathogen, typically controlled by many genes and
Vertical Resistance, e.g., complete resistance to some races of a
pathogen but not to other races, typically controlled by a few
genes. In a Gene-for-Gene level, plants and pathogens evolve
together, and the genetic changes in one balance changes in other.
Accordingly, using Natural Variability, breeders combine most
useful genes for Yield, Quality, Uniformity, Hardiness, Resistance.
The sources of resistance genes include native or foreign
Varieties, Heirloom Varieties, Wild Plant Relatives, and Induced
Mutations, e.g., treating plant material with mutagenic agents.
Using the present invention, plant breeders are provided with a new
tool to induce mutations. Accordingly, one skilled in the art can
analyze the genome of sources of resistance genes, and in Varieties
having desired characteristics or traits employ the present
invention to induce the rise of resistance genes, with more
precision than previous mutagenic agents and hence accelerate and
improve plant breeding programs.
[0152] In one aspect, the invention provides kits containing any
one or more of the elements disclosed in the above methods and
compositions. In some embodiments, the kit comprises a vector
system and instructions for using the kit. In some embodiments, the
vector system comprises (a) a first regulatory element operably
linked to a tracr mate sequence and one or more insertion sites for
inserting a guide sequence upstream of the tracr mate sequence,
wherein when expressed, the guide sequence directs
sequence-specific binding of a CRISPR complex to a target sequence
in a eukaryotic cell, wherein the CRISPR complex comprises a CRISPR
enzyme complexed with (1) the guide sequence that is hybridized to
the target sequence, and (2) the tracr mate sequence that is
hybridized to the tracr sequence; and/or (b) a second regulatory
element operably linked to an enzyme-coding sequence encoding said
CRISPR enzyme comprising a nuclear localization sequence. Elements
may be provide individually or in combinations, and may be provided
in any suitable container, such as a vial, a bottle, or a tube. In
some embodiments, the kit includes instructions in one or more
languages, for example in more than one language.
[0153] In some embodiments, a kit comprises one or more reagents
for use in a process utilizing one or more of the elements
described herein. Reagents may be provided in any suitable
container. For example, a kit may provide one or more reaction or
storage buffers. Reagents may be provided in a form that is usable
in a particular assay, or in a form that requires addition of one
or more other components before use (e.g. in concentrate or
lyophilized form). A buffer can be any buffer, including but not
limited to a sodium carbonate buffer, a sodium bicarbonate buffer,
a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and
combinations thereof. In some embodiments, the buffer is alkaline.
In some embodiments, the buffer has a pH from about 7 to about 10.
In some embodiments, the kit comprises one or more oligonucleotides
corresponding to a guide sequence for insertion into a vector so as
to operably link the guide sequence and a regulatory element. In
some embodiments, the kit comprises a homologous recombination
template polynucleotide.
[0154] In one aspect, the invention provides methods for using one
or more elements of a CRISPR system. The CRISPR complex of the
invention provides an effective means for modifying a target
polynucleotide. The CRISPR complex of the invention has a wide
variety of utility including modifying (e.g., deleting, inserting,
translocating, inactivating, activating) a target polynucleotide in
a multiplicity of cell types. As such the CRISPR complex of the
invention has a broad spectrum of applications in, e.g., gene
therapy, drug screening, disease diagnosis, and prognosis. An
exemplary CRISPR complex comprises a CRISPR enzyme complexed with a
guide sequence hybridized to a target sequence within the target
polynucleotide. The guide sequence is linked to a tracr mate
sequence, which in turn hybridizes to a tracr sequence.
[0155] The target polynucleotide of a CRISPR complex can be any
polynucleotide endogenous or exogenous to the eukaryotic cell. For
example, the target polynucleotide can be a polynucleotide residing
in the nucleus of the eukaryotic cell. The target polynucleotide
can be a sequence coding a gene product (e.g., a protein) or a
non-coding sequence (e.g., a regulatory polynucleotide or a junk
DNA). Without wishing to be bound by theory, it is believed that
the target sequence should be associated with a PAM (protospacer
adjacent motif); that is, a short sequence recognized by the CRISPR
complex. The precise sequence and length requirements for the PAM
differ depending on the CRISPR enzyme used, but PAMs are typically
2-5 base pair sequences adjacent the protospacer (that is, the
target sequence) Examples of PAM sequences are given in the
examples section below, and the skilled person will be able to
identify further PAM sequences for use with a given CRISPR
enzyme.
[0156] The target polynucleotide of a CRISPR complex may include a
number of disease-associated genes and polynucleotides as well as
signaling biochemical pathway-associated genes and polynucleotides
as listed in U.S. provisional patent applications 61/736,527 and
61/748,427 having Broad reference BI-2011/008/WSGR Docket No.
44063-701.101 and BI-2011/008/WSGR Docket No. 44063-701.102
respectively, both entitled SYSTEMS METHODS AND COMPOSITIONS FOR
SEQUENCE MANIPULATION filed on Dec. 12, 2012 and Jan. 2, 2013,
respectively, the contents of all of which are herein incorporated
by reference in their entirety.
[0157] Examples of target polynucleotides include a sequence
associated with a signaling biochemical pathway, e.g., a signaling
biochemical pathway-associated gene or polynucleotide. Examples of
target polynucleotides include a disease associated gene or
polynucleotide. A "disease-associated" gene or polynucleotide
refers to any gene or polynucleotide which is yielding
transcription or translation products at an abnormal level or in an
abnormal form in cells derived from a disease-affected tissues
compared with tissues or cells of a non disease control. It may be
a gene that becomes expressed at an abnormally high level; it may
be a gene that becomes expressed at an abnormally low level, where
the altered expression correlates with the occurrence and/or
progression of the disease. A disease-associated gene also refers
to a gene possessing mutation(s) or genetic variation that is
directly responsible or is in linkage disequilibrium with a gene(s)
that is responsible for the etiology of a disease. The transcribed
or translated products may be known or unknown, and may be at a
normal or abnormal level.
[0158] Examples of disease-associated genes and polynucleotides are
available from McKusick-Nathans Institute of Genetic Medicine,
Johns Hopkins University (Baltimore, Md.) and National Center for
Biotechnology Information, National Library of Medicine (Bethesda,
Md.), available on the World Wide Web.
[0159] Examples of disease-associated genes and polynucleotides are
listed in Tables A and B. Disease specific information is available
from McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins
University (Baltimore, Md.) and National Center for Biotechnology
Information, National Library of Medicine (Bethesda, Md.),
available on the World Wide Web. Examples of signaling biochemical
pathway-associated genes and polynucleotides are listed in Table
C.
[0160] Mutations in these genes and pathways can result in
production of improper proteins or proteins in improper amounts
which affect function. Further examples of genes, diseases and
proteins are hereby incorporated by reference from U.S. Provisional
applications 61/736,527 filed on Dec. 12, 2012 and 61/748,427 filed
Jan. 2, 2013. Such genes, proteins and pathways may be the target
polynucleotide of a CRISPR complex.
TABLE-US-00001 TABLE A DISEASE/DISORDERS GENE(S) Neoplasia PTEN;
ATM; ATR; EGFR; ERBB2; ERBB3; ERBB4; Notch1; Notch2; Notch3;
Notch4; AKT; AKT2; AKT3; H1F; H1F1a; H1F3a; Met; HRG; Bcl2; PPAR
alpha; PPAR gamma; WT1 (Wilms Tumor); FGF Receptor Family members
(5 members: 1, 2, 3, 4, 5); CDKN2a; APC; RB (retinoblastoma); MEN1;
VHL; BRCA1; BRCA2; AR (Androgen Receptor); TSG101; IGF; IGF
Receptor; Igfl (4 variants); Igf2 (3 variants); Igf 1 Receptor; Igf
2 Receptor; Bax; Bcl2; caspases family (9 members: 1, 2, 3, 4, 6,
7, 8, 9, 12); Kras; Apc Age-related Macular Abcr; Ccl2; Cc2; cp
(ceruloplasmin); Timp3; cathepsinD; Degeneration Vldlr; Ccr2
Schizophrenia Neuregulin1 (Nrg1); Erb4 (receptor for Neuregulin);
Complexin1 (Cplx1); Tph1 Tryptophan hydroxylase; Tph2 Tryptophan
hydroxylase 2; Neurexin 1; GSK3; GSK3a; GSK3b Disorders 5-HTT
(Slc6a4); COMT; DRD (Drd1a); SLC6A3; DAOA; DTNBP1; Dao (Dao1)
Trinucleotide Repeat HTT (Huntington's Dx); SBMA/SMAX1/AR
(Kennedy's Disorders Dx); FWX25 (Friedrich's Ataxia); ATX3
(Machado- Joseph's Dx); ATXN1 and ATXN2 (spinocerebellar ataxias);
DNIPK (myotonic dystrophy); Atrophin-1 and Atn1 (DRPLA Dx); CBP
(Creb-BP - global instability); VLDLR (Alzheimer's); Atxn7; Atxn10
Fragile X Syndrome FMR2; FXR1; FXR2; mGLUR5 Secretase Related APH-1
(alpha and beta); Presenilin (Psen1); nicastrin Disorders (Ncstn);
PEN-2 Others Nos1; Parp1; Nat1; Nat2 Prion - related disorders Prp
ALS SOD1; ALS2; STEX; FUS; TARDBP; VEGF (VEGF-a; VEGF-b; VEGF-c)
Drug addiction Prkce (alcohol); Drd2; Drd4; ABAT (alcohol); GRIA2;
Grm5; Grin1; Htr1b; Grin2a; Drd3; Pdyn; Gria1 (alcohol) Autism
Mecp2; BZRAP1; MDGA2; Sema5A; Neurexin 1; Fragile X (FMR2 (AFF2);
FXR1; FXR2; Mglur5) Alzheimer's Disease E1; CHIP; UCH; UBB; Tau;
LRP; PICALM; Clusterin; PS1; SORL1; CR1; Vldlr; Uba1; Uba3; CHIP28
(Aqpl, Aquaporin 1); Uchl1; Uchl3; APP Inflammation IL-10; IL-1
(IL-1a; IL-1b); IL-13; IL-17 (IL-17a (CTLA8); IL- 17b; IL-17c;
IL-17d; IL-17f); 11-23; Cx3cr1; ptpn22; TNFa; NOD2/CARD15 for IBD;
IL-6; IL-12 (IL-12a; IL-12b); CTLA4; Cx3cl1 Parkinson's Disease
x-Synuclein; DJ-1; LRRK2; Parkin; PINK1
TABLE-US-00002 TABLE B Blood and Anemia (CDAN1, CDA1, RPS19, DBA,
PKLR, PK1, NT5C3, UMPH1, coagulation diseases PSN1, RHAG, RH50A,
NRAMP2, SPTB, ALAS2, ANH1, ASB, and disorders ABCB7, ABC7, ASAT);
Bare lymphocyte syndrome (TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11,
MHC2TA, C2TA, RFX5, RFXAP, RFX5), Bleeding disorders (TBXA2R,
P2RX1, P2X1); Factor H and factor H-like 1 (HF1, CFH, HUS); Factor
V and factor VIII (MCFD2); Factor VII deficiency (F7); Factor X
deficiency (F10); Factor XI deficiency (F11); Factor XII deficiency
(F12, HAF); Factor XIIIA deficiency (F13A1, F13A); Factor XIIIB
deficiency (F13B); Fanconi anemia (FANCA, FACA, FA1, FA, FAA,
FAAP95, FAAP90, F1134064, FANCB, FANCC, FACC, BRCA2, FANCD1,
FANCD2, FANCD, FACD, FAD, FANCE, FACE, FANCF, XRCC9, FANCG, BRIP1,
BACH1, FANCJ, PHF9, FANCL, FANCM, KIAA1596); Hemophagocytic
lymphohistiocytosis disorders (PRF1, HPLH2, UNC13D, MUNC13-4,
HPLH3, HLH3, FHL3); Hemophilia A (F8, F8C, HEMA); Hemophilia B (F9,
HEMB), Hemorrhagic disorders (PI, ATT, F5); Leukocyde deficiencies
and disorders (ITGB2, CD18, LCAMB, LAD, E1F2B1, E1F2BA, E1F2B2,
E1F2B3, E1F2B5, LVWM, CACH, CLE, E1F2B4); Sickle cell anemia (HBB);
Thalassemia (HBA2, HBB, HBD, LCRB, HBA1). Cell dysregulation B-cell
non-Hodgkin lymphoma (BCL7A, BCL7); Leukemia (TAL1, and oncology
TCL5, SCL, TAL2, FLT3, NBS1, NBS, ZNFN1A1, IK1, LYF1, diseases and
disorders HOXD4, HOX4B, BCR, CML, PHL, ALL, ARNT, KRAS2, RASK2,
GMPS, AF10, ARHGEF12, LARG, KIAA0382, CALM, CLTH, CEBPA, CEBP,
CHIC2, BTL, FLT3, KIT, PBT, LPP, NPM1, NUP214, D9546E, CAN, CAIN,
RUNX1, CBFA2, AML1, WHSC1L1, NSD3, FLT3, AF1Q, NPM1, NUMA1, ZNF145,
PLZF, PML, MYL, STAT5B, AF10, CALM, CLTH, ARL11, ARLTS1, P2RX7,
P2X7, BCR, CML, PHL, ALL, GRAF, NF1, VRNF, WSS, NFNS, PTPN11,
PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCRA, GATA1, GF1,
ERYF1, NFE1, ABL1, NQO1, DIA4, NMOR1, NUP214, D9S46E, CAN, CAIN).
Inflammation and AIDS (KIR3DL1, NKAT3, NKB1, AMB11, K1R3DS1, IFNG,
CXCL12, immune related SDF1); Autoimmune lymphoproliferative
syndrome (TNFRSF6, APT1, diseases and disorders FAS, CD95, ALPS1A);
Combined immunodeficiency, (IL2RG, SCIDX1, SCIDX, IMD4); HIV-1
(CCL5, SCYA5, D17S136E, TCP228), HIV susceptibility or infection
(IL10, CSIF, CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5));
Immunodeficiencies (CD3E, CD3G, AICDA, AID, HIGM2, TNFRSF5, CD40,
UNG, DGU, HIGM4, TNFSF5, CD40LG, HIGM1, IGM, FOXP3, IPEX, AIID,
XPID, PIDX, TNFRSF14B, TACI); Inflammation (IL-10, IL-1 (IL-1a,
IL-1b), IL-13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d,
IL-17f), 11-23, Cx3cr1, ptpn22, TNFa, NOD2/CARD15 for IBD, IL-6,
IL-12 (IL-12a, IL-12b), CTLA4, Cx3c11); Severe combined
immunodeficiencies (SCIDs)(JAK3, JAKL, DCLRE1C, ARTEMIS, SCIDA,
RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDX1,
SCIDX, IMD4). Metabolic, liver, Amyloid neuropathy (TTR, PALB);
Amyloidosis (APOA1, APP, AAA, kidney and protein CVAP, AD1, GSN,
FGA, LYZ, TTR, PALB); Cirrhosis (KRT18, KRT8, diseases and
disorders CIRH1A, NAIC, TEX292, KIAA1988); Cystic fibrosis (CFTR,
ABCC7, CF, MRP7); Glycogen storage diseases (SLC2A2, GLUT2, G6PC,
G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM);
Hepatic adenoma, 142330 (TCF1, HNF1A, MODY3), Hepatic failure,
early onset, and neurologic disorder (SCOD1, SCO1), Hepatic lipase
deficiency (LIPC), Hepatoblastoma, cancer and carcinomas (CTNNB1,
PDGFRL, PDGRL, PRLTS, AXIN1, AXIN, CTNNB1, TP53, P53, LFS1, IGF2R,
MPRI, MET, CASP8, MCH5; Medullary cystic kidney disease (UMOD,
HNFJ, FJHN, MCKD2, ADMCKD2); Phenylketonuria (PAH, PKU1, QDPR,
DHPR, PTS); Polycystic kidney and hepatic disease (FCYT, PKHD1,
ARPKD, PKD1, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63).
Muscular/Skeletal Becker muscular dystrophy (DMD, BMD, MYF6),
Duchenne Muscular diseases and disorders Dystrophy (DMD, BMD);
Emery-Dreifuss muscular dystrophy (LMNA, LMN1, EMD2, FPLD, CMD1A,
HGPS, LGMD1B, LMNA, LMN1, EMD2, FPLD, CMD1A); Facioscapulohumeral
muscular dystrophy (FSHMD1A, FSHD1A); Muscular dystrophy (FKRP,
MDC1C, LGMD2I, LAMA2, LAMM, LARGE, KIAA0609, MDC1D, FCMD, TTID,
MYOT, CAPN3, CANP3, DYSF, LGMD2B, SGCG, LGMD2C, DMDA1, SCG3, SGCA,
ADL, DAG2, LGMD2D, DMDA2, SGCB, LGMD2E, SGCD, SGD, LGMD2F, CMD1L,
TCAP, LGMD2G, CMD1N, TRIM32, IIT2A, LGMD2II, FKRP, MDC1C, LGMD2I,
TTN, CMD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C, SEPN1, SELN, RSMD1,
PLEC1, PLTN, EBS1); Osteopetrosis (LRP5, BMND1, LRP7, LR3, OPPG,
VBCH2, CLCN7, CLC7, OPTA2, OSTM1, GL, TCIRG1, TIRC7, OC116, OPTB1);
Muscular atrophy (VAPB, VAPC, ALS8, SMN1, SMA1, SMA2, SMA3, SMA4,
BSCL2, SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2, SMUBP2, CATF1,
SMARD1). Neurological and ALS (SOD1, ALS2, STEX, FUS, TARDBP, VEGF
(VEGF-a, VEGF-b, neuronal diseases and VEGF-c); Alzheimer disease
(APP, AAA, CVAP, AD1, APOE, AD2, disorders PSEN2, AD4, STM2, APBB2,
FE65L1, NOS3, PLAU, URK, ACE, DCP1, ACE1, MPO, PACIP1, PAXIP1L,
PTIP, A2M, BLMH, BMH, PSEN1, AD3); Autism (Mecp2, BZRAP1, MDGA2,
Sema5A, Neurexin 1, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3,
NLGN4, KIAA1260, AUTSX2); Fragile X Syndrome (FMR2, FXR1, FXR2,
mGLUR5); Huntington's disease and disease like disorders (HD, IT15,
PRNP, PRIP, JPH3, JP3, HDL2, TBP, SCA17); Parkinson disease (NR4A2,
NURR1, NOT, TINUR, SNCAIP, TBP, SCA17, SNCA, NACP, PARK1, PARK4,
DJ1, PARK7, LRRK2, PARK8, PINK1, PARK6, UCHL1, PARKS, SNCA, NACP,
PARK1, PARK4, PRKN, PARK2, PDJ, DBH, NDUFV2); Rett syndrome (MECP2,
RTT, PPMX, MRX16, MRX79, CDKL5, STK9, MECP2, RTT, PPMX, MRX16,
MRX79, x-Synuclein, DJ-1); Schizophrenia (Neuregulin1 (Nrg1), Erb4
(receptor for Neuregulin), Complexin1 (Cplx1), Tph1 Tryptophan
hydroxylase, Tph2, Tryptophan hydroxylase 2, Neurexin 1, GSK3,
GSK3a, GSK3b, 5-HTT (Slc6a4), COMT, DRD (Drd1a), SLC6A3, DAOA,
DTNBP1, Dao (Dao1)); Secretase Related Disorders (APH-1 (alpha and
beta), Presenilin (Psen1), nicastrin, (Ncstn), PEN-2, Nos1, Parp1,
Nat1, Nat2); Trinucleotide Repeat Disorders (HTT (Huntington's Dx),
SBMA/SMAX1/AR (Kennedy's Dx), FXN/X25 (Friedrich's Ataxia), ATX3
(Machado- Joseph's Dx), ATXN1 and ATXN2 (spinocerebellar ataxias),
DMPK (myotonic dystrophy), Atrophin-1 and Atn1 (DRPLA Dx), CBP
(Creb-BP - global instability), VLDLR (Alzheimer's), Atxn7,
Atxn10). Occular diseases and Age-related macular degeneration
(Abcr, Ccl2, Cc2, cp (ceruloplasmin), disorders Timp3, cathepsinD,
Vldlr, Ccr2); Cataract (CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2,
CP49, CP47, CRYAA, CRYA1, PAX6, AN2, MGDA, CRYBA1, CRYB1, CRYGC,
CRYG3, CCL, LIM2, MP19, CRYGD, CRYG4, BFSP2, CP49, CP47, HSF4, CTM,
HSF4, CTM, MIP, AQP0, CRYAB, CRYA2, CTPP2, CRYBB1, CRYGD, CRYG4,
CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA, CRYA1, GJA8, CX50, CAE1,
GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1); Corneal clouding and
dystrophy (APOA1, TGFBI, CSD2, CDGG1, CSD, BIGH3, CDG2, TACSTD2,
TROP2, M1S1, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD, PPCD2,
PIP5K3, CFD); Cornea plana congenital (KERA, CNA2); Glaucoma (MYOC,
TIGR, GLC1A, JOAG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1B1,
GLC3A, OPA1, NTG, NPG, CYP1B1, GLC3A); Leber congenital amaurosis
(CRB1, RP12, CRX, CORD2, CRD, RPGRIP1, LCA6, CORD9, RPE65, RP20,
AIPL1, LCA4, GUCY2D, GUC2D, LCA1, CORD6, RDH12, LCA3); Macular
dystrophy (ELOVL4, ADMD, STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD,
AOFMD, VMD2).
TABLE-US-00003 TABLE C CELLULAR FUNCTION GENES PI3K/AKT Signaling
PRKCE; ITGAM; ITGA5; IRAK1; PRKAA2; EIF2AK2; PTEN; EIF4E; PRKCZ;
GRK6; MAPK1; TSC1; PLK1; AKT2; IKBKB; PIK3CA; CDK8; CDKN1B; NFKB2;
BCL2; PIK3CB; PPP2R1A; MAPK8; BCL2L1; MAPK3; TSC2; ITGA1; KRAS;
EIF4EBP1; RELA; PRKCD; NOS3; PRKAA1; MAPK9; CDK2; PPP2CA; PIM1;
ITGB7; YWHAZ; ILK; TP53; RAF1; IKBKG; RELB; DYRK1A; CDKN1A; ITGB1;
MAP2K2; JAK1; AKT1; JAK2; PIK3R1; CHUK; PDPK1; PPP2R5C; CTNNB1;
MAP2K1; NFKB1; PAK3; ITGB3; CCND1; GSK3A; FRAP1; SFN; ITGA2; TTK;
CSNK1A1; BRAF; GSK3B; AKT3; FOXO1; SGK; HSP90AA1; RPS6KB1 ERK/MAPK
Signaling PRKCE; ITGAM; ITGA5; HSPB1; IRAK1; PRKAA2; EIF2AK2; RAC1;
RAP1A; TLN1; EIF4E; ELK1; GRK6; MAPK1; RAC2; PLK1; AKT2; PIK3CA;
CDK8; CREB1; PRKCI; PTK2; FOS; RPS6KA4; PIK3CB; PPP2R1A; PIK3C3;
MAPK8; MAPK3; ITGA1; ETS1; KRAS; MYCN; EIF4EBP1; PPARG; PRKCD;
PRKAA1; MAPK9; SRC; CDK2; PPP2CA; PIM1; PIK3C2A; ITGB7; YWHAZ;
PPP1CC; KSR1; PXN; RAF1; FYN; DYRK1A; ITGB1; MAP2K2; PAK4; PIK3R1;
STAT3; PPP2R5C; MAP2K1; PAK3; ITGB3; ESR1; ITGA2; MYC; TTK;
CSNK1A1; CRKL; BRAF; ATF4; PRKCA; SRF; STAT1; SGK Glucocorticoid
Receptor RAC1; TAF4B; EP300; SMAD2; TRAF6; PCAF; ELK1; Signaling
MAPK1; SMAD3; AKT2; IKBKB; NCOR2; UBE2I; PIK3CA; CREB1; FOS; HSPA5;
NFKB2; BCL2; MAP3K14; STAT5B; PIK3CB; PIK3C3; MAPK8; BCL2L1; MAPK3;
TSC22D3; MAPK10; NRIP1; KRAS; MAPK13; RELA; STAT5A; MAPK9; NOS2A;
PBX1; NR3C1; PIK3C2A; CDKN1C; TRAF2; SERPINE1; NCOA3; MAPK14; TNF;
RAF1; IKBKG; MAP3K7; CREBBP; CDKN1A; MAP2K2; JAK1; IL8; NCOA2;
AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; TGFBR1; ESR1;
SMAD4; CEBPB; JUN; AR; AKT3; CCL2; MMP1; STAT1; IL6; HSP90AA1
Axonal Guidance PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; ADAM12;
Signaling IGF1; RAC1; RAP1A; EIF4E; PRKCZ; NRP1; NTRK2; ARHGEF7;
SMO; ROCK2; MAPK1; PGF; RAC2; PTPN11; GNAS; AKT2; PIK3CA; ERBB2;
PRKCI; PTK2; CFL1; GNAQ; PIK3CB; CXCL12; PIK3C3; WNT11; PRKD1;
GNB2L1; ABL1; MAPK3; ITGA1; KRAS; RHOA; PRKCD; PIK3C2A; ITGB7;
GLI2; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2; PAK4; ADAM17; AKT1;
PIK3R1; GLI1; WNT5A; ADAM10; MAP2K1; PAK3; ITGB3; CDC42; VEGFA;
ITGA2; EPHA8; CRKL; RND1; GSK3B; AKT3; PRKCA Ephrin Receptor PRKCE;
ITGAM; ROCK1; ITGA5; CXCR4; IRAK1; Signaling PRKAA2; EIF2AK2; RAC1;
RAP1A; GRK6; ROCK2; MAPK1; PGF; RAC2; PTPN11; GNAS; PLK1; AKT2;
DOK1; CDK8; CREB1; PTK2; CFL1; GNAQ; MAP3K14; CXCL12; MAPK8;
GNB2L1; ABL1; MAPK3; ITGA1; KRAS; RHOA; PRKCD; PRKAA1; MAPK9; SRC;
CDK2; PIM1; ITGB7; PXN; RAF1; FYN; DYRK1A; ITGB1; MAP2K2; PAK4;
AKT1; JAK2; STAT3; ADAM10; MAP2K1; PAK3; ITGB3; CDC42; VEGFA;
ITGA2; EPHA8; TTK; CSNK1A1; CRKL; BRAF; PTPN13; ATF4; AKT3; SGK
Actin Cytoskeleton ACTN4; PRKCE; ITGAM; ROCK1; ITGA5; IRAK1;
Signaling PRKAA2; EIF2AK2; RAC1; INS; ARHGEF7; GRK6; ROCK2; MAPK1;
RAC2; PLK1; AKT2; PIK3CA; CDK8; PTK2; CFL1; PIK3CB; MYH9; DIAPH1;
PIK3C3; MAPK8; F2R; MAPK3; SLC9A1; ITGA1; KRAS; RHOA; PRKCD;
PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; ITGB7; PPP1CC; PXN; VIL2; RAF1;
GSN; DYRK1A; ITGB1; MAP2K2; PAK4; PIP5K1A; PIK3R1; MAP2K1; PAK3;
ITGB3; CDC42; APC; ITGA2; TTK; CSNK1A1; CRKL; BRAF; VAV3; SGK
Huntington's Disease PRKCE; IGF1; EP300; RCOR1; PRKCZ; HDAC4; TGM2;
Signaling MAPK1; CAPNS1; AKT2; EGFR; NCOR2; SP1; CAPN2; PIK3CA;
HDAC5; CREB1; PRKCI; HSPA5; REST; GNAQ; PIK3CB; PIK3C3; MAPK8;
IGF1R; PRKD1; GNB2L1; BCL2L1; CAPN1; MAPK3; CASP8; HDAC2; HDAC7A;
PRKCD; HDAC11; MAPK9; HDAC9; PIK3C2A; HDAC3; TP53; CASP9; CREBBP;
AKT1; PIK3R1; PDPK1; CASP1; APAF1; FRAP1; CASP2; JUN; BAX; ATF4;
AKT3; PRKCA; CLTC; SGK; HDAC6; CASP3 Apoptosis Signaling PRKCE;
ROCK1; BID; IRAK1; PRKAA2; EIF2AK2; BAK1; BIRC4; GRK6; MAPK1;
CAPNS1; PLK1; AKT2; IKBKB; CAPN2; CDK8; FAS; NFKB2; BCL2; MAP3K14;
MAPK8; BCL2L1; CAPN1; MAPK3; CASP8; KRAS; RELA; PRKCD; PRKAA1;
MAPK9; CDK2; PIM1; TP53; TNF; RAF1; IKBKG; RELB; CASP9; DYRK1A;
MAP2K2; CHUK; APAF1; MAP2K1; NFKB1; PAK3; LMNA; CASP2; BIRC2; TTK;
CSNK1A1; BRAF; BAX; PRKCA; SGK; CASP3; BIRC3; PARP1 B Cell Receptor
RAC1; PTEN; LYN; ELK1; MAPK1; RAC2; PTPN11; Signaling AKT2; IKBKB;
PIK3CA; CREB1; SYK; NFKB2; CAMK2A; MAP3K14; PIK3CB; PIK3C3; MAPK8;
BCL2L1; ABL1; MAPK3; ETS1; KRAS; MAPK13; RELA; PTPN6; MAPK9; EGR1;
PIK3C2A; BTK; MAPK14; RAF1; IKBKG; RELB; MAP3K7; MAP2K2; AKT1;
PIK3R1; CHUK; MAP2K1; NFKB1; CDC42; GSK3A; FRAP1; BCL6; BCL10; JUN;
GSK3B; ATF4; AKT3; VAV3; RPS6KB1 Leukocyte Extravasation ACTN4;
CD44; PRKCE; ITGAM; ROCK1; CXCR4; CYBA; Signaling RAC1; RAP1A;
PRKCZ; ROCK2; RAC2; PTPN11; MMP14; PIK3CA; PRKCI; PTK2; PIK3CB;
CXCL12; PIK3C3; MAPK8; PRKD1; ABL1; MAPK10; CYBB; MAPK13; RHOA;
PRKCD; MAPK9; SRC; PIK3C2A; BTK; MAPK14; NOX1; PXN; VIL2; VASP;
ITGB1; MAP2K2; CTNND1; PIK3R1; CTNNB1; CLDN1; CDC42; F11R; ITK;
CRKL; VAV3; CTTN; PRKCA; IVIMP1; MMP9 Integrin Signaling ACTN4;
ITGAM; ROCK1; ITGA5; RAC1; PTEN; RAP1A; TLN1; ARHGEF7; MAPK1; RAC2;
CAPNS1; AKT2; CAPN2; PIK3CA; PTK2; PIK3CB; PIK3C3; MAPK8; CAV1;
CAPN1; ABL1; MAPK3; ITGAl; KRAS; RHOA; SRC; PIK3C2A; ITGB7; PPP1CC;
ILK; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2; PAK4; AKT1; PIK3R1; TNK2;
MAP2K1; PAK3; ITGB3; CDC42; RND3; ITGA2; CRKL; BRAF; GSK3B; AKT3
Acute Phase Response IRAK1; SOD2; MYD88; TRAF6; ELK1; MAPK1;
PTPN11; Signaling AKT2; IKBKB; PIK3CA; FOS; NFKB2; MAP3K14; PIK3CB;
MAPK8; RIPK1; MAPK3; IL6ST; KRAS; MAPK13; IL6R; RELA; SOCS1; MAPK9;
FTL; NR3C1; TRAF2; SERPINE1; MAPK14; TNF; RAF1; PDK1; IKBKG; RELB;
MAP3K7; MAP2K2; AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1;
FRAP1; CEBPB; JUN; AKT3; IL1R1; IL6 PTEN Signaling ITGAM; ITGA5;
RAC1; PTEN; PRKCZ; BCL2L11; MAPK1; RAC2; AKT2; EGFR; IKBKB; CBL;
PIK3CA; CDKN1B; PTK2; NFKB2; BCL2; PIK3CB; BCL2L1; MAPK3; ITGAl;
KRAS; ITGB7; ILK; PDGFRB; INSR; RAF1; IKBKG; CASP9; CDKN1A; ITGB1;
MAP2K2; AKT1; PIK3R1; CHUK; PDGFRA; PDPK1; MAP2K1; NFKB1; ITGB3;
CDC42; CCND1; GSK3A; ITGA2; GSK3B; AKT3; FOXO1; CASP3; RPS6KB1 p53
Signaling PTEN; EP300; BBC3; PCAF; FASN; BRCA1; GADD45A; BIRC5;
AKT2; PIK3CA; CHEK1; TP53INP1; BCL2; PIK3CB; PIK3C3; MAPK8; THBS1;
ATR; BCL2L1; E2F1; PMAIP1; CHEK2; TNFRSF10B; TP73; RB1; HDAC9;
CDK2; PIK3C2A; MAPK14; TP53; LRDD; CDKN1A; HIPK2; AKT1; PIK3R1;
RRM2B; APAF1; CTNNB1; SIRT1; CCND1; PRKDC; ATM; SFN; CDKN2A; JUN;
SNAI2; GSK3B; BAX; AKT3 Aryl Hydrocarbon HSPB1; EP300; FASN; TGM2;
RXRA; MAPK1; NQO1; Receptor NCOR2; SP1; ARNT; CDKN1B; FOS; CHEK1;
Signaling SMARCA4; NFKB2; MAPK8; ALDH1A1; ATR; E2F1; MAPK3; NRIP1;
CHEK2; RELA; TP73; GSTP1; RBI; SRC; CDK2; AHR; NFE2L2; NCOA3; TP53;
TNF; CDKN1A; NCOA2; APAF1; NFKB1; CCND1; ATM; ESR1; CDKN2A; MYC;
JUN; ESR2; BAX; IL6; CYP1B1; HSP90AA1 Xenobiotic Metabolism PRKCE;
EP300; PRKCZ; RXRA; MAPK1; NQO1; Signaling NCOR2; PIK3CA; ARNT;
PRKCI; NFKB2; CAMK2A; PIK3CB; PPP2R1A; PIK3C3; MAPK8; PRKD1;
ALDH1A1; MAPK3; NRIP1; KRAS; MAPK13; PRKCD; GSTP1; MAPK9; NOS2A;
ABCB1; AHR; PPP2CA; FTL; NFE2L2; PIK3C2A; PPARGC1A; MAPK14; TNF;
RAF1; CREBBP; MAP2K2; PIK3R1; PPP2R5C; MAP2K1; NFKB1; KEAP1; PRKCA;
EIF2AK3; IL6; CYP1B1; HSP90AA1 SAPK/JNK Signaling PRKCE; IRAK1;
PRKAA2; EIF2AK2; RAC1; ELK1; GRK6; MAPK1; GADD45A; RAC2; PLK1;
AKT2; PIK3CA; FADD; CDK8; PIK3CB; PIK3C3; MAPK8; RIPK1; GNB2L1;
IRS1; MAPK3; MAPK10; DAXX; KRAS; PRKCD; PRKAA1; MAPK9; CDK2; PIM1;
PIK3C2A; TRAF2; TP53; LCK; MAP3K7; DYRK1A; MAP2K2; PIK3R1; MAP2K1;
PAK3; CDC42; JUN; TTK; CSNK1A1; CRKL; BRAF; SGK PPAr/RXR Signaling
PRKAA2; EP300; INS; SMAD2; TRAF6; PPARA; FASN; RXRA; MAPK1; SMAD3;
GNAS; IKBKB; NCOR2; ABCA1; GNAQ; NFKB2; MAP3K14; STAT5B; MAPK8;
IRS1; MAPK3; KRAS; RELA; PRKAA1; PPARGC1A; NCOA3; MAPK14; INSR;
RAF1; IKBKG; RELB; MAP3K7; CREBBP; MAP2K2; JAK2; CHUK; MAP2K1;
NFKB1; TGFBR1; SMAD4; JUN; IL1R1; PRKCA; IL6; HSP90AA1; ADIPOQ
NF-KB Signaling IRAK1; EIF2AK2; EP300; INS; MYD88; PRKCZ; TRAF6;
TBK1; AKT2; EGFR; IKBKB; PIK3CA; BTRC; NFKB2; MAP3K14; PIK3CB;
PIK3C3; MAPK8; RIPK1; HDAC2; KRAS; RELA; PIK3C2A; TRAF2; TLR4;
PDGFRB; TNF; INSR; LCK; IKBKG; RELB; MAP3K7; CREBBP; AKT1; PIK3R1;
CHUK; PDGFRA; NFKB1; TLR2; BCL10; GSK3B; AKT3; TNFAIP3; IL1R1
Neuregulin Signaling ERBB4; PRKCE; ITGAM; ITGA5; PTEN; PRKCZ; ELK1;
MAPK1; PTPN11; AKT2; EGFR; ERBB2; PRKCI; CDKN1B; STAT5B; PRKD1;
MAPK3; ITGAl; KRAS; PRKCD; STAT5A; SRC; ITGB7; RAF1; ITGB1; MAP2K2;
ADAM17; AKT1; PIK3R1; PDPK1; MAP2K1; ITGB3; EREG; FRAP1; PSEN1;
ITGA2; MYC; NRG1; CRKL; AKT3; PRKCA; HSP90AA1; RPS6KB1 Wnt &
Beta catenin CD44; EP300; LRP6; DVL3; CSNK1E; GJA1; SMO; Signaling
AKT2; PIN1; CDH1; BTRC; GNAQ; MARK2; PPP2R1A; WNT11; SRC; DKK1;
PPP2CA; SOX6; SFRP2; ILK; LEF1; SOX9; TP53; MAP3K7; CREBBP; TCF7L2;
AKT1; PPP2R5C; WNT5A; LRP5; CTNNB1; TGFBR1; CCND1; GSK3A; DVL1;
APC; CDKN2A; MYC; CSNK1A1; GSK3B; AKT3; SOX2 Insulin Receptor PTEN;
INS; EIF4E; PTPN1; PRKCZ; MAPK1; TSC1; Signaling PTPN11; AKT2; CBL;
PIK3CA; PRKCI; PIK3CB; PIK3C3; MAPK8; IRS1; MAPK3; TSC2; KRAS;
EIF4EBP1; SLC2A4; PIK3C2A; PPP1CC; INSR; RAF1; FYN; MAP2K2; JAK1;
AKT1; JAK2; PIK3R1; PDPK1; MAP2K1; GSK3A; FRAP1; CRKL; GSK3B; AKT3;
FOXO1; SGK; RPS6KB1 IL-6 Signaling HSPB1; TRAF6; MAPKAPK2; ELK1;
MAPK1; PTPN11; IKBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK3; MAPK10;
IL6ST; KRAS; MAPK13; IL6R; RELA; SOCS1; MAPK9; ABCB1; TRAF2;
MAPK14; TNF; RAF1; IKBKG; RELB; MAP3K7; MAP2K2; IL8; JAK2; CHUK;
STAT3; MAP2K1; NFKB1; CEBPB; JUN; IL1R1; SRF; IL6 Hepatic
Cholestasis PRKCE; IRAK1; INS; MYD88; PRKCZ; TRAF6; PPARA; RXRA;
IKBKB; PRKCI; NFKB2; MAP3K14; MAPK8; PRKD1; MAPK10; RELA; PRKCD;
MAPK9; ABCB1; TRAF2; TLR4; TNF; INSR; IKBKG; RELB; MAP3K7; IL8;
CHUK; NR1H2; TJP2; NFKB1; ESR1; SREBF1; FGFR4; JUN; IL1R1; PRKCA;
IL6 IGF-1 Signaling IGF1; PRKCZ; ELK1; MAPK1; PTPN11; NEDD4; AKT2;
PIK3CA; PRKCI; PTK2; FOS; PIK3CB; PIK3C3; MAPK8; IGF1R; IRS1;
MAPK3; IGFBP7; KRAS; PIK3C2A; YWHAZ; PXN; RAF1; CASP9; MAP2K2;
AKT1; PIK3R1; PDPK1; MAP2K1; IGFBP2; SFN; JUN; CYR61; AKT3; FOXO1;
SRF; CTGF; RPS6KB1 NRF2-mediated PRKCE; EP300; SOD2; PRKCZ; MAPK1;
SQSTM1; Oxidative NQO1; PIK3CA; PRKCI; FOS; PIK3CB; PIK3C3; MAPK8;
Stress Response PRKD1; MAPK3; KRAS; PRKCD; GSTP1; MAPK9; FTL;
NFE2L2; PIK3C2A; MAPK14; RAF1; MAP3K7; CREBBP; MAP2K2; AKT1;
PIK3R1; MAP2K1; PPM; JUN; KEAP1; GSK3B; ATF4; PRKCA; EIF2AK3;
HSP90AA1 Hepatic Fibrosis/Hepatic EDN1; IGF1; KDR; FLT1; SMAD2;
FGFR1; MET; PGF; Stellate Cell Activation SMAD3; EGFR; FAS; CSF1;
NFKB2; BCL2; MYH9; IGF1R; IL6R; RELA; TLR4; PDGFRB; TNF; RELB; IL8;
PDGFRA; NFKB1; TGFBR1; SMAD4; VEGFA; BAX; IL1R1; CCL2; HGF; MMP1;
STAT1; IL6; CTGF; MIMP9 PPAR Signaling EP300; INS; TRAF6; PPARA;
RXRA; MAPK1; IKBKB; NCOR2; FOS; NFKB2; MAP3K14; STAT5B; MAPK3;
NRIP1; KRAS; PPARG; RELA; STAT5A; TRAF2; PPARGC1A; PDGFRB; TNF;
INSR; RAF1; IKBKG; RELB; MAP3K7; CREBBP; MAP2K2; CHUK; PDGFRA;
MAP2K1; NFKB1; JUN; IL1R1; HSP90AA1 Fc Epsilon RI Signaling PRKCE;
RAC1; PRKCZ; LYN; MAPK1; RAC2; PTPN11; AKT2; PIK3CA; SYK; PRKCI;
PIK3CB; PIK3C3; MAPK8; PRKD1; MAPK3; MAPK10; KRAS; MAPK13; PRKCD;
MAPK9; PIK3C2A; BTK; MAPK14; TNF; RAF1; FYN; MAP2K2; AKT1; PIK3R1;
PDPK1; MAP2K1; AKT3; VAV3; PRKCA G-Protein Coupled PRKCE; RAP1A;
RGS16; MAPK1; GNAS; AKT2; IKBKB; Receptor Signaling PIK3CA; CREB1;
GNAQ; NFKB2; CAMK2A; PIK3CB; PIK3C3; MAPK3; KRAS; RELA; SRC;
PIK3C2A; RAF1; IKBKG; RELB; FYN; MAP2K2; AKT1; PIK3R1; CHUK; PDPK1;
STAT3; MAP2K1; NFKB1; BRAF; ATF4; AKT3; PRKCA Inositol Phosphate
PRKCE; IRAK1; PRKAA2; EIF2AK2; PTEN; GRK6; Metabolism MAPK1; PLK1;
AKT2; PIK3CA; CDK8; PIK3CB; PIK3C3; MAPK8; MAPK3; PRKCD; PRKAA1;
MAPK9; CDK2; PIM1; PIK3C2A; DYRK1A; MAP2K2; PIP5K1A; PIK3R1;
MAP2K1; PAK3; ATM; TTK; CSNK1A1; BRAF; SGK
PDGF Signaling EIF2AK2; ELK1; ABL2; MAPK1; PIK3CA; FOS; PIK3CB;
PIK3C3; MAPK8; CAV1; ABL1; MAPK3; KRAS; SRC; PIK3C2A; PDGFRB; RAF1;
MAP2K2; JAK1; JAK2; PIK3R1; PDGFRA; STAT3; SPHK1; MAP2K1; MYC; JUN;
CRKL; PRKCA; SRF; STAT1; SPHK2 VEGF Signaling ACTN4; ROCK1; KDR;
FLT1; ROCK2; MAPK1; PGF; AKT2; PIK3CA; ARNT; PTK2; BCL2; PIK3CB;
PIK3C3; BCL2L1; MAPK3; KRAS; HIF1A; NOS3; PIK3C2A; PXN; RAF1;
MAP2K2; ELAVL1; AKT1; PIK3R1; MAP2K1; SFN; VEGFA; AKT3; FOXO1;
PRKCA Natural Killer Cell PRKCE; RAC1; PRKCZ; MAPK1; RAC2; PTPN11;
Signaling KIR2DL3; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3; PRKD1;
MAPK3; KRAS; PRKCD; PTPN6; PIK3C2A; LCK; RAF1; FYN; MAP2K2; PAK4;
AKT1; PIK3R1; MAP2K1; PAK3; AKT3; VAV3; PRKCA Cell Cycle: G1/S
HDAC4; SMAD3; SUV39H1; HDAC5; CDKN1B; BTRC; Checkpoint Regulation
ATR; ABL1; E2F1; HDAC2; HDAC7A; RB1; HDAC11; HDAC9; CDK2; E2F2;
HDAC3; TP53; CDKN1A; CCND1; E2F4; ATM; RBL2; SMAD4; CDKN2A; MYC;
NRG1; GSK3B; RBL1; HDAC6 T Cell Receptor RAC1; ELK1; MAPK1; IKBKB;
CBL; PIK3CA; FOS; Signaling NFKB2; PIK3CB; PIK3C3; MAPK8; MAPK3;
KRAS; RELA; PIK3C2A; BTK; LCK; RAF1; IKBKG; RELB; FYN; MAP2K2;
PIK3R1; CHUK; MAP2K1; NFKB1; ITK; BCL10; JUN; VAV3 Death Receptor
Signaling CRADD; HSPB1; BID; BIRC4; TBK1; IKBKB; FADD; FAS; NFKB2;
BCL2; MAP3K14; MAPK8; RIPK1; CASP8; DAXX; TNFRSF10B; RELA; TRAF2;
TNF; IKBKG; RELB; CASP9; CHUK; APAF1; NFKB1; CASP2; BIRC2; CASP3;
BIRC3 FGF Signaling RAC1; FGFR1; MET; MAPKAPK2; MAPK1; PTPN11;
AKT2; PIK3CA; CREB1; PIK3CB; PIK3C3; MAPK8; MAPK3; MAPK13; PTPN6;
PIK3C2A; MAPK14; RAF1; AKT1; PIK3R1; STAT3; MAP2K1; FGFR4; CRKL;
ATF4; AKT3; PRKCA; HGF GM-CSF Signaling LYN; ELK1; MAPK1; PTPN11;
AKT2; PIK3CA; CAMK2A; STAT5B; PIK3CB; PIK3C3; GNB2L1; BCL2L1;
MAPK3; ETS1; KRAS; RUNX1; PIM1; PIK3C2A; RAF1; MAP2K2; AKT1; JAK2;
PIK3R1; STAT3; MAP2K1; CCND1; AKT3; STAT1 Amyotrophic Lateral BID;
IGF1; RAC1; BIRC4; PGF; CAPNS1; CAPN2; Sclerosis Signaling PIK3CA;
BCL2; PIK3CB; PIK3C3; BCL2L1; CAPN1; PIK3C2A; TP53; CASP9; PIK3R1;
RAB5A; CASP1; APAF1; VEGFA; BIRC2; BAX; AKT3; CASP3; BIRC3 JAK/Stat
Signaling PTPN1; MAPK1; PTPN11; AKT2; PIK3CA; STAT5B; PIK3CB;
PIK3C3; MAPK3; KRAS; SOCS1; STAT5A; PTPN6; PIK3C2A; RAF1; CDKN1A;
MAP2K2; JAK1; AKT1; JAK2; PIK3R1; STAT3; MAP2K1; FRAP1; AKT3; STAT1
Nicotinate and PRKCE; IRAK1; PRKAA2; EIF2AK2; GRK6; MAPK1;
Nicotinamide Metabolism PLK1; AKT2; CDK8; MAPK8; MAPK3; PRKCD;
PRKAA1; PBEF1; MAPK9; CDK2; PIM1; DYRK1A; MAP2K2; MAP2K1; PAK3;
NT5E; TTK; CSNK1A1; BRAF; SGK Chemokine Signaling CXCR4; ROCK2;
MAPK1; PTK2; FOS; CFL1; GNAQ; CAMK2A; CXCL12; MAPK8; MAPK3; KRAS;
MAPK13; RHOA; CCR3; SRC; PPP1CC; MAPK14; NOX1; RAF1; MAP2K2;
MAP2K1; JUN; CCL2; PRKCA IL-2 Signaling ELK1; MAPK1; PTPN11; AKT2;
PIK3CA; SYK; FOS; STAT5B; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS;
SOCS1; STAT5A; PIK3C2A; LCK; RAF1; MAP2K2; JAK1; AKT1; PIK3R1;
MAP2K1; JUN; AKT3 Synaptic Long Term PRKCE; IGF1; PRKCZ; PRDX6;
LYN; MAPK1; GNAS; Depression PRKCI; GNAQ; PPP2R1A; IGF1R; PRKD1;
MAPK3; KRAS; GRN; PRKCD; NOS3; NOS2A; PPP2CA; YWHAZ; RAF1; MAP2K2;
PPP2R5C; MAP2K1; PRKCA Estrogen Receptor TAF4B; EP300; CARM1; PCAF;
MAPK1; NCOR2; Signaling SMARCA4; MAPK3; NRIP1; KRAS; SRC; NR3C1;
HDAC3; PPARGC1A; RBM9; NCOA3; RAF1; CREBBP; MAP2K2; NCOA2; MAP2K1;
PRKDC; ESR1; ESR2 Protein Ubiquitination TRAF6; SMURF1; BIRC4;
BRCA1; UCHL1; NEDD4; Pathway CBL; UBE2I; BTRC; HSPA5; USP7; USP10;
FBW7; USP9X; STUB1; USP22; B2M; BIRC2; PARK2; USP8; USP1; VHL;
HSP90AA1; BIRC3 IL-10 Signaling TRAF6; CCR1; ELK1; IKBKB; SP1; FOS;
NFKB2; MAP3K14; MAPK8; MAPK13; RELA; MAPK14; TNF; IKBKG; RELB;
MAP3K7; JAK1; CHUK; STAT3; NFKB1; JUN; IL1R1; IL6 VDR/RXR
Activation PRKCE; EP300; PRKCZ; RXRA; GADD45A; HES1; NCOR2; SP1;
PRKCI; CDKN1B; PRKD1; PRKCD; RUNX2; KLF4; YY1; NCOA3; CDKN1A;
NCOA2; SPP1; LRP5; CEBPB; FOXO1; PRKCA TGF-beta Signaling EP300;
SMAD2; SMURF1; MAPK1; SMAD3; SMAD1; FOS; MAPK8; MAPK3; KRAS; MAPK9;
RUNX2; SERPINE1; RAF1; MAP3K7; CREBBP; MAP2K2; MAP2K1; TGFBR1;
SMAD4; JUN; SMAD5 Toll-like Receptor IRAK1; EIF2AK2; MYD88; TRAF6;
PPARA; ELK1; Signaling IKBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK13;
RELA; TLR4; MAPK14; IKBKG; RELB; MAP3K7; CHUK; NFKB1; TLR2; JUN p38
MAPK Signaling HSPB1; IRAK1; TRAF6; MAPKAPK2; ELK1; FADD; FAS;
CREB1; DDIT3; RPS6KA4; DAXX; MAPK13; TRAF2; MAPK14; TNF; MAP3K7;
TGFBR1; MYC; ATF4; IL1R1; SRF; STAT1 Neurotrophin/TRK NTRK2; MAPK1;
PTPN11; PIK3CA; CREB1; FOS; Signaling PIK3CB; PIK3C3; MAPK8; MAPK3;
KRAS; PIK3C2A; RAF1; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; CDC42;
JUN; ATF4 FXR/RXR Activation INS; PPARA; FASN; RXRA; AKT2; SDC1;
MAPK8; APOB; MAPK10; PPARG; MTTP; MAPK9; PPARGC1A; TNF; CREBBP;
AKT1; SREBF1; FGFR4; AKT3; FOXO1 Synaptic Long Term PRKCE; RAP1A;
EP300; PRKCZ; MAPK1; CREB1; Potentiation PRKCI; GNAQ; CAMK2A;
PRKD1; MAPK3; KRAS; PRKCD; PPP1CC; RAF1; CREBBP; MAP2K2; MAP2K1;
ATF4; PRKCA Calcium Signaling RAP1A; EP300; HDAC4; MAPK1; HDAC5;
CREB1; CAMK2A; MYH9; MAPK3; HDAC2; HDAC7A; HDAC11; HDAC9; HDAC3;
CREBBP; CALR; CAMKK2; ATF4; HDAC6 EGF Signaling ELK1; MAPK1; EGFR;
PIK3CA; FOS; PIK3CB; PIK3C3; MAPK8; MAPK3; PIK3C2A; RAF1; JAK1;
PIK3R1; STAT3; MAP2K1; JUN; PRKCA; SRF; STAT1 Hypoxia Signaling in
the EDN1; PTEN; EP300; NQO1; UBE2I; CREB1; ARNT; Cardiovascular
System HIF1A; SLC2A4; NOS3; TP53; LDHA; AKT1; ATM; VEGFA; JUN;
ATF4; VHL; HSP90AA1 LPS/IL-1 Mediated IRAK1; MYD88; TRAF6; PPARA;
RXRA; ABCA1; Inhibition MAPK8; ALDH1A1; GSTP1; MAPK9; ABCB1; TRAF2;
of RXR Function TLR4; TNF; MAP3K7; NR1H2; SREBF1; JUN; IL1R1
LXR/RXR Activation FASN; RXRA; NCOR2; ABCA1; NFKB2; IRF3; RELA;
NOS2A; TLR4; TNF; RELB; LDLR; NR1H2; NFKB1; SREBF1; IL1R1; CCL2;
IL6; MIMP9 Amyloid Processing PRKCE; CSNK1E; MAPK1; CAPNS1; AKT2;
CAPN2; CAPN1; MAPK3; MAPK13; MAPT; MAPK14; AKT1; PSEN1; CSNK1A1;
GSK3B; AKT3; APP IL-4 Signaling AKT2; PIK3CA; PIK3CB; PIK3C3; IRS1;
KRAS; SOCS1; PTPN6; NR3C1; PIK3C2A; JAK1; AKT1; JAK2; PIK3R1;
FRAP1; AKT3; RPS6KB1 Cell Cycle: G2/M DNA EP300; PCAF; BRCA1;
GADD45A; PLK1; BTRC; Damage Checkpoint CHEK1; ATR; CHEK2; YWHAZ;
TP53; CDKN1A; Regulation PRKDC; ATM; SFN; CDKN2A Nitric Oxide
Signaling in KDR; FLT1; PGF; AKT2; PIK3CA; PIK3CB; PIK3C3; the
CAV1; PRKCD; NOS3; PIK3C2A; AKT1; PIK3R1; Cardiovascular System
VEGFA; AKT3; HSP90AA1 Purine Metabolism NME2; SMARCA4; MYH9; RRM2;
ADAR; EIF2AK4; PKM2; ENTPD1; RAD51; RRM2B; TJP2; RAD51C; NT5E;
POLD1; NME1 cAMP-mediated RAP1A; MAPK1; GNAS; CREB1; CAMK2A; MAPK3;
Signaling SRC; RAF1; MAP2K2; STAT3; MAP2K1; BRAF; ATF4
Mitochondrial SOD2; MAPK8; CASP8; MAPK10; MAPK9; CASP9; Dysfunction
PARK7; PSEN1; PARK2; APP; CASP3 Notch Signaling HES1; JAG1; NUMB;
NOTCH4; ADAM17; NOTCH2; PSEN1; NOTCH3; NOTCH1; DLL4 Endoplasmic
Reticulum HSPA5; MAPK8; XBP1; TRAF2; ATF6; CASP9; ATF4; Stress
Pathway EIF2AK3; CASP3 Pyrimidine Metabolism NME2; AICDA; RRM2;
EIF2AK4; ENTPD1; RRM2B; NT5E; POLD1; NME1 Parkinson's Signaling
UCHL1; MAPK8; MAPK13; MAPK14; CASP9; PARK7; PARK2; CASP3 Cardiac
& Beta GNAS; GNAQ; PPP2R1A; GNB2L1; PPP2CA; PPP1CC; Adrenergic
Signaling PPP2R5C Glycolysis/Gluconeogenesis HK2; GCK; GPI;
ALDH1A1; PKM2; LDHA; HK1 Interferon Signaling IRF1; SOCS1; JAK1;
JAK2; IFITM1; STAT1; IFIT3 Sonic Hedgehog ARRB2; SMO; GLI2; DYRK1A;
Gill; GSK3B; DYRK1B Signaling Glycerophospholipid PLD1; GRN; GPAM;
YWHAZ; SPHK1; SPHK2 Metabolism Phospholipid PRDX6; PLD1; GRN;
YWHAZ; SPHK1; SPHK2 Degradation Tryptophan Metabolism SIAH2; PRMT5;
NEDD4; ALDH1A1; CYP1B1; SIAH1 Lysine Degradation SUV39H1; EHMT2;
NSD1; SETD7; PPP2R5C Nucleotide Excision ERCC5; ERCC4; XPA; XPC;
ERCC1 Repair Pathway Starch and Sucrose UCHL1; HK2; GCK; GPI; HK1
Metabolism Aminosugars Metabolism NQO1; HK2; GCK; HK1 Arachidonic
Acid PRDX6; GRN; YWHAZ; CYP1B1 Metabolism Circadian Rhythm CSNK1E;
CREB1; ATF4; NR1D1 Signaling Coagulation System BDKRB1; F2R;
SERPINE1; F3 Dopamine Receptor PPP2R1A; PPP2CA; PPP1CC; PPP2R5C
Signaling Glutathione Metabolism IDH2; GSTP1; ANPEP; IDH1
Glycerolipid Metabolism ALDH1A1; GPAM; SPHK1; SPHK2 Linoleic Acid
PRDX6; GRN; YWHAZ; CYP1B1 Metabolism Methionine Metabolism DNMT1;
DNMT3B; AHCY; DNMT3A Pyruvate Metabolism GLO1; ALDH1A1; PKM2; LDHA
Arginine and Proline ALDH1A1; NOS3; NOS2A Metabolism Eicosanoid
Signaling PRDX6; GRN; YWHAZ Fructose and Mannose HK2; GCK; HK1
Metabolism Galactose Metabolism HK2; GCK; HK1 Stilbene, Coumarine
and PRDX6; PRDX1; TYR Lignin Biosynthesis Antigen Presentation
CALR; B2M Pathway Biosynthesis of Steroids NQO1; DHCR7 Butanoate
Metabolism ALDH1A1; NLGN1 Citrate Cycle IDH2; IDH1 Fatty Acid
Metabolism ALDH1A1; CYP1B1 Glycerophospholipid PRDX6; CHKA
Metabolism Histidine Metabolism PRMT5; ALDH1A1 Inositol Metabolism
ERO1L; APEX1 Metabolism of GSTP1; CYP1B1 Xenobiotics by Cytochrome
p450 Methane Metabolism PRDX6; PRDX1 Phenylalanine PRDX6; PRDX1
Metabolism Propanoate Metabolism ALDH1A1; LDHA Selenoamino Acid
PRMT5; AHCY Metabolism Sphingolipid Metabolism SPHK1; SPHK2
Aminophosphonate PRMT5 Metabolism Androgen and Estrogen PRMT5
Metabolism Ascorbate and Aldarate ALDH1A1 Metabolism Bile Acid
Biosynthesis ALDH1A1 Cysteine Metabolism LDHA Fatty Acid
Biosynthesis FASN Glutamate Receptor GNB2L1 Signaling NRF2-mediated
PRDX1 Oxidative Stress Response Pentose Phosphate GPI Pathway
Pentose and Glucuronate UCHL1 Interconversions Retinol Metabolism
ALDH1A1 Riboflavin Metabolism TYR Tyrosine Metabolism PRMT5, TYR
Ubiquinone Biosynthesis PRMT5 Valine, Leucine and ALDH1A1
Isoleucine Degradation Glycine, Serine and CHKA Threonine
Metabolism Lysine Degradation ALDH1A1 Pain/Taste TRPM5; TRPA1 Pain
TRPM7; TRPC5; TRPC6; TRPC1; Cnr1; cnr2; Grk2; Trpa1; Pomc; Cgrp;
Crf; Pka; Era; Nr2b; TRPM5; Prkaca; Prkacb; Prkar1a; Prkar2a
Mitochondrial Function AIF; CytC; SMAC (Diablo); Aifm-1; Aifm-2
Developmental BMP-4; Chordin (Chrd); Noggin (Nog); WNT (Wnt2;
Neurology Wnt2b; Wnt3a; Wnt4; Wnt5a; Wnt6; Wnt7b; Wnt8b; Wnt9a;
Wnt9b; Wnt10a; Wnt10b; Wnt16); beta-catenin; Dkk-1; Frizzled
related proteins; Otx-2; Gbx2; FGF-8; Reelin; Dab 1; unc-86 (Pou4f1
or Brn3a); Numb; Reln
[0161] Embodiments of the invention also relate to methods and
compositions related to knocking out genes, amplifying genes and
repairing particular mutations associated with DNA repeat
instability and neurological disorders (Robert D. Wells, Tetsuo
Ashizawa, Genetic Instabilities and Neurological Diseases, Second
Edition, Academic Press, Oct. 13, 2011--Medical). Specific aspects
of tandem repeat sequences have been found to be responsible for
more than twenty human diseases (New insights into repeat
instability: role of RNA.DNA hybrids. McIvor E I, Polak U,
Napierala M. RNA Biol. 2010 September-October; 7(5):551-8). The
CRISPR-Cas system may be harnessed to correct these defects of
genomic instability.
[0162] A further aspect of the invention relates to utilizing the
CRISPR-Cas system for correcting defects in the EMP2A and EMP2B
genes that have been identified to be associated with Lafora
disease. Lafora disease is an autosomal recessive condition which
is characterized by progressive myoclonus epilepsy which may start
as epileptic seizures in adolescence. A few cases of the disease
may be caused by mutations in genes yet to be identified. The
disease causes seizures, muscle spasms, difficulty walking,
dementia, and eventually death. There is currently no therapy that
has proven effective against disease progression. Other genetic
abnormalities associated with epilepsy may also be targeted by the
CRISPR-Cas system and the underlying genetics is further described
in Genetics of Epilepsy and Genetic Epilepsies, edited by Giuliano
Avanzini, Jeffrey L. Noebels, Mariani Foundation Paediatric
Neurology:20; 2009).
[0163] In yet another aspect of the invention, the CRISPR-Cas
system may be used to correct ocular defects that arise from
several genetic mutations further described in Genetic Diseases of
the Eye, Second Edition, edited by Elias I. Traboulsi, Oxford
University Press, 2012.
[0164] Several further aspects of the invention relate to
correcting defects associated with a wide range of genetic diseases
which are further described on the website of the National
Institutes of Health under the topic subsection Genetic Disorders
(website at health.nih.gov/topic/GeneticDisorders). The genetic
brain diseases may include but are not limited to
Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Aicardi
Syndrome, Alpers' Disease, Alzheimer's Disease, Barth Syndrome,
Batten Disease, CADASIL, Cerebellar Degeneration, Fabry's Disease,
Gerstmann-Straussler-Scheinker Disease, Huntington's Disease and
other Triplet Repeat Disorders, Leigh's Disease, Lesch-Nyhan
Syndrome, Menkes Disease, Mitochondrial Myopathies and NINDS
Colpocephaly. These diseases are further described on the website
of the National Institutes of Health under the subsection Genetic
Brain Disorders.
[0165] In some embodiments, the condition may be neoplasia. In some
embodiments, where the condition is neoplasia, the genes to be
targeted are any of those listed in Table A (in this case PTEN and
so forth). In some embodiments, the condition may be Age-related
Macular Degeneration. In some embodiments, the condition may be a
Schizophrenic Disorder. In some embodiments, the condition may be a
Trinucleotide Repeat Disorder. In some embodiments, the condition
may be Fragile X Syndrome. In some embodiments, the condition may
be a Secretase Related Disorder. In some embodiments, the condition
may be a Prion--related disorder. In some embodiments, the
condition may be ALS. In some embodiments, the condition may be a
drug addiction. In some embodiments, the condition may be Autism.
In some embodiments, the condition may be Alzheimer's Disease. In
some embodiments, the condition may be inflammation. In some
embodiments, the condition may be Parkinson's Disease.
[0166] Examples of proteins associated with Parkinson's disease
include but are not limited to .alpha.-synuclein, DJ-1, LRRK2,
PINK1, Parkin, UCHL1, Synphilin-1, and NURR1.
[0167] Examples of addiction-related proteins may include ABAT for
example.
[0168] Examples of inflammation-related proteins may include the
monocyte chemoattractant protein-1 (MCP1) encoded by the Ccr2 gene,
the C-C chemokine receptor type 5 (CCR5) encoded by the Ccr5 gene,
the IgG receptor IIB (FCGR2b, also termed CD32) encoded by the
Fcgr2b gene, or the Fc epsilon R1g (FCER1g) protein encoded by the
Fcer1g gene, for example.
[0169] Examples of cardiovascular diseases associated proteins may
include IL1B (interleukin 1, beta), XDH (xanthine dehydrogenase),
TP53 (tumor protein p53), PTGIS (prostaglandin 12 (prostacyclin)
synthase), MB (myoglobin), IL4 (interleukin 4), ANGPT1
(angiopoietin 1), ABCG8 (ATP-binding cassette, sub-family G
(WHITE), member 8), or CTSK (cathepsin K), for example.
[0170] Examples of Alzheimer's disease associated proteins may
include the very low density lipoprotein receptor protein (VLDLR)
encoded by the VLDLR gene, the ubiquitin-like modifier activating
enzyme 1 (UBA1) encoded by the UBA1 gene, or the NEDD8-activating
enzyme E1 catalytic subunit protein (UBE1C) encoded by the UBA3
gene, for example.
[0171] Examples of proteins associated Autism Spectrum Disorder may
include the benzodiazapine receptor (peripheral) associated protein
1 (BZRAP1) encoded by the BZRAP1 gene, the AF4/FMR2 family member 2
protein (AFF2) encoded by the AFF2 gene (also termed MFR2), the
fragile X mental retardation autosomal homolog 1 protein (FXR1)
encoded by the FXR1 gene, or the fragile X mental retardation
autosomal homolog 2 protein (FXR2) encoded by the FXR2 gene, for
example.
[0172] Examples of proteins associated Macular Degeneration may
include the ATP-binding cassette, sub-family A (ABC1) member 4
protein (ABCA4) encoded by the ABCR gene, the apolipoprotein E
protein (APOE) encoded by the APOE gene, or the chemokine (C-C
motif) Ligand 2 protein (CCL2) encoded by the CCL2 gene, for
example.
[0173] Examples of proteins associated Schizophrenia may include
NRG1, ErbB4, CPLX1, TPH1, TPH2, NRXN1, GSK3A, BDNF, DISC1, GSK3B,
and combinations thereof.
[0174] Examples of proteins involved in tumor suppression may
include ATM (ataxia telangiectasia mutated), ATR (ataxia
telangiectasia and Rad3 related), EGFR (epidermal growth factor
receptor), ERBB2 (v-erb-b2 erythroblastic leukemia viral oncogene
homolog 2), ERBB3 (v-erb-b2 erythroblastic leukemia viral oncogene
homolog 3), ERBB4 (v-erb-b2 erythroblastic leukemia viral oncogene
homolog 4), Notch 1, Notch2, Notch 3, or Notch 4, for example.
[0175] Examples of proteins associated with a secretase disorder
may include PSENEN (presenilin enhancer 2 homolog (C. elegans)),
CTSB (cathepsin B), PSEN1 (presenilin 1), APP (amyloid beta (A4)
precursor protein), APH1B (anterior pharynx defective 1 homolog B
(C. elegans)), PSEN2 (presenilin 2 (Alzheimer disease 4)), or BACE1
(beta-site APP-cleaving enzyme 1), for example.
[0176] Examples of proteins associated with Amyotrophic Lateral
Sclerosis may include SOD1 (superoxide dismutase 1), ALS2
(amyotrophic lateral sclerosis 2), FUS (fused in sarcoma), TARDBP
(TAR DNA binding protein), VAGFA (vascular endothelial growth
factor A), VAGFB (vascular endothelial growth factor B), and VAGFC
(vascular endothelial growth factor C), and any combination
thereof.
[0177] Examples of proteins associated with prion diseases may
include SOD1 (superoxide dismutase 1), ALS2 (amyotrophic lateral
sclerosis 2), FUS (fused in sarcoma), TARDBP (TAR DNA binding
protein), VAGFA (vascular endothelial growth factor A), VAGFB
(vascular endothelial growth factor B), and VAGFC (vascular
endothelial growth factor C), and any combination thereof.
[0178] Examples of proteins related to neurodegenerative conditions
in prion disorders may include A2M (Alpha-2-Macroglobulin), AATF
(Apoptosis antagonizing transcription factor), ACPP (Acid
phosphatase prostate), ACTA2 (Actin alpha 2 smooth muscle aorta),
ADAM22 (ADAM metallopeptidase domain), ADORA3 (Adenosine A3
receptor), or ADRA1D (Alpha-1D adrenergic receptor for Alpha-1D
adrenoreceptor), for example.
[0179] Examples of proteins associated with Immunodeficiency may
include A2M [alpha-2-macroglobulin]; AANAT [arylalkylamine
N-acetyltransferase]; ABCA1 [ATP-binding cassette, sub-family A
(ABC1), member 1]; ABCA2 [ATP-binding cassette, sub-family A
(ABC1), member 2]; or ABCA3 [ATP-binding cassette, sub-family A
(ABC1), member 3]; for example.
[0180] Examples of proteins associated with Trinucleotide Repeat
Disorders include AR (androgen receptor), FMR1 (fragile X mental
retardation 1), HTT (huntingtin), or DMPK (dystrophia
myotonica-protein kinase), FXN (frataxin), ATXN2 (ataxin 2), for
example.
[0181] Examples of proteins associated with Neurotransmission
Disorders include SST (somatostatin), NOS1 (nitric oxide synthase 1
(neuronal)), ADRA2A (adrenergic, alpha-2A-, receptor), ADRA2C
(adrenergic, alpha-2C-, receptor), TACR1 (tachykinin receptor 1),
or HTR2c (5-hydroxytryptamine (serotonin) receptor 2C), for
example.
[0182] Examples of neurodevelopmental-associated sequences include
A2BP1 [ataxin 2-binding protein 1], AADAT [aminoadipate
aminotransferase], AANAT [arylalkylamine N-acetyltransferase], ABAT
[4-aminobutyrate aminotransferase], ABCA1 [ATP-binding cassette,
sub-family A (ABC1), member 1], or ABCA13 [ATP-binding cassette,
sub-family A (ABC1), member 13], for example.
[0183] Further examples of preferred conditions treatable with the
present system include may be selected from: Aicardi-Goutieres
Syndrome; Alexander Disease; Allan-Herndon-Dudley Syndrome;
POLG-Related Disorders; Alpha-Mannosidosis (Type II and III);
Alstrom Syndrome; Angelman; Syndrome; Ataxia-Telangiectasia;
Neuronal Ceroid-Lipofuscinoses; Beta-Thalassemia; Bilateral Optic
Atrophy and (Infantile) Optic Atrophy Type 1; Retinoblastoma
(bilateral); Canavan Disease; Cerebrooculofacioskeletal Syndrome 1
[COFS1]; Cerebrotendinous Xanthomatosis; Cornelia de Lange
Syndrome; MAPT-Related Disorders; Genetic Prion Diseases; Dravet
Syndrome; Early-Onset Familial Alzheimer Disease; Friedreich Ataxia
[FRDA]; Fryns Syndrome; Fucosidosis; Fukuyama Congenital Muscular
Dystrophy; Galactosialidosis; Gaucher Disease; Organic Acidemias;
Hemophagocytic Lymphohistiocytosis; Hutchinson-Gilford Progeria
Syndrome; Mucolipidosis II; Infantile Free Sialic Acid Storage
Disease; PLA2G6-Associated Neurodegeneration; Jervell and
Lange-Nielsen Syndrome; Junctional Epidermolysis Bullosa;
Huntington Disease; Krabbe Disease (Infantile); Mitochondrial
DNA-Associated Leigh Syndrome and NARP; Lesch-Nyhan Syndrome;
LIS1-Associated Lissencephaly; Lowe Syndrome; Maple Syrup Urine
Disease; MECP2 Duplication Syndrome; ATP7A-Related Copper Transport
Disorders; LAMA2-Related Muscular Dystrophy; Arylsulfatase A
Deficiency; Mucopolysaccharidosis Types I, II or III; Peroxisome
Biogenesis Disorders, Zellweger Syndrome Spectrum;
Neurodegeneration with Brain Iron Accumulation Disorders; Acid
Sphingomyelinase Deficiency; Niemann-Pick Disease Type C; Glycine
Encephalopathy; ARX-Related Disorders; Urea Cycle Disorders;
COL1A1/2-Related Osteogenesis Imperfecta; Mitochondrial DNA
Deletion Syndromes; PLP1-Related Disorders; Perry Syndrome;
Phelan-McDermid Syndrome; Glycogen Storage Disease Type II (Pompe
Disease) (Infantile); MAPT-Related Disorders; MECP2-Related
Disorders; Rhizomelic Chondrodysplasia Punctata Type 1; Roberts
Syndrome; Sandhoff Disease; Schindler Disease--Type 1; Adenosine
Deaminase Deficiency; Smith-Lemli-Opitz Syndrome; Spinal Muscular
Atrophy; Infantile-Onset Spinocerebellar Ataxia; Hexosaminidase A
Deficiency; Thanatophoric Dysplasia Type 1; Collagen Type
VI-Related Disorders; Usher Syndrome Type I; Congenital Muscular
Dystrophy; Wolf-Hirschhorn Syndrome; Lysosomal Acid Lipase
Deficiency; and Xeroderma Pigmentosum.
[0184] As will be apparent, it is envisaged that the present system
can be used to target any polynucleotide sequence of interest. Some
examples of conditions or diseases that might be usefully treated
using the present system are included in the Tables above and
examples of genes currently associated with those conditions are
also provided there. However, the genes exemplified are not
exhaustive.
EXAMPLES
[0185] The following examples are given for the purpose of
illustrating various embodiments of the invention and are not meant
to limit the present invention in any fashion. The present
examples, along with the methods described herein are presently
representative of preferred embodiments, are exemplary, and are not
intended as limitations on the scope of the invention. Changes
therein and other uses which are encompassed within the spirit of
the invention as defined by the scope of the claims will occur to
those skilled in the art.
Example 1: CRISPR Complex Activity in the Nucleus of a Eukaryotic
Cell
[0186] An example type II CRISPR system is the type II CRISPR locus
from Streptococcus pyogenes SF370, which contains a cluster of four
genes Cas9, Cas1, Cas2, and Csn1, as well as two non-coding RNA
elements, tracrRNA and a characteristic array of repetitive
sequences (direct repeats) interspaced by short stretches of
non-repetitive sequences (spacers, about 30 bp each). In this
system, targeted DNA double-strand break (DSB) is generated in four
sequential steps (FIG. 2A). First, two non-coding RNAs, the
pre-crRNA array and tracrRNA, are transcribed from the CRISPR
locus. Second, tracrRNA hybridizes to the direct repeats of
pre-crRNA, which is then processed into mature crRNAs containing
individual spacer sequences. Third, the mature crRNA:tracrRNA
complex directs Cas9 to the DNA target consisting of the
protospacer and the corresponding PAM via heteroduplex formation
between the spacer region of the crRNA and the protospacer DNA.
Finally, Cas9 mediates cleavage of target DNA upstream of PAM to
create a DSB within the protospacer (FIG. 2A). This example
describes an example process for adapting this RNA-programmable
nuclease system to direct CRISPR complex activity in the nuclei of
eukaryotic cells.
[0187] Cell Culture and Transfection
[0188] Human embryonic kidney (HEK) cell line HEK 293FT (Life
Technologies) was maintained in Dulbecco's modified Eagle's Medium
(DMEM) supplemented with 10% fetal bovine serum (HyClone), 2 mM
GlutaMAX (Life Technologies), 100 U/mL penicillin, and 100 .mu.g/mL
streptomycin at 37.degree. C. with 5% CO.sub.2 incubation. Mouse
neuro2A (N2A) cell line (ATCC) was maintained with DMEM
supplemented with 5% fetal bovine serum (HyClone), 2 mM GlutaMAX
(Life Technologies), 100 U/mL penicillin, and 100 .mu.g/mL
streptomycin at 37.degree. C. with 5% CO.sub.2.
[0189] HEK 293FT or N2A cells were seeded into 24-well plates
(Corning) one day prior to transfection at a density of 200,000
cells per well. Cells were transfected using Lipofectamine 2000
(Life Technologies) following the manufacturer's recommended
protocol. For each well of a 24-well plate a total of 800 ng of
plasmids were used.
[0190] Surveyor Assay and Sequencing Analysis for Genome
Modification
[0191] HEK 293FT or N2A cells were transfected with plasmid DNA as
described above. After transfection, the cells were incubated at
37.degree. C. for 72 hours before genomic DNA extraction. Genomic
DNA was extracted using the QuickExtract DNA extraction kit
(Epicentre) following the manufacturer's protocol. Briefly, cells
were resuspended in QuickExtract solution and incubated at
65.degree. C. for 15 minutes and 98.degree. C. for 10 minutes.
Extracted genomic DNA was immediately processed or stored at
-20.degree. C.
[0192] The genomic region surrounding a CRISPR target site for each
gene was PCR amplified, and products were purified using QiaQuick
Spin Column (Qiagen) following manufacturer's protocol. A total of
400 ng of the purified PCR products were mixed with 2 .mu.l
10.times. Taq polymerase PCR buffer (Enzymatics) and ultrapure
water to a final volume of 20 .mu.l, and subjected to a
re-annealing process to enable heteroduplex formation: 95.degree.
C. for 10 min, 95.degree. C. to 85.degree. C. ramping at -2.degree.
C./s, 85.degree. C. to 25.degree. C. at -0.25.degree. C./s, and
25.degree. C. hold for 1 minute. After re-annealing, products were
treated with Surveyor nuclease and Surveyor enhancer S
(Transgenomics) following the manufacturer's recommended protocol,
and analyzed on 4-20% Novex TBE poly-acrylamide gels (Life
Technologies). Gels were stained with SYBR Gold DNA stain (Life
Technologies) for 30 minutes and imaged with a Gel Doc gel imaging
system (Bio-rad). Quantification was based on relative band
intensities, as a measure of the fraction of cleaved DNA. FIG. 8
provides a schematic illustration of this Surveyor assay.
[0193] Restriction Fragment Length Polymorphism Assay for Detection
of Homologous Recombination
[0194] HEK 293FT and N2A cells were transfected with plasmid DNA,
and incubated at 37.degree. C. for 72 hours before genomic DNA
extraction as described above. The target genomic region was PCR
amplified using primers outside the homology arms of the homologous
recombination (HR) template. PCR products were separated on a 1%
agarose gel and extracted with MinElute GelExtraction Kit (Qiagen).
Purified products were digested with HindIII (Fermentas) and
analyzed on a 6% Novex TBE poly-acrylamide gel (Life
Technologies).
[0195] RNA Secondary Structure Prediction and Analysis
[0196] RNA secondary structure prediction was performed using the
online webserver RNAfold developed at Institute for Theoretical
Chemistry at the University of Vienna, using the centroid structure
prediction algorithm (see e.g. A. R. Gruber et al., 2008, Cell
106(1): 23-24; and PA Carr and GM Church, 2009, Nature
Biotechnology 27(12): 1151-62).
[0197] Bacterial Plasmid Transformation Interference Assay
[0198] Elements of the S. pyogenes CRISPR locus 1 sufficient for
CRISPR activity were reconstituted in E. coli using pCRISPR plasmid
(schematically illustrated in FIG. 10A). pCRISPR contained
tracrRNA, SpCas9, and a leader sequence driving the crRNA array.
Spacers (also referred to as "guide sequences") were inserted into
the crRNA array between BsaI sites using annealed oligonucleotides,
as illustrated. Challenge plasmids used in the interference assay
were constructed by inserting the protospacer (also referred to as
a "target sequence") sequence along with an adjacent CRISPR motif
sequence (PAM) into pUC19 (see FIG. 10B). The challenge plasmid
contained ampicillin resistance. FIG. 10C provides a schematic
representation of the interference assay. Chemically competent E.
coli strains already carrying pCRISPR and the appropriate spacer
were transformed with the challenge plasmid containing the
corresponding protospacer-PAM sequence. pUC19 was used to assess
the transformation efficiency of each pCRISPR-carrying competent
strain. CRISPR activity resulted in cleavage of the pPSP plasmid
carrying the protospacer, precluding ampicillin resistance
otherwise conferred by pUC19 lacking the protospacer. FIG. 10D
illustrates competence of each pCRISPR-carrying E. coli strain used
in assays illustrated in FIG. 4C.
[0199] RNA Purification
[0200] HEK 293FT cells were maintained and transfected as stated
above. Cells were harvested by trypsinization followed by washing
in phosphate buffered saline (PBS). Total cell RNA was extracted
with TRI reagent (Sigma) following manufacturer's protocol.
Extracted total RNA was quantified using Naonodrop (Thermo
Scientific) and normalized to same concentration.
[0201] Northern Blot Analysis of crRNA and tracrRNA Expression in
Mammalian Cells
[0202] RNAs were mixed with equal volumes of 2.times. loading
buffer (Ambion), heated to 95.degree. C. for 5 min, chilled on ice
for 1 min, and then loaded onto 8% denaturing polyacrylamide gels
(SequaGel, National Diagnostics) after pre-running the gel for at
least 30 minutes. The samples were electrophoresed for 1.5 hours at
40W limit. Afterwards, the RNA was transferred to Hybond N+
membrane (GE Healthcare) at 300 mA in a semi-dry transfer apparatus
(Bio-rad) at room temperature for 1.5 hours. The RNA was
crosslinked to the membrane using autocrosslink button on
Stratagene UV Crosslinker the Stratalinker (Stratagene). The
membrane was pre-hybridized in ULTRAhyb-Oligo Hybridization Buffer
(Ambion) for 30 min with rotation at 42.degree. C., and probes were
then added and hybridized overnight. Probes were ordered from IDT
and labeled with [gamma-.sup.32P] ATP (Perkin Elmer) with T4
polynucleotide kinase (New England Biolabs). The membrane was
washed once with pre-warmed (42.degree. C.) 2.times.SSC, 0.5% SDS
for 1 min followed by two 30 minute washes at 42.degree. C. The
membrane was exposed to a phosphor screen for one hour or overnight
at room temperature and then scanned with a phosphorimager
(Typhoon).
[0203] Bacterial CRISPR System Construction and Evaluation
[0204] CRISPR locus elements, including tracrRNA, Cas9, and leader
were PCR amplified from Streptococcus pyogenes SF370 genomic DNA
with flanking homology arms for Gibson Assembly. Two BsaI type IIS
sites were introduced in between two direct repeats to facilitate
easy insertion of spacers (FIG. 9). PCR products were cloned into
EcoRV-digested pACYC184 downstream of the tet promoter using Gibson
Assembly Master Mix (NEB). Other endogenous CRISPR system elements
were omitted, with the exception of the last 50 bp of Csn2. Oligos
(Integrated DNA Technology) encoding spacers with complimentary
overhangs were cloned into the BsaI-digested vector pDC000 (NEB)
and then ligated with T7 ligase (Enzymatics) to generate pCRISPR
plasmids. Challenge plasmids containing spacers with PAM sequences
(also referred to herein as "CRISPR motif sequences") were created
by ligating hybridized oligos carrying compatible overhangs
(Integrated DNA Technology) into BamHI-digested pUC19. Cloning for
all constructs was performed in E. coli strain JM109 (Zymo
Research).
[0205] pCRISPR-carrying cells were made competent using the
Z-Competent E. coli Transformation Kit and Buffer Set (Zymo
Research, T3001) according to manufacturer's instructions. In the
transformation assay, 50 uL aliquots of competent cells carrying
pCRISPR were thawed on ice and transformed with 1 ng of spacer
plasmid or pUC19 on ice for 30 minutes, followed by 45 second heat
shock at 42.degree. C. and 2 minutes on ice. Subsequently, 250 ul
SOC (Invitrogen) was added followed by shaking incubation at
37.degree. C. for 1 hr, and 100 uL of the post-SOC outgrowth was
plated onto double selection plates (12.5 ug/ml chloramphenicol,
100 ug/ml ampicillin). To obtain cfu/ng of DNA, total colony
numbers were multiplied by 3.
[0206] To improve expression of CRISPR components in mammalian
cells, two genes from the SF370 locus 1 of Streptococcus pyogenes
(S. pyogenes) were codon-optimized, Cas9 (SpCas9) and RNase III
(SpRNase III). To facilitate nuclear localization, a nuclear
localization signal (NLS) was included at the amino (N)- or
carboxyl (C)-termini of both SpCas9 and SpRNase III (FIG. 2B). To
facilitate visualization of protein expression, a fluorescent
protein marker was also included at the N- or C-termini of both
proteins (FIG. 2B). A version of SpCas9 with an NLS attached to
both N- and C-termini (2.times.NLS-SpCas9) was also generated.
Constructs containing NLS-fused SpCas9 and SpRNase III were
transfected into 293FT human embryonic kidney (HEK) cells, and the
relative positioning of the NLS to SpCas9 and SpRNase III was found
to affect their nuclear localization efficiency. Whereas the
C-terminal NLS was sufficient to target SpRNase III to the nucleus,
attachment of a single copy of these particular NLS's to either the
N- or C-terminus of SpCas9 was unable to achieve adequate nuclear
localization in this system. In this example, the C-terminal NLS
was that of nucleoplasmin (KRPAATKKAGQAKKKK (SEQ ID NO: 2)), and
the C-terminal NLS was that of the SV40 large T-antigen (PKKKRKV
(SEQ ID NO: 1)). Of the versions of SpCas9 tested, only
2.times.NLS-SpCas9 exhibited nuclear localization (FIG. 2B).
[0207] The tracrRNA from the CRISPR locus of S. pyogenes SF370 has
two transcriptional start sites, giving rise to two transcripts of
89-nucleotides (nt) and 171 nt that are subsequently processed into
identical 75 nt mature tracrRNAs. The shorter 89 nt tracrRNA was
selected for expression in mammalian cells (expression constructs
illustrated in FIG. 7A, with functionality as determined by results
of the Surveyor assay shown in FIG. 7B). Transcription start sites
are marked as +1, and transcription terminator and the sequence
probed by northern blot are also indicated. Expression of processed
tracrRNA was also confirmed by Northern blot. FIG. 7C shows results
of a Northern blot analysis of total RNA extracted from 293FT cells
transfected with U6 expression constructs carrying long or short
tracrRNA, as well as SpCas9 and DR-EMX1(1)-DR. Left and right
panels are from 293FT cells transfected without or with SpRNase
III, respectively. U6 indicate loading control blotted with a probe
targeting human U6 snRNA. Transfection of the short tracrRNA
expression construct led to abundant levels of the processed form
of tracrRNA (.about.75 bp). Very low amounts of long tracrRNA are
detected on the Northern blot.
[0208] To promote precise transcriptional initiation, the RNA
polymerase III-based U6 promoter was selected to drive the
expression of tracrRNA (FIG. 2C). Similarly, a U6 promoter-based
construct was developed to express a pre-crRNA array consisting of
a single spacer flanked by two direct repeats (DRs, also
encompassed by the term "tracr-mate sequences"; FIG. 2C). The
initial spacer was designed to target a 33-base-pair (bp) target
site (30-bp protospacer plus a 3-bp CRISPR motif (PAM) sequence
satisfying the NGG recognition motif of Cas9) in the human EMX1
locus (FIG. 2C), a key gene in the development of the cerebral
cortex.
[0209] To test whether heterologous expression of the CRISPR system
(SpCas9, SpRNase III, tracrRNA, and pre-crRNA) in mammalian cells
can achieve targeted cleavage of mammalian chromosomes, HEK 293FT
cells were transfected with combinations of CRISPR components.
Since DSBs in mammalian nuclei are partially repaired by the
non-homologous end joining (NHEJ) pathway, which leads to the
formation of indels, the Surveyor assay was used to detect
potential cleavage activity at the target EMX1 locus (FIG. 8) (see
e.g. Guschin et al., 2010, Methods Mol Biol 649: 247).
Co-transfection of all four CRISPR components was able to induce up
to 5.0% cleavage in the protospacer (see FIG. 2D). Co-transfection
of all CRISPR components minus SpRNase III also induced up to 4.7%
indel in the protospacer, suggesting that there may be endogenous
mammalian RNases that are capable of assisting with crRNA
maturation, such as for example the related Dicer and Drosha
enzymes. Removing any of the remaining three components abolished
the genome cleavage activity of the CRISPR system (FIG. 2D). Sanger
sequencing of amplicons containing the target locus verified the
cleavage activity: in 43 sequenced clones, 5 mutated alleles
(11.6%) were found. Similar experiments using a variety of guide
sequences produced indel percentages as high as 29% (see FIGS. 4-7,
12, and 13). These results define a three-component system for
efficient CRISPR-mediated genome modification in mammalian cells.
To optimize the cleavage efficiency, Applicants also tested whether
different isoforms of tracrRNA affected the cleavage efficiency and
found that, in this example system, only the short (89-bp)
transcript form was able to mediate cleavage of the human EMX1
genomic locus (FIG. 7B).
[0210] FIG. 14 provides an additional Northern blot analysis of
crRNA processing in mammalian cells. FIG. 14A illustrates a
schematic showing the expression vector for a single spacer flanked
by two direct repeats (DR-EMX1(1)-DR). The 30 bp spacer targeting
the human EMX1 locus protospacer 1 (see FIG. 6) and the direct
repeat sequences are shown in the sequence beneath FIG. 14A. The
line indicates the region whose reverse-complement sequence was
used to generate Northern blot probes for EMX1(1) crRNA detection.
FIG. 14B shows a Northern blot analysis of total RNA extracted from
293FT cells transfected with U6 expression constructs carrying
DR-EMX1(1)-DR. Left and right panels are from 293FT cells
transfected without or with SpRNase III respectively. DR-EMX1(1)-DR
was processed into mature crRNAs only in the presence of SpCas9 and
short tracrRNA and was not dependent on the presence of SpRNase
III. The mature crRNA detected from transfected 293FT total RNA is
.about.33 bp and is shorter than the 39-42 bp mature crRNA from S.
pyogenes. These results demonstrate that a CRISPR system can be
transplanted into eukaryotic cells and reprogrammed to facilitate
cleavage of endogenous mammalian target polynucleotides.
[0211] FIG. 2 illustrates the bacterial CRISPR system described in
this example. FIG. 2A illustrates a schematic showing the CRISPR
locus 1 from Streptococcus pyogenes SF370 and a proposed mechanism
of CRISPR-mediated DNA cleavage by this system. Mature crRNA
processed from the direct repeat-spacer array directs Cas9 to
genomic targets consisting of complimentary protospacers and a
protospacer-adjacent motif (PAM). Upon target-spacer base pairing,
Cas9 mediates a double-strand break in the target DNA. FIG. 2B
illustrates engineering of S. pyogenes Cas9 (SpCas9) and RNase III
(SpRNase III) with nuclear localization signals (NLSs) to enable
import into the mammalian nucleus. FIG. 2C illustrates mammalian
expression of SpCas9 and SpRNase III driven by the constitutive
EF1a promoter and tracrRNA and pre-crRNA array (DR-Spacer-DR)
driven by the RNA Pol3 promoter U6 to promote precise transcription
initiation and termination. A protospacer from the human EMX1 locus
with a satisfactory PAM sequence is used as the spacer in the
pre-crRNA array. FIG. 2D illustrates surveyor nuclease assay for
SpCas9-mediated minor insertions and deletions. SpCas9 was
expressed with and without SpRNase III, tracrRNA, and a pre-crRNA
array carrying the EMX1-target spacer. FIG. 2E illustrates a
schematic representation of base pairing between target locus and
EMX1-targeting crRNA, as well as an example chromatogram showing a
micro deletion adjacent to the SpCas9 cleavage site. FIG. 2F
illustrates mutated alleles identified from sequencing analysis of
43 clonal amplicons showing a variety of micro insertions and
deletions. Dashes indicate deleted bases, and non-aligned or
mismatched bases indicate insertions or mutations. Scale bar=10
.mu.m.
[0212] To further simplify the three-component system, a chimeric
crRNA-tracrRNA hybrid design was adapted, where a mature crRNA
(comprising a guide sequence) is fused to a partial tracrRNA via a
stem-loop to mimic the natural crRNA:tracrRNA duplex (FIG. 3A). To
increase co-delivery efficiency, a bicistronic expression vector
was created to drive co-expression of a chimeric RNA and SpCas9 in
transfected cells (FIGS. 3A and 8). In parallel, the bicistronic
vectors were used to express a pre-crRNA (DR-guide sequence-DR)
with SpCas9, to induce processing into crRNA with a separately
expressed tracrRNA (compare FIG. 13B top and bottom). FIG. 9
provides schematic illustrations of bicistronic expression vectors
for pre-crRNA array (FIG. 9A) or chimeric crRNA (represented by the
short line downstream of the guide sequence insertion site and
upstream of the EF1.alpha. promoter in FIG. 9B) with hSpCas9,
showing location of various elements and the point of guide
sequence insertion. The expanded sequence around the location of
the guide sequence insertion site in FIG. 9B also shows a partial
DR sequence (GTTTTAGAGCTA (SEQ ID NO: 27)) and a partial tracrRNA
sequence (TAGCAAGTTAAAATAAGGCTAGTCCGTTTTT (SEQ ID NO: 28)). Guide
sequences can be inserted between BbsI sites using annealed
oligonucleotides. Sequence design for the oligonucleotides are
shown below the schematic illustrations in FIG. 9, with appropriate
ligation adapters indicated. WPRE represents the Woodchuck
hepatitis virus post-transcriptional regulatory element. The
efficiency of chimeric RNA-mediated cleavage was tested by
targeting the same EMX1 locus described above. Using both Surveyor
assay and Sanger sequencing of amplicons, Applicants confirmed that
the chimeric RNA design facilitates cleavage of human EMX1 locus
with approximately a 4.7% modification rate (FIG. 4).
[0213] Generalizability of CRISPR-mediated cleavage in eukaryotic
cells was tested by targeting additional genomic loci in both human
and mouse cells by designing chimeric RNA targeting multiple sites
in the human EMX1 and PVALB, as well as the mouse Th loci. FIG. 15
illustrates the selection of some additional targeted protospacers
in human PVALB (FIG. 15A) and mouse Th (FIG. 15B) loci. Schematics
of the gene loci and the location of three protospacers within the
last exon of each are provided. The underlined sequences include 30
bp of protospacer sequence and 3 bp at the 3' end corresponding to
the PAM sequences. Protospacers on the sense and anti-sense strands
are indicated above and below the DNA sequences, respectively. A
modification rate of 6.3% and 0.75% was achieved for the human
PVALB and mouse Th loci respectively, demonstrating the broad
applicability of the CRISPR system in modifying different loci
across multiple organisms (FIGS. 3B and 6). While cleavage was only
detected with one out of three spacers for each locus using the
chimeric constructs, all target sequences were cleaved with
efficiency of indel production reaching 27% when using the
co-expressed pre-crRNA arrangement (FIG. 6).
[0214] FIG. 13 provides a further illustration that SpCas9 can be
reprogrammed to target multiple genomic loci in mammalian cells.
FIG. 13A provides a schematic of the human EMX1 locus showing the
location of five protospacers, indicated by the underlined
sequences. FIG. 13B provides a schematic of the pre-crRNA/trcrRNA
complex showing hybridization between the direct repeat region of
the pre-crRNA and tracrRNA (top), and a schematic of a chimeric RNA
design comprising a 20 bp guide sequence, and tracr mate and tracr
sequences consisting of partial direct repeat and tracrRNA
sequences hybridized in a hairpin structure (bottom). Results of a
Surveyor assay comparing the efficacy of Cas9-mediated cleavage at
five protospacers in the human EMX1 locus is illustrated in FIG.
13C. Each protospacer is targeted using either processed
pre-crRNA/tracrRNA complex (crRNA) or chimeric RNA (chiRNA).
[0215] Since the secondary structure of RNA can be crucial for
intermolecular interactions, a structure prediction algorithm based
on minimum free energy and Boltzmann-weighted structure ensemble
was used to compare the putative secondary structure of all guide
sequences used in our genome targeting experiment (FIG. 3B) (see
e.g. Gruber et al., 2008, Nucleic Acids Research, 36: W70).
Analysis revealed that in most cases, the effective guide sequences
in the chimeric crRNA context were substantially free of secondary
structure motifs, whereas the ineffective guide sequences were more
likely to form internal secondary structures that could prevent
base pairing with the target protospacer DNA. It is thus possible
that variability in the spacer secondary structure might impact the
efficiency of CRISPR-mediated interference when using a chimeric
crRNA.
[0216] FIG. 3 illustrates example expression vectors. FIG. 3A
provides a schematic of a bi-cistronic vector for driving the
expression of a synthetic crRNA-tracrRNA chimera (chimeric RNA) as
well as SpCas9. The chimeric guide RNA contains a 20-bp guide
sequence corresponding to the protospacer in the genomic target
site. FIG. 3B provides a schematic showing guide sequences
targeting the human EMX1, PVALB, and mouse Th loci, as well as
their predicted secondary structures. The modification efficiency
at each target site is indicated below the RNA secondary structure
drawing (EMX1, n=216 amplicon sequencing reads; PVALB, n=224 reads;
Th, n=265 reads). The folding algorithm produced an output with
each base colored according to its probability of assuming the
predicted secondary structure, as indicated by a rainbow scale that
is reproduced in FIG. 3B in gray scale. Further vector designs for
SpCas9 are shown in FIG. 44, which illustrates single expression
vectors incorporating a U6 promoter linked to an insertion site for
a guide oligo, and a Cbh promoter linked to SpCas9 coding sequence.
The vector shown in FIG. 44b includes a tracrRNA coding sequence
linked to an H1 promoter.
[0217] To test whether spacers containing secondary structures are
able to function in prokaryotic cells where CRISPRs naturally
operate, transformation interference of protospacer-bearing
plasmids were tested in an E. coli strain heterologously expressing
the S. pyogenes SF370 CRISPR locus 1 (FIG. 10). The CRISPR locus
was cloned into a low-copy E. coli expression vector and the crRNA
array was replaced with a single spacer flanked by a pair of DRs
(pCRISPR). E. coli strains harboring different pCRISPR plasmids
were transformed with challenge plasmids containing the
corresponding protospacer and PAM sequences (FIG. 10C). In the
bacterial assay, all spacers facilitated efficient CRISPR
interference (FIG. 4C). These results suggest that there may be
additional factors affecting the efficiency of CRISPR activity in
mammalian cells.
[0218] To investigate the specificity of CRISPR-mediated cleavage,
the effect of single-nucleotide mutations in the guide sequence on
protospacer cleavage in the mammalian genome was analyzed using a
series of EMX1-targeting chimeric crRNAs with single point
mutations (FIG. 4A). FIG. 4B illustrates results of a Surveyor
nuclease assay comparing the cleavage efficiency of Cas9 when
paired with different mutant chimeric RNAs. Single-base mismatch up
to 12-bp 5' of the PAM substantially abrogated genomic cleavage by
SpCas9, whereas spacers with mutations at farther upstream
positions retained activity against the original protospacer target
(FIG. 4B). In addition to the PAM, SpCas9 has single-base
specificity within the last 12-bp of the spacer. Furthermore,
CRISPR is able to mediate genomic cleavage as efficiently as a pair
of TALE nucleases (TALEN) targeting the same EMX1 protospacer. FIG.
4C provides a schematic showing the design of TALENs targeting
EMX1, and FIG. 4D shows a Surveyor gel comparing the efficiency of
TALEN and Cas9 (n=3).
[0219] Having established a set of components for achieving
CRISPR-mediated gene editing in mammalian cells through the
error-prone NHEJ mechanism, the ability of CRISPR to stimulate
homologous recombination (HR), a high fidelity gene repair pathway
for making precise edits in the genome, was tested. The wild type
SpCas9 is able to mediate site-specific DSBs, which can be repaired
through both NHEJ and HR. In addition, an aspartate-to-alanine
substitution (D10A) in the RuvC I catalytic domain of SpCas9 was
engineered to convert the nuclease into a nickase (SpCas9n;
illustrated in FIG. 5A) (see e.g. Sapranauskas et al., 2011,
Nucleic Acids Research, 39: 9275; Gasiunas et al., 2012, Proc.
Natl. Acad. Sci. USA, 109:E2579), such that nicked genomic DNA
undergoes the high-fidelity homology-directed repair (HDR).
Surveyor assay confirmed that SpCas9n does not generate indels at
the EMX1 protospacer target. As illustrated in FIG. 5B,
co-expression of EMX1-targeting chimeric crRNA with SpCas9 produced
indels in the target site, whereas co-expression with SpCas9n did
not (n=3). Moreover, sequencing of 327 amplicons did not detect any
indels induced by SpCas9n. The same locus was selected to test
CRISPR-mediated HR by co-transfecting HEK 293FT cells with the
chimeric RNA targeting EMX1, hSpCas9 or hSpCas9n, as well as a HR
template to introduce a pair of restriction sites (HindIII and
NheI) near the protospacer. FIG. 5C provides a schematic
illustration of the HR strategy, with relative locations of
recombination points and primer annealing sequences (arrows).
SpCas9 and SpCas9n indeed catalyzed integration of the HR template
into the EMX1 locus. PCR amplification of the target region
followed by restriction digest with HindIII revealed cleavage
products corresponding to expected fragment sizes (arrows in
restriction fragment length polymorphism gel analysis shown in FIG.
5D), with SpCas9 and SpCas9n mediating similar levels of HR
efficiencies. Applicants further verified HR using Sanger
sequencing of genomic amplicons (FIG. 5E). These results
demonstrate the utility of CRISPR for facilitating targeted gene
insertion in the mammalian genome. Given the 14-bp (12-bp from the
spacer and 2-bp from the PAM) target specificity of the wild type
SpCas9, the availability of a nickase can significantly reduce the
likelihood of off-target modifications, since single strand breaks
are not substrates for the error-prone NHEJ pathway.
[0220] Expression constructs mimicking the natural architecture of
CRISPR loci with arrayed spacers (FIG. 2A) were constructed to test
the possibility of multiplexed sequence targeting. Using a single
CRISPR array encoding a pair of EMX1- and PVALB-targeting spacers,
efficient cleavage at both loci was detected (FIG. 4F, showing both
a schematic design of the crRNA array and a Surveyor blot showing
efficient mediation of cleavage). Targeted deletion of larger
genomic regions through concurrent DSBs using spacers against two
targets within EMX1 spaced by 119 bp was also tested, and a 1.6%
deletion efficacy (3 out of 182 amplicons; FIG. 4G) was detected.
This demonstrates that the CRISPR system can mediate multiplexed
editing within a single genome.
Example 2: CRISPR System Modifications and Alternatives
[0221] The ability to use RNA to program sequence-specific DNA
cleavage defines a new class of genome engineering tools for a
variety of research and industrial applications. Several aspects of
the CRISPR system can be further improved to increase the
efficiency and versatility of CRISPR targeting. Optimal Cas9
activity may depend on the availability of free Mg.sup.2+ at levels
higher than that present in the mammalian nucleus (see e.g. Jinek
et al., 2012, Science, 337:816), and the preference for an NGG
motif immediately downstream of the protospacer restricts the
ability to target on average every 12-bp in the human genome (FIG.
11, evaluating both plus and minus strands of human chromosomal
sequences). Some of these constraints can be overcome by exploring
the diversity of CRISPR loci across the microbial metagenome (see
e.g. Makarova et al., 2011, Nat Rev Microbiol, 9:467). Other CRISPR
loci may be transplanted into the mammalian cellular milieu by a
process similar to that described in Example 1. For example, FIG.
12 illustrates adaptation of the Type II CRISPR system from CRISPR
1 of Streptococcus thermophilus LMD-9 for heterologous expression
in mammalian cells to achieve CRISPR-mediated genome editing. FIG.
12A provides a Schematic illustration of CRISPR 1 from S.
thermophilus LMD-9. FIG. 12B illustrates the design of an
expression system for the S. thermophilus CRISPR system. Human
codon-optimized hStCas9 is expressed using a constitutive
EF1.alpha. promoter. Mature versions of tracrRNA and crRNA are
expressed using the U6 promoter to promote precise transcription
initiation. Sequences from the mature crRNA and tracrRNA are
illustrated. A single base indicated by the lower case "a" in the
crRNA sequence is used to remove the polyU sequence, which serves
as a RNA polIII transcriptional terminator. FIG. 12C provides a
schematic showing guide sequences targeting the human EMX1 locus as
well as their predicted secondary structures. The modification
efficiency at each target site is indicated below the RNA secondary
structures. The algorithm generating the structures colors each
base according to its probability of assuming the predicted
secondary structure, which is indicated by a rainbow scale
reproduced in FIG. 12C in gray scale. FIG. 12D shows the results of
hStCas9-mediated cleavage in the target locus using the Surveyor
assay. RNA guide spacers 1 and 2 induced 14% and 6.4%,
respectively. Statistical analysis of cleavage activity across
biological replica at these two protospacer sites is also provided
in FIG. 6. FIG. 16 provides a schematic of additional protospacer
and corresponding PAM sequence targets of the S. thermophilus
CRISPR system in the human EMX1 locus. Two protospacer sequences
are highlighted and their corresponding PAM sequences satisfying
NNAGAAW motif are indicated by underlining 3' with respect to the
corresponding highlighted sequence. Both protospacers target the
anti-sense strand.
Example 3: Sample Target Sequence Selection Algorithm
[0222] A software program is designed to identify candidate CRISPR
target sequences on both strands of an input DNA sequence based on
desired guide sequence length and a CRISPR motif sequence (PAM) for
a specified CRISPR enzyme. For example, target sites for Cas9 from
S. pyogenes, with PAM sequences NGG, may be identified by searching
for 5'-N.sub.x-NGG-3' both on the input sequence and on the
reverse-complement of the input. Likewise, target sites for Cas9 of
S. thermophilus CRISPR1, with PAM sequence NNAGAAW, may be
identified by searching for 5'-N.sub.x-NNAGAAW-3' (SEQ ID NO: 29)
both on the input sequence and on the reverse-complement of the
input. Likewise, target sites for Cas9 of S. thermophilus CRISPR3,
with PAM sequence NGGNG, may be identified by searching for
5'-N.sub.x-NGGNG-3' both on the input sequence and on the
reverse-complement of the input. The value "x" in N.sub.x may be
fixed by the program or specified by the user, such as 20.
[0223] Since multiple occurrences in the genome of the DNA target
site may lead to nonspecific genome editing, after identifying all
potential sites, the program filters out sequences based on the
number of times they appear in the relevant reference genome. For
those CRISPR enzymes for which sequence specificity is determined
by a `seed` sequence, such as the 11-12 bp 5' from the PAM
sequence, including the PAM sequence itself, the filtering step may
be based on the seed sequence. Thus, to avoid editing at additional
genomic loci, results are filtered based on the number of
occurrences of the seed:PAM sequence in the relevant genome. The
user may be allowed to choose the length of the seed sequence. The
user may also be allowed to specify the number of occurrences of
the seed:PAM sequence in a genome for purposes of passing the
filter. The default is to screen for unique sequences. Filtration
level is altered by changing both the length of the seed sequence
and the number of occurrences of the sequence in the genome. The
program may in addition or alternatively provide the sequence of a
guide sequence complementary to the reported target sequence(s) by
providing the reverse complement of the identified target
sequence(s).
[0224] Further details of methods and algorithms to optimize
sequence selection can be found in U.S. application Ser. No.
61/836,080 (attorney docket 44790.11.2022); incorporated herein by
reference.
Example 4: Evaluation of Multiple Chimeric crRNA-tracrRNA
Hybrids
[0225] This example describes results obtained for chimeric RNAs
(chiRNAs; comprising a guide sequence, a tracr mate sequence, and a
tracr sequence in a single transcript) having tracr sequences that
incorporate different lengths of wild-type tracrRNA sequence. FIG.
18A illustrates a schematic of a bicistronic expression vector for
chimeric RNA and Cas9. Cas9 is driven by the CBh promoter and the
chimeric RNA is driven by a U6 promoter. The chimeric guide RNA
consists of a 20 bp guide sequence (Ns) joined to the tracr
sequence (running from the first "U" of the lower strand to the end
of the transcript), which is truncated at various positions as
indicated. The guide and tracr sequences are separated by the
tracr-mate sequence GUUUUAGAGCUA (SEQ ID NO: 30) followed by the
loop sequence GAAA. Results of SURVEYOR assays for Cas9-mediated
indels at the human EMX1 and PVALB loci are illustrated in FIG. 18B
and FIG. 18C, respectively. Arrows indicate the expected SURVEYOR
fragments. ChiRNAs are indicated by their "+n" designation, and
crRNA refers to a hybrid RNA where guide and tracr sequences are
expressed as separate transcripts. Quantification of these results,
performed in triplicate, are illustrated by histogram in FIGS. 19a
and 19b, corresponding to FIGS. 18b and 18c, respectively ("N.D."
indicates no indels detected). Protospacer IDs and their
corresponding genomic target, protospacer sequence, PAM sequence,
and strand location are provided in Table D. Guide sequences were
designed to be complementary to the entire protospacer sequence in
the case of separate transcripts in the hybrid system, or only to
the underlined portion in the case of chimeric RNAs.
TABLE-US-00004 TABLE D protospacer protospacer genomic sequence ID
target (5' to 3') PAM strand 1 EMX1 GGACATCGATGTCAC TGG +
CTCCAATGACTAGGG (SEQ ID NO: 31) 2 EMX1 CATTGGAGGTGACAT TGG -
CGATGTCCTCCCCAT (SEQ ID NO: 32) 3 EMX1 GGAAGGGCCTGAGTC GGG +
CGAGCAGAAGAAGAA (SEQ ID NO: 33) 4 PVALB GGTGGCGAGAGGGGC AGG +
CGAGATTGGGTGTTC (SEQ ID NO: 34) 5 PVALB ATGCAGGAGGGTGGC TGG +
GAGAGGGGCCGAGAT (SEQ ID NO: 35)
[0226] Cell Culture and Transfection
[0227] Human embryonic kidney (HEK) cell line 293FT (Life
Technologies) was maintained in Dulbecco's modified Eagle's Medium
(DMEM) supplemented with 10% fetal bovine serum (HyClone), 2 mM
GlutaMAX (Life Technologies), 100 U/mL penicillin, and 100 .mu.g/mL
streptomycin at 37.degree. C. with 5% CO.sub.2 incubation. 293FT
cells were seeded onto 24-well plates (Corning) 24 hours prior to
transfection at a density of 150,000 cells per well. Cells were
transfected using Lipofectamine 2000 (Life Technologies) following
the manufacturer's recommended protocol. For each well of a 24-well
plate, a total of 500 ng plasmid was used.
[0228] SURVEYOR Assay for Genome Modification
[0229] 293FT cells were transfected with plasmid DNA as described
above. Cells were incubated at 37.degree. C. for 72 hours
post-transfection prior to genomic DNA extraction. Genomic DNA was
extracted using the QuickExtract DNA Extraction Solution
(Epicentre) following the manufacturer's protocol. Briefly,
pelleted cells were resuspended in QuickExtract solution and
incubated at 65.degree. C. for 15 minutes and 98.degree. C. for 10
minutes. The genomic region flanking the CRISPR target site for
each gene was PCR amplified (primers listed in Table E), and
products were purified using QiaQuick Spin Column (Qiagen)
following the manufacturer's protocol. 400 ng total of the purified
PCR products were mixed with 2 .mu.l 10.times.Taq DNA Polymerase
PCR buffer (Enzymatics) and ultrapure water to a final volume of 20
.mu.l, and subjected to a re-annealing process to enable
heteroduplex formation: 95.degree. C. for 10 min, 95.degree. C. to
85.degree. C. ramping at -2.degree. C./s, 85.degree. C. to
25.degree. C. at -0.25.degree. C./s, and 25.degree. C. hold for 1
minute. After re-annealing, products were treated with SURVEYOR
nuclease and SURVEYOR enhancer S (Transgenomics) following the
manufacturer's recommended protocol, and analyzed on 4-20% Novex
TBE poly-acrylamide gels (Life Technologies). Gels were stained
with SYBR Gold DNA stain (Life Technologies) for 30 minutes and
imaged with a Gel Doc gel imaging system (Bio-rad). Quantification
was based on relative band intensities.
TABLE-US-00005 TABLE E genomic primer sequence primer name target
(5' to 3') Sp-EMX1-F EMX1 AAAACCACCCTTCTCTCTGGC (SEQ ID NO: 36)
Sp-EMX1-R EMX1 GGAGATTGGAGACACGGAGAG (SEQ ID NO: 37) Sp-PVALB-F
PVALB CTGGAAAGCCAATGCCTGAC (SEQ ID NO: 38) Sp-PVALB-R PVALB
GGCAGCAAACTCCTTGTCCT (SEQ ID NO: 39)
[0230] Computational Identification of Unique CRISPR Target
Sites
[0231] To identify unique target sites for the S. pyogenes SF370
Cas9 (SpCas9) enzyme in the human, mouse, rat, zebrafish, fruit
fly, and C. elegans genome, we developed a software package to scan
both strands of a DNA sequence and identify all possible SpCas9
target sites. For this example, each SpCas9 target site was
operationally defined as a 20 bp sequence followed by an NGG
protospacer adjacent motif (PAM) sequence, and we identified all
sequences satisfying this 5'-N20-NGG-3' (SEQ ID NO: 542) definition
on all chromosomes. To prevent non-specific genome editing, after
identifying all potential sites, all target sites were filtered
based on the number of times they appear in the relevant reference
genome. To take advantage of sequence specificity of Cas9 activity
conferred by a `seed` sequence, which can be, for example,
approximately 11-12 bp sequence 5' from the PAM sequence, 5'-NGG-3'
(SEQ ID NO: 543) sequences were selected to be unique in the
relevant genome. All genomic sequences were downloaded from the
UCSC Genome Browser (Human genome hg19, Mouse genome mm9, Rat
genome rn5, Zebrafish genome danRer7, D. melanogaster genome dm4
and C. elegans genome ce10). The full search results are available
to browse using UCSC Genome Browser information. An example
visualization of some target sites in the human genome is provided
in FIG. 21.
[0232] Initially, three sites within the EMX1 locus in human HEK
293FT cells were targeted. Genome modification efficiency of each
chiRNA was assessed using the SURVEYOR nuclease assay, which
detects mutations resulting from DNA double-strand breaks (DSBs)
and their subsequent repair by the non-homologous end joining
(NHEJ) DNA damage repair pathway. Constructs designated chiRNA(+n)
indicate that up to the +n nucleotide of wild-type tracrRNA is
included in the chimeric RNA construct, with values of 48, 54, 67,
and 85 used for n. Chimeric RNAs containing longer fragments of
wild-type tracrRNA (chiRNA(+67) and chiRNA(+85)) mediated DNA
cleavage at all three EMX1 target sites, with chiRNA(+85) in
particular demonstrating significantly higher levels of DNA
cleavage than the corresponding crRNA/tracrRNA hybrids that
expressed guide and tracr sequences in separate transcripts (FIGS.
18b and 19a). Two sites in the PVALB locus that yielded no
detectable cleavage using the hybrid system (guide sequence and
tracr sequence expressed as separate transcripts) were also
targeted using chiRNAs. chiRNA(+67) and chiRNA(+85) were able to
mediate significant cleavage at the two PVALB protospacers (FIGS.
18c and 19b).
[0233] For all five targets in the EMX1 and PVALB loci, a
consistent increase in genome modification efficiency with
increasing tracr sequence length was observed. Without wishing to
be bound by any theory, the secondary structure formed by the 3'
end of the tracrRNA may play a role in enhancing the rate of CRISPR
complex formation. An illustration of predicted secondary
structures for each of the chimeric RNAs used in this example is
provided in FIG. 21. The secondary structure was predicted using
RNAfold using minimum free energy and partition function algorithm.
Pseudocolor for each based (reproduced in grayscale) indicates the
probability of pairing. Because chiRNAs with longer tracr sequences
were able to cleave targets that were not cleaved by native CRISPR
crRNA/tracrRNA hybrids, it is possible that chimeric RNA may be
loaded onto Cas9 more efficiently than its native hybrid
counterpart. To facilitate the application of Cas9 for
site-specific genome editing in eukaryotic cells and organisms, all
predicted unique target sites for the S. pyogenes Cas9 were
computationally identified in the human, mouse, rat, zebra fish, C.
elegans, and D. melanogaster genomes. Chimeric RNAs can be designed
for Cas9 enzymes from other microbes to expand the target space of
CRISPR RNA-programmable nucleases.
[0234] FIG. 22 illustrates an exemplary bicistronic expression
vector for expression of chimeric RNA including up to the +85
nucleotide of wild-type tracr RNA sequence, and SpCas9 with nuclear
localization sequences. SpCas9 is expressed from a CBh promoter and
terminated with the bGH polyA signal (bGH pA). The expanded
sequence illustrated immediately below the schematic corresponds to
the region surrounding the guide sequence insertion site, and
includes, from 5' to 3', 3'-portion of the U6 promoter (first
shaded region), BbsI cleavage sites (arrows), partial direct repeat
(tracr mate sequence GTTTTAGAGCTA (SEQ ID NO: 27), underlined),
loop sequence GAAA, and +85 tracr sequence (underlined sequence
following loop sequence). An exemplary guide sequence insert is
illustrated below the guide sequence insertion site, with
nucleotides of the guide sequence for a selected target represented
by an "N".
[0235] Sequences described in the above examples are as follows
(polynucleotide sequences are 5' to 3'):
TABLE-US-00006 U6-short tracrRNA (Streptococcus pyogenes SF370):
(SEQ ID NO: 40) GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGG
CTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATT
AGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCA
GTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTT
GAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAA
CACCGGAACCATTCAAAACAGCATAGCAAGTTAAAATAAGGCTAGTCCG
TTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT (bold = tracrRNA
sequence; underline = terminator sequence) U6-long tracrRNA
(Streptococcus pyogenes SF370): (SEQ ID NO: 41)
GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGG
CTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATT
AGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCA
GTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTT
GAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAA
CACCGGTAGTATTAAGTATTGTTTTATGGCTGATAAATTTCTTTGAATT
TCTCCTTGATTATTTGTTATAAAAGTTATAAAATAATCTTGTTGGAACC
ATTCAAAACAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT
GAAAAAGTGGCACCGAGTCGGTGCTTTTTTT U6-DR-BbsI backbone-DR
(Streptococcus pyogenes SF370): (SEQ ID NO: 42)
GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGG
CTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATT
AGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCA
GTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTT
GAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAA
CACCGGGTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAACGGGTCTT
CGAGAAGACGTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAAC U6-chimeric RNA-BbsI
backbone (Streptococcus pyogenes SF370) (SEQ ID NO: 43)
GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGG
CTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATT
AGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCA
GTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTT
GAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAA
CACCGGGTCTTCGAGAAGACCTGTTTTAGAGCTAGAAATAGCAAGTTAA AATAAGGCTAGTCCG
NLS-SpCas9-EGFP: (SEQ ID NO: 44)
MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLD
IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA
EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE
EDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLA
LAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV
DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSN
FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD
ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD
QSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ
RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY
VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK
NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV
DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL
KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKV
MKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQL
IHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVD
ELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQ
ILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVP
QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLI
TQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNT
KYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFY
SNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSP
TVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKG
YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNF
LYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDR
KRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDAAAVSKGEELFTGV
VPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTL
VTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTR
AEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQK
NGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSAL
SKDPNEKRDHMVLLEFVTAAGITLGMDELYK SpCas9-EGFP-NLS: (SEQ ID NO: 45)
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIG
ALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFF
HRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTD
KADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF
EENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS
LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAK
NLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL
PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVK
LNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIE
KILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQS
FIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAF
LSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFN
ASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLK
TYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD
GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKK
GILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRI
EEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRL
SDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY
WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINN
YHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEI
GKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGR
DFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD
PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEK
NPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS
EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAA
FKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDAAAV
SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTT
GKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSH
NVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPD
NHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKKRPAATKK AGQAKKKK
NLS-SpCas9-EGFP-NLS: (SEQ ID NO: 46)
MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLD
IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA
EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE
EDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLA
LAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV
DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSN
FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD
ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD
QSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ
RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY
VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK
NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV
DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL
KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKV
MKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQL
IHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVD
ELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQ
ILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVP
QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLI
TQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNT
KYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFY
SNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSP
TVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKG
YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNF
LYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDR
KRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDAAAVSKGEELFTGV
VPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTL
VTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTR
AEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQK
NGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSAL
SKDPNEKRDHMVLLEFVTAAGITLGMDELYKKRPAATKKAGQAKKKK NLS-SpCas9-NLS:
(SEQ ID NO: 47) MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLD
IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA
EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE
EDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLA
LAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV
DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSN
FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD
ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD
QSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ
RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY
VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK
NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV
DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL
KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKV
MKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQL
IHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVD
ELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQ
ILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVP
QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLI
TQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNT
KYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFY
SNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSP
TVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKG
YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNF
LYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDR
KRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKK KK
NLS-mCherry-SpRNase3: (SEQ ID NO: 48)
MFLFLSLTSFLSSSRTLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFE
IEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPA
DIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRG
TNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDA
EVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTG
GMDELYKGSKQLEELLSTSFDIQFNDLTLLETAFTHTSYANEHRLLNVS
HNERLEFLGDAVLQLIISEYLFAKYPKKTEGDMSKLRSMIVREESLAGF
SRFCSFDAYIKLGKGEEKSGGRRRDTILGDLFEAFLGALLLDKGIDAVR
RFLKQVMIPQVEKGNFERVKDYKTCLQEFLQTKGDVAIDYQVISEKGPA
HAKQFEVSIVVNGAVLSKGLGKSKKLAEQDAAKNALAQLSEV SpRNase3-mCherry-NLS:
(SEQ ID NO: 49) MKQLEELLSTSFDIQFNDLTLLETAFTHTSYANEHRLLNVSHNERLEFL
GDAVLQLIISEYLFAKYPKKTEGDMSKLRSMIVREESLAGFSRFCSFDA
YIKLGKGEEKSGGRRRDTILGDLFEAFLGALLLDKGIDAVRRFLKQVMI
PQVEKGNFERVKDYKTCLQEFLQTKGDVAIDYQVISEKGPAHAKQFEVS
IVVNGAVLSKGLGKSKKLAEQDAAKNALAQLSEVGSVSKGEEDNMAIIK
EFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAW
DILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTV
TQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGA
LKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHN
EDYTIVEQYERAEGRHSTGGMDELYKKRPAATKKAGQAKKKK NLS-SpCas9n-NLS (the
D10A nickase mutation is lowercase): (SEQ ID NO: 50)
MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLa
IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA
EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE
EDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLA
LAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV
DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSN
FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD
ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD
QSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ
RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY
VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK
NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV
DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL
KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKV
MKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQL
IHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVD
ELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQ
ILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVP
QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLI
TQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNT
KYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFY
SNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSP
TVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKG
YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNF
LYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDR
KRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKK KK hEMX1-HR
Template-HindII-NheI: (SEQ ID NO: 51)
GAATGCTGCCCTCAGACCCGCTTCCTCCCTGTCCTTGTCTGTCCAAGGA
GAATGAGGTCTCACTGGTGGATTTCGGACTACCCTGAGGAGCTGGCACC
TGAGGGACAAGGCCCCCCACCTGCCCAGCTCCAGCCTCTGATGAGGGGT
GGGAGAGAGCTACATGAGGTTGCTAAGAAAGCCTCCCCTGAAGGAGACC
ACACAGTGTGTGAGGTTGGAGTCTCTAGCAGCGGGTTCTGTGCCCCCAG
GGATAGTCTGGCTGTCCAGGCACTGCTCTTGATATAAACACCACCTCCT
AGTTATGAAACCATGCCCATTCTGCCTCTCTGTATGGAAAAGAGCATGG
GGCTGGCCCGTGGGGTGGTGTCCACTTTAGGCCCTGTGGGAGATCATGG
GAACCCACGCAGTGGGTCATAGGCTCTCTCATTTACTACTCACATCCAC
TCTGTGAAGAAGCGATTATGATCTCTCCTCTAGAAACTCGTAGAGTCCC
ATGTCTGCCGGCTTCCAGAGCCTGCACTCCTCCACCTTGGCTTGGCTTT
GCTGGGGCTAGAGGAGCTAGGATGCACAGCAGCTCTGTGACCCTTTGTT
TGAGAGGAACAGGAAAACCACCCTTCTCTCTGGCCCACTGTGTCCTCTT
CCTGCCCTGCCATCCCCTTCTGTGAATGTTAGACCCATGGGAGCAGCTG
GTCAGAGGGGACCCCGGCCTGGGGCCCCTAACCCTATGTAGCCTCAGTC
TTCCCATCAGGCTCTCAGCTCAGCCTGAGTGTTGAGGCCCCAGTGGCTG
CTCTGGGGGCCTCCTGAGTTTCTCATCTGTGCCCCTCCCTCCCTGGCCC
AGGTGAAGGTGTGGTTCCAGAACCGGAGGACAAAGTACAAACGGCAGAA
GCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAAGAAGGGCTCCCAT
CACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACA
TCGATGTCACCTCCAATGACaagcttgctagcGGTGGGCAACCACAAAC
CCACGAGGGCAGAGTGCTGCTTGCTGCTGGCCAGGCCCCTGCGTGGGCC
CAAGCTGGACTCTGGCCACTCCCTGGCCAGGCTTTGGGGAGGCCTGGAG
TCATGGCCCCACAGGGCTTGAAGCCCGGGGCCGCCATTGACAGAGGGAC
AAGCAATGGGCTGGCTGAGGCCTGGGACCACTTGGCCTTCTCCTCGGAG
AGCCTGCCTGCCTGGGCGGGCCCGCCCGCCACCGCAGCCTCCCAGCTGC
TCTCCGTGTCTCCAATCTCCCTTTTGTTTTGATGCATTTCTGTTTTAAT
TTATTTTCCAGGCACCACTGTAGTTTAGTGATCCCCAGTGTCCCCCTTC
CCTATGGGAATAATAAAAGTCTCTCTCTTAATGACACGGGCATCCAGCT
CCAGCCCCAGAGCCTGGGGTGGTAGATTCCGGCTCTGAGGGCCAGTGGG
GGCTGGTAGAGCAAACGCGTTCAGGGCCTGGGAGCCTGGGGTGGGGTAC
TGGTGGAGGGGGTCAAGGGTAATTCATTAACTCCTCTCTTTTGTTGGGG
GACCCTGGTCTCTACCTCCAGCTCCACAGCAGGAGAAACAGGCTAGACA
TAGGGAAGGGCCATCCTGTATCTTGAGGGAGGACAGGCCCAGGTCTTTC
TTAACGTATTGAGAGGTGGGAATCAGGCCCAGGTAGTTCAATGGGAGAG
GGAGAGTGCTTCCCTCTGCCTAGAGACTCTGGTGGCTTCTCCAGTTGAG
GAGAAACCAGAGGAAAGGGGAGGATTGGGGTCTGGGGGAGGGAACACCA
TTCACAAAGGCTGACGGTTCCAGTCCGAAGTCGTGGGCCCACCAGGATG
CTCACCTGTCCTTGGAGAACCGCTGGGCAGGTTGAGACTGCAGAGACAG
GGCTTAAGGCTGAGCCTGCAACCAGTCCCCAGTGACTCAGGGCCTCCTC
AGCCCAAGAAAGAGCAACGTGCCAGGGCCCGCTGAGCTCTTGTGTTCAC CTG
NLS-StCsn1-NLS: (SEQ ID NO: 52)
MKRPAATKKAGQAKKKKSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSR
IFPAAQAENNLVRRTNRQGRRLARRKKHRRVRLNRLFEESGLITDETKI
SINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISYLDDASDDGNSS
VGDYAQIVKENSKQLETKTPGQIQLERYQTYGQLRGDFTVEKDGKKHRL
INVEPTSAYRSEALRILQTQQEFNPQITDEFINRYLEILTGKRKYYHGP
GNEKSRTDYGRYRTSGETLDNIEGILIGKCTFYPDEFRAAKASYTAQEF
NLLNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLEKYIAKLL
SCDVADIKGYRIDKSGKAEIHTFEAYRKMKTLETLDIEQMDRETLDKLA
YVLTLNTEREGIQEALEHEFADGSFSQKQVDELVQFRKANSSIFGKGWH
NFSVKLMMELIPELYETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLT
EEIYNPVVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNEDDEKKAI
QKIQKANKDEKDAAMLKAANQYNGKAELPHSVEHGHKQLATKIRLWHQQ
GERCLYTGKTISIHDLINNSNQFEVDHILPLSITEDDSLANKVLVYATA
NQEKGQRTPYQALDSMDDAWSFRELKAFVRESKTLSNKKKEYLLTEEDI
SKEDVRKKFIERNLVDTRYASRVVLNALQEHFRAHKIDTKVSVVRGQFT
SQLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNTLVSYSEDQ
LLDIETGELISDDEYKESVFKAPYQHFVDTLKSKEFEDSILFSYQVDSK
ENRKISDATIYATRQAKVGKDKADETYVLGKIKDIYTQDGYDAFMKIYK
KDKSKFLMYRHDPQTFEKVIEPILENYPNKQINEKGKEVPCNPFLKYKE
EHGYIRKYSKKGNGPEIKSLKYYDSKLGNHIDITPKDSNNKVVLQSVSP
WRADVYFNKTTGKYEILGLKYADLQFEKGTGTYKISQEKYNDIKKKEGV
DSDSEFKFTLYKNDLLLVKDTETKEQQLFRFLSRTMPKQKHYVELKPYD
KQKFEGGEALIKVLGNVANSGQCKKGLGKSNISIYKVRTDVLGNQHIIK
NEGDKPKLDFKRPAATKKAGQAKKKK U6-St_tracrRNA(7-97): (SEQ ID NO: 53)
GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGG
CTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATT
AGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCA
GTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTT
GAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAA
CACCGTTACTTAAATCTTGCAGAAGCTACAAAGATAAGGCTTCATGCCG
AAATCAACACCCTGTCATTTTATGGCAGGGTGTTTTCGTTATTTAA U6-DR-spacer-DR (S.
pyogenes SF370) (SEQ ID NO: 54)
gagggcctatttcccatgattccttcatatttgcatatacgatacaagg
ctgttagagagataattggaattaatttgactgtaaacacaaagatatt
agtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgca
gttttaaaattatgttttaaaatggactatcatatgcttaccgtaactt
gaaagtatttcgatttatggattatatatcttgtggaaaggacgaaaca
ccgggttttagagctatgctgttttgaatggtcccaaaacNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNgttttagagctatgctgttttgaatggt cccaaaacTTTTTTT
(lowercase underline = direct repeat; N = guide sequence; bold =
terminator) Chimeric RNA containing +48 tracr RNA (S. pyogenes
SF370) (SEQ ID NO: 55)
gagggcctatttcccatgattccttcatatttgcatatacgatacaagg
ctgttagagagataattggaattaatttgactgtaaacacaaagatatt
agtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgca
gttttaaaattatgttttaaaatggactatcatatgcttaccgtaactt
gaaagtatttcgatttatggattatatatcttgtggaaaggacgaaaca
ccNNNNNNNNNNNNNNNNNNNNgttttagagctagaaatagcaagttaa
aataaggctagtccgTTTTTTT (N = guide sequence; first underline = tracr
mate sequence; second underline = tracr sequence; bold =
terminator) Chimeric RNA containing +54 tracr RNA (S. pyogenes
SF370) (SEQ ID NO: 56)
gagggcctatttcccatgattccttcatatttgcatatacgatacaagg
ctgttagagagataattggaattaatttgactgtaaacacaaagatatt
agtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgca
gttttaaaattatgttttaaaatggactatcatatgcttaccgtaactt
gaaagtatttcgatttatggattatatatcttgtggaaaggacgaaaca
ccNNNNNNNNNNNNNNNNNNNNgttttagagctagaaatagcaagttaa
aataaggctagtccgttatcaTTTTTTTT (N = guide sequence; first underline
= tracr mate sequence; second underline = tracr sequence; bold =
terminator) Chimeric RNA containing +67 tracr RNA (S. pyogenes
SF370) (SEQ ID NO: 57)
gagggcctatttcccatgattccttcatatttgcatatacgatacaagg
ctgttagagagataattggaattaatttgactgtaaacacaaagatatt
agtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgca
gttttaaaattatgttttaaaatggactatcatatgcttaccgtaactt
gaaagtatttcgatttatggattatatatcttgtggaaaggacgaaaca
ccNNNNNNNNNNNNNNNNNNNNgttttagagctagaaatagcaagttaa
aataaggctagtccgttatcaacttgaaaaagtgTTTTTTT (N = guide sequence;
first underline = tracr mate sequence; second underline = tracr
sequence; bold = terminator) Chimeric RNA containing +85 tracr RNA
(S. pyogenes SF370) (SEQ ID NO: 58)
gagggcctatttcccatgattccttcatatttgcatatacgatacaagg
ctgttagagagataattggaattaatttgactgtaaacacaaagatatt
agtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgca
gttttaaaattatgttttaaaatggactatcatatgcttaccgtaactt
gaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaa
caccNNNNNNNNNNNNNNNNNNNNgttttagagctagaaatagcaagtt
aaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggt gcTTTTTTT (N =
guide sequence; first underline = tracr mate sequence; second
underline = tracr sequence; bold = terminator) CBh-NLS-SpCas9-NLS
(SEQ ID NO: 59) CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGAC
CCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAA
TAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGC
CCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATT
GACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGA
CCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGC
TATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATC
TCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTAT
TTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGG
GGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCG
GCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGC
GGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGT
CGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCG
CCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGG
CGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAAG
GGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGA
GCACCTGCCTGAAATCACTTTTTTTCAGGTTGGaccggtgccaccATGG
ACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAA
AGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATC
CACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCG
GCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCC
CAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAG
AAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGG
CCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAA
CCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTG
GACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGG
ATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGT
GGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTG
GTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGG
CCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAA
CCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACC
TACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACG
CCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAA
TCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAAC
CTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCG
ACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGA
CGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTG
TTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCC
TGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGAT
CAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTC
GTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGA
GCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGA
GTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAG
GAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGA
CCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCA
CGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAAC
CGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGG
GCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAG
CGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGC
GCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACC
TGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTT
CACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATG
AGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACC
TGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGA
CTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTG
GAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAA
TTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCT
GGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATC
GAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGA
AGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAA
GCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGAT
TTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCC
ACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTC
CGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGC
CCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGC
TCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAAT
GGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAG
AGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCC
TGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTA
CCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTG
GACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGA
GCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGA
CAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAG
AAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCC
AGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGA
ACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAG
ATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGT
ACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAA
GTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTG
CGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCG
TCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTT
CGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAG
AGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCA
ACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGAT
CCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTG
TGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGC
CCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAG
CAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGA
AAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCG
TGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAA
GAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGA
AGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACA
AAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTT
CGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTG
CAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGT
ACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGA
GCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATC
ATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTA
ATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCAT
CAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTG
GGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGA
GGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAG
CATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGC
GACTTTCTTTTTCTTAGCTTGACCAGCTTTCTTAGTAGCAGCAGGACGC TTTAA (underline
= NLS-hSpCas9-NLS) Example chimeric RNA for S. thermophilus LMD-9
CRISPR1 Cas9 (with PAM of NNAGAAW) (SEQ ID NO: 21)
NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaagatttaGAAAtaa
atcttgcagaagctacaaagataaggatcatgccgaaatcaacaccctg
tcattttatggcagggtgttttcgttatttaaTTTTTT (N = guide sequence; first
underline = tracr mate sequence; second underline = tracr sequence;
bold = terminator) Example chimeric RNA for S. thermophilus LMD-9
CRISPR1 Cas9 (with PAM of NNAGAAW) (SEQ ID NO: 22)
NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagct
acaaagataaggcttcatgccgaaatcaacaccctgtcattttatggca
gggtgttttcgttatttaaTTTTTT (N = guide sequence; first underline =
tracr mate sequence; second underline = tracr sequence; bold =
terminator) Example chimeric RNA for S. thermophilus LMD-9 CRISPR1
Cas9 (with PAM of NNAGAAW) (SEQ ID NO: 23)
NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagct
acaaagataaggcttcatgccgaaatcaacaccctgtcattttatggca gggtgtTTTTTT (N =
guide sequence; first underline = tracr mate sequence; second
underline = tracr sequence; bold = terminator) Example chimeric RNA
for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM of NNAGAAW) (SEQ
ID NO: 60) NNNNNNNNNNNNNNNNNNNNgttattgtactctcaagatttaGAAAtaa
atcttgcagaagctacaaagataaggcttcatgccgaaatcaacaccct
gtcattttatggcagggtgttttcgttatttaaTTTTTT (N = guide sequence; first
underline = tracr mate sequence; second underline = tracr sequence;
bold = terminator) Example chimeric RNA for S. thermophilus LMD-9
CRISPR1 Cas9 (with PAM of NNAGAAW) (SEQ ID NO: 61)
NNNNNNNNNNNNNNNNNNNNgttattgtactctcaGAAAgcagaagcta
caaagataaggcttcatgccgaaatcaacaccctgtcattttatggcag
ggtgttttcgttatttaaTTTTTT (N = guide sequence; first underline =
tracr mate sequence; second underline = tracr sequence; bold =
terminator) Example chimeric RNA for S. thermophilus LMD-9 CRISPR1
Cas9 (with PAM of NNAGAAW) (SEQ ID NO: 62)
NNNNNNNNNNNNNNNNNNNNgttattgtactctcaGAAAtgcagaagct
acaaagataaggcttcatgccgaaatcaacaccctgtcattttatggca gggtgtTTTTTT (N =
guide sequence; first underline = tracr mate sequence; second
underline = tracr sequence; bold = terminator) Example chimeric RNA
for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM of NNAGAAW) (SEQ
ID NO: 63) NNNNNNNNNNNNNNNNNNNNgttattgtactctcaagatttaGAAAtaa
atcttgcagaagctacaatgataaggatcatgccgaaatcaacaccctg
tcattttatggcagggtgttttcgttatttaaTTTTTT (N = guide sequence; first
underline = tracr mate sequence; second underline = tracr sequence;
bold = terminator) Example chimeric RNA for S. thermophilus
LMD-9
CRISPR1 Cas9 (with PAM of NNAGAAW) (SEQ ID NO: 64)
NNNNNNNNNNNNNNNNNNNNgttattgtactctcaGAAAtgcagaagct
acaatgataaggcttcatgccgaaatcaacaccctgtcattttatggca
gggtgttttcgttatttaaTTTTTT (N = guide sequence; first underline =
tracr mate sequence; second underline = tracr sequence; bold =
terminator) Example chimeric RNA for S. thermophilus LMD-9 CRISPR1
Cas9 (with PAM of NNAGAAW) (SEQ ID NO: 65)
NNNNNNNNNNNNNNNNNNNNgttattgtactctcaGAAAtgcagaagct
acaatgataaggcttcatgccgaaatcaacaccctgtcattttatggca gggtgtTTTTTT (N =
guide sequence; first underline = tracr mate sequence; second
underline = tracr sequence; bold = terminator) Example chimeric RNA
for S. thermophilus LMD-9 CRISPR3 Cas9 (with PAM of NGGNG) (SEQ ID
NO: 66) NNNNNNNNNNNNNNNNNNNNgttttagagctgtgGAAAcacagcgagtt
aaaataaggcttagtccgtactcaacttgaaaaggtggcaccgattcgg tgtTTTTTT (N =
guide sequence; first underline = tracr mate sequence; second
underline = tracr sequence; bold = terminator) Codon-optimized
version of Cas9 from S. thermophilus LMD-9 CRISPR3 locus (with an
NLS at both 5' and 3' ends) (SEQ ID NO: 67)
ATGAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAA
AGACCAAGCCCTACAGCATCGGCCTGGACATCGGCACCAATAGCGTGGG
CTGGGCCGTGACCACCGACAACTACAAGGTGCCCAGCAAGAAAATGAAG
GTGCTGGGCAACACCTCCAAGAAGTACATCAAGAAAAACCTGCTGGGCG
TGCTGCTGTTCGACAGCGGCATTACAGCCGAGGGCAGACGGCTGAAGAG
AACCGCCAGACGGCGGTACACCCGGCGGAGAAACAGAATCCTGTATCTG
CAAGAGATCTTCAGCACCGAGATGGCTACCCTGGACGACGCCTTCTTCC
AGCGGCTGGACGACAGCTTCCTGGTGCCCGACGACAAGCGGGACAGCAA
GTACCCCATCTTCGGCAACCTGGTGGAAGAGAAGGCCTACCACGACGAG
TTCCCCACCATCTACCACCTGAGAAAGTACCTGGCCGACAGCACCAAGA
AGGCCGACCTGAGACTGGTGTATCTGGCCCTGGCCCACATGATCAAGTA
CCGGGGCCACTTCCTGATCGAGGGCGAGTTCAACAGCAAGAACAACGAC
ATCCAGAAGAACTTCCAGGACTTCCTGGACACCTACAACGCCATCTTCG
AGAGCGACCTGTCCCTGGAAAACAGCAAGCAGCTGGAAGAGATCGTGAA
GGACAAGATCAGCAAGCTGGAAAAGAAGGACCGCATCCTGAAGCTGTTC
CCCGGCGAGAAGAACAGCGGAATCTTCAGCGAGTTTCTGAAGCTGATCG
TGGGCAACCAGGCCGACTTCAGAAAGTGCTTCAACCTGGACGAGAAAGC
CAGCCTGCACTTCAGCAAAGAGAGCTACGACGAGGACCTGGAAACCCTG
CTGGGATATATCGGCGACGACTACAGCGACGTGTTCCTGAAGGCCAAGA
AGCTGTACGACGCTATCCTGCTGAGCGGCTTCCTGACCGTGACCGACAA
CGAGACAGAGGCCCCACTGAGCAGCGCCATGATTAAGCGGTACAACGAG
CACAAAGAGGATCTGGCTCTGCTGAAAGAGTACATCCGGAACATCAGCC
TGAAAACCTACAATGAGGTGTTCAAGGACGACACCAAGAACGGCTACGC
CGGCTACATCGACGGCAAGACCAACCAGGAAGATTTCTATGTGTACCTG
AAGAAGCTGCTGGCCGAGTTCGAGGGGGCCGACTACTTTCTGGAAAAAA
TCGACCGCGAGGATTTCCTGCGGAAGCAGCGGACCTTCGACAACGGCAG
CATCCCCTACCAGATCCATCTGCAGGAAATGCGGGCCATCCTGGACAAG
CAGGCCAAGTTCTACCCATTCCTGGCCAAGAACAAAGAGCGGATCGAGA
AGATCCTGACCTTCCGCATCCCTTACTACGTGGGCCCCCTGGCCAGAGG
CAACAGCGATTTTGCCTGGTCCATCCGGAAGCGCAATGAGAAGATCACC
CCCTGGAACTTCGAGGACGTGATCGACAAAGAGTCCAGCGCCGAGGCCT
TCATCAACCGGATGACCAGCTTCGACCTGTACCTGCCCGAGGAAAAGGT
GCTGCCCAAGCACAGCCTGCTGTACGAGACATTCAATGTGTATAACGAG
CTGACCAAAGTGCGGTTTATCGCCGAGTCTATGCGGGACTACCAGTTCC
TGGACTCCAAGCAGAAAAAGGACATCGTGCGGCTGTACTTCAAGGACAA
GCGGAAAGTGACCGATAAGGACATCATCGAGTACCTGCACGCCATCTAC
GGCTACGATGGCATCGAGCTGAAGGGCATCGAGAAGCAGTTCAACTCCA
GCCTGAGCACATACCACGACCTGCTGAACATTATCAACGACAAAGAATT
TCTGGACGACTCCAGCAACGAGGCCATCATCGAAGAGATCATCCACACC
CTGACCATCTTTGAGGACCGCGAGATGATCAAGCAGCGGCTGAGCAAGT
TCGAGAACATCTTCGACAAGAGCGTGCTGAAAAAGCTGAGCAGACGGCA
CTACACCGGCTGGGGCAAGCTGAGCGCCAAGCTGATCAACGGCATCCGG
GACGAGAAGTCCGGCAACACAATCCTGGACTACCTGATCGACGACGGCA
TCAGCAACCGGAACTTCATGCAGCTGATCCACGACGACGCCCTGAGCTT
CAAGAAGAAGATCCAGAAGGCCCAGATCATCGGGGACGAGGACAAGGGC
AACATCAAAGAAGTCGTGAAGTCCCTGCCCGGCAGCCCCGCCATCAAGA
AGGGAATCCTGCAGAGCATCAAGATCGTGGACGAGCTCGTGAAAGTGAT
GGGCGGCAGAAAGCCCGAGAGCATCGTGGTGGAAATGGCTAGAGAGAAC
CAGTACACCAATCAGGGCAAGAGCAACAGCCAGCAGAGACTGAAGAGAC
TGGAAAAGTCCCTGAAAGAGCTGGGCAGCAAGATTCTGAAAGAGAATAT
CCCTGCCAAGCTGTCCAAGATCGACAACAACGCCCTGCAGAACGACCGG
CTGTACCTGTACTACCTGCAGAATGGCAAGGACATGTATACAGGCGACG
ACCTGGATATCGACCGCCTGAGCAACTACGACATCGACCATATTATCCC
CCAGGCCTTCCTGAAAGACAACAGCATTGACAACAAAGTGCTGGTGTCC
TCCGCCAGCAACCGCGGCAAGTCCGATGATGTGCCCAGCCTGGAAGTCG
TGAAAAAGAGAAAGACCTTCTGGTATCAGCTGCTGAAAAGCAAGCTGAT
TAGCCAGAGGAAGTTCGACAACCTGACCAAGGCCGAGAGAGGCGGCCTG
AGCCCTGAAGATAAGGCCGGCTTCATCCAGAGACAGCTGGTGGAAACCC
GGCAGATCACCAAGCACGTGGCCAGACTGCTGGATGAGAAGTTTAACAA
CAAGAAGGACGAGAACAACCGGGCCGTGCGGACCGTGAAGATCATCACC
CTGAAGTCCACCCTGGTGTCCCAGTTCCGGAAGGACTTCGAGCTGTATA
AAGTGCGCGAGATCAATGACTTTCACCACGCCCACGACGCCTACCTGAA
TGCCGTGGTGGCTTCCGCCCTGCTGAAGAAGTACCCTAAGCTGGAACCC
GAGTTCGTGTACGGCGACTACCCCAAGTACAACTCCTTCAGAGAGCGGA
AGTCCGCCACCGAGAAGGTGTACTTCTACTCCAACATCATGAATATCTT
TAAGAAGTCCATCTCCCTGGCCGATGGCAGAGTGATCGAGCGGCCCCTG
ATCGAAGTGAACGAAGAGACAGGCGAGAGCGTGTGGAACAAAGAAAGCG
ACCTGGCCACCGTGCGGCGGGTGCTGAGTTATCCTCAAGTGAATGTCGT
GAAGAAGGTGGAAGAACAGAACCACGGCCTGGATCGGGGCAAGCCCAAG
GGCCTGTTCAACGCCAACCTGTCCAGCAAGCCTAAGCCCAACTCCAACG
AGAATCTCGTGGGGGCCAAAGAGTACCTGGACCCTAAGAAGTACGGCGG
ATACGCCGGCATCTCCAATAGCTTCACCGTGCTCGTGAAGGGCACAATC
GAGAAGGGCGCTAAGAAAAAGATCACAAACGTGCTGGAATTTCAGGGGA
TCTCTATCCTGGACCGGATCAACTACCGGAAGGATAAGCTGAACTTTCT
GCTGGAAAAAGGCTACAAGGACATTGAGCTGATTATCGAGCTGCCTAAG
TACTCCCTGTTCGAACTGAGCGACGGCTCCAGACGGATGCTGGCCTCCA
TCCTGTCCACCAACAACAAGCGGGGCGAGATCCACAAGGGAAACCAGAT
CTTCCTGAGCCAGAAATTTGTGAAACTGCTGTACCACGCCAAGCGGATC
TCCAACACCATCAATGAGAACCACCGGAAATACGTGGAAAACCACAAGA
AAGAGTTTGAGGAACTGTTCTACTACATCCTGGAGTTCAACGAGAACTA
TGTGGGAGCCAAGAAGAACGGCAAACTGCTGAACTCCGCCTTCCAGAGC
TGGCAGAACCACAGCATCGACGAGCTGTGCAGCTCCTTCATCGGCCCTA
CCGGCAGCGAGCGGAAGGGACTGTTTGAGCTGACCTCCAGAGGCTCTGC
CGCCGACTTTGAGTTCCTGGGAGTGAAGATCCCCCGGTACAGAGACTAC
ACCCCCTCTAGTCTGCTGAAGGACGCCACCCTGATCCACCAGAGCGTGA
CCGGCCTGTACGAAACCCGGATCGACCTGGCTAAGCTGGGCGAGGGAAA
GCGTCCTGCTGCTACTAAGAAAGCTGGTCAAGCTAAGAAAAAGAAATAA
Example 5: RNA Guided Editing of Bacterial Genomes Using CRISPR-Cas
Systems
[0236] Applicants used the CRISPR-associated endonuclease Cas9 to
introduce precise mutations in the genomes of Streptococcus
pneumoniae and Escherichia coli. The approach relied on
Cas9-directed cleavage at the targeted site to kill unmutated cells
and circumvented the need for selectable markers or
counter-selection systems. Cas9 specificity was reprogrammed by
changing the sequence of short CRISPR RNA (crRNA) to make single-
and multi-nucleotide changes carried on editing templates.
Simultaneous use of two crRNAs enabled multiplex mutagenesis. In S.
pneumoniae, nearly 100% of cells that survived Cas9 cleavage
contained the desired mutation, and 65% when used in combination
with recombineering in E. coli. Applicants exhaustively analyzed
Cas9 target requirements to define the range of targetable
sequences and showed strategies for editing sites that do not meet
these requirements, suggesting the versatility of this technique
for bacterial genome engineering.
[0237] The understanding of gene function depends on the
possibility of altering DNA sequences within the cell in a
controlled fashion. Site-specific mutagenesis in eukaryotes is
achieved by the use of sequence-specific nucleases that promote
homologous recombination of a template DNA containing the mutation
of interest. Zinc finger nucleases (ZFNs), transcription
activator-like effector nucleases (TALENs) and homing meganucleases
can be programmed to cleave genomes in specific locations, but
these approaches require engineering of new enzymes for each target
sequence. In prokaryotic organisms, mutagenesis methods either
introduce a selection marker in the edited locus or require a
two-step process that includes a counter-selection system. More
recently, phage recombination proteins have been used for
recombineering, a technique that promotes homologuous recombination
of linear DNA or oligonucleotides. However, because there is no
selection of mutations, recombineering efficiency can be relatively
low (0.1-10% for point mutations down to 10.sup.-5-10.sup.-6 for
larger modifications), in many cases requiring the screening of a
large number of colonies. Therefore new technologies that are
affordable, easy to use and efficient are still in need for the
genetic engineering of both eukaryotic and prokaryotic
organisms.
[0238] Recent work on the CRISPR (clustered, regularly interspaced,
short palindromic repeats) adaptive immune system of prokaryotes
has led to the identification of nucleases whose sequence
specificity is programmed by small RNAs. CRISPR loci are composed
of a series of repeats separated by `spacer` sequences that match
the genomes of bacteriophages and other mobile genetic elements.
The repeat-spacer array is transcribed as a long precursor and
processed within repeat sequences to generate small crRNA that
specify the target sequences (also known as protospacers) cleaved
by CRISPR systems. Essential for cleavage is the presence of a
sequence motif immediately downstream of the target region, known
as the protospacer-adjacent motif (PAM). CRISPR-associated (cas)
genes usually flank the repeat-spacer array and encode the
enzymatic machinery responsible for crRNA biogenesis and targeting.
Cas9 is a dsDNA endonuclease that uses a crRNA guide to specify the
site of cleavage. Loading of the crRNA guide onto Cas9 occurs
during the processing of the crRNA precursor and requires a small
RNA antisense to the precursor, the tracrRNA, and RNAse III. In
contrast to genome editing with ZFNs or TALENs, changing Cas9
target specificity does not require protein engineering but only
the design of the short crRNA guide.
[0239] Applicants recently showed in S. pneumoniae that the
introduction of a CRISPR system targeting a chromosomal locus leads
to the killing of the transformed cells. It was observed that
occasional survivors contained mutations in the target region,
suggesting that Cas9 dsDNA endonuclease activity against endogenous
targets could be used for genome editing. Applicants showed that
marker-less mutations can be introduced through the transformation
of a template DNA fragment that will recombine in the genome and
eliminate Cas9 target recognition. Directing the specificity of
Cas9 with several different crRNAs allows for the introduction of
multiple mutations at the same time. Applicants also characterized
in detail the sequence requirements for Cas9 targeting and show
that the approach can be combined with recombineering for genome
editing in E. coli.
[0240] Results: Genome Editing by Cas9 Cleavage of a Chromosomal
Target
[0241] S. pneumoniae strain crR6 contains a Cas9-based CRISPR
system that cleaves a target sequence present in the bacteriophage
.PHI.8232.5. This target was integrated into the srtA chromosomal
locus of a second strain R6.sup.8232.5. An altered target sequence
containing a mutation in the PAM region was integrated into the
srtA locus of a third strain R6.sup.370.1, rendering this strain
`immune` to CRISPR cleavage (FIG. 28A). Applicants transformed
R6.sup.8232.5 and R6.sup.370.1 cells with genomic DNA from crR6
cells, expecting that successful transformation of R6.sup.8232.5
cells should lead to cleavage of the target locus and cell death.
Contrary to this expectation, Applicants isolated R6.sup.8232.5
transformants, albeit with approximately 10-fold less efficiency
than R6.sup.370.1 transformants (FIG. 28B). Genetic analysis of
eight R6.sup.8232.5 transformants (FIG. 28) revealed that the great
majority are the product of a double recombination event that
eliminates the toxicity of Cas9 targeting by replacing the
.PHI.8232.5 target with the crR6 genome's wild-type srtA locus,
which does not contain the protospacer required for Cas9
recognition. These results were proof that the concurrent
introduction of a CRISPR system targeting a genomic locus (the
targeting construct) together with a template for recombination
into the targeted locus (the editing template) led to targeted
genome editing (FIG. 23A).
[0242] To create a simplified system for genome editing, Applicants
modified the CRISPR locus in strain crR6 by deleting cas1, cas2 and
csn2, genes which have been shown to be dispensable for CRISPR
targeting, yielding strain crR6M (FIG. 28A). This strain retained
the same properties of crR6 (FIG. 28B). To increase the efficiency
of Cas9-based editing and demonstrate that a template DNA of choice
can be used to control the mutation introduced, Applicants
co-transformed R6.sup.8232.5 cells with PCR products of the
wild-type srtA gene or the mutant R6.sup.370.1 target, either of
which should be resistant to cleavage by Cas9. This resulted in a
5- to 10-fold increase of the frequency of transformation compared
with genomic crR6 DNA alone (FIG. 23B). The efficiency of editing
was also substantially increased, with 8/8 transformants tested
containing a wild-type srtA copy and 7/8 containing the PAM
mutation present in the R6.sup.3701 target (FIG. 23B and FIG. 29A).
Taken together, these results showed the potential of genome
editing assisted by Cas9.
[0243] Analysis of Cas9 Target Requirements:
[0244] To introduce specific changes in the genome, one must use an
editing template carrying mutations that abolish Cas9-mediated
cleavage, thereby preventing cell death. This is easy to achieve
when the deletion of the target or its replacement by another
sequence (gene insertion) is sought. When the goal is to produce
gene fusions or to generate single-nucleotide mutations, the
abolishment of Cas9 nuclease activity will only be possible by
introducing mutations in the editing template that alter either the
PAM or the protospacer sequences. To determine the constraints of
CRISPR-mediated editing, Applicants performed an exhaustive
analysis of PAM and protospacer mutations that abrogate CRISPR
targeting.
[0245] Previous studies proposed that S. pyogenes Cas9 requires an
NGG PAM immediately downstream of the protospacer. However, because
only a very limited number of PAM-inactivating mutations have been
described so far, Applicants conducted a systematic analysis to
find all 5-nucleotide sequences following the protospacer that
eliminate CRISPR cleavage. Applicants used randomized
oligonucleotides to generate all possible 1,024 PAM sequences in a
heterogeneous PCR product that was transformed into crR6 or R6
cells. Constructs carrying functional PAMs were expected to be
recognized and destroyed by Cas9 in crR6 but not R6 cells (FIG.
24A). More than 2.times.10.sup.5 colonies were pooled together to
extract DNA for use as template for the co-amplification of all
targets. PCR products were deep sequenced and found to contain all
1,024 sequences, with coverage ranging from 5 to 42,472 reads (See
section "Analysis of deep sequencing data"). The functionality of
each PAM was estimated by the relative proportion of its reads in
the crR6 sample over the R6 sample. Analysis of the first three
bases of the PAM, averaging over the two last bases, clearly showed
that the NGG pattern was under-represented in crR6 transformants
(FIG. 24B). Furthermore, the next two bases had no detectable
effect on the NGG PAM (See section "Analysis of deep sequencing
data"), demonstrating that the NGGNN sequence was sufficient to
license Cas9 activity. Partial targeting was observed for NAG PAM
sequences (FIG. 24B). Also the NNGGN pattern partially inactivated
CRISPR targeting (Table G), indicating that the NGG motif can still
be recognized by Cas9 with reduced efficiency when shifted by 1 bp.
These data shed light onto the molecular mechanism of Cas9 target
recognition, and they revealed that NGG (or CCN on the
complementary strand) sequences are sufficient for Cas9 targeting
and that NGG to NAG or NNGGN mutations in the editing template
should be avoided. Owing to the high frequency of these
tri-nucleotide sequences (once every 8 bp), this means that almost
any position of the genome can be edited. Indeed, Applicants tested
ten randomly chosen targets carrying various PAMs and all were
found to be functional (FIG. 30).
[0246] Another way to disrupt Cas9-mediated cleavage is to
introduce mutations in the protospacer region of the editing
template. It is known that point mutations within the `seed
sequence` (the 8 to 10 protospacer nucleotides immediately adjacent
to the PAM) can abolish cleavage by CRISPR nucleases. However, the
exact length of this region is not known, and it is unclear whether
mutations to any nucleotide in the seed can disrupt Cas9 target
recognition. Applicants followed the same deep sequencing approach
described above to randomize the entire protospacer sequence
involved in base pair contacts with the crRNA and to determine all
sequences that disrupt targeting. Each position of the 20 matching
nucleotides (14) in the spc1 target present in R6.sup.8232.5 cells
(FIG. 23A) was randomized and transformed into crR6 and R6 cells
(FIG. 24A). Consistent with the presence of a seed sequence, only
mutations in the 12 nucleotides immediately upstream of the PAM
abrogated cleavage by Cas9 (FIG. 24C). However, different mutations
displayed markedly different effects. The distal (from the PAM)
positions of the seed (12 to 7) tolerated most mutations and only
one particular base substitution abrogated targeting. In contrast,
mutations to any nucleotide in the proximal positions (6 to 1,
except 3) eliminated Cas9 activity, although at different levels
for each particular substitution. At position 3, only two
substitutions affected CRISPR activity and with different strength.
Applicants concluded that, although seed sequence mutations can
prevent CRISPR targeting, there are restrictions regarding the
nucleotide changes that can be made in each position of the seed.
Moreover, these restrictions can most likely vary for different
spacer sequences. Therefore Applicants believe that mutations in
the PAM sequence, if possible, should be the preferred editing
strategy. Alternatively, multiple mutations in the seed sequence
may be introduced to prevent Cas9 nuclease activity.
[0247] Cas9-Mediated Genome Editing in S. pneumonia:
[0248] To develop a rapid and efficient method for targeted genome
editing, Applicants engineered strain crR6Rk, a strain in which
spacers can be easily introduced by PCR (FIG. 33). Applicants
decided to edit the .beta.-galactosidase (bgaA) gene of S.
pneumoniae, whose activity can be easily measured. Applicants
introduced alanine substitutions of amino acids in the active site
of this enzyme: R481A (R.fwdarw.A) and N563A,E564A (NE.fwdarw.AA)
mutations. To illustrate different editing strategies, Applicants
designed mutations of both the PAM sequence and the protospacer
seed. In both cases the same targeting construct with a crRNA
complementary to a region of the .beta.-galactosidase gene that is
adjacent to a TGG PAM sequence (CCA in the complementary strand,
FIG. 26) was used. The R.fwdarw.A editing template created a
three-nucleotide mismatch on the protospacer seed sequence (CGT to
GCA, also introducing a BtgZI restriction site). In the
NE.fwdarw.AA editing template Applicants simultaneously introduced
a synonymous mutation that created an inactive PAM (TGG to TTG)
along with mutations that are 218 nt downstream of the protospacer
region (AAT GAA to GCT GCA, also generating a TseI restriction
site). This last editing strategy demonstrated the possibility of
using a remote PAM to make mutations in places where a proper
target may be hard to choose. For example, although the S.
pneumoniae R6 genome, which has a 39.7% GC content, contains on
average one PAM motif every 12 bp, some PAM motifs are separated by
up to 194 bp (FIG. 33). In addition Applicants designed a
.DELTA.bgaA in-frame deletion of 6,664 bp. In all three cases,
co-transformation of the targeting and editing templates produced
10-times more kanamycin-resistant cells than co-transformation with
a control editing template containing wild-type bgaA sequences
(FIG. 25B). Applicants genotyped 24 transformants (8 for each
editing experiment) and found that all but one incorporated the
desired change (FIG. 25C). DNA sequencing also confirmed not only
the presence of the introduced mutations but also the absence of
secondary mutations in the target region (FIG. 29B and FIG. 29C).
Finally, Applicants measured .beta.-galactosidase activity to
confirm that all edited cells displayed the expected phenotype
(FIG. 25D).
[0249] Cas9-mediated editing can also be used to generate multiple
mutations for the study of biological pathways. Applicants decided
to illustrate this for the sortase-dependent pathway that anchors
surface proteins to the envelope of Gram-positive bacteria.
Applicants introduced a sortase deletion by co-transformation of a
chloramphenicol-resistant targeting construct and a .DELTA.srtA
editing template (FIG. 33A, FIG. 33B), followed by a .DELTA.bgaA
deletion using a kanamycin-resistant targeting construct that
replaced the previous one. In S. pneumoniae, .beta.-galactosidase
is covalently linked to the cell wall by sortase. Therefore,
deletion of srtA results in the release of the surface protein into
the supernatant, whereas the double deletion has no detectable
.beta.-galactosidase activity (FIG. 34C). Such a sequential
selection can be iterated as many times as required to generate
multiple mutations.
[0250] These two mutations may also be introduced at the same time.
Applicants designed a targeting construct containing two spacers,
one matching srtA and the other matching bgaA, and co-transformed
it with both editing templates at the same time (FIG. 25E). Genetic
analysis of transformants showed that editing occurred in 6/8 cases
(FIG. 25F). Notably, the remaining two clones each contained either
a .DELTA.srtA or a .DELTA.bgaA deletion, suggesting the possibility
of performing combinatorial mutagenesis using Cas9. Finally, to
eliminate the CRISPR sequences, Applicants introduced a plasmid
containing the bgaA target and a spectinomycin resistance gene
along with genomic DNA from the wild-type strain R6.
Spectinomycin-resistant transformants that retain the plasmid
eliminated the CRISPR sequences (FIG. 34A, FIG. 34D).
[0251] Mechanism and Efficiency of Editing:
[0252] To understand the mechanisms underlying genome editing with
Cas9, Applicants designed an experiment in which the editing
efficiency was measured independently of Cas9 cleavage. Applicants
integrated the ermAM erythromycin resistance gene in the srtA
locus, and introduced a premature stop codon using Cas9-mediated
editing (FIG. 33). The resulting strain (JEN53) contains an
ermAM(stop) allele and is sensitive to erythromycin. This strain
may be used to assess the efficiency at which the ermAM gene is
repaired by measuring the fraction of cells that restore antibiotic
resistance with or without the use of Cas9 cleavage. JEN53 was
transformed with an editing template that restores the wild-type
allele, together with either a kanamycin-resistant CRISPR construct
targeting the ermAM(stop) allele (CRISPR::ermAM(stop)) or a control
construct without a spacer (CRISPR::O) (FIG. 26A and FIG. 26B). In
the absence of kanamycin selection, the fraction of edited colonies
was on the order of 10.sup.-2 (erythromycin-resistant cfu/total
cfu) (FIG. 26C), representing the baseline frequency of
recombination without Cas9-mediated selection against unedited
cells. However, if kanamycin selection was applied and the control
CRISPR construct was co-transformed, the fraction of edited
colonies increased to about 10.sup.-1 (kanamycin- and
erythromycin-resistant cfu/kanamycin-resistant cfu) (FIG. 26C).
This result shows that selection for the recombination of the
CRISPR locus co-selected for recombination in the ermAM locus
independently of Cas9 cleavage of the genome, suggesting that a
subpopulation of cells is more prone to transformation and/or
recombination. Transformation of the CRISPR::ermAM(stop) construct
followed by kanamycin selection resulted in an increase of the
fraction of erythromycin-resistant, edited cells to 99% (FIG. 26C).
To determine if this increase is caused by the killing of
non-edited cells, Applicants compared the kanamycin-resistant
colony forming units (cfu) obtained after co-transformation of
JEN53 cells with the CRISPR::ermAM(stop) or CRISPR::O
constructs.
[0253] Applicants counted 5.3 times less kanamycin-resistant
colonies after transformation of the ermAM(stop) construct
(2.5.times.10.sup.4/4.7.times.10.sup.3, FIG. 35A), a result that
suggests that indeed targeting of a chromosomal locus by Cas9 leads
to the killing of non-edited cells. Finally, because the
introduction of dsDNA breaks in the bacterial chromosome is known
to trigger repair mechanisms that increase the rate of
recombination of the damaged DNA, Applicants investigated whether
cleavage by Cas9 induces recombination of the editing template.
Applicants counted 2.2 times more colonies after co-transformation
with the CRISPR::erm(stop) construct than with the CRISPR::O
construct (FIG. 26D), indicating that there was a modest induction
of recombination. Taken together, these results showed that
co-selection of transformable cells, induction of recombination by
Cas9-mediated cleavage and selection against non-edited cells, each
contributed to the high efficiency of genome editing in S.
pneumoniae.
[0254] As cleavage of the genome by Cas9 should kill non-edited
cells, one would not expect to recover any cells that received the
kanamycin resistance-containing Cas9 cassette but not the editing
template. However, in the absence of the editing template
Applicants recovered many kanamycin-resistant colonies after
transformation of the CRISPR::ermAM(stop) construct (FIG. 35A).
These cells that `escape` CRISPR-induced death produced a
background that determined a limit of the method. This background
frequency may be calculated as the ratio of
CRISPR::ermAM(stop)/CRISPR::O cfu, 2.6.times.10.sup.-3
(7.1.times.10.sup.1/2.7.times.10.sup.4) in this experiment, meaning
that if the recombination frequency of the editing template is less
than this value, CRISPR selection may not efficiently recover the
desired mutants above the background. To understand the origin of
these cells, Applicants genotyped 8 background colonies and found
that 7 contained deletions of the targeting spacer (FIG. 35B) and
one harbored a presumably inactivating mutation in Cas9 (FIG.
35C).
[0255] Genome Editing with Cas9 in E. coli:
[0256] The activation of Cas9 targeting through the chromosomal
integration of a CRISPR-Cas system is only possible in organisms
that are highly recombinogenic. To develop a more general method
that is applicable to other microbes, Applicants decided to perform
genome editing in E. coli using a plasmid-based CRISPR-Cas system.
Two plasmids were constructed: a pCas9 plasmid carrying the
tracrRNA, Cas9 and a chloramphenicol resistance cassette (FIG. 36),
and a pCRISPR kanamycin-resistant plasmid carrying the array of
CRISPR spacers. To measure the efficiency of editing independently
of CRISPR selection, Applicants sought to introduce an A to C
transversion in the rpsL gene that confers streptomycin resistance.
Applicants constructed a pCRISPR::rpsL plasmid harboring a spacer
that would guide Cas9 cleavage of the wild-type, but not the mutant
rpsL allele (FIG. 27B). The pCas9 plasmid was first introduced into
E. coli MG1655 and the resulting strain was co-transformed with the
pCRISPR::rpsL plasmid and W542, an editing oligonucleotide
containing the A to C mutation. streptomycin-resistant colonies
after transformation of the pCRISPR::rpsL plasmid were only
recovered, suggesting that Cas9 cleavage induces recombination of
the oligonucleotide (FIG. 37). However, the number of
streptomycin-resistant colonies was two orders of magnitude lower
than the number of kanamycin-resistant colonies, which are
presumably cells that escape cleavage by Cas9. Therefore, in these
conditions, cleavage by Cas9 facilitated the introduction of the
mutation, but with an efficiency that was not enough to select the
mutant cells above the background of `escapers`.
[0257] To improve the efficiency of genome editing in E. coli,
Applicants applied their CRISPR system with recombineering, using
Cas9-induced cell death to select for the desired mutations. The
pCas9 plasmid was introduced into the recombineering strain H1VIE63
(31), which contains the Gam, Exo and Beta functions of the
.quadrature.-red phage. The resulting strain was co-transformed
with the pCRISPR::rpsL plasmid (or a pCRISPR::O control) and the
W542 oligonucleotide (FIG. 27A). The recombineering efficiency was
5.3.times.10.sup.-5, calculated as the fraction of total cells that
become streptomycin-resistant when the control plasmid was used
(FIG. 27C). In contrast, transformation with the pCRISPR::rpsL
plasmid increased the percentage of mutant cells to 65.+-.14%
(FIGS. 27C and 29F). Applicants observed that the number of cfu was
reduced by about three orders of magnitude after transformation of
the pCRISPR::rpsL plasmid than the control plasmid
(4.8.times.10.sup.5/5.3.times.10.sup.2, FIG. 38A), suggesting that
selection results from CRISPR-induced death of non-edited cells. To
measure the rate at which Cas9 cleavage was inactivated, an
important parameter of Applicants' method, Applicants transformed
cells with either pCRISPR::rpsL or the control plasmid without the
W542 editing oligonucleotide (FIG. 38A). This background of CRISPR
`escapers`, measured as the ratio of pCRISPR::rpsL/pCRISPR::O cfu,
was 2.5.times.10.sup.-4 (1.2.times.10.sup.2/4.8.times.10.sup.5).
Genotyping eight of these escapers revealed that in all cases there
was a deletion of the targeting spacer (FIG. 38B). This background
was higher than the recombineering efficiency of the rpsL mutation,
5.3.times.10.sup.-5, which suggested that to obtain 65% of edited
cells, Cas9 cleavage must induce oligonucleotide recombination. To
confirm this, Applicants compared the number of kanamycin- and
streptomycin-resistant cfu after transformation of pCRISPR::rpsL or
pCRISPR::O (FIG. 27D). As in the case for S. pneumoniae, Applicants
observed a modest induction of recombination, about 6.7 fold
(2.0.times.10.sup.-4/3.0.times.10.sup.-5). Taken together, these
results indicated that the CRISPR system provided a method for
selecting mutations introduced by recombineering.
[0258] Applicants showed that CRISPR-Cas systems may be used for
targeted genome editing in bacteria by the co-introduction of a
targeting construct that killed wild-type cells and an editing
template that both eliminated CRISPR cleavage and introduced the
desired mutations. Different types of mutations (insertions,
deletions or scar-less single-nucleotide substitutions) may be
generated. Multiple mutations may be introduced at the same time.
The specificity and versatility of editing using the CRISPR system
relied on several unique properties of the Cas9 endonuclease: (i)
its target specificity may be programmed with a small RNA, without
the need for enzyme engineering, (ii) target specificity was very
high, determined by a 20 bp RNA-DNA interaction with low
probability of non-target recognition, (iii) almost any sequence
may be targeted, the only requirement being the presence of an
adjacent NGG sequence, (iv) almost any mutation in the NGG
sequence, as well as mutations in the seed sequence of the
protospacer, eliminates targeting.
[0259] Applicants showed that genome engineering using the CRISPR
system worked not only in highly recombinogenic bacteria such as S.
pneumoniae, but also in E. coli. Results in E. coli suggested that
the method may be applicable to other microorganisms for which
plasmids may be introduced. In E. coli, the approach complements
recombineering of mutagenic oligonucleotides. To use this
methodology in microbes where recombineering is not a possible, the
host homologous recombination machinery may be used by providing
the editing template on a plasmid. In addition, because accumulated
evidence indicates that CRISPR-mediated cleavage of the chromosome
leads to cell death in many bacteria and archaea, it is possible to
envision the use of endogenous CRISPR-Cas systems for editing
purposes.
[0260] In both S. pneumoniae and E. coli, Applicants observed that
although editing was facilitated by a co-selection of transformable
cells and a small induction of recombination at the target site by
Cas9 cleavage, the mechanism that contributed the most to editing
was the selection against non-edited cells. Therefore the major
limitation of the method was the presence of a background of cells
that escape CRISPR-induced cell death and lack the desired
mutation. Applicants showed that these `escapers` arose primarily
through the deletion of the targeting spacer, presumably after the
recombination of the repeat sequences that flank the targeting
spacer. Future improvements may focus on the engineering of
flanking sequences that can still support the biogenesis of
functional crRNAs but that are sufficiently different from one
another to eliminate recombination. Alternatively, the direct
transformation of chimeric crRNAs may be explored. In the
particular case of E. coli, the construction of the CRISPR-Cas
system was not possible if this organism was also used as a cloning
host. Applicants solved this issue by placing Cas9 and the tracrRNA
on a different plasmid than the CRISPR array. The engineering of an
inducible system may also circumvent this limitation.
[0261] Although new DNA synthesis technologies provide the ability
to cost-effectively create any sequence with a high throughput, it
remains a challenge to integrate synthetic DNA in living cells to
create functional genomes. Recently, the co-selection MAGE strategy
was shown to improve the mutation efficiency of recombineering by
selecting a subpopulation of cells that has an increased
probability to achieve recombination at or around a given locus. In
this method, the introduction of selectable mutations is used to
increase the chances of generating nearby non-selectable mutations.
As opposed to the indirect selection provided by this strategy, the
use of the CRISPR system makes it possible to directly select for
the desired mutation and to recover it with a high efficiency.
These technologies add to the toolbox of genetic engineers, and
together with DNA synthesis, they may substantially advance both
the ability to decipher gene function and to manipulate organisms
for biotechnological purposes. Two other studies also relate to
CRISPR-assisted engineering of mammalian genomes. It is expected
that these crRNA-directed genome editing technologies may be
broadly useful in the basic and medical sciences.
[0262] Strains and Culture Conditions.
[0263] S. pneumoniae strain R6 was provided by Dr. Alexander
Tomasz. Strain crR6 was generated in a previous study. Liquid
cultures of S. pneumoniae were grown in THYE medium (30 g/l
Todd-Hewitt agar, 5 g/l yeast extract). Cells were plated on
tryptic soy agar (TSA) supplemented with 5% defibrinated sheep
blood. When appropriate, antibiotics were added as followings:
kanamycin (400 .mu.g/ml), chloramphenicol (5 .mu.g/ml),
erythromycin (1 .mu.g/ml) streptomycin (100 .mu.g/ml) or
spectinomycin (100 .mu.g/ml). Measurements of .beta.-galactosidase
activity were made using the Miller assay as previously
described.
[0264] E. coli strains MG1655 and HME63 (derived from MG1655,
.DELTA.(argF-lac) U169 .lamda., cI857 .DELTA.cro-bioA galK tyr 145
UAG mutS< >amp) (31) were provided by Jeff Roberts and Donald
Court, respectively. Liquid cultures of E. coli were grown in LB
medium (Difco). When appropriate, antibiotics were added as
followings: chloramphenicol (25 .mu.g/ml), kanamycin (25 .mu.g/ml)
and streptomycin (50 .mu.g/ml).
[0265] S. pneumoniae Transformation.
[0266] Competent cells were prepared as described previously (23).
For all genome editing transformations, cells were gently thawed on
ice and resuspended in 10 volumes of M2 medium supplemented with
100 ng/ml of competence-stimulating peptide CSP1(40), and followed
by addition of editing constructs (editing constructs were added to
cells at a final concentration between 0.7 ng/.mu.1 to 2.5
.mu.g/ul). Cells were incubated 20 min at 37.degree. C. before the
addition of 2 .mu.l of targeting constructs and then incubated 40
min at 37.degree. C. Serial dilutions of cells were plated on the
appropriate medium to determine the colony forming units (cfu)
count.
[0267] E. coli Lambda-Red Recombineering.
[0268] Strain HME63 was used for all recombineering experiments.
Recombineering cells were prepared and handled according to a
previously published protocol (6). Briefly, a 2 ml overnight
culture (LB medium) inoculated from a single colony obtained from a
plate was grown at 30.degree. C. The overnight culture was diluted
100-fold and grown at 30.degree. C. with shaking (200 rpm) until
the OD.sub.600 is from 0.4-0.5 (approximately 3 hrs). For
Lambda-red induction, the culture was transferred to a 42.degree.
C. water bath to shake at 200 rpm for 15 min. Immediately after
induction, the culture was swirled in an ice-water slurry and
chilled on ice for 5-10 min. Cells were then washed and aliquoted
according to the protocol. For electro-transformation, 50 .mu.l of
cells were mixed with 1 mM of salt-free oligos (IDT) or 100-150 ng
of plasmid DNA (prepared by QIAprep Spin Miniprep Kit, Qiagen).
Cells were electroporated using 1 mm Gene Pulser cuvette (Bio-rad)
at 1.8 kV and were immediately resuspended in 1 ml of room
temperature LB medium. Cells were recovered at 30.degree. C. for
1-2 hrs before being plated on LB agar with appropriate antibiotic
resistance and incubated at 32.degree. C. overnight.
[0269] Preparation of S. pneumoniae Genomic DNA.
[0270] For transformation purposes, S. pneumoniae genomic DNA was
extracted using the Wizard Genomic DNA Purification Kit, following
instructions provided by the manufacturer (Promega). For genotyping
purposes, 700 ul of overnight S. pneumoniae cultures were pelleted,
resuspended in 60 ul of lysozyme solution (2 mg/ml) and incubated
30 min at 37.degree. C. The genomic DNA was extracted using QIAprep
Spin Miniprep Kit (Qiagen).
[0271] Strain Construction.
[0272] All primers used in this study are provided in Table G. To
generate S. pneumoniae crR6M, an intermediate strain, LAM226, was
made. In this strain the aphA-3 gene (providing kanamycin
resistance) adjacent to the CRISPR array of S. pneumoniae crR6
strain was replaced by a cat gene (providing chloramphenicol
resistance). Briefly, crR6 genomic DNA was amplified using primers
L448/L444 and L447/L481, respectively. The cat gene was amplified
from plasmid pC194 using primers L445/L446. Each PCR product was
gel-purified and all three were fused by SOEing PCR with primers
L448/L481. The resulting PCR product was transformed into competent
S. pneumoniae crR6 cells and chloramphenicol-resistant
transformants were selected. To generate S. pneumoniae crR6M, S.
pneumoniae crR6 genomic DNA was amplified by PCR using primers
L409/L488 and L448/L481, respectively. Each PCR product was
gel-purified and they were fused by SOEing PCR with primers
L409/L481. The resulting PCR product was transformed into competent
S. pneumoniae LAM226 cells and kanamycin-resistant transformants
were selected.
[0273] To generate S. pneumoniae crR6Rc, S. pneumoniae crR6M
genomic DNA was amplified by PCR using primers L430/W286, and S.
pneumoniae LAM226 genomic DNA was amplified by PCR using primers
W288/L481. Each PCR product was gel-purified and they were fused by
SOEing PCR with primers L430/L481. The resulting PCR product was
transformed into competent S. pneumoniae crR6M cells and
chloramphenicol-resistant transformants were selected.
[0274] To generate S. pneumoniae crR6Rk, S. pneumoniae crR6M
genomic DNA was amplified by PCR using primers L430/W286 and
W287/L481, respectively. Each PCR product was gel-purified and they
were fused by SOEing PCR with primers L430/L481. The resulting PCR
product was transformed into competent S. pneumoniae crR6Rc cells
and kanamycin-resistant transformants were selected.
[0275] To generate JEN37, S. pneumoniae crR6Rk genomic DNA was
amplified by PCR using primers L430/W356 and W357/L481,
respectively. Each PCR product was gel-purified and they were fused
by SOEing PCR with primers L430/L481. The resulting PCR product was
transformed into competent S. pneumoniae crR6Rc cells and
kanamycin-resistant transformants were selected.
[0276] To generate JEN38, R6 genomic DNA was amplified using
primers L422/L461 and L459/L426, respectively. The ermAM gene
(specifying erythromycin resistance) was amplified from plasmid
pFW15.sup.43 using primers L457/L458. Each PCR product was
gel-purified and all three were fused by SOEing PCR with primers
L422/L426. The resulting PCR product was transformed into competent
S. pneumoniae crR6Rc cells and erythromycin-resistant transformants
were selected.
[0277] S. pneumoniae JEN53 was generated in two steps. First JEN43
was constructed as illustrated in FIG. 33. JEN53 was generated by
transforming genomic DNA of JEN25 into competent JEN43 cells and
selecting on both chloramphenicol and erythromycin.
[0278] To generate S. pneumoniae JEN62, S. pneumoniae crR6Rk
genomic DNA was amplified by PCR using primers W256/W365 and
W366/L403, respectively. Each PCR product was purified and ligated
by Gibson assembly. The assembly product was transformed into
competent S. pneumoniae crR6Rc cells and kanamycin-resistant
transformants were selected.
[0279] Plasmid Construction.
[0280] pDB97 was constructed through phosphorylation and annealing
of oligonucleotides B296/B297, followed by ligation in pLZ12spec
digested by EcoRI/BamHI. Applicants fully sequenced pLZ12spec and
deposited its sequence in genebank (accession: KC112384).
[0281] pDB98 was obtained after cloning the CRISPR leader sequence
was cloned together with a repeat-spacer-repeat unit into
pLZ12spec. This was achieved through amplification of crR6Rc DNA
with primers B298/B320 and B299/B321, followed by SOEing PCR of
both products and cloning in pLZ12spec with restriction sites
BamHI/EcoRI. In this way the spacer sequence in pDB98 was
engineered to contain two BsaI restriction sites in opposite
directions that allow for the scar-less cloning of new spacers.
[0282] pDB99 to pDB108 were constructed by annealing of
oligonucleotides B300/B301 (pDB99), B302/B303 (pDB100), B304/B305
(pDB101), B306/B307 (pDB102), B308/B309 (pDB103), B310/B311
(pDB104), B312/B313 (pDB105), B314/B315 (pDB106), B315/B317
(pDB107), B318/B319 (pDB108), followed by ligation in pDB98 cut by
BsaI.
[0283] The pCas9 plasmid was constructed as follow. Essential
CRISPR elements were amplified from Streptococcus pyogenes SF370
genomic DNA with flanking homology arms for Gibson Assembly. The
tracrRNA and Cas9 were amplified with oligos HC008 and HC010. The
leader and CRISPR sequences were amplified HC011/HC014 and
HC015/HC009, so that two BsaI type IIS sites were introduced in
between two direct repeats to facilitate easy insertion of
spacers.
[0284] pCRISPR was constructed by subcloning the pCas9 CRISPR array
in pZE21-MCS1 through amplification with oligos B298+B299 and
restriction with EcoRI and BamHI. The rpsL targeting spacer was
cloned by annealing of oligos B352+B353 and cloning in the BsaI cut
pCRISPR giving pCRISPR::rpsL.
[0285] Generation of Targeting and Editing Constructs.
[0286] Targeting constructs used for genome editing were made by
Gibson assembly of Left PCRs and Right PCRs (Table G). Editing
constructs were made by SOEing PCR fusing PCR products A (PCR A),
PCR products B (PCR B) and PCR products C (PCR C) when applicable
(Table G). The CRISPR::O and CRISPR::ermAM(stop) targeting
constructs were generated by PCR amplification of JEN62 and crR6
genomic DNA respectively, with oligos L409 and L481.
[0287] Generation of Targets with Randomized PAM or Protospacer
Sequences.
[0288] The 5 nucleotides following the spacer 1 target were
randomized through amplification of R6.sup.8232.5 genomic DNA with
primers W377/L426. This PCR product was then assembled with the cat
gene and the srtA upstream region that were amplified from the same
template with primers L422/W376. 80 ng of the assembled DNA was
used to transform strains R6 and crR6. Samples for the randomized
targets were prepared using the following primers: B280-B290/L426
to randomize bases 1-10 of the target and B269-B278/L426 to
randomize bases 10-20. Primers L422/B268 and L422/B279 were used to
amplify the cat gene and srtA upstream region to be assembled with
the first and last 10 PCR products respectively. The assembled
constructs were pooled together and 30 ng was transformed in R6 and
crR6. After transformation, cells were plated on chloramphenicol
selection. For each sample more than 2.times.10.sup.5 cells were
pooled together in 1 ml of THYE and genomic DNA was extracted with
the Promega Wizard kit. Primers B250/B251 were used to amplify the
target region. PCR products were tagged and run on one Illumina
MiSeq paired-end lane using 300 cycles.
[0289] Analysis of Deep Sequencing Data.
[0290] Randomized PAM: For the randomized PAM experiment 3,429,406
reads were obtained for crR6 and 3,253,998 for R6. It is expected
that only half of them will correspond to the PAM-target while the
other half will sequence the other end of the PCR product.
1,623,008 of the crR6 reads and 1,537,131 of the R6 reads carried
an error-free target sequence. The occurrence of each possible PAM
among these reads is shown in supplementary file. To estimate the
functionality of a PAM, its relative proportion in the crR6 sample
over the R6 sample was computed and is denoted r.sub.ijklm where
I,j,k,l,m are one of the 4 possible bases. The following
statistical model was constructed:
log(r.sub.ijklm=.mu.+b2.sub.i+b3.sub.j+b4.sub.k+b2b3.sub.i,j+b3b4.sub.j,-
k+.epsilon..sub.ijklm,
[0291] where .epsilon. is the residual error, b2 is the effect of
the 2.sup.nd base of the PAM, b3 of the third, b4 of the fourth,
b2b3 is the interaction between the second and third bases, b3b4
between the third and fourth bases. An analysis of variance was
performed:
TABLE-US-00007 Anova table Df Sum Sq Mean Sq F value Pr(>F) b3 3
151.693 50.564 601.8450 <2.2e-16 *** b2 3 90.521 30.174 359.1454
<2.2e-16 *** b4 3 1.881 0.627 7.4623 6.070e-05 *** b3:b2 9
228.940 25.438 302.7738 <2.2e-16 *** b3:b4 9 3.010 0.334 3.9809
5.227e-05 *** Residuals 996 83.680 0.084
[0292] When added to this model, b1 or b5 do not appear to be
significant and other interactions than the ones included can also
be discarded. The model choice was made through successive
comparisons of more or less complete models using the anova method
in R. Tukey's honest significance test was used to determine if
pairwise differences between effects are significant.
[0293] NGGNN patterns are significantly different from all other
patterns and carry the strongest effect (see table below).
[0294] In order to show that positions 1, 4 or 5 do not affect the
NGGNN pattern Applicants looked at theses sequences only. Their
effect appears to be normally distributed (see QQ plot in FIG. 71),
and model comparisons using the anova method in R shows that the
null model is the best one, i.e. there is no significant role of
b1, b4 and b5.
[0295] Model Comparison Using the Anova Method in R for the NGGNN
Sequences
TABLE-US-00008 Model 1: ratio.log~1 Model 2: ratio.log~b1 + b4 + b5
Res. Df RSS DF Sum of Sq F Pr(>F) 1 63 14.579 2 54 11.295 9
3.2836 1.7443 0.01013
[0296] Partial interference of NAGNN and NNGGN patterns
[0297] NAGNN patterns are significantly different from all other
patterns but carry a much smaller effect than NGGNN (see Tukey's
honest significance test below).
[0298] Finally, NTGGN and NCGGN patterns are similar and show
significantly more CRISPR interference than NTGHN and NCGHN
patterns (where H is A, T or C), as shown by a bonferroni adjusted
pairwise student-test.
[0299] Pairwise Comparisons of the Effect of b4 on NYGNN Sequences
Using t Tests with Pooled SD
TABLE-US-00009 Data: b4 A C G C 1.00 -- -- G 9.2e-05 2.4e-06 -- T
0.31 1.00 1.2e-08
[0300] Taken together, these results allow concluding that NNGGN
patterns in general produce either a complete interference in the
case of NGGGN, or a partial interference in the case of NAGGN,
NTGGN or NCGGN.
[0301] Tukey multiple comparisons of means: 95% family-wise
confidence level
TABLE-US-00010 diff 1wr upr p adj $b2:b3 G:G-A:A -2.76475 -2.94075
-2.58875 <1E-07 G:G-C:A -2.79911 -2.97511 -2.62311 <1E-07
G:G-T:A -2.7809 -2.9569 -2.6049 <1E-07 G:G-A:C -2.81643 -2.99244
-2.64043 <1E-07 G:G-C:C -2.77903 -2.95504 -2.60303 <1E-07
G:G-G:C -2.64867 -2.82468 -2.47267 <1E-07 G:G-T:C -2.79718
-2.97319 -2.62118 <1E-07 G:G-A:G -2.67068 -2.84668 -2.49463
<1E-07 G:G-C:G -2.73525 -2.91135 -2.55925 <1E-07 G:G-T:G
-2.7976 -2.62159 -2.9736 <1E-07 G:G-A:T -2.76727 -2.59127
-2.94328 <1E-07 G:G-C:T -2.84114 -2.66513 -3.01714 <1E-07
G:G-G:T -2.76409 -2.58809 -2.94009 <1E-07 G:G-T:T -2.76781
-2.59181 -2.94381 <1E-07 G:G-G:A -2.13964 -2.31565 -1.96364
<1E-07 G:A-A:A -0.62511 -0.80111 -0.4491 <1E-07 G:A-C:A
-0.65947 -0.83547 -0.48346 <1E-07 G:A-T:A -0.64126 -0.46525
-0.81726 <1E-07 G:A-A:C -0.67679 -0.50078 -0.85279 <1E-07
G:A-C:C -0.63939 -0.46339 -0.81539 <1E-07 G:A-G:C -0.50903
-0.33303 -0.68503 <1E-07 G:A-T:C -0.65754 -0.48154 -0.83354
<1E-07 G:A-A:G -0.53104 -0.35503 -0.70704 <1E-07 G:A-C:G
-0.59561 -0.4196 -0.77161 <1E-07 G:A-T:G -0.65795 -0.48195
-0.83396 <1E-07 G:A-A:T -0.62763 -0.45163 -0.80363 <1E-07
G:A-C:T -0.70149 -0.52549 -0.8775 <1E-07 G:A-G:T -0.62445
-0.44844 -0.80045 <1E-07 G:A-T:T -0.62817 -0.45216 -0.80417
<1E-07 $b3:b4 G:G-G:A -0.33532 -0.51133 -0.15932 <1E-07
G:G-G:C -0.18118 -0.35719 -0.00518 0.036087 G:G-G:T -0.31626
-0.14026 -0.49226 <1E-07
[0302] Randomized Target
[0303] For the randomized target experiment 540,726 reads were
obtained for crR6 and 753,570 for R6. As before, only half of the
reads are expected to sequence the interesting end of the PCR
product. After filtering for reads that carry a target that is
error-free or with a single point mutation, 217,656 and 353,141
reads remained for crR6 and R6 respectively. The relative
proportion of each mutant in the crR6 sample over the R6 sample was
computed (FIG. 24C). All mutations outside of the seed sequence
(13-20 bases away from the PAM) show full interference. Those
sequences were used as a reference to determine if other mutations
inside the seed sequence can be said to significantly disrupt
interference. A normal distribution was fitted to theses sequences
using the fitdistr function of the MASS R package. The 0.99
quantile of the fitted distribution is shown as a dotted line in
FIG. 24C. FIG. 72 shows a histogram of the data density with fitted
normal distribution (black line) and 0.99 quantile (dotted
line).
TABLE-US-00011 TABLE F Relative abundance of PAM sequences in the
crR6/R6 samples averaged over bases 1 and 5. 2nd position A C G T
4th position A AAA 1.04 ACA 1.12 AGA 0.73 ATA 1.10 A AAC 1.07 ACC
1.04 AGC 0.64 ATC 0.97 C AAG 1.00 ACG 1.09 AGG 0.61 ATG 1.07 G AAT
0.98 ACT 1.02 AGT 0.65 ATT 1.01 T C CAA 1.05 CCA 1.05 CGA 0.99 CTA
1.07 A CAC 1.04 CCC 1.02 CGC 1.08 CTC 1.04 C CAG 1.08 CCG 1.08 CGG
0.81 CTG 1.05 G CAT 1.13 CCT 1.05 CGT 1.07 CTT 1.08 T G GAA 0.97
GCA 1.05 GGA 0.08 GTA 0.99 A GAC 0.92 GCC 1.00 GGC 0.05 GTC 1.15 C
GAG 0.96 GCG 0.98 GGG 0.07 GTG 0.98 G GAT 0.98 GCT 0.99 GGT 0.06
GTT 1.05 T T TAA 1.08 TCA 1.16 TGA 1.05 TTA 1.14 A TAC 1.00 TCC
1.08 TGC 1.08 TTC 1.05 C TAG 1.02 TCG 1.11 TGG 0.77 TTG 1.01 G TAT
1.01 TCT 1.12 TGT 1.21 TTT 1.02 T
TABLE-US-00012 TABLE G Primers used in this study (SEQ ID NOS
68-183, respectively, in order of appearance). Primer Sequence
5'-3' B217 TCCTAGCAGGATTTCTGATATTACTGTCACGTTTTAGA GCTATGCTGTTTTGA
B218 GTGACAGTAATATCAGAAATCCTGCTAGGAGTTTTGGG ACCATTCAAAACAGC B229
GGGTTTCAAGTCTTTGTAGCAAGAG B230 GCCAATGAACGGGAACCCTTGGTC B250
NNNNGACGAGGCAATGGCTGAAATC B251 NNNNTTATTTGGCTCATATTTGCTG B255
CTTTACACCAATCGCTGCAACAGAC B256
CAAAATTTCTAGTCTTCTTTGCCTTTCCCCATAAAACC CTCCTTA B257
AGGGTTTTATGGGGAAAGGCAAAGAAGACTAGAAATTT TGATACC B258
CTTACGGTGCATAAAGTCAATTTCC B269 TGGCTCGATTTCAGCCATTGC B270
CTTTGACGAGGCAATGGCTGAAATCGAGCCAANAAAGC GCAAG B271
CTTTGACGAGGCAATGGCTGAAATCGAGCCAAAMAAGC GCAAG B272
CTTTGACGAGGCAATGGCTGAAATCGAGCCAAAANAGC GCAAG B273
CTTTGACGAGGCAATGGCTGAAATCGAGCCAAAAANGC GCAAG B274
CTTTGACGAGGCAATGGCTGAAATCGAGCCAAAAAANC GCAAG B275
CTTTGACGAGGCAATGGCTGAAATCGAGCCAAAAAAGN GCAAG B276
CTTTGACGAGGCAATGGCTGAAATCGAGCCAAAAAAGC NCAAGAAG B277
CTTTGACGAGGCAATGGCTGAAATCGAGCCAAAAAAGC GNAAGAAG B278
CTTTGACGAGGCAATGGCTGAAATCGAGCCAAAAAAGC GCNAGAAG B279
GCGCTTTTTTGGCTCGATTTCAG B280 CAATGGCTGAAATCGAGCCAAAAAAGCGCANGAAGAAA
TC B281 CAATGGCTGAAATCGAGCCAAAAAAGCGCAANAAGAAA TC B282
CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGNAGAAA TC B283
CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGANGAAA TC B284
CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGAANAAA TC B285
CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGAAGNAA TCAACC B286
CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGAAGANA TCAACC B287
CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGAAGAAN TCAACC B288
CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGAAGAAA NCAACC B289
CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGAAGAAA TNAACCAGC B290
CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGAAGAAA TCNACCAGC B296
gatccTCCATCCGTACAACCCACAACCCTGg B297
aattcCAGGGTTGTGGGTTGTACGGATGGAg B298
CATGGATCCTATTTCTTAATAACTAAAAATATGG B299
CATGAATTCAACTCAACAAGTCTCAGTGTGCTG B300
AAACATTTTTTCTCCATTTAGGAAAAAGGATGCTG B301
AAAACAGCATCCTTTTTCCTAAATGGAGAAAAAAT B302
AAACCTTAAATCAGTCACAAATAGCAGCAAAATTG B303
AAAACAATTTTGCTGCTATTTGTGACTGATTTAAG B304
AAACTTTTCATCATACGACCAATCTGCTTTATTTG B305
AAAACAAATAAAGCAGATTGGTCGTATGATGAAAA B306
AAACTCGTCCAGAAGTTATCGTAAAAGAAATCGAG B307
AAAACTCGATTTCTTTTACGATAACTTCTGGACGA B308
AAACAATCTCTCCAAGGTTTCCTTAAAAATCTCTG B309
AAAACAGAGATTTTTAAGGAAACCTTGGAGAGATT B310
AAACGCCATCGTCAGGAAGAAGCTATGCTTGAGTG B311
AAAACACTCAAGCATAGCTTCTTCCTGACGATGGC B312
AAACATCTCTATACTTATTGAAATTTCTTTGTATG B313
AAAACATACAAAGAAATTTCAATAAGTATAGAGAT B314
AAACTAGCTGTGATAGTCCGCAAAACCAGCCTTCG B315
AAAACGAAGGCTGGTTTTGCGGACTATCACAGCTA B316
AAACATCGGAAGGTCGAGCAAGTAATTATCTTTTG B317
AAAACAAAAGATAATTACTTGCTCGACCTTCCGAT B318
AAACAAGATGGTATCGCAAAGTAAGTGACAATAAG B319
AAAACTTATTGTCACTTACTTTGCGATACCATCTT B320
GAGACCTTTGAGCTTCCGAGACTGGTCTCAGTTTTGGG ACCATTCAAAACAG B321
TGAGACCAGTCTCGGAAGCTCAAAGGTCTCGTTTTAGA GCTATGCTGTTTTG B352
aaacTACTTTACGCAGCGCGGAGTTCGGTTTTTTg B353
aaaacAAAAAACCGAACTCCGCGCTGCGTAAAGTA HC008_SP
ATGCCGGTACTGCCGGGCCTCTTGCGGGATTACGAAAT CATCCTG HC009_SP
GTGACTGGCGATGCTGTCGGAATGGACGATCACACTAC TCTTCTT HC010_SP
TTAAGAAATAATCTTCATCTAAAATATACTTCAGTCAC CTCCTAGCTGAC HC011_SP
ATTGATTTGAGTCAGCTAGGAGGTGACTGAAGTATATT TTAGATGAAG HC014_SP
GAGACCTTTGAGCTTCCGAGACTGGTCTCAGTTTTGGG
ACCATTCAAAACAGCATAGCTCTAAAACCTCGTAGACT ATTTTTGTC HC015_SP
GAGACCAGTCTCGGAAGCTCAAAGGTCTCGTTTTAGAG
CTATGCTGTTTTGAATGGTCCCAAAACTTCAGCACACT GAGACTTG L403
AGTCATCCCAGCAACAAATGG L409 CGTGGTAAATCGGATAACGTTCCAAGTGAAG L422
Tgctcttcttcacaaacaaggg L426 AAGCCAAAGTTTGGCACCACC L430
GTAGCTTATTCAGTCCTAGTGG L444 CGTTTGTTGAACTAATGGGTGCAAATTACGAATCTTCT
CCTGACG L445 CGTCAGGAGAAGATTCGTAATTTGCACCCATTAGTTCA ACAAACG L446
GATATTATGGAGCCTATTTTTGTGGGTTTTTAGGCATA AAACTATATG L447
CATATAGTTTTATGCCTAAAAACCcACAAAAATAGGCT CCATAATATC L448
ATTATTTCTTAATAACTAAAAATATGG L457 CGTgtacaattgctagcgtacggc L458
GCACCGGTGATCACTAGTCCTAGG L459
cctaggactagtgatcaccggtGCAAATATGAGCCAAA TAAATATAT L461
GCCGTACGCTAGCAATTGTACACGTTTGTTGAACTAAT GGGTGC L481
TTCAAATTTTCCCATTTGATTCTCC L488
CCATATTTTTAGTTATTAAGAAATAATACCAGCCATCA GTCACCTCC W256
AGACGATTCAATAGACAATAAGG W286 GTTTTGGGACCATTCAAAACAGCATAGCTCTAAAACCT
CGTAGAC W287 GCTATGCTGTTTTGAATGGTCCCAAAACcattatttta acacacgaggtg
W288 GCTATGCTGTTTTGAATGGTCCCAAAACGCACCCATTA GTTCAACAAACG W326
AATTCTTTTCTTCATCATCGGTC W327 AAGAAAGAATGAAGATTGTTCATG W341
GGTACTAATCAAAATAGTGAGGAGG W354 GTTTTTCAAAATCTGCGGTTGCG W355
AAAAATTGAAAAAATGGTGGAAACAC W356
ATTTCGTAAACGGTATCGGTTTCTTTTAAAGTTTTGGG ACCATTCAAAACAGC W357
TTTAAAAGAAACCGATACCGTTTACGAAATGTTTTAGA GCTATGCTGTTTTGA W365
AAACGGTATCGGTTTCTTTTAAATTCAATTGTTTTGGG ACCATTCAAAACAGC W366
AATTGAATTTAAAAGAAACCGATACCGTTTGTTTTAGA GCTATGCTGTTTTGA W370
GTTCCTTAAACCAAAACGGTATCGGTTTCTTTTAAATT C W371
GAAACCGATACCGTTTTGGTTTAAGGAACAGGTAAAGG GCATTTAAC
W376 CGATTTCAGCCATTGCCTCGTC W377
GCCTTTGACGAGGCAATGGCTGAAATCGNNNNNAAAAA GCGCAAGAAGAAATCAAC W391
TCCGTACAACCCACAACCCTGCTAGTGAGCGTTTTGGG ACCATTCAAAACAGC W392
GCTCACTAGCAGGGTTGTGGGTTGTACGGAGTTTTAGA GCTATGCTGTTTTGA W393
TTGTTGCCACTCTTCCTTCTTTC W397 CAGGGTTGTGGGTTGTTGCGATGGAGTTAACTCCCATC
TCC W398 GGGAGTTAACTCCATCGCAACAACCCACAACCCTGCTA GTG W403
GTGGTATCTATCGTGATGTGAC W404 TTACCGAAACGGAATTTATCTGC W405
AAAGCTAGAGTTCCGCAATTGG W431 GTGGGTTGTACGGATTGAGTTAACTCCCATCTCCTTC
W432 GATGGGAGTTAACTCAATCCGTACAACCCACAACCCTG W433
GCTTCACCTATTGCAGCACCAATTGACCACATGAAGAT AG W434
GTGGTCAATTGGTGCTGCAATAGGTGAAGCTAATGGTG ATG W463
CTGATTTGTATTAATTTTGAGACATTATGCTTCACCTT C W464
GCATAATGTCTCAAAATTAATACAAATCAGTGAAATCA TG W465
GTTTTGGGACCATTCAAAACAGCATAGCTCTAAAACGT GACAGTAATATCAG W466
GTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAACGC TCACTAGCAGGGTTG W542
ATACTTTACGCAGCGCGGAGTTCGGTTTTgTAGGAGTG GTAGTATATACACGAGTACAT
TABLE-US-00013 TABLE H Design of targeting and editing constructs
used in this study (SEQ ID NOS 184, 184, 184, 185 and 186,
respectively, in order of appearance). LEFT RIGHT PCR PCR PCR PCR
PCR PAM A B C PCR TGG TGG TGG TGG TGG TGG indicates data missing or
illegible when filed
Example 6: Optimization of the Guide RNA for Streptococcus pyogenes
Cas9 (Referred to as SpCas9)
[0304] Applicants mutated the tracrRNA and direct repeat sequences,
or mutated the chimeric guide RNA to enhance the RNAs in cells.
[0305] The optimization is based on the observation that there were
stretches of thymines (Ts) in the tracrRNA and guide RNA, which
might lead to early transcription termination by the pol 3
promoter. Therefore Applicants generated the following optimized
sequences. Optimized tracrRNA and corresponding optimized direct
repeat are presented in pairs.
TABLE-US-00014 Optimized tracrRNA 1 (mutation underlined): (SEQ ID
NO: 187) GGAACCATTCAtAACAGCATAGCAAGTTAtAATAAGGCTAGTCCGTTAT
CAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTT Optimized direct repeat 1
(mutation underlined): (SEQ ID NO: 188)
GTTaTAGAGCTATGCTGTTaTGAATGGTCCCAAAAC Optimized tracrRNA 2 (mutation
underlined): (SEQ ID NO: 189)
GGAACCATTCAAtACAGCATAGCAAGTTAAtATAAGGCTAGTCCGTTAT
CAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTT Optimized direct repeat 2
(mutation underlined): (SEQ ID NO: 190)
GTaTTAGAGCTATGCTGTaTTGAATGGTCCCAAAAC Applicants also optimized the
chimeric guideRNA for optimal activity in eukaryotic cells.
Original guide RNA: (SEQ ID NO: 191)
NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAA
TAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTT TTTTT Optimized
chimeric guide RNA sequence 1: (SEQ ID NO: 192)
NNNNNNNNNNNNNNNNNNNNGTATTAGAGCTAGAAATAGCAAGTTAATA
TAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTT TTTTT Optimized
chimeric guide RNA sequence 2: (SEQ ID NO: 193)
NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTATGCTGTTTTGGAAACAA
AACAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA
GTGGCACCGAGTCGGTGCTTTTTTT Optimized chimeric guide RNA sequence 3:
(SEQ ID NO: 194) NNNNNNNNNNNNNNNNNNNNGTATTAGAGCTATGCTGTATTGGAAACAA
TACAGCATAGCAAGTTAATATAAGGCTAGTCCGTTATCAACTTGAAAAA
GTGGCACCGAGTCGGTGCTTTTTTT
[0306] Applicants showed that optimized chimeric guide RNA works
better as indicated in FIG. 3. The experiment was conducted by
co-transfecting 293FT cells with Cas9 and a U6-guide RNA DNA
cassette to express one of the four RNA forms shown above. The
target of the guide RNA is the same target site in the human Emx1
locus: "GTCACCTCCAATGACTAGGG (SEQ ID NO: 195)"
Example 7: Optimization of Streptococcus Thermophiles MD-9 CRISPR1
Cas9 (Referred to as St1Cas9)
[0307] Applicants designed guide chimeric RNAs as shown in FIG.
4.
[0308] The St1Cas9 guide RNAs can undergo the same type of
optimization as for SpCas9 guide RNAs, by breaking the stretches of
poly thymines (Ts)
Example 8: Cas9 Diversity and Mutations
[0309] The CRISPR-Cas system is an adaptive immune mechanism
against invading exogenous DNA employed by diverse species across
bacteria and archaea. The type II CRISPR-Cas9 system consists of a
set of genes encoding proteins responsible for the "acquisition" of
foreign DNA into the CRISPR locus, as well as a set of genes
encoding the "execution" of the DNA cleavage mechanism; these
include the DNA nuclease (Cas9), a non-coding transactivating
cr-RNA (tracrRNA), and an array of foreign DNA-derived spacers
flanked by direct repeats (crRNAs). Upon maturation by Cas9, the
tracRNA and crRNA duplex guide the Cas9 nuclease to a target DNA
sequence specified by the spacer guide sequences, and mediates
double-stranded breaks in the DNA near a short sequence motif in
the target DNA that is required for cleavage and specific to each
CRISPR-Cas system. The type II CRISPR-Cas systems are found
throughout the bacterial kingdom and highly diverse in in Cas9
protein sequence and size, tracrRNA and crRNA direct repeat
sequence, genome organization of these elements, and the motif
requirement for target cleavage. One species may have multiple
distinct CRISPR-Cas systems.
[0310] Applicants evaluated 207 putative Cas9s from bacterial
species identified based on sequence homology to known Cas9s and
structures orthologous to known subdomains, including the HNH
endonuclease domain and the RuvC endonuclease domains [information
from the Eugene Koonin and Kira Makarova]. Phylogenetic analysis
based on the protein sequence conservation of this set revealed
five families of Cas9s, including three groups of large Cas9s
(.about.1400 amino acids) and two of small Cas9s (.about.1100 amino
acids) (FIGS. 39 and 40A-F).
[0311] In this example, Applicants show that the following
mutations can convert SpCas9 into a nicking enzyme: D10A, E762A,
H840A, N854A, N863A, D986A.
[0312] Applicants provide sequences showing where the mutation
points are located within the SpCas9 gene (FIG. 41). Applicants
also show that the nickases are still able to mediate homologous
recombination (Assay indicated in FIG. 2). Furthermore, Applicants
show that SpCas9 with these mutations (individually) do not induce
double strand break (FIG. 47).
[0313] Furthermore, potential nicking mutation sites were chosen
based on sequence homology between Cas9 orthologs (named original
set below). The nickase mutant Cas9s were re-cloned to incorporate
both N' and C'-NLS sequences as in Cong, L et al., Multiplex genome
engineering using CRISPR/Cas systems, Science. 2013 Feb. 15;
339(6121):819-23. (sequences for NLS-E762A-NLS and
>NLS-D986A-NLS listed below).
[0314] Nuclease and double-nicking activities for these potential
nickases were tested in HEK 293FT cells as follows: co-transfection
of 400 ng of nickase and 100 ng of U6-driven sgRNA (100 ng for one
guide, or 50 ng each for a pair of sgRNAs) by Lipofectamine 2000
into .about.200,000 cells. DNAs from transfected cells were
collected for SURVEYOR analysis. Nickases do not result in indel
mutations when co-transfected with a single sgRNA, but do when
co-transfected with a pair of appropriately off-set sgRNAs. Based
on data from the original D10A SpCas9 nickase, the pair of sgRNA
chosen (A1/C1) for RuvC domain mutants have 0-bp offset and
5'-overhang for maximal cleavage.
TABLE-US-00015 Original set: Mutant domain Functional?
Cbh-hSpCas9(D10A)-NLS RuvCI nickase activity Cbh-hSpCas9(E762A)-NLS
RuvCII Cbh-hSpCas9(H840A)-NLS HNH no activity
Cbh-hSpCas9(N854A)-NLS HNH wt nuclease activity
Cbh-hSpCas9(N863A)-NLS HNH nickase activity Cbh-hSpCas9(D986A)-NLS
RuvCIII
TABLE-US-00016 >NLS-E762A-NLS (SEQ ID NO: 196)
ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAG
CCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGG
CTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAG
GTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAG
CCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAG
AACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTG
CAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCC
ACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCG
GCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAG
TACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACA
AGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTT
CCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGAC
GTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCG
AGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTC
TGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTG
CCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCC
TGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGC
CAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTG
CTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGA
ACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGA
GATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAG
CACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGC
CTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGC
CGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATC
AAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGC
TGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAG
CATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGG
CAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGA
AGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGG
AAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACC
CCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCT
TCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGT
GCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAG
CTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCC
TGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAA
CCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATC
GAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACG
CCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGA
CTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTG
ACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAA
CCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCG
GAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATC
CGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACG
GCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGAC
CTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGC
CTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGG
GCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGG
CCGGCACAAGCCCGAGAACATCGTGATCGccATGGCCAGAGAGAACCAG
ACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCG
AAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGT
GGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAG
AATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGT
CCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGA
CTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAG
AGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACT
GGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAA
TCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGC
TTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGG
CACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAA
GCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCC
GATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACT
ACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCT
GATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTAC
AAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCG
GCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTT
CAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTG
ATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGG
ATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGT
GAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTG
CCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACC
CTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCT
GGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTG
AAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGA
ATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGA
CCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGC
CGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAAC
TGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTA
TGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTT
GTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCG
AGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCT
GTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAG
AATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCT
TCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAA
AGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTAC
GAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGG
CCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGtaa >NLS-D986A-NLS (SEQ ID
NO: 197) ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAG
CCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGG
CTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAG
GTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAG
CCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAG
AACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTG
CAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCC
ACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCG
GCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAG
TACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACA
AGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTT
CCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGAC
GTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCG
AGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTC
TGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTG
CCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCC
TGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGC
CAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTG
CTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGA
ACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGA
GATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAG
CACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGC
CTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGC
CGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATC
AAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGC
TGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAG
CATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGG
CAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGA
AGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGG
AAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACC
CCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCT
TCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGT
GCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAG
CTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCC
TGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAA
CCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATC
GAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACG
CCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGA
CTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTG
ACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAA
CCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCG
GAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATC
CGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACG
GCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGAC
CTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGC
CTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGG
GCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGG
CCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAG
ACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCG
AAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGT
GGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAG
AATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGT
CCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGA
CTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAG
AGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACT
GGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAA
TCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGC
TTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGG
CACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAA
GCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCC
GATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACT
ACCACCACGCCCACGcCGCCTACCTGAACGCCGTCGTGGGAACCGCCCT
GATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTAC
AAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCG
GCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTT
CAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTG
ATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGG
ATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGT
GAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTG
CCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACC
CTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCT
GGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTG
AAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGA
ATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGA
CCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGC
CGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAAC
TGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTA
TGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTT
GTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCG
AGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCT
GTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAG
AATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCT
TCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAA
AGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTAC
GAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGG
CCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAG
Example 9: Supplement to DNA Targeting Specificity of the
RNA-Guided Cas9 Nuclease
[0315] Cell Culture and Transfection
[0316] Human embryonic kidney (HEK) cell line 293FT (Life
Technologies) was maintained in Dulbecco's modified Eagle's Medium
(DMEM) supplemented with 10% fetal bovine serum (HyClone), 2 mM
GlutaMAX (Life Technologies), 100 U/mL penicillin, and 100 .mu.g/mL
streptomycin at 37.degree. C. with 5% CO.sub.2 incubation.
[0317] 293FT cells were seeded either onto 6-well plates, 24-well
plates, or 96-well plates (Corning) 24 hours prior to transfection.
Cells were transfected using Lipofectamine 2000 (Life Technologies)
at 80-90% confluence following the manufacturer's recommended
protocol. For each well of a 6-well plate, a total of 1 ug of
Cas9+sgRNA plasmid was used. For each well of a 24-well plate, a
total of 500 ng Cas9+sgRNA plasmid was used unless otherwise
indicated. For each well of a 96-well plate, 65 ng of Cas9 plasmid
was used at a 1:1 molar ratio to the U6-sgRNA PCR product.
[0318] Human embryonic stem cell line HUES9 (Harvard Stem Cell
Institute core) was maintained in feeder-free conditions on GelTrex
(Life Technologies) in mTesR medium (Stemcell Technologies)
supplemented with 100 ug/ml Normocin (InvivoGen). HUES9 cells were
transfected with Amaxa P3 Primary Cell 4-D Nucleofector Kit (Lonza)
following the manufacturer's protocol.
[0319] SURVEYOR Nuclease Assay for Genome Modification
[0320] 293FT cells were transfected with plasmid DNA as described
above. Cells were incubated at 37.degree. C. for 72 hours
post-transfection prior to genomic DNA extraction. Genomic DNA was
extracted using the QuickExtract DNA Extraction Solution
(Epicentre) following the manufacturer's protocol. Briefly,
pelleted cells were resuspended in QuickExtract solution and
incubated at 65.degree. C. for 15 minutes and 98.degree. C. for 10
minutes.
[0321] The genomic region flanking the CRISPR target site for each
gene was PCR amplified (primers listed in Tables J and K), and
products were purified using QiaQuick Spin Column (Qiagen)
following the manufacturer's protocol. 400 ng total of the purified
PCR products were mixed with 2 .mu.l 10.times. Taq DNA Polymerase
PCR buffer (Enzymatics) and ultrapure water to a final volume of 20
.mu.l, and subjected to a re-annealing process to enable
heteroduplex formation: 95.degree. C. for 10 min, 95.degree. C. to
85.degree. C. ramping at -2.degree. C./s, 85.degree. C. to
25.degree. C. at -0.25.degree. C./s, and 25.degree. C. hold for 1
minute. After re-annealing, products were treated with SURVEYOR
nuclease and SURVEYOR enhancer S (Transgenomics) following the
manufacturer's recommended protocol, and analyzed on 4-20% Novex
TBE poly-acrylamide gels (Life Technologies). Gels were stained
with SYBR Gold DNA stain (Life Technologies) for 30 minutes and
imaged with a Gel Doc gel imaging system (Bio-rad). Quantification
was based on relative band intensities.
[0322] Northern Blot Analysis of tracrRNA Expression in Human
Cells
[0323] Northern blots were performed as previously described.
Briefly, RNAs were heated to 95.degree. C. for 5 min before loading
on 8% denaturing polyacrylamide gels (SequaGel, National
Diagnostics). Afterwards, RNA was transferred to a pre-hybridized
Hybond N+ membrane (GE Healthcare) and crosslinked with Stratagene
UV Crosslinker (Stratagene). Probes were labeled with [gamma-32P]
ATP (Perkin Elmer) with T4 polynucleotide kinase (New England
Biolabs). After washing, membrane was exposed to phosphor screen
for one hour and scanned with phosphorimager (Typhoon).
[0324] Bisulfite Sequencing to Assess DNA Methylation Status
[0325] HEK 293FT cells were transfected with Cas9 as described
above. Genomic DNA was isolated with the DNeasy Blood & Tissue
Kit (Qiagen) and bisulfite converted with EZ DNA
Methylation-Lightning Kit (Zymo Research). Bisulfite PCR was
conducted using KAPA2G Robust HotStart DNA Polymerase (KAPA
Biosystems) with primers designed using the Bisulfite Primer Seeker
(Zymo Research, Tables J and K). Resulting PCR amplicons were
gel-purified, digested with EcoRI and HindIII, and ligated into a
pUC19 backbone prior to transformation. Individual clones were then
Sanger sequenced to assess DNA methylation status.
[0326] In Vitro Transcription and Cleavage Assay
[0327] HEK 293FT cells were transfected with Cas9 as described
above. Whole cell lysates were then prepared with a lysis buffer
(20 mM HEPES, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol, 0.1%
Triton X-100) supplemented with Protease Inhibitor Cocktail
(Roche). T7-driven sgRNA was in vitro transcribed using custom
oligos (Example 10) and HiScribe T7 In Vitro Transcription Kit
(NEB), following the manufacturer's recommended protocol. To
prepare methylated target sites, pUC19 plasmid was methylated by
M.SssI and then linearized by NheI. The in vitro cleavage assay was
performed as follows: for a 20 uL cleavage reaction, 10 uL of cell
lysate with incubated with 2 uL cleavage buffer (100 mM HEPES, 500
mM KCl, 25 mM MgCl2, 5 mM DTT, 25% glycerol), the in vitro
transcribed RNA, and 300 ng pUC19 plasmid DNA.
[0328] Deep Sequencing to Assess Targeting Specificity
[0329] HEK 293FT cells plated in 96-well plates were transfected
with Cas9 plasmid DNA and single guide RNA (sgRNA) PCR cassette 72
hours prior to genomic DNA extraction (FIG. 72). The genomic region
flanking the CRISPR target site for each gene was amplified (FIG.
74, FIG. 80, (Example 10) by a fusion PCR method to attach the
Illumina P5 adapters as well as unique sample-specific barcodes to
the target amplicons (schematic described in FIG. 73). PCR products
were purified using EconoSpin 96-well Filter Plates (Epoch Life
Sciences) following the manufacturer's recommended protocol.
[0330] Barcoded and purified DNA samples were quantified by
Quant-iT PicoGreen dsDNA Assay Kit or Qubit 2.0 Fluorometer (Life
Technologies) and pooled in an equimolar ratio. Sequencing
libraries were then deep sequenced with the Illumina MiSeq Personal
Sequencer (Life Technologies).
[0331] Sequencing Data Analysis and Indel Detection
[0332] MiSeq reads were filtered by requiring an average Phred
quality (Q score) of at least 23, as well as perfect sequence
matches to barcodes and amplicon forward primers. Reads from on-
and off-target loci were analyzed by first performing
Smith-Waterman alignments against amplicon sequences that included
50 nucleotides upstream and downstream of the target site (a total
of 120 bp). Alignments, meanwhile, were analyzed for indels from 5
nucleotides upstream to 5 nucleotides downstream of the target site
(a total of 30 bp). Analyzed target regions were discarded if part
of their alignment fell outside the MiSeq read itself, or if
matched base-pairs comprised less than 85% of their total
length.
[0333] Negative controls for each sample provided a gauge for the
inclusion or exclusion of indels as putative cutting events. For
each sample, an indel was counted only if its quality score
exceeded .mu.-.sigma., where .mu. was the mean quality-score of the
negative control corresponding to that sample and .sigma. was the
standard deviation of same. This yielded whole target-region indel
rates for both negative controls and their corresponding samples.
Using the negative control's per-target-region-per-read error rate,
q, the sample's observed indel count n, and its read-count R, a
maximum-likelihood estimate for the fraction of reads having
target-regions with true-indels, p, was derived by applying a
binomial error model, as follows.
[0334] Letting the (unknown) number of reads in a sample having
target regions incorrectly counted as having at least 1 indel be E,
we can write (without making any assumptions about the number of
true indels)
Prob ( E p ) = ( R ( 1 - p ) E ) q E ( 1 - q ) R ( 1 - p ) - E
##EQU00001##
[0335] since R(1-p) is the number of reads having target-regions
with no true indels. Meanwhile, because the number of reads
observed to have indels is n, n=E+Rp, in other words the number of
reads having target-regions with errors but no true indels plus the
number of reads whose target-regions correctly have indels. We can
then re-write the above
Prob ( E p ) = Prob ( n = E + R p p ) = ( R ( 1 - p ) n - Rp ) q n
- Rp ( 1 - q ) R - n ##EQU00002##
[0336] Taking all values of the frequency of target-regions with
true-indels p to be equally probable a priori,
Prob(n|p).varies.Prob(p|n). The maximum-likelihood estimate (MLE)
for the frequency of target regions with true-indels was therefore
set as the value of p that maximized Prob(n|p). This was evaluated
numerically.
[0337] In order to place error bounds on the true-indel read
frequencies in the sequencing libraries themselves, Wilson score
intervals (2) were calculated for each sample, given the
MLE-estimate for true-indel target-regions, RP, and the number of
reads R. Explicitly, the lower bound l and upper bound u were
calculated as
l = ( Rp + z 2 2 + z Rp ( 1 - p ) + z 2 / 4 ) / ( R + z 2 )
##EQU00003## u = ( Rp + z 2 2 + z Rp ( 1 - p ) + z 2 / 4 ) / ( R +
z 2 ) ##EQU00003.2##
[0338] where z, the standard score for the confidence required in
normal distribution of variance 1, was set to 1.96, meaning a
confidence of 95%. The maximum upper bounds and minimum lower
bounds for each biological replicate are listed in FIGS. 80-83.
[0339] qRT-PCR analysis of relative Cas9 and sgRNA expression
[0340] 293FT cells plated in 24-well plates were transfected as
described above. 72 hours post-transfection, total RNA was
harvested with miRNeasy Micro Kit (Qiagen). Reverse-strand
synthesis for sgRNAs was performed with qScript Flex cDNA kit (VWR)
and custom first-strand synthesis primers (Tables J and K). qPCR
analysis was performed with Fast SYBR Green Master Mix (Life
Technologies) and custom primers (Tables J and K), using GAPDH as
an endogenous control. Relative quantification was calculated by
the .DELTA..DELTA.CT method.
TABLE-US-00017 TABLE I Target site sequences. Tested target sites
for S. pyogenes type II CRISPR system with the requisite PAM. Cells
were transfected with Cas9 and either crRNA-tracrRNA or chimeric
sgRNA for each target. Target site genomic Target site sequence ID
target (5' to 3') PAM strand 1 EMX1 GTCACCTCCAATGACTAGGG TGG + (SEQ
ID NO: 321) 2 EMX1 GACATCGATGTCCTCCCCAT TGG - (SEQ ID NO: 198) 3
EMX1 GAGTCCGAGCAGAAGAAGAA GGG + (SEQ ID NO: 199) 6 EMX1
GCGCCACCGGTTGATGTGAT GGG - (SEQ ID NO: 200) 10 EMX1
GGGGCACAGATGAGAAACTC AGG - (SEQ ID NO: 201) 11 EMX1
GTACAAACGGCAGAAGCTGG AGG + (SEQ ID NO: 202) 12 EMX1
GGCAGAAGCTGGAGGAGGAA GGG + (SEQ ID NO: 203) 13 EMX1
GGAGCCCTTCTTCTTCTGCT CGG - (SEQ ID NO: 204) 14 EMX1
GGGCAACCACAAACCCACGA GGG + (SEQ ID NO: 205) 15 EMX1
GCTCCCATCACATCAACCGG TGG + (SEQ ID NO: 206) 16 EMX1
GTGGCGCATTGCCACGAAGC AGG + (SEQ ID NO: 207) 17 EMX1
GGCAGAGTGCTGCTTGCTGC TGG + (SEQ ID NO: 208) 18 EMX1
GCCCCTGCGTGGGCCCAAGC TGG + (SEQ ID NO: 209) 19 EMX1
GAGTGGCCAGAGTCCAGCTT GGG - (SEQ ID NO: 210) 20 EMX1
GGCCTCCCCAAAGCCTGGCC AGG - (SEQ ID NO: 211) 4 PVALB
GGGGCCGAGATTGGGTGTTC AGG + (SEQ ID NO: 212) 5 PVALB
GTGGCGAGAGGGGCCGAGAT TGG + (SEQ ID NO: 213) 1 SERPINB5
GAGTGCCGCCGAGGCGGGGC GGG + (SEQ ID NO: 214) 2 SERPINB5
GGAGTGCCGCCGAGGCGGGG CGG + (SEQ ID NO: 215) 3 SERPINB5
GGAGAGGAGTGCCGCCGAGG CGG + (SEQ ID NO: 216)
TABLE-US-00018 TABLE J Primer sequences SURVEYOR assay genomic
primer sequence primer name target (5' to 3') Sp-EMX1-F1 EMX1
AAAACCACCCTTCTCTCTGGC (SEQ ID NO: 36) Sp-EMX1-R1 EMX1
GGAGATTGGAGACACGGAGAG (SEQ ID NO: 37) Sp-EMX1-F2 EMX1
CCATCCCCTTCTGTGAATGT (SEQ ID NO: 217) Sp-EMX1-R2 EMX1
GGAGATTGGAGACACGGAGA (SEQ ID NO: 218) Sp-PVALB-F PVALB
CTGGAAAGCCAATGCCTGAC (SEQ ID NO: 38) Sp-PVALB-R PVALB
GGCAGCAAACTCCTTGTCCT (SEQ ID NO: 39) qRT-PCR for Cas9 and sgRNA
expression primer name primer sequence (5' to 3') sgRNA reverse-
AAGCACCGACTCGGTGCCAC strand synthesis (SEQ ID NO: 219) EMX1.1 sgRNA
TCACCTCCAATGACTAGGGG qPCR F (SEQ ID NO: 220) EMX1.1 sgRNA
CAAGTTGATAACGGACTAGCCT qPCR R (SEQ ID NO: 221) EMX1.3 sgRNA
AGTCCGAGCAGAAGAAGAAGTTT qPCR F (SEQ ID NO: 222) EMX1.3 sgRNA
TTTCAAGTTGATAACGGACTAGCCT qPCR R (SEQ ID NO: 223) Cas9 qPCR F
AAACAGCAGATTGCCTGGA (SEQ ID NO: 224) Cas9 qPCR R
TCATCCGCTCGATGAAGCTC (SEQ ID NO: 225) GAPDH qPCR F
TCCAAAATCAAGTGGGGCGA (SEQ ID NO: 226) GAPDH qPCR R
TGATGACCCTTTTGGCTCCC (SEQ ID NO: 227) Bisulfite PCR and sequencing
primer name primer sequence (5' to 3') Bisulfite PCR F
GAGGAATTCTTTTTTTGTTYGAATAT (SERPINB5 locus) GTTGGAGGTTTTTTGGAAG
(SEQ ID NO: 228) Bisulfite PCR R GAGAAGCTTAAATAAAAAACRACAAT
(SERPINB5 locus) ACTCAACCCAACAACC (SEQ ID NO: 229) pUC19 sequencing
CAGGAAACAGCTATGAC (SEQ ID NO: 230)
TABLE-US-00019 TABLE K Sequences for primers to test sgRNA
architecture. Primers hybridize to the reverse strand of the U6
promoter unless otherwise indicated. The U6 priming site is in
italics, the guide sequence is indicated as a stretch of Ns, the
direct repeat sequence is highlighted in bold, and the tracrRNA
sequence underlined. The secondary structure of each sgRNA
architecture is shown in FIG. 43. primer name primer sequence (5'
to 3') U6-Forward GCCTCTAGAGGTACCTGAGGGCCTATTT CCCATGATTCC (SEQ ID
NO: 231) I: sgRNA(DR +12, ACCTCTAGAAAAAAAGCACCGACTCGGT tracrRNA
+85) GCCACTTTTTCAAGTTGATAACGGACTA GCCTTATTTTAACTTGCTATTTCTAGCT
CTAAAACNNNNNNNNNNNNNNNNNNNNG GTGTTTCGTCCTTTCCACAAG (SEQ ID NO: 232)
II: sgRNA(DR +12 ACCTCTAGAAAAAAAGCACCGACTCGGT tracrRNA +85)
GCCACTTTTTCAAGTTGATAACGGACTA mut2 GCCTTATATTAACTTGCTATTTCTAGCT
CTAATACNNNNNNNNNNNNNNNNNNNNG GTGTTTCGTCCTTTCCACAAG (SEQ ID NO: 233)
III: sgRNA(DR +22, ACCTCTAGAAAAAAAGCACCGACTCGGT tracrRNA +85)
GCCACTTTTTCAAGTTGATAACGGACTA GCCTTATTTTAACTTGCTATGCTGTTTT
GTTTCCAAAACAGCATAGCTCTAAAACN NNNNNNNNNNNNNNNNNNNGGTGTTTCG
TCCTTTCCACAAG (SEQ ID NO: 234) IV: sgRNA(DR +22,
ACCTCTAGAAAAAAAGCACCGACTCGGT tracrRNA +85)
GCCACTTTTTCAAGTTGATAACGGACTA mut4 GCCTTATATTAACTTGCTATGCTGTATT
GTTTCCAATACAGCATAGCTCTAATACN NNNNNNNNNNNNNNNNNNNGGTGTTTCG
TCCTTTCCACAAG (SEQ ID NO: 235)
TABLE-US-00020 TABLE L Target sites with alternate PAMs for testing
PAM specificity of Cas9. All target sites for PAM specificity
testing are found within the human EMX1 locus. Target site sequence
(5' to 3') PAM AGGCCCCAGTGGCTGCTCT (SEQ ID NO: 236) NAA
ACATCAACCGGTGGCGCAT (SEQ ID NO: 237) NAT AAGGTGTGGTTCCAGAACC (SEQ
ID NO: 238) NAC CCATCACATCAACCGGTGG (SEQ ID NO: 239) NAG
AAACGGCAGAAGCTGGAGG (SEQ ID NO: 240) NTA GGCAGAAGCTGGAGGAGGA (SEQ
ID NO: 241) NTT GGTGTGGTTCCAGAACCGG (SEQ ID NO: 242) NTC
AACCGGAGGACAAAGTACA (SEQ ID NO: 243) NTG TTCCAGAACCGGAGGACAA (SEQ
ID NO: 244) NCA GTGTGGTTCCAGAACCGGA (SEQ ID NO: 245) NCT
TCCAGAACCGGAGGACAAA (SEQ ID NO: 246) NCC CAGAAGCTGGAGGAGGAAG (SEQ
ID NO: 247) NCG CATCAACCGGTGGCGCATT (SEQ ID NO: 248) NGA
GCAGAAGCTGGAGGAGGAA (SEQ ID NO: 249) NGT CCTCCCTCCCTGGCCCAGG (SEQ
ID NO: 250) NGC TCATCTGTGCCCCTCCCTC (SEQ ID NO: 251) NAA
GGGAGGACATCGATGTCAC (SEQ ID NO: 252) NAT CAAACGGCAGAAGCTGGAG (SEQ
ID NO: 253) NAC GGGTGGGCAACCACAAACC (SEQ ID NO: 254) NAG
GGTGGGCAACCACAAACCC (SEQ ID NO: 255) NTA GGCTCCCATCACATCAACC (SEQ
ID NO: 256) NTT GAAGGGCCTGAGTCCGAGC (SEQ ID NO: 257) NTC
CAACCGGTGGCGCATTGCC (SEQ ID NO: 258) NTG AGGAGGAAGGGCCTGAGTC (SEQ
ID NO: 259) NCA AGCTGGAGGAGGAAGGGCC (SEQ ID NO: 260) NCT
GCATTGCCACGAAGCAGGC (SEQ ID NO: 261) NCC ATTGCCACGAAGCAGGCCA (SEQ
ID NO: 262) NCG AGAACCGGAGGACAAAGTA (SEQ ID NO: 263) NGA
TCAACCGGTGGCGCATTGC (SEQ ID NO: 264) NGT GAAGCTGGAGGAGGAAGGG (SEQ
ID NO: 265) NGC
Example 10: Supplementary Sequences
[0341] All sequences are in the 5' to 3' direction. For U6
transcription, the string of underlined Ts serve as the
transcriptional terminator.
TABLE-US-00021 > U6-short tracrRNA (Streptococcus pyogenes
SF370) (SEQ ID NO: 40)
gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaa-
tttgactgtaaa
cacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattat-
gttttaaaatggactatcatat
gcttaccgtaacttgaaagtatttcgatttatggattatatatcttgtggaaaggacgaaacaccGGAACCATT-
CAAAACAGC ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
GTCGGTGCTTTTTTT (tracrRNA sequence in bold) > U6-DR-guide
sequence-DR (Streptococcus pyogenes SF370) (SEQ ID NO: 54)
gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaa-
tttgactgtaaa
cacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattat-
gttttaaaatggactatcatat ##STR00001## ##STR00002## ##STR00003##
(direct repeat sequence is highlighted in gray and the guide
sequence is in bold Ns) > sgRNA containing +48 tracrRNA
(Streptococcus pyogenes SF370) (SEQ ID NO: 55)
gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaa-
tttgactgtaaa
cacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattat-
gttttaaaatggactatcatat
gcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccNNNNNNN-
NNNNNNNNNNN ##STR00004## (guide sequence is in bold Ns and the
tracrRNA fragment is in bold) > sgRNA containing +54 tracrRNA
(Streptococcus pyogenes SF370) (SEQ ID NO: 56)
gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaa-
tttgactgtaaa
cacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattat-
gttttaaaatggactatcatat
gcttaccgtaacttgaaagtatttcgatttatggattatatatcttgtggaaaggacgaaacaccNNNNNNNNN-
NNNNNNNNN ##STR00005## (guide sequence is in bold Ns and the
tracrRNA fragment is in bold) > sgRNA containing +67 tracrRNA
(Streptococcus pyogenes SF370) (SEQ ID NO: 57)
gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaa-
tttgactgtaaa
cacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattat-
gttttaaaatggactatcatat
gcttaccgtaacttgaaagtatttcgatttatggattatatatcttgtggaaaggacgaaacaccNNNNNNNNN-
NNNNNNNNN ##STR00006## (guide sequence is in bold Ns and the
tracrRNA fragment is in bold) > sgRNA containing +85 tracrRNA
(Streptococcus pyogenes SF370) (SEQ ID NO: 58)
gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaa-
tttgactgtaaa
cacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattat-
gttttaaaatggactatcatat
gcttaccgtaacttgaaagtatttcgatttatggattatatatcttgtggaaaggacgaaacaccNNNNNNNNN-
NNNNNNNNN ##STR00007## (guide sequence is in bold Ns and the
tracrRNA fragment is in bold) > CBh-NLS-SpCas9-NLS (SEQ ID NO:
59) CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACC
CCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTT
TCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATC
AAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCG
CCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTA
CGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTC
CCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTG
TGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGG
GGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCG
GCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAA
AAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCC
GCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCAC
AGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCTGAGCAAGAGG
TAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCAC
CTGCCTGAAATCACTTTTTTTCAGGTTGGaccggtgccaccATGGACTATAAGGACCACG
ACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATG
GCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAA
GAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGA
TCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACC
GACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGG
CGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCA
GACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCC
AAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGA
GGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGG
CCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGAC
AGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGAT
CAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCG
ACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAG
GAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAG
ACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGA
AGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCC
AACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAA
GGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGT
ACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGC
GACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTAT
GATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCG
TGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAG
AACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAA
GTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGA
AGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAG
CATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGG
AAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTG
ACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATT
CGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGG
AAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAAC
TTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTA
CGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGG
GAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGAC
CTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTA
CTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATC
GGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGAC
AAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCT
GACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCT
ATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATAC
ACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGC
AGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGA
AACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCA
GAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATC
TGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTG
GACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGA
AATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAG
AGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAA
AGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACT
ACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGG
CTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGA
CTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCG
ACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAG
CTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGC
CGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAG
CTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCG
GATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGA
TCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACA
AAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCC
GTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGT
GTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGC
AGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACT
TTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTG
ATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATT
TTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAG
ACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAA
CAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCG
GCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAA
AAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCAT
CATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGG
GCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTG
TTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCA
GAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGG
CCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAG
CTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAG
CGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGT
CCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATC
ATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTT
GACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGC
CACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGT
CTCAGCTGGGAGGCGACTTTCTTTTTCTTAGCTTGACCAGCTTTCTTAGTAGCA
GCAGGACGCTTTAA (NLS-hSpCas9-NLS is highlighted in bold) >
Sequencing amplicon for EMX1 guides 1.1, 1.14, 1.17 (SEQ ID NO:
266) CCAATGGGGAGGACATCGATGTCACCTCCAATGACTAGGGTGGGCAACC
ACAAACCCACGAGGGCAGAGTGCTGCTTGCTGCTGGCCAGGCCCCTGCGTGGGCCC
AAGCTGGACTCTGGCCAC > Sequencing amplicon for EMX1 guides 1.2,
1.16 (SEQ ID NO: 267)
CGAGCAGAAGAAGAAGGGCTCCCATCACATCAACCGGTGGCGCATTGCC
ACGAAGCAGGCCAATGGGGAGGACATCGATGTCACCTCCAATGACTAGGGTGGGCA
ACCACAAACCCACGAG > Sequencing amplicon for EMX1 guides 1.3,
1.13, 1.15 (SEQ ID NO: 268)
GGAGGACAAAGTACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTC
CGAGCAGAAGAAGAAGGGCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGC
AGGCCAATGGGGAGGACATCGAT > Sequencing amplicon for EMX1 guides
1.6 (SEQ ID NO: 269)
AGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAAGAAGGGCTC
CCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCG
ATGTCACCTCCAATGACTAGGGTGG > Sequencing amplicon for EMX1 guides
1.10 (SEQ ID NO: 270)
CCTCAGTCTTCCCATCAGGCTCTCAGCTCAGCCTGAGTGTTGAGGCCCCAG
TGGCTGCTCTGGGGGCCTCCTGAGTTTCTCATCTGTGCCCCTCCCTCCCTGGCCCAGG
TGAAGGTGTGGTTCCA > Sequencing amplicon for EMX1 guides 1.11,
1.12 (SEQ ID NO: 271)
TCATCTGTGCCCCTCCCTCCCTGGCCCAGGTGAAGGTGTGGTTCCAGAACC
GGAGGACAAAGTACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCA
GAAGAAGAAGGGCTCCCATCACA > Sequencing amplicon for EMX1 guides
1.18, 1.19 (SEQ ID NO: 272)
CTCCAATGACTAGGGTGGGCAACCACAAACCCACGAGGGCAGAGTGCTG
CTTGCTGCTGGCCAGGCCCCTGCGTGGGCCCAAGCTGGACTCTGGCCACTCCCTGGC
CAGGCTTTGGGGAGGCCTGGAGT > Sequencing amplicon for EMX1 guides
1.20 (SEQ ID NO: 273)
CTGCTTGCTGCTGGCCAGGCCCCTGCGTGGGCCCAAGCTGGACTCTGGCC
ACTCCCTGGCCAGGCTTTGGGGAGGCCTGGAGTCATGGCCCCACAGGGCTTGAAGC
CCGGGGCCGCCATTGACAGAG > T7 promoter F primer for annealing with
target strand (SEQ ID NO: 274) GAAATTAATACGACTCACTATAGGG > oligo
containing pUC19 target site 1 for methylation (T7 reverse) (SEQ ID
NO: 275) AAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCC
TTATTTTAACTTGCTATTTCTAGCTCTAAAACAACGACGAGCGTGACACCACCCTAT
AGTGAGTCGTATTAATTTC > oligo containing pUC19 target site 2 for
methylation (T7 reverse) (SEQ ID NO: 276)
AAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCC
TTATTTTAACTTGCTATTTCTAGCTCTAAAACGCAACAATTAATAGACTGGACCTATA
GTGAGTCGTATTAATTTC
Example 11: Oligo-Mediated Cas9-Induced Homologous
Recombination
[0342] The oligo homologous recombination test is a comparison of
efficiency across different Cas9 variants and different HR template
(oligo vs. plasmid).
[0343] 293FT cells were used. SpCas9=Wildtype Cas9 and
SpCas9n=nickase Cas9 (D10A). The chimeric RNA target is the same
EMX1Protospacer Target 1 as in Examples 5, 9 and 10 and oligos
synthesized by IDT using PAGE purification.
[0344] FIG. 44 depicts a design of the oligo DNA used as Homologous
Recombination (HR) template in this experiment. Long oligos contain
100 bp homology to the EMX1 locus and a HindIII restriction site.
293FT cells were co-transfected with: first, a plasmid containing a
chimeric RNA targeting human EMX1 locus and wild-type cas9 protein,
and second, the oligo DNA as HR template. Samples are from 293FT
cells collected 96 hours post transfection with Lipofectamine 2000.
All products were amplified with an EMX1 HR Primer, gel purified,
followed by digestion with HindIII to detect the efficiency of
integration of HR template into the human genome.
[0345] FIGS. 45 and 46 depict a comparison of HR efficiency induced
by different combination of Cas9 protein and HR template. The Cas9
construct used were either wild-type Cas9 or the nickase version of
Cas9 (Cas9n). The HR template used were: antisense oligo DNA
(Antisense-Oligo in above figure), or sense oligo DNA (Sense-Oligo
in above figure), or plasmid HR template (HR template in above
figure). The sense/anti-sense definition is that the
actively-transcribed strand with sequence corresponding to the
transcribed mRNA is defined as the sense strand of genome. HR
Efficiency is shown as percentage of HindIII digestion band as
against all genomic PCR amplified product (bottom numbers).
Example 12: Autistic Mouse
[0346] Recent large-scale sequencing initiatives have produced a
large number of genes associated with disease. Discovering the
genes is only the beginning in understanding what the gene does and
how it leads to a diseased phenotype. Current technologies and
approaches to study candidate genes are slow and laborious. The
gold standards, gene targeting and genetic knockouts, require a
significant investment in time and resources, both monetary and in
terms of research personnel. Applicants set out to utilize the
hSpCas9 nuclease to target many genes and do so with higher
efficiency and lower turnaround compared to any other technology.
Because of the high efficiency of hSpCas9 Applicants can do RNA
injection into mouse zygotes and immediately get genome-modified
animals without the need to do any preliminary gene targeting in
mESCs.
[0347] Chromodomain helicase DNA binding protein 8 (CHD8) is a
pivotal gene in involved in early vertebrate development and
morphogenesis. Mice lacking CHD8 die during embryonic development.
Mutations in the CHD8 gene have been associated with autism
spectrum disorder in humans. This association was made in three
different papers published simultaneously in Nature. The same three
studies identified a plethora of genes associated with autism
spectrum disorder. Applicants' aim was to create knockout mice for
the four genes that were found in all papers, Chd8, Katnal2,
Kctd13, and Scn2a. In addition, Applicants chose two other genes
associated with autism spectrum disorder, schizophrenia, and ADHD,
GIT1, CACNA1C, and CACNB2. And finally, as a positive control
Applicants decide to target MeCP2.
[0348] For each gene Applicants designed three gRNAs that would
likely knockout the gene. A knockout would occur after the hSpCas9
nuclease makes a double strand break and the error prone DNA repair
pathway, non-homologous end joining, corrects the break, creating a
mutation. The most likely result is a frameshift mutation that
would knockout the gene. The targeting strategy involved finding
proto-spacers in the exons of the gene that had a PAM sequence,
NGG, and was unique in the genome. Preference was given to
proto-spacers in the first exon, which would be most deleterious to
the gene.
[0349] Each gRNA was validated in the mouse cell line, Neuro-N2a,
by liposomal transient co-transfection with hSpCas9. 72 hours
post-transfection genomic DNA was purified using QuickExtract DNA
from Epicentre. PCR was performed to amplify the locus of interest.
Subsequently the SURVEYOR Mutation Detection Kit from Transgenomics
was followed. The SURVEYOR results for each gRNA and respective
controls are shown in Figure A1. A positive SURVEYOR result is one
large band corresponding to the genomic PCR and two smaller bands
that are the product of the SURVEYOR nuclease making a
double-strand break at the site of a mutation. The average cutting
efficiency of each gRNA was also determined for each gRNA. The gRNA
that was chosen for injection was the highest efficiency gRNA that
was the most unique within the genome.
[0350] RNA (hSpCas9+gRNA RNA) was injected into the pronucleus of a
zygote and later transplanted into a foster mother. Mothers were
allowed to go full term and pups were sampled by tail snip 10 days
postnatal. DNA was extracted and used as a template for PCR, which
was then processed by SURVEYOR. Additionally, PCR products were
sent for sequencing. Animals that were detected as being positive
in either the SURVEYOR assay or PCR sequencing would have their
genomic PCR products cloned into a pUC19 vector and sequenced to
determine putative mutations from each allele.
[0351] So far, mice pups from the Chd8 targeting experiment have
been fully processed up to the point of allele sequencing. The
Surveyor results for 38 live pups (lanes 1-38) 1 dead pup (lane 39)
and 1 wild-type pup for comparison (lane 40) are shown in Figure
A2. Pups 1-19 were injected with gRNA Chd8.2 and pups 20-38 were
injected with gRNA Chd8.3. Of the 38 live pups, 13 were positive
for a mutation. The one dead pup also had a mutation. There was no
mutation detected in the wild-type sample. Genomic PCR sequencing
was consistent with the SURVEYOR assay findings.
Example 13: CRISPR/Cas-Mediated Transcriptional Modulation
[0352] FIG. 67 depicts a design of the CRISPR-TF (Transcription
Factor) with transcriptional activation activity. The chimeric RNA
is expressed by U6 promoter, while a human-codon-optimized,
double-mutant version of the Cas9 protein (hSpCas9m), operably
linked to triple NLS and a VP64 functional domain is expressed by a
EF1a promoter. The double mutations, D10A and H840A, renders the
cas9 protein unable to introduce any cleavage but maintained its
capacity to bind to target DNA when guided by the chimeric RNA.
[0353] FIG. 68 depicts transcriptional activation of the human SOX2
gene with CRISPR-TF system (Chimeric RNA and the Cas9-NLS-VP64
fusion protein). 293FT cells were transfected with plasmids bearing
two components: (1) U6-driven different chimeric RNAs targeting
20-bp sequences within or around the human SOX2 genomic locus, and
(2) EF1a-driven hSpCas9m (double mutant)-NLS-VP64 fusion protein.
96 hours post transfection, 293FT cells were harvested and the
level of activation is measured by the induction of mRNA expression
using a qRT-PCR assay. All expression levels are normalized against
the control group (grey bar), which represents results from cells
transfected with the CRISPR-TF backbone plasmid without chimeric
RNA. The qRT-PCR probes used for detecting the SOX2 mRNA is Taqman
Human Gene Expression Assay (Life Technologies). All experiments
represents data from 3 biological replicates, n=3, error bars show
s.e.m.
Example 14: NLS: Cas9 NLS
[0354] 293FT cells were transfected with plasmid containing two
components: (1) EF1.alpha. promoter driving the expression of Cas9
(wild-type human-codon-optimized Sp Cas9) with different NLS
designs (2) U6 promoter driving the same chimeric RNA targeting
human EMX1 locus.
[0355] Cells were collect at 72 h time point post transfection, and
then extracted with 50 .mu.l of the QuickExtract genomic DNA
extraction solution following manufacturer's protocol. Target EMX1
genomic DNA were PCR amplified and then Gel-purify with 1% agarose
gel. Genomic PCR product were re-anneal and subjected to the
Surveyor assay following manufacturer's protocol. The genomic
cleavage efficiency of different constructs were measured using
SDS-PAGE on a 4-12% TBE-PAGE gel (Life Technologies), analyzed and
quantified with ImageLab (Bio-rad) software, all following
manufacturer's protocol.
[0356] FIG. 69 depicts a design of different Cas9 NLS constructs.
All Cas9 were the human-codon-optimized version of the Sp Cas9. NLS
sequences are linked to the cas9 gene at either N-terminus or
C-terminus. All Cas9 variants with different NLS designs were
cloned into a backbone vector containing so it is driven by
EF1.alpha. promoter. On the same vector there is a chimeric RNA
targeting human EMX1 locus driven by U6 promoter, together forming
a two-component system.
TABLE-US-00022 TABLE M Cas9 NLS Design Test Results. Quantification
of genomic cleavage of different cas9-nls constructs by surveyor
assay. Percentage Error Genome Biological Biological Biological
(S.E.M., Cleavage as Replicate Replicate Replicate standard
measured by 1 2 3 Average error of Surveyor assay (%) (%) (%) (%)
the mean) Cas9 (No NLS) 2.50 3.30 2.73 2.84 0.24 Cas9 with 7.61
6.29 5.46 6.45 0.63 N-term NLS Cas9 with 5.75 4.86 4.70 5.10 0.33
C-term NLS Cas9 with 9.08 9.85 7.78 8.90 0.60 Double (N-term and
C-term) NLS
[0357] FIG. 70 depicts the efficiency of genomic cleavage induced
by Cas9 variants bearing different NLS designs. The percentage
indicate the portion of human EMX1 genomic DNA that were cleaved by
each construct. All experiments are from 3 biological replicates.
n=3, error indicates S.E.M.
Example 15: Engineering of Microalgae Using Cas9
[0358] Methods of Delivering Cas9
[0359] Method 1: Applicants deliver Cas9 and guide RNA using a
vector that expresses Cas9 under the control of a constitutive
promoter such as Hsp70A-Rbc S2 or Beta2-tubulin.
[0360] Method 2: Applicants deliver Cas9 and T7 polymerase using
vectors that expresses Cas9 and T7 polymerase under the control of
a constitutive promoter such as Hsp70A-Rbc S2 or Beta2-tubulin.
Guide RNA will be delivered using a vector containing T7 promoter
driving the guide RNA.
[0361] Method 3: Applicants deliver Cas9 mRNA and in vitro
transcribed guide RNA to algae cells. RNA can be in vitro
transcribed. Cas9 mRNA will consist of the coding region for Cas9
as well as 3'UTR from Cop1 to ensure stabilization of the Cas9
mRNA.
[0362] For Homologous recombination, Applicants provide an
additional homology directed repair template.
[0363] Sequence for a cassette driving the expression of Cas9 under
the control of beta-2 tubulin promoter, followed by the 3' UTR of
Cop1.
TABLE-US-00023 (SEQ ID NO: 277)
TCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGA
CGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAG
CTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTG
TTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAA
GCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAG
CTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTC
ACAACCCGCAAACATGTACCCATACGATGTTCCAGATTACGCTTCGCCGA
AGAAAAAGCGCAAGGTCGAAGCGTCCGACAAGAAGTACAGCATCGGCCTG
GACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAA
GGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCA
TCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCC
GAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAA
GAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGG
TGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAG
GATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGT
GGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGG
TGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCC
CACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCC
CGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACA
ACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAG
GCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGAT
CGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTG
CCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCC
GAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGA
CAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCG
CCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAAC
ACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGA
CGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGC
TGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTAC
GCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCAT
CAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGC
TGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGC
ATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCA
GGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGA
TCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAAC
AGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTG
GAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCG
AGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCC
AAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAA
AGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCG
AGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTG
ACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGA
CTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCA
CATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAAT
GAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTT
TGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGT
TCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGG
GGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGG
CAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACT
TCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAG
AAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAA
TCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGG
TGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATC
GTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAA
CAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCA
GCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAG
AAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCA
GGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGC
CTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGA
AGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGT
GAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTA
CCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGC
GAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCA
GATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGT
ACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAG
TCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCG
CGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCG
TGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTG
TACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGA
GCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCA
TGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAG
CGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAA
GGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGA
ATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCT
ATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTG
GGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTG
TGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGT
GTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAA
GAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGG
ACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGC
CGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACT
GGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATG
AGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTG
GAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTT
CTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCG
CCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATC
ATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTA
CTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGC
TGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGG
ATCGACCTGTCTCAGCTGGGAGGCGACAGCCCCAAGAAGAAGAGAAAGGT
GGAGGCCAGCTAAGGATCCGGCAAGACTGGCCCCGCTTGGCAACGCAACA
GTGAGCCCCTCCCTAGTGTGTTTGGGGATGTGACTATGTATTCGTGTGTT
GGCCAACGGGTCAACCCGAACAGATTGATACCCGCCTTGGCATTTCCTGT
CAGAATGTAACGTCAGTTGATGGTACT
[0364] Sequence for a cassette driving the expression of T7
polymerase under the control of beta-2 tubulin promoter, followed
by the 3' UTR of Cop1:
TABLE-US-00024 (SEQ ID NO: 278)
TCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGA
CGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAG
CTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTG
TTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAA
GCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAG
CTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTC
ACAACCCGCAAACatgcctaagaagaagaggaaggttaacacgattaaca
tcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaac
actctggctgaccattacggtgagcgtttagctcgcgaacagttggccct
tgagcatgagtcttacgagatgggtgaagcacgcttccgcaagatgtttg
agcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcct
ctcatcactaccctactccctaagatgattgcacgcatcaacgactggtt
tgaggaagtgaaagctaagcgcggcaagcgcccgacagccttccagttcc
tgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccact
ctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaag
cgcaatcggtcgggccattgaggacgaggctcgcttcggtcgtatccgtg
accttgaagctaagcacttcaagaaaaacgttgaggaacaactcaacaag
cgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctga
catgctctctaagggtctactcggtggcgaggcgtggtcttcgtggcata
aggaagactctattcatgtaggagtacgctgcatcgagatgctcattgag
tcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtca
agactctgagactatcgaactcgcacctgaatacgctgaggctatcgcaa
cccgtgcaggtgcgctggctggcatctctccgatgttccaaccttgcgta
gttcctcctaagccgtggactggcattactggtggtggctattgggctaa
cggtcgtcgtcctctggcgctggtgcgtactcacagtaagaaagcactga
tgcgctacgaagacgtttacatgcctgaggtgtacaaagcgattaacatt
gcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaa
cgtaatcaccaagtggaagcattgtccggtcgaggacatccctgcgattg
agcgtgaagaactcccgatgaaaccggaagacatcgacatgaatcctgag
gctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaa
ggctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagcca
ataagtttgctaaccataaggccatctggttcccttacaacatggactgg
cgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgatat
gaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaag
gttactactggctgaaaatccacggtgcaaactgtgcgggtgtcgacaag
gttccgttccctgagcgcatcaagttcattgaggaaaaccacgagaacat
catggcttgcgctaagtctccactggagaacacttggtgggctgagcaag
attctccgttctgcttccttgcgttctgctttgagtacgctggggtacag
caccacggcctgagctataactgctcccttccgctggcgtttgacgggtc
ttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtg
gtcgcgcggttaacttgcttcctagtgaaaccgttcaggacatctacggg
attgttgctaagaaagtcaacgagattctacaagcagacgcaatcaatgg
gaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatct
ctgagaaagtcaagctgggcactaaggcactggctggtcaatggctggct
tacggtgttactcgcagtgtgactaagcgttcagtcatgacgctggctta
cgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattc
agccagctattgattccggcaagggtctgatgttcactcagccgaatcag
gctgctggatacatggctaagctgatttgggaatctgtgagcgtgacggt
ggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgc
tggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgt
tgcgctgtgcattgggtaactcctgatggtttccctgtgtggcaggaata
caagaagcctattcagacgcgcttgaacctgatgttcctcggtcagttcc
gcttacagcctaccattaacaccaacaaagatagcgagattgatgcacac
aaacaggagtctggtatcgctcctaactttgtacacagccaagacggtag
ccaccttcgtaagactgtagtgtgggcacacgagaagtacggaatcgaat
cttttgcactgattcacgactccttcggtacgattccggctgacgctgcg
aacctgttcaaagcagtgcgcgaaactatggttgacacatatgagtcttg
tgatgtactggctgatttctacgaccagttcgctgaccagttgcacgagt
ctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctc
cgtgacatcttagagtcggacttcgcgttcgcgtaaGGATCCGGCAAGAC
TGGCCCCGCTTGGCAACGCAACAGTGAGCCCCTCCCTAGTGTGTTTGGGG
ATGTGACTATGTATTCGTGTGTTGGCCAACGGGTCAACCCGAACAGATTG
ATACCCGCCTTGGCATTTCCTGTCAGAATGTAACGTCAGTTGATGGTACT
[0365] Sequence of guide RNA driven by the T7 promoter (T7
promoter, Ns represent targeting sequence):
TABLE-US-00025 (SEQ ID NO: 279)
gaaatTAATACGACTCACTATANNNNNNNNNNNNNNNNNNNNgttttaga
gctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaa
gtggcaccgagtcggtgcttttttt
[0366] Gene Delivery:
[0367] Chlamydomonas reinhardtii strain CC-124 and CC-125 from the
Chlamydomonas Resource Center will be used for electroporation.
Electroporation protocol follows standard recommended protocol from
the GeneArt Chlamydomonas Engineering kit.
[0368] Also, Applicants generate a line of Chlamydomonas
reinhardtii that expresses Cas9 constitutively. This can be done by
using pChlamy1 (linearized using Pvu1) and selecting for hygromycin
resistant colonies. Sequence for pChlamy1 containing Cas9 is below.
In this way to achieve gene knockout one simply needs to deliver
RNA for the guideRNA. For homologous recombination Applicants
deliver guideRNA as well as a linearized homologous recombination
template.
TABLE-US-00026 pChlamy1-Cas9: (SEQ ID NO: 280)
TGCGGTATTTCACACCGCATCAGGTGGCACTTTTCGGGGAAATGTGCGCG
GAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTC
ATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATG
AAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTT
ACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGT
TCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGA
GGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCT
CACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAG
CGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTG
TTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACG
TTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATG
GCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCC
CATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCA
GAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCAT
AATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGA
GTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCT
CTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTA
AAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGAT
CTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACT
GATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACA
GGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTG
AATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTT
ATTGTCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCG
TCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCT
GCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGG
TTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGC
TTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTT
AGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGC
TAATCCTGTTACCAGTGGCTGTTGCCAGTGGCGATAAGTCGTGTCTTACC
GGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTG
AACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCG
AACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAA
GGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGA
GCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTG
TCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCA
GGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTT
CCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCC
CTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCT
CGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGT
CGCTGAGGCTTGACATGATTGGTGCGTATGTTTGTATGAAGCTACAGGAC
TGATTTGGCGGGCTATGAGGGCGGGGGAAGCTCTGGAAGGGCCGCGATGG
GGCGCGCGGCGTCCAGAAGGCGCCATACGGCCCGCTGGCGGCACCCATCC
GGTATAAAAGCCCGCGACCCCGAACGGTGACCTCCACTTTCAGCGACAAA
CGAGCACTTATACATACGCGACTATTCTGCCGCTATACATAACCACTCAG
CTAGCTTAAGATCCCATCAAGCTTGCATGCCGGGCGCGCCAGAAGGAGCG
CAGCCAAACCAGGATGATGTTTGATGGGGTATTTGAGCACTTGCAACCCT
TATCCGGAAGCCCCCTGGCCCACAAAGGCTAGGCGCCAATGCAAGCAGTT
CGCATGCAGCCCCTGGAGCGGTGCCCTCCTGATAAACCGGCCAGGGGGCC
TATGTTCTTTACTTTTTTACAAGAGAAGTCACTCAACATCTTAAAATGGC
CAGGTGAGTCGACGAGCAAGCCCGGCGGATCAGGCAGCGTGCTTGCAGAT
TTGACTTGCAACGCCCGCATTGTGTCGACGAAGGCTTTTGGCTCCTCTGT
CGCTGTCTCAAGCAGCATCTAACCCTGCGTCGCCGTTTCCATTTGCAGGA
GATTCGAGGTACCATGTACCCATACGATGTTCCAGATTACGCTTCGCCGA
AGAAAAAGCGCAAGGTCGAAGCGTCCGACAAGAAGTACAGCATCGGCCTG
GACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAA
GGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCA
TCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCC
GAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAA
GAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGG
TGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAG
GATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGT
GGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGG
TGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCC
CACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCC
CGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACA
ACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAG
GCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGAT
CGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTG
CCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCC
GAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGA
CAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCG
CCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAAC
ACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGA
CGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGC
TGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTAC
GCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCAT
CAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGC
TGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGC
ATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCA
GGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGA
TCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAAC
AGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTG
GAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCG
AGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCC
AAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAA
AGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCG
AGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTG
ACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGA
CTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCA
CATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAAT
GAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTT
TGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGT
TCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGG
GGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGG
CAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACT
TCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAG
AAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAA
TCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGG
TGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATC
GTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAA
CAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCA
GCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAG
AAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCA
GGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGC
CTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGA
AGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGT
GAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTA
CCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGC
GAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCA
GATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGT
ACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAG
TCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCG
CGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCG
TGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTG
TACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGA
GCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCA
TGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAG
CGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAA
GGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGA
ATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCT
ATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTG
GGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTG
TGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGT
GTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAA
GAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGG
ACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGC
CGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACT
GGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATG
AGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTG
GAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTT
CTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCG
CCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATC
ATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTA
CTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGC
TGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGG
ATCGACCTGTCTCAGCTGGGAGGCGACAGCCCCAAGAAGAAGAGAAAGGT
GGAGGCCAGCTAACATATGATTCGAATGTCTTTCTTGCGCTATGACACTT
CCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCA
ACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCG
CTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGAT
TGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATC
ACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAG
GGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACATGACACAA
GAATCCCTGTTACTTCTCGACCGTATTGATTCGGATGATTCCTACGCGAG
CCTGCGGAACGACCAGGAATTCTGGGAGGTGAGTCGACGAGCAAGCCCGG
CGGATCAGGCAGCGTGCTTGCAGATTTGACTTGCAACGCCCGCATTGTGT
CGACGAAGGCTTTTGGCTCCTCTGTCGCTGTCTCAAGCAGCATCTAACCC
TGCGTCGCCGTTTCCATTTGCAGCCGCTGGCCCGCCGAGCCCTGGAGGAG
CTCGGGCTGCCGGTGCCGCCGGTGCTGCGGGTGCCCGGCGAGAGCACCAA
CCCCGTACTGGTCGGCGAGCCCGGCCCGGTGATCAAGCTGTTCGGCGAGC
ACTGGTGCGGTCCGGAGAGCCTCGCGTCGGAGTCGGAGGCGTACGCGGTC
CTGGCGGACGCCCCGGTGCCGGTGCCCCGCCTCCTCGGCCGCGGCGAGCT
GCGGCCCGGCACCGGAGCCTGGCCGTGGCCCTACCTGGTGATGAGCCGGA
TGACCGGCACCACCTGGCGGTCCGCGATGGACGGCACGACCGACCGGAAC
GCGCTGCTCGCCCTGGCCCGCGAACTCGGCCGGGTGCTCGGCCGGCTGCA
CAGGGTGCCGCTGACCGGGAACACCGTGCTCACCCCCCATTCCGAGGTCT
TCCCGGAACTGCTGCGGGAACGCCGCGCGGCGACCGTCGAGGACCACCGC
GGGTGGGGCTACCTCTCGCCCCGGCTGCTGGACCGCCTGGAGGACTGGCT
GCCGGACGTGGACACGCTGCTGGCCGGCCGCGAACCCCGGTTCGTCCACG
GCGACCTGCACGGGACCAACATCTTCGTGGACCTGGCCGCGACCGAGGTC
ACCGGGATCGTCGACTTCACCGACGTCTATGCGGGAGACTCCCGCTACAG
CCTGGTGCAACTGCATCTCAACGCCTTCCGGGGCGACCGCGAGATCCTGG
CCGCGCTGCTCGACGGGGCGCAGTGGAAGCGGACCGAGGACTTCGCCCGC
GAACTGCTCGCCTTCACCTTCCTGCACGACTTCGAGGTGTTCGAGGAGAC
CCCGCTGGATCTCTCCGGCTTCACCGATCCGGAGGAACTGGCGCAGTTCC
TCTGGGGGCCGCCGGACACCGCCCCCGGCGCCTGATAAGGATCCGGCAAG
ACTGGCCCCGCTTGGCAACGCAACAGTGAGCCCCTCCCTAGTGTGTTTGG
GGATGTGACTATGTATTCGTGTGTTGGCCAACGGGTCAACCCGAACAGAT
TGATACCCGCCTTGGCATTTCCTGTCAGAATGTAACGTCAGTTGATGGTA CT
[0369] For all modified Chlamydomonas reinhardtii cells, Applicants
used PCR, SURVEYOR nuclease assay, and DNA sequencing to verify
successful modification.
Example 16: Use of Cas9 as a Transcriptional Repressor in
Bacteria
[0370] The ability to artificially control transcription is
essential both to the study of gene function and to the
construction of synthetic gene networks with desired properties.
Applicants describe here the use of the RNA-guided Cas9 protein as
a programmable transcriptional repressor.
[0371] Applicants have previously demonstrated how the Cas9 protein
of Streptococcus pyogenes SF370 can be used to direct genome
editing in Streptococcus pneumoniae. In this study Applicants
engineered the crR6Rk strain containing a minimal CRISPR system,
consisting of cas9, the tracrRNA and a repeat. The D10A-H840
mutations were introduced into cas9 in this strain, giving strain
crR6Rk**. Four spacers targeting different positions of the bgaA
.beta.-galactosidase gene promoter were cloned in the CRISPR array
carried by the previously described pDB98 plasmid. Applicants
observed a X to Y fold reduction in .beta.-galactosidase activity
depending on the targeted position, demonstrating the potential of
Cas9 as a programmable repressor (FIG. 73).
[0372] To achieve Cas9** repression in Escherichia coli a green
fluorescence protein (GFP) reporter plasmimd (pDB127) was
constructred to express the gfpmut2 gene from a constituitive
promoter. The promoter was designed to carry several NPP PAMs on
both strands, to measure the effect of Cas9** binding at various
positions. Applicants introduced the D10A-H840 mutations into
pCas9, a plasmid described carrying the tracrRNA, cas9 and a
minimal CRISPR array designed for the easy cloning of new spacers.
Twenty-two different spacers were designed to target different
regions of the gfpmut2 promoter and open reading frame. An
approximately 20-fold reduction of fluorescence of was observed
upon targeting regions overlapping or adjacent to the -35 and -10
promoter elements and to the Shine-Dalgarno sequence. Targets on
both strands showed similar repression levels. These results
suggest that the binding of Cas9** to any position of the promoter
region prevents transcription initiation, presumably through steric
inhibition of RNAP binding.
[0373] To determine whether Cas9** could prevent transcription
elongation, Applicants directed it to the reading frame of gpfmut2.
A reduction in fluorescence was observed both when the coding and
non-coding strands where targeted, suggesting that Cas9 binding is
actually strong enough to represent an obstacle to the running
RNAP. However, while a 40% reduction in expression was observed
when the coding strand was the target, a 20-fold reduction was
observed for the non-coding strand (FIG. 21b, compare T9, T10 and
T11 to B9, B10 and B11). To directly determine the effects of
Cas9** binding on transcription, Applicants extracted RNA from
strains carrying either the T5, T10, B10 or a control construct
that does not target pDB127 and subjected it to Northern blot
analysis using either a probe binding before (B477) or after (B510)
the B10 and T10 target sites. Consistent with Applicants'
fluorescence methods, no gfpmut2 transcription was detected when
Cas9** was directed to the promoter region (T5 target) and a
transcription was observed after the targeting of the T10 region.
Interestingly, a smaller transcript was observed with the B477
probe. This band corresponds to the expected size of a transcript
that would be interrupted by Cas9**, and is a direct indication of
a transcriptional termination caused by dgRNA::Cas9** binding to
the coding strand. Surprisingly, Applicants detected no transcript
when the non-coding strand was targeted (B10). Since Cas9** binding
to the B10 region is unlikely to interfere with transcription
initiation, this result suggests that the mRNA was degraded.
DgRNA::Cas9 was shown to bind ssRNA in vitro. Applicants speculate
that binding may trigger degradation of the mRNA by host nucleases.
Indeed, ribosome stalling can induce cleavage on the translated
mRNA in E. coli.
[0374] Some applications require a precise tuning gene expression
rather than its complete repression. Applicants sought to achieve
intermediate repression levels through the introduction of
mismatches that will weaken the crRNA/target interactions.
Applicants created a series of spacers based on the B1, T5 and B10
constructs with increasing numbers of mutations in the 5' end of
the crRNA. Up to 8 mutations in B1 and T5 did not affect the
repression level, and a progressive increased in fluorescence was
observed for additional mutations.
[0375] The observed repression with only an 8 nt match between the
crRNA and its target raises the question of off-targeting effects
of the use of Cas9** as a transcriptional regulator. Since a good
PAM (NGG) is also required for Cas9 binding, the number of
nucleotides to match to obtain some level of respiration is 10. A
10 nt match occurs randomly once every .about.1 Mbp, and such sites
are thus likely to be found even in small bacterial genomes.
However, to effectively repress transcription, such site needs to
be in the promoter region of gene, which makes off-targeting much
less likely. Applicants also showed that gene expression can be
affected if the non-coding strand of a gene is targeted. For this
to happen, a random target would have to be in the right
orientation, but such events relatively more likely to happen. As a
matter of fact, during the course of this study Applicants were
unable to construct one of the designed spacer on pCas9**.
Applicants later found this spacer showed a 12 bp match next to a
good PAM in the essential murC gene. Such off-targeting could
easily be avoided by a systematic blast of the designed
spacers.
[0376] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
REFERENCES
[0377] 1. Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S.
& Gregory, P. D. Genome editing with engineered zinc finger
nucleases. Nat. Rev. Genet. 11, 636-646 (2010). [0378] 2.
Bogdanove, A. J. & Voytas, D. F. TAL effectors: customizable
proteins for DNA targeting. Science 333, 1843-1846 (2011). [0379]
3. Stoddard, B. L. Homing endonuclease structure and function. Q.
Rev. Biophys. 38, 49-95 (2005). [0380] 4. Bae, T. & Schneewind,
O. Allelic replacement in Staphylococcus aureus with inducible
counter-selection. Plasmid 55, 58-63 (2006). [0381] 5. Sung, C. K.,
Li, H., Claverys, J. P. & Morrison, D. A. An rpsL cassette,
janus, for gene replacement through negative selection in
Streptococcus pneumoniae. Appl. Environ. Microbiol. 67, 5190-5196
(2001). [0382] 6. Sharan, S. K., Thomason, L. C., Kuznetsov, S. G.
& Court, D. L. Recombineering: a homologous recombination-based
method of genetic engineering. Nat. Protoc. 4, 206-223 (2009).
[0383] 7. Jinek, M. et al. A programmable dual-RNA-guided DNA
endonuclease in adaptive bacterial immunity. Science 337, 816-821
(2012). [0384] 8. Deveau, H., Garneau, J. E. & Moineau, S.
CRISPR/Cas system and its role in phage-bacteria interactions.
Annu. Rev. Microbiol. 64, 475-493 (2010). [0385] 9. Horvath, P.
& Barrangou, R. CRISPR/Cas, the immune system of bacteria and
archaea. Science 327, 167-170 (2010). [0386] 10. Terns, M. P. &
Terns, R. M. CRISPR-based adaptive immune systems. Curr. Opin.
Microbiol. 14, 321-327 (2011). [0387] 11. van der Oost, J., Jore,
M. M., Westra, E. R., Lundgren, M. & Brouns, S. J. CRISPR-based
adaptive and heritable immunity in prokaryotes. Trends. Biochem.
Sci. 34, 401-407 (2009). [0388] 12. Brouns, S. J. et al. Small
CRISPR RNAs guide antiviral defense in prokaryotes. Science 321,
960-964 (2008). [0389] 13. Carte, J., Wang, R., Li, H., Terns, R.
M. & Terns, M. P. Cas6 is an endoribonuclease that generates
guide RNAs for invader defense in prokaryotes. Genes Dev. 22,
3489-3496 (2008). [0390] 14. Deltcheva, E. et al. CRISPR RNA
maturation by trans-encoded small RNA and host factor RNase III.
Nature 471, 602-607 (2011). [0391] 15. Hatoum-Aslan, A., Maniv, I.
& Marraffini, L. A. Mature clustered, regularly interspaced,
short palindromic repeats RNA (crRNA) length is measured by a ruler
mechanism anchored at the precursor processing site. Proc. Natl.
Acad. Sci. U.S.A. 108, 21218-21222 (2011). [0392] 16. Haurwitz, R.
E., Jinek, M., Wiedenheft, B., Zhou, K. & Doudna, J. A.
Sequence- and structure-specific RNA processing by a CRISPR
endonuclease. Science 329, 1355-1358 (2010). [0393] 17. Deveau, H.
et al. Phage response to CRISPR-encoded resistance in Streptococcus
thermophilus. J. Bacteriol. 190, 1390-1400 (2008). [0394] 18.
Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V.
Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage
for adaptive immunity in bacteria. Proc. Natl. Acad. Sci. U.S.A.
(2012). [0395] 19. Makarova, K. S., Aravind, L., Wolf, Y. I. &
Koonin, E. V. Unification of Cas protein families and a simple
scenario for the origin and evolution of CRISPR-Cas systems. Biol.
Direct. 6, 38 (2011). [0396] 20. Barrangou, R. RNA-mediated
programmable DNA cleavage. Nat. Biotechnol. 30, 836-838 (2012).
[0397] 21. Brouns, S. J. Molecular biology. A Swiss army knife of
immunity. Science 337, 808-809 (2012). [0398] 22. Carroll, D. A
CRISPR Approach to Gene Targeting. Mol. Ther. 20, 1658-1660 (2012).
[0399] 23. Bikard, D., Hatoum-Aslan, A., Mucida, D. &
Marraffini, L. A. CRISPR interference can prevent natural
transformation and virulence acquisition during in vivo bacterial
infection. Cell Host Microbe 12, 177-186 (2012). [0400] 24.
Sapranauskas, R. et al. The Streptococcus thermophilus CRISPR/Cas
system provides immunity in Escherichia coli. Nucleic Acids Res.
(2011). [0401] 25. Semenova, E. et al. Interference by clustered
regularly interspaced short palindromic repeat (CRISPR) RNA is
governed by a seed sequence. Proc. Natl. Acad. Sci. U.S.A. (2011).
[0402] 26. Wiedenheft, B. et al. RNA-guided complex from a
bacterial immune system enhances target recognition through seed
sequence interactions. Proc. Natl. Acad. Sci. U.S.A. (2011). [0403]
27. Zahner, D. & Hakenbeck, R. The Streptococcus pneumoniae
beta-galactosidase is a surface protein. J. Bacteriol. 182,
5919-5921 (2000). [0404] 28. Marraffini, L. A., Dedent, A. C. &
Schneewind, O. Sortases and the art of anchoring proteins to the
envelopes of gram-positive bacteria. Microbiol. Mol. Biol. Rev. 70,
192-221 (2006). [0405] 29. Motamedi, M. R., Szigety, S. K. &
Rosenberg, S. M. Double-strand-break repair recombination in
Escherichia coli: physical evidence for a DNA replication mechanism
in vivo. Genes Dev. 13, 2889-2903 (1999). [0406] 30. Hosaka, T. et
al. The novel mutation K87E in ribosomal protein S12 enhances
protein synthesis activity during the late growth phase in
Escherichia coli. Mol. Genet. Genomics 271, 317-324 (2004). [0407]
31. Costantino, N. & Court, D. L. Enhanced levels of lambda
Red-mediated recombinants in mismatch repair mutants. Proc. Natl.
Acad. Sci. U.S.A. 100, 15748-15753 (2003). [0408] 32. Edgar, R.
& Qimron, U. The Escherichia coli CRISPR system protects from
lambda lysogenization, lysogens, and prophage induction. J.
Bacteriol. 192, 6291-6294 (2010). [0409] 33. Marraffini, L. A.
& Sontheimer, E. J. Self versus non-self discrimination during
CRISPR RNA-directed immunity. Nature 463, 568-571 (2010). [0410]
34. Fischer, S. et al. An archaeal immune system can detect
multiple Protospacer Adjacent Motifs (PAMs) to target invader DNA.
J. Biol. Chem. 287, 33351-33363 (2012). [0411] 35. Gudbergsdottir,
S. et al. Dynamic properties of the Sulfolobus CRISPR/Cas and
CRISPR/Cmr systems when challenged with vector-borne viral and
plasmid genes and protospacers. Mol. Microbiol. 79, 35-49 (2011).
[0412] 36. Wang, H. H. et al. Genome-scale promoter engineering by
coselection MAGE. Nat Methods 9, 591-593 (2012). [0413] 37. Cong,
L. et al. Multiplex Genome Engineering Using CRISPR/Cas Systems.
Science In press (2013). [0414] 38. Mali, P. et al. RNA-Guided
Human Genome Engineering via Cas9. Science In press (2013). [0415]
39. Hoskins, J. et al. Genome of the bacterium Streptococcus
pneumoniae strain R6. J. Bacteriol. 183, 5709-5717 (2001). [0416]
40. Havarstein, L. S., Coomaraswamy, G. & Morrison, D. A. An
unmodified heptadecapeptide pheromone induces competence for
genetic transformation in Streptococcus pneumoniae. Proc. Natl.
Acad. Sci. U.S.A. 92, 11140-11144 (1995). [0417] 41. Horinouchi, S.
& Weisblum, B. Nucleotide sequence and functional map of pC194,
a plasmid that specifies inducible chloramphenicol resistance. J.
Bacteriol. 150, 815-825 (1982). [0418] 42. Horton, R. M. In Vitro
Recombination and Mutagenesis of DNA: SOEing Together Tailor-Made
Genes. Methods Mol. Biol. 15, 251-261 (1993). [0419] 43.
Podbielski, A., Spellerberg, B., Woischnik, M., Pohl, B. &
Lutticken, R. Novel series of plasmid vectors for gene inactivation
and expression analysis in group A streptococci (GAS). Gene 177,
137-147 (1996). [0420] 44. Husmann, L. K., Scott, J. R., Lindahl,
G. & Stenberg, L. Expression of the Arp protein, a member of
the M protein family, is not sufficient to inhibit phagocytosis of
Streptococcus pyogenes. Infection and immunity 63, 345-348 (1995).
[0421] 45. Gibson, D. G. et al. Enzymatic assembly of DNA molecules
up to several hundred kilobases. Nat Methods 6, 343-345 (2009).
PARTIES TO A JOINT RESEARCH AGREEMENT
[0422] The claimed invention was made by, on behalf of, and/or in
connection with one or more of the following parties to a joint
research agreement: The Broad Institute, Inc., Massachusetts
Institute of Technology, and President and Fellows of Harvard
College. The joint research agreement was in effect on and before
the date the claimed invention was made, and the claimed invention
was made as a result of activities undertaken within the scope of
the joint research agreement.
Sequence CWU 1
1
54317PRTSimian virus 40 1Pro Lys Lys Lys Arg Lys Val1
5216PRTUnknownDescription of Unknown Nucleoplasmin bipartite NLS
sequence 2Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys
Lys Lys1 5 10 1539PRTUnknownDescription of Unknown C-myc NLS
sequence 3Pro Ala Ala Lys Arg Val Lys Leu Asp1
5411PRTUnknownDescription of Unknown C-myc NLS sequence 4Arg Gln
Arg Arg Asn Glu Leu Lys Arg Ser Pro1 5 10538PRTHomo sapiens 5Asn
Gln Ser Ser Asn Phe Gly Pro Met Lys Gly Gly Asn Phe Gly Gly1 5 10
15Arg Ser Ser Gly Pro Tyr Gly Gly Gly Gly Gln Tyr Phe Ala Lys Pro
20 25 30Arg Asn Gln Gly Gly Tyr 35642PRTUnknownDescription of
Unknown IBB domain from importin-alpha sequence 6Arg Met Arg Ile
Glx Phe Lys Asn Lys Gly Lys Asp Thr Ala Glu Leu1 5 10 15Arg Arg Arg
Arg Val Glu Val Ser Val Glu Leu Arg Lys Ala Lys Lys 20 25 30Asp Glu
Gln Ile Leu Lys Arg Arg Asn Val 35 4078PRTUnknownDescription of
Unknown Myoma T protein sequence 7Val Ser Arg Lys Arg Pro Arg Pro1
588PRTUnknownDescription of Unknown Myoma T protein sequence 8Pro
Pro Lys Lys Ala Arg Glu Asp1 598PRTHomo sapiens 9Pro Gln Pro Lys
Lys Lys Pro Leu1 51012PRTMus musculus 10Ser Ala Leu Ile Lys Lys Lys
Lys Lys Met Ala Pro1 5 10115PRTInfluenza virus 11Asp Arg Leu Arg
Arg1 5127PRTInfluenza virus 12Pro Lys Gln Lys Lys Arg Lys1
51310PRTHepatitus delta virus 13Arg Lys Leu Lys Lys Lys Ile Lys Lys
Leu1 5 101410PRTMus musculus 14Arg Glu Lys Lys Lys Phe Leu Lys Arg
Arg1 5 101520PRTHomo sapiens 15Lys Arg Lys Gly Asp Glu Val Asp Gly
Val Asp Glu Val Ala Lys Lys1 5 10 15Lys Ser Lys Lys 201617PRTHomo
sapiens 16Arg Lys Cys Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys
Thr Lys1 5 10 15Lys1727DNAArtificial SequenceDescription of
Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(20)a, c, t or
gmodified_base(21)..(22)a, c, t, g, unknown or other 17nnnnnnnnnn
nnnnnnnnnn nnagaaw 271819DNAArtificial SequenceDescription of
Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(12)a, c, t or
gmodified_base(13)..(14)a, c, t, g, unknown or other 18nnnnnnnnnn
nnnnagaaw 191927DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotidemodified_base(1)..(20)a, c, t or
gmodified_base(21)..(22)a, c, t, g, unknown or other 19nnnnnnnnnn
nnnnnnnnnn nnagaaw 272018DNAArtificial SequenceDescription of
Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(11)a, c, t or
gmodified_base(12)..(13)a, c, t, g, unknown or other 20nnnnnnnnnn
nnnagaaw 1821137DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotidemodified_base(1)..(20)a, c, t, g,
unknown or other 21nnnnnnnnnn nnnnnnnnnn gtttttgtac tctcaagatt
tagaaataaa tcttgcagaa 60gctacaaaga taaggcttca tgccgaaatc aacaccctgt
cattttatgg cagggtgttt 120tcgttattta atttttt 13722123DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
polynucleotidemodified_base(1)..(20)a, c, t, g, unknown or other
22nnnnnnnnnn nnnnnnnnnn gtttttgtac tctcagaaat gcagaagcta caaagataag
60gcttcatgcc gaaatcaaca ccctgtcatt ttatggcagg gtgttttcgt tatttaattt
120ttt 12323110DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotidemodified_base(1)..(20)a, c, t, g,
unknown or other 23nnnnnnnnnn nnnnnnnnnn gtttttgtac tctcagaaat
gcagaagcta caaagataag 60gcttcatgcc gaaatcaaca ccctgtcatt ttatggcagg
gtgttttttt 11024102DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotidemodified_base(1)..(20)a, c, t, g,
unknown or other 24nnnnnnnnnn nnnnnnnnnn gttttagagc tagaaatagc
aagttaaaat aaggctagtc 60cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
tt 1022588DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotidemodified_base(1)..(20)a, c, t, g, unknown
or other 25nnnnnnnnnn nnnnnnnnnn gttttagagc tagaaatagc aagttaaaat
aaggctagtc 60cgttatcaac ttgaaaaagt gttttttt 882676DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(20)a, c, t, g, unknown or other
26nnnnnnnnnn nnnnnnnnnn gttttagagc tagaaatagc aagttaaaat aaggctagtc
60cgttatcatt tttttt 762712DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 27gttttagagc ta
122831DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 28tagcaagtta aaataaggct agtccgtttt t
312927DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotidemodified_base(1)..(22)a, c, t, g, unknown
or other 29nnnnnnnnnn nnnnnnnnnn nnagaaw 273012RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 30guuuuagagc ua 123133DNAHomo sapiens 31ggacatcgat
gtcacctcca atgactaggg tgg 333233DNAHomo sapiens 32cattggaggt
gacatcgatg tcctccccat tgg 333333DNAHomo sapiens 33ggaagggcct
gagtccgagc agaagaagaa ggg 333433DNAHomo sapiens 34ggtggcgaga
ggggccgaga ttgggtgttc agg 333533DNAHomo sapiens 35atgcaggagg
gtggcgagag gggccgagat tgg 333621DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 36aaaaccaccc ttctctctgg c
213721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 37ggagattgga gacacggaga g 213820DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
38ctggaaagcc aatgcctgac 203920DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 39ggcagcaaac tccttgtcct
2040335DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 40gagggcctat ttcccatgat tccttcatat
ttgcatatac gatacaaggc tgttagagag 60ataattggaa ttaatttgac tgtaaacaca
aagatattag tacaaaatac gtgacgtaga 120aagtaataat ttcttgggta
gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180atgcttaccg
taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
240cgaaacaccg gaaccattca aaacagcata gcaagttaaa ataaggctag
tccgttatca 300acttgaaaaa gtggcaccga gtcggtgctt ttttt
33541423DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 41gagggcctat ttcccatgat tccttcatat
ttgcatatac gatacaaggc tgttagagag 60ataattggaa ttaatttgac tgtaaacaca
aagatattag tacaaaatac gtgacgtaga 120aagtaataat ttcttgggta
gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180atgcttaccg
taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
240cgaaacaccg gtagtattaa gtattgtttt atggctgata aatttctttg
aatttctcct 300tgattatttg ttataaaagt tataaaataa tcttgttgga
accattcaaa acagcatagc 360aagttaaaat aaggctagtc cgttatcaac
ttgaaaaagt ggcaccgagt cggtgctttt 420ttt 42342339DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
42gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
60ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
120aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
ggactatcat 180atgcttaccg taacttgaaa gtatttcgat ttcttggctt
tatatatctt gtggaaagga 240cgaaacaccg ggttttagag ctatgctgtt
ttgaatggtc ccaaaacggg tcttcgagaa 300gacgttttag agctatgctg
ttttgaatgg tcccaaaac 33943309DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 43gagggcctat
ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60ataattggaa
ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
120aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
ggactatcat 180atgcttaccg taacttgaaa gtatttcgat ttcttggctt
tatatatctt gtggaaagga 240cgaaacaccg ggtcttcgag aagacctgtt
ttagagctag aaatagcaag ttaaaataag 300gctagtccg
309441648PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 44Met Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys
Asp His Asp Ile Asp1 5 10 15Tyr Lys Asp Asp Asp Asp Lys Met Ala Pro
Lys Lys Lys Arg Lys Val 20 25 30Gly Ile His Gly Val Pro Ala Ala Asp
Lys Lys Tyr Ser Ile Gly Leu 35 40 45Asp Ile Gly Thr Asn Ser Val Gly
Trp Ala Val Ile Thr Asp Glu Tyr 50 55 60Lys Val Pro Ser Lys Lys Phe
Lys Val Leu Gly Asn Thr Asp Arg His65 70 75 80Ser Ile Lys Lys Asn
Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu 85 90 95Thr Ala Glu Ala
Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr 100 105 110Arg Arg
Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu 115 120
125Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe
130 135 140Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe
Gly Asn145 150 155 160Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr
Pro Thr Ile Tyr His 165 170 175Leu Arg Lys Lys Leu Val Asp Ser Thr
Asp Lys Ala Asp Leu Arg Leu 180 185 190Ile Tyr Leu Ala Leu Ala His
Met Ile Lys Phe Arg Gly His Phe Leu 195 200 205Ile Glu Gly Asp Leu
Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe 210 215 220Ile Gln Leu
Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile225 230 235
240Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser
245 250 255Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly
Glu Lys 260 265 270Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser
Leu Gly Leu Thr 275 280 285Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala
Glu Asp Ala Lys Leu Gln 290 295 300Leu Ser Lys Asp Thr Tyr Asp Asp
Asp Leu Asp Asn Leu Leu Ala Gln305 310 315 320Ile Gly Asp Gln Tyr
Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser 325 330 335Asp Ala Ile
Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr 340 345 350Lys
Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His 355 360
365Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu
370 375 380Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr
Ala Gly385 390 395 400Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe
Tyr Lys Phe Ile Lys 405 410 415Pro Ile Leu Glu Lys Met Asp Gly Thr
Glu Glu Leu Leu Val Lys Leu 420 425 430Asn Arg Glu Asp Leu Leu Arg
Lys Gln Arg Thr Phe Asp Asn Gly Ser 435 440 445Ile Pro His Gln Ile
His Leu Gly Glu Leu His Ala Ile Leu Arg Arg 450 455 460Gln Glu Asp
Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu465 470 475
480Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg
485 490 495Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu
Thr Ile 500 505 510Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly
Ala Ser Ala Gln 515 520 525Ser Phe Ile Glu Arg Met Thr Asn Phe Asp
Lys Asn Leu Pro Asn Glu 530 535 540Lys Val Leu Pro Lys His Ser Leu
Leu Tyr Glu Tyr Phe Thr Val Tyr545 550 555 560Asn Glu Leu Thr Lys
Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro 565 570 575Ala Phe Leu
Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe 580 585 590Lys
Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe 595 600
605Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp
610 615 620Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys
Ile Ile625 630 635 640Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn
Glu Asp Ile Leu Glu 645 650 655Asp Ile Val Leu Thr Leu Thr Leu Phe
Glu Asp Arg Glu Met Ile Glu 660 665 670Glu Arg Leu Lys Thr Tyr Ala
His Leu Phe Asp Asp Lys Val Met Lys 675 680 685Gln Leu Lys Arg Arg
Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys 690 695 700Leu Ile Asn
Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp705 710 715
720Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile
725 730 735His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala
Gln Val 740 745 750Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala
Asn Leu Ala Gly 755 760 765Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln
Thr Val Lys Val Val Asp 770 775 780Glu Leu Val Lys Val Met Gly Arg
His Lys Pro Glu Asn Ile Val Ile785 790 795 800Glu Met Ala Arg Glu
Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser 805 810 815Arg Glu Arg
Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser 820 825 830Gln
Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu 835 840
845Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp
850 855 860Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp
His Ile865 870 875 880Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
Asp Asn Lys Val Leu 885 890 895Thr Arg Ser Asp Lys Asn Arg Gly Lys
Ser Asp Asn Val Pro Ser Glu 900 905 910Glu Val Val Lys Lys Met Lys
Asn Tyr Trp Arg Gln Leu Leu Asn Ala 915 920 925Lys Leu Ile Thr Gln
Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg 930 935 940Gly Gly Leu
Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu945 950 955
960Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser
965 970 975Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg
Glu Val 980 985 990Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp
Phe Arg Lys Asp 995 1000 1005Phe Gln Phe Tyr Lys Val Arg Glu Ile
Asn Asn Tyr His His Ala 1010 1015 1020His Asp Ala Tyr Leu Asn Ala
Val Val Gly Thr Ala Leu Ile Lys 1025 1030 1035Lys Tyr Pro Lys Leu
Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys 1040 1045 1050Val Tyr Asp
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile 1055 1060 1065Gly
Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn 1070 1075
1080Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys
1085 1090 1095Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile
Val Trp 1100 1105 1110Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys
Val Leu Ser Met 1115 1120 1125Pro Gln Val Asn Ile Val Lys Lys Thr
Glu Val Gln Thr Gly Gly 1130 1135 1140Phe Ser Lys Glu Ser Ile Leu
Pro Lys Arg Asn Ser Asp Lys Leu 1145 1150 1155Ile Ala Arg Lys
Lys
Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe 1160 1165 1170Asp Ser Pro
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val 1175 1180 1185Glu
Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu 1190 1195
1200Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile
1205 1210 1215Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys
Asp Leu 1220 1225 1230Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu
Leu Glu Asn Gly 1235 1240 1245Arg Lys Arg Met Leu Ala Ser Ala Gly
Glu Leu Gln Lys Gly Asn 1250 1255 1260Glu Leu Ala Leu Pro Ser Lys
Tyr Val Asn Phe Leu Tyr Leu Ala 1265 1270 1275Ser His Tyr Glu Lys
Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln 1280 1285 1290Lys Gln Leu
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile 1295 1300 1305Ile
Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp 1310 1315
1320Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp
1325 1330 1335Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu
Phe Thr 1340 1345 1350Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys
Tyr Phe Asp Thr 1355 1360 1365Thr Ile Asp Arg Lys Arg Tyr Thr Ser
Thr Lys Glu Val Leu Asp 1370 1375 1380Ala Thr Leu Ile His Gln Ser
Ile Thr Gly Leu Tyr Glu Thr Arg 1385 1390 1395Ile Asp Leu Ser Gln
Leu Gly Gly Asp Ala Ala Ala Val Ser Lys 1400 1405 1410Gly Glu Glu
Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu 1415 1420 1425Asp
Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly 1430 1435
1440Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys
1445 1450 1455Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val
Thr Thr 1460 1465 1470Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr
Pro Asp His Met 1475 1480 1485Lys Gln His Asp Phe Phe Lys Ser Ala
Met Pro Glu Gly Tyr Val 1490 1495 1500Gln Glu Arg Thr Ile Phe Phe
Lys Asp Asp Gly Asn Tyr Lys Thr 1505 1510 1515Arg Ala Glu Val Lys
Phe Glu Gly Asp Thr Leu Val Asn Arg Ile 1520 1525 1530Glu Leu Lys
Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly 1535 1540 1545His
Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met 1550 1555
1560Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg
1565 1570 1575His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His
Tyr Gln 1580 1585 1590Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu
Leu Pro Asp Asn 1595 1600 1605His Tyr Leu Ser Thr Gln Ser Ala Leu
Ser Lys Asp Pro Asn Glu 1610 1615 1620Lys Arg Asp His Met Val Leu
Leu Glu Phe Val Thr Ala Ala Gly 1625 1630 1635Ile Thr Leu Gly Met
Asp Glu Leu Tyr Lys 1640 1645451625PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
45Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val1
5 10 15Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys
Phe 20 25 30Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn
Leu Ile 35 40 45Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala
Thr Arg Leu 50 55 60Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys
Asn Arg Ile Cys65 70 75 80Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met
Ala Lys Val Asp Asp Ser 85 90 95Phe Phe His Arg Leu Glu Glu Ser Phe
Leu Val Glu Glu Asp Lys Lys 100 105 110His Glu Arg His Pro Ile Phe
Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125His Glu Lys Tyr Pro
Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140Ser Thr Asp
Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His145 150 155
160Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln
Thr Tyr 180 185 190Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser
Gly Val Asp Ala 195 200 205Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys
Ser Arg Arg Leu Glu Asn 210 215 220Leu Ile Ala Gln Leu Pro Gly Glu
Lys Lys Asn Gly Leu Phe Gly Asn225 230 235 240Leu Ile Ala Leu Ser
Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255Asp Leu Ala
Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270Asp
Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280
285Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser
Ala Ser305 310 315 320Met Ile Lys Arg Tyr Asp Glu His His Gln Asp
Leu Thr Leu Leu Lys 325 330 335Ala Leu Val Arg Gln Gln Leu Pro Glu
Lys Tyr Lys Glu Ile Phe Phe 340 345 350Asp Gln Ser Lys Asn Gly Tyr
Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365Gln Glu Glu Phe Tyr
Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380Gly Thr Glu
Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg385 390 395
400Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr
Pro Phe 420 425 430Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu
Thr Phe Arg Ile 435 440 445Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly
Asn Ser Arg Phe Ala Trp 450 455 460Met Thr Arg Lys Ser Glu Glu Thr
Ile Thr Pro Trp Asn Phe Glu Glu465 470 475 480Val Val Asp Lys Gly
Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495Asn Phe Asp
Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510Leu
Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520
525Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys
Val Thr545 550 555 560Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys
Ile Glu Cys Phe Asp 565 570 575Ser Val Glu Ile Ser Gly Val Glu Asp
Arg Phe Asn Ala Ser Leu Gly 580 585 590Thr Tyr His Asp Leu Leu Lys
Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605Asn Glu Glu Asn Glu
Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620Leu Phe Glu
Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala625 630 635
640His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile
Arg Asp 660 665 670Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys
Ser Asp Gly Phe 675 680 685Ala Asn Arg Asn Phe Met Gln Leu Ile His
Asp Asp Ser Leu Thr Phe 690 695 700Lys Glu Asp Ile Gln Lys Ala Gln
Val Ser Gly Gln Gly Asp Ser Leu705 710 715 720His Glu His Ile Ala
Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735Ile Leu Gln
Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750Arg
His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760
765Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu
His Pro785 790 795 800Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu
Tyr Leu Tyr Tyr Leu 805 810 815Gln Asn Gly Arg Asp Met Tyr Val Asp
Gln Glu Leu Asp Ile Asn Arg 820 825 830Leu Ser Asp Tyr Asp Val Asp
His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845Asp Asp Ser Ile Asp
Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860Gly Lys Ser
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys865 870 875
880Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu
Leu Asp 900 905 910Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr
Arg Gln Ile Thr 915 920 925Lys His Val Ala Gln Ile Leu Asp Ser Arg
Met Asn Thr Lys Tyr Asp 930 935 940Glu Asn Asp Lys Leu Ile Arg Glu
Val Lys Val Ile Thr Leu Lys Ser945 950 955 960Lys Leu Val Ser Asp
Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975Glu Ile Asn
Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990Val
Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995
1000 1005Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile
Ala 1010 1015 1020Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys
Tyr Phe Phe 1025 1030 1035Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr
Glu Ile Thr Leu Ala 1040 1045 1050Asn Gly Glu Ile Arg Lys Arg Pro
Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065Thr Gly Glu Ile Val Trp
Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080Arg Lys Val Leu
Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095Glu Val
Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105
1110Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro
1115 1120 1125Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr
Ser Val 1130 1135 1140Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser
Lys Lys Leu Lys 1145 1150 1155Ser Val Lys Glu Leu Leu Gly Ile Thr
Ile Met Glu Arg Ser Ser 1160 1165 1170Phe Glu Lys Asn Pro Ile Asp
Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185Glu Val Lys Lys Asp
Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200Phe Glu Leu
Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215Glu
Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225
1230Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln
His Lys 1250 1255 1260His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser
Glu Phe Ser Lys 1265 1270 1275Arg Val Ile Leu Ala Asp Ala Asn Leu
Asp Lys Val Leu Ser Ala 1280 1285 1290Tyr Asn Lys His Arg Asp Lys
Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305Ile Ile His Leu Phe
Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320Phe Lys Tyr
Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335Thr
Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345
1350Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365Ala Ala Ala Val Ser Lys Gly Glu Glu Leu Phe Thr Gly
Val Val 1370 1375 1380Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn
Gly His Lys Phe 1385 1390 1395Ser Val Ser Gly Glu Gly Glu Gly Asp
Ala Thr Tyr Gly Lys Leu 1400 1405 1410Thr Leu Lys Phe Ile Cys Thr
Thr Gly Lys Leu Pro Val Pro Trp 1415 1420 1425Pro Thr Leu Val Thr
Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser 1430 1435 1440Arg Tyr Pro
Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala 1445 1450 1455Met
Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp 1460 1465
1470Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp
1475 1480 1485Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe
Lys Glu 1490 1495 1500Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
Asn Tyr Asn Ser 1505 1510 1515His Asn Val Tyr Ile Met Ala Asp Lys
Gln Lys Asn Gly Ile Lys 1520 1525 1530Val Asn Phe Lys Ile Arg His
Asn Ile Glu Asp Gly Ser Val Gln 1535 1540 1545Leu Ala Asp His Tyr
Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 1550 1555 1560Val Leu Leu
Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 1565 1570 1575Ser
Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu 1580 1585
1590Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr
1595 1600 1605Lys Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala
Lys Lys 1610 1615 1620Lys Lys 1625461664PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
46Met Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp1
5 10 15Tyr Lys Asp Asp Asp Asp Lys Met Ala Pro Lys Lys Lys Arg Lys
Val 20 25 30Gly Ile His Gly Val Pro Ala Ala Asp Lys Lys Tyr Ser Ile
Gly Leu 35 40 45Asp Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr
Asp Glu Tyr 50 55 60Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn
Thr Asp Arg His65 70 75 80Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu
Leu Phe Asp Ser Gly Glu 85 90 95Thr Ala Glu Ala Thr Arg Leu Lys Arg
Thr Ala Arg Arg Arg Tyr Thr 100 105 110Arg Arg Lys Asn Arg Ile Cys
Tyr Leu Gln Glu Ile Phe Ser Asn Glu 115 120 125Met Ala Lys Val Asp
Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe 130 135 140Leu Val Glu
Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn145 150 155
160Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His
165 170 175Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu
Arg Leu 180 185 190Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg
Gly His Phe Leu 195 200 205Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser
Asp Val Asp Lys Leu Phe 210 215 220Ile Gln Leu Val Gln Thr Tyr Asn
Gln Leu Phe Glu Glu Asn Pro Ile225 230 235 240Asn Ala Ser Gly Val
Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser 245 250 255Lys Ser Arg
Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys 260 265 270Lys
Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr 275 280
285Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln
290 295 300Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu
Ala Gln305 310 315 320Ile Gly Asp Gln
Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser 325 330 335Asp Ala
Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr 340 345
350Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His
355 360 365Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu
Pro Glu 370 375 380Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn
Gly Tyr Ala Gly385 390 395 400Tyr Ile Asp Gly Gly Ala Ser Gln Glu
Glu Phe Tyr Lys Phe Ile Lys 405 410 415Pro Ile Leu Glu Lys Met Asp
Gly Thr Glu Glu Leu Leu Val Lys Leu 420 425 430Asn Arg Glu Asp Leu
Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser 435 440 445Ile Pro His
Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg 450 455 460Gln
Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu465 470
475 480Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala
Arg 485 490 495Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu
Glu Thr Ile 500 505 510Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys
Gly Ala Ser Ala Gln 515 520 525Ser Phe Ile Glu Arg Met Thr Asn Phe
Asp Lys Asn Leu Pro Asn Glu 530 535 540Lys Val Leu Pro Lys His Ser
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr545 550 555 560Asn Glu Leu Thr
Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro 565 570 575Ala Phe
Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe 580 585
590Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe
595 600 605Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val
Glu Asp 610 615 620Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu
Leu Lys Ile Ile625 630 635 640Lys Asp Lys Asp Phe Leu Asp Asn Glu
Glu Asn Glu Asp Ile Leu Glu 645 650 655Asp Ile Val Leu Thr Leu Thr
Leu Phe Glu Asp Arg Glu Met Ile Glu 660 665 670Glu Arg Leu Lys Thr
Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys 675 680 685Gln Leu Lys
Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys 690 695 700Leu
Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp705 710
715 720Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu
Ile 725 730 735His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys
Ala Gln Val 740 745 750Ser Gly Gln Gly Asp Ser Leu His Glu His Ile
Ala Asn Leu Ala Gly 755 760 765Ser Pro Ala Ile Lys Lys Gly Ile Leu
Gln Thr Val Lys Val Val Asp 770 775 780Glu Leu Val Lys Val Met Gly
Arg His Lys Pro Glu Asn Ile Val Ile785 790 795 800Glu Met Ala Arg
Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser 805 810 815Arg Glu
Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser 820 825
830Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu
835 840 845Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr
Val Asp 850 855 860Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
Val Asp His Ile865 870 875 880Val Pro Gln Ser Phe Leu Lys Asp Asp
Ser Ile Asp Asn Lys Val Leu 885 890 895Thr Arg Ser Asp Lys Asn Arg
Gly Lys Ser Asp Asn Val Pro Ser Glu 900 905 910Glu Val Val Lys Lys
Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala 915 920 925Lys Leu Ile
Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg 930 935 940Gly
Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu945 950
955 960Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp
Ser 965 970 975Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile
Arg Glu Val 980 985 990Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser
Asp Phe Arg Lys Asp 995 1000 1005Phe Gln Phe Tyr Lys Val Arg Glu
Ile Asn Asn Tyr His His Ala 1010 1015 1020His Asp Ala Tyr Leu Asn
Ala Val Val Gly Thr Ala Leu Ile Lys 1025 1030 1035Lys Tyr Pro Lys
Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys 1040 1045 1050Val Tyr
Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile 1055 1060
1065Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn
1070 1075 1080Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile
Arg Lys 1085 1090 1095Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly
Glu Ile Val Trp 1100 1105 1110Asp Lys Gly Arg Asp Phe Ala Thr Val
Arg Lys Val Leu Ser Met 1115 1120 1125Pro Gln Val Asn Ile Val Lys
Lys Thr Glu Val Gln Thr Gly Gly 1130 1135 1140Phe Ser Lys Glu Ser
Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu 1145 1150 1155Ile Ala Arg
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe 1160 1165 1170Asp
Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val 1175 1180
1185Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu
1190 1195 1200Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn
Pro Ile 1205 1210 1215Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val
Lys Lys Asp Leu 1220 1225 1230Ile Ile Lys Leu Pro Lys Tyr Ser Leu
Phe Glu Leu Glu Asn Gly 1235 1240 1245Arg Lys Arg Met Leu Ala Ser
Ala Gly Glu Leu Gln Lys Gly Asn 1250 1255 1260Glu Leu Ala Leu Pro
Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala 1265 1270 1275Ser His Tyr
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln 1280 1285 1290Lys
Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile 1295 1300
1305Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp
1310 1315 1320Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His
Arg Asp 1325 1330 1335Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile
His Leu Phe Thr 1340 1345 1350Leu Thr Asn Leu Gly Ala Pro Ala Ala
Phe Lys Tyr Phe Asp Thr 1355 1360 1365Thr Ile Asp Arg Lys Arg Tyr
Thr Ser Thr Lys Glu Val Leu Asp 1370 1375 1380Ala Thr Leu Ile His
Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg 1385 1390 1395Ile Asp Leu
Ser Gln Leu Gly Gly Asp Ala Ala Ala Val Ser Lys 1400 1405 1410Gly
Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu 1415 1420
1425Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly
1430 1435 1440Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe
Ile Cys 1445 1450 1455Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr
Leu Val Thr Thr 1460 1465 1470Leu Thr Tyr Gly Val Gln Cys Phe Ser
Arg Tyr Pro Asp His Met 1475 1480 1485Lys Gln His Asp Phe Phe Lys
Ser Ala Met Pro Glu Gly Tyr Val 1490 1495 1500Gln Glu Arg Thr Ile
Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 1505 1510 1515Arg Ala Glu
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile 1520 1525 1530Glu
Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly 1535 1540
1545His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met
1550 1555 1560Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys
Ile Arg 1565 1570 1575His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala
Asp His Tyr Gln 1580 1585 1590Gln Asn Thr Pro Ile Gly Asp Gly Pro
Val Leu Leu Pro Asp Asn 1595 1600 1605His Tyr Leu Ser Thr Gln Ser
Ala Leu Ser Lys Asp Pro Asn Glu 1610 1615 1620Lys Arg Asp His Met
Val Leu Leu Glu Phe Val Thr Ala Ala Gly 1625 1630 1635Ile Thr Leu
Gly Met Asp Glu Leu Tyr Lys Lys Arg Pro Ala Ala 1640 1645 1650Thr
Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 1655
1660471423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 47Met Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys
Asp His Asp Ile Asp1 5 10 15Tyr Lys Asp Asp Asp Asp Lys Met Ala Pro
Lys Lys Lys Arg Lys Val 20 25 30Gly Ile His Gly Val Pro Ala Ala Asp
Lys Lys Tyr Ser Ile Gly Leu 35 40 45Asp Ile Gly Thr Asn Ser Val Gly
Trp Ala Val Ile Thr Asp Glu Tyr 50 55 60Lys Val Pro Ser Lys Lys Phe
Lys Val Leu Gly Asn Thr Asp Arg His65 70 75 80Ser Ile Lys Lys Asn
Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu 85 90 95Thr Ala Glu Ala
Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr 100 105 110Arg Arg
Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu 115 120
125Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe
130 135 140Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe
Gly Asn145 150 155 160Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr
Pro Thr Ile Tyr His 165 170 175Leu Arg Lys Lys Leu Val Asp Ser Thr
Asp Lys Ala Asp Leu Arg Leu 180 185 190Ile Tyr Leu Ala Leu Ala His
Met Ile Lys Phe Arg Gly His Phe Leu 195 200 205Ile Glu Gly Asp Leu
Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe 210 215 220Ile Gln Leu
Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile225 230 235
240Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser
245 250 255Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly
Glu Lys 260 265 270Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser
Leu Gly Leu Thr 275 280 285Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala
Glu Asp Ala Lys Leu Gln 290 295 300Leu Ser Lys Asp Thr Tyr Asp Asp
Asp Leu Asp Asn Leu Leu Ala Gln305 310 315 320Ile Gly Asp Gln Tyr
Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser 325 330 335Asp Ala Ile
Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr 340 345 350Lys
Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His 355 360
365Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu
370 375 380Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr
Ala Gly385 390 395 400Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe
Tyr Lys Phe Ile Lys 405 410 415Pro Ile Leu Glu Lys Met Asp Gly Thr
Glu Glu Leu Leu Val Lys Leu 420 425 430Asn Arg Glu Asp Leu Leu Arg
Lys Gln Arg Thr Phe Asp Asn Gly Ser 435 440 445Ile Pro His Gln Ile
His Leu Gly Glu Leu His Ala Ile Leu Arg Arg 450 455 460Gln Glu Asp
Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu465 470 475
480Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg
485 490 495Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu
Thr Ile 500 505 510Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly
Ala Ser Ala Gln 515 520 525Ser Phe Ile Glu Arg Met Thr Asn Phe Asp
Lys Asn Leu Pro Asn Glu 530 535 540Lys Val Leu Pro Lys His Ser Leu
Leu Tyr Glu Tyr Phe Thr Val Tyr545 550 555 560Asn Glu Leu Thr Lys
Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro 565 570 575Ala Phe Leu
Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe 580 585 590Lys
Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe 595 600
605Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp
610 615 620Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys
Ile Ile625 630 635 640Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn
Glu Asp Ile Leu Glu 645 650 655Asp Ile Val Leu Thr Leu Thr Leu Phe
Glu Asp Arg Glu Met Ile Glu 660 665 670Glu Arg Leu Lys Thr Tyr Ala
His Leu Phe Asp Asp Lys Val Met Lys 675 680 685Gln Leu Lys Arg Arg
Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys 690 695 700Leu Ile Asn
Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp705 710 715
720Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile
725 730 735His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala
Gln Val 740 745 750Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala
Asn Leu Ala Gly 755 760 765Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln
Thr Val Lys Val Val Asp 770 775 780Glu Leu Val Lys Val Met Gly Arg
His Lys Pro Glu Asn Ile Val Ile785 790 795 800Glu Met Ala Arg Glu
Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser 805 810 815Arg Glu Arg
Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser 820 825 830Gln
Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu 835 840
845Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp
850 855 860Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp
His Ile865 870 875 880Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
Asp Asn Lys Val Leu 885 890 895Thr Arg Ser Asp Lys Asn Arg Gly Lys
Ser Asp Asn Val Pro Ser Glu 900 905 910Glu Val Val Lys Lys Met Lys
Asn Tyr Trp Arg Gln Leu Leu Asn Ala 915 920 925Lys Leu Ile Thr Gln
Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg 930 935 940Gly Gly Leu
Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu945 950 955
960Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser
965 970 975Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg
Glu Val 980 985 990Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp
Phe Arg Lys Asp 995 1000 1005Phe Gln Phe Tyr Lys Val Arg Glu Ile
Asn Asn Tyr His His Ala 1010 1015 1020His Asp Ala Tyr Leu Asn Ala
Val Val Gly Thr Ala Leu Ile Lys 1025 1030 1035Lys Tyr Pro Lys Leu
Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys 1040 1045 1050Val Tyr Asp
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile 1055 1060 1065Gly
Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn 1070 1075
1080Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys
1085 1090 1095Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile
Val Trp 1100
1105 1110Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser
Met 1115 1120 1125Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln
Thr Gly Gly 1130 1135 1140Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg
Asn Ser Asp Lys Leu 1145 1150 1155Ile Ala Arg Lys Lys Asp Trp Asp
Pro Lys Lys Tyr Gly Gly Phe 1160 1165 1170Asp Ser Pro Thr Val Ala
Tyr Ser Val Leu Val Val Ala Lys Val 1175 1180 1185Glu Lys Gly Lys
Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu 1190 1195 1200Gly Ile
Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile 1205 1210
1215Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu
1220 1225 1230Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu
Asn Gly 1235 1240 1245Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu
Gln Lys Gly Asn 1250 1255 1260Glu Leu Ala Leu Pro Ser Lys Tyr Val
Asn Phe Leu Tyr Leu Ala 1265 1270 1275Ser His Tyr Glu Lys Leu Lys
Gly Ser Pro Glu Asp Asn Glu Gln 1280 1285 1290Lys Gln Leu Phe Val
Glu Gln His Lys His Tyr Leu Asp Glu Ile 1295 1300 1305Ile Glu Gln
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp 1310 1315 1320Ala
Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp 1325 1330
1335Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr
1340 1345 1350Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe
Asp Thr 1355 1360 1365Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys
Glu Val Leu Asp 1370 1375 1380Ala Thr Leu Ile His Gln Ser Ile Thr
Gly Leu Tyr Glu Thr Arg 1385 1390 1395Ile Asp Leu Ser Gln Leu Gly
Gly Asp Lys Arg Pro Ala Ala Thr 1400 1405 1410Lys Lys Ala Gly Gln
Ala Lys Lys Lys Lys 1415 142048483PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 48Met Phe Leu Phe Leu
Ser Leu Thr Ser Phe Leu Ser Ser Ser Arg Thr1 5 10 15Leu Val Ser Lys
Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe 20 25 30Met Arg Phe
Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe 35 40 45Glu Ile
Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr 50 55 60Ala
Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp65 70 75
80Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His
85 90 95Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly
Phe 100 105 110Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val
Val Thr Val 115 120 125Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe
Ile Tyr Lys Val Lys 130 135 140Leu Arg Gly Thr Asn Phe Pro Ser Asp
Gly Pro Val Met Gln Lys Lys145 150 155 160Thr Met Gly Trp Glu Ala
Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly 165 170 175Ala Leu Lys Gly
Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly 180 185 190His Tyr
Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val 195 200
205Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser
210 215 220His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala
Glu Gly225 230 235 240Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr
Lys Gly Ser Lys Gln 245 250 255Leu Glu Glu Leu Leu Ser Thr Ser Phe
Asp Ile Gln Phe Asn Asp Leu 260 265 270Thr Leu Leu Glu Thr Ala Phe
Thr His Thr Ser Tyr Ala Asn Glu His 275 280 285Arg Leu Leu Asn Val
Ser His Asn Glu Arg Leu Glu Phe Leu Gly Asp 290 295 300Ala Val Leu
Gln Leu Ile Ile Ser Glu Tyr Leu Phe Ala Lys Tyr Pro305 310 315
320Lys Lys Thr Glu Gly Asp Met Ser Lys Leu Arg Ser Met Ile Val Arg
325 330 335Glu Glu Ser Leu Ala Gly Phe Ser Arg Phe Cys Ser Phe Asp
Ala Tyr 340 345 350Ile Lys Leu Gly Lys Gly Glu Glu Lys Ser Gly Gly
Arg Arg Arg Asp 355 360 365Thr Ile Leu Gly Asp Leu Phe Glu Ala Phe
Leu Gly Ala Leu Leu Leu 370 375 380Asp Lys Gly Ile Asp Ala Val Arg
Arg Phe Leu Lys Gln Val Met Ile385 390 395 400Pro Gln Val Glu Lys
Gly Asn Phe Glu Arg Val Lys Asp Tyr Lys Thr 405 410 415Cys Leu Gln
Glu Phe Leu Gln Thr Lys Gly Asp Val Ala Ile Asp Tyr 420 425 430Gln
Val Ile Ser Glu Lys Gly Pro Ala His Ala Lys Gln Phe Glu Val 435 440
445Ser Ile Val Val Asn Gly Ala Val Leu Ser Lys Gly Leu Gly Lys Ser
450 455 460Lys Lys Leu Ala Glu Gln Asp Ala Ala Lys Asn Ala Leu Ala
Gln Leu465 470 475 480Ser Glu Val49483PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
49Met Lys Gln Leu Glu Glu Leu Leu Ser Thr Ser Phe Asp Ile Gln Phe1
5 10 15Asn Asp Leu Thr Leu Leu Glu Thr Ala Phe Thr His Thr Ser Tyr
Ala 20 25 30Asn Glu His Arg Leu Leu Asn Val Ser His Asn Glu Arg Leu
Glu Phe 35 40 45Leu Gly Asp Ala Val Leu Gln Leu Ile Ile Ser Glu Tyr
Leu Phe Ala 50 55 60Lys Tyr Pro Lys Lys Thr Glu Gly Asp Met Ser Lys
Leu Arg Ser Met65 70 75 80Ile Val Arg Glu Glu Ser Leu Ala Gly Phe
Ser Arg Phe Cys Ser Phe 85 90 95Asp Ala Tyr Ile Lys Leu Gly Lys Gly
Glu Glu Lys Ser Gly Gly Arg 100 105 110Arg Arg Asp Thr Ile Leu Gly
Asp Leu Phe Glu Ala Phe Leu Gly Ala 115 120 125Leu Leu Leu Asp Lys
Gly Ile Asp Ala Val Arg Arg Phe Leu Lys Gln 130 135 140Val Met Ile
Pro Gln Val Glu Lys Gly Asn Phe Glu Arg Val Lys Asp145 150 155
160Tyr Lys Thr Cys Leu Gln Glu Phe Leu Gln Thr Lys Gly Asp Val Ala
165 170 175Ile Asp Tyr Gln Val Ile Ser Glu Lys Gly Pro Ala His Ala
Lys Gln 180 185 190Phe Glu Val Ser Ile Val Val Asn Gly Ala Val Leu
Ser Lys Gly Leu 195 200 205Gly Lys Ser Lys Lys Leu Ala Glu Gln Asp
Ala Ala Lys Asn Ala Leu 210 215 220Ala Gln Leu Ser Glu Val Gly Ser
Val Ser Lys Gly Glu Glu Asp Asn225 230 235 240Met Ala Ile Ile Lys
Glu Phe Met Arg Phe Lys Val His Met Glu Gly 245 250 255Ser Val Asn
Gly His Glu Phe Glu Ile Glu Gly Glu Gly Glu Gly Arg 260 265 270Pro
Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys Val Thr Lys Gly Gly 275 280
285Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln Phe Met Tyr Gly
290 295 300Ser Lys Ala Tyr Val Lys His Pro Ala Asp Ile Pro Asp Tyr
Leu Lys305 310 315 320Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg
Val Met Asn Phe Glu 325 330 335Asp Gly Gly Val Val Thr Val Thr Gln
Asp Ser Ser Leu Gln Asp Gly 340 345 350Glu Phe Ile Tyr Lys Val Lys
Leu Arg Gly Thr Asn Phe Pro Ser Asp 355 360 365Gly Pro Val Met Gln
Lys Lys Thr Met Gly Trp Glu Ala Ser Ser Glu 370 375 380Arg Met Tyr
Pro Glu Asp Gly Ala Leu Lys Gly Glu Ile Lys Gln Arg385 390 395
400Leu Lys Leu Lys Asp Gly Gly His Tyr Asp Ala Glu Val Lys Thr Thr
405 410 415Tyr Lys Ala Lys Lys Pro Val Gln Leu Pro Gly Ala Tyr Asn
Val Asn 420 425 430Ile Lys Leu Asp Ile Thr Ser His Asn Glu Asp Tyr
Thr Ile Val Glu 435 440 445Gln Tyr Glu Arg Ala Glu Gly Arg His Ser
Thr Gly Gly Met Asp Glu 450 455 460Leu Tyr Lys Lys Arg Pro Ala Ala
Thr Lys Lys Ala Gly Gln Ala Lys465 470 475 480Lys Lys
Lys501423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 50Met Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys
Asp His Asp Ile Asp1 5 10 15Tyr Lys Asp Asp Asp Asp Lys Met Ala Pro
Lys Lys Lys Arg Lys Val 20 25 30Gly Ile His Gly Val Pro Ala Ala Asp
Lys Lys Tyr Ser Ile Gly Leu 35 40 45Ala Ile Gly Thr Asn Ser Val Gly
Trp Ala Val Ile Thr Asp Glu Tyr 50 55 60Lys Val Pro Ser Lys Lys Phe
Lys Val Leu Gly Asn Thr Asp Arg His65 70 75 80Ser Ile Lys Lys Asn
Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu 85 90 95Thr Ala Glu Ala
Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr 100 105 110Arg Arg
Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu 115 120
125Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe
130 135 140Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe
Gly Asn145 150 155 160Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr
Pro Thr Ile Tyr His 165 170 175Leu Arg Lys Lys Leu Val Asp Ser Thr
Asp Lys Ala Asp Leu Arg Leu 180 185 190Ile Tyr Leu Ala Leu Ala His
Met Ile Lys Phe Arg Gly His Phe Leu 195 200 205Ile Glu Gly Asp Leu
Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe 210 215 220Ile Gln Leu
Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile225 230 235
240Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser
245 250 255Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly
Glu Lys 260 265 270Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser
Leu Gly Leu Thr 275 280 285Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala
Glu Asp Ala Lys Leu Gln 290 295 300Leu Ser Lys Asp Thr Tyr Asp Asp
Asp Leu Asp Asn Leu Leu Ala Gln305 310 315 320Ile Gly Asp Gln Tyr
Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser 325 330 335Asp Ala Ile
Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr 340 345 350Lys
Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His 355 360
365Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu
370 375 380Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr
Ala Gly385 390 395 400Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe
Tyr Lys Phe Ile Lys 405 410 415Pro Ile Leu Glu Lys Met Asp Gly Thr
Glu Glu Leu Leu Val Lys Leu 420 425 430Asn Arg Glu Asp Leu Leu Arg
Lys Gln Arg Thr Phe Asp Asn Gly Ser 435 440 445Ile Pro His Gln Ile
His Leu Gly Glu Leu His Ala Ile Leu Arg Arg 450 455 460Gln Glu Asp
Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu465 470 475
480Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg
485 490 495Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu
Thr Ile 500 505 510Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly
Ala Ser Ala Gln 515 520 525Ser Phe Ile Glu Arg Met Thr Asn Phe Asp
Lys Asn Leu Pro Asn Glu 530 535 540Lys Val Leu Pro Lys His Ser Leu
Leu Tyr Glu Tyr Phe Thr Val Tyr545 550 555 560Asn Glu Leu Thr Lys
Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro 565 570 575Ala Phe Leu
Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe 580 585 590Lys
Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe 595 600
605Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp
610 615 620Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys
Ile Ile625 630 635 640Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn
Glu Asp Ile Leu Glu 645 650 655Asp Ile Val Leu Thr Leu Thr Leu Phe
Glu Asp Arg Glu Met Ile Glu 660 665 670Glu Arg Leu Lys Thr Tyr Ala
His Leu Phe Asp Asp Lys Val Met Lys 675 680 685Gln Leu Lys Arg Arg
Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys 690 695 700Leu Ile Asn
Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp705 710 715
720Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile
725 730 735His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala
Gln Val 740 745 750Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala
Asn Leu Ala Gly 755 760 765Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln
Thr Val Lys Val Val Asp 770 775 780Glu Leu Val Lys Val Met Gly Arg
His Lys Pro Glu Asn Ile Val Ile785 790 795 800Glu Met Ala Arg Glu
Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser 805 810 815Arg Glu Arg
Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser 820 825 830Gln
Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu 835 840
845Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp
850 855 860Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp
His Ile865 870 875 880Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
Asp Asn Lys Val Leu 885 890 895Thr Arg Ser Asp Lys Asn Arg Gly Lys
Ser Asp Asn Val Pro Ser Glu 900 905 910Glu Val Val Lys Lys Met Lys
Asn Tyr Trp Arg Gln Leu Leu Asn Ala 915 920 925Lys Leu Ile Thr Gln
Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg 930 935 940Gly Gly Leu
Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu945 950 955
960Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser
965 970 975Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg
Glu Val 980 985 990Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp
Phe Arg Lys Asp 995 1000 1005Phe Gln Phe Tyr Lys Val Arg Glu Ile
Asn Asn Tyr His His Ala 1010 1015 1020His Asp Ala Tyr Leu Asn Ala
Val Val Gly Thr Ala Leu Ile Lys 1025 1030 1035Lys Tyr Pro Lys Leu
Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys 1040 1045 1050Val Tyr Asp
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile 1055 1060 1065Gly
Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn 1070 1075
1080Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys
1085 1090 1095Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile
Val Trp 1100 1105 1110Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys
Val Leu Ser Met 1115 1120 1125Pro Gln Val Asn Ile Val Lys Lys Thr
Glu Val Gln Thr Gly Gly 1130 1135 1140Phe Ser Lys Glu Ser Ile Leu
Pro Lys Arg Asn Ser Asp Lys Leu 1145
1150 1155Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly
Phe 1160 1165 1170Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val
Ala Lys Val 1175 1180 1185Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser
Val Lys Glu Leu Leu 1190 1195 1200Gly Ile Thr Ile Met Glu Arg Ser
Ser Phe Glu Lys Asn Pro Ile 1205 1210 1215Asp Phe Leu Glu Ala Lys
Gly Tyr Lys Glu Val Lys Lys Asp Leu 1220 1225 1230Ile Ile Lys Leu
Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly 1235 1240 1245Arg Lys
Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn 1250 1255
1260Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala
1265 1270 1275Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn
Glu Gln 1280 1285 1290Lys Gln Leu Phe Val Glu Gln His Lys His Tyr
Leu Asp Glu Ile 1295 1300 1305Ile Glu Gln Ile Ser Glu Phe Ser Lys
Arg Val Ile Leu Ala Asp 1310 1315 1320Ala Asn Leu Asp Lys Val Leu
Ser Ala Tyr Asn Lys His Arg Asp 1325 1330 1335Lys Pro Ile Arg Glu
Gln Ala Glu Asn Ile Ile His Leu Phe Thr 1340 1345 1350Leu Thr Asn
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr 1355 1360 1365Thr
Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp 1370 1375
1380Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg
1385 1390 1395Ile Asp Leu Ser Gln Leu Gly Gly Asp Lys Arg Pro Ala
Ala Thr 1400 1405 1410Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 1415
1420512012DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 51gaatgctgcc ctcagacccg cttcctccct
gtccttgtct gtccaaggag aatgaggtct 60cactggtgga tttcggacta ccctgaggag
ctggcacctg agggacaagg ccccccacct 120gcccagctcc agcctctgat
gaggggtggg agagagctac atgaggttgc taagaaagcc 180tcccctgaag
gagaccacac agtgtgtgag gttggagtct ctagcagcgg gttctgtgcc
240cccagggata gtctggctgt ccaggcactg ctcttgatat aaacaccacc
tcctagttat 300gaaaccatgc ccattctgcc tctctgtatg gaaaagagca
tggggctggc ccgtggggtg 360gtgtccactt taggccctgt gggagatcat
gggaacccac gcagtgggtc ataggctctc 420tcatttacta ctcacatcca
ctctgtgaag aagcgattat gatctctcct ctagaaactc 480gtagagtccc
atgtctgccg gcttccagag cctgcactcc tccaccttgg cttggctttg
540ctggggctag aggagctagg atgcacagca gctctgtgac cctttgtttg
agaggaacag 600gaaaaccacc cttctctctg gcccactgtg tcctcttcct
gccctgccat ccccttctgt 660gaatgttaga cccatgggag cagctggtca
gaggggaccc cggcctgggg cccctaaccc 720tatgtagcct cagtcttccc
atcaggctct cagctcagcc tgagtgttga ggccccagtg 780gctgctctgg
gggcctcctg agtttctcat ctgtgcccct ccctccctgg cccaggtgaa
840ggtgtggttc cagaaccgga ggacaaagta caaacggcag aagctggagg
aggaagggcc 900tgagtccgag cagaagaaga agggctccca tcacatcaac
cggtggcgca ttgccacgaa 960gcaggccaat ggggaggaca tcgatgtcac
ctccaatgac aagcttgcta gcggtgggca 1020accacaaacc cacgagggca
gagtgctgct tgctgctggc caggcccctg cgtgggccca 1080agctggactc
tggccactcc ctggccaggc tttggggagg cctggagtca tggccccaca
1140gggcttgaag cccggggccg ccattgacag agggacaagc aatgggctgg
ctgaggcctg 1200ggaccacttg gccttctcct cggagagcct gcctgcctgg
gcgggcccgc ccgccaccgc 1260agcctcccag ctgctctccg tgtctccaat
ctcccttttg ttttgatgca tttctgtttt 1320aatttatttt ccaggcacca
ctgtagttta gtgatcccca gtgtccccct tccctatggg 1380aataataaaa
gtctctctct taatgacacg ggcatccagc tccagcccca gagcctgggg
1440tggtagattc cggctctgag ggccagtggg ggctggtaga gcaaacgcgt
tcagggcctg 1500ggagcctggg gtggggtact ggtggagggg gtcaagggta
attcattaac tcctctcttt 1560tgttggggga ccctggtctc tacctccagc
tccacagcag gagaaacagg ctagacatag 1620ggaagggcca tcctgtatct
tgagggagga caggcccagg tctttcttaa cgtattgaga 1680ggtgggaatc
aggcccaggt agttcaatgg gagagggaga gtgcttccct ctgcctagag
1740actctggtgg cttctccagt tgaggagaaa ccagaggaaa ggggaggatt
ggggtctggg 1800ggagggaaca ccattcacaa aggctgacgg ttccagtccg
aagtcgtggg cccaccagga 1860tgctcacctg tccttggaga accgctgggc
aggttgagac tgcagagaca gggcttaagg 1920ctgagcctgc aaccagtccc
cagtgactca gggcctcctc agcccaagaa agagcaacgt 1980gccagggccc
gctgagctct tgtgttcacc tg 2012521153PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
52Met Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys1
5 10 15Lys Ser Asp Leu Val Leu Gly Leu Asp Ile Gly Ile Gly Ser Val
Gly 20 25 30Val Gly Ile Leu Asn Lys Val Thr Gly Glu Ile Ile His Lys
Asn Ser 35 40 45Arg Ile Phe Pro Ala Ala Gln Ala Glu Asn Asn Leu Val
Arg Arg Thr 50 55 60Asn Arg Gln Gly Arg Arg Leu Ala Arg Arg Lys Lys
His Arg Arg Val65 70 75 80Arg Leu Asn Arg Leu Phe Glu Glu Ser Gly
Leu Ile Thr Asp Phe Thr 85 90 95Lys Ile Ser Ile Asn Leu Asn Pro Tyr
Gln Leu Arg Val Lys Gly Leu 100 105 110Thr Asp Glu Leu Ser Asn Glu
Glu Leu Phe Ile Ala Leu Lys Asn Met 115 120 125Val Lys His Arg Gly
Ile Ser Tyr Leu Asp Asp Ala Ser Asp Asp Gly 130 135 140Asn Ser Ser
Val Gly Asp Tyr Ala Gln Ile Val Lys Glu Asn Ser Lys145 150 155
160Gln Leu Glu Thr Lys Thr Pro Gly Gln Ile Gln Leu Glu Arg Tyr Gln
165 170 175Thr Tyr Gly Gln Leu Arg Gly Asp Phe Thr Val Glu Lys Asp
Gly Lys 180 185 190Lys His Arg Leu Ile Asn Val Phe Pro Thr Ser Ala
Tyr Arg Ser Glu 195 200 205Ala Leu Arg Ile Leu Gln Thr Gln Gln Glu
Phe Asn Pro Gln Ile Thr 210 215 220Asp Glu Phe Ile Asn Arg Tyr Leu
Glu Ile Leu Thr Gly Lys Arg Lys225 230 235 240Tyr Tyr His Gly Pro
Gly Asn Glu Lys Ser Arg Thr Asp Tyr Gly Arg 245 250 255Tyr Arg Thr
Ser Gly Glu Thr Leu Asp Asn Ile Phe Gly Ile Leu Ile 260 265 270Gly
Lys Cys Thr Phe Tyr Pro Asp Glu Phe Arg Ala Ala Lys Ala Ser 275 280
285Tyr Thr Ala Gln Glu Phe Asn Leu Leu Asn Asp Leu Asn Asn Leu Thr
290 295 300Val Pro Thr Glu Thr Lys Lys Leu Ser Lys Glu Gln Lys Asn
Gln Ile305 310 315 320Ile Asn Tyr Val Lys Asn Glu Lys Ala Met Gly
Pro Ala Lys Leu Phe 325 330 335Lys Tyr Ile Ala Lys Leu Leu Ser Cys
Asp Val Ala Asp Ile Lys Gly 340 345 350Tyr Arg Ile Asp Lys Ser Gly
Lys Ala Glu Ile His Thr Phe Glu Ala 355 360 365Tyr Arg Lys Met Lys
Thr Leu Glu Thr Leu Asp Ile Glu Gln Met Asp 370 375 380Arg Glu Thr
Leu Asp Lys Leu Ala Tyr Val Leu Thr Leu Asn Thr Glu385 390 395
400Arg Glu Gly Ile Gln Glu Ala Leu Glu His Glu Phe Ala Asp Gly Ser
405 410 415Phe Ser Gln Lys Gln Val Asp Glu Leu Val Gln Phe Arg Lys
Ala Asn 420 425 430Ser Ser Ile Phe Gly Lys Gly Trp His Asn Phe Ser
Val Lys Leu Met 435 440 445Met Glu Leu Ile Pro Glu Leu Tyr Glu Thr
Ser Glu Glu Gln Met Thr 450 455 460Ile Leu Thr Arg Leu Gly Lys Gln
Lys Thr Thr Ser Ser Ser Asn Lys465 470 475 480Thr Lys Tyr Ile Asp
Glu Lys Leu Leu Thr Glu Glu Ile Tyr Asn Pro 485 490 495Val Val Ala
Lys Ser Val Arg Gln Ala Ile Lys Ile Val Asn Ala Ala 500 505 510Ile
Lys Glu Tyr Gly Asp Phe Asp Asn Ile Val Ile Glu Met Ala Arg 515 520
525Glu Thr Asn Glu Asp Asp Glu Lys Lys Ala Ile Gln Lys Ile Gln Lys
530 535 540Ala Asn Lys Asp Glu Lys Asp Ala Ala Met Leu Lys Ala Ala
Asn Gln545 550 555 560Tyr Asn Gly Lys Ala Glu Leu Pro His Ser Val
Phe His Gly His Lys 565 570 575Gln Leu Ala Thr Lys Ile Arg Leu Trp
His Gln Gln Gly Glu Arg Cys 580 585 590Leu Tyr Thr Gly Lys Thr Ile
Ser Ile His Asp Leu Ile Asn Asn Ser 595 600 605Asn Gln Phe Glu Val
Asp His Ile Leu Pro Leu Ser Ile Thr Phe Asp 610 615 620Asp Ser Leu
Ala Asn Lys Val Leu Val Tyr Ala Thr Ala Asn Gln Glu625 630 635
640Lys Gly Gln Arg Thr Pro Tyr Gln Ala Leu Asp Ser Met Asp Asp Ala
645 650 655Trp Ser Phe Arg Glu Leu Lys Ala Phe Val Arg Glu Ser Lys
Thr Leu 660 665 670Ser Asn Lys Lys Lys Glu Tyr Leu Leu Thr Glu Glu
Asp Ile Ser Lys 675 680 685Phe Asp Val Arg Lys Lys Phe Ile Glu Arg
Asn Leu Val Asp Thr Arg 690 695 700Tyr Ala Ser Arg Val Val Leu Asn
Ala Leu Gln Glu His Phe Arg Ala705 710 715 720His Lys Ile Asp Thr
Lys Val Ser Val Val Arg Gly Gln Phe Thr Ser 725 730 735Gln Leu Arg
Arg His Trp Gly Ile Glu Lys Thr Arg Asp Thr Tyr His 740 745 750His
His Ala Val Asp Ala Leu Ile Ile Ala Ala Ser Ser Gln Leu Asn 755 760
765Leu Trp Lys Lys Gln Lys Asn Thr Leu Val Ser Tyr Ser Glu Asp Gln
770 775 780Leu Leu Asp Ile Glu Thr Gly Glu Leu Ile Ser Asp Asp Glu
Tyr Lys785 790 795 800Glu Ser Val Phe Lys Ala Pro Tyr Gln His Phe
Val Asp Thr Leu Lys 805 810 815Ser Lys Glu Phe Glu Asp Ser Ile Leu
Phe Ser Tyr Gln Val Asp Ser 820 825 830Lys Phe Asn Arg Lys Ile Ser
Asp Ala Thr Ile Tyr Ala Thr Arg Gln 835 840 845Ala Lys Val Gly Lys
Asp Lys Ala Asp Glu Thr Tyr Val Leu Gly Lys 850 855 860Ile Lys Asp
Ile Tyr Thr Gln Asp Gly Tyr Asp Ala Phe Met Lys Ile865 870 875
880Tyr Lys Lys Asp Lys Ser Lys Phe Leu Met Tyr Arg His Asp Pro Gln
885 890 895Thr Phe Glu Lys Val Ile Glu Pro Ile Leu Glu Asn Tyr Pro
Asn Lys 900 905 910Gln Ile Asn Glu Lys Gly Lys Glu Val Pro Cys Asn
Pro Phe Leu Lys 915 920 925Tyr Lys Glu Glu His Gly Tyr Ile Arg Lys
Tyr Ser Lys Lys Gly Asn 930 935 940Gly Pro Glu Ile Lys Ser Leu Lys
Tyr Tyr Asp Ser Lys Leu Gly Asn945 950 955 960His Ile Asp Ile Thr
Pro Lys Asp Ser Asn Asn Lys Val Val Leu Gln 965 970 975Ser Val Ser
Pro Trp Arg Ala Asp Val Tyr Phe Asn Lys Thr Thr Gly 980 985 990Lys
Tyr Glu Ile Leu Gly Leu Lys Tyr Ala Asp Leu Gln Phe Glu Lys 995
1000 1005Gly Thr Gly Thr Tyr Lys Ile Ser Gln Glu Lys Tyr Asn Asp
Ile 1010 1015 1020Lys Lys Lys Glu Gly Val Asp Ser Asp Ser Glu Phe
Lys Phe Thr 1025 1030 1035Leu Tyr Lys Asn Asp Leu Leu Leu Val Lys
Asp Thr Glu Thr Lys 1040 1045 1050Glu Gln Gln Leu Phe Arg Phe Leu
Ser Arg Thr Met Pro Lys Gln 1055 1060 1065Lys His Tyr Val Glu Leu
Lys Pro Tyr Asp Lys Gln Lys Phe Glu 1070 1075 1080Gly Gly Glu Ala
Leu Ile Lys Val Leu Gly Asn Val Ala Asn Ser 1085 1090 1095Gly Gln
Cys Lys Lys Gly Leu Gly Lys Ser Asn Ile Ser Ile Tyr 1100 1105
1110Lys Val Arg Thr Asp Val Leu Gly Asn Gln His Ile Ile Lys Asn
1115 1120 1125Glu Gly Asp Lys Pro Lys Leu Asp Phe Lys Arg Pro Ala
Ala Thr 1130 1135 1140Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 1145
115053340DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 53gagggcctat ttcccatgat tccttcatat
ttgcatatac gatacaaggc tgttagagag 60ataattggaa ttaatttgac tgtaaacaca
aagatattag tacaaaatac gtgacgtaga 120aagtaataat ttcttgggta
gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180atgcttaccg
taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
240cgaaacaccg ttacttaaat cttgcagaag ctacaaagat aaggcttcat
gccgaaatca 300acaccctgtc attttatggc agggtgtttt cgttatttaa
34054360DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotidemodified_base(288)..(317)a, c, t, g,
unknown or other 54gagggcctat ttcccatgat tccttcatat ttgcatatac
gatacaaggc tgttagagag 60ataattggaa ttaatttgac tgtaaacaca aagatattag
tacaaaatac gtgacgtaga 120aagtaataat ttcttgggta gtttgcagtt
ttaaaattat gttttaaaat ggactatcat 180atgcttaccg taacttgaaa
gtatttcgat ttcttggctt tatatatctt gtggaaagga 240cgaaacaccg
ggttttagag ctatgctgtt ttgaatggtc ccaaaacnnn nnnnnnnnnn
300nnnnnnnnnn nnnnnnngtt ttagagctat gctgttttga atggtcccaa
aacttttttt 36055318DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotidemodified_base(250)..(269)a, c, t,
g, unknown or other 55gagggcctat ttcccatgat tccttcatat ttgcatatac
gatacaaggc tgttagagag 60ataattggaa ttaatttgac tgtaaacaca aagatattag
tacaaaatac gtgacgtaga 120aagtaataat ttcttgggta gtttgcagtt
ttaaaattat gttttaaaat ggactatcat 180atgcttaccg taacttgaaa
gtatttcgat ttcttggctt tatatatctt gtggaaagga 240cgaaacaccn
nnnnnnnnnn nnnnnnnnng ttttagagct agaaatagca agttaaaata
300aggctagtcc gttttttt 31856325DNAArtificial SequenceDescription of
Artificial Sequence Synthetic
polynucleotidemodified_base(250)..(269)a, c, t, g, unknown or other
56gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
60ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
120aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
ggactatcat 180atgcttaccg taacttgaaa gtatttcgat ttcttggctt
tatatatctt gtggaaagga 240cgaaacaccn nnnnnnnnnn nnnnnnnnng
ttttagagct agaaatagca agttaaaata 300aggctagtcc gttatcattt ttttt
32557337DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotidemodified_base(250)..(269)a, c, t, g,
unknown or other 57gagggcctat ttcccatgat tccttcatat ttgcatatac
gatacaaggc tgttagagag 60ataattggaa ttaatttgac tgtaaacaca aagatattag
tacaaaatac gtgacgtaga 120aagtaataat ttcttgggta gtttgcagtt
ttaaaattat gttttaaaat ggactatcat 180atgcttaccg taacttgaaa
gtatttcgat ttcttggctt tatatatctt gtggaaagga 240cgaaacaccn
nnnnnnnnnn nnnnnnnnng ttttagagct agaaatagca agttaaaata
300aggctagtcc gttatcaact tgaaaaagtg ttttttt 33758352DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
polynucleotidemodified_base(250)..(269)a, c, t, g, unknown or other
58gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
60ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
120aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
ggactatcat 180atgcttaccg taacttgaaa gtatttcgat ttcttggctt
tatatatctt gtggaaagga 240cgaaacaccn nnnnnnnnnn nnnnnnnnng
ttttagagct agaaatagca agttaaaata 300aggctagtcc gttatcaact
tgaaaaagtg gcaccgagtc ggtgcttttt tt 352595101DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
59cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
60gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
120atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
atcatatgcc 180aagtacgccc cctattgacg tcaatgacgg taaatggccc
gcctggcatt atgcccagta 240catgacctta tgggactttc ctacttggca
gtacatctac gtattagtca tcgctattac 300catggtcgag gtgagcccca
cgttctgctt cactctcccc atctcccccc cctccccacc 360cccaattttg
tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg
420gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg
cgaggcggag 480aggtgcggcg gcagccaatc agagcggcgc gctccgaaag
tttcctttta tggcgaggcg 540gcggcggcgg cggccctata aaaagcgaag
cgcgcggcgg gcgggagtcg ctgcgacgct 600gccttcgccc cgtgccccgc
tccgccgccg cctcgcgccg cccgccccgg ctctgactga 660ccgcgttact
cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc
720tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg
tttaattacc 780tggagcacct gcctgaaatc actttttttc aggttggacc
ggtgccacca tggactataa 840ggaccacgac ggagactaca aggatcatga
tattgattac aaagacgatg acgataagat 900ggccccaaag aagaagcgga
aggtcggtat ccacggagtc ccagcagccg acaagaagta 960cagcatcggc
ctggacatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta
1020caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca
gcatcaagaa 1080gaacctgatc ggagccctgc tgttcgacag cggcgaaaca
gccgaggcca cccggctgaa 1140gagaaccgcc agaagaagat acaccagacg
gaagaaccgg atctgctatc tgcaagagat 1200cttcagcaac gagatggcca
aggtggacga cagcttcttc cacagactgg aagagtcctt 1260cctggtggaa
gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga
1320ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac
tggtggacag 1380caccgacaag gccgacctgc ggctgatcta tctggccctg
gcccacatga tcaagttccg 1440gggccacttc ctgatcgagg gcgacctgaa
ccccgacaac agcgacgtgg acaagctgtt 1500catccagctg gtgcagacct
acaaccagct gttcgaggaa aaccccatca acgccagcgg 1560cgtggacgcc
aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct
1620gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggcaacctga
ttgccctgag 1680cctgggcctg acccccaact tcaagagcaa cttcgacctg
gccgaggatg ccaaactgca 1740gctgagcaag gacacctacg acgacgacct
ggacaacctg ctggcccaga tcggcgacca 1800gtacgccgac ctgtttctgg
ccgccaagaa cctgtccgac gccatcctgc tgagcgacat 1860cctgagagtg
aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata
1920cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc
agctgcctga 1980gaagtacaaa gagattttct tcgaccagag caagaacggc
tacgccggct acattgacgg 2040cggagccagc caggaagagt tctacaagtt
catcaagccc atcctggaaa agatggacgg 2100caccgaggaa ctgctcgtga
agctgaacag agaggacctg ctgcggaagc agcggacctt 2160cgacaacggc
agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg
2220gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga
agatcctgac 2280cttccgcatc ccctactacg tgggccctct ggccagggga
aacagcagat tcgcctggat 2340gaccagaaag agcgaggaaa ccatcacccc
ctggaacttc gaggaagtgg tggacaaggg 2400cgcttccgcc cagagcttca
tcgagcggat gaccaacttc gataagaacc tgcccaacga 2460gaaggtgctg
cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac
2520caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg
gcgagcagaa 2580aaaggccatc gtggacctgc tgttcaagac caaccggaaa
gtgaccgtga agcagctgaa 2640agaggactac ttcaagaaaa tcgagtgctt
cgactccgtg gaaatctccg gcgtggaaga 2700tcggttcaac gcctccctgg
gcacatacca cgatctgctg aaaattatca aggacaagga 2760cttcctggac
aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact
2820gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc
tgttcgacga 2880caaagtgatg aagcagctga agcggcggag atacaccggc
tggggcaggc tgagccggaa 2940gctgatcaac ggcatccggg acaagcagtc
cggcaagaca atcctggatt tcctgaagtc 3000cgacggcttc gccaacagaa
acttcatgca gctgatccac gacgacagcc tgacctttaa 3060agaggacatc
cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc
3120caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga
aggtggtgga 3180cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac
atcgtgatcg aaatggccag 3240agagaaccag accacccaga agggacagaa
gaacagccgc gagagaatga agcggatcga 3300agagggcatc aaagagctgg
gcagccagat cctgaaagaa caccccgtgg aaaacaccca 3360gctgcagaac
gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga
3420ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg
tgcctcagag 3480ctttctgaag gacgactcca tcgacaacaa ggtgctgacc
agaagcgaca agaaccgggg 3540caagagcgac aacgtgccct ccgaagaggt
cgtgaagaag atgaagaact actggcggca 3600gctgctgaac gccaagctga
ttacccagag aaagttcgac aatctgacca aggccgagag 3660aggcggcctg
agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg
3720gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta
agtacgacga 3780gaatgacaag ctgatccggg aagtgaaagt gatcaccctg
aagtccaagc tggtgtccga 3840tttccggaag gatttccagt tttacaaagt
gcgcgagatc aacaactacc accacgccca 3900cgacgcctac ctgaacgccg
tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga 3960aagcgagttc
gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag
4020cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca
tcatgaactt 4080tttcaagacc gagattaccc tggccaacgg cgagatccgg
aagcggcctc tgatcgagac 4140aaacggcgaa accggggaga tcgtgtggga
taagggccgg gattttgcca ccgtgcggaa 4200agtgctgagc atgccccaag
tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt 4260cagcaaagag
tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga
4320ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt
ctgtgctggt 4380ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag
agtgtgaaag agctgctggg 4440gatcaccatc atggaaagaa gcagcttcga
gaagaatccc atcgactttc tggaagccaa 4500gggctacaaa gaagtgaaaa
aggacctgat catcaagctg cctaagtact ccctgttcga 4560gctggaaaac
ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga
4620actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact
atgagaagct 4680gaagggctcc cccgaggata atgagcagaa acagctgttt
gtggaacagc acaagcacta 4740cctggacgag atcatcgagc agatcagcga
gttctccaag agagtgatcc tggccgacgc 4800taatctggac aaagtgctgt
ccgcctacaa caagcaccgg gataagccca tcagagagca 4860ggccgagaat
atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa
4920gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg
tgctggacgc 4980caccctgatc caccagagca tcaccggcct gtacgagaca
cggatcgacc tgtctcagct 5040gggaggcgac tttctttttc ttagcttgac
cagctttctt agtagcagca ggacgcttta 5100a 510160137DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
polynucleotidemodified_base(1)..(20)a, c, t, g, unknown or other
60nnnnnnnnnn nnnnnnnnnn gttattgtac tctcaagatt tagaaataaa tcttgcagaa
60gctacaaaga taaggcttca tgccgaaatc aacaccctgt cattttatgg cagggtgttt
120tcgttattta atttttt 13761123DNAArtificial SequenceDescription of
Artificial Sequence Synthetic
polynucleotidemodified_base(1)..(20)a, c, t, g, unknown or other
61nnnnnnnnnn nnnnnnnnnn gttattgtac tctcagaaat gcagaagcta caaagataag
60gcttcatgcc gaaatcaaca ccctgtcatt ttatggcagg gtgttttcgt tatttaattt
120ttt 12362110DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotidemodified_base(1)..(20)a, c, t, g,
unknown or other 62nnnnnnnnnn nnnnnnnnnn gttattgtac tctcagaaat
gcagaagcta caaagataag 60gcttcatgcc gaaatcaaca ccctgtcatt ttatggcagg
gtgttttttt 11063137DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotidemodified_base(1)..(20)a, c, t, g,
unknown or other 63nnnnnnnnnn nnnnnnnnnn gttattgtac tctcaagatt
tagaaataaa tcttgcagaa 60gctacaatga taaggcttca tgccgaaatc aacaccctgt
cattttatgg cagggtgttt 120tcgttattta atttttt 13764123DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
polynucleotidemodified_base(1)..(20)a, c, t, g, unknown or other
64nnnnnnnnnn nnnnnnnnnn gttattgtac tctcagaaat gcagaagcta caatgataag
60gcttcatgcc gaaatcaaca ccctgtcatt ttatggcagg gtgttttcgt tatttaattt
120ttt 12365110DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotidemodified_base(1)..(20)a, c, t, g,
unknown or other 65nnnnnnnnnn nnnnnnnnnn gttattgtac tctcagaaat
gcagaagcta caatgataag 60gcttcatgcc gaaatcaaca ccctgtcatt ttatggcagg
gtgttttttt 11066107DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotidemodified_base(1)..(20)a, c, t, g,
unknown or other 66nnnnnnnnnn nnnnnnnnnn gttttagagc tgtggaaaca
cagcgagtta aaataaggct 60tagtccgtac tcaacttgaa aaggtggcac cgattcggtg
ttttttt 107674263DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 67atgaaaaggc cggcggccac
gaaaaaggcc ggccaggcaa aaaagaaaaa gaccaagccc 60tacagcatcg gcctggacat
cggcaccaat agcgtgggct gggccgtgac caccgacaac 120tacaaggtgc
ccagcaagaa aatgaaggtg ctgggcaaca cctccaagaa gtacatcaag
180aaaaacctgc tgggcgtgct gctgttcgac agcggcatta cagccgaggg
cagacggctg 240aagagaaccg ccagacggcg gtacacccgg cggagaaaca
gaatcctgta tctgcaagag 300atcttcagca ccgagatggc taccctggac
gacgccttct tccagcggct ggacgacagc 360ttcctggtgc ccgacgacaa
gcgggacagc aagtacccca tcttcggcaa cctggtggaa 420gagaaggcct
accacgacga gttccccacc atctaccacc tgagaaagta cctggccgac
480agcaccaaga aggccgacct gagactggtg tatctggccc tggcccacat
gatcaagtac 540cggggccact tcctgatcga gggcgagttc aacagcaaga
acaacgacat ccagaagaac 600ttccaggact tcctggacac ctacaacgcc
atcttcgaga gcgacctgtc cctggaaaac 660agcaagcagc tggaagagat
cgtgaaggac aagatcagca agctggaaaa gaaggaccgc 720atcctgaagc
tgttccccgg cgagaagaac agcggaatct tcagcgagtt tctgaagctg
780atcgtgggca accaggccga cttcagaaag tgcttcaacc tggacgagaa
agccagcctg 840cacttcagca aagagagcta cgacgaggac ctggaaaccc
tgctgggata tatcggcgac 900gactacagcg acgtgttcct gaaggccaag
aagctgtacg acgctatcct gctgagcggc 960ttcctgaccg tgaccgacaa
cgagacagag gccccactga gcagcgccat gattaagcgg 1020tacaacgagc
acaaagagga tctggctctg ctgaaagagt acatccggaa catcagcctg
1080aaaacctaca atgaggtgtt caaggacgac accaagaacg gctacgccgg
ctacatcgac 1140ggcaagacca accaggaaga tttctatgtg tacctgaaga
agctgctggc cgagttcgag 1200ggggccgact actttctgga aaaaatcgac
cgcgaggatt tcctgcggaa gcagcggacc 1260ttcgacaacg gcagcatccc
ctaccagatc catctgcagg aaatgcgggc catcctggac 1320aagcaggcca
agttctaccc attcctggcc aagaacaaag agcggatcga gaagatcctg
1380accttccgca tcccttacta cgtgggcccc ctggccagag gcaacagcga
ttttgcctgg 1440tccatccgga agcgcaatga gaagatcacc ccctggaact
tcgaggacgt gatcgacaaa 1500gagtccagcg ccgaggcctt catcaaccgg
atgaccagct tcgacctgta cctgcccgag 1560gaaaaggtgc tgcccaagca
cagcctgctg tacgagacat tcaatgtgta taacgagctg 1620accaaagtgc
ggtttatcgc cgagtctatg cgggactacc agttcctgga ctccaagcag
1680aaaaaggaca tcgtgcggct gtacttcaag gacaagcgga aagtgaccga
taaggacatc 1740atcgagtacc tgcacgccat ctacggctac gatggcatcg
agctgaaggg catcgagaag 1800cagttcaact ccagcctgag cacataccac
gacctgctga acattatcaa cgacaaagaa 1860tttctggacg actccagcaa
cgaggccatc atcgaagaga tcatccacac cctgaccatc 1920tttgaggacc
gcgagatgat caagcagcgg ctgagcaagt tcgagaacat cttcgacaag
1980agcgtgctga aaaagctgag cagacggcac tacaccggct ggggcaagct
gagcgccaag 2040ctgatcaacg gcatccggga cgagaagtcc ggcaacacaa
tcctggacta cctgatcgac 2100gacggcatca gcaaccggaa cttcatgcag
ctgatccacg acgacgccct gagcttcaag 2160aagaagatcc agaaggccca
gatcatcggg gacgaggaca agggcaacat caaagaagtc 2220gtgaagtccc
tgcccggcag ccccgccatc aagaagggaa tcctgcagag catcaagatc
2280gtggacgagc tcgtgaaagt gatgggcggc agaaagcccg agagcatcgt
ggtggaaatg 2340gctagagaga accagtacac caatcagggc aagagcaaca
gccagcagag actgaagaga 2400ctggaaaagt ccctgaaaga gctgggcagc
aagattctga aagagaatat ccctgccaag 2460ctgtccaaga tcgacaacaa
cgccctgcag aacgaccggc tgtacctgta ctacctgcag 2520aatggcaagg
acatgtatac aggcgacgac ctggatatcg accgcctgag caactacgac
2580atcgaccata ttatccccca ggccttcctg aaagacaaca gcattgacaa
caaagtgctg 2640gtgtcctccg ccagcaaccg cggcaagtcc gatgatgtgc
ccagcctgga agtcgtgaaa 2700aagagaaaga ccttctggta tcagctgctg
aaaagcaagc tgattagcca gaggaagttc 2760gacaacctga ccaaggccga
gagaggcggc ctgagccctg aagataaggc cggcttcatc 2820cagagacagc
tggtggaaac ccggcagatc accaagcacg tggccagact gctggatgag
2880aagtttaaca acaagaagga cgagaacaac cgggccgtgc ggaccgtgaa
gatcatcacc 2940ctgaagtcca ccctggtgtc ccagttccgg aaggacttcg
agctgtataa agtgcgcgag 3000atcaatgact ttcaccacgc ccacgacgcc
tacctgaatg ccgtggtggc ttccgccctg 3060ctgaagaagt accctaagct
ggaacccgag ttcgtgtacg gcgactaccc caagtacaac 3120tccttcagag
agcggaagtc cgccaccgag aaggtgtact tctactccaa catcatgaat
3180atctttaaga agtccatctc cctggccgat ggcagagtga tcgagcggcc
cctgatcgaa 3240gtgaacgaag agacaggcga gagcgtgtgg aacaaagaaa
gcgacctggc caccgtgcgg 3300cgggtgctga gttatcctca agtgaatgtc
gtgaagaagg tggaagaaca gaaccacggc 3360ctggatcggg gcaagcccaa
gggcctgttc aacgccaacc tgtccagcaa gcctaagccc 3420aactccaacg
agaatctcgt gggggccaaa gagtacctgg accctaagaa gtacggcgga
3480tacgccggca tctccaatag cttcaccgtg ctcgtgaagg gcacaatcga
gaagggcgct 3540aagaaaaaga tcacaaacgt gctggaattt caggggatct
ctatcctgga ccggatcaac 3600taccggaagg ataagctgaa ctttctgctg
gaaaaaggct acaaggacat tgagctgatt 3660atcgagctgc ctaagtactc
cctgttcgaa ctgagcgacg gctccagacg gatgctggcc 3720tccatcctgt
ccaccaacaa caagcggggc gagatccaca agggaaacca gatcttcctg
3780agccagaaat ttgtgaaact gctgtaccac gccaagcgga tctccaacac
catcaatgag 3840aaccaccgga aatacgtgga aaaccacaag aaagagtttg
aggaactgtt ctactacatc 3900ctggagttca acgagaacta tgtgggagcc
aagaagaacg gcaaactgct gaactccgcc 3960ttccagagct ggcagaacca
cagcatcgac gagctgtgca gctccttcat cggccctacc 4020ggcagcgagc
ggaagggact gtttgagctg acctccagag gctctgccgc cgactttgag
4080ttcctgggag tgaagatccc ccggtacaga gactacaccc cctctagtct
gctgaaggac 4140gccaccctga tccaccagag cgtgaccggc ctgtacgaaa
cccggatcga cctggctaag 4200ctgggcgagg gaaagcgtcc tgctgctact
aagaaagctg gtcaagctaa gaaaaagaaa 4260taa 42636853DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
68tcctagcagg atttctgata ttactgtcac gttttagagc tatgctgttt tga
536953DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 69gtgacagtaa tatcagaaat cctgctagga gttttgggac
cattcaaaac agc 537025DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 70gggtttcaag tctttgtagc aagag
257124DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 71gccaatgaac gggaaccctt ggtc 247225DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
primermodified_base(1)..(4)a, c, t, g, unknown or other
72nnnngacgag gcaatggctg aaatc 257325DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
primermodified_base(1)..(4)a, c, t, g, unknown or other
73nnnnttattt ggctcatatt tgctg 257425DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
74ctttacacca atcgctgcaa cagac 257545DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
75caaaatttct agtcttcttt gcctttcccc ataaaaccct cctta
457645DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 76agggttttat ggggaaaggc aaagaagact agaaattttg
atacc 457725DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 77cttacggtgc ataaagtcaa tttcc
257821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 78tggctcgatt tcagccattg c 217943DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
primermodified_base(33)..(33)a, c, t, g, unknown or other
79ctttgacgag gcaatggctg aaatcgagcc aanaaagcgc aag
438043DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primermodified_base(34)..(34)a, c, t, g, unknown or other
80ctttgacgag gcaatggctg aaatcgagcc aaanaagcgc aag
438143DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primermodified_base(35)..(35)a, c, t, g, unknown or other
81ctttgacgag gcaatggctg aaatcgagcc aaaanagcgc aag
438243DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primermodified_base(36)..(36)a, c, t, g, unknown or other
82ctttgacgag gcaatggctg aaatcgagcc aaaaangcgc aag
438343DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primermodified_base(37)..(37)a, c, t, g, unknown or other
83ctttgacgag gcaatggctg aaatcgagcc aaaaaancgc aag
438443DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primermodified_base(38)..(38)a, c, t, g, unknown or other
84ctttgacgag gcaatggctg aaatcgagcc aaaaaagngc aag
438546DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primermodified_base(39)..(39)a, c, t, g, unknown or other
85ctttgacgag gcaatggctg aaatcgagcc aaaaaagcnc aagaag
468646DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primermodified_base(40)..(40)a, c, t, g, unknown or other
86ctttgacgag gcaatggctg aaatcgagcc aaaaaagcgn aagaag
468746DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primermodified_base(41)..(41)a, c, t, g, unknown or other
87ctttgacgag gcaatggctg aaatcgagcc aaaaaagcgc nagaag
468823DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 88gcgctttttt ggctcgattt cag 238940DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
primermodified_base(31)..(31)a, c, t, g, unknown or other
89caatggctga aatcgagcca aaaaagcgca ngaagaaatc 409040DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
primermodified_base(32)..(32)a, c, t, g, unknown or other
90caatggctga aatcgagcca aaaaagcgca anaagaaatc 409140DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
primermodified_base(33)..(33)a, c, t, g, unknown or other
91caatggctga aatcgagcca aaaaagcgca agnagaaatc 409240DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
primermodified_base(34)..(34)a, c, t, g, unknown or other
92caatggctga aatcgagcca aaaaagcgca agangaaatc 409340DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
primermodified_base(35)..(35)a, c, t, g, unknown or other
93caatggctga aatcgagcca aaaaagcgca agaanaaatc 409444DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
primermodified_base(36)..(36)a, c, t, g, unknown or other
94caatggctga aatcgagcca aaaaagcgca agaagnaatc aacc
449544DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primermodified_base(37)..(37)a, c, t, g, unknown or other
95caatggctga aatcgagcca aaaaagcgca agaaganatc aacc
449644DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primermodified_base(38)..(38)a, c, t, g, unknown or other
96caatggctga aatcgagcca aaaaagcgca agaagaantc aacc
449744DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primermodified_base(39)..(39)a, c, t, g, unknown or other
97caatggctga aatcgagcca aaaaagcgca agaagaaanc aacc
449847DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primermodified_base(40)..(40)a, c, t, g, unknown or other
98caatggctga aatcgagcca aaaaagcgca agaagaaatn aaccagc
479947DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primermodified_base(41)..(41)a, c, t, g, unknown or other
99caatggctga aatcgagcca aaaaagcgca agaagaaatc naccagc
4710031DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 100gatcctccat ccgtacaacc cacaaccctg g
3110131DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 101aattccaggg ttgtgggttg tacggatgga g
3110234DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 102catggatcct atttcttaat aactaaaaat atgg
3410333DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 103catgaattca actcaacaag tctcagtgtg ctg
3310435DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 104aaacattttt tctccattta ggaaaaagga tgctg
3510535DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 105aaaacagcat cctttttcct aaatggagaa aaaat
3510635DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 106aaaccttaaa tcagtcacaa atagcagcaa aattg
3510735DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 107aaaacaattt tgctgctatt tgtgactgat ttaag
3510835DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 108aaacttttca tcatacgacc aatctgcttt atttg
3510935DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 109aaaacaaata aagcagattg gtcgtatgat gaaaa
3511035DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 110aaactcgtcc agaagttatc gtaaaagaaa tcgag
3511135DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 111aaaactcgat ttcttttacg ataacttctg gacga
3511235DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 112aaacaatctc tccaaggttt ccttaaaaat ctctg
3511335DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 113aaaacagaga tttttaagga aaccttggag agatt
3511435DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 114aaacgccatc gtcaggaaga agctatgctt gagtg
3511535DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 115aaaacactca agcatagctt cttcctgacg atggc
3511635DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 116aaacatctct atacttattg aaatttcttt gtatg
3511735DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 117aaaacataca aagaaatttc aataagtata gagat
3511835DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 118aaactagctg tgatagtccg caaaaccagc cttcg
3511935DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 119aaaacgaagg ctggttttgc ggactatcac agcta
3512035DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 120aaacatcgga aggtcgagca agtaattatc ttttg
3512135DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 121aaaacaaaag ataattactt gctcgacctt ccgat
3512235DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 122aaacaagatg gtatcgcaaa gtaagtgaca ataag
3512335DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 123aaaacttatt gtcacttact ttgcgatacc atctt
3512452DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 124gagacctttg agcttccgag actggtctca gttttgggac
cattcaaaac ag 5212552DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 125tgagaccagt ctcggaagct
caaaggtctc gttttagagc tatgctgttt tg 5212635DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
126aaactacttt acgcagcgcg gagttcggtt ttttg 3512735DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
127aaaacaaaaa accgaactcc gcgctgcgta aagta 3512845DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
128atgccggtac tgccgggcct cttgcgggat tacgaaatca tcctg
4512945DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 129gtgactggcg atgctgtcgg aatggacgat cacactactc
ttctt 4513050DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 130ttaagaaata atcttcatct aaaatatact
tcagtcacct cctagctgac 5013148DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 131attgatttga gtcagctagg
aggtgactga agtatatttt agatgaag 4813285DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
132gagacctttg agcttccgag actggtctca gttttgggac cattcaaaac
agcatagctc 60taaaacctcg tagactattt ttgtc 8513384DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
133gagaccagtc tcggaagctc aaaggtctcg ttttagagct atgctgtttt
gaatggtccc 60aaaacttcag cacactgaga cttg 8413421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
134agtcatccca gcaacaaatg g 2113531DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 135cgtggtaaat cggataacgt
tccaagtgaa g 3113622DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 136tgctcttctt cacaaacaag gg
2213721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 137aagccaaagt ttggcaccac c 2113822DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
138gtagcttatt cagtcctagt gg 2213945DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
139cgtttgttga actaatgggt gcaaattacg aatcttctcc tgacg
4514045DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 140cgtcaggaga agattcgtaa tttgcaccca ttagttcaac
aaacg 4514148DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 141gatattatgg agcctatttt tgtgggtttt
taggcataaa actatatg 4814248DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 142catatagttt tatgcctaaa
aacccacaaa aataggctcc ataatatc 4814327DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
143attatttctt aataactaaa aatatgg 2714424DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
144cgtgtacaat tgctagcgta cggc 2414524DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
145gcaccggtga tcactagtcc tagg 2414647DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
146cctaggacta gtgatcaccg gtgcaaatat gagccaaata aatatat
4714744DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 147gccgtacgct agcaattgta cacgtttgtt gaactaatgg
gtgc 4414825DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 148ttcaaatttt cccatttgat tctcc
2514947DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 149ccatattttt agttattaag aaataatacc agccatcagt
cacctcc 4715023DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 150agacgattca atagacaata agg
2315145DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 151gttttgggac cattcaaaac agcatagctc taaaacctcg
tagac 4515250DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 152gctatgctgt tttgaatggt cccaaaacca
ttattttaac acacgaggtg 5015350DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 153gctatgctgt tttgaatggt
cccaaaacgc acccattagt tcaacaaacg 5015423DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
154aattcttttc ttcatcatcg gtc 2315524DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
155aagaaagaat gaagattgtt catg 2415625DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
156ggtactaatc aaaatagtga ggagg 2515723DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
157gtttttcaaa atctgcggtt gcg 2315826DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
158aaaaattgaa aaaatggtgg aaacac 2615953DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
159atttcgtaaa cggtatcggt ttcttttaaa gttttgggac cattcaaaac agc
5316053DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 160tttaaaagaa accgataccg tttacgaaat gttttagagc
tatgctgttt tga 5316153DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 161aaacggtatc ggtttctttt
aaattcaatt gttttgggac cattcaaaac agc 5316253DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
162aattgaattt aaaagaaacc gataccgttt gttttagagc tatgctgttt tga
5316339DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 163gttccttaaa ccaaaacggt atcggtttct tttaaattc
3916447DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 164gaaaccgata ccgttttggt ttaaggaaca ggtaaagggc
atttaac 4716522DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 165cgatttcagc cattgcctcg tc
2216656DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primermodified_base(29)..(33)a, c, t, g, unknown or other
166gcctttgacg aggcaatggc tgaaatcgnn nnnaaaaagc gcaagaagaa atcaac
5616753DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 167tccgtacaac ccacaaccct gctagtgagc gttttgggac
cattcaaaac agc 5316853DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 168gctcactagc agggttgtgg
gttgtacgga gttttagagc tatgctgttt tga 5316923DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
169ttgttgccac tcttccttct ttc 2317041DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
170cagggttgtg ggttgttgcg atggagttaa ctcccatctc c
4117141DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 171gggagttaac tccatcgcaa caacccacaa ccctgctagt g
4117222DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 172gtggtatcta tcgtgatgtg ac 2217323DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
173ttaccgaaac ggaatttatc tgc 2317422DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
174aaagctagag ttccgcaatt gg 2217537DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
175gtgggttgta cggattgagt taactcccat ctccttc 3717638DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
176gatgggagtt aactcaatcc gtacaaccca caaccctg 3817740DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
177gcttcaccta ttgcagcacc aattgaccac atgaagatag 4017841DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
178gtggtcaatt ggtgctgcaa taggtgaagc taatggtgat g
4117939DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 179ctgatttgta ttaattttga gacattatgc ttcaccttc
3918040DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 180gcataatgtc tcaaaattaa tacaaatcag tgaaatcatg
4018152DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 181gttttgggac cattcaaaac agcatagctc taaaacgtga
cagtaatatc ag 5218253DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 182gttttagagc tatgctgttt
tgaatggtcc caaaacgctc actagcaggg ttg 5318359DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
183atactttacg cagcgcggag ttcggttttg taggagtggt agtatataca cgagtacat
5918433DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 184gctcactagc agggttgtgg gttgtacgga tgg
3318533DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 185tcctagcagg atttctgata ttactgtcac tgg
3318633DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 186tttaaaagaa accgataccg tttacgaaat tgg
3318784DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 187ggaaccattc ataacagcat agcaagttat
aataaggcta gtccgttatc aacttgaaaa 60agtggcaccg agtcggtgct tttt
8418836DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 188gttatagagc tatgctgtta tgaatggtcc
caaaac 3618984DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 189ggaaccattc aatacagcat
agcaagttaa tataaggcta gtccgttatc aacttgaaaa 60agtggcaccg agtcggtgct
tttt 8419036DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 190gtattagagc tatgctgtat
tgaatggtcc caaaac 36191103DNAArtificial SequenceDescription of
Artificial Sequence Synthetic
polynucleotidemodified_base(1)..(20)a, c, t, g, unknown or other
191nnnnnnnnnn nnnnnnnnnn gttttagagc tagaaatagc aagttaaaat
aaggctagtc 60cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttt
103192103DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotidemodified_base(1)..(20)a, c, t, g, unknown
or other 192nnnnnnnnnn nnnnnnnnnn gtattagagc tagaaatagc aagttaatat
aaggctagtc 60cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttt
103193123DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotidemodified_base(1)..(20)a, c, t, g, unknown
or other 193nnnnnnnnnn nnnnnnnnnn gttttagagc tatgctgttt tggaaacaaa
acagcatagc 60aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
cggtgctttt 120ttt 123194123DNAArtificial SequenceDescription of
Artificial Sequence Synthetic
polynucleotidemodified_base(1)..(20)a, c, t, g, unknown or other
194nnnnnnnnnn nnnnnnnnnn gtattagagc tatgctgtat tggaaacaat
acagcatagc 60aagttaatat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
cggtgctttt 120ttt 12319520DNAHomo sapiens 195gtcacctcca atgactaggg
201964203DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 196atggccccaa agaagaagcg gaaggtcggt
atccacggag tcccagcagc cgacaagaag 60tacagcatcg gcctggacat cggcaccaac
tctgtgggct gggccgtgat caccgacgag 120tacaaggtgc ccagcaagaa
attcaaggtg ctgggcaaca ccgaccggca cagcatcaag 180aagaacctga
tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg
240aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta
tctgcaagag 300atcttcagca acgagatggc caaggtggac gacagcttct
tccacagact ggaagagtcc 360ttcctggtgg aagaggataa gaagcacgag
cggcacccca tcttcggcaa catcgtggac
420gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa
actggtggac 480agcaccgaca aggccgacct gcggctgatc tatctggccc
tggcccacat gatcaagttc 540cggggccact tcctgatcga gggcgacctg
aaccccgaca acagcgacgt ggacaagctg 600ttcatccagc tggtgcagac
ctacaaccag ctgttcgagg aaaaccccat caacgccagc 660ggcgtggacg
ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat
720ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct
gattgccctg 780agcctgggcc tgacccccaa cttcaagagc aacttcgacc
tggccgagga tgccaaactg 840cagctgagca aggacaccta cgacgacgac
ctggacaacc tgctggccca gatcggcgac 900cagtacgccg acctgtttct
ggccgccaag aacctgtccg acgccatcct gctgagcgac 960atcctgagag
tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga
1020tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca
gcagctgcct 1080gagaagtaca aagagatttt cttcgaccag agcaagaacg
gctacgccgg ctacattgac 1140ggcggagcca gccaggaaga gttctacaag
ttcatcaagc ccatcctgga aaagatggac 1200ggcaccgagg aactgctcgt
gaagctgaac agagaggacc tgctgcggaa gcagcggacc 1260ttcgacaacg
gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg
1320cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga
gaagatcctg 1380accttccgca tcccctacta cgtgggccct ctggccaggg
gaaacagcag attcgcctgg 1440atgaccagaa agagcgagga aaccatcacc
ccctggaact tcgaggaagt ggtggacaag 1500ggcgcttccg cccagagctt
catcgagcgg atgaccaact tcgataagaa cctgcccaac 1560gagaaggtgc
tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg
1620accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag
cggcgagcag 1680aaaaaggcca tcgtggacct gctgttcaag accaaccgga
aagtgaccgt gaagcagctg 1740aaagaggact acttcaagaa aatcgagtgc
ttcgactccg tggaaatctc cggcgtggaa 1800gatcggttca acgcctccct
gggcacatac cacgatctgc tgaaaattat caaggacaag 1860gacttcctgg
acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca
1920ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca
cctgttcgac 1980gacaaagtga tgaagcagct gaagcggcgg agatacaccg
gctggggcag gctgagccgg 2040aagctgatca acggcatccg ggacaagcag
tccggcaaga caatcctgga tttcctgaag 2100tccgacggct tcgccaacag
aaacttcatg cagctgatcc acgacgacag cctgaccttt 2160aaagaggaca
tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt
2220gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt
gaaggtggtg 2280gacgagctcg tgaaagtgat gggccggcac aagcccgaga
acatcgtgat cgccatggcc 2340agagagaacc agaccaccca gaagggacag
aagaacagcc gcgagagaat gaagcggatc 2400gaagagggca tcaaagagct
gggcagccag atcctgaaag aacaccccgt ggaaaacacc 2460cagctgcaga
acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg
2520gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat
cgtgcctcag 2580agctttctga aggacgactc catcgacaac aaggtgctga
ccagaagcga caagaaccgg 2640ggcaagagcg acaacgtgcc ctccgaagag
gtcgtgaaga agatgaagaa ctactggcgg 2700cagctgctga acgccaagct
gattacccag agaaagttcg acaatctgac caaggccgag 2760agaggcggcc
tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc
2820cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac
taagtacgac 2880gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc
tgaagtccaa gctggtgtcc 2940gatttccgga aggatttcca gttttacaaa
gtgcgcgaga tcaacaacta ccaccacgcc 3000cacgacgcct acctgaacgc
cgtcgtggga accgccctga tcaaaaagta ccctaagctg 3060gaaagcgagt
tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag
3120agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa
catcatgaac 3180tttttcaaga ccgagattac cctggccaac ggcgagatcc
ggaagcggcc tctgatcgag 3240acaaacggcg aaaccgggga gatcgtgtgg
gataagggcc gggattttgc caccgtgcgg 3300aaagtgctga gcatgcccca
agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc 3360ttcagcaaag
agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag
3420gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta
ttctgtgctg 3480gtggtggcca aagtggaaaa gggcaagtcc aagaaactga
agagtgtgaa agagctgctg 3540gggatcacca tcatggaaag aagcagcttc
gagaagaatc ccatcgactt tctggaagcc 3600aagggctaca aagaagtgaa
aaaggacctg atcatcaagc tgcctaagta ctccctgttc 3660gagctggaaa
acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac
3720gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca
ctatgagaag 3780ctgaagggct cccccgagga taatgagcag aaacagctgt
ttgtggaaca gcacaagcac 3840tacctggacg agatcatcga gcagatcagc
gagttctcca agagagtgat cctggccgac 3900gctaatctgg acaaagtgct
gtccgcctac aacaagcacc gggataagcc catcagagag 3960caggccgaga
atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc
4020aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga
ggtgctggac 4080gccaccctga tccaccagag catcaccggc ctgtacgaga
cacggatcga cctgtctcag 4140ctgggaggcg acaaaaggcc ggcggccacg
aaaaaggccg gccaggcaaa aaagaaaaag 4200taa 42031974200DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
197atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc
cgacaagaag 60tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat
caccgacgag 120tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca
ccgaccggca cagcatcaag 180aagaacctga tcggagccct gctgttcgac
agcggcgaaa cagccgaggc cacccggctg 240aagagaaccg ccagaagaag
atacaccaga cggaagaacc ggatctgcta tctgcaagag 300atcttcagca
acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc
360ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa
catcgtggac 420gaggtggcct accacgagaa gtaccccacc atctaccacc
tgagaaagaa actggtggac 480agcaccgaca aggccgacct gcggctgatc
tatctggccc tggcccacat gatcaagttc 540cggggccact tcctgatcga
gggcgacctg aaccccgaca acagcgacgt ggacaagctg 600ttcatccagc
tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc
660ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg
gctggaaaat 720ctgatcgccc agctgcccgg cgagaagaag aatggcctgt
tcggaaacct gattgccctg 780agcctgggcc tgacccccaa cttcaagagc
aacttcgacc tggccgagga tgccaaactg 840cagctgagca aggacaccta
cgacgacgac ctggacaacc tgctggccca gatcggcgac 900cagtacgccg
acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac
960atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat
gatcaagaga 1020tacgacgagc accaccagga cctgaccctg ctgaaagctc
tcgtgcggca gcagctgcct 1080gagaagtaca aagagatttt cttcgaccag
agcaagaacg gctacgccgg ctacattgac 1140ggcggagcca gccaggaaga
gttctacaag ttcatcaagc ccatcctgga aaagatggac 1200ggcaccgagg
aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc
1260ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc
cattctgcgg 1320cggcaggaag atttttaccc attcctgaag gacaaccggg
aaaagatcga gaagatcctg 1380accttccgca tcccctacta cgtgggccct
ctggccaggg gaaacagcag attcgcctgg 1440atgaccagaa agagcgagga
aaccatcacc ccctggaact tcgaggaagt ggtggacaag 1500ggcgcttccg
cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac
1560gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta
taacgagctg 1620accaaagtga aatacgtgac cgagggaatg agaaagcccg
ccttcctgag cggcgagcag 1680aaaaaggcca tcgtggacct gctgttcaag
accaaccgga aagtgaccgt gaagcagctg 1740aaagaggact acttcaagaa
aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa 1800gatcggttca
acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag
1860gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct
gaccctgaca 1920ctgtttgagg acagagagat gatcgaggaa cggctgaaaa
cctatgccca cctgttcgac 1980gacaaagtga tgaagcagct gaagcggcgg
agatacaccg gctggggcag gctgagccgg 2040aagctgatca acggcatccg
ggacaagcag tccggcaaga caatcctgga tttcctgaag 2100tccgacggct
tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt
2160aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca
cgagcacatt 2220gccaatctgg ccggcagccc cgccattaag aagggcatcc
tgcagacagt gaaggtggtg 2280gacgagctcg tgaaagtgat gggccggcac
aagcccgaga acatcgtgat cgaaatggcc 2340agagagaacc agaccaccca
gaagggacag aagaacagcc gcgagagaat gaagcggatc 2400gaagagggca
tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc
2460cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga
tatgtacgtg 2520gaccaggaac tggacatcaa ccggctgtcc gactacgatg
tggaccatat cgtgcctcag 2580agctttctga aggacgactc catcgacaac
aaggtgctga ccagaagcga caagaaccgg 2640ggcaagagcg acaacgtgcc
ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg 2700cagctgctga
acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag
2760agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct
ggtggaaacc 2820cggcagatca caaagcacgt ggcacagatc ctggactccc
ggatgaacac taagtacgac 2880gagaatgaca agctgatccg ggaagtgaaa
gtgatcaccc tgaagtccaa gctggtgtcc 2940gatttccgga aggatttcca
gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc 3000cacgccgcct
acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg
3060gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat
gatcgccaag 3120agcgagcagg aaatcggcaa ggctaccgcc aagtacttct
tctacagcaa catcatgaac 3180tttttcaaga ccgagattac cctggccaac
ggcgagatcc ggaagcggcc tctgatcgag 3240acaaacggcg aaaccgggga
gatcgtgtgg gataagggcc gggattttgc caccgtgcgg 3300aaagtgctga
gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc
3360ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc
cagaaagaag 3420gactgggacc ctaagaagta cggcggcttc gacagcccca
ccgtggccta ttctgtgctg 3480gtggtggcca aagtggaaaa gggcaagtcc
aagaaactga agagtgtgaa agagctgctg 3540gggatcacca tcatggaaag
aagcagcttc gagaagaatc ccatcgactt tctggaagcc 3600aagggctaca
aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc
3660gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca
gaagggaaac 3720gaactggccc tgccctccaa atatgtgaac ttcctgtacc
tggccagcca ctatgagaag 3780ctgaagggct cccccgagga taatgagcag
aaacagctgt ttgtggaaca gcacaagcac 3840tacctggacg agatcatcga
gcagatcagc gagttctcca agagagtgat cctggccgac 3900gctaatctgg
acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag
3960caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc
tgccgccttc 4020aagtactttg acaccaccat cgaccggaag aggtacacca
gcaccaaaga ggtgctggac 4080gccaccctga tccaccagag catcaccggc
ctgtacgaga cacggatcga cctgtctcag 4140ctgggaggcg acaaaaggcc
ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag 420019823DNAHomo
sapiens 198gacatcgatg tcctccccat tgg 2319923DNAHomo sapiens
199gagtccgagc agaagaagaa ggg 2320023DNAHomo sapiens 200gcgccaccgg
ttgatgtgat ggg 2320123DNAHomo sapiens 201ggggcacaga tgagaaactc agg
2320223DNAHomo sapiens 202gtacaaacgg cagaagctgg agg 2320323DNAHomo
sapiens 203ggcagaagct ggaggaggaa ggg 2320423DNAHomo sapiens
204ggagcccttc ttcttctgct cgg 2320523DNAHomo sapiens 205gggcaaccac
aaacccacga ggg 2320623DNAHomo sapiens 206gctcccatca catcaaccgg tgg
2320723DNAHomo sapiens 207gtggcgcatt gccacgaagc agg 2320823DNAHomo
sapiens 208ggcagagtgc tgcttgctgc tgg 2320923DNAHomo sapiens
209gcccctgcgt gggcccaagc tgg 2321023DNAHomo sapiens 210gagtggccag
agtccagctt ggg 2321123DNAHomo sapiens 211ggcctcccca aagcctggcc agg
2321223DNAHomo sapiens 212ggggccgaga ttgggtgttc agg 2321323DNAHomo
sapiens 213gtggcgagag gggccgagat tgg 2321423DNAHomo sapiens
214gagtgccgcc gaggcggggc ggg 2321523DNAHomo sapiens 215ggagtgccgc
cgaggcgggg cgg 2321623DNAHomo sapiens 216ggagaggagt gccgccgagg cgg
2321720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 217ccatcccctt ctgtgaatgt 2021820DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
218ggagattgga gacacggaga 2021920DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 219aagcaccgac tcggtgccac
2022020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 220tcacctccaa tgactagggg 2022122DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
221caagttgata acggactagc ct 2222223DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
222agtccgagca gaagaagaag ttt 2322325DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
223tttcaagttg ataacggact agcct 2522420DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
224aaacagcaga ttcgcctgga 2022520DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 225tcatccgctc gatgaagctc
2022620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 226tccaaaatca agtggggcga 2022720DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
227tgatgaccct tttggctccc 2022845DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 228gaggaattct ttttttgtty
gaatatgttg gaggtttttt ggaag 4522942DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
229gagaagctta aataaaaaac racaatactc aacccaacaa cc
4223017DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 230caggaaacag ctatgac 1723139DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
231gcctctagag gtacctgagg gcctatttcc catgattcc 39232133DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
primermodified_base(92)..(111)a, c, t, g, unknown or other
232acctctagaa aaaaagcacc gactcggtgc cactttttca agttgataac
ggactagcct 60tattttaact tgctatttct agctctaaaa cnnnnnnnnn nnnnnnnnnn
nggtgtttcg 120tcctttccac aag 133233133DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
primermodified_base(92)..(111)a, c, t, g, unknown or other
233acctctagaa aaaaagcacc gactcggtgc cactttttca agttgataac
ggactagcct 60tatattaact tgctatttct agctctaata cnnnnnnnnn nnnnnnnnnn
nggtgtttcg 120tcctttccac aag 133234153DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
primermodified_base(112)..(131)a, c, t, g, unknown or other
234acctctagaa aaaaagcacc gactcggtgc cactttttca agttgataac
ggactagcct 60tattttaact tgctatgctg ttttgtttcc aaaacagcat agctctaaaa
cnnnnnnnnn 120nnnnnnnnnn nggtgtttcg tcctttccac aag
153235153DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primermodified_base(112)..(131)a, c, t, g, unknown or
other 235acctctagaa aaaaagcacc gactcggtgc cactttttca agttgataac
ggactagcct 60tatattaact tgctatgctg tattgtttcc aatacagcat agctctaata
cnnnnnnnnn 120nnnnnnnnnn nggtgtttcg tcctttccac aag 15323622DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
236aggccccagt ggctgctctn aa 2223722DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
237acatcaaccg gtggcgcatn at 2223822DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
238aaggtgtggt tccagaaccn ac 2223922DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
239ccatcacatc aaccggtggn ag 2224022DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
240aaacggcaga agctggaggn ta 2224122DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
241ggcagaagct ggaggaggan tt 2224222DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
242ggtgtggttc cagaaccggn tc 2224322DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
243aaccggagga caaagtacan tg 2224422DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
244ttccagaacc ggaggacaan ca 2224522DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
245gtgtggttcc agaaccggan ct 2224622DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
246tccagaaccg gaggacaaan cc 2224722DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
247cagaagctgg aggaggaagn cg 2224822DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
248catcaaccgg tggcgcattn ga 2224922DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
249gcagaagctg gaggaggaan gt 2225022DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
250cctccctccc tggcccaggn gc 2225122DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
251tcatctgtgc ccctccctcn aa 2225222DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
252gggaggacat cgatgtcacn at 2225322DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
253caaacggcag aagctggagn ac
2225422DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or
other 254gggtgggcaa ccacaaaccn ag 2225522DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
255ggtgggcaac cacaaacccn ta 2225622DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
256ggctcccatc acatcaaccn tt 2225722DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
257gaagggcctg agtccgagcn tc 2225822DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
258caaccggtgg cgcattgccn tg 2225922DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
259aggaggaagg gcctgagtcn ca 2226022DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
260agctggagga ggaagggccn ct 2226122DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
261gcattgccac gaagcaggcn cc 2226222DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
262attgccacga agcaggccan cg 2226322DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
263agaaccggag gacaaagtan ga 2226422DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
264tcaaccggtg gcgcattgcn gt 2226522DNAHomo
sapiensmodified_base(20)..(20)a, c, t, g, unknown or other
265gaagctggag gaggaagggn gc 22266123DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
266ccaatgggga ggacatcgat gtcacctcca atgactaggg tgggcaacca
caaacccacg 60agggcagagt gctgcttgct gctggccagg cccctgcgtg ggcccaagct
ggactctggc 120cac 123267121DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 267cgagcagaag
aagaagggct cccatcacat caaccggtgg cgcattgcca cgaagcaggc 60caatggggag
gacatcgatg tcacctccaa tgactagggt gggcaaccac aaacccacga 120g
121268128DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 268ggaggacaaa gtacaaacgg cagaagctgg
aggaggaagg gcctgagtcc gagcagaaga 60agaagggctc ccatcacatc aaccggtggc
gcattgccac gaagcaggcc aatggggagg 120acatcgat 128269130DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
269agaagctgga ggaggaaggg cctgagtccg agcagaagaa gaagggctcc
catcacatca 60accggtggcg cattgccacg aagcaggcca atggggagga catcgatgtc
acctccaatg 120actagggtgg 130270125DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 270cctcagtctt
cccatcaggc tctcagctca gcctgagtgt tgaggcccca gtggctgctc 60tgggggcctc
ctgagtttct catctgtgcc cctccctccc tggcccaggt gaaggtgtgg 120ttcca
125271129DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 271tcatctgtgc ccctccctcc ctggcccagg
tgaaggtgtg gttccagaac cggaggacaa 60agtacaaacg gcagaagctg gaggaggaag
ggcctgagtc cgagcagaag aagaagggct 120cccatcaca
129272129DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 272ctccaatgac tagggtgggc aaccacaaac
ccacgagggc agagtgctgc ttgctgctgg 60ccaggcccct gcgtgggccc aagctggact
ctggccactc cctggccagg ctttggggag 120gcctggagt
129273127DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 273ctgcttgctg ctggccaggc ccctgcgtgg
gcccaagctg gactctggcc actccctggc 60caggctttgg ggaggcctgg agtcatggcc
ccacagggct tgaagcccgg ggccgccatt 120gacagag 12727425DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 274gaaattaata cgactcacta taggg
25275126DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 275aaaaaagcac cgactcggtg ccactttttc
aagttgataa cggactagcc ttattttaac 60ttgctatttc tagctctaaa acaacgacga
gcgtgacacc accctatagt gagtcgtatt 120aatttc 126276126DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
276aaaaaagcac cgactcggtg ccactttttc aagttgataa cggactagcc
ttattttaac 60ttgctatttc tagctctaaa acgcaacaat taatagactg gacctatagt
gagtcgtatt 120aatttc 1262774677DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 277tctttcttgc
gctatgacac ttccagcaaa aggtagggcg ggctgcgaga cggcttcccg 60gcgctgcatg
caacaccgat gatgcttcga ccccccgaag ctccttcggg gctgcatggg
120cgctccgatg ccgctccagg gcgagcgctg tttaaatagc caggcccccg
attgcaaaga 180cattatagcg agctaccaaa gccatattca aacacctaga
tcactaccac ttctacacag 240gccactcgag cttgtgatcg cactccgcta
agggggcgcc tcttcctctt cgtttcagtc 300acaacccgca aacatgtacc
catacgatgt tccagattac gcttcgccga agaaaaagcg 360caaggtcgaa
gcgtccgaca agaagtacag catcggcctg gacatcggca ccaactctgt
420gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca
aggtgctggg 480caacaccgac cggcacagca tcaagaagaa cctgatcgga
gccctgctgt tcgacagcgg 540cgaaacagcc gaggccaccc ggctgaagag
aaccgccaga agaagataca ccagacggaa 600gaaccggatc tgctatctgc
aagagatctt cagcaacgag atggccaagg tggacgacag 660cttcttccac
agactggaag agtccttcct ggtggaagag gataagaagc acgagcggca
720ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc
ccaccatcta 780ccacctgaga aagaaactgg tggacagcac cgacaaggcc
gacctgcggc tgatctatct 840ggccctggcc cacatgatca agttccgggg
ccacttcctg atcgagggcg acctgaaccc 900cgacaacagc gacgtggaca
agctgttcat ccagctggtg cagacctaca accagctgtt 960cgaggaaaac
cccatcaacg ccagcggcgt ggacgccaag gccatcctgt ctgccagact
1020gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga
agaagaatgg 1080cctgttcggc aacctgattg ccctgagcct gggcctgacc
cccaacttca agagcaactt 1140cgacctggcc gaggatgcca aactgcagct
gagcaaggac acctacgacg acgacctgga 1200caacctgctg gcccagatcg
gcgaccagta cgccgacctg tttctggccg ccaagaacct 1260gtccgacgcc
atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc
1320cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga
ccctgctgaa 1380agctctcgtg cggcagcagc tgcctgagaa gtacaaagag
attttcttcg accagagcaa 1440gaacggctac gccggctaca ttgacggcgg
agccagccag gaagagttct acaagttcat 1500caagcccatc ctggaaaaga
tggacggcac cgaggaactg ctcgtgaagc tgaacagaga 1560ggacctgctg
cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct
1620gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc
tgaaggacaa 1680ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc
tactacgtgg gccctctggc 1740caggggaaac agcagattcg cctggatgac
cagaaagagc gaggaaacca tcaccccctg 1800gaacttcgag gaagtggtgg
acaagggcgc ttccgcccag agcttcatcg agcggatgac 1860caacttcgat
aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga
1920gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg
gaatgagaaa 1980gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg
gacctgctgt tcaagaccaa 2040ccggaaagtg accgtgaagc agctgaaaga
ggactacttc aagaaaatcg agtgcttcga 2100ctccgtggaa atctccggcg
tggaagatcg gttcaacgcc tccctgggca cataccacga 2160tctgctgaaa
attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct
2220ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg
aggaacggct 2280gaaaacctat gcccacctgt tcgacgacaa agtgatgaag
cagctgaagc ggcggagata 2340caccggctgg ggcaggctga gccggaagct
gatcaacggc atccgggaca agcagtccgg 2400caagacaatc ctggatttcc
tgaagtccga cggcttcgcc aacagaaact tcatgcagct 2460gatccacgac
gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca
2520gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca
ttaagaaggg 2580catcctgcag acagtgaagg tggtggacga gctcgtgaaa
gtgatgggcc ggcacaagcc 2640cgagaacatc gtgatcgaaa tggccagaga
gaaccagacc acccagaagg gacagaagaa 2700cagccgcgag agaatgaagc
ggatcgaaga gggcatcaaa gagctgggca gccagatcct 2760gaaagaacac
cccgtggaaa acacccagct gcagaacgag aagctgtacc tgtactacct
2820gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc
tgtccgacta 2880cgatgtggac catatcgtgc ctcagagctt tctgaaggac
gactccatcg acaacaaggt 2940gctgaccaga agcgacaaga accggggcaa
gagcgacaac gtgccctccg aagaggtcgt 3000gaagaagatg aagaactact
ggcggcagct gctgaacgcc aagctgatta cccagagaaa 3060gttcgacaat
ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt
3120catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac
agatcctgga 3180ctcccggatg aacactaagt acgacgagaa tgacaagctg
atccgggaag tgaaagtgat 3240caccctgaag tccaagctgg tgtccgattt
ccggaaggat ttccagtttt acaaagtgcg 3300cgagatcaac aactaccacc
acgcccacga cgcctacctg aacgccgtcg tgggaaccgc 3360cctgatcaaa
aagtacccta agctggaaag cgagttcgtg tacggcgact acaaggtgta
3420cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta
ccgccaagta 3480cttcttctac agcaacatca tgaacttttt caagaccgag
attaccctgg ccaacggcga 3540gatccggaag cggcctctga tcgagacaaa
cggcgaaacc ggggagatcg tgtgggataa 3600gggccgggat tttgccaccg
tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa 3660aaagaccgag
gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag
3720cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg
gcttcgacag 3780ccccaccgtg gcctattctg tgctggtggt ggccaaagtg
gaaaagggca agtccaagaa 3840actgaagagt gtgaaagagc tgctggggat
caccatcatg gaaagaagca gcttcgagaa 3900gaatcccatc gactttctgg
aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat 3960caagctgcct
aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc
4020tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg
tgaacttcct 4080gtacctggcc agccactatg agaagctgaa gggctccccc
gaggataatg agcagaaaca 4140gctgtttgtg gaacagcaca agcactacct
ggacgagatc atcgagcaga tcagcgagtt 4200ctccaagaga gtgatcctgg
ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa 4260gcaccgggat
aagcccatca gagagcaggc cgagaatatc atccacctgt ttaccctgac
4320caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc
ggaagaggta 4380caccagcacc aaagaggtgc tggacgccac cctgatccac
cagagcatca ccggcctgta 4440cgagacacgg atcgacctgt ctcagctggg
aggcgacagc cccaagaaga agagaaaggt 4500ggaggccagc taaggatccg
gcaagactgg ccccgcttgg caacgcaaca gtgagcccct 4560ccctagtgtg
tttggggatg tgactatgta ttcgtgtgtt ggccaacggg tcaacccgaa
4620cagattgata cccgccttgg catttcctgt cagaatgtaa cgtcagttga tggtact
46772783150DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 278tctttcttgc gctatgacac ttccagcaaa
aggtagggcg ggctgcgaga cggcttcccg 60gcgctgcatg caacaccgat gatgcttcga
ccccccgaag ctccttcggg gctgcatggg 120cgctccgatg ccgctccagg
gcgagcgctg tttaaatagc caggcccccg attgcaaaga 180cattatagcg
agctaccaaa gccatattca aacacctaga tcactaccac ttctacacag
240gccactcgag cttgtgatcg cactccgcta agggggcgcc tcttcctctt
cgtttcagtc 300acaacccgca aacatgccta agaagaagag gaaggttaac
acgattaaca tcgctaagaa 360cgacttctct gacatcgaac tggctgctat
cccgttcaac actctggctg accattacgg 420tgagcgttta gctcgcgaac
agttggccct tgagcatgag tcttacgaga tgggtgaagc 480acgcttccgc
aagatgtttg agcgtcaact taaagctggt gaggttgcgg ataacgctgc
540cgccaagcct ctcatcacta ccctactccc taagatgatt gcacgcatca
acgactggtt 600tgaggaagtg aaagctaagc gcggcaagcg cccgacagcc
ttccagttcc tgcaagaaat 660caagccggaa gccgtagcgt acatcaccat
taagaccact ctggcttgcc taaccagtgc 720tgacaataca accgttcagg
ctgtagcaag cgcaatcggt cgggccattg aggacgaggc 780tcgcttcggt
cgtatccgtg accttgaagc taagcacttc aagaaaaacg ttgaggaaca
840actcaacaag cgcgtagggc acgtctacaa gaaagcattt atgcaagttg
tcgaggctga 900catgctctct aagggtctac tcggtggcga ggcgtggtct
tcgtggcata aggaagactc 960tattcatgta ggagtacgct gcatcgagat
gctcattgag tcaaccggaa tggttagctt 1020acaccgccaa aatgctggcg
tagtaggtca agactctgag actatcgaac tcgcacctga 1080atacgctgag
gctatcgcaa cccgtgcagg tgcgctggct ggcatctctc cgatgttcca
1140accttgcgta gttcctccta agccgtggac tggcattact ggtggtggct
attgggctaa 1200cggtcgtcgt cctctggcgc tggtgcgtac tcacagtaag
aaagcactga tgcgctacga 1260agacgtttac atgcctgagg tgtacaaagc
gattaacatt gcgcaaaaca ccgcatggaa 1320aatcaacaag aaagtcctag
cggtcgccaa cgtaatcacc aagtggaagc attgtccggt 1380cgaggacatc
cctgcgattg agcgtgaaga actcccgatg aaaccggaag acatcgacat
1440gaatcctgag gctctcaccg cgtggaaacg tgctgccgct gctgtgtacc
gcaaggacaa 1500ggctcgcaag tctcgccgta tcagccttga gttcatgctt
gagcaagcca ataagtttgc 1560taaccataag gccatctggt tcccttacaa
catggactgg cgcggtcgtg tttacgctgt 1620gtcaatgttc aacccgcaag
gtaacgatat gaccaaagga ctgcttacgc tggcgaaagg 1680taaaccaatc
ggtaaggaag gttactactg gctgaaaatc cacggtgcaa actgtgcggg
1740tgtcgacaag gttccgttcc ctgagcgcat caagttcatt gaggaaaacc
acgagaacat 1800catggcttgc gctaagtctc cactggagaa cacttggtgg
gctgagcaag attctccgtt 1860ctgcttcctt gcgttctgct ttgagtacgc
tggggtacag caccacggcc tgagctataa 1920ctgctccctt ccgctggcgt
ttgacgggtc ttgctctggc atccagcact tctccgcgat 1980gctccgagat
gaggtaggtg gtcgcgcggt taacttgctt cctagtgaaa ccgttcagga
2040catctacggg attgttgcta agaaagtcaa cgagattcta caagcagacg
caatcaatgg 2100gaccgataac gaagtagtta ccgtgaccga tgagaacact
ggtgaaatct ctgagaaagt 2160caagctgggc actaaggcac tggctggtca
atggctggct tacggtgtta ctcgcagtgt 2220gactaagcgt tcagtcatga
cgctggctta cgggtccaaa gagttcggct tccgtcaaca 2280agtgctggaa
gataccattc agccagctat tgattccggc aagggtctga tgttcactca
2340gccgaatcag gctgctggat acatggctaa gctgatttgg gaatctgtga
gcgtgacggt 2400ggtagctgcg gttgaagcaa tgaactggct taagtctgct
gctaagctgc tggctgctga 2460ggtcaaagat aagaagactg gagagattct
tcgcaagcgt tgcgctgtgc attgggtaac 2520tcctgatggt ttccctgtgt
ggcaggaata caagaagcct attcagacgc gcttgaacct 2580gatgttcctc
ggtcagttcc gcttacagcc taccattaac accaacaaag atagcgagat
2640tgatgcacac aaacaggagt ctggtatcgc tcctaacttt gtacacagcc
aagacggtag 2700ccaccttcgt aagactgtag tgtgggcaca cgagaagtac
ggaatcgaat cttttgcact 2760gattcacgac tccttcggta cgattccggc
tgacgctgcg aacctgttca aagcagtgcg 2820cgaaactatg gttgacacat
atgagtcttg tgatgtactg gctgatttct acgaccagtt 2880cgctgaccag
ttgcacgagt ctcaattgga caaaatgcca gcacttccgg ctaaaggtaa
2940cttgaacctc cgtgacatct tagagtcgga cttcgcgttc gcgtaaggat
ccggcaagac 3000tggccccgct tggcaacgca acagtgagcc cctccctagt
gtgtttgggg atgtgactat 3060gtattcgtgt gttggccaac gggtcaaccc
gaacagattg atacccgcct tggcatttcc 3120tgtcagaatg taacgtcagt
tgatggtact 3150279125DNAArtificial SequenceDescription of
Artificial Sequence Synthetic
polynucleotidemodified_base(23)..(42)a, c, t, g, unknown or other
279gaaattaata cgactcacta tannnnnnnn nnnnnnnnnn nngttttaga
gctagaaata 60gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
gtcggtgctt 120ttttt 1252808452DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 280tgcggtattt
cacaccgcat caggtggcac ttttcgggga aatgtgcgcg gaacccctat 60ttgtttattt
ttctaaatac attcaaatat gtatccgctc atgagattat caaaaaggat
120cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
gtatatatga 180gtaaacttgg tctgacagtt accaatgctt aatcagtgag
gcacctatct cagcgatctg 240tctatttcgt tcatccatag ttgcctgact
ccccgtcgtg tagataacta cgatacggga 300gggcttacca tctggcccca
gtgctgcaat gataccgcga gacccacgct caccggctcc 360agatttatca
gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
420tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
gtagttcgcc 480agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
atcgtggtgt cacgctcgtc 540gtttggtatg gcttcattca gctccggttc
ccaacgatca aggcgagtta catgatcccc 600catgttgtgc aaaaaagcgg
ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 660ggccgcagtg
ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
720atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
gagaatagtg 780tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
gataataccg cgccacatag 840cagaacttta aaagtgctca tcattggaaa
acgttcttcg gggcgaaaac tctcaaggat 900cttaccgctg ttgagatcca
gttcgatgta acccactcgt gcacccaact gatcttcagc 960atcttttact
ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
1020aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
ttcaatatta 1080ttgaagcatt tatcagggtt attgtctcat gaccaaaatc
ccttaacgtg agttttcgtt 1140ccactgagcg tcagaccccg tagaaaagat
caaaggatct tcttgagatc ctttttttct 1200gcgcgtaatc tgctgcttgc
aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc 1260ggatcaagag
ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
1320aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
ctgtagcacc 1380gcctacatac ctcgctctgc taatcctgtt accagtggct
gttgccagtg gcgataagtc 1440gtgtcttacc gggttggact caagacgata
gttaccggat aaggcgcagc ggtcgggctg 1500aacggggggt tcgtgcacac
agcccagctt ggagcgaacg acctacaccg aactgagata 1560cctacagcgt
gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
1620tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
ggggaaacgc 1680ctggtatctt tatagtcctg tcgggtttcg ccacctctga
cttgagcgtc gatttttgtg 1740atgctcgtca ggggggcgga gcctatggaa
aaacgccagc aacgcggcct ttttacggtt 1800cctggccttt tgctggcctt
ttgctcacat gttctttcct gcgttatccc ctgattctgt 1860ggataaccgt
attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
1920gcgcagcgag tcagtgagcg aggaagcggt cgctgaggct tgacatgatt
ggtgcgtatg 1980tttgtatgaa gctacaggac tgatttggcg ggctatgagg
gcgggggaag ctctggaagg 2040gccgcgatgg ggcgcgcggc gtccagaagg
cgccatacgg cccgctggcg gcacccatcc 2100ggtataaaag cccgcgaccc
cgaacggtga cctccacttt cagcgacaaa cgagcactta 2160tacatacgcg
actattctgc cgctatacat aaccactcag ctagcttaag atcccatcaa
2220gcttgcatgc cgggcgcgcc agaaggagcg cagccaaacc aggatgatgt
ttgatggggt 2280atttgagcac ttgcaaccct
tatccggaag ccccctggcc cacaaaggct aggcgccaat 2340gcaagcagtt
cgcatgcagc ccctggagcg gtgccctcct gataaaccgg ccagggggcc
2400tatgttcttt acttttttac aagagaagtc actcaacatc ttaaaatggc
caggtgagtc 2460gacgagcaag cccggcggat caggcagcgt gcttgcagat
ttgacttgca acgcccgcat 2520tgtgtcgacg aaggcttttg gctcctctgt
cgctgtctca agcagcatct aaccctgcgt 2580cgccgtttcc atttgcagga
gattcgaggt accatgtacc catacgatgt tccagattac 2640gcttcgccga
agaaaaagcg caaggtcgaa gcgtccgaca agaagtacag catcggcctg
2700gacatcggca ccaactctgt gggctgggcc gtgatcaccg acgagtacaa
ggtgcccagc 2760aagaaattca aggtgctggg caacaccgac cggcacagca
tcaagaagaa cctgatcgga 2820gccctgctgt tcgacagcgg cgaaacagcc
gaggccaccc ggctgaagag aaccgccaga 2880agaagataca ccagacggaa
gaaccggatc tgctatctgc aagagatctt cagcaacgag 2940atggccaagg
tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag
3000gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt
ggcctaccac 3060gagaagtacc ccaccatcta ccacctgaga aagaaactgg
tggacagcac cgacaaggcc 3120gacctgcggc tgatctatct ggccctggcc
cacatgatca agttccgggg ccacttcctg 3180atcgagggcg acctgaaccc
cgacaacagc gacgtggaca agctgttcat ccagctggtg 3240cagacctaca
accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag
3300gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat
cgcccagctg 3360cccggcgaga agaagaatgg cctgttcggc aacctgattg
ccctgagcct gggcctgacc 3420cccaacttca agagcaactt cgacctggcc
gaggatgcca aactgcagct gagcaaggac 3480acctacgacg acgacctgga
caacctgctg gcccagatcg gcgaccagta cgccgacctg 3540tttctggccg
ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac
3600accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga
cgagcaccac 3660caggacctga ccctgctgaa agctctcgtg cggcagcagc
tgcctgagaa gtacaaagag 3720attttcttcg accagagcaa gaacggctac
gccggctaca ttgacggcgg agccagccag 3780gaagagttct acaagttcat
caagcccatc ctggaaaaga tggacggcac cgaggaactg 3840ctcgtgaagc
tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc
3900atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca
ggaagatttt 3960tacccattcc tgaaggacaa ccgggaaaag atcgagaaga
tcctgacctt ccgcatcccc 4020tactacgtgg gccctctggc caggggaaac
agcagattcg cctggatgac cagaaagagc 4080gaggaaacca tcaccccctg
gaacttcgag gaagtggtgg acaagggcgc ttccgcccag 4140agcttcatcg
agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc
4200aagcacagcc tgctgtacga gtacttcacc gtgtataacg agctgaccaa
agtgaaatac 4260gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg
agcagaaaaa ggccatcgtg 4320gacctgctgt tcaagaccaa ccggaaagtg
accgtgaagc agctgaaaga ggactacttc 4380aagaaaatcg agtgcttcga
ctccgtggaa atctccggcg tggaagatcg gttcaacgcc 4440tccctgggca
cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat
4500gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt
tgaggacaga 4560gagatgatcg aggaacggct gaaaacctat gcccacctgt
tcgacgacaa agtgatgaag 4620cagctgaagc ggcggagata caccggctgg
ggcaggctga gccggaagct gatcaacggc 4680atccgggaca agcagtccgg
caagacaatc ctggatttcc tgaagtccga cggcttcgcc 4740aacagaaact
tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag
4800aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa
tctggccggc 4860agccccgcca ttaagaaggg catcctgcag acagtgaagg
tggtggacga gctcgtgaaa 4920gtgatgggcc ggcacaagcc cgagaacatc
gtgatcgaaa tggccagaga gaaccagacc 4980acccagaagg gacagaagaa
cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa 5040gagctgggca
gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag
5100aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca
ggaactggac 5160atcaaccggc tgtccgacta cgatgtggac catatcgtgc
ctcagagctt tctgaaggac 5220gactccatcg acaacaaggt gctgaccaga
agcgacaaga accggggcaa gagcgacaac 5280gtgccctccg aagaggtcgt
gaagaagatg aagaactact ggcggcagct gctgaacgcc 5340aagctgatta
cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc
5400gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca
gatcacaaag 5460cacgtggcac agatcctgga ctcccggatg aacactaagt
acgacgagaa tgacaagctg 5520atccgggaag tgaaagtgat caccctgaag
tccaagctgg tgtccgattt ccggaaggat 5580ttccagtttt acaaagtgcg
cgagatcaac aactaccacc acgcccacga cgcctacctg 5640aacgccgtcg
tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg
5700tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga
gcaggaaatc 5760ggcaaggcta ccgccaagta cttcttctac agcaacatca
tgaacttttt caagaccgag 5820attaccctgg ccaacggcga gatccggaag
cggcctctga tcgagacaaa cggcgaaacc 5880ggggagatcg tgtgggataa
gggccgggat tttgccaccg tgcggaaagt gctgagcatg 5940ccccaagtga
atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct
6000atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg
ggaccctaag 6060aagtacggcg gcttcgacag ccccaccgtg gcctattctg
tgctggtggt ggccaaagtg 6120gaaaagggca agtccaagaa actgaagagt
gtgaaagagc tgctggggat caccatcatg 6180gaaagaagca gcttcgagaa
gaatcccatc gactttctgg aagccaaggg ctacaaagaa 6240gtgaaaaagg
acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc
6300cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact
ggccctgccc 6360tccaaatatg tgaacttcct gtacctggcc agccactatg
agaagctgaa gggctccccc 6420gaggataatg agcagaaaca gctgtttgtg
gaacagcaca agcactacct ggacgagatc 6480atcgagcaga tcagcgagtt
ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa 6540gtgctgtccg
cctacaacaa gcaccgggat aagcccatca gagagcaggc cgagaatatc
6600atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta
ctttgacacc 6660accatcgacc ggaagaggta caccagcacc aaagaggtgc
tggacgccac cctgatccac 6720cagagcatca ccggcctgta cgagacacgg
atcgacctgt ctcagctggg aggcgacagc 6780cccaagaaga agagaaaggt
ggaggccagc taacatatga ttcgaatgtc tttcttgcgc 6840tatgacactt
ccagcaaaag gtagggcggg ctgcgagacg gcttcccggc gctgcatgca
6900acaccgatga tgcttcgacc ccccgaagct ccttcggggc tgcatgggcg
ctccgatgcc 6960gctccagggc gagcgctgtt taaatagcca ggcccccgat
tgcaaagaca ttatagcgag 7020ctaccaaagc catattcaaa cacctagatc
actaccactt ctacacaggc cactcgagct 7080tgtgatcgca ctccgctaag
ggggcgcctc ttcctcttcg tttcagtcac aacccgcaaa 7140catgacacaa
gaatccctgt tacttctcga ccgtattgat tcggatgatt cctacgcgag
7200cctgcggaac gaccaggaat tctgggaggt gagtcgacga gcaagcccgg
cggatcaggc 7260agcgtgcttg cagatttgac ttgcaacgcc cgcattgtgt
cgacgaaggc ttttggctcc 7320tctgtcgctg tctcaagcag catctaaccc
tgcgtcgccg tttccatttg cagccgctgg 7380cccgccgagc cctggaggag
ctcgggctgc cggtgccgcc ggtgctgcgg gtgcccggcg 7440agagcaccaa
ccccgtactg gtcggcgagc ccggcccggt gatcaagctg ttcggcgagc
7500actggtgcgg tccggagagc ctcgcgtcgg agtcggaggc gtacgcggtc
ctggcggacg 7560ccccggtgcc ggtgccccgc ctcctcggcc gcggcgagct
gcggcccggc accggagcct 7620ggccgtggcc ctacctggtg atgagccgga
tgaccggcac cacctggcgg tccgcgatgg 7680acggcacgac cgaccggaac
gcgctgctcg ccctggcccg cgaactcggc cgggtgctcg 7740gccggctgca
cagggtgccg ctgaccggga acaccgtgct caccccccat tccgaggtct
7800tcccggaact gctgcgggaa cgccgcgcgg cgaccgtcga ggaccaccgc
gggtggggct 7860acctctcgcc ccggctgctg gaccgcctgg aggactggct
gccggacgtg gacacgctgc 7920tggccggccg cgaaccccgg ttcgtccacg
gcgacctgca cgggaccaac atcttcgtgg 7980acctggccgc gaccgaggtc
accgggatcg tcgacttcac cgacgtctat gcgggagact 8040cccgctacag
cctggtgcaa ctgcatctca acgccttccg gggcgaccgc gagatcctgg
8100ccgcgctgct cgacggggcg cagtggaagc ggaccgagga cttcgcccgc
gaactgctcg 8160ccttcacctt cctgcacgac ttcgaggtgt tcgaggagac
cccgctggat ctctccggct 8220tcaccgatcc ggaggaactg gcgcagttcc
tctgggggcc gccggacacc gcccccggcg 8280cctgataagg atccggcaag
actggccccg cttggcaacg caacagtgag cccctcccta 8340gtgtgtttgg
ggatgtgact atgtattcgt gtgttggcca acgggtcaac ccgaacagat
8400tgatacccgc cttggcattt cctgtcagaa tgtaacgtca gttgatggta ct
8452281102DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 281gttttagagc tatgctgttt tgaatggtcc
caaaacggaa gggcctgagt ccgagcagaa 60gaagaagttt tagagctatg ctgttttgaa
tggtcccaaa ac 102282100DNAHomo sapiens 282cggaggacaa agtacaaacg
gcagaagctg gaggaggaag ggcctgagtc cgagcagaag 60aagaagggct cccatcacat
caaccggtgg cgcattgcca 10028350DNAHomo sapiens 283agctggagga
ggaagggcct gagtccgagc agaagaagaa gggctcccac 5028430RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 284gaguccgagc agaagaagaa guuuuagagc
3028549DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 285agctggagga ggaagggcct gagtccgagc
agaagagaag ggctcccat 4928653DNAHomo sapiens 286ctggaggagg
aagggcctga gtccgagcag aagaagaagg gctcccatca cat 5328752DNAHomo
sapiens 287ctggaggagg aagggcctga gtccgagcag aagagaaggg ctcccatcac
at 5228854DNAHomo sapiens 288ctggaggagg aagggcctga gtccgagcag
aagaaagaag ggctcccatc acat 5428950DNAHomo sapiens 289ctggaggagg
aagggcctga gtccgagcag aagaagggct cccatcacat 5029047DNAHomo sapiens
290ctggaggagg aagggcctga gcccgagcag aagggctccc atcacat
4729166DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotidemodified_base(1)..(20)a, c, t, g, unknown
or other 291nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc aaguuaaaau
aaggctagtc 60cguuuu 6629220RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 292gaguccgagc
agaagaagaa 2029320RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 293gacaucgaug uccuccccau
2029420RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 294gucaccucca augacuaggg
2029520RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 295auuggguguu cagggcagag
2029620RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 296guggcgagag gggccgagau
2029720RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 297ggggccgaga uuggguguuc
2029820RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 298gugccauuag cuaaaugcau
2029920RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 299guaccaccca caggugccag
2030020RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 300gaaagccucu gggccaggaa 2030148DNAHomo
sapiens 301ctggaggagg aagggcctga gtccgagcag aagaagaagg gctcccat
4830220RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 302gaguccgagc agaagaagau
2030320RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 303gaguccgagc agaagaagua
2030420RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 304gaguccgagc agaagaacaa
2030520RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 305gaguccgagc agaagaugaa
2030620RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 306gaguccgagc agaaguagaa
2030720RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 307gaguccgagc agaugaagaa
2030820RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 308gaguccgagc acaagaagaa
2030920RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 309gaguccgagg agaagaagaa
2031020RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 310gaguccgugc agaagaagaa
2031120RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 311gagucggagc agaagaagaa
2031220RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 312gagaccgagc agaagaagaa
2031324DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 313aatgacaagc ttgctagcgg tggg
2431439DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 314aaaacggaag ggcctgagtc cgagcagaag
aagaagttt 3931539DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 315aaacaggggc cgagattggg
tgttcagggc agaggtttt 3931638DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 316aaaacggaag
ggcctgagtc cgagcagaag aagaagtt 3831740DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 317aacggaggga ggggcacaga tgagaaactc agggttttag
4031838DNAHomo sapiens 318agcccttctt cttctgctcg gactcaggcc cttcctcc
3831940DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 319cagggaggga ggggcacaga tgagaaactc
aggaggcccc 4032080DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 320ggcaatgcgc caccggttga
tgtgatggga gcccttctag gaggccccca gagcagccac 60tggggcctca acactcaggc
8032123DNAHomo sapiens 321gtcacctcca atgactaggg tgg
2332225DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotidemodified_base(6)..(25)a, c, t, g, unknown
or other 322caccgnnnnn nnnnnnnnnn nnnnn 2532325DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotidemodified_base(5)..(24)a, c, t, g, unknown or other
323aaacnnnnnn nnnnnnnnnn nnnnc 2532433DNAHomo sapiens 324catcgatgtc
ctccccattg gcctgcttcg tgg 3332533DNAHomo sapiens 325ttcgtggcaa
tgcgccaccg gttgatgtga tgg 3332633DNAHomo sapiens 326tcgtggcaat
gcgccaccgg ttgatgtgat ggg 3332733DNAHomo sapiens 327tccagcttct
gccgtttgta ctttgtcctc cgg 3332833DNAHomo sapiens 328ggagggaggg
gcacagatga gaaactcagg agg 3332933DNAHomo sapiens 329aggggccgag
attgggtgtt cagggcagag agg 3333054DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 330aacaccgggt
cttcgagaag acctgtttta gagctagaaa tagcaagtta aaat
5433154DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 331caaaacgggt cttcgagaag acgttttaga
gctatgctgt tttgaatggt ccca 5433233DNAMus musculus 332caagcactga
gtgccattag ctaaatgcat agg 3333333DNAMus musculus 333aatgcatagg
gtaccaccca caggtgccag ggg 3333433DNAMus musculus 334acacacatgg
gaaagcctct gggccaggaa agg 3333537DNAHomo sapiens 335ggaggaggta
gtatacagaa acacagagaa gtagaat 3733637DNAHomo sapiens 336agaatgtaga
ggagtcacag aaactcagca ctagaaa 3733798DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 337ggacgaaaca ccggaaccat tcaaaacagc atagcaagtt
aaaataaggc tagtccgtta 60tcaacttgaa aaagtggcac cgagtcggtg cttttttt
98338186DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 338ggacgaaaca ccggtagtat taagtattgt
tttatggctg ataaatttct ttgaatttct 60ccttgattat ttgttataaa agttataaaa
taatcttgtt ggaaccattc aaaacagcat 120agcaagttaa aataaggcta
gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 180tttttt
18633995DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 339gggttttaga gctatgctgt tttgaatggt
cccaaaacgg gtcttcgaga agacgtttta 60gagctatgct gttttgaatg gtcccaaaac
ttttt 9534036DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotidemodified_base(5)..(34)a, c, t, g,
unknown or other 340aaacnnnnnn nnnnnnnnnn nnnnnnnnnn nnnngt
3634136DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotidemodified_base(7)..(36)a, c, t, g, unknown
or other 341taaaacnnnn nnnnnnnnnn nnnnnnnnnn nnnnnn
3634284DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 342gtggaaagga cgaaacaccg ggtcttcgag
aagacctgtt ttagagctag aaatagcaag 60ttaaaataag gctagtccgt tttt
8434324DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotidemodified_base(6)..(24)a, c, t, g, unknown
or other 343caccgnnnnn nnnnnnnnnn nnnn 2434424DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotidemodified_base(5)..(23)a, c, t, g, unknown or
other
344aaacnnnnnn nnnnnnnnnn nnnc 2434588DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 345gttttagagc tatgctgttt tgaatggtcc caaaactgag
accaaaggtc tcgttttaga 60gctatgctgt tttgaatggt cccaaaac
8834635DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 346aaacggaagg gcctgagtcc gagcagaaga agaag
3534735DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 347aaaacttctt cttctgctcg gactcaggcc cttcc
3534846RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotidemodified_base(1)..(19)a, c, u, g, unknown
or other 348nnnnnnnnnn nnnnnnnnng uuauuguacu cucaagauuu auuuuu
4634991RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 349guuacuuaaa ucuugcagaa gcuacaaaga
uaaggcuuca ugccgaaauc aacacccugu 60cauuuuaugg caggguguuu ucguuauuua
a 9135070DNAHomo sapiens 350ttttctagtg ctgagtttct gtgactcctc
tacattctac ttctctgtgt ttctgtatac 60tacctcctcc 70351122DNAHomo
sapiens 351ggaggaaggg cctgagtccg agcagaagaa gaagggctcc catcacatca
accggtggcg 60cattgccacg aagcaggcca atggggagga catcgatgtc acctccaatg
actagggtgg 120gc 12235248RNAArtificial SequenceDescription of
Artificial Sequence Synthetic
oligonucleotidemodified_base(3)..(32)a, c, u, g, unknown or other
352acnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnguuuuaga gcuaugcu
4835367DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 353agcauagcaa guuaaaauaa ggctaguccg
uuaucaacuu gaaaaagugg caccgagucg 60gugcuuu 6735462RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(20)a, c, u, g, unknown or other
354nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc aaguuaaaau
aaggcuaguc 60cg 6235573DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 355tgaatggtcc
caaaacggaa gggcctgagt ccgagcagaa gaagaagttt tagagctatg 60ctgttttgaa
tgg 7335669DNAHomo sapiens 356ctggtcttcc acctctctgc cctgaacacc
caatctcggc ccctctcgcc accctcctgc 60atttctgtt 69357138DNAMus
musculus 357acccaagcac tgagtgccat tagctaaatg catagggtac cacccacagg
tgccaggggc 60ctttcccaaa gttcccagcc ccttctccaa cctttcctgg cccagaggct
ttcccatgtg 120tgtggctgga ccctttga 13835820DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
358gtgctttgca gaggcctacc 2035920DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 359cctggagcgc atgcagtagt
2036022DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 360accttctgtg tttccaccat tc 2236120DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
361ttggggagtg cacagacttc 2036220DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 362ggctccctgg gttcaaagta
2036321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 363agaggggtct ggatgtcgta a 2136430DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
364tagctctaaa acttcttctt ctgctcggac 3036530DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
365ctagccttat tttaacttgc tatgctgttt 3036699RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(20)a, c, u, g, unknown or other
366nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc aaguuaaaau
aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuu
9936712DNAHomo sapiens 367tagcgggtaa gc 1236812DNAHomo sapiens
368tcggtgacat gt 1236912DNAHomo sapiens 369actccccgta gg
1237012DNAHomo sapiens 370actgcgtgtt aa 1237112DNAHomo sapiens
371acgtcgcctg at 1237212DNAHomo sapiens 372taggtcgacc ag
1237312DNAHomo sapiens 373ggcgttaatg at 1237412DNAHomo sapiens
374tgtcgcatgt ta 1237512DNAHomo sapiens 375atggaaacgc at
1237612DNAHomo sapiens 376gccgaattcc tc 1237712DNAHomo sapiens
377gcatggtacg ga 1237812DNAHomo sapiens 378cggtactctt ac
1237912DNAHomo sapiens 379gcctgtgccg ta 1238012DNAHomo sapiens
380tacggtaagt cg 1238112DNAHomo sapiens 381cacgaaatta cc
1238212DNAHomo sapiens 382aaccaagata cg 1238312DNAHomo sapiens
383gagtcgatac gc 1238412DNAHomo sapiens 384gtctcacgat cg
1238512DNAHomo sapiens 385tcgtcgggtg ca 1238612DNAHomo sapiens
386actccgtagt ga 1238712DNAHomo sapiens 387caggacgtcc gt
1238812DNAHomo sapiens 388tcgtatccct ac 1238912DNAHomo sapiens
389tttcaaggcc gg 1239012DNAHomo sapiens 390cgccggtgga at
1239112DNAHomo sapiens 391gaacccgtcc ta 1239212DNAHomo sapiens
392gattcatcag cg 1239312DNAHomo sapiens 393acaccggtct tc
1239412DNAHomo sapiens 394atcgtgccct aa 1239512DNAHomo sapiens
395gcgtcaatgt tc 1239612DNAHomo sapiens 396ctccgtatct cg
1239712DNAHomo sapiens 397ccgattcctt cg 1239812DNAHomo sapiens
398tgcgcctcca gt 1239912DNAHomo sapiens 399taacgtcgga gc
1240012DNAHomo sapiens 400aaggtcgccc at 1240112DNAHomo sapiens
401gtcggggact at 1240212DNAHomo sapiens 402ttcgagcgat tt
1240312DNAHomo sapiens 403tgagtcgtcg ag 1240412DNAHomo sapiens
404tttacgcaga gg 1240512DNAHomo sapiens 405aggaagtatc gc
1240612DNAHomo sapiens 406actcgatacc at 1240712DNAHomo sapiens
407cgctacatag ca 1240812DNAHomo sapiens 408ttcataaccg gc
1240912DNAHomo sapiens 409ccaaacggtt aa 1241012DNAHomo sapiens
410cgattccttc gt 1241112DNAHomo sapiens 411cgtcatgaat aa
1241212DNAHomo sapiens 412agtggcgatg ac 1241312DNAHomo sapiens
413cccctacggc ac 1241412DNAHomo sapiens 414gccaacccgc ac
1241512DNAHomo sapiens 415tgggacaccg gt 1241612DNAHomo sapiens
416ttgactgcgg cg 1241712DNAHomo sapiens 417actatgcgta gg
1241812DNAHomo sapiens 418tcacccaaag cg 1241912DNAHomo sapiens
419gcaggacgtc cg 1242012DNAHomo sapiens 420acaccgaaaa cg
1242112DNAHomo sapiens 421cggtgtattg ag 1242212DNAHomo sapiens
422cacgaggtat gc 1242312DNAHomo sapiens 423taaagcgacc cg
1242412DNAHomo sapiens 424cttagtcggc ca 1242512DNAHomo sapiens
425cgaaaacgtg gc 1242612DNAHomo sapiens 426cgtgccctga ac
1242712DNAHomo sapiens 427tttaccatcg aa 1242812DNAHomo sapiens
428cgtagccatg tt 1242912DNAHomo sapiens 429cccaaacggt ta
1243012DNAHomo sapiens 430gcgttatcag aa 1243112DNAHomo sapiens
431tcgatggtaa ac 1243212DNAHomo sapiens 432cgactttttg ca
1243312DNAHomo sapiens 433tcgacgactc ac 1243412DNAHomo sapiens
434acgcgtcaga ta 1243512DNAHomo sapiens 435cgtacggcac ag
1243612DNAHomo sapiens 436ctatgccgtg ca 1243712DNAHomo sapiens
437cgcgtcagat at 1243812DNAHomo sapiens 438aagatcggta gc
1243912DNAHomo sapiens 439cttcgcaagg ag 1244012DNAHomo sapiens
440gtcgtggact ac 1244112DNAHomo sapiens 441ggtcgtcatc aa
1244212DNAHomo sapiens 442gttaacagcg tg 1244312DNAHomo sapiens
443tagctaaccg tt 1244412DNAHomo sapiens 444agtaaaggcg ct
1244512DNAHomo sapiens 445ggtaatttcg tg 1244669RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 446gucaccucca augacuaggg guuuuagagc uagaaauagc
aaguuaaaau aaggcuaguc 60cguuuuuuu 6944769RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 447gacaucgaug uccuccccau guuuuagagc uagaaauagc
aaguuaaaau aaggcuaguc 60cguuuuuuu 6944869RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 448gaguccgagc agaagaagaa guuuuagagc uagaaauagc
aaguuaaaau aaggcuaguc 60cguuuuuuu 6944969RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 449ggggccgaga uuggguguuc guuuuagagc uagaaauagc
aaguuaaaau aaggcuaguc 60cguuuuuuu 6945069RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 450guggcgagag gggccgagau guuuuagagc uagaaauagc
aaguuaaaau aaggcuaguc 60cguuuuuuu 6945176RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 451gucaccucca augacuaggg guuuuagagc uagaaauagc
aaguuaaaau aaggcuaguc 60cguuaucauu uuuuuu 7645276RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 452gacaucgaug uccuccccau guuuuagagc uagaaauagc
aaguuaaaau aaggcuaguc 60cguuaucauu uuuuuu 7645376RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 453gaguccgagc agaagaagaa guuuuagagc uagaaauagc
aaguuaaaau aaggcuaguc 60cguuaucauu uuuuuu 7645476RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 454ggggccgaga uuggguguuc guuuuagagc uagaaauagc
aaguuaaaau aaggcuaguc 60cguuaucauu uuuuuu 7645576RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 455guggcgagag gggccgagau guuuuagagc uagaaauagc
aaguuaaaau aaggcuaguc 60cguuaucauu uuuuuu 7645688RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 456gucaccucca augacuaggg guuuuagagc uagaaauagc
aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu guuuuuuu
8845788RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 457gacaucgaug uccuccccau guuuuagagc
uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu guuuuuuu
8845888RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 458gaguccgagc agaagaagaa guuuuagagc
uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu guuuuuuu
8845988RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 459ggggccgaga uuggguguuc guuuuagagc
uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu guuuuuuu
8846088RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 460guggcgagag gggccgagau guuuuagagc
uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu guuuuuuu
88461103RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 461gucaccucca augacuaggg guuuuagagc
uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu
cggugcuuuu uuu 103462103RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 462gacaucgaug
uccuccccau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac
uugaaaaagu ggcaccgagu cggugcuuuu uuu 103463103RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 463gaguccgagc agaagaagaa guuuuagagc uagaaauagc
aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu
uuu 103464103RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 464ggggccgaga uuggguguuc
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu
ggcaccgagu cggugcuuuu uuu 103465103RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 465guggcgagag gggccgagau guuuuagagc uagaaauagc
aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu
uuu 103466120DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 466gtggaaagga cgaaacaccg
ggtcttcgag aagacctgtt ttagagctag aaatagcaag 60ttaaaataag gctagtccgt
tatcaacttg aaaaagtggc accgagtcgg tgcttttttt 12046740DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 467tcggtgcgct ggttgatttc ttcttgcgct tttttggctt
4046826RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 468gauuucuucu ugcgcuuuuu guuuua
2646926DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotidemodified_base(22)..(26)a, c, t, g, unknown
or other
469tgatttcttc ttgcgctttt tnnnnn 2647026DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotidemodified_base(21)..(21)a, c, t, g, unknown or other
470tgatttcttc ttgcgctttt ntggct 2647126DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotidemodified_base(2)..(2)a, c, t, g, unknown or other
471tnatttcttc ttgcgctttt ttggct 2647223DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 472gatttcttct tgcgcttttt tgg 2347334DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideCDS(1)..(33) 473tcc atc cgt aca acc cac aac cct gct
agt gag c 34Ser Ile Arg Thr Thr His Asn Pro Ala Ser Glu1 5
1047411PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 474Ser Ile Arg Thr Thr His Asn Pro Ala Ser Glu1 5
1047534DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideCDS(1)..(33) 475tcc atc gca aca acc cac
aac cct gct agt gag c 34Ser Ile Ala Thr Thr His Asn Pro Ala Ser
Glu1 5 1047611PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 476Ser Ile Ala Thr Thr His Asn Pro Ala
Ser Glu1 5 1047734DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotideCDS(1)..(33) 477tca atc cgt aca
acc cac aac cct gct agt gag c 34Ser Ile Arg Thr Thr His Asn Pro Ala
Ser Glu1 5 1047842DNAHomo sapiensCDS(1)..(36) 478caa ttg aat tta
aaa gaa acc gat acc gtt ttg gtt taagga 42Gln Leu Asn Leu Lys Glu
Thr Asp Thr Val Leu Val1 5 1047912PRTHomo sapiens 479Gln Leu Asn
Leu Lys Glu Thr Asp Thr Val Leu Val1 5 1048042DNAHomo
sapiensCDS(1)..(42) 480caa ttg aat tta aaa gaa acc gat acc gtt tac
gaa att gga 42Gln Leu Asn Leu Lys Glu Thr Asp Thr Val Tyr Glu Ile
Gly1 5 1048114PRTHomo sapiens 481Gln Leu Asn Leu Lys Glu Thr Asp
Thr Val Tyr Glu Ile Gly1 5 1048234DNAHomo sapiensCDS(2)..(34) 482t
cct aaa aaa ccg aac tcc gcg ctg cgt aaa gta 34 Pro Lys Lys Pro Asn
Ser Ala Leu Arg Lys Val 1 5 1048311PRTHomo sapiens 483Pro Lys Lys
Pro Asn Ser Ala Leu Arg Lys Val1 5 1048434DNAHomo
sapiensCDS(2)..(34) 484t cct aca aaa ccg aac tcc gcg ctg cgt aaa
gta 34 Pro Thr Lys Pro Asn Ser Ala Leu Arg Lys Val 1 5
1048511PRTHomo sapiens 485Pro Thr Lys Pro Asn Ser Ala Leu Arg Lys
Val1 5 1048633DNAHomo sapiens 486tgcgctggtt gatttcttct tgcgcttttt
tgg 3348733DNAHomo sapiens 487tacgctggtt gatttcttct tgcgcttttt ttg
3348827DNAHomo sapiens 488ggagggtttt atggggaaag gccattg
2748929DNAHomo sapiens 489gtaaaaaaga agactagaaa ttttgatac
2949046DNAHomo sapiens 490ggagggtttt atggggaaag gcaaagaaga
ctagaaattt tgatac 4649127DNAHomo sapiens 491aggtgaagca taatgtctca
aaaaata 2749229DNAHomo sapiens 492attttattaa tacaaatcag tgaaatcat
2949346DNAHomo sapiens 493aggtgaagca taatgtctca aaattaatac
aaatcagtga aatcat 4649436DNAHomo sapiensCDS(1)..(36) 494aat tta aaa
gaa acc gat acc gtt tac gaa att gga 36Asn Leu Lys Glu Thr Asp Thr
Val Tyr Glu Ile Gly1 5 1049512PRTHomo sapiens 495Asn Leu Lys Glu
Thr Asp Thr Val Tyr Glu Ile Gly1 5 1049636DNAHomo
sapiensCDS(1)..(30) 496aat tta aaa gaa acc gat acc gtt ttg gtt
taagga 36Asn Leu Lys Glu Thr Asp Thr Val Leu Val1 5 1049710PRTHomo
sapiens 497Asn Leu Lys Glu Thr Asp Thr Val Leu Val1 5
1049836DNAHomo sapiensCDS(1)..(36) 498tgg gat cca aaa aaa tat ggt
ggt ttt gat agt cca 36Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser
Pro1 5 1049912PRTHomo sapiens 499Trp Asp Pro Lys Lys Tyr Gly Gly
Phe Asp Ser Pro1 5 1050036DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotideCDS(1)..(36) 500tgg
gat cca aaa aaa tat tgt ggt ttt gat agt cca 36Trp Asp Pro Lys Lys
Tyr Cys Gly Phe Asp Ser Pro1 5 1050112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 501Trp
Asp Pro Lys Lys Tyr Cys Gly Phe Asp Ser Pro1 5 1050235DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 502aaactacttt acgcagcgcg gagttcggtt ttttg
355034104DNAHomo sapiensCDS(1)..(4104) 503atg gac aag aag tac agc
atc ggc ctg gac atc ggc acc aac tct gtg 48Met Asp Lys Lys Tyr Ser
Ile Gly Leu Asp Ile Gly Thr Asn Ser Val1 5 10 15ggc tgg gcc gtg atc
acc gac gag tac aag gtg ccc agc aag aaa ttc 96Gly Trp Ala Val Ile
Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30aag gtg ctg ggc
aac acc gac cgg cac agc atc aag aag aac ctg atc 144Lys Val Leu Gly
Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45gga gcc ctg
ctg ttc gac agc ggc gaa aca gcc gag gcc acc cgg ctg 192Gly Ala Leu
Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60aag aga
acc gcc aga aga aga tac acc aga cgg aag aac cgg atc tgc 240Lys Arg
Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys65 70 75
80tat ctg caa gag atc ttc agc aac gag atg gcc aag gtg gac gac agc
288Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95ttc ttc cac aga ctg gaa gag tcc ttc ctg gtg gaa gag gat aag
aag 336Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys
Lys 100 105 110cac gag cgg cac ccc atc ttc ggc aac atc gtg gac gag
gtg gcc tac 384His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu
Val Ala Tyr 115 120 125cac gag aag tac ccc acc atc tac cac ctg aga
aag aaa ctg gtg gac 432His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg
Lys Lys Leu Val Asp 130 135 140agc acc gac aag gcc gac ctg cgg ctg
atc tat ctg gcc ctg gcc cac 480Ser Thr Asp Lys Ala Asp Leu Arg Leu
Ile Tyr Leu Ala Leu Ala His145 150 155 160atg atc aag ttc cgg ggc
cac ttc ctg atc gag ggc gac ctg aac ccc 528Met Ile Lys Phe Arg Gly
His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175gac aac agc gac
gtg gac aag ctg ttc atc cag ctg gtg cag acc tac 576Asp Asn Ser Asp
Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190aac cag
ctg ttc gag gaa aac ccc atc aac gcc agc ggc gtg gac gcc 624Asn Gln
Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200
205aag gcc atc ctg tct gcc aga ctg agc aag agc aga cgg ctg gaa aat
672Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220ctg atc gcc cag ctg ccc ggc gag aag aag aat ggc ctg ttc
ggc aac 720Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe
Gly Asn225 230 235 240ctg att gcc ctg agc ctg ggc ctg acc ccc aac
ttc aag agc aac ttc 768Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn
Phe Lys Ser Asn Phe 245 250 255gac ctg gcc gag gat gcc aaa ctg cag
ctg agc aag gac acc tac gac 816Asp Leu Ala Glu Asp Ala Lys Leu Gln
Leu Ser Lys Asp Thr Tyr Asp 260 265 270gac gac ctg gac aac ctg ctg
gcc cag atc ggc gac cag tac gcc gac 864Asp Asp Leu Asp Asn Leu Leu
Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285ctg ttt ctg gcc gcc
aag aac ctg tcc gac gcc atc ctg ctg agc gac 912Leu Phe Leu Ala Ala
Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300atc ctg aga
gtg aac acc gag atc acc aag gcc ccc ctg agc gcc tct 960Ile Leu Arg
Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser305 310 315
320atg atc aag aga tac gac gag cac cac cag gac ctg acc ctg ctg aaa
1008Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335gct ctc gtg cgg cag cag ctg cct gag aag tac aaa gag att
ttc ttc 1056Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile
Phe Phe 340 345 350gac cag agc aag aac ggc tac gcc ggc tac att gac
ggc gga gcc agc 1104Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp
Gly Gly Ala Ser 355 360 365cag gaa gag ttc tac aag ttc atc aag ccc
atc ctg gaa aag atg gac 1152Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro
Ile Leu Glu Lys Met Asp 370 375 380ggc acc gag gaa ctg ctc gtg aag
ctg aac aga gag gac ctg ctg cgg 1200Gly Thr Glu Glu Leu Leu Val Lys
Leu Asn Arg Glu Asp Leu Leu Arg385 390 395 400aag cag cgg acc ttc
gac aac ggc agc atc ccc cac cag atc cac ctg 1248Lys Gln Arg Thr Phe
Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415gga gag ctg
cac gcc att ctg cgg cgg cag gaa gat ttt tac cca ttc 1296Gly Glu Leu
His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430ctg
aag gac aac cgg gaa aag atc gag aag atc ctg acc ttc cgc atc 1344Leu
Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440
445ccc tac tac gtg ggc cct ctg gcc agg gga aac agc aga ttc gcc tgg
1392Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460atg acc aga aag agc gag gaa acc atc acc ccc tgg aac ttc
gag gaa 1440Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe
Glu Glu465 470 475 480gtg gtg gac aag ggc gct tcc gcc cag agc ttc
atc gag cgg atg acc 1488Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe
Ile Glu Arg Met Thr 485 490 495aac ttc gat aag aac ctg ccc aac gag
aag gtg ctg ccc aag cac agc 1536Asn Phe Asp Lys Asn Leu Pro Asn Glu
Lys Val Leu Pro Lys His Ser 500 505 510ctg ctg tac gag tac ttc acc
gtg tat aac gag ctg acc aaa gtg aaa 1584Leu Leu Tyr Glu Tyr Phe Thr
Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525tac gtg acc gag gga
atg aga aag ccc gcc ttc ctg agc ggc gag cag 1632Tyr Val Thr Glu Gly
Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540aaa aag gcc
atc gtg gac ctg ctg ttc aag acc aac cgg aaa gtg acc 1680Lys Lys Ala
Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr545 550 555
560gtg aag cag ctg aaa gag gac tac ttc aag aaa atc gag tgc ttc gac
1728Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575tcc gtg gaa atc tcc ggc gtg gaa gat cgg ttc aac gcc tcc
ctg ggc 1776Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser
Leu Gly 580 585 590aca tac cac gat ctg ctg aaa att atc aag gac aag
gac ttc ctg gac 1824Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys
Asp Phe Leu Asp 595 600 605aat gag gaa aac gag gac att ctg gaa gat
atc gtg ctg acc ctg aca 1872Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp
Ile Val Leu Thr Leu Thr 610 615 620ctg ttt gag gac aga gag atg atc
gag gaa cgg ctg aaa acc tat gcc 1920Leu Phe Glu Asp Arg Glu Met Ile
Glu Glu Arg Leu Lys Thr Tyr Ala625 630 635 640cac ctg ttc gac gac
aaa gtg atg aag cag ctg aag cgg cgg aga tac 1968His Leu Phe Asp Asp
Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655acc ggc tgg
ggc agg ctg agc cgg aag ctg atc aac ggc atc cgg gac 2016Thr Gly Trp
Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670aag
cag tcc ggc aag aca atc ctg gat ttc ctg aag tcc gac ggc ttc 2064Lys
Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680
685gcc aac aga aac ttc atg cag ctg atc cac gac gac agc ctg acc ttt
2112Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700aaa gag gac atc cag aaa gcc cag gtg tcc ggc cag ggc gat
agc ctg 2160Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp
Ser Leu705 710 715 720cac gag cac att gcc aat ctg gcc ggc agc ccc
gcc att aag aag ggc 2208His Glu His Ile Ala Asn Leu Ala Gly Ser Pro
Ala Ile Lys Lys Gly 725 730 735atc ctg cag aca gtg aag gtg gtg gac
gag ctc gtg aaa gtg atg ggc 2256Ile Leu Gln Thr Val Lys Val Val Asp
Glu Leu Val Lys Val Met Gly 740 745 750cgg cac aag ccc gag aac atc
gtg atc gcc atg gcc aga gag aac cag 2304Arg His Lys Pro Glu Asn Ile
Val Ile Ala Met Ala Arg Glu Asn Gln 755 760 765acc acc cag aag gga
cag aag aac agc cgc gag aga atg aag cgg atc 2352Thr Thr Gln Lys Gly
Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780gaa gag ggc
atc aaa gag ctg ggc agc cag atc ctg aaa gaa cac ccc 2400Glu Glu Gly
Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro785 790 795
800gtg gaa aac acc cag ctg cag aac gag aag ctg tac ctg tac tac ctg
2448Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815cag aat ggg cgg gat atg tac gtg gac cag gaa ctg gac atc
aac cgg 2496Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile
Asn Arg 820 825 830ctg tcc gac tac gat gtg gac gcc atc gtg cct cag
agc ttt ctg aag 2544Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln
Ser Phe Leu Lys 835 840 845gac gac tcc atc gac gcc aag gtg ctg acc
aga agc gac aag gcc cgg 2592Asp Asp Ser Ile Asp Ala Lys Val Leu Thr
Arg Ser Asp Lys Ala Arg 850 855 860ggc aag agc gac aac gtg ccc tcc
gaa gag gtc gtg aag aag atg aag 2640Gly Lys Ser Asp Asn Val Pro Ser
Glu Glu Val Val Lys Lys Met Lys865 870 875 880aac tac tgg cgg cag
ctg ctg aac gcc aag ctg att acc cag aga aag 2688Asn Tyr Trp Arg Gln
Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895ttc gac aat
ctg acc aag gcc gag aga ggc ggc ctg agc gaa ctg gat 2736Phe Asp Asn
Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910aag
gcc ggc ttc atc aag aga cag ctg gtg gaa acc cgg cag atc aca 2784Lys
Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920
925aag cac gtg gca cag atc ctg gac tcc cgg atg aac act aag tac gac
2832Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940gag aat gac aag ctg atc cgg gaa gtg aaa gtg atc acc ctg
aag tcc 2880Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu
Lys Ser945 950 955 960aag ctg gtg tcc gat ttc cgg aag gat ttc cag
ttt tac aaa gtg cgc 2928Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln
Phe Tyr Lys Val Arg 965 970 975gag atc aac aac tac cac cac gcc cac
gcc gcc tac ctg aac gcc gtc 2976Glu Ile Asn Asn Tyr His His Ala His
Ala Ala Tyr Leu Asn Ala Val 980 985 990gtg gga acc gcc ctg atc aaa
aag tac cct aag ctg gaa agc gag ttc 3024Val Gly Thr Ala Leu Ile Lys
Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005gtg tac ggc gac
tac aag gtg tac gac gtg cgg aag atg atc gcc 3069Val Tyr Gly Asp Tyr
Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020aag agc gag
cag gaa atc ggc aag gct acc gcc aag tac ttc ttc 3114Lys Ser Glu Gln
Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035tac agc
aac atc atg aac ttt ttc aag acc gag att acc ctg gcc 3159Tyr Ser Asn
Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050aac
ggc gag atc cgg aag cgg cct ctg atc gag aca aac ggc gaa 3204Asn Gly
Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060
1065acc ggg gag atc gtg tgg gat aag ggc cgg gat ttt gcc acc gtg
3249Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val
1070
1075 1080cgg aaa gtg ctg agc atg ccc caa gtg aat atc gtg aaa aag
acc 3294Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr
1085 1090 1095gag gtg cag aca ggc ggc ttc agc aaa gag tct atc ctg
ccc aag 3339Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro
Lys 1100 1105 1110agg aac agc gat aag ctg atc gcc aga aag aag gac
tgg gac cct 3384Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp
Asp Pro 1115 1120 1125aag aag tac ggc ggc ttc gac agc ccc acc gtg
gcc tat tct gtg 3429Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala
Tyr Ser Val 1130 1135 1140ctg gtg gtg gcc aaa gtg gaa aag ggc aag
tcc aag aaa ctg aag 3474Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser
Lys Lys Leu Lys 1145 1150 1155agt gtg aaa gag ctg ctg ggg atc acc
atc atg gaa aga agc agc 3519Ser Val Lys Glu Leu Leu Gly Ile Thr Ile
Met Glu Arg Ser Ser 1160 1165 1170ttc gag aag aat ccc atc gac ttt
ctg gaa gcc aag ggc tac aaa 3564Phe Glu Lys Asn Pro Ile Asp Phe Leu
Glu Ala Lys Gly Tyr Lys 1175 1180 1185gaa gtg aaa aag gac ctg atc
atc aag ctg cct aag tac tcc ctg 3609Glu Val Lys Lys Asp Leu Ile Ile
Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200ttc gag ctg gaa aac ggc
cgg aag aga atg ctg gcc tct gcc ggc 3654Phe Glu Leu Glu Asn Gly Arg
Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215gaa ctg cag aag gga
aac gaa ctg gcc ctg ccc tcc aaa tat gtg 3699Glu Leu Gln Lys Gly Asn
Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230aac ttc ctg tac
ctg gcc agc cac tat gag aag ctg aag ggc tcc 3744Asn Phe Leu Tyr Leu
Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245ccc gag gat
aat gag cag aaa cag ctg ttt gtg gaa cag cac aag 3789Pro Glu Asp Asn
Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260cac tac
ctg gac gag atc atc gag cag atc agc gag ttc tcc aag 3834His Tyr Leu
Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275aga
gtg atc ctg gcc gac gct aat ctg gac aaa gtg ctg tcc gcc 3879Arg Val
Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285
1290tac aac aag cac cgg gat aag ccc atc aga gag cag gcc gag aat
3924Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn
1295 1300 1305atc atc cac ctg ttt acc ctg acc aat ctg gga gcc cct
gcc gcc 3969Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala
Ala 1310 1315 1320ttc aag tac ttt gac acc acc atc gac cgg aag agg
tac acc agc 4014Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr
Thr Ser 1325 1330 1335acc aaa gag gtg ctg gac gcc acc ctg atc cac
cag agc atc acc 4059Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln
Ser Ile Thr 1340 1345 1350ggc ctg tac gag aca cgg atc gac ctg tct
cag ctg gga ggc gac 4104Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln
Leu Gly Gly Asp 1355 1360 13655041368PRTHomo sapiens 504Met Asp Lys
Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val1 5 10 15Gly Trp
Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30Lys
Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40
45Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile
Cys65 70 75 80Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val
Asp Asp Ser 85 90 95Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu
Glu Asp Lys Lys 100 105 110His Glu Arg His Pro Ile Phe Gly Asn Ile
Val Asp Glu Val Ala Tyr 115 120 125His Glu Lys Tyr Pro Thr Ile Tyr
His Leu Arg Lys Lys Leu Val Asp 130 135 140Ser Thr Asp Lys Ala Asp
Leu Arg Leu Ile Tyr Leu Ala Leu Ala His145 150 155 160Met Ile Lys
Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175Asp
Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185
190Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu
Glu Asn 210 215 220Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly
Leu Phe Gly Asn225 230 235 240Leu Ile Ala Leu Ser Leu Gly Leu Thr
Pro Asn Phe Lys Ser Asn Phe 245 250 255Asp Leu Ala Glu Asp Ala Lys
Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270Asp Asp Leu Asp Asn
Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285Leu Phe Leu
Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300Ile
Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser305 310
315 320Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu
Lys 325 330 335Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu
Ile Phe Phe 340 345 350Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile
Asp Gly Gly Ala Ser 355 360 365Gln Glu Glu Phe Tyr Lys Phe Ile Lys
Pro Ile Leu Glu Lys Met Asp 370 375 380Gly Thr Glu Glu Leu Leu Val
Lys Leu Asn Arg Glu Asp Leu Leu Arg385 390 395 400Lys Gln Arg Thr
Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415Gly Glu
Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425
430Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe
Ala Trp 450 455 460Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp
Asn Phe Glu Glu465 470 475 480Val Val Asp Lys Gly Ala Ser Ala Gln
Ser Phe Ile Glu Arg Met Thr 485 490 495Asn Phe Asp Lys Asn Leu Pro
Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510Leu Leu Tyr Glu Tyr
Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525Tyr Val Thr
Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540Lys
Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr545 550
555 560Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe
Asp 565 570 575Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala
Ser Leu Gly 580 585 590Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp
Lys Asp Phe Leu Asp 595 600 605Asn Glu Glu Asn Glu Asp Ile Leu Glu
Asp Ile Val Leu Thr Leu Thr 610 615 620Leu Phe Glu Asp Arg Glu Met
Ile Glu Glu Arg Leu Lys Thr Tyr Ala625 630 635 640His Leu Phe Asp
Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655Thr Gly
Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665
670Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu
Thr Phe 690 695 700Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln
Gly Asp Ser Leu705 710 715 720His Glu His Ile Ala Asn Leu Ala Gly
Ser Pro Ala Ile Lys Lys Gly 725 730 735Ile Leu Gln Thr Val Lys Val
Val Asp Glu Leu Val Lys Val Met Gly 740 745 750Arg His Lys Pro Glu
Asn Ile Val Ile Ala Met Ala Arg Glu Asn Gln 755 760 765Thr Thr Gln
Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780Glu
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro785 790
795 800Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr
Leu 805 810 815Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp
Ile Asn Arg 820 825 830Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro
Gln Ser Phe Leu Lys 835 840 845Asp Asp Ser Ile Asp Ala Lys Val Leu
Thr Arg Ser Asp Lys Ala Arg 850 855 860Gly Lys Ser Asp Asn Val Pro
Ser Glu Glu Val Val Lys Lys Met Lys865 870 875 880Asn Tyr Trp Arg
Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895Phe Asp
Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905
910Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys
Tyr Asp 930 935 940Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile
Thr Leu Lys Ser945 950 955 960Lys Leu Val Ser Asp Phe Arg Lys Asp
Phe Gln Phe Tyr Lys Val Arg 965 970 975Glu Ile Asn Asn Tyr His His
Ala His Ala Ala Tyr Leu Asn Ala Val 980 985 990Val Gly Thr Ala Leu
Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005Val Tyr
Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015
1020Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe
1025 1030 1035Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr
Leu Ala 1040 1045 1050Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu
Thr Asn Gly Glu 1055 1060 1065Thr Gly Glu Ile Val Trp Asp Lys Gly
Arg Asp Phe Ala Thr Val 1070 1075 1080Arg Lys Val Leu Ser Met Pro
Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095Glu Val Gln Thr Gly
Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110Arg Asn Ser
Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125Lys
Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135
1140Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys
1145 1150 1155Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg
Ser Ser 1160 1165 1170Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala
Lys Gly Tyr Lys 1175 1180 1185Glu Val Lys Lys Asp Leu Ile Ile Lys
Leu Pro Lys Tyr Ser Leu 1190 1195 1200Phe Glu Leu Glu Asn Gly Arg
Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215Glu Leu Gln Lys Gly
Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230Asn Phe Leu
Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245Pro
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255
1260His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys
1265 1270 1275Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu
Ser Ala 1280 1285 1290Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu
Gln Ala Glu Asn 1295 1300 1305Ile Ile His Leu Phe Thr Leu Thr Asn
Leu Gly Ala Pro Ala Ala 1310 1315 1320Phe Lys Tyr Phe Asp Thr Thr
Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335Thr Lys Glu Val Leu
Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350Gly Leu Tyr
Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360
136550515DNAHomo sapiens 505cagaagaaga agggc 1550651DNAHomo sapiens
506ccaatgggga ggacatcgat gtcacctcca atgactaggg tggtgggcaa c
5150715DNAHomo sapiens 507ctctggccac tccct 1550852DNAHomo sapiens
508acatcgatgt cacctccaat gacaagcttg ctagcggtgg gcaaccacaa ac
525091733DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 509ccgtttaaac aattctgcag gaatctagtt
attaatagta atcaattacg gggtcattag 60ttcatagccc atatatggag ttccgcgtta
cataacttac ggtaaatggc ccgcctggct 120gaccgcccaa cgacccccgc
ccattgacgt caataatgac gtatgttccc atagtaacgc 180caatagggac
tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
240cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
gacggtaaat 300ggcccgcctg gcattatgcc cagtacatga ccttatggga
ctttcctact tggcagtaca 360tctacgtatt agtcatcgct attaccatgg
tcgaggtgag ccccacgttc tgcttcactc 420tccccatctc ccccccctcc
ccacccccaa ttttgtattt atttattttt taattatttt 480gtgcagcgat
gggggcgggg gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg
540aggggcgggg cggggcgagg cggagaggtg cggcggcagc caatcagagc
ggcgcgctcc 600gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc
ctataaaaag cgaagcgcgc 660ggcgggcgga agtcgctgcg cgctgccttc
gccccgtgcc ccgctccgcc gccgcctcgc 720gccgcccgcc ccggctctga
ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc 780cttctcctcc
gggctgtaat tagcgcttgg tttaatgacg gcttgtttct tttctgtggc
840tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg cggggggagc
ggctcggggg 900gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg
ctccgcgctg cccggcggct 960gtgagcgctg cgggcgcggc gcggggcttt
gtgcgctccg cagtgtgcgc gaggggagcg 1020cggccggggg cggtgccccg
cggtgcgggg ggggctgcga ggggaacaaa ggctgcgtgc 1080ggggtgtgtg
cgtggggggg tgagcagggg gtgtgggcgc gtcggtcggg ctgcaacccc
1140ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg
gggctccgta 1200cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg
gcggcaggtg ggggtgccgg 1260gcggggcggg gccgcctcgg gccggggagg
gctcggggga ggggcgcggc ggcccccgga 1320gcgccggcgg ctgtcgaggc
gcggcgagcc gcagccattg ccttttatgg taatcgtgcg 1380agagggcgca
gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc
1440gccgcacccc ctctagcggg cgcggggcga agcggtgcgg cgccggcagg
aaggaaatgg 1500gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct
tctccctctc cagcctcggg 1560gctgtccgcg gggggacggc tgccttcggg
ggggacgggg cagggcgggg ttcggcttct 1620ggcgtgtgac cggcggctct
agagcctctg ctaaccatgt tcatgccttc ttctttttcc 1680tacagctcct
gggcaacgtg ctggttattg tgctgtctca tcattttggc aaa
17335104269DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 510atggactata aggaccacga cggagactac
aaggatcatg atattgatta caaagacgat 60gacgataaga tggccccaaa gaagaagcgg
aaggtcggta tccacggagt cccagcagcc 120gacaagaagt acagcatcgg
cctggacatc ggcaccaact ctgtgggctg ggccgtgatc 180accgacgagt
acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac
240agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac
agccgaggcc 300acccggctga agagaaccgc cagaagaaga tacaccagac
ggaagaaccg gatctgctat 360ctgcaagaga tcttcagcaa cgagatggcc
aaggtggacg acagcttctt ccacagactg 420gaagagtcct tcctggtgga
agaggataag aagcacgagc ggcaccccat cttcggcaac 480atcgtggacg
aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa
540ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct
ggcccacatg 600atcaagttcc ggggccactt cctgatcgag ggcgacctga
accccgacaa cagcgacgtg 660gacaagctgt tcatccagct ggtgcagacc
tacaaccagc tgttcgagga aaaccccatc 720aacgccagcg gcgtggacgc
caaggccatc ctgtctgcca gactgagcaa gagcagacgg 780ctggaaaatc
tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg
840attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct
ggccgaggat 900gccaaactgc agctgagcaa ggacacctac gacgacgacc
tggacaacct gctggcccag 960atcggcgacc agtacgccga cctgtttctg
gccgccaaga acctgtccga cgccatcctg 1020ctgagcgaca tcctgagagt
gaacaccgag atcaccaagg cccccctgag cgcctctatg 1080atcaagagat
acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag
1140cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg
ctacgccggc 1200tacattgacg gcggagccag ccaggaagag ttctacaagt
tcatcaagcc catcctggaa 1260aagatggacg gcaccgagga actgctcgtg
aagctgaaca gagaggacct gctgcggaag 1320cagcggacct tcgacaacgg
cagcatcccc caccagatcc acctgggaga gctgcacgcc 1380attctgcggc
ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag
1440aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg
aaacagcaga
1500ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt
cgaggaagtg 1560gtggacaagg gcgcttccgc ccagagcttc atcgagcgga
tgaccaactt cgataagaac 1620ctgcccaacg agaaggtgct gcccaagcac
agcctgctgt acgagtactt caccgtgtat 1680aacgagctga ccaaagtgaa
atacgtgacc gagggaatga gaaagcccgc cttcctgagc 1740ggcgagcaga
aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg
1800aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt
ggaaatctcc 1860ggcgtggaag atcggttcaa cgcctccctg ggcacatacc
acgatctgct gaaaattatc 1920aaggacaagg acttcctgga caatgaggaa
aacgaggaca ttctggaaga tatcgtgctg 1980accctgacac tgtttgagga
cagagagatg atcgaggaac ggctgaaaac ctatgcccac 2040ctgttcgacg
acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg
2100ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac
aatcctggat 2160ttcctgaagt ccgacggctt cgccaacaga aacttcatgc
agctgatcca cgacgacagc 2220ctgaccttta aagaggacat ccagaaagcc
caggtgtccg gccagggcga tagcctgcac 2280gagcacattg ccaatctggc
cggcagcccc gccattaaga agggcatcct gcagacagtg 2340aaggtggtgg
acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc
2400gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg
cgagagaatg 2460aagcggatcg aagagggcat caaagagctg ggcagccaga
tcctgaaaga acaccccgtg 2520gaaaacaccc agctgcagaa cgagaagctg
tacctgtact acctgcagaa tgggcgggat 2580atgtacgtgg accaggaact
ggacatcaac cggctgtccg actacgatgt ggaccatatc 2640gtgcctcaga
gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac
2700aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa
gatgaagaac 2760tactggcggc agctgctgaa cgccaagctg attacccaga
gaaagttcga caatctgacc 2820aaggccgaga gaggcggcct gagcgaactg
gataaggccg gcttcatcaa gagacagctg 2880gtggaaaccc ggcagatcac
aaagcacgtg gcacagatcc tggactcccg gatgaacact 2940aagtacgacg
agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag
3000ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat
caacaactac 3060caccacgccc acgacgccta cctgaacgcc gtcgtgggaa
ccgccctgat caaaaagtac 3120cctaagctgg aaagcgagtt cgtgtacggc
gactacaagg tgtacgacgt gcggaagatg 3180atcgccaaga gcgagcagga
aatcggcaag gctaccgcca agtacttctt ctacagcaac 3240atcatgaact
ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct
3300ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg
ggattttgcc 3360accgtgcgga aagtgctgag catgccccaa gtgaatatcg
tgaaaaagac cgaggtgcag 3420acaggcggct tcagcaaaga gtctatcctg
cccaagagga acagcgataa gctgatcgcc 3480agaaagaagg actgggaccc
taagaagtac ggcggcttcg acagccccac cgtggcctat 3540tctgtgctgg
tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa
3600gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc
catcgacttt 3660ctggaagcca agggctacaa agaagtgaaa aaggacctga
tcatcaagct gcctaagtac 3720tccctgttcg agctggaaaa cggccggaag
agaatgctgg cctctgccgg cgaactgcag 3780aagggaaacg aactggccct
gccctccaaa tatgtgaact tcctgtacct ggccagccac 3840tatgagaagc
tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag
3900cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa
gagagtgatc 3960ctggccgacg ctaatctgga caaagtgctg tccgcctaca
acaagcaccg ggataagccc 4020atcagagagc aggccgagaa tatcatccac
ctgtttaccc tgaccaatct gggagcccct 4080gccgccttca agtactttga
caccaccatc gaccggaaga ggtacaccag caccaaagag 4140gtgctggacg
ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac
4200ctgtctcagc tgggaggcga caaaaggccg gcggccacga aaaaggccgg
ccaggcaaaa 4260aagaaaaag 4269511780DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
511ggaagcggag ccactaactt ctccctgttg aaacaagcag gggatgtcga
agagaatccc 60gggccagtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
ggtcgagctg 120gacggcgacg taaacggcca caagttcagc gtgtccggcg
agggcgaggg cgatgccacc 180tacggcaagc tgaccctgaa gttcatctgc
accaccggca agctgcccgt gccctggccc 240accctcgtga ccaccctgac
ctacggcgtg cagtgcttca gccgctaccc cgaccacatg 300aagcagcacg
acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
360ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
gggcgacacc 420ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
aggacggcaa catcctgggg 480cacaagctgg agtacaacta caacagccac
aacgtctata tcatggccga caagcagaag 540aacggcatca aggtgaactt
caagatccgc cacaacatcg aggacggcag cgtgcagctc 600gccgaccact
accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
660cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
cgatcacatg 720gtcctgctgg agttcgtgac cgccgccggg atcactctcg
gcatggacga gctgtacaag 780512597DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 512cgataatcaa
cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt 60tgctcctttt
acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
120ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
tttatgagga 180gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
gtgtttgctg acgcaacccc 240cactggttgg ggcattgcca ccacctgtca
gctcctttcc gggactttcg ctttccccct 300ccctattgcc acggcggaac
tcatcgccgc ctgccttgcc cgctgctgga caggggctcg 360gctgttgggc
actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
420gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg
tcccttcggc 480cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
gctctgcggc ctcttccgcg 540tcttcgcctt cgccctcaga cgagtcggat
ctccctttgg gccgcctccc cgcatcg 597513210DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
513cgacctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
cccgtgcctt 60ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
gaaattgcat 120cgcattgtct gagtaggtgt cattctattc tggggggtgg
ggtggggcag gacagcaagg 180gggaggattg ggaagacaat ggcaggcatg
210514906DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotidemodified_base(109)..(109)a, c, t, g,
unknown or othermodified_base(135)..(135)a, c, t, g, unknown or
other 514ataacttcgt ataatgtatg ctatacgaag ttattcgcga tgaataaatg
aaagcttgca 60gatctgcgac tctagaggat ctgcgactct agaggatcat aatcagccnt
accacatttt 120gtagaggttt tactngcttt aaaaaacctc ccacacctcc
ccctgaacct gaaacataaa 180atgaatgcaa ttgttgttgt taacttgttt
attgcagctt ataatggtta caaataaagc 240aatagcatca caaatttcac
aaataaagca tttttttcac tgcattctag ttgtggtttg 300tccaaactca
tcaatgtatc ttatcatgtc tggatctgcg actctagagg atcataatca
360gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
ctccccctga 420acctgaaaca taaaatgaat gcaattgttg ttgttaactt
gtttattgca gcttataatg 480gttacaaata aagcaatagc atcacaaatt
tcacaaataa agcatttttt tcactgcatt 540ctagttgtgg tttgtccaaa
ctcatcaatg tatcttatca tgtctggatc tgcgactcta 600gaggatcata
atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
660acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta
acttgtttat 720tgcagcttat aatggttaca aataaagcaa tagcatcaca
aatttcacaa ataaagcatt 780tttttcactg cattctagtt gtggtttgtc
caaactcatc aatgtatctt atcatgtctg 840gatccccatc aagctgatcc
ggaaccctta atataacttc gtataatgta tgctatacga 900agttat
9065151079DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 515caggccctcc gagcgtggtg gagccgttct
gtgagacagc cgggtacgag tcgtgacgct 60ggaaggggca agcgggtggt gggcaggaat
gcggtccgcc ctgcagcaac cggaggggga 120gggagaaggg agcggaaaag
tctccaccgg acgcggccat ggctcggggg ggggggggca 180gcggaggagc
gcttccggcc gacgtctcgt cgctgattgg cttcttttcc tcccgccgtg
240tgtgaaaaca caaatggcgt gttttggttg gcgtaaggcg cctgtcagtt
aacggcagcc 300ggagtgcgca gccgccggca gcctcgctct gcccactggg
tggggcggga ggtaggtggg 360gtgaggcgag ctggacgtgc gggcgcggtc
ggcctctggc ggggcggggg aggggaggga 420gggtcagcga aagtagctcg
cgcgcgagcg gccgcccacc ctccccttcc tctgggggag 480tcgttttacc
cgccgccggc cgggcctcgt cgtctgattg gctctcgggg cccagaaaac
540tggcccttgc cattggctcg tgttcgtgca agttgagtcc atccgccggc
cagcgggggc 600ggcgaggagg cgctcccagg ttccggccct cccctcggcc
ccgcgccgca gagtctggcc 660gcgcgcccct gcgcaacgtg gcaggaagcg
cgcgctgggg gcggggacgg gcagtagggc 720tgagcggctg cggggcgggt
gcaagcacgt ttccgacttg agttgcctca agaggggcgt 780gctgagccag
acctccatcg cgcactccgg ggagtggagg gaaggagcga gggctcagtt
840gggctgtttt ggaggcagga agcacttgct ctcccaaagt cgctctgagt
tgttatcagt 900aagggagctg cagtggagta ggcggggaga aggccgcacc
cttctccgga ggggggaggg 960gagtgttgca atacctttct gggagttctc
tgctgcctcc tggcttctga ggaccgccct 1020gggcctggga gaatcccttc
cccctcttcc ctcgtgatct gcaactccag tctttctag 10795164336DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
516agatgggcgg gagtcttctg ggcaggctta aaggctaacc tggtgtgtgg
gcgttgtcct 60gcaggggaat tgaacaggtg taaaattgga gggacaagac ttcccacaga
ttttcggttt 120tgtcgggaag ttttttaata ggggcaaata aggaaaatgg
gaggataggt agtcatctgg 180ggttttatgc agcaaaacta caggttatta
ttgcttgtga tccgcctcgg agtattttcc 240atcgaggtag attaaagaca
tgctcacccg agttttatac tctcctgctt gagatcctta 300ctacagtatg
aaattacagt gtcgcgagtt agactatgta agcagaattt taatcatttt
360taaagagccc agtacttcat atccatttct cccgctcctt ctgcagcctt
atcaaaaggt 420attttagaac actcatttta gccccatttt catttattat
actggcttat ccaaccccta 480gacagagcat tggcattttc cctttcctga
tcttagaagt ctgatgactc atgaaaccag 540acagattagt tacatacacc
acaaatcgag gctgtagctg gggcctcaac actgcagttc 600ttttataact
ccttagtaca ctttttgttg atcctttgcc ttgatcctta attttcagtg
660tctatcacct ctcccgtcag gtggtgttcc acatttgggc ctattctcag
tccagggagt 720tttacaacaa tagatgtatt gagaatccaa cctaaagctt
aactttccac tcccatgaat 780gcctctctcc tttttctcca tttataaact
gagctattaa ccattaatgg tttccaggtg 840gatgtctcct cccccaatat
tacctgatgt atcttacata ttgccaggct gatattttaa 900gacattaaaa
ggtatatttc attattgagc cacatggtat tgattactgc ttactaaaat
960tttgtcattg tacacatctg taaaaggtgg ttccttttgg aatgcaaagt
tcaggtgttt 1020gttgtctttc ctgacctaag gtcttgtgag cttgtatttt
ttctatttaa gcagtgcttt 1080ctcttggact ggcttgactc atggcattct
acacgttatt gctggtctaa atgtgatttt 1140gccaagcttc ttcaggacct
ataattttgc ttgacttgta gccaaacaca agtaaaatga 1200ttaagcaaca
aatgtatttg tgaagcttgg tttttaggtt gttgtgttgt gtgtgcttgt
1260gctctataat aatactatcc aggggctgga gaggtggctc ggagttcaag
agcacagact 1320gctcttccag aagtcctgag ttcaattccc agcaaccaca
tggtggctca caaccatctg 1380taatgggatc tgatgccctc ttctggtgtg
tctgaagacc acaagtgtat tcacattaaa 1440taaataaatc ctccttcttc
ttcttttttt tttttttaaa gagaatactg tctccagtag 1500aatttactga
agtaatgaaa tactttgtgt ttgttccaat atggtagcca ataatcaaat
1560tactctttaa gcactggaaa tgttaccaag gaactaattt ttatttgaag
tgtaactgtg 1620gacagaggag ccataactgc agacttgtgg gatacagaag
accaatgcag actttaatgt 1680cttttctctt acactaagca ataaagaaat
aaaaattgaa cttctagtat cctatttgtt 1740taaactgcta gctttactta
acttttgtgc ttcatctata caaagctgaa agctaagtct 1800gcagccatta
ctaaacatga aagcaagtaa tgataatttt ggatttcaaa aatgtagggc
1860cagagtttag ccagccagtg gtggtgcttg cctttatgcc tttaatccca
gcactctgga 1920ggcagagaca ggcagatctc tgagtttgag cccagcctgg
tctacacatc aagttctatc 1980taggatagcc aggaatacac acagaaaccc
tgttggggag gggggctctg agatttcata 2040aaattataat tgaagcattc
cctaatgagc cactatggat gtggctaaat ccgtctacct 2100ttctgatgag
atttgggtat tattttttct gtctctgctg ttggttgggt cttttgacac
2160tgtgggcttt ctttaaagcc tccttcctgc catgtggtct cttgtttgct
actaacttcc 2220catggcttaa atggcatggc tttttgcctt ctaagggcag
ctgctgagat ttgcagcctg 2280atttccaggg tggggttggg aaatctttca
aacactaaaa ttgtccttta attttttttt 2340taaaaaatgg gttatataat
aaacctcata aaatagttat gaggagtgag gtggactaat 2400attaaatgag
tccctcccct ataaaagagc tattaaggct ttttgtctta tacttaactt
2460tttttttaaa tgtggtatct ttagaaccaa gggtcttaga gttttagtat
acagaaactg 2520ttgcatcgct taatcagatt ttctagtttc aaatccagag
aatccaaatt cttcacagcc 2580aaagtcaaat taagaatttc tgacttttaa
tgttaatttg cttactgtga atataaaaat 2640gatagctttt cctgaggcag
ggtctcacta tgtatctctg cctgatctgc aacaagatat 2700gtagactaaa
gttctgcctg cttttgtctc ctgaatacta aggttaaaat gtagtaatac
2760ttttggaact tgcaggtcag attcttttat aggggacaca ctaagggagc
ttgggtgata 2820gttggtaaat gtgtttaagt gatgaaaact tgaattatta
tcaccgcaac ctacttttta 2880aaaaaaaaag ccaggcctgt tagagcatgc
ttaagggatc cctaggactt gctgagcaca 2940caagagtagt tacttggcag
gctcctggtg agagcatatt tcaaaaaaca aggcagacaa 3000ccaagaaact
acagttaagg ttacctgtct ttaaaccatc tgcatataca cagggatatt
3060aaaatattcc aaataatatt tcattcaagt tttcccccat caaattggga
catggatttc 3120tccggtgaat aggcagagtt ggaaactaaa caaatgttgg
ttttgtgatt tgtgaaattg 3180ttttcaagtg atagttaaag cccatgagat
acagaacaaa gctgctattt cgaggtctct 3240tggtttatac tcagaagcac
ttctttgggt ttccctgcac tatcctgatc atgtgctagg 3300cctaccttag
gctgattgtt gttcaaataa acttaagttt cctgtcaggt gatgtcatat
3360gatttcatat atcaaggcaa aacatgttat atatgttaaa catttgtact
taatgtgaaa 3420gttaggtctt tgtgggtttg atttttaatt ttcaaaacct
gagctaaata agtcattttt 3480acatgtctta catttggtgg aattgtataa
ttgtggtttg caggcaagac tctctgacct 3540agtaacccta cctatagagc
actttgctgg gtcacaagtc taggagtcaa gcatttcacc 3600ttgaagttga
gacgttttgt tagtgtatac tagtttatat gttggaggac atgtttatcc
3660agaagatatt caggactatt tttgactggg ctaaggaatt gattctgatt
agcactgtta 3720gtgagcattg agtggccttt aggcttgaat tggagtcact
tgtatatctc aaataatgct 3780ggcctttttt aaaagccctt gttctttatc
accctgtttt ctacataatt tttgttcaaa 3840gaaatacttg tttggatctc
cttttgacaa caatagcatg ttttcaagcc atattttttt 3900tccttttttt
tttttttttt ggtttttcga gacagggttt ctctgtatag ccctggctgt
3960cctggaactc actttgtaga ccaggctggc ctcgaactca gaaatccgcc
tgcctctgcc 4020tcctgagtgc cgggattaaa ggcgtgcacc accacgcctg
gctaagttgg atattttgtt 4080atataactat aaccaatact aactccactg
ggtggatttt taattcagtc agtagtctta 4140agtggtcttt attggccctt
cattaaaatc tactgttcac tctaacagag gctgttggta 4200ctagtggcac
ttaagcaact tcctacggat atactagcag attaagggtc agggatagaa
4260actagtctag cgttttgtat acctaccagc tttatactac cttgttctga
tagaaatatt 4320tcaggacatc tagctt 43365171846DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
517aattctaccg ggtaggggag gcgcttttcc caaggcagtc tggagcatgc
gctttagcag 60ccccgctggg cacttggcgc tacacaagtg gcctctggcc tcgcacacat
tccacatcca 120ccggtaggcg ccaaccggct ccgttctttg gtggcccctt
cgcgccacct tctactcctc 180ccctagtcag gaagttcccc cccgccccgc
agctcgcgtc gtgcaggacg tgacaaatgg 240aagtagcacg tctcactagt
ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg 300taggcctttg
gggcagcggc caatagcagc tttgctcctt cgctttctgg gctcagaggc
360tgggaagggg tgggtccggg ggcgggctca ggggcgggct caggggcggg
gcgggcgccc 420gaaggtcctc cggaggcccg gcattctgca cgcttcaaaa
gcgcacgtct gccgcgctgt 480tctcctcttc ctcatctccg ggcctttcga
cctgcaatcg ccgctagcga agttcctatt 540ctctagaaag tataggaact
tcgccaccat gggatcggcc attgaacaag atggattgca 600cgcaggttct
ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
660aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
cggttctttt 720tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
gacgaggcag cgcggctatc 780gtggctggcc acgacgggcg ttccttgcgc
agctgtgctc gacgttgtca ctgaagcggg 840aagggactgg ctgctattgg
gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc 900tcctgccgag
aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
960ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
gtactcggat 1020ggaagccggt cttgtcgatc aggatgatct ggacgaagag
catcaggggc tcgcgccagc 1080cgaactgttc gccaggctca aggcgcgcat
gcccgacggc gatgatctcg tcgtgaccca 1140tggcgatgcc tgcttgccga
atatcatggt ggaaaatggc cgcttttctg gattcatcga 1200ctgtggccgg
ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
1260tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
gtatcgccgc 1320tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
gagttcttct gaggggatcc 1380gctgtaagtc tgcagaaatt gatgatctat
taaacaataa agatgtccac taaaatggaa 1440gtttttcctg tcatactttg
ttaagaaggg tgagaacaga gtacctacat tttgaatgga 1500aggattggag
ctacgggggt gggggtgggg tgggattaga taaatgcctg ctctttactg
1560aaggctcttt actattgctt tatgataatg tttcatagtt ggatatcata
atttaaacaa 1620gcaaaaccaa attaagggcc agctcattcc tcccactcat
gatctataga tctatagatc 1680tctcgtggga tcattgtttt tctcttgatt
cccactttgt ggttctaagt actgtggttt 1740ccaaatgtgt cagtttcata
gcctgaagaa cgagatcagc agcctctgtt ccacatacac 1800ttcattctca
gtattgtttt gccaagttct aattccatca gaaagc 18465181519DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
518taccgggtag gggaggcgct tttcccaagg cagtctggag catgcgcttt
agcagccccg 60ctgggcactt ggcgctacac aagtggcctc tggcctcgca cacattccac
atccaccggt 120aggcgccaac cggctccgtt ctttggtggc cccttcgcgc
caccttctac tcctccccta 180gtcaggaagt tcccccccgc cccgcagctc
gcgtcgtgca ggacgtgaca aatggaagta 240gcacgtctca ctagtctcgt
gcagatggac agcaccgctg agcaatggaa gcgggtaggc 300ctttggggca
gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga
360aggggtgggt ccgggggcgg gctcaggggc gggctcaggg gcggggcggg
cgcccgaagg 420tcctccggag gcccggcatt ctgcacgctt caaaagcgca
cgtctgccgc gctgttctcc 480tcttcctcat ctccgggcct ttcgacctgc
aggtcctcgc catggatcct gatgatgttg 540ttgattcttc taaatctttt
gtgatggaaa acttttcttc gtaccacggg actaaacctg 600gttatgtaga
ttccattcaa aaaggtatac aaaagccaaa atctggtaca caaggaaatt
660atgacgatga ttggaaaggg ttttatagta ccgacaataa atacgacgct
gcgggatact 720ctgtagataa tgaaaacccg ctctctggaa aagctggagg
cgtggtcaaa gtgacgtatc 780caggactgac gaaggttctc gcactaaaag
tggataatgc cgaaactatt aagaaagagt 840taggtttaag tctcactgaa
ccgttgatgg agcaagtcgg aacggaagag tttatcaaaa 900ggttcggtga
tggtgcttcg cgtgtagtgc tcagccttcc cttcgctgag gggagttcta
960gcgttgaata tattaataac tgggaacagg cgaaagcgtt aagcgtagaa
cttgagatta 1020attttgaaac ccgtggaaaa cgtggccaag atgcgatgta
tgagtatatg gctcaagcct 1080gtgcaggaaa tcgtgtcagg cgatctcttt
gtgaaggaac cttacttctg tggtgtgaca 1140taattggaca aactacctac
agagatttaa agctctaagg taaatataaa atttttaagt 1200gtataatgtg
ttaaactact gattctaatt gtttgtgtat tttagattcc aacctatgga
1260actgatgaat gggagcagtg gtggaatgca gatcctagag ctcgctgatc
agcctcgact 1320gtgccttcta gttgccagcc atctgttgtt tgcccctccc
ccgtgccttc cttgaccctg 1380gaaggtgcca ctcccactgt cctttcctaa
taaaatgagg aaattgcatc gcattgtctg 1440agtaggtgtc attctattct
ggggggtggg gtggggcagg acagcaaggg ggaggattgg
1500gaagacaata gcaggcatg 151951923DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 519gagggcctat ttcccatgat
tcc 2352022DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 520cttgtggaaa ggacgaaaca cc
2252145DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotidemodified_base(4)..(23)a, c, t, g, unknown
or other 521aacnnnnnnn nnnnnnnnnn nnnggtgttt cgtcctttcc acaag
4552228DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 522cctgagtgtt gaggccccag tggctgct
2852337DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 523acgagggcag agtgctgctt gctgctggcc
aggcccc 3752468DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 524catcaggctc tcagctcagc
ctgagtgttg aggccctgct ggccaggccc ctgcgtgggc 60ccaagctg
6852527DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 525gagggcctat ttcccatgat tccttca
27526125DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotidemodified_base(84)..(103)a, c, t, g, unknown
or other 526aaaaaaagca ccgactcggt gccacttttt caagttgata acggactagc
cttattttaa 60cttgctattt ctagctctaa aacnnnnnnn nnnnnnnnnn nnnggtgttt
cgtcctttcc 120acaag 125527111DNAArtificial SequenceDescription of
Artificial Sequence Synthetic
polynucleotidemodified_base(9)..(28)a, c, t, g, unknown or other
527gaaacaccnn nnnnnnnnnn nnnnnnnngt tttagagcta gaaatagcaa
gttaaaataa 60ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
t 11152820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 528agctgtttta ctggtcggct
2052920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 529aatggataca cctggtcgaa
2053020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 530caatggatac acctggtcga 2053168DNAHomo
sapiens 531accatgtata ccacttgggc tttggcagta gctaactgca ctaaatataa
tataaggagg 60gttttatg 6853226RNAArtificial SequenceDescription of
Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(24)a, c, u, g, unknown or other
532nnnnnnnnnn nnnnnnnnnn nnnngg 2653326RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotidemodified_base(3)..(26)a, c, u, g, unknown or other
533ccnnnnnnnn nnnnnnnnnn nnnnnn 2653423DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(20)a, c, t or
gmodified_base(21)..(21)a, c, t, g, unknown or other 534nnnnnnnnnn
nnnnnnnnnn ngg 2353515DNAArtificial SequenceDescription of
Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(12)a, c, t or
gmodified_base(13)..(13)a, c, t, g, unknown or other 535nnnnnnnnnn
nnngg 1553623DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotidemodified_base(1)..(20)a, c, t or
gmodified_base(21)..(21)a, c, t, g, unknown or other 536nnnnnnnnnn
nnnnnnnnnn ngg 2353714DNAArtificial SequenceDescription of
Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(11)a, c, t or
gmodified_base(12)..(12)a, c, t, g, unknown or other 537nnnnnnnnnn
nngg 1453825DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotidemodified_base(1)..(20)a, c, t or
gmodified_base(21)..(21)a, c, t, g, unknown or
othermodified_base(24)..(24)a, c, t, g, unknown or other
538nnnnnnnnnn nnnnnnnnnn nggng 2553917DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(12)a, c, t or
gmodified_base(13)..(13)a, c, t, g, unknown or
othermodified_base(16)..(16)a, c, t, g, unknown or other
539nnnnnnnnnn nnnggng 1754025DNAArtificial SequenceDescription of
Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(20)a, c, t or
gmodified_base(21)..(21)a, c, t, g, unknown or
othermodified_base(24)..(24)a, c, t, g, unknown or other
540nnnnnnnnnn nnnnnnnnnn nggng 2554116DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(11)a, c, t or
gmodified_base(12)..(12)a, c, t, g, unknown or
othermodified_base(15)..(15)a, c, t, g, unknown or other
541nnnnnnnnnn nnggng 1654223DNAArtificial SequenceDescription of
Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(21)a, c, t, g, unknown or other
542nnnnnnnnnn nnnnnnnnnn ngg 2354313DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(11)a, c, t, g, unknown or other
543nnnnnnnnnn ngg 13
* * * * *