U.S. patent application number 15/953141 was filed with the patent office on 2018-08-23 for artificially-manipulated neovascularization regulatory system.
This patent application is currently assigned to TOOLGEN INCORPORATED. The applicant listed for this patent is SEOUL NATIONAL UNIVERSITY HOSPITAL, SEOUL NATIONAL UNIVERSITY R&DB FOUNDATION, TOOLGEN INCORPORATED. Invention is credited to Jeong Hun KIM, Seokjoong KIM, Sung Wook PARK, Dong Woo SONG.
Application Number | 20180237771 15/953141 |
Document ID | / |
Family ID | 61196886 |
Filed Date | 2018-08-23 |
United States Patent
Application |
20180237771 |
Kind Code |
A1 |
KIM; Jeong Hun ; et
al. |
August 23, 2018 |
Artificially-Manipulated Neovascularization Regulatory System
Abstract
The present invention relates to an artificially manipulated
neovascularization-associated factor for regulating
neovascularization and a use thereof. More particularly, the
present invention relates to a system for artificially regulating
neovascularization, which includes an artificially manipulated
neovascularization-associated factor for regulating
neovascularization and/or a composition for artificially
manipulating the neovascularization-associated factor. In a
specific aspect, a neovascularization regulatory system including a
neovascularization-associated factor such as artificially
manipulated VEGFA, HIF1A, ANGPT2, EPAS1, or ANGPTL4 and/or an
expression product thereof is provided.
Inventors: |
KIM; Jeong Hun; (Seoul,
KR) ; PARK; Sung Wook; (Seoul, KR) ; KIM;
Seokjoong; (Seoul, KR) ; SONG; Dong Woo;
(Seoul, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TOOLGEN INCORPORATED
SEOUL NATIONAL UNIVERSITY R&DB FOUNDATION
SEOUL NATIONAL UNIVERSITY HOSPITAL |
Seoul
Seoul
Seoul |
|
KR
KR
KR |
|
|
Assignee: |
TOOLGEN INCORPORATED
SEOUL NATIONAL UNIVERSITY R&DB FOUNDATION
SEOUL NATIONAL UNIVERSITY HOSPITAL
|
Family ID: |
61196886 |
Appl. No.: |
15/953141 |
Filed: |
April 13, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/KR2017/009078 |
Aug 21, 2017 |
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15953141 |
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62376998 |
Aug 19, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 9/00 20180101; Y02A
50/30 20180101; A61K 9/0048 20130101; A01K 2267/03 20130101; A61P
27/02 20180101; C12N 15/86 20130101; C07K 14/4702 20130101; A61K
38/465 20130101; A61P 43/00 20180101; A01K 2227/105 20130101; C12N
15/113 20130101; C12N 2310/20 20170501; A61K 48/00 20130101; C07K
14/515 20130101; C12N 9/22 20130101; C12N 15/1136 20130101; A61K
48/005 20130101; C07K 14/52 20130101; C12N 2750/14143 20130101;
C12N 15/11 20130101 |
International
Class: |
C12N 15/11 20060101
C12N015/11; C12N 9/22 20060101 C12N009/22; C12N 15/86 20060101
C12N015/86; A61K 38/46 20060101 A61K038/46; A61K 9/00 20060101
A61K009/00 |
Claims
1. A composition for gene manipulation, comprising: a guide nucleic
acid capable of targeting at least one of the target sequence
selected from SEQ ID NOs: 1 to 1522 in nucleic acid sequences of
one or more genes selected from the group consisting of a VEGFA
gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4
gene, or a nucleic acid sequence encoding the same; and an editor
protein or a nucleic acid sequence encoding the same.
2. The composition for gene manipulation of claim 1, wherein the
editor protein includes one or more proteins selected from the
group consisting of a Streptococcus pyogenes-derived Cas9 protein,
a Campylobacter jejuni-derived Cas9 protein, a Streptococcus
thermophilus-derived Cas9 protein, a Streptocuccus aureus-derived
Cas9 protein, a Neisseria meningitidis-derived Cas9 protein, and a
Cpf1 protein.
3. The composition for gene manipulation of claim 2, wherein the
editor protein is a Streptococcus pyogenes-derived Cas9 protein or
Campylobacter jejuni-derived Cas9 protein.
4. The composition for gene manipulation of claim 1, wherein the
gene manipulation includes one or more modifications of nucleic
acids which is at least one of a deletion or insertion of one or
more nucleotides, a substitution with one or more nucleotides
different from a wild-type gene, and an insertion of one or more
foreign nucleotide, in a proto-spacer-adjacent motif (PAM) sequence
in a nucleic acid sequence constituting the
neovascularization-associated factor or in a continuous 1 bp to 50
bp the base sequence region adjacent to the 5' end and/or 3' end
thereof, or a chemical modification of one or more nucleotides in a
nucleic acid sequence constituting the
neovascularization-associated factor.
5. The composition for gene manipulation of claim 4, wherein the
PAM sequence includes one or more of the following sequences
(described in the 5' to 3' direction): NGG (N is A, T, C or G);
NNNNRYAC (each N is independently A, T, C or G, R is A or G, and Y
is C or T); NNAGAAW (each N is independently A, T, C or G, and W is
A or T); NNNNGATT (each N is independently A, T, C or G); NNGRR(T)
(each N is independently A, T, C or G, R is A or G, and Y is C or
T); and TTN (N is A, T, C or G).
6. The composition for gene manipulation of claim 1, wherein the
composition for gene manipulation is formed in a viral vector
system.
7. The composition for gene manipulation of claim 6, wherein the
viral vector includes one or more selected from a retrovirus, a
lentivirus, an adenovirus, adeno-associated virus (AAV), vaccinia
virus, a poxvirus and a herpes simplex virus.
8. The composition for gene manipulation of claim 1, wherein the
composition is formed in a ribonucleoprotein which is a complex of
a guide nucleic acid and an editor protein, and wherein the guide
nucleic acid is guide RNA.
9. The composition for gene manipulation of claim 1, wherein the
composition is used for treating an angiovascular disorder.
10. The composition for treating of claim 9, wherein the
angiovascular disorder is ischemic retinopathy or retinopathy of
prematurity.
11. A guide nucleic acid, which is capable of targeting at least
one of group consisting of target sequences of SEQ ID NOs: 1 to
1522 for artificially manipulating one or more genes selected from
the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2
gene, an EPAS1 gene and an ANGPTL4 gene, wherein the target
sequences of SEQ ID NOs: 1 to 1522 are in the nucleic acid
sequences of the genes, and wherein the guide nucleic acid is
complexed with an editor protein for artificially manipulating the
genes.
12. The guide nucleic acid of claim 11, which includes one or more
guide nucleic acids selected from the group consisting of: guide
nucleic acids capable of targeting at least one of the target
sequence selected from SEQ ID NOs: 3, 4, 7, 9, 10 and 11 in the
nucleic acid sequence of the VEGFA gene; guide nucleic acids
capable of targeting at least one of the target sequence selected
from SEQ ID NOs: 14, 18, 19, 20, 26, 29 and 31 in the nucleic acid
sequence of the HIF1A gene; guide nucleic acids capable of
targeting at least one of the target sequence selected from SEQ ID
NOs: 33, 34, 37, 38, 39 and 43 in the nucleic acid sequence of the
ANGPT2 gene; guide nucleic acids capable of targeting at least one
of the target sequence selected from SEQ ID NOs: 47, 48, 49, 50,
53, 54 and 55 in the nucleic acid sequence of the EPAS1 gene; and
guide nucleic acids capable of targeting at least one of the target
sequence selected from SEQ ID NOs: 64, 66, 67, 73, 76 and 79 in the
nucleic acid sequence of the ANGPTL4 gene.
13. The guide nucleic acid of claim 11, wherein the guide nucleic
acid is a nucleotide of 18 to 23 bp.
14. A method for treating an angiovascular disorder, comprising:
introducing (administering) a composition to a subject, wherein the
composition comprising: a guide nucleic acid capable of targeting
at least one of the target sequence selected from SEQ ID NOs: 1 to
1522 in nucleic acid sequences of one or more genes selected from
the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2
gene, an EPAS1 gene and an ANGPTL4 gene, or a nucleic acid sequence
encoding the same; and an editor protein or a nucleic acid sequence
encoding the same.
15. The method of claim 14, wherein the angiovascular disorder is
ischemic retinopathy or retinopathy of prematurity.
16. The method of claim 14, wherein the editor protein includes one
or more proteins selected from the group consisting of a
Streptococcus pyogenes-derived Cas9 protein, a Campylobacter
jejuni-derived Cas9 protein, a Streptococcus thermophilus-derived
Cas9 protein, a Streptocuccus aureus-derived Cas9 protein, a
Neisseria meningitidis-derived Cas9 protein, and a Cpf1
protein.
17. The method of claim 14, wherein the introducing is conducted
through eyeball.
18. The method of claim 14, wherein the composition is formed in a
viral vector system.
19. The method of claim 18, wherein the viral vector includes one
or more selected from a retrovirus, a lentivirus, an adenovirus,
adeno-associated virus (AAV), vaccinia virus, a poxvirus and a
herpes simplex virus.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation application of
International Patent Application No. PCT/KR2017/009078, filed 21
Aug. 2017, which claims the priority and the benefit of U.S.
Provisional Patent Application No. 62/376,998, filed on Aug. 19,
2016, the disclosures of which are incorporated herein by reference
in their entirety.
FIELD
[0002] The present invention relates to an artificially manipulated
neovascularization-associated factor for regulating
neovascularization and a use thereof. More particularly, the
present invention relates to a system capable of artificially
regulating neovascularization, which includes an artificially
manipulated neovascularization-associated factor for regulating
neovascularization and/or a composition able to be used in
artificial manipulation of the neovascularization-associated
factor.
BACKGROUND
[0003] Excessive neovascularization found in many cases of severe
diseases occurs in diseases such as cancer, macular degeneration,
diabetic retinopathy, arthritis and psoriasis. In such a state, new
blood vessels are provided to tissue with a disease, resulting in
destroyed normal tissue, and in the case of cancer, new blood
vessels allow tumor cells to enter the circulation system and thus
settle in another organ (tumor metastasis).
[0004] Particularly, since cancer cells receive nutrients through
neovascularization and are metastasized to another organ,
neovascularization is essential for growth and metastasis of
cancer. It has been known that there is an actual close
relationship between the density of capillaries generated in cancer
tissue and probability of cancer metastasis in various types of
cancer. In addition, rheumatoid arthritis, which is the
representative disease among inflammatory diseases, is caused by an
autoimmune disorder, however, during the development of the
disease, chronic inflammation generated in the synovial cavity
between joints leads to neovascularization, resulting in destroyed
cartilage. Various ophthalmologic diseases leading to blindness in
several million people in the world every year are also caused by
neovascularization. As a representative example, diabetic blindness
is a diabetic complication, and refers to an invasion of
capillaries generated in the retina to the vitreous body through
neovascularization, ending up in blindness. Therefore,
neovascularization inhibitory substances may be usefully employed
as therapeutic agents and preventive agents for various diseases
such as cancer, rheumatoid arthritis and diabetic blindness, in
which continuous neovascularization occurs.
[0005] Meanwhile, conventionally, inhibition of signaling of
vascular endothelial growth factors (VEGFs) in order to inhibit
neovascularization had been actively studied. However, in the
conventional art, initially, neovascularization seemed to be
inhibited, and then there was a side effect in which cancer cells
became more aggressive because the pathway of an anticancer agent
to the cancer cells was also inhibited.
[0006] As such, while a variety of studies to treat diseases
induced by neovascularization are progressing, there is almost no
fundamental method for treating such a disease.
[0007] Particularly, there is no method for treating a severe
disease such as cancer or cancer metastasis caused by
neovascularization, blindness caused by retinal or corneal
degeneration, and therefore, there is an urgent demand for
developing such a fundamental method for treating a
neovascularization-associated disease.
SUMMARY
Technical Problem
[0008] To solve the above problems, the present invention relates
to an artificially manipulated neovascularization system, which has
an improved neovascularizing effect. More particularly, the present
invention relates to an artificially manipulated
neovascularization-associated factor and a neovascularization
system which has a function artificially modified by the
factor.
[0009] The present invention also relates to a
neovascularization-associated factor genetically manipulated or
modified for a specific purpose.
[0010] As an exemplary embodiment, the present invention is
directed to providing an artificially manipulated
neovascularization-regulating system.
[0011] As an exemplary embodiment, the present invention is
directed to providing an artificially manipulated
neovascularization-associated factor and an expression product
thereof.
[0012] As an exemplary embodiment, the present invention is
directed to providing a composition for manipulating a gene to
manipulate a neovascularization-associated factor and a method for
utilizing the same.
[0013] As an exemplary embodiment, the present invention is
directed to providing a method for regulating
neovascularization.
[0014] As an exemplary embodiment, the present invention is
directed to providing a pharmaceutical composition for treating a
neovascularization-associated disease and various uses thereof.
[0015] As an exemplary embodiment, the present invention is
directed to providing an artificially manipulated
neovascularization-associated factor, for example, VEGFA, HIF1A,
ANGPT2, EPAS1, ANGPTL4, etc., and/or expression products
thereof.
[0016] As an exemplary embodiment, the present invention is
directed to providing a composition for manipulating a gene to
enable artificial manipulation of a neovascularization-associated
factor, for example, VEGFA, HIF1A, ANGPT2, EPAS1, ANGPTL4, etc.
[0017] As an exemplary embodiment, the present invention is
directed to providing a therapeutic use of an artificially
manipulated neovascularization-associated factor, for example,
VEGFA, HIF1A, ANGPT2, EPAS1, ANGPTL4, etc., and/or a composition
for manipulating a gene to enable the artificial manipulation.
[0018] As an exemplary embodiment, the present invention is
directed to providing an additional use of an artificially
manipulated neovascularization-associated factor, for example,
VEGFA, HIF1A, ANGPT2, EPAS1, ANGPTL4, etc., and/or a composition
for manipulating a gene to enable the artificial manipulation.
Technical Solution
[0019] To solve these problems, the present invention relates to a
system for artificially regulating neovascularization, which
includes an artificially manipulated neovascularization-associated
factor for regulating neovascularization and/or a composition for
artificially manipulating the neovascularization-associated
factor.
[0020] The present invention provides an artificially manipulated
neovascularization-associated factor for a specific purpose.
[0021] The term "neovascularization-associated factor" encompasses
a variety of non-natural, artificially manipulated substances
capable of having a neovascularization regulating function, which
directly participate in or indirectly affect neovascularization.
The substances may be DNA, RNA, genes, peptides, polypeptides or
proteins. For example, the substances may be genetically
manipulated or modified genes or proteins expressed in an immune
cells. The neovascularization-associated factor may promote or
increase neovascularization, or conversely, suppress or inhibit
neovascularization.
[0022] In addition, it may induce, activate or inactivate a
neovascularization environment or a neovascularization-inhibiting
environment.
[0023] In an exemplary embodiment of the present invention, the
neovascularization-associated factor may be, for example, an
artificially manipulated VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1
gene or ANGPTL4 gene.
[0024] In an exemplary embodiment of the present invention, the
neovascularization-associated factor may include two or more
artificially manipulated genes. For example, two or more genes
selected from the group consisting of a VEGFA gene, an HIF1A gene,
an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene may be
artificially manipulated.
[0025] Therefore, in an exemplary embodiment of the present
invention, one or more artificially manipulated
neovascularization-associated factors selected from the group
consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1
gene and an ANGPTL4 gene, which have undergone modification in a
nucleic acid sequence, are provided.
[0026] The modification in a nucleic acid sequence may be
non-limitedly, artificially manipulated by a guide nucleic
acid-editor protein complex.
[0027] The term "guide nucleic acid-editor protein complex" refers
to a complex formed through the interaction between a guide nucleic
acid and an editor protein, and the nucleic acid-protein complex
includes the guide nucleic acid and the editor protein.
[0028] The guide nucleic acid-editor protein complex may serve to
modify a subject. The subject may be a target nucleic acid, a gene,
a chromosome or a protein.
[0029] For example, the gene may be a neovascularization-associated
factor, artificially manipulated by a guide nucleic acid-editor
protein complex, wherein the neovascularization-associated factor
artificially manipulated includes one or more modifications of
nucleic acids which is at least one of a deletion or insertion of
one or more nucleotides, a substitution with one or more
nucleotides different from a wild-type gene, and an insertion of
one or more foreign nucleotide, in a proto-spacer-adjacent motif
(PAM) sequence in a nucleic acid sequence constituting the
neovascularization-associated factor or in a continuous 1 bp to 50
bp the base sequence region adjacent to the 5' end and/or 3' end
thereof, or a chemical modification of one or more nucleotides in a
nucleic acid sequence constituting the
neovascularization-associated factor.
[0030] The modification of nucleic acids may occur in a promoter
region of the gene.
[0031] The modification of nucleic acids may occur in an exon
region of the gene. In one exemplary embodiment, 50% of the
modifications may occur in the upstream section of the coding
regions of the gene.
[0032] The modification of nucleic acids may occur in an intron
region of the gene.
[0033] The modification of nucleic acids may occur in an enhancer
region of the gene.
[0034] The PAM sequence may be, for example, one or more of the
following sequences (described in the 5' to 3' direction): [0035]
NGG (N is A, T, C or G); [0036] NNNNRYAC (each of N is
independently A, T, C or G, R is A or G, and Y is C or T); [0037]
NNAGAAW (each of N is independently A, T, C or G, and W is A or T);
[0038] NNNNGATT (each of N is independently A, T, C or G); [0039]
NNGRR(T) (each of N is independently A, T, C or G, R is A or G,);
and [0040] TTN (N is A, T, C or G).
[0041] The editor protein may be derived from Streptococcus
pyogenes, Streptococcus thermophilus, Streptococcus sp.,
Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces
pristinaespiralis, Streptomyces viridochromogenes,
Streptosporangium roseum, AlicyclobacHlus acidocaldarius, Bacillus
pseudomycoides, Bacillus selenitireducens, Exiguobacterium
sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius,
Microscilla marina, Burkholderiales bacterium, Polaromonas
naphthalenivorans, Polaromonas sp., Crocosphaera watsonii,
Cyanothece sp., Microcystis aeruginosa, Synechococcus sp.,
Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor
bescii, Candidatus Desulforudis, Clostridium botulinum, Clostridium
difficile, Finegoldia magna, Natranaerobius thermophilus,
Pelotomaculum thermopropionicum, Acidithiobacillus caldus,
Acidithiobacillus ferrooxidans, Allochromatium vinosum,
Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsonii,
Pseudoalteromonas haloplanktis, Ktedonobacter racemifer,
Methanohalobium evestigatum, Anabaena variabilis, Nodularia
spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis,
Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes,
Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, or
Acaryochloris marina.
[0042] In one exemplary embodiment, the editor protein may be one
or more selected from the group consisting of a Streptococcus
pyogenes-derived Cas9 protein, a Campylobacter jejuni-derived Cas9
protein, a Streptococcus thermophilus-derived Cas9 protein, a
Streptococcus aureus-derived Cas9 protein, a Neisseria
meningitidis-derived Cas9 protein, and a Cpf1 protein. As an
example, the editor protein may be a Streptococcus pyogenes-derived
Cas9 protein or a Campylobacter jejuni-derived Cas9 protein.
[0043] In addition, in another embodiment, the present invention
provides a guide nucleic acid, which is capable of forming a
complementary bond with respect to target sequences of SEQ ID NOs:
1 to 1522, for example, SEQ ID Nos: 1 to 79 in the nucleic acid
sequences of one or more genes selected from the group consisting
of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and
an ANGPTL4 gene, respectively.
[0044] The guide nucleic acid may form a complementary bond with a
part of nucleic acid sequences of one or more genes selected from
the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2
gene, an EPAS1 gene and an ANGPTL4 gene. It may create 0 to 5, 0 to
4, 0 to 3, or 0 to 2 mismatches. As an exemplary example, the guide
nucleic acid may be nucleotides forming a complementary bond with
one or more of the target sequences of SEQ ID NOs: 1 to 1522, for
example, SEQ ID NOs: 1 to 79, respectively.
[0045] For example, the present invention may provide one or more
guide nucleic acids selected from the group as described below:
[0046] a guide nucleic acid capable of forming a complementary bond
with respect to the target sequences of SEQ ID NOs: 3, 4, 7, 9, 10
and 11 in the nucleic acid sequence of the VEGFA gene,
respectively; [0047] a guide nucleic acid capable of forming a
complementary bond with respect to the target sequences of SEQ ID
NOs: 14, 18, 19, 20, 26, 29 and 31 in the nucleic acid sequence of
the HIF1A gene, respectively; [0048] a guide nucleic acid capable
of forming a complementary bond with respect to the target
sequences of SEQ ID NOs: 33, 34, 37, 38, 39 and 43 in the nucleic
acid sequence of the ANGPT2 gene, respectively; [0049] a guide
nucleic acid capable of forming a complementary bond with respect
to the target sequences of SEQ ID NOs: 47, 48, 49, 50, 53, 54 and
55 in the nucleic acid sequence of the EPAS1 gene, respectively;
and [0050] a guide nucleic acid capable of forming a complementary
bond with respect to the target sequences of SEQ ID NOs: 64, 66,
67, 73, 76 and 79 in the nucleic acid sequence of the ANGPTL4 gene,
respectively.
[0051] The guide nucleic acid may be non-limitedly 18 to 25 bp, 18
to 24 bp, 18 to 23 bp, 19 to 23 bp, or 20 to 23 bp nucleotides.
[0052] In addition, the present invention provides a composition
for gene manipulation, which may be employed in artificial
manipulation of a neovascularization-associated factor for a
specific purpose.
[0053] The composition for gene manipulation may include a guide
nucleic acid-editor protein complex or a nucleic acid sequence
encoding the same.
[0054] The composition for gene manipulation may include: [0055]
(a) a guide nucleic acid capable of forming a complementary bond
with respect to each of target sequences of SEQ ID NOs: 1 to 1522,
for example, SEQ ID NOs: 1 to 79 in the nucleic acid sequences of
one or more genes selected from the group consisting of a VEGFA
gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4
gene, respectively or a nucleic acid sequence encoding the guide
nucleic acid; [0056] (b) an editor protein including one or more
proteins selected from the group consisting of a Streptococcus
pyogenes-derived Cas9 protein, a Campylobacter jejuni-derived Cas9
protein, a Streptococcus thermophilus-derived Cas9 protein, a
Streptococcus aureus-derived Cas9 protein, a Neisseria
meningitidis-derived Cas9 protein, and a Cpf1 protein or a nucleic
acid sequence encoding the same.
[0057] In one exemplary embodiment, the guide nucleic acid may be a
nucleic acid sequence which forms a complementary bond with respect
to one or more of the target sequences of SEQ ID NOs: 3, 4, 7, 9,
10 and 11 (VEGFA), SEQ ID NOs: 14, 18, 19, 20, 26, 29 and 31
(HIF1A), SEQ ID NOs: 33, 34, 37, 38, 39 and 43 (ANGPT2), SEQ ID
NOs: 47, 48, 49, 50, 53, 54 and 55 (EPAS1), and SEQ ID NOs: 64, 66,
67, 73, 76 and 79 (ANGPTL4), respectively.
[0058] In one exemplary embodiment, the composition for gene
manipulation may be a viral vector system.
[0059] The viral vector may include one or more selected from the
group consisting of a retrovirus, a lentivirus, an adenovirus, an
adeno-associated virus (AAV), a vaccinia virus, a poxvirus and a
herpes simplex virus.
[0060] In an exemplary embodiment, the present invention provides a
method for artificially manipulating cells, which includes:
introducing [0061] (a) a guide nucleic acid which is capable of
forming a complementary bond with respect to the target sequences
of SEQ ID NOs: 1 to 1522, for example, SEQ ID NOs: 1 to 79 in the
nucleic acid sequences of one or more genes selected from the group
consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1
gene and an ANGPTL4 gene, respectively, or a nucleic acid sequence
encoding the same; and [0062] (b) an editor protein including one
or more proteins selected from the group consisting of a
Streptococcus pyogenes-derived Cas9 protein, a Campylobacter
jejuni-derived Cas9 protein, a Streptococcus thermophilus-derived
Cas9 protein, a Streptococcus aureus-derived Cas9 protein, a
Neisseria meningitidis-derived Cas9 protein, and a Cpf1 protein,
respectively, or a nucleic acid sequence encoding the same to
cells.
[0063] The guide nucleic acid and the editor protein may be present
in one or more vectors in the form of a nucleic acid sequence, or
may be present in a complex formed by coupling the guide nucleic
acid with the editor protein.
[0064] The introduction may be performed in vivo or ex vivo.
[0065] The introduction may be performed by one or more methods
selected from electroporation, liposomes, plasmids, viral vectors,
nanoparticles and a protein translocation domain (PTD) fusion
protein method.
[0066] The viral vector may be one or more selected from the group
consisting of a retrovirus, a lentivirus, an adenovirus, an
adeno-associated virus (AAV), a vaccinia virus, a poxvirus and a
herpes simplex virus.
[0067] In another exemplary embodiment, the present invention
provides a pharmaceutical composition for treating a
neovascularization-associated disease.
[0068] The pharmaceutical composition may include a composition for
gene manipulation which may be employed in artificial manipulation
of a neovascularization-associated factor.
[0069] The formulation of the composition for gene manipulation is
the same as described above.
[0070] In an exemplary embodiment, the present invention provides a
method for obtaining information about the sequences of target
sites that are artificially manipulated from a subject by
sequencing one or more genes selected from the group consisting of
a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an
ANGPTL4 gene.
[0071] In addition, the present invention provides a method for
constructing libraries using the information obtained thereby.
[0072] In an exemplary embodiment, the present invention provides a
kit for gene manipulation, which includes the following components:
[0073] (a) a guide nucleic acid capable of forming a complementary
bond with respect to the target sequences of SEQ ID NOs: 1 to 1522,
for example, SEQ ID NOs: 1 to 79 in the nucleic acid sequences of
one or more genes selected from the group consisting of a VEGFA
gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4
gene, respectively, or a nucleic acid sequence encoding the same;
and [0074] (b) an editor protein including one or more proteins
selected from the group consisting of a Streptococcus
pyogenes-derived Cas9 protein, a Campylobacter jejuni-derived Cas9
protein, a Streptococcus thermophilus-derived Cas9 protein, a
Streptococcus aureus-derived Cas9 protein, a Neisseria
meningitidis-derived Cas9 protein, and a Cpf1 protein,
respectively, or a nucleic acid sequence encoding the same.
[0075] The gene of interest may be artificially manipulated using
such a kit.
[0076] In one exemplary embodiment, the present invention may
provide a composition for treating a neovascularization-related
disorder, which includes: [0077] a guide nucleic acid capable of
forming a complementary bond with one or more target sequences in
the nucleic acid sequences of one or more genes selected from the
group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an
EPAS1 gene and an ANGPTL4 gene, respectively, or a nucleic acid
sequence encoding the same; and an editor protein or a nucleic acid
sequence encoding the same.
[0078] The target sequences may be one or more sequences of SEQ ID
NOs: 1 to 1522, for example, SEQ ID NOs: 1 to 79.
[0079] In one exemplary embodiment, a Campylobacter jejuni-derived
Cas9 protein may be employed as the editor protein.
[0080] In one exemplary embodiment, the neovascularization-related
disorder may be ischemic retinopathy or retinopathy of
prematurity.
[0081] In one exemplary embodiment, the present invention provides
all aspects of uses of an artificially manipulated
neovascularization-associated factor or a composition for gene
manipulation which is employed in artificial manipulation of the
neovascularization-associated factor for treating a disease in a
target.
[0082] Targets for treatment may be mammals including primates such
as humans, monkeys, etc., rodents such as mice, rats, etc., and the
like.
Advantageous Effects
[0083] An artificially manipulated neovascularization-associated
factor and a neovascularization system whose function is
artificially modified thereby can be effectively used to treat a
neovascularization-related disease, for example, a
neovascularization-related ocular disease. The efficiency of the
neovascularization system can be improved by modulation of a
variety of in vivo mechanisms in which various
neovascularization-associated factors are involved.
[0084] For example, one or more genes of a VEGFA gene, an HIF1A
gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene can be
utilized.
BRIEF DESCRIPTION OF DRAWINGS
[0085] FIGS. 1, 2, 3, 4, 5, 6, and 7 show the reducing effect on a
laser-induced choroidal neovascularization (CNV) area due to CjCas9
targeting Vegfa or Hif1a in mouse models with age-related macular
degeneration (AMD): FIG. 1 CjCas9 target sequences in Vegfa and
Hif1a/HIF1A genes (the PAM sequence and the target sequence of
sgRNA are marked with a dotted line and solid line, respectively),
FIG. 2 the all-in-one AAV vector encoding CjCas9 and an in vivo
test schedule, FIG. 3 graphs of indel frequencies at Rosa26, Vegfa,
and Hif1a target sites in RPE cells (Error bars=s.e.m. (a control
not injected with AAV: n=4, AAV-CjCa9-injected test group: n=5),
Student's t-tests, * p<0.05, *** p<0.001), FIG. 4a graph of
Vegfa expression levels measured by ELISA in RPE cells (Error
bars=s.e.m. (a control not injected with AAV: n=4,
AAV-CjCa9-injected test group: n=5), One-way ANOVA and Tukey
post-hoc tests, * p<0.05, *** p<0.001), FIG. 5a graph of
indel frequencies at off-target sites (the mismatched base sequence
is marked with a solid line, and the PAM sequence is marked with a
dotted line), FIG. 6 laser-induced CNV areas of eyeballs of mice
injected with AAV9-CjCa9 targeting Rosa26, Vegfa, or Hif1a, stained
with isolectin B4 (Scale bar=200 .mu.m), and FIG. 7 a graph of the
laser-induced CNV areas (%) (Error bars=s.e.m.(n=17-18), One-way
ANOVA and Tukey post-hoc tests, * p<0.05, ** p<0.01, ***
p<0.001, ns: not significant).
[0086] FIG. 8 shows images of in vivo expression of eGFP with
CjCas9 in mouse RPE cells (n=6, anti-GFP antibody (green) and DAPI
(blue), Scale bar=20 .mu.m).
[0087] FIGS. 9 and 10 show opsin-positive areas in the retinas of
AAV/CjCas9-injected mice: FIG. 9 images of opsin-positive areas
corresponding to RPE cells expressing HA-tagged CjCas9 in Rosa26-,
Vegfa-, or Hif1a-specific CjCas9-injected mice (Opsin: red, HA:
green, and DAPI: blue, Scale bar=20 .mu.m, ONL: outer nuclear
layer, IS: inner segment of photoreceptor cells, OS: outer segment
of photoreceptor cells); and FIG. 10 a graph of relative
opsin-positive areas (%) (Error bars=s.e.m.(n=4), One-way ANOVA and
Tukey post-hoc tests, * p<0.05).
[0088] FIGS. 11 and 12 show reduced expression of VEGF due to
CjCas9 targeting Vegfa or Hif1a in retinal tissue: FIG. 11a graph
of indel frequencies at target sites of Rosa26, Vegfa and Hif1a in
retinal cells (Error bars=s.e.m. (a control not injected with AAV:
n=4, AAV-CjCa9-injected test group: n=5), Student's t-tests, *
p<0.05, ** p<0.01, *** p<0.001), FIG. 12a graph of Vegfa
expression levels in retinal cells, measured using ELISA (Error
bars=s.e.m. (n=6-7), One-way ANOVA and Tukey post-hoc tests, *
p<0.05, *** p<0.001).
[0089] FIG. 13 shows the reducing effect of CjCas9 targeting Vegfa
on vascular leakage and blood leakage in retinas of diabetic
retinopathy mouse models, (a) an in vivo test schedule, (b) images
of reduced effects on vascular leakage and blood leakage in mouse
retinas injected with AAV2-CjCa9 targeting Vegfa.
[0090] FIGS. 14 and 15 show the reducing effect of CjCas9 targeting
Vegfa on vascular leakage and blood leakage in retinas of diabetic
retinopathy mouse models: FIG. 14 is an in vivo test schedule (a),
images of the reducing effect on vascular leakage and blood leakage
in mouse retinas injected with AAV2-CjCa9 targeting Vegfa (b), and
FIG. 15 is a graph of relative vascular leakage (%) due to CjCas9
targeting Rosa26 or Vegfa.
[0091] FIG. 16 shows CjCas9 target site screening results and indel
frequencies of human VEGFAs for gene manipulation.
[0092] FIGS. 17 and 18 show the result of CjCas9 target site
screening of human HIF1As for gene manipulation: FIG. 17 CjCas9
target site screening results and indel frequencies of human
HIF1As, and FIG. 18 target sites of HIF1As, conserved between
various mammals.
[0093] FIG. 19 shows CjCas9 target site screening results and indel
frequencies of human ANGPT2s for gene manipulation.
[0094] FIG. 20 shows CjCas9 target site screening results and indel
frequencies of human EPAS1s for gene manipulation.
[0095] FIG. 21 shows CjCas9 target site screening results and indel
frequencies of human ANGPTL4s for gene manipulation.
DETAILED DESCRIPTION
[0096] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by those
of ordinary skill in the art to which the present invention
belongs. Although methods and materials similar or identical to
those described herein can be used in practice or testing of the
present invention, suitable methods and materials are described
below. All publications, patent applications, patents and other
references mentioned herein are incorporated by reference in their
entirety. In addition, materials, methods and examples are merely
illustrative, and not intended to be limitive.
[0097] One aspect of the present invention relates to an
artificially manipulated neovascularization system, which has a
regulated neovascularization effect.
[0098] Specifically, the present invention relates to combination
of various aspects capable of regulating neovascularization or
improving or treating a neovascularization-associated disease by
artificially manipulating a neovascularization-associated factor.
The present invention includes a neovascularization-associated
factor whose function is artificially modified, a method of
manufacturing the same, a composition including the same, and a
therapeutic use thereof.
[0099] Another aspect of the present invention relates to an
additional regulating system with a third in vivo mechanism,
concomitant with various functions of a specific factor (e.g., a
gene known as a neovascularization-associated factor, etc.) whose
function is artificially modified.
[0100] Specifically, targeting of a third in vivo function as well
as a neovascularization function in which artificially manipulated
specific factors are involved may lead to the regulation of
corresponding mechanisms. The present invention includes a
neovascularization-associated factor whose function is artificially
modified, a method for manufacturing the same, a composition
including the same, and therapeutic uses thereof for improving or
treating a disease associated with the third function.
[0101] Neovascularization
[0102] An exemplary embodiment of the present invention relates to
improvement and modification of a neovascularization-associated
system.
[0103] The term "neovascularization" refers to a process of tissue
vascularization, including generation, development and/or
differentiation of new blood vessels. Here, neovascularization
encompasses angiogenesis and vasculogenesis. Neovascularization may
be closely related to various factors to promote or inhibit
proliferation of vascular endothelial cells.
[0104] Neovascularization encompasses all mechanisms for extension
from existing blood vessels, new generation of blood vessels from
precursor cells, and/or an increase in diameter of an existing
small blood vessel.
[0105] In addition, neovascularization encompasses all mechanisms
associated with formation of new vessels, which is involved in
vascular leakage or repair of damaged blood vessels.
[0106] The vascularization includes mechanisms concomitant with
excessive and/or abnormal neovascularization in various severe
disease states.
[0107] For example, in diseases such as cancer, macular
degeneration, diabetic retinopathy, arthritis and psoriasis,
excessive neovascularization occurs. In such a disease state, new
blood vessels are provided to tissue with a disease, and normal
tissue is damaged. In the case of cancer, new blood vessels allow
tumor cells to enter into the circulation system and thus enable
them to settle in another organ (tumor metastasis).
[0108] In one exemplary embodiment, the neovascularization may be
ocular vascularization.
[0109] For example, the neovascularization may be found in eye
diseases such as AMD, diabetic retinopathy and the like.
Particularly, AMD is the most common cause of legal irreversible
blindness in older people over the age of 65 in the US, Canada,
England, Wales, Scotland and Australia, and about 10% to 15% of the
patients show exudative (wet) diseases. The exudative AMD is
characterized by neovascularization and disease-causing
angiogenesis.
[0110] For example, ocular neovascularization may include choroidal
neovascularization (CNV), corneal neovascularization and/or
rubeosis iridis.
[0111] CNV is the vascularization in the choroid layer, and rapidly
occurs in people having a defect in the Bruch's membrane, which is
the innermost layer of the choroid. In addition, CNV is associated
with an excessive amount of vascular endothelial growth factor
(VEGF). CNV may cause excessive myopia, malignant myopia, or
neovascular degenerative macular degeneration (e.g., wet macular
degeneration).
[0112] Corneal neovascularization is the growth of new blood
vessels from the pericorneal plexus in the periphery of the cornea
into avascular corneal tissue due to oxygen deprivation, and may be
mainly caused by congenital or inflammatory (e.g., rejection after
corneal transplantation, grafted tissue or host diseases, atopic
conjunctivitis, injections, ocular pemphigoid, Lyell's syndrome,
and Stevens-Johnson syndrome), infectious (e.g., bacterial
(chlamydia, syphilis, pseduomonas), viral (herpes simplex virus and
herpes zoster virus), fungal (candida, aspergillus, fusarium),
parasistic (onchocerca volvolus), degenerative, traumatic and
iatrogenic (e.g., the wearing of contact lenses) diseases.
[0113] Rubeosis iridis is the vascularization on the surface of the
iris, associated with diabetic retinopathy, and also known to be
caused by central retinal vein occlusion, ocular ischemic syndrome,
chronic retinal detachment, and the like.
[0114] In a certain embodiment, the neovascularization may be
associated with survival, proliferation, persistency, cytotoxicity,
and a cytokine-release function of vascular endothelial cells.
[0115] In a certain embodiment, neovascularization may be
associated with an increase in the expression of an angiogenic
cytokine.
[0116] In a certain embodiment, the neovascularization may be
associated with functions of a receptor of vascular endothelial
cells.
[0117] In a certain embodiment, the neovascularization may be
associated with a migration ability of vascular endothelial
cells.
[0118] In a certain embodiment, the neovascularization may be
associated with an attachment ability of vascular endothelial
cells.
[0119] Neovascularization-Associated Factor
[0120] Neovascularization-Associated Factor
[0121] One embodiment of the present invention relates to an
artificially manipulated or modified neovascularization-associated
factor.
[0122] The term "neovascularization-associated factor" includes all
elements directly participating in or indirectly affecting
vasculogenesis or angiogenesis. Here, the elements may include DNA,
RNA, genes, peptides, polypeptides or proteins.
[0123] In an exemplary embodiment, the
neovascularization-associated factor may include various substances
which can have a non-natural, that is, artificially manipulated,
neovascularization regulating function. For example, it may be a
genetically manipulated or modified genes or proteins expressed in
an immune cells.
[0124] The term "artificially manipulated" means an artificially
modified state, which is not a naturally occurring state.
[0125] The term "genetically manipulated" means that a genetic
modification is artificially introduced to biological or
non-biological substances cited in the present invention, and may
be, for example, genes and/or gene products (polypeptides,
proteins, etc.) in which their genomes are artificially modified
for a specific purpose.
[0126] As an exemplary example, the present invention provides a
neovascularization-associated factor which is genetically
manipulated or modified for a specific purpose.
[0127] Genes or proteins having the functions listed below may have
multiple types of functions, not only one type of
neovascularization-associated function. In addition, as needed, two
or more neovascularization functions and factors may be
provided.
[0128] The neovascularization-associated factor may promote or
increase neovascularization.
[0129] The neovascularization-associated factor may suppress or
inhibit neovascularization.
[0130] The neovascularization-associated factor may induce or
activate a neovascularization environment.
[0131] The neovascularization-associated factor may induce a
neovascularization-inhibited environment or inactivate a
neovascularization environment.
[0132] The neovascularization-associated factor may regulate
(promote, increase, suppress and/or inhibit etc.)
neovascularization.
[0133] The neovascularization-associated factor may be utilized in
improvement and treatment of a neovascularization-associated
disease.
[0134] In an exemplary embodiment, the
neovascularization-associated factor may be one or more selected
from the group consisting of ABCA1, ACAT, ACC2, ADAMTS12, ADCY2,
ADIPOQ, ADIPOR1, ADIPOR2, ADRB2, AGPAT5, AIP4, AKAP2, AKR1C2, AMPK,
ANG2, ANGPT2, ANGPTL4, ANK1, ANXA1, APOA1, ARHGAP17, ATP10A, AUH,
AUTOTAXIN, BAI3, BCAR1, BIN1, BMP3A, CA10, CAMK1D, CAMKK2, CD36,
CD44, CDC42, CDH13, CHAT, CNTFR, COL4A2, CPT, CSH1, CTNN, CUBN,
CYP7B1, CYSLTR1, CYSLTR2, DGKB, DGKH, DGKZ, DHCR7, DHFR, DRD2,
DRD5, EDG1, EDG2, EDG3, EDG4, EDG5, EDGE, EDG7, EDGE, EDNRA,
EHHADH, ENPP6, EPAS1, ERBB4, ERK1, ERK2, ESRRG, ETFA, F2, FDPS,
FGF2, FLNA, FLT4, FOXO1, FOXO3A, FTO, GABBR2, GATA3, GH1, GNA12,
GNA13, GRK2, GRK5, GRM5, HAPLN1, HAS1, HAS2, HAS3, HCRTR2, HIF1A,
HSD11B1, HYAL1, HYAL2, HYAL3, IL20RA, IL20RB, IL6ST, IL8, ITGA6,
ITGB1, KDR, LAMA1, LDLR, LEPR, LEPTIN, LIFR, LIPL2, LKB1, LRP,
LTBP2, MAT2B, ME1, MEGALIN, MERLIN, MET, MGST2, MMP2, MMP9, MTOR,
MTR, NCK2, NEDD9, NFKB1, NFKBIB, NOS2A, NOS3, NR112, NR3C2, NRG1,
NRP1, NRP2, OPRS1, OSBPL10, OSBPL3, OSTEOPONTIN, P2RY1, P2RY12,
PAI1, PAI2, PAK1, PAK6, PALLD, PAP1, PAR1, PAXILLIN, PC, PCTP,
PDE11A, PDE1A, PDE3A, PDE4D, PDE5, PDGFA, PDGFB, PDGFRA, PDGFRB,
PI3K, PITPNC1, PKA, PKCD, PLA1A, PLA2, PLAT, PLAU, PLCB1, PLD1,
PLD2, PLG, PLXDC2, PPARA, PPARG, PPARGC1B, PRKG1, PRL, PTGS2, PTN,
PTPN11, PYK2, RAC1, RAS, RHEB, RHOA, ROCK1, ROCK2, RPS6KA1,
RPS6KB2, SCARB1, SCHIP1, SGPP2, SLC25A21, SMAD3, SMAD4, SNCA,
SORBS2, SPLA2, SPOCK1, SRD5A1, SREBF1, SREBF2, STAT3, TGFBR1,
TGFBR2, TGFBR3, THBS1, THBS2, THEM2, THRB, TIAM1, TIMP2, TLL2,
TSC1, TSC2, TSPO, VEGFA, VEGFR1, and YES1.
[0135] As an exemplary example of the present invention, the
neovascularization-associated factor may be one or more selected
from the group consisting of VEGFA, HIF1A, ANGPT2, EPAS1, and
ANGPTL4.
[0136] In a certain embodiment, the neovascularization-associated
factor may be VEGFA.
[0137] A vascular endothelial growth factor A (VEGFA) gene is a
gene (full-length DNA, cDNA or mRNA) encoding a VEGFA protein also
called MVCD1, VEGF or VPF. In one example, the VEGFA gene may be
one or more selected from the group consisting of the following
genes, but the present invention is not limited thereto: genes
encoding human VEGFA (e.g., NCBI Accession No. NP_001020537.2 or
NP_001020538.2), for example, VEGFA genes represented by NCBI
Accession No. NM_001025366.2, NM_001025367.2, NM_003376, or
NG_008732.1.
[0138] In a certain embodiment, the neovascularization-associated
factor may be HIF1A.
[0139] A hypoxia-inducible factor 1-alpha (HIF-1-alpha; HIF1A) gene
refers to a gene (full-length DNA, cDNA or mRNA) encoding a HIF1A
protein also called HIF1, MOP1, PASD8 or bHLHe78. In an example,
the HIF1A gene may be one or more selected from the group
consisting of the following genes, but the present invention is not
limited thereto: genes encoding human HIF1A (e.g., NCBI Accession
No. NP_001230013.1 or NP_001521.1), for example, HIF1A genes
represented by NCBI Accession No. NM_001243084.1, NM_001530.3,
NM_181054.2, or NG_029606.1.
[0140] In a certain embodiment, the neovascularization-associated
factor may be ANGPT2.
[0141] An angiopoietin-2 (ANGPT2) gene refers to a gene
(full-length DNA, cDNA or mRNA) encoding an ANGPT2 protein also
called AGPT2 or ANG2. In an example, the ANGPT2 gene may be one or
more selected from the group consisting of the following genes, but
the present invention is not limited thereto: genes encoding human
ANGPT2 (e.g., NCBI Accession No. NP_001112359.1, NP_001112360.1 or
NP_001138.1), for example, ANGPT2 genes represented by NCBI
Accession No. NM_001118887.1, NM_001118888.1, NM_001147.2 or
NG_029483.1.
[0142] In a certain embodiment, the neovascularization-associated
factor may be EPAS1.
[0143] An endothelial PAS domain-containing protein 1 (EPAS1) gene
refers to a gene (full-length DNA, cDNA or mRNA) encoding an EPAS1
protein also called ECYT4, HIF2A, HLF, MOP2, PASD2 or bHLHe73. In
an example, the EPAS1 gene may be one or more selected from the
group consisting of the following genes, but the present invention
is not limited thereto: genes encoding human EPAS1 (e.g., NCBI
Accession No. NP_001421.2, etc.), for example, EPAS1 genes
represented by NCBI Accession No. NM_001430.4 or NG_016000.1.
[0144] In a certain embodiment, the neovascularization-associated
factor may be ANGPTL4.
[0145] An angiopoietin-like 4 (ANGPTL4) gene refers to a gene
(full-length DNA, cDNA or mRNA) encoding an ANGPTL4 protein also
called ARP4, FIAF, HARP, HFARP, NL2, PGAR, TGQTL or UNQ171. In an
example, the ANGPTL4 gene may be one or more selected from the
group consisting of the following genes, but the present invention
is not limited thereto: genes encoding human ANGPTL4 (e.g., NCBI
Accession No. NP_001034756.1 or NP_647475.1), for example, ANGPTL4
genes represented by NCBI Accession No. NM_001039667.2,
NM_139314.2, or NG_012169.1.
[0146] The neovascularization-associated factor may be derived from
mammals including primates such as human, monkeys and the like,
rodents such as rats, mice and the like, etc.
[0147] Information about the genes may be obtained from a known
database such as GeneBank of the National Center for Biotechnology
Information (NCBI).
[0148] In an exemplary embodiment of the present invention, the
neovascularization-associated factor, for example, VEGFA, HIF1A,
ANGPT2, EPAS1 or ANGPTL4, may be an artificially manipulated
neovascularization-associated factor.
[0149] In a certain embodiment, the artificially manipulated
neovascularization-associated factor may be genetically
manipulated.
[0150] The gene manipulation or modification may be achieved by
artificial insertion, deletion, substitution or inversion occurring
in a partial or entire region of the genomic sequence of a wild
type gene. In addition, the gene manipulation or modification may
be achieved by fusion of manipulation or modification of two or
more genes.
[0151] For example, the gene is inactivated by such gene
manipulation or modification, such that a protein encoded from the
gene may not be expressed in the form of a protein having an innate
function.
[0152] For example, the gene may be further activated by such gene
manipulation or modification, such that a protein encoded from the
gene is to be expressed in the form of a protein having an improved
function, compared to the innate function. In an example, when a
function of the protein encoded by a specific gene is A, a function
of a protein expressed by a manipulated gene may be totally
different from A or may have an additional function (A+B) including
A.
[0153] For example, a fusion of two or more proteins may be
expressed using two or more genes having different or complementary
functions due to such gene manipulation or modification.
[0154] For example, two or more proteins may be expressed
separately or independently in cells by using two or more genes
having different or complementary functions due to such gene
manipulation or modification.
[0155] The manipulated neovascularization-associated factor may
promote or increase neovascularization.
[0156] The manipulated neovascularization-associated factor may
suppress or inhibit neovascularization.
[0157] The manipulated neovascularization-associated factor may
induce or activate a neovascularization environment.
[0158] The manipulated neovascularization-associated factor may
induce a neovascularization inhibiting environment or inactivate a
neovascularization environment.
[0159] The manipulated neovascularization-associated factor may
regulate (promote, increase, suppress and/or inhibit)
neovascularization.
[0160] The manipulated neovascularization-associated factor may be
utilized in improvement and treatment of a
neovascularization-associated disease.
[0161] The manipulation includes all types of structural or
functional modifications of the neovascularization-associated
factor.
[0162] The structural modification of the
neovascularization-associated factor includes all types of
modifications, which are not the same as those of a wild type
existing in a natural state.
[0163] For example, when the neovascularization-associated factor
is DNA, RNA or a gene, the structural modification may be the loss
of one or more nucleotides.
[0164] The structural modification may be the insertion of one or
more nucleotides.
[0165] Here, the inserted nucleotides include all of a subject
including a neovascularization-associated factor and nucleotides
entering from the outside of the subject.
[0166] The structural modification may be the substitution of one
or more nucleotides.
[0167] The structural modification may include the chemical
modification of one or more nucleotides.
[0168] Here, the chemical modification includes all of the
addition, removal and substitution of chemical functional
groups.
[0169] As another example, when the neovascularization-associated
factor is a peptide, a polypeptide or a protein, the structural
modification may be the loss of one or more amino acids.
[0170] The structural modification may be the insertion of one or
more amino acids.
[0171] Here, the inserted amino acids include all of a subject
including a neovascularization-associated factor and amino acids
entering from the outside of the subject.
[0172] The structural modification may be the substitution of one
or more amino acids.
[0173] The structural modification may include the chemical
modification of one or more amino acids.
[0174] Here, the chemical modification includes all of the
addition, removal and substitution of chemical functional
groups.
[0175] The structural modification may be the partial or entire
attachment of a different peptide, polypeptide or protein.
[0176] Here, the different peptide, polypeptide or protein may be a
neovascularization-associated factor, or a peptide, polypeptide or
protein having a different function.
[0177] The functional modification of the
neovascularization-associated factor may include all types having
an improved or reduced function, compared to that of a wild type
existing in a natural state, and having a third different
function.
[0178] For example, when the neovascularization-associated factor
is a peptide, polypeptide or protein, the functional modification
may be a mutation of the neovascularization-associated factor.
[0179] Here, the mutation may be a mutation that enhances or
suppresses a function of the neovascularization-associated
factor.
[0180] The functional modification may have an additional function
of the neovascularization-associated factor.
[0181] Here, the additional function may be the same or a different
function. In addition, the neovascularization-associated factor
having the additional function may be fused with a different
peptide, polypeptide or protein.
[0182] The functional modification may be the enhancement in
functionality due to increased expression of the
neovascularization-associated factor.
[0183] The functional modification may be the degradation in
functionality due to decreased expression of the
neovascularization-associated factor.
[0184] In an exemplary embodiment, the manipulated
neovascularization-associated factor may be induced by one or more
of the following mutations: [0185] all or partial deletions of the
neovascularization-associated factor, that is, a gene to be
manipulated (hereinafter, referred to as a target gene), for
example, deletion of 1 bp or longer nucleotides, for example, 1 to
30, 1 to 27, 1 to 25, 1 to 23, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1
to 3, or 1 nucleotide of the target gene, [0186] substitution of 1
bp or longer nucleotides, for example, 1 to 30, 1 to 27, 1 to 25, 1
to 23, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 nucleotide
of the target gene with a nucleotide different from a wild type,
and [0187] insertion of one or more nucleotides, for example, 1 to
30, 1 to 27, 1 to 25, 1 to 23, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1
to 3, or 1 nucleotide (each independently selected from A, T, C and
G) into a certain position of the target gene.
[0188] A part of the modified target gene ("target region") may be
a continuous 1 bp or more, 3 bp or more, 5 bp or more, 7 bp or
more, 10 bp or more, 12 bp or more, 15 bp or more, 17 bp or more,
or 20 bp or more, for example, 1 bp to 30 bp, 3 bp to 30 bp, 5 bp
to 30 bp, 7 bp to 30 bp, 10 bp to 30 bp, 12 bp to 30 bp, 15 bp to
30 bp, 17 bp to 30 bp, 20 bp to 30 bp, 1 bp to 27 bp, 3 bp to 27
bp, 5 bp to 27 bp, 7 bp to 27 bp, 10 bp to 27 bp, 12 bp to 27 bp,
15 bp to 27 bp, 17 bp to 27 bp, 20 bp to 27 bp, 1 bp to 25 bp, 3 bp
to 25 bp, 5 bp to 25 bp, 7 bp to 25 bp, 10 bp to 25 bp, 12 bp to 25
bp, 15 bp to 25 bp, 17 bp to 25 bp, 20 bp to 25 bp, 1 bp to 23 bp,
3 bp to 23 bp, 5 bp to 23 bp, 7 bp to 23 bp, 10 bp to 23 bp, 12 bp
to 23 bp, 15 bp to 23 bp, 17 bp to 23 bp, 20 bp to 23 bp, 1 bp to
20 bp, 3 bp to 20 bp, 5 bp to 20 bp, 7 bp to 20 bp, 10 bp to 20 bp,
12 bp to 20 bp, 15 bp to 20 bp, 17 bp to 20 bp, 21 bp to 25 bp, 18
bp to 22 bp, or 21 bp to 23 bp region of the base sequence of the
gene.
[0189] Meanwhile, a different embodiment of the present invention
relates to an additional system for regulating a third in vivo
mechanism, concomitant with various functions of the
above-described neovascularization-associated factors whose
functions are artificially modified.
[0190] In one exemplary embodiment, VEGF may be involved in
regulation of the third in vivo mechanism.
[0191] Since an increase in vascular permeability by VEGF may be a
cause of edema, as well as tumor growth, artificially manipulated
VEGF may increase a survival rate in various types of tumors (e.g.,
brain tumor, uterine cancer, vestibular schwannomas, etc.) or
recover hearing loss, for example, by manipulation to inactivate
VEGF. In addition, a decrease in vascular permeability by
artificially manipulated VEGF may impart therapeutic effects on
renal failure, arthritis, psoriasis, coronary disease, etc.
[0192] In addition, the artificially manipulated VEGF may impart a
therapeutic effect on an autoimmune disease. For example,
inflammation-inducing activity may be artificially regulated by
VEGF, thereby imparting therapeutic effects on uveitis, rheumatoid
arthritis, systemic lupus erythematosus, an inflammatory bowel
disease, psoriasis, systemic sclerosis, multiple sclerosis,
etc.
[0193] In addition, the artificially manipulated VEGF may impart a
therapeutic effect on a mental disease. For example, a therapeutic
effect on depression may be imparted by artificially regulating the
expression of a neurotransmission-associated factor by VEGF.
[0194] In another embodiment, the artificially manipulated VEGF may
be involved in the regulation of a third in vivo mechanism of HIF.
The HIF may be HIF1 or HIF2.
[0195] The artificially manipulated HIF may regulate
inflammation-inducing activity, thereby imparting therapeutic
effects on uveitis, rheumatoid arthritis, systemic lupus
erythematosus, an inflammatory bowel disease, psoriasis, systemic
sclerosis, multiple sclerosis, etc.
[0196] In addition, the artificially manipulated HIF may provide a
therapeutic effect on an autoimmune disease.
[0197] Likewise, the illustrative factors of the present invention
which are artificially manipulated may regulate corresponding
mechanisms by targeting the third in vivo function as well as the
neovascularization function. One exemplary embodiment of the
present invention includes such a neovascularization factor whose
function is artificially modified and a method for manufacturing
the same, a composition including the same, and uses of the factor
and the composition for improving or treating a disease associated
with a third function.
[0198] Neovascularization System
[0199] Neovascularization-Regulating System
[0200] In one aspect of the present invention, a
neovascularization-regulating system for regulating
neovascularization by artificially manipulating a
neovascularization-associated factor is provided.
[0201] The term "neovascularization-regulating system" used herein
includes all phenomena affecting the promotion, increase,
suppression and/or inhibition of neovascularization by change of a
function of an artificially manipulated
neovascularization-associated factor, and also includes all
substances, compositions, methods, and uses which are directly or
indirectly involved in such a neovascularization-regulating
system.
[0202] Each factor constituting such a
neovascularization-regulating system is also generally called
"neovascularization-regulating factor."
[0203] The system of the present invention includes a modified in
vivo mechanism, associated with an artificially manipulated
neovascularization-associated factor.
[0204] In a certain embodiment, the expression of hematopoietic
stem cell surface antigens such as CD34, CD117, CD133, etc. and
vascular endothelial cell antigens such as Flk-1/KDR, Tie-2, etc.
may be regulated by the artificially manipulated
neovascularization-associated factor.
[0205] In a certain embodiment, angiogenesis in which new vessels
are formed by sprouting and the growth of cells constituting a
blood vessel may be regulated.
[0206] In a certain embodiment, the activity of direct angiogenic
factors (DAFs) directly stimulating endothelial cells may be
regulated. The growth and/or migration of endothelial cells may be
promoted or inhibited.
[0207] For example, the DAFs may include vascular endothelial
growth factors (VEGFs), basic fibroblast growth factors (bFGFs),
hepatocyte growth factors (HGFs), epidermal growth factors (EGFs),
thymidine phosphorylase (PD-ECGF), placental growth factors
(PIGFs), transforming growth factors (TGFs), proliferin,
interleukin-8 of a cytokine, angiogenin (angiogenesis-inducing
protein), fibrin, nicotinamide (vitamin B complex), angiopoietin
(angiogenesis-promoting protein), platelet activating factors
(PAFs), 12-hydroxy eicosatetraenoate (12-HETE; a toxic degradation
product of arachidonic acid, which is an angiogenesis-promoting
factor of epithelial cells), matrix metalloproteases (MMPs),
sphingosine 1-phosphate (S1P), and leptin.
[0208] In a certain embodiment, two different intercellular
signaling pathways operating in blood vessel cells, that is, PDGF
and VEGF signaling pathways may be utilized.
[0209] In a certain embodiment, the activity of indirect angiogenic
factors (IAFs) inducing angiogenesis by formation of DAFs may be
regulated by stimulating vascular pericytes.
[0210] In a certain embodiment, vascular endothelial cells may be
differentiated from endothelial progenitor cells (EPCs), and thus a
mechanism of forming a primary vascular plexus may be
regulated.
[0211] In a certain embodiment, the degradability of extracellular
matrix components for the migration of endothelial cells may be
regulated.
[0212] In a certain embodiment, a cell migration-associated
signaling pathway may be regulated.
[0213] In a certain embodiment, the activity of VEGF receptors such
as VEGFR-1 (flt-1; fmslike-tyrosine kinase-1), VEGFR-2 (flk-1/KDR),
and VEGFR-3, and a platelet derived growth factor (PDGF) receptor,
or neuropilin-1 (NP-1) may be regulated.
[0214] In an exemplary embodiment, the
neovascularization-regulating system includes a composition for
manipulating a neovascularization-associated factor.
[0215] The composition for manipulation may be a composition
capable of artificially manipulating a
neovascularization-associated factor, and preferably, a composition
for gene manipulation.
[0216] Hereinafter, the composition for gene manipulation will be
described.
[0217] Composition for Manipulating Neovascularization-Associated
Factor
[0218] Manipulation or modification of substances involved in the
neovascularization-associated factor and the neovascularization
system of the present invention is preferably accomplished by
genetic manipulation.
[0219] In one aspect, composition and method for manipulating a
gene by targeting a partial or entire non-coding or coding region
of the neovascularization-associated factor may be provided.
[0220] In an exemplary embodiment, the composition and method may
be used in manipulation or modification of one or more
neovascularization regulating genes involved in the formation of a
desired neovascularization system. The manipulation or modification
may be performed by modification of nucleic acids constituting a
gene. As a result of the manipulation, all of knockdown, knockout,
and knockin are included.
[0221] In an exemplary embodiment, the manipulation may be
performed by targeting a promoter region, or a transcription
sequence, for example, an intron or exon sequence. A coding
sequence, for example, a coding region, specifically, an initial
coding region may be targeted for the modification of expression
and knockout.
[0222] In an exemplary embodiment, the modification of nucleic
acids may be substitution, deletion, and/or insertion of one or
more nucleotides, for example, 1 to 30 bp, 1 to 27 bp, 1 to 25 bp,
1 to 23 bp, 1 to 20 bp, 1 to 15 bp, 1 to 10 bp, 1 to 5 bp, 1 to 3
bp, or 1 bp nucleotides.
[0223] In an exemplary embodiment, for the knockout of one or more
neovascularization-associated genes, elimination of expression of
one or more of the genes, or one or more knockouts of one or two
alleles, the above-mentioned region may be targeted such that one
or more neovascularization-associated genes contain a deletion or
mutation.
[0224] In an exemplary embodiment, the knockdown of a gene may be
used to decrease the expression of undesired alleles or
transcriptomes.
[0225] In an exemplary embodiment, non-coding sequences of a
promoter, an enhancer, an intron, a 3'UTR, and/or a polyadenylation
signal may be targeted to be used in modifying a
neovascularization-associated gene affecting a neovascularization
function.
[0226] In an exemplary embodiment, the activity of a
neovascularization-associated gene may be regulated, for example,
activated or inactivated by the modification of nucleic acids of
the gene.
[0227] In an exemplary embodiment, the modification of nucleic
acids of the gene may catalyze cleavage of a single strand or
double strands, that is, breaks of nucleic acid strands in a
specific region of the target gene by a guide nucleic acid-editor
protein complex, resulting in inactivation of the target gene.
[0228] In an exemplary embodiment, the nucleic acid strand breaks
may be repaired through a mechanism such as homologous
recombination or non-homologous end joining (NHEJ).
[0229] In this case, when the NHEJ mechanism takes place, a change
in DNA sequence is induced at the cleavage site, resulting in
inactivation of the gene. The repair by NHEJ may induce
substitution, insertion or deletion of a short gene fragment, and
may be used in the induction of a corresponding gene knockout.
[0230] In another aspect, the present invention provides a
composition for manipulating a neovascularization-associated
factor.
[0231] The composition for manipulation is a composition that is
able to artificially manipulate a neovascularization-associated
factor, and preferably, a composition for gene manipulation.
[0232] The composition may be employed in gene manipulation for one
or more neovascularization-associated factors involved in formation
of a desired neovascularization-regulating system.
[0233] The gene manipulation may be performed in consideration of a
gene expression regulating process.
[0234] In an exemplary embodiment, it may be performed by selecting
a suitable manipulation means for each stage of transcription, RNA
processing, RNA transporting, RNA degradation, translation, and
protein modification regulating stages.
[0235] In an exemplary embodiment, small RNA (sRNA) interferes with
mRNA or reduces stability thereof using RNA interference (RNAi) or
RNA silencing, and in some cases, breaks up mRNA to interrupt the
delivery of protein synthesis information, resulting in regulation
of the expression of genetic information.
[0236] The gene manipulation may be performed by modification of
nucleic acids constituting a neovascularization-associated factor.
As manipulation results, all of knockdown, knockout, and knockin
are included.
[0237] In a certain embodiment, the modification of nucleic acids
may be substitution, deletion, and/or insertion of one or more
nucleotides, for example, 1 to 30 bp, 1 to 27 bp, 1 to 25 bp, 1 to
23 bp, 1 to 20 bp, 1 to 15 bp, 1 to 10 bp, 1 to 5 bp, 1 to 3 bp, or
1 bp nucleotides.
[0238] In a certain embodiment, for knockout of one or more
neovascularization-associated factors, elimination of the
expression of one or more factors, or one or more knockouts of one
or two alleles, the gene may be manipulated such that one or more
neovascularization-associated factors contain a deletion or
mutation.
[0239] In a certain embodiment, knockdown of the
neovascularization-associated factor may be used to decrease
expression of undesired alleles or transcriptomes.
[0240] In a certain embodiment, the modification of nucleic acids
may be insertion of one or more nucleic acid fragments or genes.
Here, the nucleic acid fragment may be a nucleic acid sequence
consisting of one or more nucleotides, and a length of the nucleic
acid fragment may be 1 to 40 bp, 1 to 50 bp, 1 to 60 bp, 1 to 70
bp, 1 to 80 bp, 1 to 90 bp, 1 to 100 bp, 1 to 500 bp or 1 to 1000
bp. Here, the inserted gene may be one of the
neovascularization-associated factors, or a gene having a different
function.
[0241] In an exemplary embodiment, the modification of nucleic
acids may employ a wild type or variant enzyme which is capable of
catalyzing hydrolysis (cleavage) of bonds between nucleic acids in
a DNA or RNA molecule, preferably, a DNA molecule. It may also
employ a guide nucleic acid-editor protein complex.
[0242] For example, the gene may be manipulated using one or more
nucleases selected from the group consisting of a meganuclease, a
zinc finger nuclease, CRISPR/Cas9 (Cas9 protein), CRISPR-Cpf1 (Cpf1
protein) and a TALE-nuclease, thereby regulating the expression of
genetic information.
[0243] In a certain embodiment, non-limitedly, the gene
manipulation may be mediated by NHEJ or homology-directed repair
(HDR) using a guide nucleic acid-editor protein complex, for
example, a CRISPR/Cas system.
[0244] In this case, when the NHEJ mechanism takes place, a change
in DNA sequence may be induced at a cleavage site, thereby
inactivating the gene. Repair by NHEJ may induce substitution,
insertion or deletion of a short gene fragment, and may be used in
the induction of the knockout of a corresponding gene.
[0245] In another aspect, the present invention may provide the
gene manipulation site.
[0246] In an exemplary embodiment, when the gene is modified by
NHEJ-mediated modification, the gene manipulation site may be a
site in the gene, triggering the decrease or elimination of
expression of a neovascularization regulating gene product.
[0247] For example, the site may be in an initial coding region,
[0248] a promoter sequence, [0249] an enhancer sequence, [0250] a
specific intron sequence, or [0251] a specific exon sequence.
[0252] In an exemplary embodiment, the composition for manipulating
a neovascularization-associated factor may target a
neovascularization-associated factor affecting the regulation of
neovascularization, such as a VEGFA gene, an HIF1A gene, an ANGPT2
gene, an EPAS1 gene, or an ANGPTL4 gene, as a manipulation
subject.
[0253] Examples of target regions, that is, target sequences for
regions in which gene manipulation occurs or which are recognized
for gene manipulation are summarized in Table 1, Table 2, Table 3,
Table 4 and Table 5.
[0254] The target sequence may target one or more genes.
[0255] The target sequence may simultaneously target two or more
genes. Here, the two or more genes may be homologous genes or
heterologous genes.
[0256] The gene may contain one or more target sequences.
[0257] The gene may be simultaneously targeted at two or more
target sequences.
[0258] The gene may be changed in the site and number of gene
manipulations according to the number of target sequences.
[0259] The gene manipulation may be designed in various forms
depending on the number and positions of the target sequences.
[0260] The gene manipulation may simultaneously occur in two or
more target sequences. Here, the two or more target sequences may
be present in the homologous gene or heterologous gene.
[0261] The gene manipulation may be simultaneously performed with
respect to the two or more genes. Here, the two or more genes may
be homologous genes or heterologous genes.
[0262] Hereinafter, examples of target sequences which are able to
be used in embodiments of the present invention are shown in the
following tables: [0263] Table 1 Target sequences of VEGFA gene
[0264] Table 2 Target sequences of HIF1A gene [0265] Table 3 Target
sequences of ANGPT2 gene [0266] Table 4 Target sequences of EPAS1
gene [0267] Table 5 Target sequences of ANGPTL4 gene
TABLE-US-00001 [0267] TABLE 1 Gene No. Target sequence VEGFA 1
GTAGAGCAGCAAGGCAAGGCTC (SEQ ID NO: 1) VEGFA 2
CTTTCTGTCCTCAGTGGTCCCA (SEQ ID NO: 2) VEGFA 3 GAGACCCGGTGGACATCTTCC
(SEQ ID NO: 3) VEGFA 4 TTCCAGGAGTACCCTGATGAGA (SEQ ID NO: 4) VEGFA
5 TTGAAGATGTACTCGATCTCAT (SEQ ID NO: 5) VEGFA 6
AGGGGCACACAGGATGGCTTGA (SEQ ID NO: 6) VEGFA 7
AGCAGCCCCCGCATCGCATCAG (SEQ ID NO: 7) VEGFA 8
GCAGCAGCCCCCGCATCGCATC (SEQ ID NO: 8) VEGFA 9
GTGATGTTGGACTCCTCAGTGG (SEQ ID NO: 9) VEGFA 10
TGGTGATGTTGGACTCCTCAGT (SEQ ID NO: 10) VEGFA 11
CATGGTGATGTTGGACTCCTCA (SEQ ID NO: 11) VEGFA 12
ATGCGGATCAAACCTCACCAAG (SEQ ID NO: 12) VEGFA 13
CACATAGGAGAGATGAGCTTCC (SEQ ID NO: 13)
TABLE-US-00002 TABLE 2 Gene No. Target sequence HIF1A 1
ACTCACCAGCATCCAGAAGTTT (SEQ ID NO: 14) HIF1A 2
ATTTGGATATTGAAGATGACAT (SEQ ID NO: 15) HIF1A 3
ATTTACATTTCTGATAATGTGA (SEQ ID NO: 16) HIF1A 4
ATGTGTTTACAGTTTGAACTAA (SEQ ID NO: 17) HIF1A 5
CTGTGTCCAGTTAGTTCAAACT (SEQ ID NO: 18) HIF1A 6
ATGGTCACATGGATGAGTAAAA (SEQ ID NO: 19) HIF1A 7
CATGAGGAAATGAGAGAAATGC (SEQ ID NO: 20) HIF1A 8
CCCAGTGAGAAAAGGGAAAGAA (SEQ ID NO: 21) HIF1A 9
TTGTGAAAAAGGGTAAAGAACA (SEQ ID NO: 22) HIF1A 10
ATAGTTCTTCCTCGGCTAGTTA (SEQ ID NO: 23) HIF1A 11
TCATAGTTCTTCCTCGGCTAGT (SEQ ID NO: 24) HIF1A 12
TGTTCTTCATACACAGGTATTG (SEQ ID NO: 25) HIF1A 13
TACGTGAATGTGGCCTGTGCAG (SEQ ID NO: 26) HIF1A 14
CTGCACAGGCCACATTCACGTA (SEQ ID NO: 27) HIF1A 15
CTGAGGTTGGTTACTGTTGGTA (SEQ ID NO: 28) HIF1A 16
CAGGTCATAGGTGGTTTCTTAT (SEQ ID NO: 29) HIF1A 17
ACCAAGCAGGTCATAGGTGGTT (SEQ ID NO: 30) HIF1A 18
TTAGATAGCAAGACTTTCCTCA (SEQ ID NO: 31)
TABLE-US-00003 TABLE 3 Gene No. Target sequence ANGPT2 1
TCAGGTCCAGCATGGGTCCTGC (SEQ ID NO: 32) ANGPT2 2
CGGCGCGTCCCTCTGCACAGCA (SEQ ID NO: 33) ANGPT2 3
GCTGTGCAGAGGGACGCGCCGC (SEQ ID NO: 34) ANGPT2 4
ATCGTATTCGAGCGGCGCGTCC (SEQ ID NO: 35) ANGPT2 5
GATGTTCTCCAGCACTTGCAGC (SEQ ID NO: 36) ANGPT2 6
AGTGCTGGAGAACATCATGGAA (SEQ ID NO: 37) ANGPT2 7
ACAACATGAAGAAAGAAATGGT (SEQ ID NO: 38) ANGPT2 8
AAATGGTAGAGATACAGCAGAA (SEQ ID NO: 39) ANGPT2 9
TTCTATCATCACAGCCGTCTGG (SEQ ID NO: 40) ANGPT2 10
AAGTTCAAGTCTCGTGGTCTGA (SEQ ID NO: 41) ANGPT2 11
ACGAGACTTGAACTTCAGCTCT (SEQ ID NO: 42) ANGPT2 12
AAGAAGGTGCTAGCTATGGAAG (SEQ ID NO: 43) ANGPT2 13
GATGATGTGCTTGTCTTCCATA (SEQ ID NO: 44)
TABLE-US-00004 TABLE 4 Gene No. Target sequence EPAS1 1
AACACCTCCGTCTCCTTGCTCC (SEQ ID NO: 45) EPAS1 2
GAAGCTGACCAGCAGATGGACA (SEQ ID NO: 46) EPAS1 3
GCAATGAAACCCTCCAAGGCTT (SEQ ID NO: 47) EPAS1 4
AAAACATCAGCAAGTTCATGGG (SEQ ID NO: 48) EPAS1 5
GCAAGTTCATGGGACTTACACA (SEQ ID NO: 49) EPAS1 6
GGTCGCAGGGATGAGTGAAGTC (SEQ ID NO: 50) EPAS1 7
GCGGGACTTCTTCATGAGGATG (SEQ ID NO: 51) EPAS1 8
GAAGTGCACGGTCACCAACAGA (SEQ ID NO: 52) EPAS1 9
ACAGTACGGCCTCTGTTGGTGA (SEQ ID NO: 53) EPAS1 10
TCCAGGTGGCTGACTTGAGGTT (SEQ ID NO: 54) EPAS1 11
CAGGACAGCAGGGGCTCCTTGT (SEQ ID NO: 55) EPAS1 12
TAGCCCCCATGCTTTGCGAGCA (SEQ ID NO: 56)
TABLE-US-00005 TABLE 5 Gene No. Target sequence ANGPTL4 1
GCATCAGGGCTGCCCCGGCCGT (SEQ ID NO: 57) ANGPTL4 2
CACGGGTCCGCCCTGAGCGCTC (SEQ ID NO: 58) ANGPTL4 3
GGACGCAAAGCGCGGCGACTTG (SEQ ID NO: 59) ANGPTL4 4
TCCTGGGACGAGATGAATGTCC (SEQ ID NO: 60) ANGPTL4 5
CTGCAGCTCGGCCAGGGGCTGC (SEQ ID NO: 61) ANGPTL4 6
CCAGGGGCTGCGCGAACACGCG (SEQ ID NO: 62) ANGPTL4 7
CCCTCGGTTCCCTGACAGGCGG (SEQ ID NO: 63) ANGPTL4 8
ACCCTGAGGTCCTTCACAGCCT (SEQ ID NO: 64) ANGPTL4 9
TTCCACAAGGTGGCCCAGCAGC (SEQ ID NO: 65) ANGPTL4 10
CAGCAGCAGCGGCACCTGGAGA (SEQ ID NO: 66) ANGPTL4 11
TCCTAGTTTGGCCTCCTGGACC (SEQ ID NO: 67) ANGPTL4 12
GACCCGGCTCACAATGTCAGCC (SEQ ID NO: 68) ANGPTL4 13
GCTGTTGCGGTCCCCCGTGATG (SEQ ID NO: 69) ANGPTL4 14
GGCGTTGCCATCCCAGTCCCGC (SEQ ID NO: 70) ANGPTL4 15
AACGCCGAGTTGCTGCAGTTCT (SEQ ID NO: 71) ANGPTL4 16
ATAGGCCGTGTCCTCGCCACCC (SEQ ID NO: 72) ANGPTL4 17
GTTCTCCGTGCACCTGGGTGGC (SEQ ID NO: 73) ANGPTL4 18
ACACGGCCTATAGCCTGCAGCT (SEQ ID NO: 74) ANGPTL4 19
CCACCGTCCCACCCAGCGGCCT (SEQ ID NO: 75) ANGPTL4 20
GTGATCCTGGTCCCAAGTGGAG (SEQ ID NO: 76) ANGPTL4 21
GACCCCGGCAGGAGGCTGGTGG (SEQ ID NO: 77) ANGPTL4 22
TGCAGCCATTCCAACCTCAACG (SEQ ID NO: 78) ANGPTL4 23
TGCCGCTGCTGTGGGATGGAGC (SEQ ID NO: 79)
[0268] Composition for Manipulation-Gene Scissors System
[0269] The neovascularization-regulating system of the present
invention may include a guide nucleic acid-editor protein complex
as a composition for manipulating a neovascularization-associated
factor.
[0270] Guide Nucleic Acid-Editor Protein Complex
[0271] The term "guide nucleic acid-editor protein complex" refers
to a complex formed through the interaction between a guide nucleic
acid and an editor protein, and the nucleic acid-protein complex
includes a guide nucleic acid and an editor protein.
[0272] The term "guide nucleic acid" refers to a nucleic acid
capable of recognizing a target nucleic acid, gene, chromosome or
protein.
[0273] The guide nucleic acid may be present in the form of DNA,
RNA or a DNA/RNA hybrid, and may have a nucleic acid sequence of 5
to 150 bases.
[0274] The guide nucleic acid may include one or more domains.
[0275] The domains may be, but are not limited to, a guide domain,
a first complementary domain, a linker domain, a second
complementary domain, a proximal domain, or a tail domain.
[0276] The guide nucleic acid may include two or more domains,
which may be the same domain repeats, or different domains.
[0277] The guide nucleic acid may have one continuous nucleic acid
sequence.
[0278] For example, the one continuous nucleic acid sequence may be
(N)m, where N represents A, T, C or G, or A, U, C or G, and m is an
integer of 1 to 150.
[0279] The guide nucleic acid may have two or more continuous
nucleic acid sequences.
[0280] For example, the two or more continuous nucleic acid
sequences may be (N)m and (N)o, where N represents A, T, C or G, or
A, U, C or G, m and o are an integer of 1 to 150, and m and o may
be the same as or different from each other.
[0281] The term "editor protein" refers to a peptide, polypeptide
or protein which is able to directly bind to or interact with,
without direct binding to, a nucleic acid.
[0282] The editor protein may be an enzyme.
[0283] The editor protein may be a fusion protein.
[0284] Here, the "fusion protein" refers to a protein that is
produced by fusing an enzyme with an additional domain, peptide,
polypeptide or protein.
[0285] The term "enzyme" refers to a protein that contains a domain
capable of cleaving a nucleic acid, gene, chromosome or
protein.
[0286] The additional domain, peptide, polypeptide or protein may
be a functional domain, peptide, polypeptide or protein, which has
a function the same as or different from the enzyme.
[0287] The fusion protein may include an additional domain,
peptide, polypeptide or protein at one or more regions of the amino
terminus (N-terminus) of the enzyme or the vicinity thereof; the
carboxyl terminus (C-terminus) or the vicinity thereof; the middle
part of the enzyme; and a combination thereof.
[0288] The fusion protein may include a functional domain, peptide,
polypeptide or protein at one or more regions of the N-terminus of
the enzyme or the vicinity thereof; the C-terminus or the vicinity
thereof; the middle part of the enzyme; and a combination
thereof.
[0289] The guide nucleic acid-editor protein complex may serve to
modify a subject.
[0290] The subject may be a target nucleic acid, gene, chromosome
or protein.
[0291] For example, the guide nucleic acid-editor protein complex
may result in final regulation (e.g., inhibition, suppression,
reduction, increase or promotion) of the expression of a protein of
interest, removal of the protein, or expression of a new
protein.
[0292] Here, the guide nucleic acid-editor protein complex may act
at a DNA, RNA, gene or chromosome level.
[0293] The guide nucleic acid-editor protein complex may act in
gene transcription and translation stages.
[0294] The guide nucleic acid-editor protein complex may act at a
protein level.
[0295] 1. Guide Nucleic Acids
[0296] The guide nucleic acid is a nucleic acid that is capable of
recognizing a target nucleic acid, gene, chromosome or protein, and
forms a guide nucleic acid-protein complex.
[0297] Here, the guide nucleic acid is configured to recognize or
target a nucleic acid, gene, chromosome or protein targeted by the
guide nucleic acid-protein complex.
[0298] The guide nucleic acid may be present in the form of DNA,
RNA or a DNA/RNA mixture, and have a 5 to 150-nucleic acid
sequence.
[0299] The guide nucleic acid may be present in a linear or
circular shape.
[0300] The guide nucleic acid may be one continuous nucleic acid
sequence.
[0301] For example, the one continuous nucleic acid sequence may be
(N).sub.m, where N is A, T, C or G, or A, U, C or G, and m is an
integer of 1 to 150.
[0302] The guide nucleic acid may be two or more continuous nucleic
acid sequences.
[0303] For example, the two or more continuous nucleic acid
sequences may be (N)m and (N)o, where N represents A, T, C or G, or
A, U, C or G, m and o are an integer of 1 to 150, and may be the
same as or different from each other.
[0304] The guide nucleic acid may include one or more domains.
[0305] Here, the domains may be, but are not limited to, a guide
domain, a first complementary domain, a linker domain, a second
complementary domain, a proximal domain, or a tail domain.
[0306] The guide nucleic acid may include two or more domains,
which may be the same domain repeats, or different domains.
[0307] The domains will be described below.
[0308] i) Guide Domain
[0309] The term "guide domain" is a domain having a complementary
guide sequence which is able to form a complementary bond with a
target sequence on a target gene or nucleic acid, and serves to
specifically interact with the target gene or nucleic acid.
[0310] The guide sequence is a nucleic acid sequence complementary
to the target sequence on a target gene or nucleic acid, which has,
for example, at least 50% or more, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90% or 95% complementarity or complete complementarity.
[0311] The guide domain may be a sequence of 5 to 50 bases.
[0312] In an example, the guide domain may be a sequence of 5 to
50, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40
to 50 or 45 to 50 bases.
[0313] In another example, the guide domain may be a sequence of 1
to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35
to 40, 40 to 45, or 45 to 50 bases.
[0314] The guide domain may have a guide sequence.
[0315] The guide sequence may be a complementary base sequence
which is able to form a complementary bond with the target sequence
on the target gene or nucleic acid.
[0316] The guide sequence may be a nucleic acid sequence
complementary to the target sequence on the target gene or nucleic
acid, which has, for example, at least 70%, 75%, 80%, 85%, 90% or
95% or more complementarity or complete complementarity.
[0317] The guide sequence may be a 5 to 50-base sequence.
[0318] In an example, the guide domain may be a 5 to 50, 10 to 50,
15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, or 45
to 50-base sequence.
[0319] In another example, the guide sequence may be a 1 to 5, 5 to
10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40
to 45, or 45 to 50-base sequence.
[0320] In addition, the guide domain may include a guide sequence
and an additional base sequence.
[0321] The additional base sequence may be utilized to improve or
degrade the function of the guide domain.
[0322] The additional base sequence may be utilized to improve or
degrade the function of the guide sequence.
[0323] The additional base sequence may be a 1 to 35-base
sequence.
[0324] In one example, the additional base sequence may be a 5 to
35, 10 to 35, 15 to 35, 20 to 35, 25 to 35 or 30 to 35-base
sequence.
[0325] In another example, the additional base sequence may be a 1
to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30 or 30 to
35-base sequence.
[0326] The additional base sequence may be located at the 5'end of
the guide sequence.
[0327] The additional base sequence may be located at the 3'end of
the guide sequence.
[0328] ii) First Complementary Domain
[0329] The term "first complementary domain" is a nucleic acid
sequence including a nucleic acid sequence complementary to a
second complementary domain, and has enough complementarity so as
to form a double strand with the second complementary domain.
[0330] The first complementary domain may be a 5 to 35-base
sequence.
[0331] In an example, the first complementary domain may be a 5 to
35, 10 to 35, 15 to 35, 20 to 35, 25 to 35, or 30 to 35-base
sequence.
[0332] In another example, the first complementary domain may be a
1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30 or 30 to
35-base sequence.
[0333] iii) Linker Domain
[0334] The term "linker domain" is a nucleic acid sequence
connecting two or more domains, which are two or more identical or
different domains. The linker domain may be connected with two or
more domains by covalent bonding or non-covalent bonding, or may
connect two or more domains by covalent bonding or non-covalent
bonding.
[0335] The linker domain may be a 1 to 30-base sequence.
[0336] In one example, the linker domain may be a 1 to 5, 5 to 10,
10 to 15, 15 to 20, 20 to 25, or 25 to 30-base sequence.
[0337] In another example, the linker domain may be a 1 to 30, 5 to
30, 10 to 30, 15 to 30, 20 to 30, or 25 to 30-base sequence.
[0338] iv) Second Complementary Domain
[0339] The term "second complementary domain" is a nucleic acid
sequence including a nucleic acid sequence complementary to the
first complementary domain, and has enough complementarity so as to
form a double strand with the first complementary domain.
[0340] The second complementary domain may have a base sequence
complementary to the first complementary domain, and a base
sequence having no complementarity to the first complementary
domain, for example, a base sequence not forming a double strand
with the first complementary domain, and may have a longer base
sequence than the first complementary domain.
[0341] The second complementary domain may have a 5 to 35-base
sequence.
[0342] In an example, the second complementary domain may be a 1 to
35, 5 to 35, 10 to 35, 15 to 35, 20 to 35, 25 to 35, or 30 to
35-base sequence.
[0343] In another example, the second complementary domain may be a
1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, or 30 to
35-base sequence.
[0344] v) Proximal Domain
[0345] The term "proximal domain" is a nucleic acid sequence
located adjacent to the second complementary domain.
[0346] The proximal domain may have a complementary base sequence
therein, and may be formed in a double strand due to a
complementary base sequence.
[0347] The proximal domain may be a 1 to 20-base sequence.
[0348] In one example, the proximal domain may be a 1 to 20, 5 to
20, 10 to 20 or 15 to 20-base sequence.
[0349] In another example, the proximal domain may be a 1 to 5, 5
to 10, 10 to 15 or 15 to 20-base sequence.
[0350] vi) Tail Domain
[0351] The term "tail domain" is a nucleic acid sequence located at
one or more ends of the both ends of the guide nucleic acid.
[0352] The tail domain may have a complementary base sequence
therein, and may be formed in a double strand due to a
complementary base sequence.
[0353] The tail domain may be a 1 to 50-base sequence.
[0354] In an example, the tail domain may be a 5 to 50, 10 to 50,
15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, or 45
to 50-base sequence.
[0355] In another example, the tail domain may be a 1 to 5, 5 to
10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40
to 45, or 45 to 50-base sequence.
[0356] Meanwhile, a part or all of the nucleic acid sequences
included in the domains, that is, the guide domain, the first
complementary domain, the linker domain, the second complementary
domain, the proximal domain and the tail domain may selectively or
additionally include a chemical modification.
[0357] The chemical modification may be, but is not limited to,
methylation, acetylation, phosphorylation, phosphorothioate
linkage, a locked nucleic acid (LNA), 2'-O-methyl
3'phosphorothioate (MS) or 2'-O-methyl 3'thioPACE (MSP).
[0358] The guide nucleic acid includes one or more domains.
[0359] The guide nucleic acid may include a guide domain.
[0360] The guide nucleic acid may include a first complementary
domain.
[0361] The guide nucleic acid may include a linker domain.
[0362] The guide nucleic acid may include a second complementary
domain.
[0363] The guide nucleic acid may include a proximal domain.
[0364] The guide nucleic acid may include a tail domain.
[0365] Here, there may be 1, 2, 3, 4, 5, 6 or more domains.
[0366] The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more
guide domains.
[0367] The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more
first complementary domains.
[0368] The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more
linker domains.
[0369] The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more
second complementary domains.
[0370] The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more
proximal domains.
[0371] The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more
tail domains.
[0372] Here, in the guide nucleic acid, one type of domain may be
duplicated.
[0373] The guide nucleic acid may include several domains with or
without duplication.
[0374] The guide nucleic acid may include the same type of domain.
Here, the same type of domain may have the same nucleic acid
sequence or different nucleic acid sequences.
[0375] The guide nucleic acid may include two types of domains.
Here, the two different types of domains may have different nucleic
acid sequences or the same nucleic acid sequence.
[0376] The guide nucleic acid may include three types of domains.
Here, the three different types of domains may have different
nucleic acid sequences or the same nucleic acid sequence.
[0377] The guide nucleic acid may include four types of domains.
Here, the four different types of domains may have different
nucleic acid sequences, or the same nucleic acid sequence.
[0378] The guide nucleic acid may include five types of domains.
Here, the five different types of domains may have different
nucleic acid sequences, or the same nucleic acid sequence.
[0379] The guide nucleic acid may include six types of domains.
Here, the six different types of domains may have different nucleic
acid sequences, or the same nucleic acid sequence.
[0380] For example, the guide nucleic acid may consist of [guide
domain]-[first complementary domain]-[linker domain]-[second
complementary domain]-[linker domain]-[guide domain]-[first
complementary domain]-[linker domain]-[second complementary
domain]. Here, the two guide domains may include guide sequences
for different or the same targets, the two first complementary
domains and the two second complementary domains may have the same
or different nucleic acid sequences. When the guide domains include
guide sequences for different targets, the guide nucleic acids may
specifically bind to two different targets, and here, the specific
bindings may be performed simultaneously or sequentially. In
addition, the linker domains may be cleaved by specific enzymes,
and the guide nucleic acids may be divided into two or three parts
in the presence of specific enzymes.
[0381] As a specific example of the guide nucleic acid of the
present invention, gRNA will be described below.
[0382] gRNA
[0383] The term "gRNA" refers to a nucleic acid capable of
specifically targeting a gRNA-CRISPR enzyme complex, that is, a
CRISPR complex, with respect to a target gene or nucleic acid. In
addition, the gRNA is a nucleic acid-specific RNA which may bind to
a CRISPR enzyme and guide the CRISPR enzyme to the target gene or
nucleic acid.
[0384] The gRNA may include multiple domains. Due to each domain,
interactions may occur in a three-dimensional structure or active
form of a gRNA strand, or between these strands.
[0385] The gRNA may be called single-stranded gRNA (single RNA
molecule); or double-stranded gRNA (including more than one,
generally, two discrete RNA molecules).
[0386] In one exemplary embodiment, the single-stranded gRNA may
include a guide domain, that is, a domain including a guide
sequence capable of forming a complementary bond with a target gene
or nucleic acid; a first complementary domain; a linker domain; a
second complementary domain, a domain having a sequence
complementary to the first complementary domain sequence, thereby
forming a double-stranded nucleic acid with the first complementary
domain; a proximal domain; and optionally a tail domain in the 5'
to 3' direction.
[0387] In another embodiment, the double-stranded gRNA may include
a first strand which includes a guide domain, that is, a domain
including a guide sequence capable of forming a complementary bond
with a target gene or nucleic acid and a first complementary
domain; and a second strand which includes a second complementary
domain, a domain having a sequence complementary to the first
complementary domain sequence, thereby forming a double-stranded
nucleic acid with the first complementary domain, a proximal
domain; and optionally a tail domain in the 5' to 3' direction.
[0388] Here, the first strand may be referred to as crRNA, and the
second strand may be referred to as tracrRNA. The crRNA may include
a guide domain and a first complementary domain, and the tracrRNA
may include a second complementary domain, a proximal domain and
optionally a tail domain.
[0389] In still another embodiment, the single-stranded gRNA may
include a guide domain, that is, a domain including a guide
sequence capable of forming a complementary bond with a target gene
or nucleic acid; a first complementary domain; a second
complementary domain, and a domain having a sequence complementary
to the first complementary domain sequence, thereby forming a
double-stranded nucleic acid with the first complementary domain in
the 5' to 3' direction.
[0390] i) Guide Domain
[0391] The guide domain includes a complementary guide sequence
capable of forming a complementary bond with a target sequence on a
target gene or nucleic acid. The guide sequence may be a nucleic
acid sequence having complementarity to the target sequence on the
target gene or nucleic acid, for example, at least 70%, 75%, 80%,
85%, 90% or 95% or more complementarity or complete
complementarity. The guide domain is considered to allow a gRNA-Cas
complex, that is, a CRISPR complex to specifically interact with
the target gene or nucleic acid.
[0392] The guide domain may be a 5 to 50-base sequence.
[0393] As an exemplary embodiment, the guide domain may be a 16,
17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
[0394] As an exemplary embodiment, the guide domain may include a
16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
[0395] Here, the guide domain may include a guide sequence.
[0396] The guide sequence may be a complementary base sequence
capable of forming a complementary bond with a target sequence on a
target gene or nucleic acid.
[0397] The guide sequence may be a nucleic acid sequence
complementary to the target sequence on the target gene or nucleic
acid, which has, for example, at least 70%, 75%, 80%, 85%, 90% or
95% or more complementarity or complete complementarity.
[0398] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target gene, that is, a
target sequence of a neovascularization-associated factor such as a
VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene or an
ANGPTL4 gene, which has, for example, at least 70%, 75%, 80%, 85%,
90% or 95% or more complementarity or complete complementarity.
[0399] The guide sequence may be a 5 to 50-base sequence.
[0400] In an exemplary embodiment, the guide sequence may be a 16,
17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
[0401] In one exemplary embodiment, the guide sequence may include
a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
[0402] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target sequence of the
VEGFA gene, which is a 16, 17, 18, 19, 20, 21, 22, 23, 24 or
25-base sequence.
[0403] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target sequence of the
HIF1A gene, which is a 16, 17, 18, 19, 20, 21, 22, 23, 24 or
25-base sequence.
[0404] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target sequence of the
ANGPT2 gene, which is a 16, 17, 18, 19, 20, 21, 22, 23, 24 or
25-base sequence.
[0405] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target sequence of the
EPAS1 gene, which is a 16, 17, 18, 19, 20, 21, 22, 23, 24 or
25-base sequence.
[0406] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target sequence of the
ANGPTL4 gene, which is a 16, 17, 18, 19, 20, 21, 22, 23, 24 or
25-base sequence.
[0407] Here, target sequences of the target genes, that is, the
neovascularization-associated factors such as the VEGFA gene, the
HIF1A gene, the ANGPT2 gene, the EPAS1 gene, and the ANGPTL4 gene
for the guide sequence are listed above in Table 1, Table 2, Table
3, Table 4 and Table 5, respectively, but the present invention is
not limited thereto.
[0408] Here, the guide domain may include a guide sequence and an
additional base sequence.
[0409] The additional base sequence may be a 1 to 35-base
sequence.
[0410] In one exemplary embodiment, the additional base sequence
may be a 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10-base sequence.
[0411] For example, the additional base sequence may be a single
base sequence, guanine (G), or a sequence of two bases, GG.
[0412] The additional base sequence may be located at the 5' end of
the guide sequence.
[0413] The additional base sequence may be located at the 3' end of
the guide sequence.
[0414] Selectively, a part or all of the base sequence of the guide
domain may include a chemical modification. The chemical
modification may be methylation, acetylation, phosphorylation,
phosphorothioate linkage, a locked nucleic acid (LNA), 2'-O-methyl
3' phosphorothioate (MS) or 2'-O-methyl 3' thioPACE (MSP), but the
present invention is not limited thereto.
[0415] ii) First Complementary Domain
[0416] The first complementary domain includes a nucleic acid
sequence complementary to a second complementary domain, and has
enough complementarity such that it is able to form a double strand
with the second complementary domain.
[0417] Here, the first complementary domain may be a 5 to 35-base
sequence. The first complementary domain may include a 5 to 35-base
sequence.
[0418] In one exemplary embodiment, the first complementary domain
may be a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, or 25-base sequence.
[0419] In another embodiment, the first complementary domain may
include a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, or 25-base sequence.
[0420] The first complementary domain may have homology with a
natural first complementary domain, or may be derived from a
natural first complementary domain. In addition, the first
complementary domain may have a difference in the base sequence of
a first complementary domain depending on the species existing in
nature, may be derived from a first complementary domain contained
in the species existing in nature, or may have partial or complete
homology with the first complementary domain contained in the
species existing in nature.
[0421] In one exemplary embodiment, the first complementary domain
may have partial, that is, at least 50% or more, or complete
homology with a first complementary domain of Streptococcus
pyogenes, Campylobacter jejuni, Streptococcus thermophilus,
Streptococcus aureus or Neisseria meningitides, or a first
complementary domain derived therefrom.
[0422] For example, when the first complementary domain is the
first complementary domain of Streptococcus pyogenes or a first
complementary domain derived therefrom, the first complementary
domain may be 5'-GUUUUAGAGCUA-3' or a base sequence having partial,
that is, at least 50% or more, or complete homology with
5'-GUUUUAGAGCUA-3'. Here, the first complementary domain may
further include (X)n, resulting in 5'-GUUUUAGAGCUA(X)n-3'. The X
may be selected from the group consisting of bases A, T, U and G,
and the n may represent the number of bases, which is an integer of
5 to 15. Here, the (X)n may be n repeats of the same base, or a
mixture of n bases of A, T, U and G.
[0423] In another embodiment, when the first complementary domain
is the first complementary domain of Campylobacter jejuni or a
first complementary domain derived therefrom, the first
complementary domain may be 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3', or a
base sequence having partial, that is, at least 50% or more, or
complete homology with 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3'. Here, the
first complementary domain may further include (X)n, resulting in
5'-GUUUUAGUCCCUUUUUAAAUUUCUU(X)n-3'. The X may be selected from the
group consisting of bases A, T, U and G, and the n may represent
the number of bases, which is an integer of 5 to 15. Here, the (X)n
may represent n repeats of the same base, or a mixture of n bases
of A, T, U and G.
[0424] In another embodiment, the first complementary domain may
have partial, that is, at least 50% or more, or complete homology
with a first complementary domain of Parcubacteria bacterium
(GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017),
Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium
(GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas
macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas
crevioricanis, Prevotella disiens, Moraxella bovoculi (237),
Smiihella sp. (SC_KO8D17), Leptospira inadai, Lachnospiraceae
bacterium (MA2020), Francisella novicida (U112), Candidatus
Methanoplasma termitum or Eubacterium eligens, or a first
complementary domain derived therefrom.
[0425] For example, when the first complementary domain is the
first complementary domain of Parcubacteria bacterium or a first
complementary domain derived therefrom, the first complementary
domain may be 5'-UUUGUAGAU-3', or a base sequence having partial,
that is, at least 50% or more homology with 5'-UUUGUAGAU-3'. Here,
the first complementary domain may further include (X)n, resulting
in 5'-(X)nUUUGUAGAU-3'. The X may be selected from the group
consisting of bases A, T, U and G, and the n may represent the
number of bases, which is an integer of 1 to 5. Here, the (X)n may
represent n repeats of the same base, or a mixture of n bases of A,
T, U and G.
[0426] Selectively, a part or all of the base sequence of the first
complementary domain may have a chemical modification. The chemical
modification may be methylation, acetylation, phosphorylation,
phosphorothioate linkage, a locked nucleic acid (LNA), 2'-O-methyl
3' phosphorothioate (MS) or 2'-O-methyl 3' thioPACE (MSP), but the
present invention is not limited thereto.
[0427] iii) Linker Domain
[0428] The linker domain is a nucleic acid sequence connecting two
or more domains, and connects two or more identical or different
domains. The linker domain may be connected with two or more
domains, or may connect two or more domains by covalent or
non-covalent bonding.
[0429] The linker domain may be a nucleic acid sequence connecting
a first complementary domain with a second complementary domain to
produce single-stranded gRNA.
[0430] The linker domain may be connected with the first
complementary domain and the second complementary domain by
covalent or non-covalent bonding.
[0431] The linker domain may connect the first complementary domain
with the second complementary domain by covalent or non-covalent
bonding
[0432] The linker domain may be a 1 to 30-base sequence. The linker
domain may include a 1 to 30-base sequence.
[0433] In an exemplary embodiment, the linker domain may be a 1 to
5, 5 to 10, 10 to 15, 15 to 20, 20 to 25 or 25 to 30-base
sequence.
[0434] In an exemplary embodiment, the linker domain may include a
1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, or 25 to 30-base
sequence.
[0435] The linker domain is suitable to be used in a
single-stranded gRNA molecule, and may be used to produce
single-stranded gRNA by being connected with a first strand and a
second strand of double-stranded gRNA or connecting the first
strand with the second strand by covalent or non-covalent bonding.
The linker domain may be used to produce single-stranded gRNA by
being connected with crRNA and tracrRNA of double-stranded gRNA or
connecting the crRNA with the tracrRNA by covalent or non-covalent
bonding.
[0436] The linker domain may have homology with a natural sequence,
for example, a partial sequence of tracrRNA, or may be derived
therefrom.
[0437] Selectively, a part or all of the base sequence of the
linker domain may have a chemical modification. The chemical
modification may be methylation, acetylation, phosphorylation,
phosphorothioate linkage, a locked nucleic acid (LNA), 2'-O-methyl
3' phosphorothioate (MS) or 2'-O-methyl 3' thioPACE (MSP), but the
present invention is not limited thereto.
[0438] iv) Second Complementary Domain
[0439] The second complementary domain includes a nucleic acid
sequence complementary to the first complementary domain, and has
enough complementarity so as to form a double strand with the first
complementary domain. The second complementary domain may include a
base sequence complementary to the first complementary domain, and
a base sequence having no complementarity with the first
complementary domain, for example, a base sequence not forming a
double strand with the first complementary domain, and may have a
longer base sequence than the first complementary domain.
[0440] Here, the second complementary domain may be a 5 to 35-base
sequence. The first complementary domain may include a 5 to 35-base
sequence.
[0441] In an exemplary embodiment, the second complementary domain
may be a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24 or 25-base sequence.
[0442] In an exemplary embodiment, the second complementary domain
may include a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24 or 25-base sequence.
[0443] In addition, the second complementary domain may have
homology with a natural second complementary domain, or may be
derived from the natural second complementary domain. In addition,
the second complementary domain may have a difference in base
sequence of a second complementary domain according to a species
existing in nature, and may be derived from a second complementary
domain contained in the species existing in nature, or may have
partial or complete homology with the second complementary domain
contained in the species existing in nature.
[0444] In an exemplary embodiment, the second complementary domain
may have partial, that is, at least 50% or more, or complete
homology with a second complementary domain of Streptococcus
pyogenes, Campylobacter jejuni, Streptococcus thermophilus,
Streptococcus aureus or Neisseria meningitides, or a second
complementary domain derived therefrom.
[0445] For example, when the second complementary domain is a
second complementary domain of Streptococcus pyogenes or a second
complementary domain derived therefrom, the second complementary
domain may be 5'-UAGCAAGUUAAAAU-3', or a base sequence having
partial, that is, at least 50% or more homology with
5'-UAGCAAGUUAAAAU-3' (a base sequence forming a double strand with
the first complementary domain is underlined). Here, the second
complementary domain may further include (X)n and/or (X)m,
resulting in 5'-(X)n UAGCAAGUUAAAAU(X)m-3'. The X may be selected
from the group consisting of bases A, T, U and G, and each of the n
and m may represent the number of bases, in which the n may be an
integer of 1 to 15, and the m may be an integer of 1 to 6. Here,
the (X)n may represent n repeats of the same base, or a mixture of
n bases of A, T, U and G. In addition, (X)m may represent m repeats
of the same base, or a mixture of m bases of A, T, U and G.
[0446] In another example, when the second complementary domain is
the second complementary domain of Campylobacter jejuni or a second
complementary domain derived therefrom, the second complementary
domain may be 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3', or a base sequence
having partial, that is, at least 50% or more homology with
5'-AAGAAAUUUAAAAAGGGACUAAAAU-3' (a base sequence forming a double
strand with the first complementary domain is underlined). Here,
the second complementary domain may further include (X)n and/or
(X)m, resulting in 5'-(X)nAAGAAAUUUAAAAAGGGACUAAAAU(X)m-3'. The X
may be selected from the group consisting of bases A, T, U and G,
and each of the n and m may represent the number of bases, in which
the n may be an integer of 1 to 15, and the m may be an integer of
1 to 6. Here, (X)n may represent n repeats of the same base, or a
mixture of n bases of A, T, U and G. In addition, (X)m may
represent m repeats of the same base, or a mixture of m bases of A,
T, U and G.
[0447] In another embodiment, the secondcomplementary domain may
have partial, that is, at least 50% or more, or complete homology
with a first complementary domain of Parcubacteria bacterium
(GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017),
Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium
(GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas
macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas
crevioricanis, Prevotella disiens, Moraxella bovoculi (237),
Smiihella sp. (SC_KO8D17), Leptospira inadai, Lachnospiraceae
bacterium (MA2020), Francisella novicida (U112), Candidatus
Methanoplasma termitum or Eubacterium eligens, or a
secondcomplementary domain derived therefrom.
[0448] For example, when the second complementary domain is a
second complementary domain of Parcubacteria bacterium or a second
complementary domain derived therefrom, the second complementary
domain may be 5'-AAAUUUCUACU-3', or a base sequence having partial,
that is, at least 50% or more homology with 5'-AAAUUUCUACU-3' (a
base sequence forming a double strand with the first complementary
domain is underlined). Here, the second complementary domain may
further include (X)n and/or (X)m, resulting in
5'-(X)nAAAUUUCUACU(X)m-3'. The X may be selected from the group
consisting of bases A, T, U and G, and each of the n and m may
represent the number of bases, in which the n may be an integer of
1 to 10, and the m may be an integer of 1 to 6. Here, the (X)n may
represent n repeats of the same base, or a mixture of n bases of A,
T, U and G. In addition, the (X)m may represent m repeats of the
same base, or a mixture of m bases of A, T, U and G.
[0449] Selectively, a part or all of the base sequence of the
second complementary domain may have a chemical modification. The
chemical modification may be methylation, acetylation,
phosphorylation, phosphorothioate linkage, a locked nucleic acid
(LNA), 2'-O-methyl 3'phosphorothioate (MS) or 2'-O-methyl
3'thioPACE (MSP), but the present invention is not limited
thereto.
[0450] v) Proximal Domain
[0451] The proximal domain is a sequence of 1 to 20 bases located
adjacent to the second complementary domain, and a domain located
at the 3'end direction of the second complementary domain. Here,
the proximal domain may be used to form a double strand between
complementary base sequences therein.
[0452] In one exemplary embodiment, the proximal domain may be a 5,
6, 7, 8, 9, 10, 11, 12, 13, 14 or 15-base sequence.
[0453] In another embodiment, the proximal domain may include a 5,
6, 7, 8, 9, 10, 11, 12, 13, 14 or 15-base sequence.
[0454] In addition, the proximal domain may have homology with a
natural proximal domain, or may be derived from the natural
proximal domain. In addition, the proximal domain may have a
difference in base sequence according to a species existing in
nature, may be derived from a proximal domain contained in the
species existing in nature, or may have partial or complete
homology with the proximal domain contained in the species existing
in nature.
[0455] In an exemplary embodiment, the proximal domain may have
partial, that is, at least 50% or more, or complete homology with a
proximal domain of Streptococcus pyogenes, Campylobacter jejuni,
Streptococcus thermophilus, Streptococcus aureus or Neisseria
meningitides, or a proximal domain derived therefrom.
[0456] For example, when the proximal domain is a proximal domain
of Streptococcus pyogenes or a proximal domain derived therefrom,
the proximal domain may be 5'-AAGGCUAGUCCG-3', or a base sequence
having partial, that is, at least 50% or more homology with
5'-AAGGCUAGUCCG-3'. Here, the proximal domain may further include
(X)n, resulting in 5'-AAGGCUAGUCCG(X)n-3'. The X may be selected
from the group consisting of bases A, T, U and G, and the n may
represent the number of bases, which is an integer of 1 to 15.
Here, the (X)n may represent n repeats of the same base, or a
mixture of n bases of A, T, U and G.
[0457] In yet another example, when the proximal domain is a
proximal domain of Campylobacter jejuni or a proximal domain
derived therefrom, the proximal domain may be 5'-AAAGAGUUUGC-3', or
a base sequence having at least 50% or more homology with
5'-AAAGAGUUUGC-3'. Here, the proximal domain may further include
(X)n, resulting in 5'-AAAGAGUUUGC(X)n-3'. The X may be selected
from the group consisting of bases A, T, U and G, and the n may
represent the number of bases, which is an integer of 1 to 40.
Here, the (X)n may represent n repeats of the same base, or a
mixture of n bases of A, T, U and G.
[0458] Selectively, a part or all of the base sequence of the
proximal domain may have a chemical modification. The chemical
modification may be methylation, acetylation, phosphorylation,
phosphorothioate linkage, a locked nucleic acid (LNA), 2'-O-methyl
3'phosphorothioate (MS) or 2'-O-methyl 3'thioPACE (MSP), but the
present invention is not limited thereto.
[0459] vi) Tail Domain
[0460] The tail domain is a domain which is able to be selectively
added to the 3' end of single-stranded gRNA or double-stranded
gRNA. The tail domain may be a 1 to 50-base sequence, or include a
1 to 50-base sequence. Here, the tail domain may be used to form a
double strand between complementary base sequences therein.
[0461] In an exemplary embodiment, the tail domain may be a 1 to 5,
5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to
40, 40 to 45, or 45 to 50-base sequence.
[0462] In an exemplary embodiment, the tail domain may include a 1
to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35
to 40, 40 to 45, or 45 to 50-base sequence.
[0463] In addition, the tail domain may have homology with a
natural tail domain, or may be derived from the natural tail
domain. In addition, the tail domain may have a difference in base
sequence according to a species existing in nature, may be derived
from a tail domain contained in a species existing in nature, or
may have partial or complete homology with a tail domain contained
in a species existing in nature.
[0464] In one exemplary embodiment, the tail domain may have
partial, that is, at least 50% or more, or complete homology with a
tail domain of Streptococcus pyogenes, Campylobacter jejuni,
Streptococcus thermophilus, Streptococcus aureus or Neisseria
meningitides or a tail domain derived therefrom.
[0465] For example, when the tail domain is a tail domain of
Streptococcus pyogenes or a tail domain derived therefrom, the tail
domain may be 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3', or a base
sequence having partial, that is, at least 50% or more homology
with 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3'. Here, the tail
domain may further include (X).sub.n, resulting in
5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(X).sub.n-3'. The X may be
selected from the group consisting of bases A, T, U and G, and the
n may represent the number of bases, which is an integer of 1 to
15. Here, the (X), may represent n repeats of the same base, or a
mixture of n bases such as A, T, U and G.
[0466] In another example, when the tail domain is a tail domain of
Campylobacter jejuni or a tail domain derived therefrom, the tail
domain may be 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3', or a
base sequence having partial, that is, at least 50% or more
homology with 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3'. Here,
the tail domain may further include (X)n, resulting in
5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU(X)n-3'. The X may be
selected from the group consisting of bases A, T, U and G, and the
n may represent the number of bases, which is an integer of 1 to
15. Here, the (X)n may represent n repeats of the same base, or a
mixture of n bases of A, T, U and G.
[0467] In another embodiment, the tail domain may include a 1 to
10-base sequence at the 3' end involved in an in vitro or in vivo
transcription method.
[0468] For example, when a T7 promoter is used in in vitro
transcription of gRNA, the tail domain may be an arbitrary base
sequence present at the 3' end of a DNA template. In addition, when
a U6 promoter is used in in vivo transcription, the tail domain may
be UUUUUU, when an H1 promoter is used in transcription, the tail
domain may be UUUU, and when a pol-III promoter is used, the tail
domain may include several uracil bases or alternative bases.
[0469] Selectively, a part or all of the base sequence of the tail
domain may have a chemical modification. The chemical modification
may be methylation, acetylation, phosphorylation, phosphorothioate
linkage, a locked nucleic acid (LNA), 2'-O-methyl
3'phosphorothioate (MS) or 2'-O-methyl 3'thioPACE (MSP), but the
present invention is not limited thereto.
[0470] The gRNA may include a plurality of domains as described
above, and therefore, the length of the nucleic acid sequence may
be regulated according to a domain contained in the gRNA, and
interactions may occur in strands in a three-dimensional structure
or active form of gRNA or between theses strands due to each
domain.
[0471] The gRNA may be referred to as single-stranded gRNA (single
RNA molecule); or double-stranded gRNA (including more than one,
generally two discrete RNA molecules).
[0472] Double-Stranded gRNA
[0473] The double-stranded gRNA consists of a first strand and a
second strand.
[0474] Here, the first strand may consist of 5'-[guide
domain]-[first complementary domain]-3', and the second strand may
consist of 5'-[second complementary domain]-[proximal domain]-3' or
5'-[second complementary domain]-[proximal domain]-[tail
domain]-3'.
[0475] Here, the first strand may be referred to as crRNA, and the
second strand may be referred to as tracrRNA.
[0476] First Strand
[0477] Guide Domain
[0478] In the first strand, the guide domain includes a
complementary guide sequence which is able to form a complementary
bond with a target sequence on a target gene or nucleic acid. The
guide sequence is a nucleic acid sequence complementary to the
target sequence on the target gene or nucleic acid, which has, for
example, at least 70%, 75%, 80%, 85%, 90% or 95% or more
complementarity or complete complementarity. The guide domain is
considered to allow a gRNA-Cas complex, that is, a CRISPR complex
to specifically interact with the target gene or nucleic acid.
[0479] Here, the guide domain may be a 5 to 50-base sequence, or
includes a 5 to 50-base sequence. For example, the guide domain may
be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base
sequence.
[0480] In addition, the guide domain may include a guide
sequence.
[0481] Here, the guide sequence may be a complementary base
sequence which is able to form a complementary bond with a target
sequence on a target gene or nucleic acid, which has, for example,
at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or
complete complementarity.
[0482] In an exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target gene, that is, a
target sequence of a neovascularization-associated factor such as a
VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene, or an
ANGPTL4 gene, which has, for example, at least 70%, 75%, 80%, 85%,
90% or 95% or more complementarity or complete complementarity.
[0483] Here, the guide sequence may be a 5 to 50-base sequence or
include a 5 to 50-base sequence. For example, the guide sequence
may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base
sequence.
[0484] In one exemplary embodiment, the guide sequence is a nucleic
acid sequence complementary to a target sequence of the VEGFA gene.
The guide sequence may be or include a 5 to 50-base sequence. For
example, the guide sequence may be or include a 16, 17, 18, 19, 20,
21, 22, 23, 24 or 25-base sequence.
[0485] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target sequence of the
HIF1A gene. The guide sequence may be or include a 5 to 50-base
sequence. For example, the guide sequence may be or include a 16,
17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
[0486] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target sequence of the
ANGPT2 gene. The guide sequence may be or include a 5 to 50-base
sequence. For example, the guide sequence may be or include a 16,
17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
[0487] In one exemplary embodiment, the guide sequence is a nucleic
acid sequence complementary to a target sequence of the EPAS1 gene.
The guide sequence may be or include a 5 to 50-base sequence. For
example, the guide sequence may be or include a 16, 17, 18, 19, 20,
21, 22, 23, 24 or 25-base sequence.
[0488] In one exemplary embodiment, the guide sequence is a nucleic
acid sequence complementary to a target sequence of the ANGPTL4
gene. The guide sequence may be or include a 5 to 50-base sequence.
For example, the guide sequence may be or include a 16, 17, 18, 19,
20, 21, 22, 23, 24 or 25-base sequence.
[0489] Here, for the guide sequence, target genes, that is, target
sequences of neovascularization-associated factors such as a VEGFA
gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene, and an ANGPTL4
gene are listed above in Table 1, Table 2, Table 3, Table 4 and
Table 5, but the present invention is not limited thereto.
[0490] Selectively, the guide domain may include a guide sequence
and an additional base sequence.
[0491] Here, the additional base sequence may be a 1 to 35-base
sequence. For example, the additional base sequence may be a 1, 2,
3, 4, 5, 6, 7, 8, 9 or 10-base sequence.
[0492] In one exemplary embodiment, the additional base sequence
may include one base, guanine (G), or two bases, GG.
[0493] Here, the additional base sequence may be located at the 5'
end of the guide domain, or at the 5' end of the guide
sequence.
[0494] The additional base sequence may be located at the 3' end of
the guide domain, or at the 3' end of the guide sequence.
[0495] First Complementary Domain
[0496] The first complementary domain includes a nucleic acid
sequence complementary to a second complementary domain of the
second strand, and is a domain having enough complementarity so as
to form a double strand with the second complementary domain.
[0497] Here, the first complementary domain may be or include a 5
to 35-base sequence. For example, the first complementary domain
may be or include a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
[0498] The first complementary domain may have homology with a
natural first complementary domain, or may be derived from a
natural first complementary domain. In addition, the first
complementary domain may have a difference in base sequence
according to a species existing in nature, may be derived from the
first complementary domain contained in the species existing in
nature, or may have partial or complete homology with the first
complementary domain contained in the species existing in
nature.
[0499] In one exemplary embodiment, the first complementary domain
may have partial, that is, at least 50% or more, or complete
homology with a first complementary domain of Streptococcus
pyogenes, Campylobacter jejuni, Streptococcus thermophilus,
Streptococcus aureus or Neisseria meningitides, or a first
complementary domain derived therefrom.
[0500] Selectively, the first complementary domain may include an
additional base sequence which does not undergo complementary
bonding with the second complementary domain of the second
strand.
[0501] Here, the additional base sequence may be a sequence of 1 to
15 bases. For example, the additional base sequence may be a
sequence of 1 to 5, 5 to 10, or 10 to 15 bases.
[0502] Selectively, a part or all of the base sequence of the guide
domain and/or first complementary domain may have a chemical
modification. The chemical modification may be methylation,
acetylation, phosphorylation, phosphorothioate linkage, a locked
nucleic acid (LNA), 2'-O-methyl 3' phosphorothioate (MS) or
2'-O-methyl 3' thioPACE (MSP), but the present invention is not
limited thereto.
[0503] Therefore, the first strand may consist of 5'-[guide
domain]-[first complementary domain]-3' as described above.
[0504] In addition, the first strand may optionally include an
additional base sequence.
[0505] In one example, the first strand may be
5'-(N.sub.target)-(Q).sub.m-3'; or
5'-(X).sub.a-(N.sub.target)-(X).sub.b-(Q).sub.m-(X).sub.c-3'.
[0506] Here, the Ntarget is a base sequence capable of forming a
complementary bond with a target sequence on a target gene or
nucleic acid, and a base sequence region which may be changed
according to a target sequence on a target gene or nucleic
acid.
[0507] In one exemplary embodiment, Ntarget may be a base sequence
capable of forming a complementary bond with a target gene, that
is, a target sequence of a neovascularization-associated factor
such as a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene
or an ANGPTL4 gene.
[0508] Here, the (Q)m is a base sequence including the first
complementary domain, which is able to form a complementary bond
with the second complementary domain of the second strand. The (Q)m
may be a sequence having partial or complete homology with the
first complementary domain of a species existing in nature, and the
base sequence of the first complementary domain may be changed
according to the species of origin. The Q may be each independently
selected from the group consisting of A, U, C and G, and the m may
be the number of bases, which is an integer of 5 to 35.
[0509] For example, when the first complementary domain has partial
or complete homology with a first complementary domain of
Streptococcus pyogenes or a Streptococcus pyogenes-derived first
complementary domain, the (Q)m may be 5'-GUUUUAGAGCUA-3', or a base
sequence having at least 50% or more homology with
5'-GUUUUAGAGCUA-3'.
[0510] In another example, when the first complementary domain has
partial or complete homology with a first complementary domain of
Campylobacter jejuni or a Campylobacter jejuni-derived first
complementary domain, the (Q)m may be
5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3', or a base sequence having at least
50% or more homology with 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3'.
[0511] In still another example, when the first complementary
domain has partial or complete homology with a first complementary
domain of Streptococcus thermophilus or a Streptococcus
thermophilus-derived first complementary domain, the (Q)m may be
5'-GUUUUAGAGCUGUGUUGUUUCG-3', or a base sequence having at least
50% or more homology with 5'-GUUUUAGAGCUGUGUUGUUUCG-3'.
[0512] In addition, each of the (X)a, (X)b and (X)c is selectively
an additional base sequence, where the X may be each independently
selected from the group consisting of A, U, C and G, and each of
the a, b and c may be the number of bases, which is 0 or an integer
of 1 to 20.
[0513] Second Strand
[0514] The second strand may consist of a second complementary
domain and a proximal domain, and selectively include a tail
domain.
[0515] Second Complementary Domain
[0516] In the second strand, the second complementary domain
includes a nucleic acid sequence complementary to the first
complementary domain of the first strand, and has enough
complementarity so as to form a double strand with the first
complementary domain. The second complementary domain may include a
base sequence complementary to the first complementary domain and a
base sequence not complementary to the first complementary domain,
for example, a base sequence not forming a double strand with the
first complementary domain, and may have a longer base sequence
than the first complementary domain.
[0517] Here, the second complementary domain may be a 5 to 35-base
sequence, or include a 5 to 35-base sequence. For example, the
second complementary domain may be or include a 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base
sequence, but the present invention is not limited thereto.
[0518] The second complementary domain may have homology with a
natural second complementary domain, or may be derived from a
natural second complementary domain. In addition, the second
complementary domain may have a difference in base sequence thereof
according to a species existing in nature, may be derived from a
second complementary domain contained in the species existing in
nature, or may have partial or complete homology with the second
complementary domain contained in the species existing in
nature.
[0519] In one exemplary embodiment, the second complementary domain
may have partial, that is, at least 50% or more, or complete
homology with a second complementary domain of Streptococcus
pyogenes, Campylobacter jejuni, Streptococcus thermophilus,
Streptococcus aureus or Neisseria meningitides, or a second
complementary domain derived therefrom.
[0520] Selectively, the second complementary domain may further
include an additional base sequence which does not undergo
complementary bonding with the first complementary domain of the
first strand.
[0521] Here, the additional base sequence may be a 1 to 25-base
sequence. For example, the additional base sequence may be a 1 to
5, 5 to 10, 10 to 15, 15 to 20 or 20 to 25-base sequence.
[0522] Proximal Domain
[0523] In the second strand, the proximal domain is a sequence of 1
to 20 bases, and a domain located at the 3' end direction of the
second complementary domain. For example, the proximal domain may
be or include a sequence of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15
bases.
[0524] Here, the proximal domain may have a double strand bond
between complementary base sequences therein.
[0525] In addition, the proximal domain may have homology with a
natural proximal domain, or may be derived from a natural proximal
domain. In addition, the proximal domain may have a difference in
base sequence according to a species existing in nature, may be
derived from a proximal domain of a species existing in nature, or
may have partial or complete homology with the proximal domain of a
species existing in nature.
[0526] In one exemplary embodiment, the proximal domain may have
partial, that is, at least 50% or more, or complete homology with a
proximal domain of Streptococcus pyogenes, Campylobacter jejuni,
Streptococcus thermophilus, Streptococcus aureus or Neisseria
meningitides, or a proximal domain derived therefrom.
[0527] Tail Domain
[0528] Selectively, in the second strand, the tail domain may be a
domain selectively added to the 3' end of the second strand, and
the tail domain may be or include a 1 to 50-base sequence. For
example, the tail domain may be or include a 1 to 5, 5 to 10, 10 to
15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45 or
45 to 50-base sequence.
[0529] Here, the tail domain may have a double strand bond between
complementary base sequences therein.
[0530] In addition, the tail domain may have homology with a
natural tail domain, or may be derived from a natural tail domain.
In addition, the tail domain may have a difference in base sequence
according to a species existing in nature, may be derived from a
tail domain contained in the species existing in nature, or may
have partial or complete homology with the tail domain contained in
the species existing in nature.
[0531] In one exemplary embodiment, the tail domain may have
partial, that is, at least 50% or more, or complete homology with a
tail domain of Streptococcus pyogenes, Campylobacter jejuni,
Streptococcus thermophilus, Streptococcus aureus or Neisseria
meningitides, or a tail domain derived therefrom.
[0532] In another embodiment, the tail domain may include a
sequence of 1 to 10 bases at the 3' end involved in an in vitro or
in vivo transcription method.
[0533] For example, when a T7 promoter is used in in vitro
transcription of gRNA, the tail domain may be an arbitrary base
sequence present at the 3' end of a DNA template. In addition, when
a U6 promoter is used in in vivo transcription, the tail domain may
be UUUUUU, when an H1 promoter is used in transcription, the tail
domain may be UUUU, and when a pol-III promoter is used, the tail
domain may include several uracil bases or alternative bases.
[0534] Selectively, a part or all of each of the base sequence of
the second complementary domain, the proximal domain and/or the
tail domain may have a chemical modification. The chemical
modification may be methylation, acetylation, phosphorylation,
phosphorothioate linkage, a locked nucleic acid (LNA), 2'-O-methyl
3'phosphorothioate (MS) or 2'-O-methyl 3'thioPACE (MSP), but the
present invention is not limited thereto.
[0535] Therefore, the second strand may consist of 5'-[second
complementary domain]-[proximal domain]-3' or 5'-[second
complementary domain]-[proximal domain]-[tail domain]-3' as
described above.
[0536] In addition, the second strand may selectively include an
additional base sequence.
[0537] In one exemplary embodiment, the second strand may be
5'-(Z)h-(P)k-3'; or 5'-(X)d-(Z)h-(X)e-(P)k-(X)f-3'.
[0538] In another embodiment, the second strand may be
5'-(Z)h-(P)k-(F)i-3'; or 5'-(X)d-(Z)h-(X)e-(P)k-(X)f-(F)i-3'.
[0539] Here, the (Z)h is a base sequence including a second
complementary domain, which is able to form a complementary bond
with the first complementary domain of the first strand. The (Z)h
may be a sequence having partial or complete homology with the
second complementary domain of a species existing in nature, and
the base sequence of the second complementary domain may be
modified according to the species of origin. The Z may be each
independently selected from the group consisting of A, U, C and G,
and the h may be the number of bases, which is an integer of 5 to
50.
[0540] For example, when the second complementary domain has
partial or complete homology with a second complementary domain of
Streptococcus pyogenes or a second complementary domain derived
therefrom, the (Z)h may be 5'-UAGCAAGUUAAAAU-3', or a base sequence
having at least 50% or more homology with 5'-UAGCAAGUUAAAAU-3'.
[0541] In another example, when the second complementary domain has
partial or complete homology with a second complementary domain of
Campylobacter jejuni or a second complementary domain derived
therefrom, the (Z)h may be 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3', or a
base sequence having at least 50% or more homology with
5'-AAGAAAUUUAAAAAGGGACUAAAAU-3'.
[0542] In still another example, when the second complementary
domain has partial or complete homology with a second complementary
domain of Streptococcus thermophilus or a second complementary
domain derived therefrom, the (Z)h may be
5'-CGAAACAACACAGCGAGUUAAAAU-3', or a base sequence having at least
50% or more homology with 5'-CGAAACAACACAGCGAGUUAAAAU-3'.
[0543] The (P)k is a base sequence including a proximal domain,
which may have partial or complete homology with a proximal domain
of a species existing in nature, and the base sequence of the
proximal domain may be modified according to the species of origin.
The P may be each independently selected from the group consisting
of A, U, C and G, and the k may be the number of bases, which is an
integer of 1 to 20.
[0544] For example, when the proximal domain has partial or
complete homology with a proximal domain of Streptococcus pyogenes
or a proximal domain derived therefrom, the (P).sub.k may be
5'-AAGGCUAGUCCG-3', or a base sequence having at least 50% or more
homology with 5'-AAGGCUAGUCCG-3'.
[0545] In another example, when the proximal domain has partial or
complete homology with a proximal domain of Campylobacter jejuni or
a proximal domain derived therefrom, the (P).sub.k may be
5'-AAAGAGUUUGC-3', or a base sequence having at least 50% or more
homology with 5'-AAAGAGUUUGC-3'.
[0546] In still another example, when the proximal domain has
partial or complete homology with a proximal domain of
Streptococcus thermophilus or a proximal domain derived therefrom,
the (P)k may be 5'-AAGGCUUAGUCCG-3', or a base sequence having at
least 50% or more homology with 5'-AAGGCUUAGUCCG-3'.
[0547] The (F)i may be a base sequence including a tail domain, and
having partial or complete homology with a tail domain of a species
existing in nature, and the base sequence of the tail domain may be
modified according to the species of origin. The F may be each
independently selected from the group consisting of A, U, C and G,
and the i may be the number of bases, which is an integer of 1 to
50.
[0548] For example, when the tail domain has partial or complete
homology with a tail domain of Streptococcus pyogenes or a tail
domain derived therefrom, the (F)i may be
5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3', or a base sequence having
at least 50% or more homology with
5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3'.
[0549] In another example, when the tail domain has partial or
complete homology with a tail domain of Campylobacter jejuni or a
tail domain derived therefrom, the (F)i may be
5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3', or a base sequence
having at least 50% or more homology with
5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3'.
[0550] In still another example, when the tail domain has partial
or complete homology with a tail domain of Streptococcus
thermophilus or a tail domain derived therefrom, the (F)i may be
5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3', or a base sequence
having at least 50% or more homology with
5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3'.
[0551] In addition, the (F)i may include a sequence of 1 to 10
bases at the 3' end involved in an in vitro or in vivo
transcription method.
[0552] For example, when a T7 promoter is used in in vitro
transcription of gRNA, the tail domain may be an arbitrary base
sequence present at the 3' end of a DNA template. In addition, when
a U6 promoter is used in in vivo transcription, the tail domain may
be UUUUUU, when an H1 promoter is used in transcription, the tail
domain may be UUUU, and when a pol-III promoter is used, the tail
domain may include several uracil bases or alternative bases.
[0553] In addition, the (X)d, (X)e and (X)f may be base sequences
selectively added, where the X may be each independently selected
from the group consisting of A, U, C and G, and each of the d, e
and f may be the number of bases, which is 0 or an integer of 1 to
20.
[0554] Single-Stranded gRNA
[0555] Single-stranded gRNA may be classified into two types.
[0556] i) Single-Stranded gRNA
[0557] First, there is single-stranded gRNA in which a first strand
or a second strand of the double-stranded gRNA is linked by a
linker domain, and here, the single-stranded gRNA consists of
5'-[first strand]-[linker domain]-[second strand]-3'.
[0558] Specifically, the single-stranded gRNA may consist of
5'-[guide domain]-[first complementary domain]-[linker
domain]-[second complementary domain]-[proximal domain]-3' or
5'-[guide domain]-[first complementary domain]-[linker
domain]-[second complementary domain]-[proximal domain]-[tail
domain]-3'.
[0559] Each domain except the linker domain is the same as the
description of each domain of the first and second strands of the
double-stranded gRNA.
[0560] Linker Domain
[0561] In the single-stranded gRNA, the linker domain is a domain
connecting a first strand and a second strand, and specifically, is
a nucleic acid sequence which connects a first complementary domain
with a second complementary domain to produce single-stranded gRNA.
Here, the linker domain may be connected with the first
complementary domain and the second complementary domain or connect
the first complementary domain with the second complementary domain
by covalent or non-covalent bonding.
[0562] The linker domain may be or include a 1 to 30-base sequence.
For example, the linker domain may be or include a 1 to 5, 5 to 10,
10 to 15, 15 to 20, 20 to 25 or 25 to 30-base sequence.
[0563] The linker domain is suitable to be used in a
single-stranded gRNA molecule, and may be connected with the first
strand and the second strand of the double-stranded gRNA, or
connect the first strand with the second strand by covalent or
non-covalent bonding to be used in production of the
single-stranded gRNA. The linker domain may be connected with crRNA
and tracrRNA of the double-stranded gRNA, or connect crRNA with
tracrRNA by covalent or non-covalent bonding to be used in
production of the single-stranded gRNA.
[0564] The linker domain may have homology with a natural sequence,
for example, a partial sequence of tracrRNA, or may be derived
therefrom.
[0565] Selectively, a part or all of the base sequence of the
linker domain may have a chemical modification. The chemical
modification may be methylation, acetylation, phosphorylation,
phosphorothioate linkage, a locked nucleic acid (LNA), 2'-O-methyl
3'phosphorothioate (MS) or 2'-O-methyl 3'thioPACE (MSP), but the
present invention is not limited thereto.
[0566] Therefore, the single-stranded gRNA may consist of 5'-[guide
domain]-[first complementary domain]-[linker domain]-[second
complementary domain]-[proximal domain]-3' or 5'-[guide
domain]-[first complementary domain]-[linker domain]-[second
complementary domain]-[proximal domain]-[tail domain]-3' as
described above.
[0567] In addition, the single-stranded gRNA may selectively
include an additional base sequence.
[0568] In one exemplary embodiment, the single-stranded gRNA may
be
[0569]
5'-(N.sub.target)-(Q).sub.m-(L).sub.j-(Z).sub.h-(P).sub.k-3';
or
[0570]
5'-(N.sub.target)-(Q).sub.m-(L).sub.j-(Z).sub.h-(P).sub.k-(F).sub.i-
-3'.
[0571] In another embodiment, the single-stranded gRNA may be
[0572]
5'-(X).sub.a-(N.sub.target)-(X).sub.b-(Q).sub.m-(X).sub.c-(L).sub.j-
-(X).sub.d-(Z).sub.h-(X).sub.e-(P).sub.k-(X).sub.f-3'; or
[0573]
5'-(X).sub.a-(N.sub.target)-(X).sub.b-(Q).sub.m-(X).sub.c-(L).sub.j-
-(X).sub.d-(Z).sub.h-(X).sub.e-(P).sub.k-(X).sub.f-(F).sub.i-3'.
[0574] Here, the N.sub.target is a base sequence capable of forming
a complementary bond with a target sequence on a target gene or
nucleic acid, and a base sequence region capable of being changed
according to a target sequence on a target gene or nucleic
acid.
[0575] In one exemplary embodiment, Ntarget is a base sequence
capable of forming a complementary bond with a target gene, that
is, a target sequence of a neovascularization-associated factor
such as a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene,
or an ANGPTL4 gene.
[0576] The (Q)m includes a base sequence including the first
complementary domain, which is able to form a complementary bond
with a second complementary domain. The (Q)m may be a sequence
having partial or complete homology with a first complementary
domain of a species existing in nature, and the base sequence of
the first complementary domain may be changed according to the
species of origin. The Q may be each independently selected from
the group consisting of A, U, C and G, and the m may be the number
of bases, which is an integer of 5 to 35.
[0577] For example, when the first complementary domain has partial
or complete homology with a first complementary domain of
Streptococcus pyogenes or a first complementary domain derived
therefrom, the (Q)m may be 5'-GUUUUAGAGCUA-3', or a base sequence
having at least 50% or more homology with 5'-GUUUUAGAGCUA-3'.
[0578] In another example, when the first complementary domain has
partial or complete homology with a first complementary domain of
Campylobacter jejuni or a first complementary domain derived
therefrom, the (Q)m may be 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3', or a
base sequence having at least 50% or more homology with
5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3'.
[0579] In still another example, when the first complementary
domain has partial or complete homology with a first complementary
domain of Streptococcus thermophilus or a first complementary
domain derived therefrom, the (Q)m may be
5'-GUUUUAGAGCUGUGUUGUUUCG-3', or a base sequence having at least
50% or more homology with 5'-GUUUUAGAGCUGUGUUGUUUCG-3'.
[0580] In addition, the (L)j is a base sequence including the
linker domain, and connecting the first complementary domain with
the second complementary domain, thereby producing single-stranded
gRNA. Here, the L may be each independently selected from the group
consisting of A, U, C and G, and the j may be the number of bases,
which is an integer of 1 to 30.
[0581] The (Z)h is a base sequence including the second
complementary domain, which is able to have a complementary bond
with the first complementary domain. The (Z)h may be a sequence
having partial or complete homology with the second complementary
domain of a species existing in nature, and the base sequence of
the second complementary domain may be changed according to the
species of origin. The Z may be each independently selected from
the group consisting of A, U, C and G, and the h is the number of
bases, which may be an integer of 5 to 50.
[0582] For example, when the second complementary domain has
partial or complete homology with a second complementary domain of
Streptococcus pyogenes or a second complementary domain derived
therefrom, the (Z)h may be 5'-UAGCAAGUUAAAAU-3', or a base sequence
having at least 50% or more homology with 5'-UAGCAAGUUAAAAU-3'.
[0583] In another example, when the second complementary domain has
partial or complete homology with a second complementary domain of
Campylobacter jejuni or a second complementary domain derived
therefrom, the (Z)h may be 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3', or a
base sequence having at least 50% or more homology with
5'-AAGAAAUUUAAAAAGGGACUAAAAU-3'.
[0584] In still another example, when the second complementary
domain has partial or complete homology with a second complementary
domain of Streptococcus thermophilus or a second complementary
domain derived therefrom, the (Z)h may be
5'-CGAAACAACACAGCGAGUUAAAAU-3', or a base sequence having at least
50% or more homology with 5'-CGAAACAACACAGCGAGUUAAAAU-3'.
[0585] The (P)k is a base sequence including a proximal domain,
which may have partial or complete homology with a proximal domain
of a species existing in nature, and the base sequence of the
proximal domain may be modified according to the species of origin.
The P may be each independently selected from the group consisting
of A, U, C and G, and the k may be the number of bases, which is an
integer of 1 to 20.
[0586] For example, when the proximal domain has partial or
complete homology with a proximal domain of Streptococcus pyogenes
or a proximal domain derived therefrom, the (P)k may be
5'-AAGGCUAGUCCG-3', or a base sequence having at least 50% or more
homology with 5'-AAGGCUAGUCCG-3'.
[0587] In another example, when the proximal domain has partial or
complete homology with a proximal domain of Campylobacter jejuni or
a proximal domain derived therefrom, the (P)k may be
5'-AAAGAGUUUGC-3', or a base sequence having at least 50% or more
homology with 5'-AAAGAGUUUGC-3'.
[0588] In still another example, when the proximal domain has
partial or complete homology with a proximal domain of
Streptococcus thermophilus or a proximal domain derived therefrom,
the (P)k may be 5'-AAGGCUUAGUCCG-3', or a base sequence having at
least 50% or more homology with 5'-AAGGCUUAGUCCG-3'.
[0589] The (F).sub.i may be a base sequence including a tail
domain, and having partial or complete homology with a tail domain
of a species existing in nature, and the base sequence of the tail
domain may be modified according to the species of origin. The F
may be each independently selected from the group consisting of A,
U, C and G, and the i may be the number of bases, which is an
integer of 1 to 50.
[0590] For example, when the tail domain has partial or complete
homology with a tail domain of Streptococcus pyogenes or a tail
domain derived therefrom, the (F)i may be
5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3', or a base sequence having
at least 50% or more homology with
5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3'
[0591] In another example, when the tail domain has partial or
complete homology with a tail domain of Campylobacter jejuni or a
tail domain derived therefrom, the (F), may be
5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3', or a base sequence
having at least 50% or more homology with
5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3'.
[0592] In still another example, when the tail domain has partial
or complete homology with a tail domain of Streptococcus
thermophilus or a tail domain derived therefrom, the (F), may be
5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3', or a base sequence
having at least 50% or more homology with
5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3'.
[0593] In addition, the (F)i may include a sequence of 1 to 10
bases at the 3' end involved in an in vitro or in vivo
transcription method.
[0594] For example, when a T7 promoter is used in in vitro
transcription of gRNA, the tail domain may be an arbitrary base
sequence present at the 3' end of a DNA template. In addition, when
a U6 promoter is used in in vivo transcription, the tail domain may
be UUUUUU, when an H1 promoter is used in transcription, the tail
domain may be UUUU, and when a pol-III promoter is used, the tail
domain may include several uracil bases or alternative bases.
[0595] In addition, the (X)a, (X)b, (X)c, (X)d, (X)e and (X)f may
be base sequences selectively added, where the X may be each
independently selected from the group consisting of A, U, C and G,
and each of the a, b, c, d, e and f may be the number of bases,
which is 0 or an integer of 1 to 20.
[0596] Single-Stranded gRNA
[0597] Second, the single-stranded gRNA may be single-stranded gRNA
consisting of a guide domain, a first complementary domain and a
second complementary domain, and here, the single-stranded gRNA may
consist of: 5'-[second complementary domain]-[first complementary
domain]-[guide domain]-3'; or 5'-[second complementary
domain]-[linker domain]-[first complementary domain]-[guide
domain]-3'.
[0598] Guide Domain
[0599] In the single-stranded gRNA, the guide domain includes a
complementary guide sequence capable of forming a complementary
bond with a target sequence on a target gene or nucleic acid. The
guide sequence may be a nucleic acid sequence having
complementarity to the target sequence on the target gene or
nucleic acid, which has, for example, at least 70%, 75%, 80%, 85%,
90% or 95% or more complementarity or complete complementarity. The
guide domain is considered to allow a gRNA-Cas complex, that is, a
CRISPR complex to specifically interact with the target gene or
nucleic acid.
[0600] Here, the guide domain may be or include a 5 to 50-base
sequence. For example, the guide domain may be or include a 16, 17,
18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
[0601] In addition, the guide domain may include a guide
sequence.
[0602] Here, the guide sequence may be a complementary base
sequence capable of forming a complementary bond with a target
sequence on a target gene or nucleic acid, which has, for example,
at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or
complete complementarity.
[0603] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target gene, that is, a
target sequence of a neovascularization-associated factor such as a
VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene or an
ANGPTL4 gene, which has, for example, at least 70%, 75%, 80%, 85%,
90% or 95% or more complementarity or complete complementarity.
[0604] Here, the guide sequence may be or include a 5 to 50-base
sequence. For example, the guide sequence may be or include a 16,
17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
[0605] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target sequence of the
VEGFA gene. The guide sequence may be or include a 5 to 50-base
sequence. For example, the guide sequence may be or include a 16,
17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
[0606] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target sequence of the
HIF1A gene. The guide sequence may be or include a 5 to 50-base
sequence. For example, the guide sequence may be or include a 16,
17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
[0607] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target sequence of the
ANGPT2 gene. The guide sequence may be or include a 5 to 50-base
sequence. For example, the guide sequence may be or include a 16,
17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
[0608] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target sequence of the
EPAS1 gene. The guide sequence may be or include a 5 to 50-base
sequence. For example, the guide sequence may be or include a 16,
17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
[0609] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target sequence of the
ANGPTL4 gene. The guide sequence may be or include a 5 to 50-base
sequence. For example, the guide sequence may be or include a 16,
17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
[0610] Here, target sequences of the target genes, that is, the
neovascularization-associated factors such as the VEGFA gene, the
HIF1A gene, the ANGPT2 gene, the EPAS1 gene, and the ANGPTL4 gene
for the guide sequence are listed above in Table 1, Table 2, Table
3, Table 4 and Table 5, respectively, but the present invention is
not limited thereto.
[0611] Selectively, the guide domain may include a guide sequence
and an additional base sequence.
[0612] Here, the additional base sequence may be a 1 to 35-base
sequence. For example, the additional base sequence may be a 1, 2,
3, 4, 5, 6, 7, 8, 9 or 10-base sequence.
[0613] In one exemplary embodiment, the additional base sequence
may be a single base sequence, guanine (G), or a sequence of two
bases, GG.
[0614] Here, the additional base sequence may be located at the 5'
end of the guide domain, or at the 5' end of the guide
sequence.
[0615] The additional base sequence may be located at the 3' end of
the guide domain, or at the 3' end of the guide sequence.
[0616] First Complementary Domain
[0617] The first complementary domain is a domain including a
nucleic acid sequence complementary to the second complementary
domain, and having enough complementarity so as to form a double
strand with the second complementary domain.
[0618] Here, the first complementary domain may be or include a 5
to 35-base sequence. For example, the first complementary domain
may be or include a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
[0619] The first complementary domain may have homology with a
natural first complementary domain, or may be derived from a
natural first complementary domain. In addition, the first
complementary domain may have a difference in the base sequence of
a first complementary domain depending on the species existing in
nature, may be derived from a first complementary domain contained
in the species existing in nature, or may have partial or complete
homology with the first complementary domain contained in the
species existing in nature.
[0620] In one exemplary embodiment, the first complementary domain
may have partial, that is, at least 50% or more, or complete
homology with a first complementary domain of Parcubacteria
bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017),
Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium
(GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas
macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas
crevioricanis, Prevotella disiens, Moraxella bovoculi (237),
Smiihella sp. (SC_KO8D17), Leptospira inadai, Lachnospiraceae
bacterium (MA2020), Francisella novicida (U112), Candidatus
Methanoplasma termitum or Eubacterium eligens, or a first
complementary domain derived therefrom.
[0621] Selectively, the first complementary domain may include an
additional base sequence which does not undergo complementary
bonding with the second complementary domain.
[0622] Here, the additional base sequence may be a 1 to 15-base
sequence. For example, the additional base sequence may be a 1 to
5, 5 to 10, or 10 to 15-base sequence.
[0623] Second Complementary Domain
[0624] The second complementary domain includes a nucleic acid
sequence complementary to the first complementary domain, and has
enough complementarity so as to form a double strand with the first
complementary domain. The second complementary domain may include a
base sequence complementary to the first complementary domain, and
a base sequence having no complementarity with the first
complementary domain, for example, a base sequence not forming a
double strand with the first complementary domain, and may have a
longer base sequence than the first complementary domain.
[0625] Here, the second complementary domain may be or include a 5
to 35-base sequence. For example, the second complementary domain
may be a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24 or 25-base sequence.
[0626] The second complementary domain may have homology with a
natural second complementary domain, or may be derived from the
natural second complementary domain. In addition, the second
complementary domain may have a difference in base sequence of the
second complementary domain according to a species existing in
nature, and may be derived from second complementary domain
contained in the species existing in nature, or may have partial or
complete homology with the second complementary domain contained in
the species existing in nature.
[0627] In one exemplary embodiment, the second complementary domain
may have partial, that is, at least 50% or more, or complete
homology with a second complementary domain of Parcubacteria
bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017),
Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium
(GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas
macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas
crevioricanis, Prevotella disiens, Moraxella bovoculi (237),
Smiihella sp. (SC_KO8D17), Leptospira inadai, Lachnospiraceae
bacterium (MA2020), Francisella novicida (U112), Candidatus
Methanoplasma termitum or Eubacterium eligens, or a second
complementary domain derived therefrom.
[0628] Selectively, the second complementary domain may include an
additional base sequence which does not undergo complementary
bonding with the first complementary domain.
[0629] Here, the additional base sequence may be a 1 to 15-base
sequence. For example, the additional base sequence may be a 1 to
5, 5 to 10, or 10 to 15-base sequence.
[0630] Linker Domain
[0631] Selectively, the linker domain is a nucleic acid sequence
connecting a first complementary domain with a second complementary
domain to produce single-stranded gRNA. Here, the linker domain may
be connected with the first complementary domain and the second
complementary domain, or may connect the first and second
complementary domains by covalent or non-covalent bonding.
[0632] The linker domain may be or include a 1 to 30-base sequence.
For example, the linker domain may be or include a 1 to 5, 5 to 10,
10 to 15, 15 to 20, 20 to 25 or 25 to 30-base sequence.
[0633] Selectively, a part or all of the base sequence of the guide
domain, the first complementary domain, the second complementary
domain and the linker domain may have a chemical modification. The
chemical modification may be methylation, acetylation,
phosphorylation, phosphorothioate linkage, a locked nucleic acid
(LNA), 2'-O-methyl 3'phosphorothioate (MS) or 2'-O-methyl
3'thioPACE (MSP), but the present invention is not limited
thereto.
[0634] Therefore, the single-stranded gRNA may consist of
5'-[second complementary domain]-[first complementary
domain]-[guide domain]-3' or 5'-[second complementary
domain]-[linker domain]-[first complementary domain]-[guide
domain]-3' as described above.
[0635] In addition, the single-stranded gRNA may selectively
include an additional base sequence.
[0636] In one exemplary embodiment, the single-stranded gRNA may be
5-(Z).sub.h-(Q).sub.m-(N.sub.target)-3'; or
5'-(X).sub.a-(Z).sub.h-(X).sub.b-(Q).sub.m-(X).sub.c-(N.sub.target)-3'.
In another embodiment, the single-stranded gRNA may be
5'-(Z).sub.h-(L).sub.j-(Q).sub.m-(N.sub.target)-3'; or
5-(X).sub.a-(Z).sub.h-(L).sub.j-(Q).sub.m-(X).sub.c-(N.sub.target)-3'.
[0637] Here, the N.sub.target is a base sequence capable of forming
a complementary bond with a target sequence on a target gene or
nucleic acid, and a base sequence region which may be changed
according to a target sequence on a target gene or nucleic
acid.
[0638] In one exemplary embodiment, Ntarget may be a base sequence
capable of forming a complementary bond with a target gene, that
is, a target sequence of a neovascularization-associated factor
such as a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene
or an ANGPTL4 gene.
[0639] The (Q)m is a base sequence including the first
complementary domain, which is able to form a complementary bond
with the second complementary domain of the second strand. The (Q)m
may be a sequence having partial or complete homology with the
first complementary domain of a species existing in nature, and the
base sequence of the first complementary domain may be changed
according to the species of origin. The Q may be each independently
selected from the group consisting of A, U, C and G, and the m may
be the number of bases, which is an integer of 5 to 35.
[0640] For example, when the first complementary domain has partial
or complete homology with a first complementary domain of
Parcubacteria bacterium or a first complementary domain derived
therefrom, the (Q)m may be 5'-UUUGUAGAU-3', or a base sequence
having at least 50% or more homology with 5'-UUUGUAGAU-3'.
[0641] The (Z)h is a base sequence including a second complementary
domain, which is able to form a complementary bond with the first
complementary domain of the first strand. The (Z)h may be a
sequence having partial or complete homology with the second
complementary domain of a species existing in nature, and the base
sequence of the second complementary domain may be modified
according to the species of origin. The Z may be each independently
selected from the group consisting of A, U, C and G, and the h may
be the number of bases, which is an integer of 5 to 50.
[0642] For example, when the second complementary domain has
partial or complete homology with a second complementary domain of
Parcubacteria bacterium or a Parcubacteria bacterium-derived second
complementary domain, the (Z)h may be 5'-AAAUUUCUACU-3', or a base
sequence having at least 50% or more homology with
5'-AAAUUUCUACU-3'.
[0643] In addition, the (L)j is a base sequence including the
linker domain, which connects the first complementary domain with
the second complementary domain. Here, the L may be each
independently selected from the group consisting of A, U, C and G,
and the j may be the number of bases, which is an integer of 1 to
30.
[0644] In addition, each of the (X)a, (X)b and (X)c is selectively
an additional base sequence, where the X may be each independently
selected from the group consisting of A, U, C and G, and the a, b
and c may be the number of bases, which is 0 or an integer of 1 to
20.
[0645] 2. Editor Protein
[0646] An editor protein refers to a peptide, polypeptide or
protein which is able to directly bind to or interact with, without
direct binding to, a nucleic acid.
[0647] The nucleic acid may be a nucleic acid contained in a target
nucleic acid, gene or chromosome.
[0648] The nucleic acid may be a guide nucleic acid.
[0649] The editor protein may be an enzyme.
[0650] The editor protein may be a fusion protein.
[0651] Here, the fusion protein refers to a protein produced by
fusing an enzyme with an additional domain, peptide, polypeptide or
protein.
[0652] The enzyme refers to a protein including a domain which is
able to cleave a nucleic acid, gene, chromosome or protein.
[0653] The enzyme may be a nuclease, protease or restriction
enzyme.
[0654] The additional domain, peptide, polypeptide or protein may
be a functional domain, peptide, polypeptide or protein, which has
a function the same as or different from the enzyme.
[0655] The fusion protein may include an additional domain,
peptide, polypeptide or protein at one or more of an N-terminus of
an enzyme or the proximity thereof; a C-terminus of the enzyme or
the proximity thereof; the middle region of an enzyme; and a
combination thereof.
[0656] The fusion protein may include a functional domain, peptide,
polypeptide or protein at one or more of an N-terminus of an enzyme
or the proximity thereof; a C-terminus of the enzyme or the
proximity thereof; the middle region of an enzyme; and a
combination thereof.
[0657] Here, the functional domain, peptide, polypeptide or protein
may be a domain, peptide, polypeptide or protein having methylase
activity, demethylase activity, transcription activation activity,
transcription repression activity, transcription release factor
activity, histone modification activity, RNA cleavage activity or
nucleic acid binding activity, or a tag or reporter gene for
isolation and purification of a protein (including a peptide), but
the present invention is not limited thereto.
[0658] The functional domain, peptide, polypeptide or protein may
be a deaminase.
[0659] The tag includes a histidine (His) tag, a V5 tag, a FLAG
tag, an influenza hemagglutinin (HA) tag, a Myc tag, a VSV-G tag
and a thioredoxin (Trx) tag, and the reporter gene includes
glutathione-S-transferase (GST), horseradish peroxidase (HRP),
chloramphenicol acetyltransferase (CAT) .beta.-galactosidase,
.beta.-glucoronidase, luciferase, autofluorescent proteins
including the green fluorescent protein (GFP), HcRed, DsRed, cyan
fluorescent protein (CFP), yellow fluorescent protein (YFP) and
blue fluorescent protein (BFP), but the present invention is not
limited thereto.
[0660] In addition, the functional domain, peptide, polypeptide or
protein may be a nuclear localization sequence or signal (NLS) or a
nuclear export sequence or signal (NES).
[0661] The NLS may be NLS of SV40 virus large T-antigen with an
amino acid sequence PKKKRKV; NLS derived from nucleoplasmin (e.g.,
nucleoplasmin bipartite NLS with a sequence KRPAATKKAGQAKKKK);
c-myc NLS with an amino acid sequence PAAKRVKLD or RQRRNELKRSP;
hRNPA1 M9 NLS with a sequence
NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; an importin-.alpha.-derived
IBB domain sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV;
myoma T protein sequences VSRKRPRP and PPKKARED; human p53 sequence
POPKKKPL; a mouse c-abl IV sequence SALIKKKKKMAP; influenza virus
NS1 sequences DRLRR and PKQKKRK; a hepatitis virus-.delta. antigen
sequence RKLKKKIKKL; a mouse Mx1 protein sequence REKKKFLKRR; a
human poly(ADP-ribose) polymerase sequence KRKGDEVDGVDEVAKKKSKK; or
steroid hormone receptor (human) glucocorticoid sequence
RKCLQAGMNLEARKTKK, but the present invention is not limited
thereto.
[0662] The editor protein may include a complete active enzyme.
[0663] Here, the "complete active enzyme" refers to an enzyme
having the same function as a function of a wild-type enzyme, and
for example, the wild-type enzyme cleaving the double strand of DNA
has complete enzyme activity of entirely cleaving the double strand
of DNA.
[0664] In addition, the complete active enzyme includes an enzyme
having an improved function compared to the function of the
wild-type enzyme, and for example, a specific modification or
manipulation type of the wild-type enzyme cleaving the double
strand of DNA has full enzyme activity which is improved compared
to the wild-type enzyme, that is, activity of cleaving the double
strand of DNA.
[0665] The editor protein may include an incomplete or partially
active enzyme.
[0666] Here, the "incomplete or partially active enzyme" refers to
an enzyme having some of the functions of the wild-type enzyme, and
for example, a specific modification or manipulation type of the
wild-type enzyme cleaving the double strand of DNA has incomplete
or partial enzyme activity of cleaving a part of the double strand,
that is, a single strand of DNA.
[0667] The editor protein may include an inactive enzyme.
[0668] Here, the "inactive enzyme" refers to an enzyme in which the
function of a wild-type enzyme is completely inactivated. For
example, a specific modification or manipulation type of the
wild-type enzyme cleaving the double strand of DNA has inactivity
so as not to completely cleave the DNA double strand.
[0669] The editor protein may be a natural enzyme or fusion
protein.
[0670] The editor protein may be present in the form of a partially
modified natural enzyme or fusion protein.
[0671] The editor protein may be an artificially produced enzyme or
fusion protein, which does not exist in nature.
[0672] The editor protein may be present in the form of a partially
modified artificial enzyme or fusion protein, which does not exist
in nature.
[0673] Here, the modification may be substitution, removal,
addition of amino acids contained in the editor protein, or a
combination thereof.
[0674] In addition, the modification may be substitution, removal,
addition of some bases in the base sequence encoding the editor
protein, or a combination thereof.
[0675] As one exemplary embodiment of the editor protein of the
present invention, a CRISPR enzyme will be described below.
[0676] CRISPR Enzyme
[0677] The term "CRISPR enzyme" is a main protein component of a
CRISPR-Cas system, and forms a complex with gRNA, resulting in the
CRISPR-Cas system.
[0678] The CRISPR enzyme is a nucleic acid or polypeptide (or a
protein) having a sequence encoding the CRISPR enzyme, and
representatively, a Type II CRISPR enzyme or Type V CRISPR enzyme
is widely used.
[0679] The Type II CRISPR enzyme is Cas9, which may be derived from
various microorganisms such as Streptococcus pyogenes,
Streptococcus thermophilus, Streptococcus sp., Staphylococcus
aureus, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis,
Streptomyces viridochromogenes, Streptosporangium roseum,
AlicyclobacHlus acidocaldarius, Bacillus pseudomycoides, Bacillus
selenitireducens, Exiguobacterium sibiricum, Lactobacillus
delbrueckii, Lactobacillus salivarius, Microscilla marina,
Burkholderiales bacterium, Polaromonas naphthalenivorans,
Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis
aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex
degensii, Caldicelulosiruptor bescii, Candidatus Desulforudis,
Clostridium botulinum, Clostridium difficile, Finegoldia magna,
Natranaerobius thermophilus, Pelotomaculum thermopropionicum,
Acidithiobacillus caldus, Acidithiobacillus ferrooxidans,
Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus,
Nitrosococcus watsoni, Pseudoalteromonas haloplanktis,
Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena
variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima,
Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus
chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho
africanus and Acaryochloris marina.
[0680] The term "Cas9" is an enzyme which binds to gRNA so as to
cleave or modify a target sequence or position on a target gene or
nucleic acid, and may consist of an HNH domain capable of cleaving
a nucleic acid strand forming a complementary bond with gRNA, an
RuvC domain capable of cleaving a nucleic acid strand forming a
complementary bond with gRNA, an REC domain recognizing a target
and a PI domain recognizing PAM. Hiroshi Nishimasu et al. (2014)
Cell 156:935-949 may be referenced for specific structural
characteristics of Cas9.
[0681] In addition, the Type V CRISPR enzyme may be Cpf1, which may
be derived from Streptococcus, Campylobacter, Nitratifractor,
Staphylococcus, Parvibaculum, Roseburia, Neisseria,
Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus,
Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria,
Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium,
Leptotrichia, Francisella, Legionella, Alicyclobacillus,
Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes,
Helcococcus, Letospira, Desulfovibrio, Desulfonatronum,
Opitutaceae, Tuberibacillus, Bacillus, Brevibacillus,
Methylobacterium or Acidaminococcus.
[0682] The Cpf1 may consist of an RuvC domain similar and
corresponding to the RuvC domain of Cas9, an Nuc domain without the
HNH domain of Cas9, an REC domain recognizing a target, a WED
domain and a PI domain recognizing PAM. For specific structural
characteristics of Cpf1, Takashi Yamano et al. (2016) Cell
165:949-962 may be referenced.
[0683] The CRISPR enzyme of the Cas9 or Cpf1 protein may be
isolated from a microorganism existing in nature or non-naturally
produced by a recombinant or synthetic method.
[0684] Type II CRISPR Enzyme
[0685] The crystal structure of the type II CRISPR enzyme was
determined according to studies on two or more types of natural
microbial type II CRISPR enzyme molecules (Jinek et al., Science,
343(6176):1247997, 2014) and studies on Streptococcus pyogenes Cas9
(SpCas9) complexed with gRNA (Nishimasu et al., Cell, 156:935-949,
2014; and Anders et al., Nature, 2014, doi:
10.1038/nature13579).
[0686] The type II CRISPR enzyme includes two lobes, that is,
recognition (REC) and nuclease (NUC) lobes, and each lobe includes
several domains.
[0687] The REC lobe includes an arginine-rich bridge helix (BH)
domain, an REC1 domain and an REC2 domain.
[0688] Here, the BH domain is a long .alpha.-helix and
arginine-rich region, and the REC1 and REC2 domains play an
important role in recognizing a double strand formed in gRNA, for
example, single-stranded gRNA, double-stranded gRNA or
tracrRNA.
[0689] The NUC lobe includes an RuvC domain, an HNH domain and a
PAM-interaction (PI) domain. Here, the RuvC domain encompasses
RuvC-like domains, or the HNH domain is used to include HNH-like
domains.
[0690] Here, the RuvC domain shares structural similarity with
members of the microorganism family existing in nature having the
type II CRISPR enzyme, and cleaves a single strand, for example, a
non-complementary strand of a target gene or nucleic acid, that is,
a strand not forming a complementary bond with gRNA. The RuvC
domain is sometimes referred to as an RuvCI domain, RuvCII domain
or RuvCIII domain in the art, and generally called an RuvC I,
RuvCII or RuvCIII. For example, in the case of SpCas9, the RuvC
domain is assembled from each of three divided RuvC domains (RuvC
I, RuvCII and RuvCIII) located at the sequences of amino acids 1 to
59, 718 to 769 and 909 to 1098 of SpCas9, respectively.
[0691] The HNH domain shares structural similarity with the HNH
endonuclease, and cleaves a single strand, for example, a
complementary strand of a target nucleic acid molecule, that is, a
strand forming a complementary bond with gRNA. The HNH domain is
located between RuvC II and III motifs. For example, in the case of
SpCas9, the HNH domain is located at amino acid sequence 775 to 908
of SpCas9.
[0692] The PI domain recognizes a specific base sequence in a
target gene or nucleic acid, that is, a protospacer adjacent motif
(PAM) or interacts with PAM. For example, in the case of SpCas9,
the PI domain is located at the sequence of amino acids1099 to 1368
of SpCas9.
[0693] Here, the PAM may vary according to the origin of the type
II CRISPR enzyme. For example, when the CRISPR enzyme is SpCas9,
PAM may be 5'-NGG-3', when the CRISPR enzyme is Streptococcus
thermophilus Cas9 (StCas9), PAM may be 5'-NNAGAAW-3'(W=A or T),
when the CRISPR enzyme is Neisseria meningitides Cas9 (NmCas9), PAM
may be 5'-NNNNGATT-3', and when the CRISPR enzyme is Campylobacter
jejuni Cas9 (CjCas9), PAM may be 5'-NNNVRYAC-3' (V=G or C or A, R=A
or G, Y=C or T), where the N may be A, T, G or C; or A, U, G or
C.
[0694] Type V CRISPR Enzyme
[0695] Type V CRISPR enzyme includes similar RuvC domains
corresponding to the RuvC domains of the type II CRISPR enzyme, and
may consist of an Nuc domain, instead of the HNH domain of the type
II CRISPR enzyme, REC and WED domains, which recognize a target,
and a PI domain recognizing PAM. For specific structural
characteristics of the type V CRISPR enzyme, Takashi Yamano et al.
(2016) Cell 165:949-962 may be referenced.
[0696] The type V CRISPR enzyme may interact with gRNA, thereby
forming a gRNA-CRISPR enzyme complex, that is, a CRISPR complex,
and may allow a guide sequence to approach a target sequence
including a PAM sequence in cooperation with gRNA. Here, the
ability of the type V CRISPR enzyme for interaction with a target
gene or nucleic acid is dependent on the PAM sequence.
[0697] The PAM sequence is a sequence present in a target gene or
nucleic acid, and may be recognized by the PI domain of the type V
CRISPR enzyme. The PAM sequence may vary according to the origin of
the type V CRISPR enzyme. That is, there are different PAM
sequences which are able to be specifically recognized depending on
a species.
[0698] In one example, the PAM sequence recognized by Cpf1 may be
5'-TTN-3' (N is A, T, C or G).
[0699] CRISPR Enzyme Activity
[0700] A CRISPR enzyme cleaves a double or single strand of a
target gene or nucleic acid, and has nuclease activity causing
breakage or deletion of the double or single strand. Generally, the
wild-type type II CRISPR enzyme or type V CRISPR enzyme cleaves the
double strand of the target gene or nucleic acid.
[0701] To manipulate or modify the above-described nuclease
activity of the CRISPR enzyme, the CRISPR enzyme may be manipulated
or modified, such a manipulated or modified CRISPR enzyme may be
modified into an incompletely or partially active or inactive
enzyme.
[0702] Incompletely or Partially Active Enzyme
[0703] A CRISPR enzyme modified to change enzyme activity, thereby
exhibiting incomplete or partial activity is called a nickase.
[0704] The term "nickase" refers to a CRISPR enzyme manipulated or
modified to cleave only one strand of the double strand of the
target gene or nucleic acid, and the nickase has nuclease activity
of cleaving a single strand, for example, a strand that is not
complementary or complementary to gRNA of the target gene or
nucleic acid. Therefore, to cleave the double strand, nuclease
activity of the two nickases is needed.
[0705] For example, the nickase may have nuclease activity by the
RuvC domain. That is, the nickase may include nuclease activity of
the HNH domain, and to this end, the HNH domain may be manipulated
or modified.
[0706] In one example, provided that the CRISPR enzyme is the type
II CRISPR enzyme, when the residue 840 in the amino acid sequence
of SpCas9 is mutated from histidine to alanine, the nuclease
activity of the HNH domain is inactivated to be used as a nickase.
Since the nickase produced thereby has nuclease activity of the
RuvC domain, it is able to cleave a strand which does not form a
complementary bond with a non-complementary strand of the target
gene or nucleic acid, that is, gRNA.
[0707] In another exemplary embodiment, when the residue 559 in the
amino acid sequence of CjCas9 is mutated from histidine to alanine,
the nuclease activity of the HNH domain is inactivated to be used
as a nickase. The nickase produced thereby has nuclease activity by
the RuvC domain, and thus is able to cleave a non-complementary
strand of the target gene or nucleic acid, that is, a strand that
does not form a complementary bond with gRNA.
[0708] For example, the nickase may have nuclease activity by the
HNH domain. That is, the nickase may include the nuclease activity
of the RuvC domain, and to this end, the RuvC domain may be
manipulated or modified.
[0709] In one example, provided that the CRISPR enzyme is the type
II CRISPR enzyme, in one exemplary embodiment, when the residue 10
in the amino acid sequence of SpCas9 is mutated from aspartic acid
to alanine, the nuclease activity of the RuvC domain is inactivated
to be used as a nickase. The nickase produced thereby has the
nuclease activity of the HNH domain, and thus is able to cleave a
complementary strand of the target gene or nucleic acid, that is, a
strand that forms a complementary bond with gRNA.
[0710] In another exemplary embodiment, when the residue 8 in the
amino acid sequence of CjCas9 is mutated from aspartic acid to
alanine, the nuclease activity of the RuvC domain is inactivated to
be used as a nickase. The nickase produced thereby has the nuclease
activity of the HNH domain, and thus is able to cleave a
complementary strand of the target gene or nucleic acid, that is, a
strand that forms a complementary bond with gRNA.
[0711] Inactive Enzyme
[0712] A CRISPR enzyme which is modified to make enzyme activity
completely inactive is called an inactive CRISPR enzyme.
[0713] The term "inactive CRISPR enzyme" refers to a CRISPR enzyme
which is modified not to completely cleave the double strand of the
target gene or nucleic acid, and the inactive CRISPR enzyme has
nuclease inactivity due to the mutation in the domain with nuclease
activity of the wild-type CRISPR enzyme. The inactive CRISPR enzyme
may be one in which the nuclease activities of the RuvC domain and
the HNH domain are inactivated.
[0714] For example, the inactive CRISPR enzyme may be manipulated
or modified in the RuvC domain and the HNH domain so as to inactive
nuclease activity.
[0715] In one example, provided that the CRISPR enzyme is the type
II CRISPR enzyme, in one exemplary embodiment, when the residues 10
and 840 in the amino acid sequence of SpCas9 are mutated from
aspartic acid and histidine to alanine, respectively, nuclease
activities by the RuvC domain and the HNH domain are inactivated,
such that the double strand may not cleave completely the double
strand of the target gene or nucleic acid.
[0716] In another exemplary embodiment, when the residues 8 and 559
in the amino acid sequence of CjCas9 are mutated from aspartic acid
and histidine to alanine, the nuclease activities by the RuvC
domain and the HNH domain are inactivated, such that the double
strand may not cleave completely the double strand of the target
gene or nucleic acid.
[0717] Other Activities
[0718] The CRISPR enzyme may have endonuclease activity,
exonuclease activity or helicase activity, that is, an ability to
anneal the helix structure of the double-stranded nucleic acid, in
addition to the above-described nuclease activity.
[0719] In addition, the CRISPR enzyme may be modified to
completely, incompletely, or partially activate the endonuclease
activity, exonuclease activity or helicase activity.
[0720] Targeting of CRISPR Enzyme
[0721] The CRISPR enzyme may interact with gRNA, thereby forming a
gRNA-CRISPR enzyme complex, that is, a CRISPR complex, and lead a
guide sequence to approach a target sequence including a PAM
sequence in cooperation with gRNA. Here, the ability of the CRISPR
enzyme to interact with the target gene or nucleic acid is
dependent on the PAM sequence.
[0722] The PAM sequence is a sequence present in the target gene or
nucleic acid, which may be recognized by the PI domain of the
CRISPR enzyme. The PAM sequence may vary depending on the origin of
the CRISPR enzyme. That is, there are various PAM sequences which
are able to be specifically recognized according to species.
[0723] In one example, provided that the CRISPR enzyme is the type
II CRISPR enzyme, [0724] in the case of SpCas9, the PAM sequence
may be 5'-NGG-3', 5'-NAG-3' and/or 5'-NGA-3', [0725] in the case of
StCas9, the PAM sequence may be 5'-NGGNG-3' and/or 5'-NNAGAAW-3'
(W=A or T), [0726] in the case of NmCas9, the PAM sequence may be
5'-NNNNGATT-3' and/or 5'-NNNGCTT-3', [0727] in the case of CjCas9,
the PAM sequence may be 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C
or T), [0728] in the case of Streptococcus mutans Cas9 (SmCas9),
the PAM sequence may be 5'-NGG-3' and/or 5'-NAAR-3' (R=A or G), and
[0729] in the case of Staphylococcus aureus Cas9 (SaCas9), the PAM
sequence may be 5'-NNGRR-3', 5'-NNGRRT-3' and/or 5'-NNGRRV-3' (R=A
or G; V=G, C or A).
[0730] In another example, provided that the CRISPR enzyme is the
type V CRISPR enzyme, in the case of Cpf1, the PAM sequence may be
5'-TTN-3'.
[0731] Here, the N may be A, T, G or C; or A, U, G or C.
[0732] The CRISPR enzyme capable of recognizing a specific PAM
sequence may be manipulated or modified using the PAM sequence
capable of being specifically recognized according to species. For
example, the PI domain of SpCas9 may be replaced with the PI domain
of CjCas9 so as to have the nuclease activity of SpCas9 and
recognize a CjCas9-specific PAM sequence, thereby producing SpCas9
recognizing the CjCas9-specific PAM sequence. A specifically
recognized PAM sequence may be changed by substitution or
replacement of the PI domain.
[0733] CRISPR Enzyme Mutant
[0734] The CRISPR enzyme may be modified to improve or inhibit
various characteristics such as nuclease activity, helicase
activity, an ability to interact with gRNA, and an ability to
approach the target gene or nucleic acid, for example, PAM
recognizing ability of the CRISPR enzyme.
[0735] In addition, the CRISPR enzyme mutant may be a CRISPR enzyme
which interacts with gRNA to form a gRNA-CRISPR enzyme complex,
that is, a CRISPR complex, and is modified or manipulated to
improve target specificity, when approaching or localized to the
target gene or nucleic acid, such that only a double or single
strand of the target gene or nucleic acid is cleaved without
cleavage of a double or single strand of a non-target gene or
nucleic acid which partially forms a complementary bond with gRNA
and a non-target gene or nucleic acid which does not form a
complementary bond therewith.
[0736] Here, an effect of cleaving the double or single strand of
the non-target gene or nucleic acid partially forming a
complementary bond with gRNA and the non-target gene or nucleic
acid not forming a complementary bond therewith is referred to as
an off-target effect, a position or base sequence of the non-target
gene or nucleic acid partially forming a complementary bond with
gRNA and the non-target gene or nucleic acid not forming a
complementary bond therewith is referred to as an off-target. Here,
there may be one or more off-targets. One the other hand, the
cleavage effect of the double or single strand of the target gene
or nucleic acid is referred to as an on-target effect, and a
location or target sequence of the target gene or nucleic acid is
referred to as an on-target.
[0737] The CRISPR enzyme mutant is modified in at least one of the
amino acids of a naturally-occurring CRISPR enzyme, and may be
modified, for example, improved or inhibited in one or more of the
various characteristics such as nuclease activity, helicase
activity, an ability to interact with gRNA, an ability to approach
the target gene or nucleic acid and target specificity, compared to
the unmodified CRISPR enzyme. Here, the modification may be
substitution, removal, addition of an amino acid, or a mixture
thereof.
[0738] In the CRISPR enzyme mutant, the modification may be a
modification of one or two or more amino acids located in a region
consisting of amino acids having positive charges, present in the
naturally-occurring CRISPR enzyme.
[0739] For example, the modification may be a modification of one
or two or more amino acids of the positively-charged amino acids
such as lysine (K), arginine (R) and histidine (H), present in the
naturally-occurring CRISPR enzyme.
[0740] The modification may be a modification of one or two or more
amino acids located in a region composed of non-positively-charged
amino acids present in the naturally-occurring CRISPR enzyme.
[0741] For example, the modification may be a modification of one
or two or more amino acids of the non-positively-charged amino
acids, that is, aspartic acid (D), glutamic acid (E), serine (S),
threonine (T), asparagine (N), glutamine (Q), cysteine (C), proline
(P), glycine (G), alanine (A), valine (V), isoleucine (I), leucine
(L), methionine (M), phenylalanine (F), tyrosine (Y) and tryptophan
(W), present in the naturally-occurring CRISPR enzyme.
[0742] In another example, the modification may be a modification
of one or two or more amino acids of non-charged amino acids, that
is, serine (S), threonine (T), asparagine (N), glutamine (Q),
cysteine (C), proline (P), glycine (G), alanine (A), valine (V),
isoleucine (I), leucine (L), methionine (M), phenylalanine (F),
tyrosine (Y) and tryptophan (W), present in the naturally-occurring
CRISPR enzyme.
[0743] In addition, the modification may be a modification of one
or two or more of the amino acids having hydrophobic residues
present in the naturally-occurring CRISPR enzyme.
[0744] For example, the modification may be a modification of one
or two or more amino acids of glycine (G), alanine (A), valine (V),
isoleucine (I), leucine (L), methionine (M), phenylalanine (F),
tyrosine (Y) and tryptophan (W), present in the naturally-occurring
CRISPR enzyme.
[0745] The modification may be a modification of one or two or more
of the amino acids having polar residues, present in the
naturally-occurring CRISPR enzyme.
[0746] For example, the modification may be a modification of one
or two or more amino acids of serine (S), threonine (T), asparagine
(N), glutamine (Q), cysteine (C), proline (P), lysine (K), arginine
(R), histidine (H), aspartic acid (D) and glutamic acid (E),
present in the naturally-occurring CRISPR enzyme.
[0747] In addition, the modification may be a modification of one
or two or more of the amino acids including lysine (K), arginine
(R) and histidine (H), present in the naturally-occurring CRISPR
enzyme.
[0748] For example, the modification may be a substitution of one
or two or more of the amino acids including lysine (K), arginine
(R) and histidine (H), present in the naturally-occurring CRISPR
enzyme.
[0749] The modification may be a modification of one or two or more
of the amino acids including aspartic acid (D) and glutamic acid
(E), present in the naturally-occurring CRISPR enzyme.
[0750] For example, the modification may be a substitution of one
or two or more of the amino acids including aspartic acid (D) and
glutamic acid (E), present in the naturally-occurring CRISPR
enzyme.
[0751] The modification may be a modification of one or two or more
of the amino acids including serine (S), threonine (T), asparagine
(N), glutamine (Q), cysteine (C), proline (P), glycine (G), alanine
(A), valine (V), isoleucine (I), leucine (L), methionine (M),
phenylalanine (F), tyrosine (Y) and tryptophan (W), present in the
naturally-occurring CRISPR enzyme.
[0752] For example, the modification may be a substitution of one
or two or more of the amino acid including serine (S), threonine
(T), asparagine (N), glutamine (Q), cysteine (C), proline (P),
glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L),
methionine (M), phenylalanine (F), tyrosine (Y) and tryptophan (W),
present in the naturally-occurring CRISPR enzyme.
[0753] In addition, the modification may be a modification of one,
two, three, four, five, six, seven or more of the amino acids
present in the naturally-occurring CRISPR enzyme.
[0754] In addition, in the CRISPR enzyme mutant, the modification
may be a modification of one or two or more of the amino acids
present in the RuvC domain of the CRISPR enzyme. Here, the RuvC
domain may be an RuvCI, RuvCII or RuvCIII domain.
[0755] The modification may be a modification of one or two or more
of the amino acids present in the HNH domain of the CRISPR
enzyme.
[0756] The modification may be a modification of one or two or more
of the amino acids present in the REC domain of the CRISPR
enzyme.
[0757] The modification may be one or two or more of the amino
acids present in the PI domain of the CRISPR enzyme.
[0758] The modification may be a modification of two or more of the
amino acids contained in at least two or more domains of the REC,
RuvC, HNH and PI domains of the CRISPR enzyme.
[0759] In one example, the modification may be a modification of
two or more of the amino acids contained in the REC and RuvC
domains of the CRISPR enzyme.
[0760] In one exemplary embodiment, in the SpCas9 mutant, the
modification may be a modification of at least two or more of the
A203, H277, G366, F539, I601, M763, D965 and F1038 amino acids
contained in the REC and RuvC domains of SpCas9.
[0761] In another example, the modification may be a modification
of two or more of the amino acids contained in the REC and HNH
domains of the CRISPR enzyme.
[0762] In one exemplary embodiment, in the SpCas9 mutant, the
modification may be a modification of at least two or more of the
A203, H277, G366, F539, I601 and K890 amino acids contained in the
REC and HNH domains of SpCas9.
[0763] In one example, the modification may be a modification of
two or more of the amino acids contained in the REC and PI domains
of the CRISPR enzyme.
[0764] In one exemplary embodiment, in the SpCas9 mutant, the
modification may be a modification of at least two or more of the
A203, H277, G366, F539, I601, T1102 and D1127 amino acids contained
in the REC and PI domains of SpCas9.
[0765] In another example, the modification may be a modification
of three or more of the amino acids contained in the REC, RuvC and
HNH domains of the CRISPR enzyme.
[0766] In one exemplary embodiment, in the SpCas9 mutant, the
modification may be a modification of at least three or more of the
A203, H277, G366, F539, I601, M763, K890, D965 and F1038 amino
acids contained in the REC, RuvC and HNH domains of SpCas9.
[0767] In one example, the modification may be a modification of
three or more of the amino acids contained in the REC, RuvC and PI
domains contained in the CRISPR enzyme.
[0768] In one exemplary embodiment, in the SpCas9 mutant, the
modification may be a modification of at least three or more of the
A203, H277, G366, F539, I601, M763, D965, F1038, T1102 and D1127
amino acids contained in the REC, RuvC and PI domains of
SpCas9.
[0769] In another example, the modification may be a modification
of three or more of the amino acids contained in the REC, HNH and
PI domains of the CRISPR enzyme.
[0770] In one exemplary embodiment, in the SpCas9 mutant, the
modification may be a modification of at least three or more of the
A203, H277, G366, F539, I601, K890, T1102 and D1127 amino acids
contained in the REC, HNH and PI domains of SpCas9.
[0771] In one example, the modification may be a modification of
three or more of the amino acids contained in the RuvC, HNH and PI
domains of the CRISPR enzyme.
[0772] In one exemplary embodiment, in the SpCas9 mutant, the
modification may be a modification of at least three or more of the
M763, K890, D965, F1038, T1102 and D1127 amino acids contained in
the RuvC, HNH and PI domains of SpCas9.
[0773] In another example, the modification may be a modification
of four or more of the amino acids contained in the REC, RuvC, HNH
and PI domains of the CRISPR enzyme.
[0774] In one exemplary embodiment, in the SpCas9 mutant, the
modification may be a modification of at least four or more of the
A203, H277, G366, F539, I601, M763, K890, D965, F1038, T1102 and
D1127 amino acids contained in the REC, RuvC, HNH and PI domains of
SpCas9.
[0775] In addition, in the CRISPR enzyme mutant,
[0776] the modification may be a modification of one or two or more
of the amino acids participating in the nuclease activity of the
CRISPR enzyme.
[0777] For example, in the SpCas9 mutant, the modification may be a
modification of one or two or more of the group consisting of the
amino acids D10, E762, H840, N854, N863 and D986, or one or two or
more of the group consisting of the amino acids corresponding to
other Cas9 orthologs.
[0778] The modification may be a modification for partially
inactivating the nuclease activity of the CRISPR enzyme, and such a
CRISPR enzyme mutant may be a nickase.
[0779] Here, the modification may be a modification for
inactivating the nuclease activity of the RuvC domain of the CRISPR
enzyme, and such a CRISPR enzyme mutant may not cleave a
non-complementary strand of a target gene or nucleic acid, that is,
a strand which does not form a complementary bond with gRNA.
[0780] In one exemplary embodiment, in the case of SpCas9, when
residue 10 of the amino acid sequence of SpCas9 is mutated from
aspartic acid to alanine, that is, when mutated to D10A, the
nuclease activity of the RuvC domain is inactivated, and thus the
SpCas9 may be used as a nickase. The nickase produced thereby may
not cleave a non-complementary strand of the target gene or nucleic
acid, that is, a strand that does not form a complementary bond
with gRNA.
[0781] In another exemplary embodiment, in the case of CjCas9, when
residue 8 of the amino acid sequence of CjCas9 is mutated from
aspartic acid to alanine, that is, when mutated to D8A, the
nuclease activity of the RuvC domain is inactivated, and thus the
CjCas9 may be used as a nickase. The nickase produced thereby may
not cleave a non-complementary strand of the target gene or nucleic
acid, that is, a strand that does not form a complementary bond
with gRNA.
[0782] In addition, here, the modification may be a modification
for inactivating the nuclease activity of the HNH domain of the
CRISPR enzyme, and such a CRISPR enzyme mutant may not cleave a
complementary strand of the target gene or nucleic acid, that is, a
strand forming a complementary bond with gRNA.
[0783] In one exemplary embodiment, in the case of SpCas9, when
residue 840 of the amino acid sequence of SpCas9 is mutated from
histidine to alanine, that is, when mutated to H840A, the nuclease
activity of the HNH domain is inactivated, and thus the SpCas9 may
be used as a nickase. The nickase produced thereby may not cleave a
complementary strand of the target gene or nucleic acid, that is, a
strand that forms a complementary bond with gRNA.
[0784] In another exemplary embodiment, in the case of CjCas9, when
residue 559 of the amino acid sequence of CjCas9 is mutated from
histidine to alanine, that is, when mutated to H559A, the nuclease
activity of the HNH domain is inactivated, and thus the CjCas9 may
be used as a nickase. The nickase produced thereby may not cleave a
complementary strand of the target gene or nucleic acid, that is, a
strand that forms a complementary bond with g RNA.
[0785] In addition, the modification may be a modification for
completely inactivating the nuclease activity of the CRISPR enzyme,
and such a CRISPR enzyme mutant may be an inactive CRISPR
enzyme.
[0786] Here, the modification may be a modification for
inactivating the nuclease activities of the RuvC and HNH domains of
the CRISPR enzyme, and such a CRISPR enzyme mutant may does not
cleave a double strand of the target gene or nucleic acid.
[0787] In one exemplary embodiment, in the case of SpCas9, when the
residues 10 and 840 in the amino acid sequence of SpCas9 are
mutated from aspartic acid and histidine to alanine, that is,
mutated to D10A and H840A, respectively, the nuclease activities of
the RuvC domain and the HNH domain are inactivated, the double
strand of the target gene or nucleic acid may not be completely
cleaved.
[0788] In another exemplary embodiment, in the case of CjCas9, when
residues 8 and 559 of the amino acid sequence of CjCas9 are mutated
from aspartic acid and histidine to alanine, that is, mutated to
D8A and H559A, respectively, the nuclease activities by the RuvC
and HNH domains are inactivated, and thus the double strand of the
target gene or nucleic acid may not be completely cleaved.
[0789] In addition, the CRISPR enzyme mutant may further include an
optionally functional domain, in addition to the innate
characteristics of the CRISPR enzyme, and such a CRISPR enzyme
mutant may have an additional characteristic in addition to the
innate characteristics.
[0790] Here, the functional domain may be a domain having methylase
activity, demethylase activity, transcription activation activity,
transcription repression activity, transcription release factor
activity, histone modification activity, RNA cleavage activity or
nucleic acid binding activity, or a tag or reporter gene for
isolating and purifying a protein (including a peptide), but the
present invention is not limited thereto.
[0791] The functional domain, peptide, polypeptide or protein may
be a deaminase.
[0792] For example, an incomplete or partial CRISPR enzyme may
additionally include a cytidine deaminase as a functional domain.
In one exemplary embodiment, a cytidine deaminase, for example,
apolipoprotein B editing complex 1 (APOBEC1) may be added to SpCas9
nickase, thereby producing a fusion protein. The [SpCas9
nickase]-[APOBEC1] formed thereby may be used in base repair or
editing of C into T or U, or G into A.
[0793] The tag includes a histidine (His) tag, a V5 tag, a FLAG
tag, an influenza hemagglutinin (HA) tag, a Myc tag, a VSV-G tag
and a thioredoxin (Trx) tag, and the reporter gene includes
glutathione-S-transferase (GST), horseradish peroxidase (HRP),
chloramphenicol acetyltransferase (CAT) .beta.-galactosidase,
.beta.-glucoronidase, luciferase, autofluorescent proteins
including the green fluorescent protein (GFP), HcRed, DsRed, cyan
fluorescent protein (CFP), yellow fluorescent protein (YFP) and
blue fluorescent protein (BFP), but the present invention is not
limited thereto.
[0794] In addition, the functional domain may be a nuclear
localization sequence or signal (NLS) or a nuclear export sequence
or signal (NES).
[0795] In one example, the CRISPR enzyme may include one or more
NLSs. Here, one or more NLSs may be included at an N-terminus of an
CRISPR enzyme or the proximity thereof; a C-terminus of the enzyme
or the proximity thereof; or a combination thereof. The NLS may be
an NLS sequence derived from the following NLSs, but the present
invention is not limited thereto: NLS of a SV40 virus large
T-antigen having the amino acid sequence PKKKRKV; NLS from
nucleoplasmin (e.g., nucleoplasmin bipartite NLS having the
sequence KRPAATKKAGQAKKKK); c-myc NLS having the amino acid
sequence PAAKRVKLD or RQRRNELKRSP; hRNPA1 M9 NLS having the
sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; the sequence
RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV of the IBB domain from
importin-.alpha.; the sequences VSRKRPRP and PPKKARED of a myoma T
protein; the sequence POPKKKPL of human p53; the sequence
SALIKKKKKMAP of mouse c-abl IV; the sequences DRLRR and PKQKKRK of
influenza virus NS1; the sequence RKLKKKIKKL of a hepatitis delta
virus antigen; the sequence REKKKFLKRR of a mouse Mx1 protein; the
sequence KRKGDEVDGVDEVAKKKSKK of a human poly (ADP-ribose)
polymerase; or the NLS sequence RKCLQAGMNLEARKTKK, derived from a
sequence of a steroid hormone receptor (human) glucocorticoid.
[0796] In addition, the CRISPR enzyme mutant may include a
split-type CRISPR enzyme prepared by dividing the CRISPR enzyme
into two or more parts. The term "split" refers to functional or
structural division of a protein or random division of a protein
into two or more parts.
[0797] Here, the split-type CRISPR enzyme may be a completely,
incompletely or partially active enzyme or inactive enzyme.
[0798] For example, the SpCas9 may be divided into two parts
between the residue 656, tyrosine, and the residue 657, threonine,
thereby generating split SpCas9.
[0799] In addition, the split-type CRISPR enzyme may selectively
include an additional domain, peptide, polypeptide or protein for
reconstitution.
[0800] Here, the "reconstitution" refers to formation of the
split-type CRISPR enzyme to be structurally the same or similar to
the wild-type CRISPR enzyme.
[0801] The additional domain, peptide, polypeptide or protein for
reconstitution may be FRB and FKBP dimerization domains; intein;
ERT and VPR domains; or domains which form a heterodimer under
specific conditions.
[0802] For example, the SpCas9 may be divided into two parts
between the residue 713, serine, and the residue 714, glycine,
thereby generating split SpCas9. The FRB domain may be connected to
one of the two parts, and the FKBP domain may be connected to the
other one. In the split SpCas9 produced thereby, the FRB domain and
the FKBP domain may be formed in a dimer in an environment in which
rapamycine is present, thereby producing a reconstituted CRISPR
enzyme.
[0803] The CRISPR enzyme or CRISPR enzyme mutant described in the
present invention may be a polypeptide, protein or nucleic acid
having a sequence encoding the same, and may be codon-optimized for
a subject to introduce the CRISPR enzyme or CRISPR enzyme
mutant.
[0804] The term "codon optimization" refers to a process of
modifying a nucleic acid sequence by maintaining a native amino
acid sequence while replacing at least one codon of the native
sequence with a codon more frequently or the most frequently used
in host cells so as to improve expression in the host cells. A
variety of species have a specific bias to a specific codon of a
specific amino acid, and the codon bias (the difference in codon
usage between organisms) is frequently correlated with efficiency
of the translation of mRNA, which is considered to be dependent on
the characteristic of a translated codon and availability of a
specific tRNA molecule. The dominance of tRNA selected in cells
generally reflects codons most frequently used in peptide
synthesis. Therefore, a gene may be customized by optimal gene
expression in a given organism based on codon optimization.
[0805] 3. Target Sequence
[0806] The term "target sequence" is a base sequence present in a
target gene or nucleic acid, and has complementarity to a guide
sequence contained in a guide domain of a guide nucleic acid. The
target sequence is a base sequence which may vary according to a
target gene or nucleic acid, that is, a subject for gene
manipulation or correction, which may be designed in various forms
according to the target gene or nucleic acid.
[0807] The target sequence may form a complementary bond with the
guide sequence contained in the guide domain of the guide nucleic
acid, and a length of the target sequence may be the same as that
of the guide sequence.
[0808] The target sequence may be a 5 to 50-base sequence.
[0809] In an embodiment, the target sequence may be a 16, 17, 18,
19, 20, 21, 22, 23, 24 or 25-base sequence.
[0810] The target sequence may be a nucleic acid sequence
complementary to the guide sequence contained in the guide domain
of the guide nucleic acid, which has, for example, at least 70%,
75%, 80%, 85%, 90% or 95% or more complementarity or complete
complementarity.
[0811] In one example, the target sequence may be or include a 1 to
8-base sequence, which is not complementary to the guide sequence
contained in the guide domain of the guide nucleic acid.
[0812] In addition, the target sequence may be a base sequence
adjacent to a nucleic acid sequence that is able to be recognized
by an editor protein.
[0813] In one example, the target sequence may be a continuous 5 to
50-base sequence adjacent to the 5' end and/or 3' end of the
nucleic acid sequence that is able to be recognized by the editor
protein.
[0814] In one exemplary embodiment, target sequences for a
gRNA-CRISPR enzyme complex will be described below.
[0815] When the target gene or nucleic acid is targeted by the
gRNA-CRISPR enzyme complex, the target sequence has complementarity
to the guide sequence contained in the guide domain of gRNA. The
target sequence is a base sequence which varies according to the
target gene or nucleic acid, that is, a subject for gene
manipulation or correction, which may be designed in various forms
according to the target gene or nucleic acid.
[0816] In addition, the target sequence may be a base sequence
adjacent to a PAM sequence which is able to be recognized by the
CRISPR enzyme, that is, Cas9 or Cpf1.
[0817] In one example, the target sequence may be a continuous 5 to
50-base sequence adjacent to the 5' end and/or 3' end of the PAM
sequence which is recognized by the CRISPR enzyme.
[0818] In one exemplary embodiment, when the CRISPR enzyme is
SpCas9, the target sequence may be a continuous 16 to 25-base
sequence adjacent to the 5' end and/or 3' end of a 5'-NGG-3',
5'-NAG-3' and/or 5'-NGA-3' (N=A, T, G or C; or A, U, G or C)
sequence.
[0819] In another exemplary embodiment, when the CRISPR enzyme is
StCas9, the target sequence may be a continuous 16 to 25-base
sequence adjacent to the 5' end and/or 3' end of a 5'-NGGNG-3'
and/or 5'-NNAGAAW-3' (W=A or T, and N=A, T, G or C; or A, U, G or
C) sequence.
[0820] In still another exemplary embodiment, when the CRISPR
enzyme is NmCas9, the target sequence may be a continuous 16 to
25-base sequence adjacent to the 5' end and/or 3' end of a
5'-NNNNGATT-3' and/or 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or
C) sequence.
[0821] In one exemplary embodiment, when the CRISPR enzyme is
CjCas9, the target sequence may be a continuous 16 to 25-base
sequence adjacent to the 5' end and/or 3' end of a 5'-NNNVRYAC-3'
(V=G, C or A; R=A or G, Y=C or T, N=A, T, G or C; or A, U, G or C)
sequence.
[0822] In another exemplary embodiment, when the CRISPR enzyme is
SmCas9, the target sequence may be a continuous 16 to 25-base
sequence adjacent to the 5' end and/or 3' end of a 5'-NGG-3' and/or
5'-NAAR-3'(R=A or G, N=A, T, G or C; or A, U, G or C) sequence.
[0823] In yet another exemplary embodiment, when the CRISPR enzyme
is SaCas9, the target sequence may be a continuous 16 to 25-base
sequence adjacent to the 5' end and/or 3' end of a 5'-NNGRR-3',
5'-NNGRRT-3' and/or 5'-NNGRRV-3' (R=A or G, V=G, C or A, N=A, T, G
or C; or A, U, G or C) sequence.
[0824] In one exemplary embodiment, when the CRISPR enzyme is Cpf1,
the target sequence may be a continuous 16 to 25-base sequence
adjacent to the 5' end and/or 3' end of a 5'-TTN-3' (N=A, T, G or
C; or A, U, G or C) sequence.
[0825] In one exemplary embodiment of the present invention, the
target sequence may be a nucleic acid sequence contained in one or
more genes selected from the group consisting of a VEGFA gene, an
HIF1A gene, an ANGPT2 gene, an EPAS1 gene, and an ANGPTL4 gene.
[0826] The target sequence may be a nucleic acid sequence contained
in the VEGFA gene.
[0827] The target sequence may be a nucleic acid sequence contained
in the HIF1A gene.
[0828] The target sequence may be a nucleic acid sequence contained
in the ANGPT2 gene.
[0829] The target sequence may be a nucleic acid sequence contained
in the EPAS1 gene.
[0830] The target sequence may be a nucleic acid sequence contained
in the ANGPTL4 gene.
[0831] Alternatively, the target sequence may be a partial nucleic
acid sequence of one or more genes selected from the group
consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1
gene, and an ANGPTL4 gene.
[0832] The target sequence may be a partial nucleic acid sequence
of the VEGFA gene.
[0833] The target sequence may be a partial nucleic acid sequence
of the HIF1A gene.
[0834] The target sequence may be a partial nucleic acid sequence
of the ANGPT2 gene.
[0835] The target sequence may be a partial nucleic acid sequence
of the EPAS1 gene.
[0836] The target sequence may be a partial nucleic acid sequence
of the ANGPTL4 gene.
[0837] Alternatively, the target sequence may be a nucleic acid
sequence of the coding or non-coding region or a mixture thereof of
one or more genes selected from the group consisting of a VEGFA
gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene, and an ANGPTL4
gene.
[0838] The target sequence may be a nucleic acid sequence of the
coding or non-coding region or a mixture thereof of the VEGFA
gene.
[0839] The target sequence may be a nucleic acid sequence of the
coding or non-coding region or a mixture thereof of the HIF1A
gene.
[0840] The target sequence may be a nucleic acid sequence of the
coding or non-coding region or a mixture thereof of the ANGPT2
gene.
[0841] The target sequence may be a nucleic acid sequence of the
coding or non-coding region or a mixture thereof of the EPAS1
gene.
[0842] The target sequence may be a nucleic acid sequence of the
coding or non-coding region or a mixture thereof of the ANGPTL4
gene.
[0843] Alternatively, the target sequence may be a nucleic acid
sequence of the promoter, enhancer, 3'UTR or polyadenyl (polyA)
region or a mixture thereof of one or more genes selected from the
group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an
EPAS1 gene, and an ANGPTL4 gene.
[0844] The target sequence may be a nucleic acid sequence of the
promoter, enhancer, 3'UTR or polyadenyl (polyA) region or a mixture
thereof of the VEGFA gene.
[0845] The target sequence may be a nucleic acid sequence of the
promoter, enhancer, 3'UTR or polyadenyl (polyA) region or a mixture
thereof of the HIF1A gene.
[0846] The target sequence may be a nucleic acid sequence of the
promoter, enhancer, 3'UTR or polyadenyl (polyA) region or a mixture
thereof of the ANGPT2 gene.
[0847] The target sequence may be a nucleic acid sequence of the
promoter, enhancer, 3'UTR or polyadenyl (polyA) region or a mixture
thereof of the EPAS1 gene.
[0848] The target sequence may be a nucleic acid sequence of the
promoter, enhancer, 3'UTR or polyadenyl (polyA) region or a mixture
thereof of the ANGPTL4 gene.
[0849] Alternatively, the target sequence may be a nucleic acid
sequence of an exon, an intron or a mixture thereof of one or more
genes selected from the group consisting of a VEGFA gene, an HIF1A
gene, an ANGPT2 gene, an EPAS1 gene, and an ANGPTL4 gene.
[0850] The target sequence may be a nucleic acid sequence of an
exon, an intron or a mixture thereof of the VEGFA gene.
[0851] The target sequence may be a nucleic acid sequence of an
exon, an intron or a mixture thereof of the HIF1A gene.
[0852] The target sequence may be a nucleic acid sequence of an
exon, an intron or a mixture thereof of the ANGPT2 gene.
[0853] The target sequence may be a nucleic acid sequence of an
exon, an intron or a mixture thereof of the EPAS1 gene.
[0854] The target sequence may be a nucleic acid sequence of an
exon, an intron or a mixture thereof of the ANGPTL4 gene.
[0855] Alternatively, The target sequence may be a nucleic acid
sequence including or adjacent to a mutated region (e.g., a region
different from a wild-type gene) of one or more genes selected from
the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2
gene, an EPAS1 gene, and an ANGPTL4 gene.
[0856] The target sequence may be a nucleic acid sequence including
or adjacent to a mutated region of the VEGFA gene.
[0857] The target sequence may be a nucleic acid sequence including
or adjacent to a mutated region of the HIF1A gene.
[0858] The target sequence may be a nucleic acid sequence including
or adjacent to a mutated region of the ANGPT2 gene.
[0859] The target sequence may be a nucleic acid sequence including
or adjacent to a mutated region of the EPAS1 gene.
[0860] The target sequence may be a nucleic acid sequence including
or adjacent to a mutated region of the ANGPTL4 gene.
[0861] Alternatively, the target sequence may be a continuous 5 to
50-nucleic acid sequence of one or more genes selected from the
group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an
EPAS1 gene, and an ANGPTL4 gene.
[0862] The target sequence may be a continuous 5 to 50-nucleic acid
sequence of the VEGFA gene.
[0863] The target sequence may be a continuous 5 to 50-nucleic acid
sequence of the HIF1A gene.
[0864] The target sequence may be a continuous 5 to 50-nucleic acid
sequence of the ANGPT2 gene.
[0865] The target sequence may be a continuous 5 to 50-nucleic acid
sequence of the EPAS1 gene.
[0866] The target sequence may be a continuous 5 to 50-nucleic acid
sequence of the ANGPTL4 gene.
[0867] As one exemplary embodiment of the present invention, the
above target sequences of the VEGFA gene, HIF1A gene, ANGPT2 gene,
EPAS1 gene, and ANGPTL4 gene are summarized in Table 1, Table 2,
Table 3, Table 4 and Table 5.
[0868] Neovascularization-Associated Factor-Manipulated Product
[0869] 4. Guide Nucleic Acid-Editor Protein Complex and Use
Thereof
[0870] A guide nucleic acid-editor protein complex may modify a
target.
[0871] The target may be a target nucleic acid, gene, chromosome or
protein.
[0872] For example, the guide nucleic acid-editor protein complex
may be used to ultimately regulate (e.g., inhibit, suppress,
reduce, increase or promote) the expression of a protein of
interest, remove a protein, regulate (e.g., inhibit, suppress,
reduce, increase or promote) protein activity, or express a new
protein.
[0873] Here, the guide nucleic acid-editor protein complex may act
at a DNA, RNA, gene or chromosomal level.
[0874] For example, the guide nucleic acid-editor protein complex
may regulate (e.g., inhibit, suppress, reduce, increase or promote)
the expression of a protein encoded by target DNA, remove a
protein, regulate (e.g., inhibit, suppress, reduce, increase or
promote) protein activity, or express a modified protein through
manipulation or modification of the target DNA.
[0875] In another example, the guide nucleic acid-editor protein
complex may regulate (e.g., inhibit, suppress, reduce, increase or
promote) the expression of a protein encoded by target DNA, remove
a protein, regulate (e.g., inhibit, suppress, reduce, increase or
promote) protein activity, or express a modified protein through
manipulation or modification of target RNA.
[0876] In one example, the guide nucleic acid-editor protein
complex may regulate (e.g., inhibit, suppress, reduce, increase or
promote) the expression of a protein encoded by target DNA, remove
a protein, regulate (e.g., inhibit, suppress, reduce, increase or
promote) protein activity, or express a modified protein through
manipulation or modification of a target gene.
[0877] In another example, the guide nucleic acid-editor protein
complex may regulate (e.g., inhibit, suppress, reduce, increase or
promote) the expression of a protein encoded by target DNA, remove
a protein, regulate (e.g., inhibit, suppress, reduce, increase or
promote) protein activity, or express a modified protein through
manipulation or modification of a target chromosome.
[0878] The guide nucleic acid-editor protein complex may act at
gene transcription and translation stages.
[0879] In one example, the guide nucleic acid-editor protein
complex may promote or suppress the transcription of a target gene,
thereby regulating (e.g., inhibiting, suppressing, reducing,
increasing or promoting) the expression of a protein encoded by the
target gene.
[0880] In another example, the guide nucleic acid-editor protein
complex may promote or suppress the translation of a target gene,
thereby regulating (e.g., inhibiting, suppressing, reducing,
increasing or promoting) the expression of a protein encoded by the
target gene.
[0881] The guide nucleic acid-editor protein complex may act at a
protein level.
[0882] In one example, the guide nucleic acid-editor protein
complex may manipulate or modify a target protein, thereby removing
the target protein or regulating (e.g., inhibiting, suppressing,
reducing, increasing or promoting) protein activity.
[0883] In one exemplary embodiment, the present invention provides
a guide nucleic acid-editor protein complex used to manipulate a
neovascularization-associated factor, for example, a VEGFA gene, an
HIF1A gene, an ANGPT2 gene, an EPAS1 gene, and/or an ANGPTL4 gene.
Preferably, a gRNA-CRISPR enzyme complex is provided.
[0884] Particularly, the present invention may provide gRNA
including a guide domain capable of forming a complementary bond
with a target sequence from a gene, for example, isolated or
non-natural gRNA and DNA encoding the same. The gRNA and the DNA
sequence encoding the same may be designed to be able to
complementarily bind to a target sequence listed in Table 1, Table
2, Table 3, Table 4 and Table 5.
[0885] In addition, a target region of the gRNA is designed to
provide a third gene, which has a nucleic acid modification, for
example, double or single strand breaks; or a specific function at
a target site in a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an
EPAS1 gene, and/or an ANGPTL4 gene.
[0886] In addition, when two or more gRNAs are used to induce two
or more cleaving events in a target gene, for example, a double or
single strand break, the two or more cleaving events may occur due
to the same or different Cas9 proteins.
[0887] The gRNA may target, for example, two or more of the VEGFA
gene, the HIF1A gene, the ANGPT2 gene, the EPAS1 gene, and/or the
ANGPTL4 gene, or two or more regions in each of the VEGFA gene,
HIF1A gene, ANGPT2 gene, EPAS1 gene, and/or ANGPTL4 gene, and may
independently induce the cleavage of a double strand and/or a
single strand of the VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1
gene, and/or ANGPTL4 gene, or may induce the insertion of one
foreign nucleotide into a cleavage site of the VEGFA gene, the
HIF1A gene, the ANGPT2 gene, the EPAS1 gene, and/or the ANGPTL4
gene.
[0888] In addition, in another exemplary embodiment of the present
invention, a nucleic acid constituting the guide nucleic
acid-editor protein complex may include: (a) a sequence encoding a
guide nucleic acid including a guide domain, which is complementary
to a target sequence of the VEGFA gene, the HIF1A gene, the ANGPT2
gene, the EPAS1 gene, and/or the ANGPTL4 gene as described herein;
and (b) a sequence encoding an editor protein.
[0889] Here, there may be two or more of the (a) according to a
target region, and the (b) may employ the same or two or more
editor proteins.
[0890] In an embodiment, the nucleic acid may be designed to target
an enzymatically inactive editor protein or a fusion protein (e.g.,
a transcription repressor domain fusion) thereof to place it
sufficiently adjacent to a knockdown target site in order to
reduce, decrease or inhibit expression of the VEGFA gene, the HIF1A
gene, the ANGPT2 gene, the EPAS1 gene, and/or the ANGPTL4 gene.
[0891] Besides, it should be obvious that the above-described
structure, function, and all applications of the guide nucleic
acid-editor protein complex will be utilized in manipulation of the
VEGFA gene, the HIF1A gene, the ANGPT2 gene, the EPAS1 gene, and/or
the ANGPTL4 gene.
[0892] Use of Guide Nucleic Acid-Editor Protein Complex
[0893] In an embodiment for the use of the guide nucleic
acid-editor protein complex of the present invention, the
manipulation or modification of target DNA, RNA, genes or
chromosomes using the gRNA-CRISPR enzyme complex will be described
below.
[0894] Gene Manipulation
[0895] A target gene or nucleic acid may be manipulated or
corrected using the above-described gRNA-CRISPR enzyme complex,
that is, the CRISPR complex. Here, the manipulation or correction
of the target gene or nucleic acid includes all of the stages of i)
cleaving or damaging the target gene or nucleic acid and ii)
repairing the damaged target gene or nucleic acid.
[0896] i) Cleavage or Damage of Target Gene or Nucleic Acid
[0897] i) The cleavage or damage of the target gene or nucleic acid
may be cleavage or damage of the target gene or nucleic acid using
the CRISPR complex, and particularly, cleavage or damage of a
target sequence in the target gene or nucleic acid.
[0898] In one example, the cleavage or damage of the target gene or
nucleic acid using the CRISPR complex may be complete cleavage or
damage to the double strand of a target sequence.
[0899] In one exemplary embodiment, when wild-type SpCas9 is used,
the double strand of a target sequence forming a complementary bond
with gRNA may be completely cleaved.
[0900] In another exemplary embodiment, when SpCas9 nickase (D10A)
and SpCas9 nickase (H840A) are used, a complementary single strand
of a target sequence forming a complementary bond with gRNA may be
cleaved by the SpCas9 nickase (D10A), and a non-complementary
single strand of the target sequence forming a complementary bond
with gRNA may be cleaved by the SpCas9 nickase (H840A), and the
cleavages may take place sequentially or simultaneously.
[0901] In still another exemplary embodiment, when SpCas9 nickase
(D10A) and SpCas9 nickase (H840A), and two gRNAs having different
target sequences are used, a complementary single strand of a
target sequence forming a complementary bond with the first gRNA
may be cleaved by the SpCas9 nickase (D10A), a non-complementary
single strand of a target sequence forming a complementary bond
with the second gRNA may be cleaved by the SpCas9 nickase (H840A),
and the cleavages may take place sequentially or
simultaneously.
[0902] In another example, the cleavage or damage of a target gene
or nucleic acid using the CRISPR complex may be cleavage or damage
to only the single strand of a target sequence. Here, the single
strand may be a complementary single strand of a target sequence
forming a complementary bond with gRNA, or a non-complementary
single strand of the target sequence forming a complementary bond
with gRNA.
[0903] In one exemplary embodiment, when SpCas9 nickase (D10A) is
used, a complementary single strand of a target sequence forming a
complementary bond with gRNA may be cleaved by the SpCas9 nickase
(D10A), but a non-complementary single strand of the target
sequence forming a complementary bond with gRNA may not be
cleaved.
[0904] In another exemplary embodiment, when SpCas9 nickase (H840A)
is used, a complementary single strand of a target sequence forming
a complementary bond with gRNA may be cleaved by the SpCas9 nickase
(H840A), but a non-complementary single strand of the target
sequence forming a complementary bond with gRNA may not be
cleaved.
[0905] In yet another example, the cleavage or damage of a target
gene or nucleic acid using the CRISPR complex may be partial
removal of a nucleic acid fragment.
[0906] In one exemplary embodiment, when two gRNAs having different
target sequences and wild-type SpCas9 are used, a double strand of
a target sequence forming a complementary bond with the first gRNA
may be cleaved, and a double strand of a target sequence forming a
complementary bond with the second gRNA may be cleaved, resulting
in the removal of nucleic acid fragments by the first and second
gRNAs and SpCas9.
[0907] In another exemplary embodiment, when two gRNAs having
different target sequences, wild-type SpCas9, SpCas9 nickase (D10A)
and SpCas9 nickase (H840A) are used, a double strand of a target
sequence forming a complementary bond with the first gRNA may be
cleaved by the wild-type SpCas9, a complementary single strand of a
target sequence forming a complementary bond with the second gRNA
may be cleaved by the SpCas9 nickase (D10A), and a
non-complementary single strand nay be cleaved by the SpCas9
nickase (H840A), resulting in the removal of nucleic acid fragments
by the first and second gRNAs, the wild-type SpCas9, the SpCas9
nickase (D10A) and the SpCas9 nickase (H840A).
[0908] In still another exemplary embodiment, when two gRNAs having
different target sequences, SpCas9 nickase (D10A) and SpCas9
nickase (H840A) are used, a complementary single strand of a target
sequence forming a complementary bond with the first gRNA may be
cleaved by the SpCas9 nickase (D10A), a non-complementary single
strand may be cleaved by the SpCas9 nickase (H840A), a
complementary double strand of a target sequence forming a
complementary bond with the second gRNA may be cleaved by the
SpCas9 nickase (D10A), and a non-complementary single strand may be
cleaved by the SpCas9 nickase (H840A), resulting in the removal of
nucleic acid fragments by the first and second gRNAs, the SpCas9
nickase (D10A) and the SpCas9 nickase (H840A).
[0909] In yet another exemplary embodiment, when three gRNAs having
different target sequences, wild-type SpCas9, SpCas9 nickase (D10A)
and SpCas9 nickase (H840A) are used, a double strand of a target
sequence forming a complementary bond with the first gRNA may be
cleaved by the wild-type SpCas9, a complementary single strand of a
target sequence forming a complementary bond with the second gRNA
may be cleaved by the SpCas9 nickase (D10A), and a
non-complementary single strand of a target sequence forming a
complementary bond with the third gRNA may be cleaved by the SpCas9
nickase (H840A), resulting in the removal of nucleic acid fragments
by the first gRNA, the second gRNA, the third gRNA, the wild-type
SpCas9, the SpCas9 nickase (D10A) and the SpCas9 nickase
(H840A).
[0910] In yet another exemplary embodiment, when four gRNAs having
different target sequences, SpCas9 nickase (D10A) and SpCas9
nickase (H840A) are used, a complementary single strand of a target
sequence forming a complementary bond with the first gRNA may be
cleaved by the SpCas9 nickase (D10A), a non-complementary single
strand of a target sequence forming a complementary bond with the
second gRNA may be cleaved by the SpCas9 nickase (H840A), a
complementary single strand of a target sequence forming a
complementary bond with the third gRNA may be cleaved by the SpCas9
nickase (D10A), and a non-complementary single strand of a target
sequence forming a complementary bond with fourth gRNA may be
cleaved by the SpCas9 nickase (H840A), resulting in the removal of
nucleic acid fragments by the first gRNA, the second gRNA, the
third gRNA, the fourth gRNA, the SpCas9 nickase (D10A) and the
SpCas9 nickase (H840A).
[0911] ii) Repair or Restoration of Damaged Target Gene or Nucleic
Acid
[0912] The target gene or nucleic acid cleaved or damaged by the
CRISPR complex may be repaired or restored through NHEJ and
homology-directed repairing (HDR).
[0913] Non-Homologous End Joining (NHEJ)
[0914] NHEJ is a method of restoration or repairing double strand
breaks in DNA by joining both ends of a cleaved double or single
strand together, and generally, when two compatible ends formed by
breaking of the double strand (for example, cleavage) are
frequently in contact with each other to completely join the two
ends, the broken double strand is recovered. The NHEJ is a
restoration method that is able to be used in the entire cell
cycle, and usually occurs when there is no homologous genome to be
used as a template in cells, like the G1 phase.
[0915] In the repair process of the damaged gene or nucleic acid
using NHEJ, some insertions and/or deletions (indels) in the
nucleic acid sequence occur in the NHEJ-repaired region, such
insertions and/or deletions cause the leading frame to be shifted,
resulting in frame-shifted transcriptome mRNA. As a result, innate
functions are lost because of nonsense-mediated decay or the
failure to synthesize normal proteins. In addition, while the
leading frame is maintained, mutations in which insertion or
deletion of a considerable amount of sequence may be caused to
destroy the functionality of the proteins. The mutation is
locus-dependent because mutations in a significant functional
domain is probably less tolerated than mutations in a
non-significant region of a protein.
[0916] While it is impossible to expect indel mutations produced by
NHEJ in a natural state, a specific indel sequence is preferred in
a given broken region, and can come from a small region of micro
homology. Conventionally, the deletion length ranges from 1 bp to
50 bp, insertions tend to be shorter, and frequently include a
short repeat sequence directly surrounding a broken region.
[0917] In addition, the NHEJ is a process causing a mutation, and
when it is not necessary to produce a specific final sequence, may
be used to delete a motif of the small sequence.
[0918] A specific knockout of a gene targeted by the CRISPR complex
may be performed using such NHEJ. A double strand or two single
strands of a target gene or nucleic acid may be cleaved using the
CRISPR enzyme such as Cas9 or Cpf1, and the broken double strand or
two single strands of the target gene or nucleic acid may have
indels through the NHEJ, thereby inducing specific knockout of the
target gene or nucleic acid. Here, the site of a target gene or
nucleic acid cleaved by the CRISPR enzyme may be a non-coding or
coding region, and in addition, the site of the target gene or
nucleic acid restored by NHEJ may be a non-coding or coding
region.
[0919] Homology Directed Repairing (HDR)
[0920] HDR is a correction method without an error, which uses a
homologous sequence as a template to repair or restoration a
damaged gene or nucleic acid, and generally, to repair or
restoration broken DNA, that is, to restore innate information of
cells, the broken DNA is repaired using information of a
complementary base sequence which is not modified or information of
a sister chromatid. The most common type of HDR is homologous
recombination (HR). HDR is a repair or restoration method usually
occurring in the S or G2/M phase of actively dividing cells.
[0921] To repair or restore damaged DNA using HDR, rather than
using a complementary base sequence or sister chromatin of the
cells, a DNA template artificially synthesized using information of
a complementary base sequence or homologous base sequence, that is,
a nucleic acid template including a complementary base sequence or
homologous base sequence may be provided to the cells, thereby
repairing the broken DNA. Here, when a nucleic acid sequence or
nucleic acid fragment is further added to the nucleic acid template
to repair the broken DNA, the nucleic acid sequence or nucleic acid
fragment further added to the broken DNA may be subjected to
knockin. The further added nucleic acid sequence or nucleic acid
fragment may be a nucleic acid sequence or nucleic acid fragment
for correcting the target gene or nucleic acid modified by a
mutation to a normal gene or nucleic acid, or a gene or nucleic
acid to be expressed in cells, but the present invention is not
limited thereto.
[0922] In one example, a double or single strand of a target gene
or nucleic acid may be cleaved using the CRISPR complex, a nucleic
acid template including a base sequence complementary to a base
sequence adjacent to the cleavage site may be provided to cells,
and the cleaved base sequence of the target gene or nucleic acid
may be repaired or restored through HDR.
[0923] Here, the nucleic acid template including the complementary
base sequence may have broken DNA, that is, a cleaved double or
single strand of a complementary base sequence, and further include
a nucleic acid sequence or nucleic acid fragment to be inserted
into the broken DNA. An additional nucleic acid sequence or nucleic
acid fragment may be inserted into a cleaved site of the broken
DNA, that is, the target gene or nucleic acid using the nucleic
acid template including a nucleic acid sequence or nucleic acid
fragment to be inserted into the complementary base sequence. Here,
the nucleic acid sequence or nucleic acid fragment to be inserted
and the additional nucleic acid sequence or nucleic acid fragment
may be a nucleic acid sequence or nucleic acid fragment for
correcting a target gene or nucleic acid modified by a mutation to
a normal gene or nucleic acid or a gene or nucleic acid to be
expressed in cells. The complementary base sequence may be a base
sequence having complementary bonds with broken DNA, that is, right
and left base sequences of the cleaved double or single strand of
the target gene or nucleic acid. Alternatively, the complementary
base sequence may be a base sequence having complementary bonds
with broken DNA, that is, 3' and 5' ends of the cleaved double or
single strand of the target gene or nucleic acid. The complementary
base sequence may be a 15 to 3000-base sequence, a length or size
of the complementary base sequence may be suitably designed
according to a size of the nucleic acid template or the target
gene. Here, as the nucleic acid template, a double- or
single-stranded nucleic acid may be used, or it may be linear or
circular, but the present invention is not limited thereto.
[0924] In another example, a double- or single-stranded target gene
or nucleic acid is cleaved using the CRISPR complex, a nucleic acid
template including a homologous base sequence with a base sequence
adjacent to a cleavage site is provided to cells, and the cleaved
base sequence of the target gene or nucleic acid may be repaired or
restored by HDR.
[0925] Here, the nucleic acid template including the homologous
base sequence may be broken DNA, that is, a cleaved double- or
single-stranded homologous base sequence, and further include a
nucleic acid sequence or nucleic acid fragment to be inserted into
the broken DNA. An additional nucleic acid sequence or nucleic acid
fragment may be inserted into broken DNA, that is, a cleaved site
of a target gene or nucleic acid using the nucleic acid template
including a homologous base sequence and a nucleic acid sequence or
nucleic acid fragment to be inserted. Here, the nucleic acid
sequence or nucleic acid fragment to be inserted and the additional
nucleic acid sequence or nucleic acid fragment may be a nucleic
acid sequence or nucleic acid fragment for correcting a target gene
or nucleic acid modified by a mutation to a normal gene or nucleic
acid or a gene or nucleic acid to be expressed in cells. The
homologous base sequence may be broken DNA, that is, a base
sequence having homology with cleaved double-stranded base sequence
or right and left single-stranded base sequences of a target gene
or nucleic acid. Alternatively, the complementary base sequence may
be a base sequence having homology with broken DNA, that is, the 3'
and 5' ends of a cleaved double or single strand of a target gene
or nucleic acid. The homologous base sequence may be a 15 to
3000-base sequence, and a length or size of the homologous base
sequence may be suitably designed according to a size of the
nucleic acid template or a target gene or nucleic acid. Here, as
the nucleic acid template, a double- or single-stranded nucleic
acid may be used and may be linear or circular, but the present
invention is not limited thereto.
[0926] Other than the NHEJ and HDR, there are methods of repairing
or restoring broken DNA.
[0927] Single-Strand Annealing (SSA)
[0928] SSA is a method of repairing double strand breaks between
two repeat sequences present in a target nucleic acid, and
generally uses a repeat sequence of more than 30 bases. The repeat
sequence is cleaved (to have sticky ends) to have a single strand
with respect to a double strand of the target nucleic acid at each
of the broken ends, and after the cleavage, a single-strand
overhang containing the repeat sequence is coated with an RPA
protein such that it is prevented from inappropriately annealing
the repeat sequences to each other. RAD52 binds to each repeat
sequence on the overhang, and a sequence capable of annealing a
complementary repeat sequence is arranged. After annealing, a
single-stranded flap of the overhang is cleaved, and synthesis of
new DNA fills a certain gap to restore a DNA double strand. As a
result of this repair, a DNA sequence between two repeats is
deleted, and a deletion length may be dependent on various factors
including the locations of the two repeats used herein, and a path
or degree of the progress of cleavage.
[0929] SSA, similar to HDR, utilizes a complementary sequence, that
is, a complementary repeat sequence, and in contrast, does not
requires a nucleic acid template for modifying or correcting a
target nucleic acid sequence.
[0930] Single-Strand Break Repair (SSBA)
[0931] Single strand breaks in a genome are repaired through a
separate mechanism, SSBR, from the above-described repair
mechanisms. In the case of single-strand DNA breaks, PARP1 and/or
PARP2 recognizes the breaks and recruits a repair mechanism. PARP1
binding and activity with respect to the DNA breaks are temporary,
and SSBR is promoted by promoting the stability of an SSBR protein
complex in the damaged regions. The most important protein in the
SSBR complex is XRCC1, which interacts with a protein promoting 3'
and 5' end processing of DNA to stabilize the DNA. End processing
is generally involved in repairing the damaged 3' end to a
hydroxylated state, and/or the damaged 5' end to a phosphatic
moiety, and after the ends are processed, DNA gap filling takes
place. There are two methods for the DNA gap filling, that is,
short patch repair and long patch repair, and the short patch
repair involves insertion of a single base. After DNA gap filling,
a DNA ligase promotes end joining.
[0932] Mismatch Repair (MMR)
[0933] MMR works on mismatched DNA bases. Each of an MSH2/6 or
MSH2/3 complex has ATPase activity and thus plays an important role
in recognizing a mismatch and initiating a repair, and the MSH2/6
primarily recognizes base-base mismatches and identifies one or two
base mismatches, but the MSH2/3 primarily recognizes a larger
mismatch.
[0934] Base Excision Repair (BER)
[0935] BER is a repair method which is active throughout the entire
cell cycle, and used to remove a small non-helix-distorting base
damaged region from the genome. In the damaged DNA, damaged bases
are removed by cleaving an N-glycoside bond joining a base to the
phosphate-deoxyribose backbone, and then the phosphodiester
backbone is cleaved, thereby generating breaks in single-strand
DNA. The broken single strand ends formed thereby were removed, a
gap generated due to the removed single strand is filled with a new
complementary base, and then an end of the newly-filled
complementary base is ligated with the backbone by a DNA ligase,
resulting in repair of the damaged DNA.
[0936] Nucleotide Excision Repair (NER)
[0937] NER is an excision mechanism important for removing large
helix-distorting damage from DNA, and when the damage is
recognized, a short single-strand DNA segment containing the
damaged region is removed, resulting in a single strand gap of 22
to 30 bases. The generated gap is filled with a new complementary
base, and an end of the newly filled complementary base is ligated
with the backbone by a DNA ligase, resulting in the repair of the
damaged DNA.
[0938] Gene Manipulation Effects
[0939] Manipulation or correction of a target gene or nucleic acid
may largely lead to effects of knockout, knockdown, and
knockin.
[0940] Knockout
[0941] The term "knockout" refers to inactivation of a target gene
or nucleic acid, and the "inactivation of a target gene or nucleic
acid" refers to a state in which transcription and/or translation
of a target gene or nucleic acid does not occur. Transcription and
translation of a gene causing a disease or a gene having an
abnormal function may be inhibited through knockout, resulting in
the prevention of protein expression.
[0942] For example, when a target gene or nucleic acid is edited or
corrected using a gRNA-CRISPR enzyme complex, that is, a CRISPR
complex, the target gene or nucleic acid may be cleaved using the
CRISPR complex. The damaged target gene or nucleic acid may be
repaired through NHEJ using the CRISPR complex. The damaged target
gene or nucleic acid may have indels due to NHEJ, and thereby,
specific knockout for the target gene or nucleic acid may be
induced.
[0943] Knockdown
[0944] The term "knockdown" refers to a decrease in transcription
and/or translation of a target gene or nucleic acid or the
expression of a target protein. The onset of a disease may be
prevented or a disease may be treated by regulating the
overexpression of a gene or protein through the knockdown.
[0945] For example, when a target gene or nucleic acid is edited or
corrected using a gRNA-CRISPR inactive enzyme-transcription
inhibitory activity domain complex, that is, a CRISPR inactive
complex including a transcription inhibitory activity domain, the
CRISPR inactive complex may specifically bind to the target gene or
nucleic acid, transcription of the target gene or nucleic acid may
be inhibited by the transcription inhibitory activity domain
included in the CRISPR inactive complex, thereby inducing knockdown
in which expression of the corresponding gene or nucleic acid is
inhibited.
[0946] Knockin
[0947] The term "knockin" refers to insertion of a specific nucleic
acid or gene into a target gene or nucleic acid, and here, the
"specific nucleic acid" refers to a gene or nucleic acid of
interest to be inserted or expressed. A mutant gene triggering a
disease may be utilized in disease treatment by correction to
normal or insertion of a normal gene to induce expression of the
normal gene through the knockin.
[0948] In addition, the knockin may further need a donor.
[0949] For example, when a target gene or nucleic acid is edited or
corrected using a gRNA-CRISPR enzyme complex, that is, a CRISPR
complex, the target gene or nucleic acid may be cleaved using the
CRISPR complex. The target gene or nucleic acid damaged using the
CRISPR complex may be repaired through HDR. Here, a specific
nucleic acid may be inserted into the damaged gene or nucleic acid
using a donor.
[0950] The term "donor" refers to a nucleic acid sequence that
helps HDR-based repair of the damaged gene or nucleic acid, and
here, the donor may include a specific nucleic acid.
[0951] The donor may be a double- or single-stranded nucleic
acid.
[0952] The donor may be present in a linear or circular shape.
[0953] The donor may include a nucleic acid sequence having
homology with a target gene or nucleic acid.
[0954] For example, the donor may include a nucleic acid sequence
having homology with each of base sequences at a location into
which a specific nucleic acid is to be inserted, for example,
upstream (left) and downstream (right) of a damaged nucleic acid.
Here, the specific nucleic acid to be inserted may be located
between a nucleic acid sequence having homology with a base
sequence downstream of the damaged nucleic acid and a nucleic acid
sequence having homology with a base sequence upstream of the
damaged nucleic acid. Here, the homologous nucleic acid sequence
may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or
95% or more homology or complete homology.
[0955] The donor may optionally include an additional nucleic acid
sequence. Here, the additional nucleic acid sequence may serve to
increase donor stability, knockin efficiency or HDR efficiency.
[0956] For example, the additional nucleic acid sequence may be an
A, T-rich nucleic acid sequence, that is, an A-T rich domain. In
addition, the additional nucleic acid sequence may be a
scaffold/matrix attachment region (SMAR).
[0957] In one exemplary embodiment relating to a gene manipulation
effect of the present invention, a manipulated target gene obtained
using a gRNA-CRISPR enzyme complex, that is, a manipulated
neovascularization-associated factor may have the following
constitution.
[0958] In one exemplary embodiment, when the
neovascularization-associated factor is a gene, the constitution of
the artificially manipulated neovascularization-associated factor
by the gRNA-CRISPR enzyme complex may include modification of one
or more nucleic acids among a deletion or insertion of one or more
nucleotides; a substitution with one or more nucleotides different
from a wild-type gene; and an insertion of one or more foreign
nucleotides in a continuous 1 bp to 50 bp, 1 bp to 40 bp or 1 bp to
30 bp, preferably, 3 bp to 25 bp region in the base sequence, which
is located in a PAM sequence in a nucleic acid sequence
constituting the neovascularization-associated factor or adjacent
to a 5' end and/or 3' end thereof.
[0959] In addition, a chemical modification of one or more
nucleotides may be included in the nucleic acid sequence
constituting the neovascularization-associated factor.
[0960] Here, the "foreign nucleotide" is the concept including all
exogeneous, for example, heterologous or artificially-synthesized
nucleotides, other than nucleotides innately included in the
neovascularization-associated factor. The foreign nucleotide also
includes a nucleotide with a size of several hundred, thousand or
tens of thousands of bp to express a protein having a specific
function, as well as a small ologonucleotide with a size of 50 bp
or less. Such a foreign nucleotide may be a donor.
[0961] The chemical modification may include methylation,
acetylation, phosphorylation, ubiquitination, ADP-ribosylation,
myristylation, and glycosylation, for example, substitution of some
functional groups contained in a nucleotide with any one of a
hydrogen atom, a fluorine atom, an --O-alkyl group, an --O-acyl
group, and an amino group, but the present invention is not limited
thereto. In addition, to increase transferability of a nucleic acid
molecule, the functional groups may also be substituted with any
one of --Br, --Cl, --R, --R'OR, --SH, --SR, --N3 and --CN (R=alkyl,
aryl, alkylene). In addition, the phosphate backbone of at least
one nucleotide may be substituted with any one of an
alkylphosphonate form, a phosphoroamidate form and a
boranophosphate form. In addition, the chemical modification may be
a substitution of at least one type of nucleotide contained in the
nucleic acid molecule with any one of a locked nucleic acid (LNA),
an unlocked nucleic acid (UNA), a morpholino, and a peptide nucleic
acid (PNA), and the chemical modification may be bonding of the
nucleic acid molecule with one or more selected from the group
consisting of a lipid, a cell-penetrating peptide and a cell-target
ligand.
[0962] To form a desired neovascularization regulating system,
artificial modification using a gRNA-CRISPR enzyme complex may be
applied to the nucleic acid constituting the
neovascularization-associated factor.
[0963] A region including the nucleic acid modification of the
neovascularization-associated factor may be a target region or
target sequence.
[0964] Such a target sequence may be a target for the gRNA-CRISPR
enzyme complex, and the target sequence may include or not include
a PAM sequence recognized by the CRISPR enzyme. Such a target
sequence may provide a critical standard in a gRNA designing stage
to those of ordinary skill in the art.
[0965] Such nucleic acid modification includes the "cleavage" of a
nucleic acid.
[0966] The term "cleavage" in a target region refers to breakage of
a covalent backbone of polynucleotides. The cleavage includes
enzymatic or chemical hydrolysis of a phosphodiester bond, but the
present invention is not limited thereto, and also include various
other methods. The cleavage is able to be performed on both of a
single strand and a double strand, and the cleavage of a double
strand may result from distinct single-strand cleavage. The
double-strand cleavage may generate blunt ends or staggered
ends.
[0967] When an inactivated CRISPR enzyme is used, it may induce a
factor possessing a specific function to approach a certain region
of the target region or neovascularization-associated factor
without the cleavage process. Chemical modification of one or more
nucleotides in the nucleic acid sequence of the
neovascularization-associated factor may be included according to
such a specific function.
[0968] In one example, various indels may occur due to target and
non-target activities through the nucleic acid cleavage formed by
the gRNA-CRISPR enzyme complex.
[0969] The term "indel" is the generic term for an insertion or
deletion mutation occurring in-between some bases in a DNA base
sequence. The indel may be introduced into a target sequence during
repair by an HDR or NHEJ mechanism when the gRNA-CRISPR enzyme
complex cleaves the nucleic acid (DNA or RNA) of the
neovascularization-associated factor as described above.
[0970] The artificially manipulated neovascularization-associated
factor of the present invention refers to modification of the
nucleic acid sequence of an original gene by cleavage, indels, or
insertion using a donor of such a nucleic acid, and contributes to
a desired neovascularization regulating system, for example,
exhibition of an effect of promoting or suppressing
neovascularization.
[0971] For example, a specific protein may be expressed and its
activity may be stimulated by the artificially manipulated
neovascularization-associated factor.
[0972] A specific protein may be inactivated by the artificially
manipulated neovascularization-associated factor.
[0973] In one example, a specific target region of each
neovascularization-associated factor of the genome, for example,
reverse regulatory genes such as a VEGFA gene, an HIF1A gene, an
ANGPT2 gene, an EPAS1 gene, and/or an ANGPTL4 gene may be cleaved,
resulting in knockdown or knockout of the gene.
[0974] In another example, targeted knockdown may be mediated using
an enzymatically inactive CRISPR enzyme fused to a transcription
repressor domain or chromatin-modified protein to change
transcription, for example, to block, negatively regulate or
decrease the transcription of a VEGFA gene, an HIF1A gene, an
ANGPT2 gene, an EPAS1 gene, and/or an ANGPTL4 gene.
[0975] The neovascularization may be regulated by the artificially
manipulated neovascularization-associated factor.
[0976] A neovascularization-associated disease may be improved or
treated by the artificially manipulated
neovascularization-associated factor.
[0977] In one exemplary embodiment of the present invention, the
artificially manipulated neovascularization-associated factor may
provide various artificially manipulated
neovascularization-associated factors according to the
constitutional characteristic of the gRNA-CRISPR enzyme complex
(e.g., included in a target region of the
neovascularization-associated factor or different in the adjacent
major PAM sequence).
[0978] Hereinafter, while representative examples of CRISPR enzymes
and a neovascularization-regulatory gene have been illustrated,
they are merely specific examples, and thus the present invention
is not limited thereto.
[0979] For example, when the CRISPR enzyme is a SpCas9 protein, the
PAM sequence is 5'-NGG-3' (N is A, T, G, or C), and the cleaved
base sequence region (target region) may be a continuous 1 bp to 25
bp, for example, 17 bp to 23 bp or 21 bp to 23 bp, region in the
base sequence adjacent to the 5' end and/or 3' end of the 5'-NGG-3'
sequence in a target gene.
[0980] The present invention may provide an artificially
manipulated neovascularization-associated factor, for example, an
artificially manipulated VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1
gene, and/or ANGPTL4 gene, which is prepared by [0981] a) deletion
of one or more nucleotides of a continuous 1 bp to 25 bp, for
example, 17 bp to 23 bp, region in the base sequence adjacent to
the 5' end and/or 3' end of the 5'-NGG-3' (N is A, T, C or G)
sequence, [0982] b) substitution of one or more nucleotides of a
continuous 1 bp to 25 bp, for example, 17 bp to 23 bp, region in
the base sequence adjacent to the 5' end and/or 3' end of the
5'-NGG-3' sequence with nucleotides different from those of the
wild-type gene, [0983] c) insertion of one or more nucleotides into
a continuous 1 bp to 25 bp, for example, 17 bp to 23 bp, region in
the base sequence adjacent to the 5' end and/or 3' end of the
5'-NGG-3' sequence, or [0984] d) a combination of two or more
selected from a) through c) [0985] in the nucleic acid sequence of
the neovascularization-associated factor.
[0986] For example, when the CRISPR enzyme is a CjCas9 protein, the
PAM sequence is 5'-NNNNRYAC-3' (each N is independently A, T, C or
G, R is A or G, and Y is C or T), and the cleaved base sequence
region (target region) may be a continuous 1 bp to 25 bp, for
example, 17 bp to 23 bp or 21 bp to 23 bp, region in the base
sequence adjacent to the 5' end and/or 3' end of the 5'-NNNNRYAC-3'
sequence in a target gene.
[0987] The present invention may provide an artificially
manipulated neovascularization-associated factor, for example, an
artificially manipulated VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1
gene, and/or ANGPTL4 gene, which is prepared by [0988] a') deletion
of one or more nucleotides of a continuous 1 bp to 25 bp, for
example, 17 bp to 23 bp, region in the base sequence adjacent to
the 5' end and/or 3' end of the 5'-NNNNRYAC-3' (each N is
independently A, T, C or G, R is A or G, and Y is C or T), [0989]
b') substitution of one or more nucleotides of a continuous 1 bp to
25 bp, for example, 17 bp to 23 bp, region in the base sequence
adjacent to the 5' end and/or 3' end of the 5'-NNNNRYAC-3' sequence
with nucleotides different from those of the wild-type gene, [0990]
c') insertion of one or more nucleotides into a continuous 1 bp to
25 bp, for example, 17 bp to 23 bp, region in the base sequence
adjacent to the 5' end and/or 3' end of the 5'-NNNNRYAC-3'
sequence, or [0991] d') a combination of two or more selected from
a') through c') in the nucleic acid sequence of the
neovascularization-associated factor.
[0992] For example, when the CRISPR enzyme is a StCas9 protein, the
PAM sequence is 5'-NNAGAAW-3' (each N is independently A, T, C or
G, and W is A or T), and the cleaved base sequence region (target
region) may be a continuous 1 bp to 25 bp, for example, 17 bp to 23
bp or 21 bp to 23 bp, region in the base sequence adjacent to the
5' end and/or 3' end of the 5'-NNAGAAW-3' sequence in a target
gene.
[0993] The present invention may provide an artificially
manipulated neovascularization-associated factor, for example, an
artificially manipulated VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1
gene, and/or ANGPTL4 gene, which is prepared by [0994] a'')
deletion of one or more nucleotides of a continuous 1 bp to 25 bp,
for example, 17 bp to 23 bp, region in the base sequence adjacent
to the 5' end and/or 3' end of the 5'-NNAGAAW-3' sequence (each N
is independently A, T, C or G, and W is A or T), [0995] b'')
substitution of one or more nucleotides of a continuous 1 bp to 25
bp, for example, 17 bp to 23 bp, region in the base sequence
adjacent to the 5' end and/or 3' end of the 5'-NNAGAAW-3' sequence
with nucleotides different from those of the wild-type gene, [0996]
c'') insertion of one or more nucleotides into a continuous 1 bp to
25 bp, for example, 17 bp to 23 bp, region in the base sequence
adjacent to the 5' end and/or 3' end of the 5'-NNAGAAW-3' sequence,
or [0997] d'') a combination of two or more selected from a'')
through c'') in the nucleic acid sequence of the
neovascularization-associated factor.
[0998] For example, when the CRISPR enzyme is an NmCas9 protein,
the PAM sequence is 5'-NNNNGATT-3'(each N is independently A, T, C
or G), and the cleaved base sequence region (target region) may be
a continuous 1 bp to 25 bp, for example, 17 bp to 23 bp or 21 bp to
23 bp, region in the base sequence adjacent to the 5' end and/or 3'
end of the 5'-NNNNGATT-3' sequence in a target gene.
[0999] The present invention may provide an artificially
manipulated neovascularization-associated factor, for example, an
artificially manipulated VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1
gene, and/or ANGPTL4 gene, which is prepared by [1000] a''')
deletion of one or more nucleotides of a continuous 1 bp to 25 bp,
for example, 17 bp to 23 bp, region in the base sequence adjacent
to the 5' end and/or the 3' end of the 5'-NNNNGATT-3' sequence
(each N is independently A, T, C or G), [1001] b''') substitution
of one or more nucleotides of a continuous 1 bp to 25 bp, for
example, 17 bp to 23 bp, region in the base sequence adjacent to
the 5' end and/or 3' end of the 5'-NNNNGATT-3' sequence with
nucleotides different from those of the wild-type gene, [1002]
c''') insertion of one or more nucleotides into a continuous 1 bp
to 25 bp, for example, 17 bp to 23 bp, region in the base sequence
adjacent to the 5'-NNNNGATT-3' sequence, or [1003] d''') a
combination of two or more selected from a''') through c''') in the
nucleic acid sequence of the neovascularization-associated
factor.
[1004] For example, when the CRISPR enzyme is an SaCas9 protein,
the PAM sequence is 5'-NNGRR(T)-3' (each N is independently A, T, C
or G, R is A or G, and (T) is a randomly addable sequence), and the
cleaved base sequence region (target region) may be a continuous 1
bp to 25 bp, for example, 17 bp to 23 bp or 21 bp to 23 bp, region
in the base sequence adjacent to the 5' end and/or 3' end of the
5'-NNGRR(T)-3' sequence in a target gene.
[1005] The present invention may provide an artificially
manipulated neovascularization-associated factor, for example, an
artificially manipulated VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1
gene, and/or ANGPTL4 gene, which is prepared by
[1006] a'''') deletion of one or more nucleotides of a continuous 1
bp to 25 bp, for example, 17 bp to 23 bp region, in the base
sequence adjacent to the 5' end and/or the 3' end of the
5'-NNGRR(T)-3' sequence (each N is independently A, T, C or G, R is
A or G, and (T) is a randomly addable sequence), [1007] b'''')
substitution of one or more nucleotides of a continuous 1 bp to 25
bp, for example, 17 bp to 23 bp, region in the base sequence
adjacent to the 5' end and/or 3' end of the 5'-NNGRR(T)-3' sequence
with nucleotides different from those of the wild-type gene, [1008]
c'''') insertion of one or more nucleotides into a continuous 1 bp
to 25 bp, for example, 17 bp to 23 bp, region in the base sequence
adjacent to the 5'-NNGRR(T)-3' sequence, or [1009] d'''') a
combination of two or more selected from a'''') through c'''') in
the nucleic acid sequence of the neovascularization-associated
factor.
[1010] For example, when the CRISPR enzyme is a Cpf1 protein, the
PAM sequence is 5'-TTN-3' (N is A, T, C or G), and the cleaved base
sequence region (target region) may be a continuous 10 bp to 30 bp,
for example, 15 bp to 26 bp, 17 bp to 30 bp or 17 bp to 26 bp,
region in the base sequence adjacent to the 5' end or the 3' end of
the 5'-TTN-3' sequence.
[1011] The Cpf1 protein may be derived from a microorganism such as
Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae
bacterium (MC2017), Butyrivibrio proteoclasiicus, Peregrinibacteria
bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6),
Porphyromonas macacae, Lachnospiraceae bacterium (ND2006),
Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi
(237), Smiihella sp. (SC_KO8D17), Leptospira inadai,
Lachnospiraceae bacterium (MA2020), Francisella novicida (U112),
Candidatus Methanoplasma termitum, or Eubacterium eligens, for
example, Parcubacteria bacterium (GWC2011_GWC2_44_17),
Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp.
(BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006),
Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi
(237), Leptospira inadai, Lachnospiraceae bacterium (MA2020),
Francisella novicida (U112), Candidatus Methanoplasma termitum, or
Eubacterium eligens, but the present invention is not limited
thereto.
[1012] The present invention may provide an artificially
manipulated neovascularization-associated factor, for example, an
artificially manipulated VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1
gene, and/or ANGPTL4 gene, which is prepared by [1013] a'''''')
deletion of one or more nucleotides of a continuous 10 bp to 30 bp,
for example, 15 bp to 26 bp, region in the base sequence adjacent
to the 5' end and/or the 3' end of the 5'-TTN-3' sequence (N is A,
T, C or G), [1014] b'''''') substitution of one or more nucleotides
of a continuous 10 bp to 30 bp, for example, 15 bp to 26 bp, region
in the base sequence adjacent to the 5' end and/or 3' end of the
5'-TTN-3' sequence with nucleotides different from those of the
wild-type gene, [1015] c'''''') insertion of one or more
nucleotides of a continuous 10 bp to 30 bp, for example, 15 bp to
26 bp, region in the base sequence adjacent to the 5' end and/or 3'
end of the 5'-TTN-3' sequence, or [1016] d'''''') a combination of
two or more selected from a'''''') through c'''''') in the nucleic
acid sequence of the neovascularization-associated factor.
[1017] In another exemplary embodiment, when the
neovascularization-associated factor is a protein, the artificially
manipulated protein includes all proteins involved in formation of
new or modified blood vessels by a direct or indirect action of the
gRNA-CRISPR enzyme complex.
[1018] For example, the artificially manipulated protein may be a
protein expressed by a neovascularization-associated factor (gene)
artificially manipulated by the gRNA-CRISPR enzyme complex or
another protein increased or reduced by an influence by such
protein activity, but the present invention is not limited
thereto.
[1019] The artificially manipulated neovascularization-associated
factor (protein) may have an amino acid composition and activity
corresponding to the composition of the artificially manipulated
neovascularization-associated factor (gene).
[1020] As an embodiment, an (i) artificially manipulated protein
which is changed in expression characteristics may be provided.
[1021] For example, protein modification may have one or more
characteristics: [1022] a decrease or increase in expression level
according to the deletion or insertion of one or more nucleotides
in a continuous 1 bp to 50 bp, 1 bp to 40 bp, 1 bp to 30 bp, and
preferably 3 bp to 25 bp region in the base sequence of the PAM
sequence in the nucleic acid sequence of the
neovascularization-associated factor or adjacent to the 5' end
and/or the 3' end thereof; [1023] a decrease or increase in
expression level according to the substitution with one or more
nucleotides different from those of a wild-type gene; [1024] a
decrease or increase in expression level, expression of a fusion
protein or independent expression of a specific protein according
to the insertion of one or more foreign nucleotides; and [1025] a
decrease or increase in expression level of a third protein
influenced by expression characteristics of the above-described
proteins.
[1026] An (ii) artificially manipulated protein which is changed in
structural characteristics may be provided.
[1027] For example, protein modification may have one or more
characteristics: [1028] a change in codons, amino acids and
three-dimensional structure according to the deletion or insertion
of one or more nucleotides in a continuous 1 bp to 50 bp, 1 bp to
40 bp, 1 bp to 30 bp, and preferably 3 bp to 25 bp region in the
base sequence of the PAM sequence in the nucleic acid sequence of
the neovascularization-associated factor or adjacent to the 5' end
and/or the 3' end thereof; [1029] a change in codons, amino acids,
and three-dimensional structure thereby according to the
substitution with one or more nucleotides different from a
wild-type gene; [1030] a change in codons, amino acids, and
three-dimensional structure, or a fusion structure with a specific
protein or independent structure from which a specific protein is
separated according to the insertion of one or more foreign
nucleotides; and [1031] a change in codons, amino acids, and
three-dimensional structure of a third protein influenced by the
above-described protein changed in structural characteristic.
[1032] An (iii) artificially manipulated protein changed in
functional characteristics may be provided.
[1033] For example, protein modification may have one or more
characteristics: [1034] the activation or inactivation of a
specific function or introduction of a new neovascularization
function by protein modification caused by a deletion or insertion
of one or more nucleotides in a continuous 1 bp to 50 bp, 1 bp to
40 bp, 1 bp to 30 bp, and preferably 3 bp to 25 bp region in the
base sequence of the PAM sequence in the nucleic acid sequence of
the neovascularization-associated factor or adjacent to the 5' end
and/or the 3' end thereof; [1035] the activation or inactivation of
a specific function or introduction of a new function by protein
modification caused by substitution with one or more nucleotides
different from those of a wild-type gene; [1036] the activation or
inactivation of a specific function or introduction of a new
function by protein modification caused by insertion of one or more
foreign nucleotides, particularly, introduction of a third function
to an existing function due to fusion or independent expression of
a specific protein; and [1037] the change in the function of a
third protein influenced by the above-described protein changed in
functional characteristics.
[1038] In addition, a protein artificially manipulated by the
chemical modification of one or more nucleotides in the nucleic
acid sequence constituting the neovascularization-associated factor
may be included.
[1039] For example, one or more of the expression, structural and
functional characteristics of a protein caused by methylation,
acetylation, phosphorylation, ubiquitination, ADP-ribosylation,
myristylation and glycosylation may be changed.
[1040] For example, the third structure and function may be
achieved by binding of a third protein into the nucleic acid
sequence of the gene due to the chemical modification of
nucleotides.
[1041] 5. Other Additional Components
[1042] An additional component may be selectively added to increase
the efficiency of a guide nucleic acid-editor protein complex or
improve the repair efficiency of a damaged gene or nucleic
acid.
[1043] The additional component may be selectively used to improve
the efficiency of the guide nucleic acid-editor protein
complex.
[1044] Activator
[1045] The additional component may be used as an activator to
increase the cleavage efficiency of a target nucleic acid, gene or
chromosome of the guide nucleic acid-editor protein complex.
[1046] The term "activator" refers to a nucleic acid serving to
stabilize the bonding between the guide nucleic acid-editor protein
complex and the target nucleic acid, gene or chromosome, or to
allow the guide nucleic acid-editor protein complex to more easily
approach the target nucleic acid, gene or chromosome.
[1047] The activator may be a double-stranded nucleic acid or
single-stranded nucleic acid.
[1048] The activator may be linear or circular.
[1049] The activator may be divided into a "helper" that stabilizes
the bonding between the guide nucleic acid-editor protein complex
and the target nucleic acid, gene or chromosome, and an "escorter"
that serves to allow the guide nucleic acid-editor protein complex
to more easily approach the target nucleic acid, gene or
chromosome.
[1050] The helper may increase the cleavage efficiency of the guide
nucleic acid-editor protein complex with respect to the target
nucleic acid, gene or chromosome.
[1051] For example, the helper includes a nucleic acid sequence
having homology with the target nucleic acid, gene or chromosome.
Therefore, when the guide nucleic acid-editor protein complex is
bonded to the target nucleic acid, gene or chromosome, the
homologous nucleic acid sequence included in the helper may form an
additional complementary bond with the target nucleic acid, gene or
chromosome to stabilize the bonding between the guide nucleic
acid-editor protein complex and the target nucleic acid, gene or
chromosome.
[1052] The escorter may increase the cleavage efficiency of the
guide nucleic acid-editor protein complex with respect to the
target nucleic acid, gene or chromosome.
[1053] For example, the escorter includes a nucleic acid sequence
having homology with the target nucleic acid, gene or chromosome.
Here, the homologous nucleic acid sequence included in the escorter
may partly form a complementary bond with a guide nucleic acid of
the guide nucleic acid-editor protein complex. Therefore, the
escorter partly forming a complementary bond with the guide nucleic
acid-editor protein complex may partly form a complementary bond
with the target nucleic acid, gene or chromosome, and as a result,
may allow the guide nucleic acid-editor protein complex to
accurately approach the position of the target nucleic acid, gene
or chromosome.
[1054] The homologous nucleic acid sequence may have at least 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more homology, or
complete homology.
[1055] In addition, the additional component may be selectively
used to improve the repair efficiency of the damaged gene or
nucleic acid.
[1056] Assistor
[1057] The additional component may be used as an assistor to
improve the repair efficiency of the damaged gene or nucleic
acid.
[1058] The term "assistor" refers to a nucleic acid that serves to
participate in a repair process or increase the repair efficiency
of the damaged gene or nucleic acid, for example, the gene or
nucleic acid cleaved by the guide nucleic acid-editor protein
complex.
[1059] The assistor may be a double-stranded nucleic acid or
single-stranded nucleic acid.
[1060] The assistor may be present in a linear or circular
shape.
[1061] The assistor may be divided into an "NHEJ assistor" that
participates in a repair process using NHEJ or improves repair
efficiency and an "HDR assistor" that participates in a repair
process using HDR or improves repair efficiency according to a
repair method.
[1062] The NHEJ assistor may participate in a repair process or
improve the repair efficiency of the damaged gene or nucleic acid
using NHEJ.
[1063] For example, the NHEJ assistor may include a nucleic acid
sequence having homology with a part of the damaged nucleic acid
sequence. Here, the homologous nucleic acid sequence may include a
nucleic acid sequence having homology with the nucleic acid
sequence at one end (e.g., the 3' end) of the damaged nucleic acid
sequence, and include a nucleic acid sequence having homology with
the nucleic acid sequence at the other end (e.g., the 5' end) of
the damaged nucleic acid sequence. In addition, a nucleic acid
sequence having homology with each of the base sequences upstream
and downstream of the damaged nucleic acid sequence may be
included. The nucleic acid sequence having such homology may assist
two parts of the damaged nucleic acid sequence to be placed in
close proximity, thereby increasing the repair efficiency of the
damaged nucleic acid by NHEJ.
[1064] The HDR assistor may participate in the repair process or
improve repair efficiency of the damaged gene or nucleic acid using
HDR.
[1065] For example, the HDR assistor may include a nucleic acid
sequence having homology with a part of the damaged nucleic acid
sequence. Here, the homologous nucleic acid sequence may include a
nucleic acid sequence having homology with the nucleic acid
sequence at one end (e.g., the 3' end) of the damaged nucleic acid
sequence, and a nucleic acid sequence having homology with the
nucleic acid sequence at the other end (e.g., the 5' end) of the
damaged nucleic acid sequence. Alternatively, a nucleic acid
sequence having homology with each of the base sequences upstream
and downstream of the damaged nucleic acid sequence may be
included. The nucleic acid sequence having such homology may serve
as a template of the damaged nucleic acid sequence to increase the
repair efficiency of the damaged nucleic acid by HDR.
[1066] In another example, the HDR assistor may include a nucleic
acid sequence having homology with a part of the damaged nucleic
acid sequence and a specific nucleic acid, for example, a nucleic
acid or gene to be inserted. Here, the homologous nucleic acid
sequence may include a nucleic acid sequence having homology with
each of the base sequences upstream and downstream of the damaged
nucleic acid sequence. The specific nucleic acid may be located
between a nucleic acid sequence having homology with a base
sequence downstream of the damaged nucleic acid and a nucleic acid
sequence having homology with a base sequence upstream of the
damaged nucleic acid. The nucleic acid sequence having such
homology and specific nucleic acid may serve as a donor to insert a
specific nucleic acid into the damaged nucleic acid, thereby
increasing HDR efficiency for knockin.
[1067] The homologous nucleic acid sequence may have at least 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more homology or
complete homology.
[1068] 6. Subject
[1069] The term "subject" refers to an organism into which a guide
nucleic acid, editor protein or guide nucleic acid-editor protein
complex is introduced, an organism in which a guide nucleic acid,
editor protein or guide nucleic acid-editor protein complex
operates, or a specimen or sample obtained from the organism.
[1070] The subject may be an organism including a target nucleic
acid, gene, chromosome or protein of the guide nucleic acid-editor
protein complex.
[1071] The organism may be cells, tissue, a plant, an animal or a
human.
[1072] The cells may be prokaryotic cells or eukaryotic cells.
[1073] The eukaryotic cells may be plant cells, animal cells or
human cells, but the present invention is not limited thereto.
[1074] The tissue may be animal or human body tissue such as skin,
liver, kidney, heart, lung, brain or muscle tissue.
[1075] The subject may be a specimen or sample including a target
nucleic acid, gene, chromosome or protein of the guide nucleic
acid-editor protein complex.
[1076] The specimen or sample may be obtained from an organism
including a target nucleic acid, gene, chromosome or protein and
may be saliva, blood, skin tissue, cancer cells or stem cells.
[1077] In the present invention, as a specific example, the subject
may include a target gene or nucleic acid of the guide nucleic
acid-editor protein complex.
[1078] Here, the target gene may be a neovascularization-associated
factor, for example, a VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1
gene, and/or ANGPTL4 gene.
[1079] The target gene may be a wild type, or a modified form in
the wild-type.
[1080] In one exemplary embodiment of the present invention, the
subject may include a gene or nucleic acid manipulated by the guide
nucleic acid-editor protein complex.
[1081] Here, the manipulated gene may be a
neovascularization-associated factor, for example, a VEGFA gene,
HIF1A gene, ANGPT2 gene, EPAS1 gene, and/or ANGPTL4 gene.
[1082] Here, the guide nucleic acid may target a
neovascularization-associated factor, for example, a VEGFA gene,
HIF1A gene, ANGPT2 gene, EPAS1 gene, and/or ANGPTL4 gene.
[1083] The guide nucleic acid may be a nucleic acid sequence
complementary to a target sequence of the VEGFA gene, HIF1A gene,
ANGPT2 gene, EPAS1 gene, and/or ANGPTL4 gene.
[1084] The guide nucleic acid may target one or more genes.
[1085] The guide nucleic acid may simultaneously target two or more
genes. Here, the two or more genes may be homologous or
heterologous genes.
[1086] The guide nucleic acid may target one or more target
sequences.
[1087] The guide nucleic acid may be designed in various forms
according to the number or locations of the target sequences.
[1088] In one exemplary embodiment of the present invention, the
guide nucleic acid may be a nucleic acid sequence complementary to
one or more target sequences of the sequences listed in Table 1,
Table 2, Table 3, Table 4 and Table 5.
[1089] In a certain embodiment, for artificial manipulation of each
gene, a guide nucleic acid sequence corresponding to any one of the
target sequences of SEQ ID NOs: 1 to 79.
[1090] In a certain embodiment, for artificial manipulation of each
gene, an editor protein that interacts with a guide nucleic acid
sequence corresponding to, for example, forming a complex with any
one of the target sequences of SEQ ID NOs: 1 to 1522, for example,
SEQ ID NOs: 1 to 79, is provided.
[1091] In a certain embodiment, a nucleic acid modification product
of each gene in which artificial manipulation occurs at a target
sequence region of any one of SEQ ID NOs: 1 to 1522, for example,
SEQ ID NOs: 1 to 79, and an expression product thereof are
provided.
[1092] 7. Delivery
[1093] The guide nucleic acid, editor protein or guide nucleic
acid-editor protein complex may be delivered or introduced into a
subject by various delivering methods and various forms.
[1094] The guide nucleic acid may be delivered or introduced into a
subject in the form of DNA, RNA or a mixed form.
[1095] The editor protein may be delivered or introduced into a
subject in the form of DNA, RNA, a DNA/RNA mixture, a peptide, a
polypeptide, which encodes the editor protein, or a protein.
[1096] The guide nucleic acid-editor protein complex may be
delivered or introduced into a target in the form of DNA, RNA or a
mixture thereof, which encodes each component, that is, a guide
nucleic acid or an editor protein.
[1097] The guide nucleic acid-editor protein complex may be
delivered or introduced into a subject as a complex of a guide
nucleic acid having a form of DNA, RNA or a mixture thereof and an
editor protein having a form of a peptide, polypeptide or
protein.
[1098] In addition, an additional component capable of increasing
or inhibiting the efficiency of the guide nucleic acid-editor
protein complex may be delivered or introduced into a subject by
various delivering methods and in various forms.
[1099] The additional component may be delivered or introduced into
a subject in the form of DNA, RNA, a DNA/RNA mixture, a peptide, a
polypeptide or a protein.
[1100] i) Delivery in Form of DNA, RNA or Mixture Thereof
[1101] The form of DNA, RNA or a mixture thereof, which encodes the
guide nucleic acid and/or editor protein may be delivered or
introduced into a subject by a method known in the art.
[1102] Or, the form of DNA, RNA or a mixture thereof, which encodes
the guide nucleic acid and/or editor protein may be delivered or
introduced into a subject by a vector, a non-vector or a
combination thereof.
[1103] The vector may be a viral or non-viral vector (e.g., a
plasmid).
[1104] The non-vector may be naked DNA, a DNA complex or mRNA.
[1105] Vector-Based Introduction
[1106] The nucleic acid sequence encoding the guide nucleic acid
and/or editor protein may be delivered or introduced into a subject
by a vector.
[1107] The vector may include a nucleic acid sequence encoding a
guide nucleic acid and/or editor protein.
[1108] For example, the vector may simultaneously include nucleic
acid sequences, which encode the guide nucleic acid and the editor
protein, respectively.
[1109] For example, the vector may include the nucleic acid
sequence encoding the guide nucleic acid.
[1110] As an example, domains included in the guide nucleic acid
may be contained all in one vector, or may be divided and then
contained in different vectors.
[1111] For example, the vector may include the nucleic acid
sequence encoding the editor protein.
[1112] In one example, in the case of the editor protein, the
nucleic acid sequence encoding the editor protein may be contained
in one vector, or may be divided and then contained in several
vectors.
[1113] The vector may include one or more regulatory/control
components.
[1114] Here, the regulatory/control components may include a
promoter, an enhancer, an intron, a polyadenylation signal, a Kozak
consensus sequence, an internal ribosome entry site (IRES), a
splice acceptor and/or a 2A sequence.
[1115] The promoter may be a promoter recognized by RNA polymerase
II.
[1116] The promoter may be a promoter recognized by RNA polymerase
III.
[1117] The promoter may be an inducible promoter.
[1118] The promoter may be a subject-specific promoter.
[1119] The promoter may be a viral or non-viral promoter.
[1120] The promoter may use a suitable promoter according to a
control region (that is, a nucleic acid sequence encoding a guide
nucleic acid or editor protein).
[1121] For example, a promoter useful for the guide nucleic acid
may be a H1, EF-1a, tRNA or U6 promoter. For example, a promoter
useful for the editor protein may be a CMV, EF-1a, EFS, MSCV, PGK
or CAG promoter.
[1122] The vector may be a viral vector or recombinant viral
vector.
[1123] The virus may be a DNA virus or an RNA virus.
[1124] Here, the DNA virus may be a double-stranded DNA (dsDNA)
virus or single-stranded DNA (ssDNA) virus.
[1125] Here, the RNA virus may be a single-stranded RNA (ssRNA)
virus.
[1126] The virus may be a retrovirus, a lentivirus, an adenovirus,
adeno-associated virus (AAV), vaccinia virus, a poxvirus or a
herpes simplex virus, but the present invention is not limited
thereto.
[1127] Generally, the virus may infect a host (e.g., cells),
thereby introducing a nucleic acid encoding the genetic information
of the virus into the host or inserting a nucleic acid encoding the
genetic information into the host genome. The guide nucleic acid
and/or editor protein may be introduced into a subject using a
virus having such a characteristic. The guide nucleic acid and/or
editor protein introduced using the virus may be temporarily
expressed in the subject (e.g., cells). Alternatively, the guide
nucleic acid and/or editor protein introduced using the virus may
be continuously expressed in a subject (e.g., cells) for a long
time (e.g., 1, 2 or 3 weeks, 1, 2, 3, 6 or 9 months, 1 or 2 years,
or permanently).
[1128] The packaging capability of the virus may vary from at least
2 kb to 50 kb according to the type of virus. Depending on such a
packaging capability, a viral vector including a guide nucleic acid
or an editor protein or a viral vector including both of a guide
nucleic acid and an editor protein may be designed. Alternatively,
a viral vector including a guide nucleic acid, an editor protein
and additional components may be designed.
[1129] In one example, a nucleic acid sequence encoding a guide
nucleic acid and/or editor protein may be delivered or introduced
by a recombinant lentivirus.
[1130] In another example, a nucleic acid sequence encoding a guide
nucleic acid and/or editor protein may be delivered or introduced
by a recombinant adenovirus.
[1131] In still another example, a nucleic acid sequence encoding a
guide nucleic acid and/or editor protein may be delivered or
introduced by recombinant AAV.
[1132] In yet another example, a nucleic acid sequence encoding a
guide nucleic acid and/or editor protein may be delivered or
introduced by a hybrid virus, for example, one or more hybrids of
the virus listed herein.
[1133] Non-Vector-Based Introduction
[1134] A nucleic acid sequence encoding a guide nucleic acid and/or
editor protein may be delivered or introduced into a subject using
a non-vector.
[1135] The non-vector may include a nucleic acid sequence encoding
a guide nucleic acid and/or editor protein.
[1136] The non-vector may be naked DNA, a DNA complex, mRNA, or a
mixture thereof.
[1137] The non-vector may be delivered or introduced into a subject
by electroporation, particle bombardment, sonoporation,
magnetofection, transient cell compression or squeezing (e.g.,
described in the literature [Lee, et al, (2012) Nano Lett., 12,
6322-6327]), lipid-mediated transfection, a dendrimer,
nanoparticles, calcium phosphate, silica, a silicate (Ormosil), or
a combination thereof.
[1138] As an example, the delivery through electroporation may be
performed by mixing cells and a nucleic acid sequence encoding a
guide nucleic acid and/or editor protein in a cartridge, chamber or
cuvette, and applying electrical stimuli with a predetermined
duration and amplitude to the cells.
[1139] In another example, the non-vector may be delivered using
nanoparticles. The nanoparticles may be inorganic nanoparticles
(e.g., magnetic nanoparticles, silica, etc.) or organic
nanoparticles (e.g., a polyethylene glycol (PEG)-coated lipid,
etc.). The outer surface of the nanoparticles may be conjugated
with a positively-charged polymer which is attachable (e.g.,
polyethyleneimine, polylysine, polyserine, etc.).
[1140] In a certain embodiment, the non-vector may be delivered
using a lipid shell.
[1141] In a certain embodiment, the non-vector may be delivered
using an exosome. The exosome is an endogenous nano-vesicle for
transferring a protein and RNA, which can deliver RNA to the brain
and another target organ.
[1142] In a certain embodiment, the non-vector may be delivered
using a liposome. The liposome is a spherical vesicle structure
which is composed of single or multiple lamellar lipid bilayers
surrounding internal aqueous compartments and an external,
lipophilic phospholipid bilayer which is relatively
non-transparent. While the liposome may be made from several
different types of lipids; phospholipids are most generally used to
produce the liposome as a drug carrier.
[1143] Other additives may be included.
[1144] ii) Delivery in Form of Peptide, Polypeptide or Protein
[1145] An editor protein in the form of a peptide, polypeptide or
protein may be delivered or introduced into a subject by a method
known in the art
[1146] The peptide, polypeptide or protein form may be delivered or
introduced into a subject by electroporation, microinjection,
transient cell compression or squeezing (e.g., described in the
literature [Lee, et al, (2012) Nano Lett., 12, 6322-6327]),
lipid-mediated transfection, nanoparticles, a liposome,
peptide-mediated delivery or a combination thereof.
[1147] The peptide, polypeptide or protein may be delivered with a
nucleic acid sequence encoding a guide nucleic acid.
[1148] In one example, the transfer through electroporation may be
performed by mixing cells into which the editor protein will be
introduced with or without a guide nucleic acid in a cartridge,
chamber or cuvette, and applying electrical stimuli with a
predetermined duration and amplitude to the cells.
[1149] iii) Delivery in Form of Nucleic Acid-Protein Mixture
[1150] The guide nucleic acid and the editor protein may be
delivered or introduced into a subject in the form of a guide
nucleic acid-editor protein complex.
[1151] For example, the guide nucleic acid may be DNA, RNA or a
mixture thereof. The editor protein may be a peptide, polypeptide
or protein.
[1152] In one example, the guide nucleic acid and the editor
protein may be delivered or introduced into a subject in the form
of a guide nucleic acid-editor protein complex containing an
RNA-type guide nucleic acid and a protein-type editor protein, that
is, a ribonucleoprotein (RNP).
[1153] In the present invention, as an embodiment of a method for
delivering the guide nucleic acid and/or editor protein into a
subject, the delivery of gRNA, a CRISPR enzyme or a gRNA-CRISPR
enzyme complex will be described below.
[1154] In an embodiment of the present invention, a nucleic acid
sequence encoding the gRNA and/or CRISPR enzyme will be delivered
or introduced into a subject using a vector.
[1155] The vector may include the nucleic acid sequence encoding
the gRNA and/or CRISPR enzyme.
[1156] For example, the vector may simultaneously include the
nucleic acid sequences encoding the gRNA and the CRISPR enzyme.
[1157] For example, the vector may include the nucleic acid
sequence encoding the gRNA.
[1158] In one example, domains contained in the gRNA may be
contained in one vector, or may be divided and then contained in
different vectors.
[1159] For example, the vector may include the nucleic acid
sequence encoding the CRISPR enzyme.
[1160] In one example, in the case of the CRISPR enzyme, the
nucleic acid sequence encoding the CRISPR enzyme may be contained
in one vector, or may be divided and then contained in several
vectors.
[1161] The vector may include one or more regulatory/control
components.
[1162] Here, the regulatory/control components may include a
promoter, an enhancer, an intron, a polyadenylation signal, a Kozak
consensus sequence, an internal ribosome entry site (IRES), a
splice acceptor and/or a 2A sequence.
[1163] The promoter may be a promoter recognized by RNA polymerase
II.
[1164] The promoter may be a promoter recognized by RNA polymerase
III.
[1165] The promoter may be an inducible promoter.
[1166] The promoter may be a subject-specific promoter.
[1167] The promoter may be a viral or non-viral promoter.
[1168] The promoter may use a suitable promoter according to a
control region (that is, a nucleic acid sequence encoding the gRNA
and/or CRISPR enzyme).
[1169] For example, a promoter useful for the gRNA may be a H1,
EF-1a, tRNA or U6 promoter. For example, a promoter useful for the
CRISPR enzyme may be a CMV, EF-1a, EFS, MSCV, PGK or CAG
promoter.
[1170] The vector may be a viral vector or recombinant viral
vector.
[1171] The virus may be a DNA virus or an RNA virus.
[1172] Here, the DNA virus may be a double-stranded DNA (dsDNA)
virus or single-stranded DNA (ssDNA) virus.
[1173] Here, the RNA virus may be a single-stranded RNA (ssRNA)
virus.
[1174] The virus may be a retrovirus, a lentivirus, an adenovirus,
adeno-associated virus (AAV), vaccinia virus, a poxvirus or a
herpes simplex virus, but the present invention is not limited
thereto.
[1175] Generally, the virus may infect a host (e.g., cells),
thereby introducing a nucleic acid encoding the genetic information
of the virus into the host or inserting a nucleic acid encoding the
genetic information into the host genome. The gRNA and/or CRISPR
enzyme may be introduced into a subject using a virus having such a
characteristic. The gRNA and/or CRISPR enzyme introduced using the
virus may be temporarily expressed in the subject (e.g., cells).
Alternatively, the gRNA and/or CRISPR enzyme introduced using the
virus may be continuously expressed in a subject (e.g., cells) for
a long time (e.g., 1, 2 or 3 weeks, 1, 2, 3, 6 or 9 months, 1 or 2
years, or permanently).
[1176] The packaging capability of the virus may vary from at least
2 kb to 50 kb according to the type of virus. Depending on such a
packaging capability, a viral vector only including gRNA or a
CRISPR enzyme or a viral vector including both of gRNA and a CRISPR
enzyme may be designed. Alternatively, a viral vector including
gRNA, a CRISPR enzyme and additional components may be
designed.
[1177] In one example, a nucleic acid sequence encoding gRNA and/or
a CRISPR enzyme may be delivered or introduced by a recombinant
lentivirus.
[1178] In another example, a nucleic acid sequence encoding gRNA
and/or a CRISPR enzyme may be delivered or introduced by a
recombinant adenovirus.
[1179] In still another example, a nucleic acid sequence encoding
gRNA and/or a CRISPR enzyme may be delivered or introduced by
recombinant AAV.
[1180] In yet another example, a nucleic acid sequence encoding
gRNA and/or a CRISPR enzyme may be delivered or introduced by one
or more hybrids of hybrid viruses, for example, the viruses
described herein.
[1181] In one exemplary embodiment of the present invention, the
gRNA-CRISPR enzyme complex may be delivered or introduced into a
subject.
[1182] For example, the gRNA may be present in the form of DNA, RNA
or a mixture thereof. The CRISPR enzyme may be present in the form
of a peptide, polypeptide or protein.
[1183] In one example, the gRNA and CRISPR enzyme may be delivered
or introduced into a subject in the form of a gRNA-CRISPR enzyme
complex including RNA-type gRNA and a protein-type CRISPR, that is,
a ribonucleoprotein (RNP).
[1184] The gRNA-CRISPR enzyme complex may be delivered or
introduced into a subject by electroporation, microinjection,
transient cell compression or squeezing (e.g., described in the
literature [Lee, et al, (2012) Nano Lett., 12, 6322-6327]),
lipid-mediated transfection, nanoparticles, a liposome,
peptide-mediated delivery or a combination thereof.
[1185] 8. Transformant
[1186] The term "transformant" refers to an organism into which a
guide nucleic acid, editor protein or guide nucleic acid-editor
protein complex is introduced, an organism in which a guide nucleic
acid, editor protein or guide nucleic acid-editor protein complex
is expressed, or a specimen or sample obtained from the
organism.
[1187] The transformant may be an organism into which a guide
nucleic acid, editor protein or guide nucleic acid-editor protein
complex is introduced in the form of DNA, RNA or a mixture
thereof.
[1188] For example, the transformant may be an organism into which
a vector including a nucleic acid sequence encoding a guide nucleic
acid and/or editor protein is introduced. Here, the vector may be a
non-viral vector, viral vector or recombinant viral vector.
[1189] In another example, the transformant may be an organism into
which a nucleic acid sequence encoding a guide nucleic acid and/or
editor protein is introduced in a non-vector form. Here, the
non-vector may be naked DNA, a DNA complex, mRNA or a mixture
thereof.
[1190] The transformant may be an organism into which a guide
nucleic acid, editor protein or guide nucleic acid-editor protein
complex is introduced in the form of a peptide, polypeptide or
protein.
[1191] The transformant may be an organism into which a guide
nucleic acid, editor protein or guide nucleic acid-editor protein
complex is introduced in the form of DNA, RNA, a peptide, a
polypeptide, a protein or a mixture thereof.
[1192] For example, the transformant may be an organism into which
a guide nucleic acid-editor protein complex including an RNA-type
guide nucleic acid and a protein-type editor protein is
introduced.
[1193] The transformant may be an organism including a target
nucleic acid, gene, chromosome or protein of the guide nucleic
acid-editor protein complex.
[1194] The organism may be cells, tissue, a plant, an animal or a
human.
[1195] The cells may be prokaryotic cells or eukaryotic cells.
[1196] The eukaryotic cells may be plant cells, animal cells, or
human cells, but the present invention is not limited thereto.
[1197] The tissue may be an animal or human body tissue such as
skin, liver, kidney, heart, lung, brain, or muscle tissue.
[1198] The transformant may be an organism in(to)to which a guide
nucleic acid, editor protein or guide nucleic acid-editor protein
complex is introduced or expressed, or a specimen or sample
obtained from the organism.
[1199] The specimen or sample may be saliva, blood, skin tissue,
cancer cells or stem cells.
[1200] Use
[1201] One exemplary embodiment of the present invention relates to
a use of treating a neovascularization-associated disease using a
method of administering a composition for artificially manipulating
a neovascularization-associated factor or an artificially
manipulated neovascularization-associated factor to a subject.
[1202] Targets for the treatment may be mammals including primates
such as a human or a monkey, rodents such as a mouse or a rat,
etc.
[1203] Diseases to be Treated
[1204] In an embodiment, diseases to be treated may be
neovascularization-associated diseases.
[1205] The term "neovascularization-associated diseases" refer to
all states including excessive and/or abnormal neovascularization.
The neovascularization-associated diseases refer to disorders
characterized by vascularization which is not changed or regulated,
except tumorigenesis or neoplastic transformation, that is, cancer.
The neovascularization-associated diseases include an ocular
neovascularization disease.
[1206] Neovascular diseases include neovascularization-dependent
cancer, for example, solid tumors, hematomas such as leukemia and
tumor metastasis; benign tumors such as hemangiomas, acoustic
neuromas, neurofibroma, trachomas and pyogenic granulomas;
rheumatoid arthritis; psoriasis; ocular neovascularization diseases
such as diabetic retinopathy, retinopathy of prematurity, macular
degenerations including dry age-related macular degeneration and
wet age-related macular degeneration, corneal graft rejection,
neovascular glaucoma, retrolental fibroplasia, rubeosis;
Osler-Webber Syndrome; myocardial neovascularization blindness;
plaque neovascularization; telangiectasia; hemophiliac joint;
angiofibromas; and wound granulation, but the present invention is
not limited thereto.
[1207] In a certain embodiment, the neovascularization-associated
diseases may be one or more diseases selected from the group
consisting of rheumatoid arthritis, psoriasis, Osler-Webber
Syndrome, myocardial neovascularization blindness, plaque
neovascularization, telangiectasia, hemophiliac joint,
angiofibromas, and wound granulation.
[1208] In an embodiment, the neovascularization-associated disease
may be a disease including excessive and/or abnormal
neovascularization.
[1209] In an embodiment, the neovascularization-associated disease
may be neovascularization-dependent cancer.
[1210] Here, the neovascularization-dependent cancer includes solid
tumors, hematologic tumors such as leukemia and tumor
metastasis.
[1211] The neovascularization-dependent cancer may be, for example,
solid tumors, hematologic tumors such as leukemia and tumor
metastasis; benign tumors such as hemangiomas, acoustic neuromas,
neurofibromas, trachomas and pyogenic granulomas.
[1212] In a certain embodiment, the neovascularization-associated
disease may be a benign tumor.
[1213] The benign tumor may include hemangiomas, acoustic neuromas,
trachomas and pyogenic granulomas.
[1214] In a certain embodiment, the neovascularization-associated
disease may be an ocular neovascularization disease.
[1215] The term "ocular neovascularization disease" refers to all
ocular diseases including excessive and/or abnormal
neovascularization. The ocular neovascularization disease includes
a disorder characterized by vascularization that is not changed or
regulated in the eyes.
[1216] As an example, the ocular neovascularization diseases may
include: ischemic retinopathy, optic papillary neovascularization,
iris neovascularization, retinal neovascularization, choroidal
neovascularization, corneal neovascularization, vitreous
neovascularization, glaucoma, panus, pterygiums, macular edemas,
diabetic retinopathy, proliferative diabetic retinopathy, diabetic
macular edemas, vascular retinopathy, retinal degeneration,
uveitis, inflammatory diseases of the retina, and proliferative
vitreoretinopathy.
[1217] In one exemplary embodiment, the ocular neovascularization
disease may be diabetic retinopathy or macular degeneration.
[1218] Diabetic Retinopathy
[1219] Diabetic retinopathy is a diabetic complication occurring in
approximately 40 to 45% of the patients diagnosed with any one of
Type 1 diabetes or Type 2 diabetes.
[1220] Diabetic retinopathy usually affects both eyes, and
generally progresses in four stages. The first stage, that is, mild
nonproliferative retinopathy is characterized by ocular
microaneurysms. Small scaled swelling occurs in a retinal capillary
tube and a small vessel. In the second stage, that is, moderate
nonproliferative retinopathy, a blood vessel provided to the retina
starts to be blocked. In the third stage, that is, severe
nonproliferative retinopathy, the occlusion leads to a decrease in
blood supply to the retina, and the retina sends a
neovascularization signal to the eye in order to provide blood
supply to the retina. In the fourth stage, that is, proliferative
retinopathy, which is the most advanced stage, angiogenesis occurs,
but the new blood vessel is abnormal, weak, and grows on the
surface of a vitreous gel contained in the retina and the eyes.
[1221] The diabetic retinopathy includes insulin-dependent
diabetes, insulin-independent diabetes, retinal detachment,
diabetic retinopathy, and vitreous hemorrhage.
[1222] Macular Degeneration
[1223] The macular degeneration refers to a disease in which visual
impairment is caused by macular degeneration, and is also called
age-related macular degeneration (AMD).
[1224] The AMD includes early, intermediate and advanced AMD, and
also includes all of dry AMD, for example, geographic atrophy, and
wet AMD which is also known as neovascular or exudative AMD.
[1225] Dry macular degeneration is a prevalent type accounting for
approximately 90% of the AMDs when a lesion such as a druse (the
state in which waste is accumulated in the macula) or retinal
pigment epithelial atrophy is generated in the retina. Wet macular
degeneration is characterized by production of choroidal
neovascularization under the retina.
[1226] Dry macular degeneration includes macular degeneration
caused by a missense mutation in an immunoregulatory complement
factor H (CFH) gene.
[1227] In another exemplary embodiment, a use of a system for
regulating an additional, third in vivo mechanism, accompanied with
various functions of specific factors artificially modified in
function (e.g., a gene known as a neovascularization-associated
factor, etc.) may be provided.
[1228] For example, the specific factors artificially modified in
function may be one or more genes of a VEGFA gene, an HIF1A gene,
an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene.
[1229] The third mechanism may be an in vivo mechanism in which the
genes are involved, other than neovascularization.
[1230] Pharmaceutical Composition
[1231] One exemplary embodiment of the present invention relates to
a composition to be used in treatment of a disease using an
artificially manipulated neovascularization-associated factor.
[1232] The composition may include an artificially manipulated
neovascularization-associated factor or a manipulation composition
capable of artificially manipulating the
neovascularization-associated factor. The composition may be
referred to as a therapeutic composition or pharmaceutical
composition.
[1233] In an exemplary embodiment, the composition may include an
artificially manipulated neovascularization-associated factor, that
is, a gene and/or protein.
[1234] In an exemplary embodiment, the composition may include a
manipulation composition capable of artificially manipulating a
neovascularization-associated factor.
[1235] The manipulation composition may include a guide nucleic
acid-editor protein complex.
[1236] The manipulation composition may include a guide nucleic
acid and/or editor protein.
[1237] The manipulation composition may include a nucleic acid
encoding the guide nucleic acid and/or editor protein.
[1238] The manipulation composition may include a virus comprising
a nucleic acid encoding the guide nucleic acid and/or editor
protein.
[1239] In another exemplary embodiment, the composition may further
include an additional element.
[1240] The additional element may include a suitable carrier for
delivery into the body of a subject.
[1241] In one exemplary embodiment, the composition may include an
expression product of a neovascularization-associated factor
manipulated in a sufficient amount to suppress an angiovascular
disorder.
[1242] The "sufficient amount to suppress an angiovascular
disorder" refers to an effective amount necessary to treat or
prevent an angiovascular disorder or a symptom thereof.
[1243] In one exemplary embodiment, the following therapeutic
compositions will be provided: [1244] a composition for treating an
angiovascular disorder, which includes a guide nucleic acid capable
of forming a complementary bond with each of one or more target
sequences in nucleic acid sequences of one or more genes selected
from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2
gene, an EPAS1 gene and an ANGPTL4 gene, or a nucleic acid sequence
encoding the same, and [1245] an editor protein or a nucleic acid
sequence encoding the same; [1246] a composition for treating an
angiovascular disorder, which includes a guide nucleic acid capable
of forming a complementary bond with each of the target sequences
of SEQ ID NOs: 1 to 1522, for example, SEQ ID NOs: 1 to 79, of
nucleic acid sequences of one or more genes selected from the group
consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1
gene and an ANGPTL4 gene, or a nucleic acid sequence encoding the
same, and [1247] an editor protein or a nucleic acid sequence
encoding the same; and [1248] a composition for treating an
angiovascular disorder, which includes a complex formed of a guide
nucleic acid capable of forming a complementary bond with each of
the target sequences of SEQ ID NOs: 1 to 1522, for example, SEQ ID
NOs: 1 to 79, of nucleic acid sequences of one or more genes
selected from the group consisting of a VEGFA gene, an HIF1A gene,
an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene or a nucleic acid
sequence encoding the same; and an editor protein.
[1249] Here, the guide nucleic acid or nucleic acid sequence
encoding the same; and a nucleic acid sequence encoding the editor
protein may be present in the form of one or more vectors. The
guide nucleic acid or nucleic acid sequence encoding the same; and
a nucleic acid sequence encoding the editor protein may be present
in the form of homologous or heterologous vectors.
[1250] Treatment Method
[1251] In another exemplary embodiment of the present invention, a
method for treating a disease in a patient, which includes
producing the above-described composition and administering an
effective amount of the composition to a patient requiring the
same, is provided.
[1252] Gene Manipulating Treatment
[1253] A treatment method for regulating neovascularization by
manipulating a gene of a living organism may be used. Such a
treatment method may be achieved by directly injecting a
composition for manipulating a gene to manipulate the gene of a
living organism into the organism.
[1254] The composition for gene manipulation may include a guide
nucleic acid-editor protein complex.
[1255] The composition for gene manipulation may be injected into a
specific location of the body.
[1256] Here, the specific location of the body may be tissue in
which neovascularization excessively and/or abnormally occurs, or a
location close thereto. For example, the specific location of the
body may be, for example, the eyeball.
[1257] Subjects for administration of the composition may be
mammals including primates such as a human or a monkey, rodents
such as a mouse or a rat, etc.
[1258] The composition may be administered by any convenient method
such as injection, transfusion, implantation or transplantation.
The composition may be administered subcutaneously, intradermally,
intraocularly, intravitreally, intratumorally, intranodally,
intramedullarily, intramuscularly, intravenously,
intralymphatically, or intraperitoneally.
[1259] A dose (pharmaceutically effective amount to obtain a
predetermined, desired effect) of the composition may be selected
from all integers in the value ranges of 104-109 cells, for
example, 105 to 106 cells/kg (body weight), per kg of the subject
of administration, but the present invention is not limited
thereto. The composition may be suitably prescribed in
consideration of the age, health condition and body weight of the
subject of administration, the types of treatments simultaneously
received, if they were, frequency of the co-treatments, and
characteristics of a desired effect.
[1260] In one aspect, the present invention provides a method for
modifying a target polynucleotide in prokaryotic cells, which may
be achieved in vivo, ex vivo, or in vitro.
[1261] In some embodiments, the method may include sampling cells
or a cell population from a human or non-human animal, and
modifying the cell or cells. Culturing may occur at any step ex
vivo. The cell or cells may also be reintroduced into a non-human
animal or plant. The reintroduced cells are most preferably stem
cells.
[1262] In still another exemplary embodiment, the present invention
may provide a method for artificially manipulating cells, which
includes: introducing (a) a guide nucleic acid capable of forming a
complementary bond with the target sequences of SEQ ID NOs: 1 to
1522, for example, SEQ ID NOs: 1 to 79, of nucleic acid sequences
of one or more genes selected from the group consisting of a VEGFA
gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4
gene or a nucleic acid sequence encoding the same; and (b) an
editor protein including one or more proteins selected from the
group consisting of a Streptococcus pyogenes-derived Cas9 protein,
a Campylobacter jejuni-derived Cas9 protein, a Streptococcus
thermophilus-derived Cas9 protein, a Streptococcus aureus-derived
Cas9 protein, a Neisseria meningitidis-derived Cas9 protein, and a
Cpf1 protein, or a nucleic acid sequence encoding the same to
cells.
[1263] The guide nucleic acid and the editor protein may be present
in one or more vectors in the form of a nucleic acid sequence, or
in a complex of a combination of the guide nucleic acid and the
editor protein.
[1264] The introduction step may be carried out in vivo or ex
vivo.
[1265] A technique of the above-described "7. Delivery" section may
be referenced before the introduction step.
[1266] For example, the introduction stage may be achieved by one
or more methods selected from electroporation, liposomes, plasmids,
viral vectors, nanoparticles, and a protein translocation domain
(PTD) fusion protein method.
[1267] For example, the viral vector may be one or more selected
from the group consisting of a retrovirus, a lentivirus, an
adenovirus, adeno-associated virus (AAV), vaccinia virus, a
poxvirus and a herpes simplex virus.
[1268] When a neovascularization-associated factor is artificially
manipulated using the method and composition of some embodiments of
the present invention, it is possible to regulate, for example,
inhibit, suppress, stimulate and/or increase neovascularization,
and therefore an effect of suppressing or improving excessive
and/or abnormal neovascularization may be obtained.
[1269] Additional Uses
[1270] In a certain embodiment, the present invention may provide a
kit for preparing a composition for treating AMD or diabetic
retinopathy.
[1271] The kit may be prepared by a conventional preparation method
known in the art.
[1272] The kit may further include a detectable label. The term
"detectable label" refers to an atom or molecule for specifically
detecting a molecule containing a label among the same type of
molecules without a label. The detectable label may be attached to
an antibody specifically binding to a protein or a fragment
thereof, an interaction protein, a ligand, nanoparticles, or an
aptamer. The detectable label may include a radionuclide, a
fluorophore, and an enzyme.
[1273] In a certain embodiment, the present invention may provide a
method for screening a material capable of regulating the
expression level of one or more genes of an artificially
manipulated VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1 gene and
ANGPTL4 gene.
[1274] In a certain embodiment, the present invention may provide a
method for providing information on the sequence of a target site
which is able to be artificially manipulated in a subject by
analyzing the sequences of one or more genes selected from the
group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an
EPAS1 gene and an ANGPTL4 gene.
[1275] In addition, a method for constructing a library using the
information provided by such a method.
[1276] Here, a known database may be used.
[1277] In specific embodiments, an animal or cells which can be
used for research using the method of the present invention may be
provided.
[1278] An animal or cells which includes chromosome editing in one
or more nucleic acid sequences associated with a disease may be
prepared using the above-described method. Such a nucleic acid
sequence may be a reference sequence which may encode a
disease-associated protein sequence or may be associated with a
disease.
[1279] In one exemplary embodiment, an effect of mutation and
occurrence and/or progression of a disease may be studied in an
animal or cells using measurements conventionally used in disease
research with the animal or cells prepared by the method of the
present invention. Alternatively, a pharmaceutical effect of an
active compound in a disease using such an animal or cells may be
studied.
[1280] In another exemplary embodiment, the effect of the strategy
of a possible gene therapy may be evaluated using the animal or
cells prepared by the method of the present invention. That is, the
development and/or progression of a corresponding disease may be
suppressed or reduced by modifying a chromosome sequence encoding a
disease-associated protein. Particularly, this method includes
forming a modified protein by editing a chromosome sequence
encoding a disease-associated protein, resulting in the achievement
of a modified response of the animal or cells. Therefore, in some
embodiments, a genetically-modified animal may be compared with an
animal vulnerable to the development of a corresponding disease,
thereby evaluating the effect of a gene therapy process.
[1281] Such uses may include a disease model, a pharmacological
model, a developmental model, a cell function model, and a
humanized model. For example, a neovascularization-associated
disease model, a pharmacological model, a developmental model, a
cell function model, and a humanized model may be included.
[1282] An artificially manipulated neovascularization-associated
factor and a neovascularization system artificially modified in
function thereby can be effectively used to treat a
neovascularization-associated disease, for example, a
neovascularization-associated ocular disease (eye disease). The
efficiency of a neovascularization system may be improved by
regulating various in vivo mechanisms in which various
neovascularization-associated factors are involved.
[1283] Hereinafter, the present invention will be described in
further detail with reference to examples.
[1284] The examples are merely provided to describe the present
invention in further detail, and it might be obvious to those of
ordinary skill in the art that the scope of the present invention
is not limited to the following examples.
[1285] Experimental Methods
[1286] 1. Design of sgRNA
[1287] CRISPR/Cas9 target regions of human VEGFA gene (NCBI
Accession No. NM_001025366.2), HIF1A gene (NCBI Accession No.
NM_001243084.1), ANGPT2 gene (NCBI Accession No. NM_001118887.1),
EPAS1 gene (NCBI Accession No. NM_001430.4) and ANGPTL4 gene (NCBI
Accession No. NM_001039667.2) were selected using CRISPR RGEN Tools
(Institute for Basic Science, Korea). The target regions of the
genes may be different according to the type of CRISPR enzyme.
Target sequences of the genes for CjCas9 are summarized in Tables 1
to 5 and Tables 6 to 9 listed above, and target sequences of the
genes for SpCas9 are summarized in Tables 10 to 14.
TABLE-US-00006 TABLE 6 Target sequences of VEGFA gene Gene No.
Target sequence VEGFA 1 AAATCCCGGTATAAGTCCTGGA (SEQ ID NO: 80)
VEGFA 2 GCACCAACGTACACGCTCCAGG (SEQ ID NO: 81) VEGFA 3
CATTAGACAGCAGCGGGCACCA (SEQ ID NO: 82) VEGFA 4
GGCATTAGACAGCAGCGGGCAC (SEQ ID NO: 83) VEGFA 5
GGCTCCAGGGCATTAGACAGCA (SEQ ID NO: 84) VEGFA 6
GCTCAGAGCGGAGAAAGCATTT (SEQ ID NO: 85) VEGFA 7
GGAACATTTACACGTCTGCGGA (SEQ ID NO: 86) VEGFA 8
GCAGACGTGTAAATGTTCCTGC (SEQ ID NO: 87) VEGFA 9
GAGTCTGTGTTTTTGCAGGAAC (SEQ ID NO: 88) VEGFA 10
GGCGAGGCAGCTTGAGTTAAAC (SEQ ID NO: 89)
TABLE-US-00007 TABLE 7 Target sequences of HIF1A gene Gene No.
Target sequence HIF1A 1 ACTAAAGGACAAGTCACCACAG (SEQ ID NO: 90)
HIF1A 2 TATCCACCTCTTTTGGCAAGCA (SEQ ID NO: 91) HIF1A 3
TGAAACTCAAGCAACTGTCATA (SEQ ID NO: 92) HIF1A 4
CTCACAACGTAATTCACACATA (SEQ ID NO: 93) HIF1A 5
TACTTACCTCACAACGTAATTC (SEQ ID NO: 94) HIF1A 6
AACTTACTTACCTCACAACGTA (SEQ ID NO: 95) HIF1A 7
TCTTGTTTTGACAGTGGTATTA (SEQ ID NO: 96) HIF1A 8
GGGAGAAAATCAAGTCGTGCTG (SEQ ID NO: 97) HIF1A 9
TATCTGAAGATTCAACCGGTTT (SEQ ID NO: 98) HIF1A 10
GCTATTCACCAAAGTTGAATCA (SEQ ID NO: 99) HIF1A 11
AACTTTGCTGGCCCCAGCCGCT (SEQ ID NO: 100) HIF1A 12
AAACTGATGACCAGCAACTTGA (SEQ ID NO: 101) HIF1A 13
GGGGAGCATTACATCATTATAT (SEQ ID NO: 102) HIF1A 14
AGCCACTTCGAAGTAGTGCTGA (SEQ ID NO: 103) HIF1A 15
CAACTTCTTGATTGAGTGCAGG (SEQ ID NO: 104) HIF1A 16
TTACCATGCCCCAGATTCAGGA (SEQ ID NO: 105) HIF1A 17
TCAGACACCTAGTCCTTCCGAT (SEQ ID NO: 106) HIF1A 18
ATTGGTAGAAAAACTTTTTGCT (SEQ ID NO: 107) HIF1A 19
AACTCATGTATTTGCTGTTTTA (SEQ ID NO: 108) HIF1A 20
AAGCCCTGAAAGCGCAAGTCCT (SEQ ID NO: 109) HIF1A 21
CAGTTACAGTATTCCAGCAGAC (SEQ ID NO: 110) HIF1A 22
AGGTTCTTGTATTTGAGTCTGC (SEQ ID NO: 111) HIF1A 23
ATGCAATCAATATTTTAATGTC (SEQ ID NO: 112) HIF1A 24
TGATTGCATCTCCATCTCCTAC (SEQ ID NO: 113) HIF1A 25
TAGTGCCACATCATCACCATAT (SEQ ID NO: 114) HIF1A 26
GAGTATCTCTATATGGTGATGA (SEQ ID NO: 115) HIF1A 27
ATACCTTTGACTCAAAGCGACA (SEQ ID NO: 116) HIF1A 28
TTCCTGAGGAAGAACTAAATCC (SEQ ID NO: 117) HIF1A 29
TCTGTTCACTAGATTTGCATCC (SEQ ID NO: 118) HIF1A 30
GAATGGAGCAAAAGACAATTAT (SEQ ID NO: 119) HIF1A 31
GTTATGATTGTGAAGTTAATGC (SEQ ID NO: 120)
TABLE-US-00008 TABLE 8 Target sequences of EPAS1 gene Gene No.
Target sequence EPAS1 1 AACACCTCCGTCTCCTTGCTCC (SEQ ID NO: 121)
EPAS1 2 TGGAGGCCTTGTCCAGATGGGA (SEQ ID NO: 122) EPAS1 3
TGCGACTGGCAATCAGCTTCCT (SEQ ID NO: 123) EPAS1 4
CGACTGGCAATCAGCTTCCTGC (SEQ ID NO: 124) EPAS1 5
GAAGCTGACCAGCAGATGGACA (SEQ ID NO: 125) EPAS1 6
GCAATGAAACCCTCCAAGGCTT (SEQ ID NO: 126) EPAS1 7
AAAACATCAGCAAGTTCATGGG (SEQ ID NO: 127) EPAS1 8
GCAAGTTCATGGGACTTACACA (SEQ ID NO: 128) EPAS1 9
GGTCGCAGGGATGAGTGAAGTC (SEQ ID NO: 129) EPAS1 10
GCGGGACTTCTTCATGAGGATG (SEQ ID NO: 130) EPAS1 11
GAAGTGCACGGTCACCAACAGA (SEQ ID NO: 131) EPAS1 12
ACAGTACGGCCTCTGTTGGTGA (SEQ ID NO: 132) EPAS1 13
TCCAGGTGGCTGACTTGAGGTT (SEQ ID NO: 133) EPAS1 14
TCCACGCCTGTCTCAGGTCTTG (SEQ ID NO: 134) EPAS1 15
CAGGACAGCAGGGGCTCCTTGT (SEQ ID NO: 135) EPAS1 16
CTCATCATCATGTGTGAACCAA (SEQ ID NO: 136) EPAS1 17
ATGTGGGATGGGTGCTGGATTG (SEQ ID NO: 137) EPAS1 18
GCCACTTACTACCTGACCCTTG (SEQ ID NO: 138) EPAS1 19
TGGCCACTTACTACCTGACCCT (SEQ ID NO: 139) EPAS1 20
ACCAAGGGTCAGGTAGTAAGTG (SEQ ID NO: 140) EPAS1 21
TAGCCCCCATGCTTTGCGAGCA (SEQ ID NO: 141) EPAS1 22
GATGACCGTCCCCTGGGTCTCC (SEQ ID NO: 142) EPAS1 23
CTCAGGACGTAGTTGACACACA (SEQ ID NO: 143) EPAS1 24
CATGCTTACCTCAGGACGTAGT (SEQ ID NO: 144) EPAS1 25
CACATGCTTACCTCAGGACGTA (SEQ ID NO: 145) EPAS1 26
CAGGGATTCAGTCTGGTCCATG (SEQ ID NO: 146) EPAS1 27
GGTGAATAGGAAGTTACTCTTC (SEQ ID NO: 147) EPAS1 28
ATGGGCCACGGAGTTGAGGAGC (SEQ ID NO: 148) EPAS1 29
CTAGCCCAATAGCCCTGAAGAC (SEQ ID NO: 149) EPAS1 30
AGTGATTGAGAAGCTCTTCGCC (SEQ ID NO: 150) EPAS1 31
GGACACAGAGGCCAAGGACCAA (SEQ ID NO: 151) EPAS1 32
CCTGATCTCCACAGCCATCTAC (SEQ ID NO: 152) EPAS1 33
CGGATTTCAATGAGCTGGACTT (SEQ ID NO: 153) EPAS1 34
TCAATGAGCTGGACTTGGAGAC (SEQ ID NO: 154) EPAS1 35
GCGGAGAACCCACAGTCCACCC (SEQ ID NO: 155) EPAS1 36
CCAGTGGCTGGAAGATGTTTGT (SEQ ID NO: 156) EPAS1 37
TTCCAGCCACTGGCCCCTGTAG (SEQ ID NO: 157) EPAS1 38
CTGGAGAGCAAGAAGACAGAGC (SEQ ID NO: 158) EPAS1 39
GAGAGAGGGGTGCTGGCCTGGC (SEQ ID NO: 159) EPAS1 40
CCTGCCACCGTGCTGTGGCCAG (SEQ ID NO: 160) EPAS1 41
TCTCTCTTCCATGGGGGGCAGA (SEQ ID NO: 161) EPAS1 42
CACAAAGTGGGCCGTCGGGGAT (SEQ ID NO: 162) EPAS1 43
GGAGAGGGCTACCATGGCCGGA (SEQ ID NO: 163) EPAS1 44
CTCAGGTCCTGGAAGGCTTGCT (SEQ ID NO: 164) EPAS1 45
CTCCCAGGGGGACCCACCTGGT (SEQ ID NO: 165) EPAS1 46
TGCCGGACAAGCCACTGAGCGC (SEQ ID NO: 166) EPAS1 47
TTCCCCCCACAGTGCTACGCCA (SEQ ID NO: 167) EPAS1 48
CTGTAGTCCTGGTACTGGGTGG (SEQ ID NO: 168) EPAS1 49
TGGGCTGACGACAGGCTGTAGT (SEQ ID NO: 169) EPAS1 50
TCCTTGCAGGAGCGTGGAGCTT (SEQ ID NO: 170)
TABLE-US-00009 TABLE 9 Target sequences of ANGPT2 gene Gene No.
Target sequence ANGPT2 1 GACTGTGCTGAAGTATTCAAAT (SEQ ID NO: 171)
ANGPT2 2 CTGTGCTGAAGTATTCAAATCA (SEQ ID NO: 172) ANGPT2 3
TGGTGTGTCCTGATTTGAATAC (SEQ ID NO: 173) ANGPT2 4
GCCATTCGTGGTGTGTCCTGAT (SEQ ID NO: 174) ANGPT2 5
ATCAGGACACACCACGAATGGC (SEQ ID NO: 175) ANGPT2 6
CTAATCAGCAACGCTATGTGCT (SEQ ID NO: 176) ANGPT2 7
AATCAGCAACGCTATGTGCTTA (SEQ ID NO: 177) ANGPT2 8
TTCCCAGTCTTTAAGGTGTATT (SEQ ID NO: 178) ANGPT2 9
TCTTCACTTGAGAGATAGAAAT (SEQ ID NO: 179) ANGPT2 10
CATCAGCCAACCAGGAAATGAT (SEQ ID NO: 180) ANGPT2 11
CTGTTAGCATTTGTGAACATTT (SEQ ID NO: 181) ANGPT2 12
TGTGGTCCTTCCAACTTGAACG (SEQ ID NO: 182) ANGPT2 13
CGGAATGTACTATCCACAGAGG (SEQ ID NO: 183) ANGPT2 14
TTATTTGTGTTCTGCCTCTGTG (SEQ ID NO: 184) ANGPT2 15
ACAAATAAGTTCAACGGCATTA (SEQ ID NO: 185) ANGPT2 16
AGCGAATAGCCTGAGCCTTTCC (SEQ ID NO: 186)
TABLE-US-00010 TABLE 10 Target sequences of VEGFA gene for SpCas9
Gene No. Target sequence VEGFA-Sp1 1 CAGCTGACCAGTCGCGCTGA (SEQ ID
NO: 187) VEGFA-Sp2 2 GTGGTAGCTGGGGCTGGGGG (SEQ ID NO: 188)
VEGFA-Sp3 3 GAGGTGGTAGCTGGGGCTGG (SEQ ID NO: 189) VEGFA-Sp4 4
GGAGGTGGTAGCTGGGGCTG (SEQ ID NO: 190) VEGFA-Sp5 5
AGGAGGTGGTAGCTGGGGCT (SEQ ID NO: 191) VEGFA-Sp6 6
GAGGAGGTGGTAGCTGGGGC (SEQ ID NO: 192) VEGFA-Sp7 7
CGGGGAGGAGGTGGTAGCTG (SEQ ID NO: 193) VEGFA-Sp8 8
CCCAGCTACCACCTCCTCCC (SEQ ID NO: 194) VEGFA-Sp9 9
CCGGGGAGGAGGTGGTAGCT (SEQ ID NO: 195) VEGFA-Sp10 10
GCCGGGGAGGAGGTGGTAGC (SEQ ID NO: 196) VEGFA-Sp11 11
GCTACCACCTCCTCCCCGGC (SEQ ID NO: 197) VEGFA-Sp12 12
ACCACCTCCTCCCCGGCCGG (SEQ ID NO: 198) VEGFA-Sp13 13
GCCGCCGGCCGGGGAGGAGG (SEQ ID NO: 199) VEGFA-Sp14 14
ACCTCCTCCCCGGCCGGCGG (SEQ ID NO: 200) VEGFA-Sp15 15
TCCGCCGCCGGCCGGGGAGG (SEQ ID NO: 201) VEGFA-Sp16 16
CTGTCCGCCGCCGGCCGGGG (SEQ ID NO: 202) VEGFA-Sp17 17
CCCCGGCCGGCGGCGGACAG (SEQ ID NO: 203) VEGFA-Sp18 18
CCACTGTCCGCCGCCGGCCG (SEQ ID NO: 204) VEGFA-Sp19 19
TCCACTGTCCGCCGCCGGCC (SEQ ID NO: 205) VEGFA-Sp20 20
GTCCACTGTCCGCCGCCGGC (SEQ ID NO: 206) VEGFA-Sp21 21
CCGGCGGCGGACAGTGGACG (SEQ ID NO: 207) VEGFA-Sp22 22
CCGCGTCCACTGTCCGCCGC (SEQ ID NO: 208) VEGFA-Sp23 23
GCGGCGGACAGTGGACGCGG (SEQ ID NO: 209) VEGFA-Sp24 24
GTGGACGCGGCGGCGAGCCG (SEQ ID NO: 210) VEGFA-Sp25 25
TGGACGCGGCGGCGAGCCGC (SEQ ID NO: 211) VEGFA-Sp26 26
CGCGGCGGCGAGCCGCGGGC (SEQ ID NO: 212) VEGFA-Sp27 27
GCGGCGGCGAGCCGCGGGCA (SEQ ID NO: 213) VEGFA-Sp28 28
CGGCGGCGAGCCGCGGGCAG (SEQ ID NO: 214) VEGFA-Sp29 29
GGCGAGCCGCGGGCAGGGGC (SEQ ID NO: 215) VEGFA-Sp30 30
CGGGCTCCGGCCCCTGCCCG (SEQ ID NO: 216) VEGFA-Sp31 31
CAGGGGCCGGAGCCCGCGCC (SEQ ID NO: 217) VEGFA-Sp32 32
GGGCCGGAGCCCGCGCCCGG (SEQ ID NO: 218) VEGFA-Sp33 33
CCGGAGCCCGCGCCCGGAGG (SEQ ID NO: 219) VEGFA-Sp34 34
CCGCCTCCGGGCGCGGGCTC (SEQ ID NO: 220) VEGFA-Sp35 35
CGGAGCCCGCGCCCGGAGGC (SEQ ID NO: 221) VEGFA-Sp36 36
GGAGCCCGCGCCCGGAGGCG (SEQ ID NO: 222) VEGFA-Sp37 37
GCCCGCGCCCGGAGGCGGGG (SEQ ID NO: 223) VEGFA-Sp38 38
TCCACCCCGCCTCCGGGCGC (SEQ ID NO: 224) VEGFA-Sp39 39
CTCCACCCCGCCTCCGGGCG (SEQ ID NO: 225) VEGFA-Sp40 40
CGCGCCCGGAGGCGGGGTGG (SEQ ID NO: 226) VEGFA-Sp41 41
GCGCCCGGAGGCGGGGTGGA (SEQ ID NO: 227) VEGFA-Sp42 42
CGCCCGGAGGCGGGGTGGAG (SEQ ID NO: 228) VEGFA-Sp43 43
GCCCGGAGGCGGGGTGGAGG (SEQ ID NO: 229) VEGFA-Sp44 44
ACCCCCTCCACCCCGCCTCC (SEQ ID NO: 230) VEGFA-Sp45 45
GACCCCCTCCACCCCGCCTC (SEQ ID NO: 331) VEGFA-Sp46 46
GGAGGCGGGGTGGAGGGGGT (SEQ ID NO: 232) VEGFA-Sp47 47
GAGGCGGGGTGGAGGGGGTC (SEQ ID NO: 233) VEGFA-Sp48 48
AGGCGGGGTGGAGGGGGTCG (SEQ ID NO: 234) VEGFA-Sp49 49
GTGGAGGGGGTCGGGGCTCG (SEQ ID NO: 235) VEGFA-Sp50 50
GAAACTTTTCGTCCAACTTC (SEQ ID NO: 236) VEGFA-Sp51 51
AAACTTTTCGTCCAACTTCT (SEQ ID NO: 237) VEGFA-Sp52 52
AGCGAGAACAGCCCAGAAGT (SEQ ID NO: 238) VEGFA-Sp53 53
CTTCTGGGCTGTTCTCGCTT (SEQ ID NO: 239) VEGFA-Sp54 54
CTGGGCTGTTCTCGCTTCGG (SEQ ID NO: 240) VEGFA-Sp55 55
TTCTCGCTTCGGAGGAGCCG (SEQ ID NO: 241) VEGFA-Sp56 56
CGGAGGAGCCGTGGTCCGCG (SEQ ID NO: 242) VEGFA-Sp57 57
GGAGGAGCCGTGGTCCGCGC (SEQ ID NO: 243) VEGFA-Sp58 58
GAGGAGCCGTGGTCCGCGCG (SEQ ID NO: 244) VEGFA-Sp59 59
AGGAGCCGTGGTCCGCGCGG (SEQ ID NO: 245) VEGFA-Sp60 60
GGCTTCCCCCGCGCGGACCA (SEQ ID NO: 246) VEGFA-Sp61 61
TCGGCTCGGCTTCCCCCGCG (SEQ ID NO: 247) VEGFA-Sp62 62
GCGGGGGAAGCCGAGCCGAG (SEQ ID NO: 248) VEGFA-Sp63 63
TCTCGCGGCTCCGCTCGGCT (SEQ ID NO: 249) VEGFA-Sp64 64
GCACTTCTCGCGGCTCCGCT (SEQ ID NO: 250) VEGFA-Sp65 65
AGCCGCGAGAAGTGCTAGCT (SEQ ID NO: 251) VEGFA-Sp66 66
GCCGCGAGAAGTGCTAGCTC (SEQ ID NO: 252) VEGFA-Sp67 67
GCCCGAGCTAGCACTTCTCG (SEQ ID NO: 253) VEGFA-Sp68 68
CGAGAAGTGCTAGCTCGGGC (SEQ ID NO: 254) VEGFA-Sp69 69
GAGAAGTGCTAGCTCGGGCC (SEQ ID NO: 255) VEGFA-Sp70 70
AAGTGCTAGCTCGGGCCGGG (SEQ ID NO: 256) VEGFA-Sp71 71
GGGCCGGGAGGAGCCGCAGC (SEQ ID NO: 257) VEGFA-Sp72 72
CCGGGAGGAGCCGCAGCCGG (SEQ ID NO: 258) VEGFA-Sp73 73
CCTCCGGCTGCGGCTCCTCC (SEQ ID NO: 259) VEGFA-Sp74 74
GGAGGAGCCGCAGCCGGAGG (SEQ ID NO: 260) VEGFA-Sp75 75
GAGGAGCCGCAGCCGGAGGA (SEQ ID NO: 261) VEGFA-Sp76 76
AGGAGCCGCAGCCGGAGGAG (SEQ ID NO: 262) VEGFA-Sp77 77
GGAGCCGCAGCCGGAGGAGG (SEQ ID NO: 263) VEGFA-Sp78 78
GCCGCAGCCGGAGGAGGGGG (SEQ ID NO: 264) VEGFA-Sp79 79
TCCTCCCCCTCCTCCGGCTG (SEQ ID NO: 265) VEGFA-Sp80 80
GCAGCCGGAGGAGGGGGAGG (SEQ ID NO: 266) VEGFA-Sp81 81
TCTTCCTCCTCCCCCTCCTC (SEQ ID NO: 267) VEGFA-Sp82 82
GAAGAGAAGGAAGAGGAGAG (SEQ ID NO: 268) VEGFA-Sp83 83
AAGAGAAGGAAGAGGAGAGG (SEQ ID NO: 269) VEGFA-Sp84 84
AAGAGGAGAGGGGGCCGCAG (SEQ ID NO: 270) VEGFA-Sp85 85
AGGGGGCCGCAGTGGCGACT (SEQ ID NO: 271) VEGFA-Sp86 86
CGAGCGCCGAGTCGCCACTG (SEQ ID NO: 272) VEGFA-Sp87 87
CGCAGTGGCGACTCGGCGCT (SEQ ID NO: 273) VEGFA-Sp88 88
GCGACTCGGCGCTCGGAAGC (SEQ ID NO: 274) VEGFA-Sp89 89
CGACTCGGCGCTCGGAAGCC (SEQ ID NO: 275) VEGFA-Sp90 90
GCGCTCGGAAGCCGGGCTCA (SEQ ID NO: 276) VEGFA-Sp91 91
TCGGAAGCCGGGCTCATGGA (SEQ ID NO: 277) VEGFA-Sp92 92
CGGAAGCCGGGCTCATGGAC (SEQ ID NO: 278) VEGFA-Sp93 93
GCCGGGCTCATGGACGGGTG (SEQ ID NO: 279) VEGFA-Sp94 94
GCCTCACCCGTCCATGAGCC (SEQ ID NO: 280) VEGFA-Sp95 95
GGGCTCATGGACGGGTGAGG (SEQ ID NO: 281) VEGFA-Sp96 96
CTCATGGACGGGTGAGGCGG (SEQ ID NO: 282) VEGFA-Sp97 97
TCCAGCCGCGCGCGCTCCCC (SEQ ID NO: 283) VEGFA-Sp98 98
GCCTGGGGAGCGCGCGCGGC (SEQ ID NO: 284) VEGFA-Sp99 99
CAGGGCCTGGGGAGCGCGCG (SEQ ID NO: 285) VEGFA-Sp100 100
CGCGCGCGCTCCCCAGGCCC (SEQ ID NO: 286) VEGFA-Sp101 101
GCGCTCCCCAGGCCCTGGCC (SEQ ID NO: 287) VEGFA-Sp102 102
CGCTCCCCAGGCCCTGGCCC (SEQ ID NO: 288) VEGFA-Sp103 103
GAGGCCCGGGCCAGGGCCTG (SEQ ID NO: 289) VEGFA-Sp104 104
CGAGGCCCGGGCCAGGGCCT (SEQ ID NO: 290) VEGFA-Sp105 105
CCAGGCCCTGGCCCGGGCCT (SEQ ID NO: 291) VEGFA-Sp106 106
CCGAGGCCCGGGCCAGGGCC (SEQ ID NO: 292) VEGFA-Sp107 107
CAGGCCCTGGCCCGGGCCTC (SEQ ID NO: 293) VEGFA-Sp108 108
CCCTGGCCCGGGCCTCGGGC (SEQ ID NO: 294) VEGFA-Sp109 109
CCGGCCCGAGGCCCGGGCCA (SEQ ID NO: 295) VEGFA-Sp110 110
CCTGGCCCGGGCCTCGGGCC (SEQ ID NO: 296) VEGFA-Sp111 111
CCCGGCCCGAGGCCCGGGCC (SEQ ID NO: 297) VEGFA-Sp112 112
CTGGCCCGGGCCTCGGGCCG (SEQ ID NO: 298) VEGFA-Sp113 113
GCCCGGGCCTCGGGCCGGGG (SEQ ID NO: 299) VEGFA-Sp114 114
TCCTCCCCGGCCCGAGGCCC (SEQ ID NO: 300) VEGFA-Sp115 115
TTCCTCCCCGGCCCGAGGCC (SEQ ID NO: 301) VEGFA-Sp116 116
TACTCTTCCTCCCCGGCCCG (SEQ ID NO: 302) VEGFA-Sp117 117
GGCGAGCTACTCTTCCTCCC (SEQ ID NO: 303) VEGFA-Sp118 118
GGAGGAAGAGTAGCTCGCCG (SEQ ID NO: 304) VEGFA-Sp119 119
AGTAGCTCGCCGAGGCGCCG (SEQ ID NO: 305) VEGFA-Sp120 120
CGCCGAGGCGCCGAGGAGAG (SEQ ID NO: 306) VEGFA-Sp121 121
GCCGAGGCGCCGAGGAGAGC (SEQ ID NO: 307) VEGFA-Sp122 122
GCCCGCTCTCCTCGGCGCCT (SEQ ID NO: 308) VEGFA-Sp123 123
GTGGGGCGGCCCGCTCTCCT (SEQ ID NO: 309)
VEGFA-Sp124 124 GGCCGCCCCACAGCCCGAGC (SEQ ID NO: 310) VEGFA-Sp125
125 CTCCGGCTCGGGCTGTGGGG (SEQ ID NO: 311) VEGFA-Sp126 126
CCCCACAGCCCGAGCCGGAG (SEQ ID NO: 312) VEGFA-Sp127 127
CCTCTCCGGCTCGGGCTGTG (SEQ ID NO: 313) VEGFA-Sp128 128
CCCACAGCCCGAGCCGGAGA (SEQ ID NO: 314) VEGFA-Sp129 129
CCCTCTCCGGCTCGGGCTGT (SEQ ID NO: 315) VEGFA-Sp130 130
TCCCTCTCCGGCTCGGGCTG (SEQ ID NO: 316) VEGFA-Sp131 131
TCGCGCTCCCTCTCCGGCTC (SEQ ID NO: 317) VEGFA-Sp132 132
CTCGCGCTCCCTCTCCGGCT (SEQ ID NO: 318) VEGFA-Sp133 133
CGCGGCTCGCGCTCCCTCTC (SEQ ID NO: 319) VEGFA-Sp134 134
AGAGGGAGCGCGAGCCGCGC (SEQ ID NO: 320) VEGFA-Sp135 135
CGAGCCGCGCCGGCCCCGGT (SEQ ID NO: 321) VEGFA-Sp136 136
GAGCCGCGCCGGCCCCGGTC (SEQ ID NO: 322) VEGFA-Sp137 137
AGGCCCGACCGGGGCCGGCG (SEQ ID NO: 323) VEGFA-Sp138 138
TTCGGAGGCCCGACCGGGGC (SEQ ID NO: 324) VEGFA-Sp139 139
TGGTTTCGGAGGCCCGACCG (SEQ ID NO: 325) VEGFA-Sp140 140
ATGGTTTCGGAGGCCCGACC (SEQ ID NO: 326) VEGFA-Sp141 141
CATGGTTTCGGAGGCCCGAC (SEQ ID NO: 327) VEGFA-Sp142 142
CAGAAAGTTCATGGTTTCGG (SEQ ID NO: 328) VEGFA-Sp143 143
CAGCAGAAAGTTCATGGTTT (SEQ ID NO: 329) VEGFA-Sp144 144
CCATGAACTTTCTGCTGTCT (SEQ ID NO: 330) VEGFA-Sp145 145
CCAAGACAGCAGAAAGTTCA (SEQ ID NO: 331) VEGFA-Sp146 146
CATGAACTTTCTGCTGTCTT (SEQ ID NO: 332) VEGFA-Sp147 147
TTCTGCTGTCTTGGGTGCAT (SEQ ID NO: 333) VEGFA-Sp148 148
GGAGGTAGAGCAGCAAGGCA (SEQ ID NO: 334) VEGFA-Sp149 149
ATGGTGGAGGTAGAGCAGCA (SEQ ID NO: 335) VEGFA-Sp150 150
GCTCTACCTCCACCATGCCA (SEQ ID NO: 336) VEGFA-Sp151 151
CGCTTACCTTGGCATGGTGG (SEQ ID NO: 337) VEGFA-Sp152 152
GACCGCTTACCTTGGCATGG (SEQ ID NO: 338) VEGFA-Sp153 153
CACGACCGCTTACCTTGGCA (SEQ ID NO: 339) VEGFA-Sp154 154
TTTCTGTCCTCAGTGGTCCC (SEQ ID NO: 340) VEGFA-Sp155 155
GGTGCAGCCTGGGACCACTG (SEQ ID NO: 341) VEGFA-Sp156 156
GTGGTCCCAGGCTGCACCCA (SEQ ID NO: 342) VEGFA-Sp157 157
TTCTGCCATGGGTGCAGCCT (SEQ ID NO: 343) VEGFA-Sp158 158
CTTCTGCCATGGGTGCAGCC (SEQ ID NO: 344) VEGFA-Sp159 159
CAGGCTGCACCCATGGCAGA (SEQ ID NO: 345) VEGFA-Sp160 160
GCTGCACCCATGGCAGAAGG (SEQ ID NO: 346) VEGFA-Sp161 161
GCACCCATGGCAGAAGGAGG (SEQ ID NO: 347) VEGFA-Sp162 162
CACCCATGGCAGAAGGAGGA (SEQ ID NO: 348) VEGFA-Sp163 163
TGCCCTCCTCCTTCTGCCAT (SEQ ID NO: 349) VEGFA-Sp164 164
CTGCCCTCCTCCTTCTGCCA (SEQ ID NO: 350) VEGFA-Sp165 165
GGAGGGCAGAATCATCACGA (SEQ ID NO: 351) VEGFA-Sp166 166
TCATGCAGTGGTGAAGTTCA (SEQ ID NO: 352) VEGFA-Sp167 167
CTGCCATCCAATCGAGACCC (SEQ ID NO: 353) VEGFA-Sp168 168
CCATCCAATCGAGACCCTGG (SEQ ID NO: 354) VEGFA-Sp169 169
CCACCAGGGTCTCGATTGGA (SEQ ID NO: 355) VEGFA-Sp170 170
ATGTCCACCAGGGTCTCGAT (SEQ ID NO: 356) VEGFA-Sp171 171
GACCCTGGTGGACATCTTCC (SEQ ID NO: 357) VEGFA-Sp172 172
CTCCTGGAAGATGTCCACCA (SEQ ID NO: 358) VEGFA-Sp173 173
ACTCCTGGAAGATGTCCACC (SEQ ID NO: 359) VEGFA-Sp174 174
CGATCTCATCAGGGTACTCC (SEQ ID NO: 360) VEGFA-Sp175 175
AGATGTACTCGATCTCATCA (SEQ ID NO: 361) VEGFA-Sp176 176
AAGATGTACTCGATCTCATC (SEQ ID NO: 362) VEGFA-Sp177 177
CGCATCAGGGGCACACAGGA (SEQ ID NO: 363) VEGFA-Sp178 178
GCATCGCATCAGGGGCACAC (SEQ ID NO: 364) VEGFA-Sp179 179
TGTGTGCCCCTGATGCGATG (SEQ ID NO: 365) VEGFA-Sp180 180
GTGTGCCCCTGATGCGATGC (SEQ ID NO: 366) VEGFA-Sp181 181
TGTGCCCCTGATGCGATGCG (SEQ ID NO: 367) VEGFA-Sp182 182
GTGCCCCTGATGCGATGCGG (SEQ ID NO: 368) VEGFA-Sp183 183
CAGCCCCCGCATCGCATCAG (SEQ ID NO: 369) VEGFA-Sp184 184
GCAGCCCCCGCATCGCATCA (SEQ ID NO: 370) VEGFA-Sp185 185
AGCAGCCCCCGCATCGCATC (SEQ ID NO: 371) VEGFA-Sp186 186
CGGGGGCTGCTGCAATGACG (SEQ ID NO: 372) VEGFA-Sp187 187
GGGGGCTGCTGCAATGACGA (SEQ ID NO: 373) VEGFA-Sp188 188
CTGCTGCAATGACGAGGGCC (SEQ ID NO: 374) VEGFA-Sp189 189
CCTGGAGTGTGTGCCCACTG (SEQ ID NO: 375) VEGFA-Sp190 190
CCTCAGTGGGCACACACTCC (SEQ ID NO: 376) VEGFA-Sp191 191
GTGATGTTGGACTCCTCAGT (SEQ ID NO: 377) VEGFA-Sp192 192
GGTGATGTTGGACTCCTCAG (SEQ ID NO: 378) VEGFA-Sp193 193
GGAGTCCAACATCACCATGC (SEQ ID NO: 379) VEGFA-Sp194 194
GTCCAACATCACCATGCAGG (SEQ ID NO: 380) VEGFA-Sp195 195
TCCAACATCACCATGCAGGT (SEQ ID NO: 381) VEGFA-Sp196 196
GCCCACCTGCATGGTGATGT (SEQ ID NO: 382) VEGFA-Sp197 197
CCCAAAGATGCCCACCTGCA (SEQ ID NO: 383) VEGFA-Sp198 198
TCCTTCCTTTCCAGATTATG (SEQ ID NO: 384) VEGFA-Sp199 199
GAGGTTTGATCCGCATAATC (SEQ ID NO: 385) VEGFA-Sp200 200
ATGCGGATCAAACCTCACCA (SEQ ID NO: 386) VEGFA-Sp201 201
CCTCACCAAGGCCAGCACAT (SEQ ID NO: 387) VEGFA-Sp202 202
CCTATGTGCTGGCCTTGGTG (SEQ ID NO: 388) VEGFA-Sp203 203
TCTCTCCTATGTGCTGGCCT (SEQ ID NO: 389) VEGFA-Sp204 204
AGCTCATCTCTCCTATGTGC (SEQ ID NO: 390) VEGFA-Sp205 205
ATTCACATTTGTTGTGCTGT (SEQ ID NO: 391) VEGFA-Sp206 206
AGCACAACAAATGTGAATGC (SEQ ID NO: 392) VEGFA-Sp207 207
AACAAATGTGAATGCAGGTG (SEQ ID NO: 393) VEGFA-Sp208 208
TGTCTTGCTCTATCTTTCTT (SEQ ID NO: 394) VEGFA-Sp209 209
TTTTCCAGAAAATCAGTTCG (SEQ ID NO: 395) VEGFA-Sp210 210
CTTTCCTCGAACTGATTTTC (SEQ ID NO: 396) VEGFA-Sp211 211
CAGAAAATCAGTTCGAGGAA (SEQ ID NO: 397) VEGFA-Sp212 212
AGAAAATCAGTTCGAGGAAA (SEQ ID NO: 398) VEGFA-Sp213 213
ATCAGTTCGAGGAAAGGGAA (SEQ ID NO: 399) VEGFA-Sp214 214
TCAGTTCGAGGAAAGGGAAA (SEQ ID NO: 400) VEGFA-Sp215 215
CAGTTCGAGGAAAGGGAAAG (SEQ ID NO: 401) VEGFA-Sp216 216
AACGAAAGCGCAAGAAATCC (SEQ ID NO: 402) VEGFA-Sp217 217
AGAAATCCCGGTATAAGTCC (SEQ ID NO: 403) VEGFA-Sp218 218
CACGCTCCAGGACTTATACC (SEQ ID NO: 404) VEGFA-Sp219 219
ACACGCTCCAGGACTTATAC (SEQ ID NO: 405) VEGFA-Sp220 220
AAGTCCTGGAGCGTGTACGT (SEQ ID NO: 406) VEGFA-Sp221 221
GGCACCAACGTACACGCTCC (SEQ ID NO: 407) VEGFA-Sp222 222
CCCGCTGCTGTCTAATGCCC (SEQ ID NO: 408) VEGFA-Sp223 223
CCAGGGCATTAGACAGCAGC (SEQ ID NO: 409) VEGFA-Sp224 224
TCCAGGGCATTAGACAGCAG (SEQ ID NO: 410) VEGFA-Sp225 225
CTAATGCCCTGGAGCCTCCC (SEQ ID NO: 411) VEGFA-Sp226 226
TGGGGGCCAGGGAGGCTCCA (SEQ ID NO: 412) VEGFA-Sp227 227
CTGGGGGCCAGGGAGGCTCC (SEQ ID NO: 413) VEGFA-Sp228 228
GGTTGTACTGGGGGCCAGGG (SEQ ID NO: 414) VEGFA-Sp229 229
GGAGGTTGTACTGGGGGCCA (SEQ ID NO: 415) VEGFA-Sp230 230
CGGAGGTTGTACTGGGGGCC (SEQ ID NO: 416) VEGFA-Sp231 231
GCAGGCGGAGGTTGTACTGG (SEQ ID NO: 417) VEGFA-Sp232 232
TTGCCTTTTTGCAGTCCCTG (SEQ ID NO: 418) VEGFA-Sp233 233
TGCCTTTTTGCAGTCCCTGT (SEQ ID NO: 419) VEGFA-Sp234 234
CGCTCTGAGCAAGGCCCACA (SEQ ID NO: 420) VEGFA-Sp235 235
CCTGTGGGCCTTGCTCAGAG (SEQ ID NO: 421) VEGFA-Sp236 236
CCGCTCTGAGCAAGGCCCAC (SEQ ID NO: 422) VEGFA-Sp237 237
TGCTTTCTCCGCTCTGAGCA (SEQ ID NO: 423) VEGFA-Sp238 238
CAGGAACATTTACACGTCTG (SEQ ID NO: 424) VEGFA-Sp239 239
ACGCGAGTCTGTGTTTTTGC (SEQ ID NO: 425) VEGFA-Sp240 240
AAACACAGACTCGCGTTGCA (SEQ ID NO: 426) VEGFA-Sp241 241
CAGACTCGCGTTGCAAGGCG (SEQ ID NO: 427) VEGFA-Sp242 242
AGTTAAACGAACGTACTTGC (SEQ ID NO: 428) VEGFA-Sp243 243
AAACGAACGTACTTGCAGGT (SEQ ID NO: 429) VEGFA-Sp244 244
CCCTCAGATGTGACAAGCCG (SEQ ID NO: 430) VEGFA-Sp245 245
GCCTCGGCTTGTCACATCTG (SEQ ID NO: 431) VEGFA-Sp246 246
TCAGATGTGACAAGCCGAGG (SEQ ID NO: 432) VEGFA-Sp247 247
ACAAGCCGAGGCGGTGAGCC (SEQ ID NO: 433) VEGFA-Sp248 248
GCCGAGGCGGTGAGCCGGGC (SEQ ID NO: 434)
VEGFA-Sp249 249 TCCTGCCCGGCTCACCGCCT (SEQ ID NO: 435)
TABLE-US-00011 TABLE 11 Target sequences of HIF1A gene for SpCas9
Gene No. Target sequence HIF1A-Sp1 1 TTTAAATGAGCTCCCAATGT (SEQ ID
NO: 436) HIF1A-Sp2 2 GAGCTCCCAATGTCGGAGTT (SEQ ID NO: 437)
HIF1A-Sp3 3 GTTTTCCAAACTCCGACATT (SEQ ID NO: 438) HIF1A-Sp4 4
TGTTTTCCAAACTCCGACAT (SEQ ID NO: 439) HIF1A-Sp5 5
AAATTTGTCTTTTTAAAAGA (SEQ ID NO: 440) HIF1A-Sp6 6
GTCTTTTTAAAAGAAGGTCT (SEQ ID NO: 441) HIF1A-Sp7 7
AAACTCAAAACCTGAAGAAT (SEQ ID NO: 442) HIF1A-Sp8 8
CTGATTTCTTCCAATTCTTC (SEQ ID NO: 443) HIF1A-Sp9 9
GAAGAAATCAGAATAGAAAA (SEQ ID NO: 444) HIF1A- 10
AAGAAATCAGAATAGAAAAT (SEQ ID NO: 445) Sp10 HIF1A- 11
ATCAGAATAGAAAATGGGTA (SEQ ID NO: 446) Sp11 HIF1A- 12
CTCGAGATGCAGCCAGATCT (SEQ ID NO: 447) Sp12 HIF1A- 13
TTCTTTACTTCGCCGAGATC (SEQ ID NO: 448) Sp13 HIF1A- 14
GAACTCACATTATGTGGAAG (SEQ ID NO: 449) Sp14 HIF1A- 15
AGATGCGAACTCACATTATG (SEQ ID NO: 450) Sp15 HIF1A- 16
TGTGAGTTCGCATCTTGATA (SEQ ID NO: 451) Sp16 HIF1A- 17
TTGATAAGGCCTCTGTGATG (SEQ ID NO: 452) Sp17 HIF1A- 18
GATGGTAAGCCTCATCACAG (SEQ ID NO: 453) Sp18 HIF1A- 19
CCATCAGCTATTTGCGTGTG (SEQ ID NO: 454) Sp19 HIF1A- 20
CCTCACACGCAAATAGCTGA (SEQ ID NO: 455) Sp20 HIF1A- 21
TTTGCGTGTGAGGAAACTTC (SEQ ID NO: 456) Sp21 HIF1A- 22
GTGAGGAAACTTCTGGATGC (SEQ ID NO: 457) Sp22 HIF1A- 23
TGTGCCCTTTTTAGGTGATT (SEQ ID NO: 458) Sp23 HIF1A- 24
TTGCTTTTATTTGAAAGCCT (SEQ ID NO: 459) Sp24 HIF1A- 25
TTTTATTTGAAAGCCTTGGA (SEQ ID NO: 460) Sp25 HIF1A- 26
AGCCTTGGATGGTTTTGTTA (SEQ ID NO: 461) Sp26 HIF1A- 27
AACCATAACAAAACCATCCA (SEQ ID NO: 462) Sp27 HIF1A- 28
GTTATGGTTCTCACAGATGA (SEQ ID NO: 463) Sp28 HIF1A- 29
TGATAATGTGAACAAATACA (SEQ ID NO: 464) Sp29 HIF1A- 30
GATAATGTGAACAAATACAT (SEQ ID NO: 465) Sp30 HIF1A- 31
CAAATACATGGGATTAACTC (SEQ ID NO: 466) Sp31 HIF1A- 32
TGTTTACAGTTTGAACTAAC (SEQ ID NO: 467) Sp32 HIF1A- 33
TACTCATCCATGTGACCATG (SEQ ID NO: 468) Sp33 HIF1A- 34
CTCATTTCCTCATGGTCACA (SEQ ID NO: 469) Sp34 HIF1A- 35
GCATTTCTCTCATTTCCTCA (SEQ ID NO: 470) Sp35 HIF1A- 36
GAAATGCTTACACACAGAAA (SEQ ID NO: 471) Sp36 HIF1A- 37
TTCATTAGGCCTTGTGAAAA (SEQ ID NO: 472) Sp37 HIF1A- 38
TCATTAGGCCTTGTGAAAAA (SEQ ID NO: 473) Sp38 HIF1A- 39
GTTCTTTACCCTTTTTCACA (SEQ ID NO: 474) Sp39 HIF1A- 40
AAGTGTACCCTAACTAGCCG (SEQ ID NO: 475) Sp40 HIF1A- 41
AGTTCTTCCTCGGCTAGTTA (SEQ ID NO: 476) Sp41 HIF1A- 42
TAGTTCTTCCTCGGCTAGTT (SEQ ID NO: 477) Sp42 HIF1A- 43
TTATGTTCATAGTTCTTCCT (SEQ ID NO: 478) Sp43 HIF1A- 44
TGAACATAAAGTCTGCAACA (SEQ ID NO: 479) Sp44 HIF1A- 45
CATAAAGTCTGCAACATGGA (SEQ ID NO: 480) Sp45 HIF1A- 46
ACACAGGTATTGCACTGCAC (SEQ ID NO: 481) Sp46 HIF1A- 47
TGGTATCATATACGTGAATG (SEQ ID NO: 482) Sp47 HIF1A- 48
ACACTGAGGTTGGTTACTGT (SEQ ID NO: 483) Sp48 HIF1A- 49
AACAGTAACCAACCTCAGTG (SEQ ID NO: 484) Sp49 HIF1A- 50
ACAGTAACCAACCTCAGTGT (SEQ ID NO: 485) Sp50 HIF1A- 51
TCTTATACCCACACTGAGGT (SEQ ID NO: 486) Sp51 HIF1A- 52
GGTTTCTTATACCCACACTG (SEQ ID NO: 487) Sp52 HIF1A- 53
GAAACCACCTATGACCTGCT (SEQ ID NO: 488) Sp53 HIF1A- 54
AGCACCAAGCAGGTCATAGG (SEQ ID NO: 489) Sp54 HIF1A- 55
ATCAGCACCAAGCAGGTCAT (SEQ ID NO: 490) Sp55 HIF1A- 56
TTCACAAATCAGCACCAAGC (SEQ ID NO: 491) Sp56 HIF1A- 57
ATATTTGATGGGTGAGGAAT (SEQ ID NO: 492) Sp57 HIF1A- 58
AATATTTGATGGGTGAGGAA (SEQ ID NO: 493) Sp58 HIF1A- 59
ATTTCAATATTTGATGGGTG (SEQ ID NO: 494) Sp59 HIF1A- 60
AAGGAATTTCAATATTTGAT (SEQ ID NO: 495) Sp60 HIF1A- 61
AAAGGAATTTCAATATTTGA (SEQ ID NO: 496) Sp61 HIF1A- 62
AGGAAAGTCTTGCTATCTAA (SEQ ID NO: 497) Sp62 HIF1A- 63
TTTCCTCAGTCGACACAGCC (SEQ ID NO: 498) Sp63 HIF1A- 64
TATCCAGGCTGTGTCGACTG (SEQ ID NO: 499) Sp64 HIF1A- 65
AATAAGAAAATTTCATATCC (SEQ ID NO: 500) Sp65 HIF1A- 66
AATTTTCTTATTGTGATGAA (SEQ ID NO: 501) Sp66 HIF1A- 67
TAACAGAATTACCGAATTGA (SEQ ID NO: 502) Sp67 HIF1A- 68
AACAGAATTACCGAATTGAT (SEQ ID NO: 503) Sp68 HIF1A- 69
TGGCTCATATCCCATCAATT (SEQ ID NO: 504) Sp69 HIF1A- 70
TATGAGCCAGAAGAACTTTT (SEQ ID NO: 505) Sp70 HIF1A- 71
GAGCGGCCTAAAAGTTCTTC (SEQ ID NO: 506) Sp71 HIF1A- 72
GATAATATTCATAAATTGAG (SEQ ID NO: 507) Sp72 HIF1A- 73
TTATGAATATTATCATGCTT (SEQ ID NO: 508) Sp73 HIF1A- 74
CTTACTATCATGATGAGTTT (SEQ ID NO: 509) Sp74 HIF1A- 75
TCCCCCCTAGTGTTTACTAA (SEQ ID NO: 510) Sp75 HIF1A- 76
TTGTCCTTTAGTAAACACTA (SEQ ID NO: 511) Sp76 HIF1A- 77
CTTGTCCTTTAGTAAACACT (SEQ ID NO: 512) Sp77 HIF1A- 78
ACTAAAGGACAAGTCACCAC (SEQ ID NO: 513) Sp78 HIF1A- 79
AAGTCACCACAGGACAGTAC (SEQ ID NO: 514) Sp79 HIF1A- 80
AAGCATCCTGTACTGTCCTG (SEQ ID NO: 515) Sp80 HIF1A- 81
TACAGGATGCTTGCCAAAAG (SEQ ID NO: 516) Sp81 HIF1A- 82
AGGATGCTTGCCAAAAGAGG (SEQ ID NO: 517) Sp82 HIF1A- 83
CCAAAAGAGGTGGATATGTC (SEQ ID NO: 518) Sp83 HIF1A- 84
CCAGACATATCCACCTCTTT (SEQ ID NO: 519) Sp84 HIF1A- 85
CAAAAGAGGTGGATATGTCT (SEQ ID NO: 520) Sp85
HIF1A- 86 GCACTGTGGTTGAGAATTCT (SEQ ID NO: 521) Sp86 HIF1A- 87
TTCACACATACAATGCACTG (SEQ ID NO: 522) Sp87 HIF1A- 88
TATGTGTGAATTACGTTGTG (SEQ ID NO: 523) Sp88 HIF1A- 89
GACACATTCTGTTTGTTGAA (SEQ ID NO: 524) Sp89 HIF1A- 90
GGACACATTCTGTTTGTTGA (SEQ ID NO: 525) Sp90 HIF1A- 91
AACAGAATGTGTCCTTAAAC (SEQ ID NO: 526) Sp91 HIF1A- 92
CTGAAGATTCAACCGGTTTA (SEQ ID NO: 527) Sp92 HIF1A- 93
TTCATATCTGAAGATTCAAC (SEQ ID NO: 528) Sp93 HIF1A- 94
TGTATCTTCTGATTCAACTT (SEQ ID NO: 529) Sp94 HIF1A- 95
CCTCTTTGACAAACTTAAGA (SEQ ID NO: 530) Sp95 HIF1A- 96
CCTTCTTAAGTTTGTCAAAG (SEQ ID NO: 531) Sp96 HIF1A- 97
ACCTGATGCTTTAACTTTGC (SEQ ID NO: 532) Sp97 HIF1A- 98
GCCAGCAAAGTTAAAGCATC (SEQ ID NO: 533) Sp98 HIF1A- 99
ACTTTGCTGGCCCCAGCCGC (SEQ ID NO: 534) Sp99 HIF1A- 100
GATTGTGTCTCCAGCGGCTG (SEQ ID NO: 535) Sp100 HIF1A- 101
TGATTGTGTCTCCAGCGGCT (SEQ ID NO: 536) Sp101 HIF1A- 102
ATGATTGTGTCTCCAGCGGC (SEQ ID NO: 537) Sp102 HIF1A- 103
AGATATGATTGTGTCTCCAG (SEQ ID NO: 538) Sp103 HIF1A- 104
ACAATCATATCTTTAGATTT (SEQ ID NO: 539) Sp104 HIF1A- 105
TCTTTAGATTTTGGCAGCAA (SEQ ID NO: 540) Sp105 HIF1A- 106
GTCATCAGTTTCTGTGTCTG (SEQ ID NO: 541) Sp106 HIF1A- 107
AACTGATGACCAGCAACTTG (SEQ ID NO: 542) Sp107 HIF1A- 108
ATGGTACTTCCTCAAGTTGC (SEQ ID NO: 543) Sp108 HIF1A- 109
AGCATTACATCATTATATAA (SEQ ID NO: 544) Sp109 HIF1A- 110
GTAATTTTTCGTTGGGTGAG (SEQ ID NO: 545) Sp110 HIF1A- 111
TGTAATTTTTCGTTGGGTGA (SEQ ID NO: 546) Sp111 HIF1A- 112
CTGTAATTTTTCGTTGGGTG (SEQ ID NO: 547) Sp112 HIF1A- 113
ATATTCTGTAATTTTTCGTT (SEQ ID NO: 548) Sp113 HIF1A- 114
TATATTCTGTAATTTTTCGT (SEQ ID NO: 549) Sp114 HIF1A- 115
AAAATTACAGAATATAAATT (SEQ ID NO: 550) Sp115 HIF1A- 116
GGCGTTTCAGCGGTGGGTAA (SEQ ID NO: 551) Sp116 HIF1A- 117
GGCTTTGGCGTTTCAGCGGT (SEQ ID NO: 552) Sp117 HIF1A- 118
TGGCTTTGGCGTTTCAGCGG (SEQ ID NO: 553) Sp118 HIF1A- 119
AAGTGGCTTTGGCGTTTCAG (SEQ ID NO: 554) Sp119 HIF1A- 120
GCACTACTTCGAAGTGGCTT (SEQ ID NO: 555) Sp120 HIF1A- 121
GGGTCAGCACTACTTCGAAG (SEQ ID NO: 556) Sp121 HIF1A- 122
CAACTTCTTGATTGAGTGCA (SEQ ID NO: 557) Sp122 HIF1A- 123
GCAACTTCTTGATTGAGTGC (SEQ ID NO: 558) Sp123 HIF1A- 124
AGAACCAAATCCAGAGTCAC (SEQ ID NO: 559) Sp124 HIF1A- 125
AGTTCCAGTGACTCTGGATT (SEQ ID NO: 560) Sp125 HIF1A- 126
AAAGAAAGTTCCAGTGACTC (SEQ ID NO: 561) Sp126 HIF1A- 127
TTTTACCATGCCCCAGATTC (SEQ ID NO: 562) Sp127 HIF1A- 128
CTGATCCTGAATCTGGGGCA (SEQ ID NO: 563) Sp128 HIF1A- 129
GGTGTCTGATCCTGAATCTG (SEQ ID NO: 564) Sp129 HIF1A- 130
AGGTGTCTGATCCTGAATCT (SEQ ID NO: 565) Sp130 HIF1A- 131
TAGGTGTCTGATCCTGAATC (SEQ ID NO: 566) Sp131 HIF1A- 132
CAGACACCTAGTCCTTCCGA (SEQ ID NO: 567) Sp132 HIF1A- 133
GTGCTTCCATCGGAAGGACT (SEQ ID NO: 568) Sp133 HIF1A- 134
TGTCTAGTCGTTCCATCGGA (SEQ ID NO: 569) Sp134 HIF1A- 135
ACTTTGTCTAGTGCTTCCAT (SEQ ID NO: 570) Sp135 HIF1A- 136
CACTAGACAAAGTTCACCTG (SEQ ID NO: 571) Sp136 HIF1A- 137
AGACAAAGTTCACCTGAGGT (SEQ ID NO: 572) Sp137 HIF1A- 138
TATATCATGACACCTACCTC (SEQ ID NO: 573) Sp138 HIF1A- 139
CAATATTCACTGGGACTATT (SEQ ID NO: 574) Sp139 HIF1A- 140
ACATAAAAACAATATTCACT (SEQ ID NO: 575) Sp140 HIF1A- 141
CACATAAAAACAATATTCAC (SEQ ID NO: 576) Sp141 HIF1A- 142
CAGTGAATATTGTTTTTATG (SEQ ID NO: 577) Sp142 HIF1A- 143
TTTTTATGTGGATAGTGATA (SEQ ID NO: 578) Sp143 HIF1A- 144
TATGGTCAATGAATTCAAGT (SEQ ID NO: 579) Sp144 HIF1A- 145
CAATGAATTCAAGTTGGAAT (SEQ ID NO: 580) Sp145 HIF1A- 146
AAAGAACCCATTTTCTACTC (SEQ ID NO: 581) Sp146 HIF1A- 147
CATATACCTGAGTAGAAAAT (SEQ ID NO: 582) Sp147 HIF1A- 148
TCATATACCTGAGTAGAAAA (SEQ ID NO: 583) Sp148 HIF1A- 149
AAAGGACACAGATTTAGACT (SEQ ID NO: 584) Sp149 HIF1A- 150
GTTAGCTCCCTATATCCCAA (SEQ ID NO: 585) Sp150 HIF1A- 151
TCATCATCCATTGGGATATA (SEQ ID NO: 586) Sp151 HIF1A- 152
GTCATCATCCATTGGGATAT (SEQ ID NO: 587) Sp152 HIF1A- 153
ACTGGAAGTCATCATCCATT (SEQ ID NO: 588) Sp153 HIF1A- 154
AACTGGAAGTCATCATCCAT (SEQ ID NO: 589) Sp154 HIF1A- 155
ACTGATCGAAGGAACGTAAC (SEQ ID NO: 590) Sp155 HIF1A- 156
TAATGGTGACAACTGATCGA (SEQ ID NO: 591) Sp156 HIF1A- 157
CTTGCGGAACTGCTTTCTAA (SEQ ID NO: 592) Sp157 HIF1A- 158
ACTTGCGCTTTCAGGGCTTG (SEQ ID NO: 593) Sp158 HIF1A- 159
TTTGAGGACTTGCGCTTTCA (SEQ ID NO: 594) Sp159 HIF1A- 160
CTTTGAGGACTTGCGCTTTC (SEQ ID NO: 595) Sp160 HIF1A- 161
AATACTGTAACTGTGCTTTG (SEQ ID NO: 596) Sp161 HIF1A- 162
GTTCTTGTATTTGAGTCTGC (SEQ ID NO: 597) Sp162 HIF1A- 163
GTAGTGGTGGCATTAGCAGT (SEQ ID NO: 598) Sp163 HIF1A- 164
AGTGGTGGCAGTGGTAGTGG (SEQ ID NO: 599) Sp164 HIF1A- 165
ATCAGTGGTGGCAGTGGTAG (SEQ ID NO: 600) Sp165 HIF1A- 166
TAATTCATCAGTGGTGGCAG (SEQ ID NO: 601) Sp166 HIF1A- 167
TGTTTTTAATTCATCAGTGG (SEQ ID NO: 602) Sp167 HIF1A- 168
CACTGTTTTTAATTCATCAG (SEQ ID NO: 603) Sp163 HIF1A- 169
AACAGTGACAAAAGACCGTA (SEQ ID NO: 604)
Sp169 HIF1A- 170 ATATTTTAATGTCTTCCATA (SEQ ID NO: 605) Sp170 HIF1A-
171 TTATGTATGTGGGTAGGAGA (SEQ ID NO: 606) Sp171 HIF1A- 172
GTTTCTTTATGTATGTGGGT (SEQ ID NO: 607) Sp172 HIF1A- 173
AGTAGTTTCTTTATGTATGT (SEQ ID NO: 608) Sp173 HIF1A- 174
TAGTAGTTTCTTTATGTATG (SEQ ID NO: 609) Sp174 HIF1A- 175
ATCTCTATATGGTGATGATG (SEQ ID NO: 610) Sp175 HIF1A- 176
CGACTTTGAGTATCTCTATA (SEQ ID NO: 611) Sp176 HIF1A- 177
CATATAGAGATACTCAAAGT (SEQ ID NO: 612) Sp177 HIF1A- 178
ACAGCCTCACCAAACAGAGC (SEQ ID NO: 613) Sp178 HIF1A- 179
TTTTCCTGCTCTGTTTGGTG (SEQ ID NO: 614) Sp179 HIF1A- 180
TCACCAAACAGAGCAGGAAA (SEQ ID NO: 615) Sp180 HIF1A- 181
ACTCCTTTTCCTGCTCTGTT (SEQ ID NO: 616) Sp181 HIF1A- 182
GATAACACGTTAGGGCTTCT (SEQ ID NO: 617) Sp182 HIF1A- 183
AAGCGACAGATAACACGTTA (SEQ ID NO: 618) Sp183 HIF1A- 184
AAAGCGACAGATAACACGTT (SEQ ID NO: 619) Sp184 HIF1A- 185
TATCTGTCGCTTTGAGTCAA (SEQ ID NO: 620) Sp185 HIF1A- 186
TTTCAGAACTACAGTTCCTG (SEQ ID NO: 621) Sp186 HIF1A- 187
TTTGGATTTAGTTCTTCCTC (SEQ ID NO: 622) Sp187 HIF1A- 188
TTCTGCAAAGCTAGTATCTT (SEQ ID NO: 623) Sp188 HIF1A- 189
TGCTCAGAGAAAGCGAAAAA (SEQ ID NO: 624) Sp189 HIF1A- 190
AAGCGAAAAATGGAACATGA (SEQ ID NO: 625) Sp190 HIF1A- 191
GTAGTAGCTGCATGATCGTC (SEQ ID NO: 626) Sp191 HIF1A- 192
CAGCTACTACATCACTTTCT (SEQ ID NO: 627) Sp192 HIF1A- 193
CTTTCTTGGAAACGTGTAAA (SEQ ID NO: 628) Sp193 HIF1A- 194
ACAATTATTTTAATACCCTC (SEQ ID NO: 629) Sp194 HIF1A- 195
AAAAGAATAAACTAACCAGA (SEQ ID NO: 630) Sp195 HIF1A- 196
AAAAAGAATAAACTAACCAG (SEQ ID NO: 631) Sp196 HIF1A- 197
AGATTTAGCATGTAGACTGC (SEQ ID NO: 632) Sp197 HIF1A- 198
GATTTAGCATGTAGACTGCT (SEQ ID NO: 633) Sp198 HIF1A- 199
ATTTAGCATGTAGACTGCTG (SEQ ID NO: 634) Sp199 HIF1A- 200
TAGACTGCTGGGGCAATCAA (SEQ ID NO: 635) Sp200 HIF1A- 201
GGGCAATCAATGGATGAAAG (SEQ ID NO: 636) Sp201 HIF1A- 202
CAATCATAACTGGTCAGCTG (SEQ ID NO: 637) Sp202 HIF1A- 203
ATTAACTTCACAATCATAAC (SEQ ID NO: 638) Sp203 HIF1A- 204
GAAGTTAATGCTCCTATACA (SEQ ID NO: 639) Sp204 HIF1A- 205
AGGTTTCTGCTGCCTTGTAT (SEQ ID NO: 640) Sp205 HIF1A- 206
AGGCAGCAGAAACCTACTGC (SEQ ID NO: 641) Sp206 HIF1A- 207
GGCAGCAGAAACCTACTGCA (SEQ ID NO: 642) Sp207 HIF1A- 208
GTAATTCTTCACCCTGCAGT (SEQ ID NO: 643) Sp208 HIF1A- 209
TGAAGAATTACTCAGAGCTT (SEQ ID NO: 644) Sp209
TABLE-US-00012 TABLE 12 Target sequences of EPAS1 gene for SpCas9
Gene No. Target sequence EPAS1- 1 AGCGACAATGACAGCTGACA (SEQ ID NO:
645) Sp1 EPAS1- 2 CAGCTGACAAGGAGAAGAAA (SEQ ID NO: 646) Sp2 EPAS1-
3 TTCTCCACTTAGGAGTAGCT (SEQ ID NO: 647) Sp3 EPAS1- 4
CACTTAGGAGTAGCTCGGAG (SEQ ID NO: 648) Sp4 EPAS1- 5
TTAGGAGTAGCTCGGAGAGG (SEQ ID NO: 649) Sp5 EPAS1- 6
GAGTAGCTCGGAGAGGAGGA (SEQ ID NO: 650) Sp6 EPAS1- 7
AGAGGAGGAAGGAGAAGTCC (SEQ ID NO: 651) Sp7 EPAS1- 8
GAGGAGGAAGGAGAAGTCCC (SEQ ID NO: 652) Sp8 EPAS1- 9
AGAAGTCCCGGGATCCTGCG (SEQ ID NO: 653) Sp9 EPAS1- 10
CCCGGGATGCTGCGCGGTGC (SEQ ID NO: 654) Sp10 EPAS1- 11
CCGGCACCGCGCAGCATCCC (SEQ ID NO: 655) Sp11 EPAS1- 12
GCCGGCACCGCGCAGCATCC (SEQ ID NO: 656) Sp12 EPAS1- 13
GGGATGCTGCGCGGTGCCGG (SEQ ID NO: 657) Sp13 EPAS1- 14
TGCGCGGTGCCGGCGGAGCA (SEQ ID NO: 658) Sp14 EPAS1- 15
GTGCCGGCGGAGCAAGGAGA (SEQ ID NO: 659) Sp15 EPAS1- 16
CCGGCGGAGCAAGGAGACGG (SEQ ID NO: 660) Sp16 EPAS1- 17
CCTCCGTCTCCTTGCTCCGC (SEQ ID NO: 661) Sp17 EPAS1- 18
GACGGAGGTGTTCTATGAGC (SEQ ID NO: 662) Sp18 EPAS1- 19
GTGGGGCAGAGGCAGCTCAT (SEQ ID NO: 663) Sp19 EPAS1- 20
TGTGGGGCAGAGGCAGCTCA (SEQ ID NO: 664) Sp20 EPAS1- 21
GAGCTCACACTGTGGGGCAG (SEQ ID NO: 665) Sp21 EPAS1- 22
AGATGGGAGCTCACACTGTG (SEQ ID NO: 666) Sp22 EPAS1- 23
CAGATGGGAGCTCACACTGT (SEQ ID NO: 667) Sp23 EPAS1- 24
CCACAGTGTGAGCTCCCATC (SEQ ID NO: 668) Sp24 EPAS1- 25
CCAGATGGGAGCTCACACTG (SEQ ID NO: 669) Sp25 EPAS1- 26
TGTGAGCTCCCATCTGGACA (SEQ ID NO: 670) Sp26 EPAS1- 27
GATGGAGGCCTTGTCCAGAT (SEQ ID NO: 671) Sp27 EPAS1- 28
TGATGGAGGCCTTGTCCAGA (SEQ ID NO: 672) Sp28 EPAS1- 29
CAAGGCCTCCATCATGCGAC (SEQ ID NO: 673) Sp29 EPAS1- 30
GATTGCCAGTCGCATGATGG (SEQ ID NO: 674) Sp30 EPAS1- 31
GCTGATTGCCAGTCGCATGA (SEQ ID NO: 675) Sp31 EPAS1- 32
AGAGGAGCTTGTGTGTTCGC (SEQ ID NO: 676) Sp32 EPAS1- 33
ACACACAAGCTCCTCTCCTC (SEQ ID NO: 677) Sp33 EPAS1- 34
CAAGCTCCTCTCCTCAGGTA (SEQ ID NO: 678) Sp34 EPAS1- 35
TGCTGGCCTTACCTGAGGAG (SEQ ID NO: 679) Sp35 EPAS1- 36
GAGCCTGCTGGCCTTACCTG (SEQ ID NO: 680) Sp36 EPAS1- 37
GACTCGTTTTCAGAGCAAAC (SEQ ID NO: 681) Sp37 EPAS1- 38
CTGCTGGTCAGCTTCGGCTT (SEQ ID NO: 682) Sp38 EPAS1- 39
AGCCGAAGCTGACCAGCAGA (SEQ ID NO: 683) Sp39 EPAS1- 40
GTCCATCTGCTGGTCAGCTT (SEQ ID NO: 684) Sp40 EPAS1- 41
GGTACAAGTTGTCCATCTGC (SEQ ID NO: 685) Sp41 EPAS1- 42
CAACTTGTACCTGAAAGCCT (SEQ ID NO: 686) Sp42 EPAS1- 43
CTTGTACCTGAAAGCCTTGG (SEQ ID NO: 687) Sp43 EPAS1- 44
TTGTACCTGAAAGCCTTGGA (SEQ ID NO: 688) Sp44 EPAS1- 45
TGAAACCCTCCAAGGCTTTC (SEQ ID NO: 689) Sp45 EPAS1- 46
CACGGCAATGAAACCCTCCA (SEQ ID NO: 690) Sp46 EPAS1- 47
CTTGGAGGGTTTCATTGCCG (SEQ ID NO: 691) Sp47 EPAS1- 48
ATTGCCGTGGTGACCCAAGA (SEQ ID NO: 692) Sp48 EPAS1- 49
GTCGCCATCTTGGGTCACCA (SEQ ID NO: 693) Sp49 EPAS1- 50
AAAGATCATGTCGCCATCTT (SEQ ID NO: 694) Sp50 EPAS1- 51
GAAAGATCATGTCGCCATCT (SEQ ID NO: 695) Sp51 EPAS1- 52
AGAAAACATCAGCAAGTTCA (SEQ ID NO: 696) Sp52 EPAS1- 53
GAAAACATCAGCAAGTTCAT (SEQ ID NO: 697) Sp53 EPAS1- 54
CAAGTTCATGGGACTTACAC (SEQ ID NO: 698) Sp54 EPAS1- 55
TTGAAACAGGTGGAGCTAAC (SEQ ID NO: 699) Sp55 EPAS1- 56
CACTCATCCCTGCGACCATG (SEQ ID NO: 700) Sp56 EPAS1- 57
CGAATCTCCTCATGGTCGCA (SEQ ID NO: 701) Sp57 EPAS1-
ACGAATCTCCTCATGGTCGC (SEQ ID NO: 702) Sp58 EPAS1- 59
GGTTCTCACGAATCTCCTCA (SEQ ID NO: 703) Sp59 EPAS1- 60
GAGAACCTGAGTCTCAAAAA (SEQ ID NO: 704) Sp60 EPAS1- 61
GGATACCATTTTTGAGACTC (SEQ ID NO: 705) Sp61 EPAS1- 62
ATCCTTCCACATCCAGGCTC (SEQ ID NO: 706) Sp62 EPAS1- 63
CCACATCCAGGCTCTGGTTT (SEQ ID NO: 707) Sp63 EPAS1- 64
CACATCCAGGCTCTGGTTTT (SEQ ID NO: 708) Sp64 EPAS1- 65
TTTTTCCCAAAACCAGAGCC (SEQ ID NO: 709) Sp65 EPAS1- 66
GCAAAGACATGTCCACAGAG (SEQ ID NO: 710) Sp66 EPAS1- 67
CAAAGACATGTCCACAGAGC (SEQ ID NO: 711) Sp67 EPAS1- 68
CATGAAGAAGTCCCGCTCTG (SEQ ID NO: 712) Sp68 EPAS1- 69
CAGAGCGGGACTTCTTCATG (SEQ ID NO: 713) Sp69 EPAS1- 70
CTTCATGAGGATGAAGTGCA (SEQ ID NO: 714) Sp70 EPAS1- 71
AAGTGCACGGTCACCAACAG (SEQ ID NO: 715) Sp71 EPAS1- 72
GTTGACAGTACGGCCTCTGT (SEQ ID NO: 716) Sp72 EPAS1- 73
CTGACTTGAGGTTGACAGTA (SEQ ID NO: 717) Sp73 EPAS1- 74
TCAACCTCAAGTCAGCCACC (SEQ ID NO: 718) Sp74 EPAS1- 75
CCTCAAGTCAGCCACCTGGA (SEQ ID NO: 719) Sp75 EPAS1- 76
CCTTCCAGGTGGCTGACTTG (SEQ ID NO: 720) Sp76 EPAS1- 77
AAGTCAGCCACCTGGAAGGT (SEQ ID NO: 721) Sp77 EPAS1- 78
AGTCAGCCACCTGGAAGGTA (SEQ ID NO: 722) Sp78 EPAS1- 79
ATGTTGCCCTACCTTCCAGG (SEQ ID NO: 723) Sp79 EPAS1- 80
CTGATGTTGCCCTACCTTCC (SEQ ID NO: 724) Sp80 EPAS1- 81
GTCTCAGGTCTTGCACTGCA (SEQ ID NO: 725) Sp81 EPAS1- 82
TCTCAGGTCTTGCACTGCAC (SEQ ID NO: 726) Sp82
EPAS1- 83 GGTCTTGCACTGCACGGGCC (SEQ ID NO: 727) Sp83 EPAS1- 84
AGTTGTTGTAGACTTTCACC (SEQ ID NO: 728) Sp84 EPAS1- 85
CACACAGACTATTGTGAGGA (SEQ ID NO: 729) Sp85 EPAS1- 86
CCTCCTCACAATAGTCTGTG (SEQ ID NO: 730) Sp86 EPAS1- 87
CCACACAGACTATTGTGAGG (SEQ ID NO: 731) Sp87 EPAS1- 88
TAGCCACACAGACTATTGTG (SEQ ID NO: 732) Sp88 EPAS1- 89
CAATAGTCTGTGTGGCTACA (SEQ ID NO: 733) Sp89 EPAS1- 90
ATGATGAGGCAGGACAGCAG (SEQ ID NO: 734) Sp90 EPAS1- 91
GATGATGAGGCAGGACAGCA (SEQ ID NO: 735) Sp91 EPAS1- 92
TGATGATGAGGCAGGACAGC (SEQ ID NO: 736) Sp92 EPAS1- 93
TTCACACATGATGATGAGGC (SEQ ID NO: 737) Sp93 EPAS1- 94
TTGGTTCACACATGATGATG (SEQ ID NO: 738) Sp94 EPAS1- 95
ATGTGGGATGGGTGCTGGAT (SEQ ID NO: 739) Sp95 EPAS1- 96
AATCCAGCACCCATCCCACA (SEQ ID NO: 740) Sp96 EPAS1- 97
TGTCCATGTGGGATGGGTGC (SEQ ID NO: 741) Sp97 EPAS1- 98
GGGGGATGTCCATGTGGGAT (SEQ ID NO: 742) Sp98 EPAS1- 99
AGGGGGATGTCCATGTGGGA (SEQ ID NO: 743) Sp99 EPAS1- 100
ATCCCACATGGACATCCCCC (SEQ ID NO: 744) Sp100 EPAS1- 101
ATCCAGGGGGATGTCCATGT (SEQ ID NO: 745) Sp101 EPAS1- 102
TATCCAGGGGGATGTCCATG (SEQ ID NO: 746) Sp102 EPAS1- 103
GGAAGGTCTTGCTATCCAGG (SEQ ID NO: 747) Sp103 EPAS1- 104
AGGAAGGTCTTGCTATCCAG (SEQ ID NO: 748) Sp104 EPAS1- 105
CAGGAAGGTCTTGCTATCCA (SEQ ID NO: 749) Sp105 EPAS1- 106
TCAGGAAGGTCTTGCTATCC (SEQ ID NO: 750) Sp106 EPAS1- 107
CATGCTGTGGCGGCTCAGGA (SEQ ID NO: 751) Sp107 EPAS1- 108
CTTCCTGAGCCGCCACAGCA (SEQ ID NO: 752) Sp108 EPAS1- 109
TGTCCATGCTGTGGCGGCTC (SEQ ID NO: 753) Sp109 EPAS1- 110
ACTTCATGTCCATGCTGTGG (SEQ ID NO: 754) Sp110 EPAS1- 111
TGAACTTCATGTCCATGCTG (SEQ ID NO: 755) Sp111 EPAS1- 112
AGTTCACCTACTGTGATGAC (SEQ ID NO: 756) Sp112 EPAS1- 113
CACCTACTGTGATGACAGGT (SEQ ID NO: 757) Sp113 EPAS1- 114
ACCTACTGTGATGACAGGTA (SEQ ID NO: 758) Sp114 EPAS1- 115
CCCCTACCTGTCATCACAGT (SEQ ID NO: 759) Sp115 EPAS1- 116
CTCAGAATCACAGAACTGAT (SEQ ID NO: 760) Sp116 EPAS1- 117
ACTGATTGGTTACCACCCTG (SEQ ID NO: 761) Sp117 EPAS1- 118
TACCACCCTGAGGAGCTGCT (SEQ ID NO: 762) Sp118 EPAS1- 119
GGCCAAGCAGCTCCTCAGGG (SEQ ID NO: 763) Sp119 EPAS1- 120
AGCGGCCAAGCAGCTCCTCA (SEQ ID NO: 764) Sp120 EPAS1- 121
GAGCGGCCAAGCAGCTCCTC (SEQ ID NO: 765) Sp121 EPAS1- 122
GGTAGAATTCATAGGCTGAG (SEQ ID NO: 766) Sp122 EPAS1- 123
TAGCGCATGGTAGAATTCAT (SEQ ID NO: 767) Sp123 EPAS1- 124
TGTTCTCGGAGTCTAGCGCA (SEQ ID NO: 768) Sp124 EPAS1- 125
GTGACTCTTGGTCATGTTCT (SEQ ID NO: 769) Sp125 EPAS1- 126
CTCACAGTTCTGGTGACTCT (SEQ ID NO: 770) Sp126 EPAS1- 127
ACTCCTGGAACTCACAGTTC (SEQ ID NO: 771) Sp127 EPAS1- 128
TCCTCCCCTAGTGTGCACCA (SEQ ID NO: 772) Sp128 EPAS1- 129
CCTCCCCTAGTGTGCACCAA (SEQ ID NO: 773) Sp129 EPAS1- 130
CTGACCCTTGGTGCACACTA (SEQ ID NO: 774) Sp130 EPAS1- 131
CCTAGTGTGCACCAAGGGTC (SEQ ID NO: 775) Sp131 EPAS1- 132
CCTGACCCTTGGTGCACACT (SEQ ID NO: 776) Sp132 EPAS1- 133
ACCAAGGGTCAGGTAGTAAG (SEQ ID NO: 777) Sp133 EPAS1- 134
GCCACTTACTACCTGACCCT (SEQ ID NO: 778) Sp134 EPAS1- 135
AGGTAGTAAGTGGCCAGTAC (SEQ ID NO: 779) Sp135 EPAS1- 136
GCTTTGCGAGCATCCGGTAC (SEQ ID NO: 780) Sp136 EPAS1- 137
TACCGGATGCTCGCAAAGCA (SEQ ID NO: 781) Sp137 EPAS1- 138
ACCGGATGCTCGCAAAGCAT (SEQ ID NO: 782) Sp138 EPAS1- 139
CCGGATGCTCGCAAAGCATG (SEQ ID NO: 783) Sp139 EPAS1- 140
CCCCATGCTTTGCGAGCATC (SEQ ID NO: 784) Sp140 EPAS1- 141
CGGATGCTCGCAAAGCATGG (SEQ ID NO: 785) Sp141 EPAS1- 142
CAAAGCATGGGGGCTACGTG (SEQ ID NO: 786) Sp142 EPAS1- 143
GCATGGGGGCTACGTGTGGC (SEQ ID NO: 787) Sp143 EPAS1- 144
CTACGTGTGGCTGGAGACCC (SEQ ID NO: 788) Sp144 EPAS1- 145
TACGTGTGGCTGGAGACCCA (SEQ ID NO: 789) Sp145 EPAS1- 146
ACGTGTGGCTGGAGACCCAG (SEQ ID NO: 790) Sp146 EPAS1- 147
GTGGCTGGAGACCCAGGGGA (SEQ ID NO: 791) Sp147 EPAS1- 148
GTTGTAGATGACCGTCCCCT (SEQ ID NO: 792) Sp148 EPAS1- 149
GGTTGTAGATGACCGTCCCC (SEQ ID NO: 793) Sp149 EPAS1- 150
ACTGGGGCTGCAGGTTGCGA (SEQ ID NO: 794) Sp150 EPAS1- 151
CACTGGGGCTGCAGGTTGCG (SEQ ID NO: 795) Sp151 EPAS1- 152
ACATGATGCACTGGGGCTGC (SEQ ID NO: 796) Sp152 EPAS1- 153
TTGACACACATGATGCACTG (SEQ ID NO: 797) Sp153 EPAS1- 154
GTTGACACACATGATGCACT (SEQ ID NO: 798) Sp154 EPAS1- 155
AGTTGACACACATGATGCAC (SEQ ID NO: 799) Sp155 EPAS1- 156
TGTGTGTCAACTACGTCCTG (SEQ ID NO: 800) Sp156 EPAS1- 157
AGCCCTCACATGCTTACCTC (SEQ ID NO: 801) Sp157 EPAS1- 158
TGAGATTGAGAAGAATGACG (SEQ ID NO: 802) Sp158 EPAS1- 159
GAATGACGTGGTGTTCTCCA (SEQ ID NO: 803) Sp159 EPAS1- 160
CAGGGATTCAGTCTGGTCCA (SEQ ID NO: 804) Sp160 EPAS1- 161
GCTTGAACAGGGATTCAGTC (SEQ ID NO: 805) Sp161 EPAS1- 162
CATCAGGTGGGGCTTGAACA (SEQ ID NO: 806) Sp162 EPAS1- 163
CCTGTTCAAGCCCCACCTGA (SEQ ID NO: 807) Sp163 EPAS1- 164
CCATCAGGTGGGGCTTGAAC (SEQ ID NO: 808) Sp164 EPAS1- 165
CTGTTCATGGCCATCAGGTG (SEQ ID NO: 809) Sp165 EPAS1- 166
GCTGTTCATGGCCATCAGGT (SEQ ID NO: 810)
Sp166 EPAS1- 167 TGCTGTTCATGGCCATCAGG (SEQ ID NO: 811) Sp167 EPAS1-
168 AGATGCTGTTCATGGCCATC (SEQ ID NO: 812) Sp168 EPAS1- 169
GCTATCAAAGATGCTGTTCA (SEQ ID NO: 813) Sp169 EPAS1- 170
AACAGCATCTTTGATAGCAG (SEQ ID NO: 814) Sp170 EPAS1- 171
CATCTTTGATAGCAGTGGCA (SEQ ID NO: 815) Sp171 EPAS1- 172
ATCTTTGATAGCAGTGGCAA (SEQ ID NO: 816) Sp172 EPAS1- 173
TCTTTGATAGCAGTGGCAAG (SEQ ID NO: 817) Sp173 EPAS1- 174
CTTTGATAGCAGTGGCAAGG (SEQ ID NO: 818) Sp174 EPAS1- 175
CTTCCTATTCACCAAGCTAA (SEQ ID NO: 819) Sp175 EPAS1- 176
CCTATTCACCAAGCTAAAGG (SEQ ID NO: 820) Sp176 EPAS1- 177
CCTCCTTTAGCTTGGTGAAT (SEQ ID NO: 821) Sp177 EPAS1- 178
CTCGGGCTCCTCCTTTAGCT (SEQ ID NO: 822) Sp178 EPAS1- 179
CAAGCTAAAGGAGGAGCCCG (SEQ ID NO: 823) Sp179 EPAS1- 180
AAAGGAGGAGCCCGAGGAGC (SEQ ID NO: 824) Sp180 EPAS1- 181
GCCCGAGGAGCTGGCCCAGC (SEQ ID NO: 825) Sp181 EPAS1- 182
GCCAGCTGGGCCAGCTCCTC (SEQ ID NO: 826) Sp182 EPAS1- 183
AGCCAGCTGGGCCAGCTCCT (SEQ ID NO: 827) Sp183 EPAS1- 184
GCCCAGCTGGCTCCCACCCC (SEQ ID NO: 828) Sp184 EPAS1- 185
TCCTGGGGTGGGAGCCAGCT (SEQ ID NO: 829) Sp185 EPAS1- 186
CTCCTGGGGTGGGAGCCAGC (SEQ ID NO: 830) Sp186 EPAS1- 187
ATGATGGCGTCTCCTGGGGT (SEQ ID NO: 831) Sp187 EPAS1- 188
GATGATGGCGTCTCCTGGGG (SEQ ID NO: 832) Sp188 EPAS1- 189
AGAGATGATGGCGTCTCCTG (SEQ ID NO: 833) Sp189 EPAS1- 190
GAGAGATGATGGCGTCTCCT (SEQ ID NO: 834) Sp190 EPAS1- 191
AGAGAGATGATGGCGTCTCC (SEQ ID NO: 835) Sp191 EPAS1- 192
AGGAGACGCCATCATCTCTC (SEQ ID NO: 836) Sp192 EPAS1- 193
GCCATCATCTCTCTGGATTT (SEQ ID NO: 837) Sp193 EPAS1- 194
ACCGAAATCCAGAGAGATGA (SEQ ID NO: 838) Sp194 EPAS1- 195
ATCATCTCTCTGGATTTCGG (SEQ ID NO: 839) Sp195 EPAS1- 196
TCATCTCTCTGGATTTCGGT (SEQ ID NO: 840) Sp196 EPAS1- 197
CTCGAAGTTCTGATTCCCTG (SEQ ID NO: 841) Sp197 EPAS1- 198
CACAGGGAATCAGAACTTCG (SEQ ID NO: 842) Sp198 EPAS1- 199
TTCGAGGAGTCCTCAGCCTA (SEQ ID NO: 843) Sp199 EPAS1- 200
GGAGTCCTCAGCCTATGGCA (SEQ ID NO: 844) Sp200 EPAS1- 201
GATGGCCTTGCCATAGGCTG (SEQ ID NO: 845) Sp201 EPAS1- 202
GGGCAGGATGGCCTTGCCAT (SEQ ID NO: 846) Sp202 EPAS1- 203
TGGCTGGCTCGGGGGCAGGA (SEQ ID NO: 847) Sp203 EPAS1- 204
TCCTGCCCCCGAGCCAGCCA (SEQ ID NO: 848) Sp204 EPAS1- 205
CCTGCCCCCGAGCCAGCCAT (SEQ ID NO: 849) Sp205 EPAS1- 206
CCCATGGCTGGCTCGGGGGC (SEQ ID NO: 850) Sp206 EPAS1- 207
GTGGCCCATGGCTGGCTCGG (SEQ ID NO: 851) Sp207 EPAS1- 208
CGTGGCCCATGGCTGGCTCG (SEQ ID NO: 852) Sp208 EPAS1- 209
CCCGAGCCAGCCATGGGCCA (SEQ ID NO: 853) Sp209 EPAS1- 210
CCGTGGCCCATGGCTGGCTC (SEQ ID NO: 854) Sp210 EPAS1- 211
TCCGTGGCCCATGGCTGGCT (SEQ ID NO: 855) Sp211 EPAS1- 212
TCAACTCCGTGGCCCATGGC (SEQ ID NO: 856) Sp212 EPAS1- 213
AGCCATGGGCCACGGAGTTG (SEQ ID NO: 857) Sp213 EPAS1- 214
CTCCTCAACTCCGTGGCCCA (SEQ ID NO: 858) Sp214 EPAS1- 215
GCTGTGGCTCCTCAACTCCG (SEQ ID NO: 859) Sp215 EPAS1- 216
GAGCCACAGCACCCAGAGCG (SEQ ID NO: 860) Sp216 EPAS1- 217
CAGCCTCGCTCTGGGTGCTG (SEQ ID NO: 861) Sp217 EPAS1- 218
CACAGCACCCAGAGCGAGGC (SEQ ID NO: 862) Sp218 EPAS1- 219
ACAGCACCCAGAGCGAGGCT (SEQ ID NO: 863) Sp219 EPAS1- 220
CAGGCTCCCAGCCTCGCTCT (SEQ ID NO: 864) Sp220 EPAS1- 221
GCAGGCTCCCAGCCTCGCTC (SEQ ID NO: 865) Sp221 EPAS1- 222
GGGGCACGGTGAAGGCAGGC (SEQ ID NO: 866) Sp222 EPAS1- 223
GCCTGCCTTCACCGTGCCCC (SEQ ID NO: 867) Sp223 EPAS1- 224
GCCTGGGGCACGGTGAAGGC (SEQ ID NO: 868) Sp224 EPAS1- 225
AGCTGCCTGGGGCACGGTGA (SEQ ID NO: 869) Sp225 EPAS1- 226
CGGGGCAGCTGCCTGGGGCA (SEQ ID NO: 870) Sp226 EPAS1- 227
CGTGCCCCAGGCAGCTGCCC (SEQ ID NO: 871) Sp227 EPAS1- 228
GTGCCCCAGGCAGCTGCCCC (SEQ ID NO: 872) Sp228 EPAS1- 229
CTGCCCGGGGCAGCTGCCTG (SEQ ID NO: 873) Sp229 EPAS1- 230
GCTGCCCGGGGCAGCTGCCT (SEQ ID NO: 874) Sp230 EPAS1- 231
TGCTGCCCGGGGCAGCTGCC (SEQ ID NO: 875) Sp231 EPAS1- 232
ACTGGGGGTGGTGCTGCCCG (SEQ ID NO: 876) Sp232 EPAS1- 233
CACTGGGGGTGGTGCTGCCC (SEQ ID NO: 877) Sp233 EPAS1- 234
GCACTGGGGGTGGTGCTGCC (SEQ ID NO: 878) Sp234 EPAS1- 235
GCTGCTGGTGGCACTGGGGG (SEQ ID NO: 879) Sp235 EPAS1- 236
GCTGCTGCTGGTGGCACTGG (SEQ ID NO: 880) Sp236 EPAS1- 237
TGCTGCTGCTGGTGGCACTG (SEQ ID NO: 881) Sp237 EPAS1- 238
CTGCTGCTGCTGGTGGCACT (SEQ ID NO: 882) Sp238 EPAS1- 239
GCTGCTGCTGCTGGTGGCAC (SEQ ID NO: 883) Sp239 EPAS1- 240
GCAGCTGCTGCTGCTGCTGG (SEQ ID NO: 884) Sp240 EPAS1- 241
CAGCAGCAGCAGCTGCTCCA (SEQ ID NO: 885) Sp241 EPAS1- 242
TCTTCAGGGCTATTGGGCTA (SEQ ID NO: 886) Sp242 EPAS1- 243
GTCTTCAGGGCTATTGGGCT (SEQ ID NO: 887) Sp243 EPAS1- 244
TAATAGTCTTCAGGGCTATT (SEQ ID NO: 888) Sp244 EPAS1- 245
GTAATAGTCTTCAGGGCTAT (SEQ ID NO: 889) Sp245 EPAS1- 246
AAGATGTGTAATAGTCTTCA (SEQ ID NO: 890) Sp246 EPAS1- 247
AAAGATGTGTAATAGTCTTC (SEQ ID NO: 891) Sp247 EPAS1- 248
TGAAGACTATTACACATCTT (SEQ ID NO: 892) Sp248 EPAS1- 249
TCTCAATCACTTCAATCTTC (SEQ ID NO: 893) Sp249
EPAS1- 250 GATTGAGAAGCTCTTCGCCA (SEQ ID NO: 894) Sp250 EPAS1- 251
GCTCTTCGCCATGGACACAG (SEQ ID NO: 895) Sp251 EPAS1- 252
CGCCATGGACACAGAGGCCA (SEQ ID NO: 896) Sp252 EPAS1- 253
GTCCTTGGCCTCTGTGTCCA (SEQ ID NO: 897) Sp253 EPAS1- 254
CTGGGTACTGCATTGGTCCT (SEQ ID NO: 898) Sp254 EPAS1- 255
CAAGGACCAATGCAGTACCC (SEQ ID NO: 899) Sp255 EPAS1- 256
CATCTACCTGGGTACTGCAT (SEQ ID NO: 900) Sp256 EPAS1- 257
AGCTCATTGAAATCCGTCTG (SEQ ID NO: 901) Sp257 EPAS1- 258
TCAGACGGATTTCAATGAGC (SEQ ID NO: 902) Sp258 EPAS1- 259
GGATTTCAATGAGCTGGACT (SEQ ID NO: 903) Sp259 EPAS1- 260
TGAGCTGGACTTGGAGACAC (SEQ ID NO: 904) Sp260 EPAS1- 261
ACTGGCACCCTATATCCCCA (SEQ ID NO: 905) Sp261 EPAS1- 262
GCACCCTATATCCCCATGGA (SEQ ID NO: 906) Sp262 EPAS1- 263
CACCCTATATCCCCATGGAC (SEQ ID NO: 907) Sp263 EPAS1- 264
ACCCTATATCCCCATGGACG (SEQ ID NO: 908) Sp264 EPAS1- 265
TCCCCGTCCATGGGGATATA (SEQ ID NO: 909) Sp265 EPAS1- 266
TTCCCCGTCCATGGGGATAT (SEQ ID NO: 910) Sp266 EPAS1- 267
GGAAGTCTTCCCCGTCCATG (SEQ ID NO: 911) Sp267 EPAS1- 268
TGGAAGTCTTCCCCGTCCAT (SEQ ID NO: 912) Sp268 EPAS1- 269
CTGGAAGTCTTCCCCGTCCA (SEQ ID NO: 913) Sp269 EPAS1- 270
CGGGGCAGATGGGGCTTAGC (SEQ ID NO: 914) Sp270 EPAS1- 271
GCTAAGCCCCATCTGCCCCG (SEQ ID NO: 915) Sp271 EPAS1- 272
GCCCCATCTGCCCCGAGGAG (SEQ ID NO: 916) Sp272 EPAS1- 273
GCCGCTCCTCGGGGCAGATG (SEQ ID NO: 917) Sp273 EPAS1- 274
AGCCGCTCCTCGGGGCAGAT (SEQ ID NO: 918) Sp274 EPAS1- 275
GAGCCGCTCCTCGGGGCAGA (SEQ ID NO: 919) Sp275 EPAS1- 276
CTGCCCCGAGGAGCGGCTCT (SEQ ID NO: 920) Sp276 EPAS1- 277
CCCCGAGGAGCGGCTCTTGG (SEQ ID NO: 921) Sp277 EPAS1- 278
CCGCCAAGAGCCGCTCCTCG (SEQ ID NO: 922) Sp278 EPAS1- 279
TCCGCCAAGAGCCGCTCCTC (SEQ ID NO: 923) Sp279 EPAS1- 280
CTCCGCCAAGAGCCGCTCCT (SEQ ID NO: 924) Sp280 EPAS1- 281
AGTGCTGGGGGGTGGACTGT (SEQ ID NO: 925) Sp281 EPAS1- 282
CAGTGCTGGGGGGTGGACTG (SEQ ID NO: 926) Sp282 EPAS1- 283
ACTGAAGCAGTGCTGGGGGG (SEQ ID NO: 927) Sp283 EPAS1- 284
GGCACTGAAGCAGTGCTGGG (SEQ ID NO: 928) Sp284 EPAS1- 285
TGGCACTGAAGCAGTGCTGG (SEQ ID NO: 929) Sp285 EPAS1- 286
ATGGCACTGAAGCAGTGCTG (SEQ ID NO: 930) Sp286 EPAS1- 287
CATGGCACTGAAGCAGTGCT (SEQ ID NO: 931) Sp287 EPAS1- 288
TCATGGCACTGAAGCAGTGC (SEQ ID NO: 932) Sp288 EPAS1- 289
TGGCTGGAAGATGTTTGTCA (SEQ ID NO: 933) Sp289 EPAS1- 290
GACAAACATCTTCCAGCCAC (SEQ ID NO: 934) Sp290 EPAS1- 291
GGGCTACAGGGGCCAGTGGC (SEQ ID NO: 935) Sp291 EPAS1- 292
TGCGGGGCTACAGGGGCCAG (SEQ ID NO: 936) Sp292 EPAS1- 293
GGGACTGTGCGGGGCTACAG (SEQ ID NO: 937) Sp293 EPAS1- 294
AGGGACTGTGCGGGGCTACA (SEQ ID NO: 938) Sp294 EPAS1- 295
AAGGGACTGTGCGGGGCTAC (SEQ ID NO: 939) Sp295 EPAS1- 296
CAGGAGGAAGGGACTGTGCG (SEQ ID NO: 940) Sp296 EPAS1- 297
CCCGCACAGTCCCTTCCTCC (SEQ ID NO: 941) Sp297 EPAS1- 298
CCAGGAGGAAGGGACTGTGC (SEQ ID NO: 942) Sp298 EPAS1- 299
TCCAGGAGGAAGGGACTGTG (SEQ ID NO: 943) Sp299 EPAS1- 300
TGAAACTTGTCCAGGAGGAA (SEQ ID NO: 944) Sp300 EPAS1- 301
CTGAAACTTGTCCAGGAGGA (SEQ ID NO: 945) Sp301 EPAS1- 302
GCTGCTGAAACTTGTCCAGG (SEQ ID NO: 946) Sp302 EPAS1- 303
GCTGCTGCTGAAACTTGTCC (SEQ ID NO: 947) Sp303 EPAS1- 304
GGACAAGTTTCAGCAGCAGC (SEQ ID NO: 948) Sp304 EPAS1- 305
AGAAGACAGAGCCCGAGCAC (SEQ ID NO: 949) Sp305 EPAS1- 306
GAGGACATGGGCCGGTGCTC (SEQ ID NO: 950) Sp306 EPAS1- 307
GGAGGACATGGGCCGGTGCT (SEQ ID NO: 951) Sp387 EPAS1- 308
AGAAGATGGAGGACATGGGC (SEQ ID NO: 952) Sp308 EPAS1- 309
TCAAAGAAGATGGAGGACAT (SEQ ID NO: 953) Sp309 EPAS1- 310
ATCAAAGAAGATGGAGGACA (SEQ ID NO: 954) Sp310 EPAS1- 311
TCCTCCATCTTCTTTGATGC (SEQ ID NO: 955) Sp311 EPAS1- 312
TCCGGCATCAAAGAAGATGG (SEQ ID NO: 956) Sp312 EPAS1- 313
GCTTCCGGCATCAAAGAAGA (SEQ ID NO: 957) Sp313 EPAS1- 314
TGGCAGGGATGCTTTGCTTC (SEQ ID NO: 958) Sp314 EPAS1- 315
GCATCCCTGCCACCGTGCTG (SEQ ID NO: 959) Sp315 EPAS1- 316
CTGGCCACAGCACGGTGGCA (SEQ ID NO: 960) Sp316 EPAS1- 317
CCTGCCACCGTGCTGTGGCC (SEQ ID NO: 961) Sp317 EPAS1- 318
CCTGGCCACAGCACGGTGGC (SEQ ID NO: 962) Sp318 EPAS1- 319
CTGGCCTGGCCACAGCACGG (SEQ ID NO: 963) Sp319 EPAS1- 320
GTGCTGGCCTGGCCACAGCA (SEQ ID NO: 964) Sp320 EPAS1- 321
AAGAGAGAGGGGTGCTGGCC (SEQ ID NO: 965) Sp321 EPAS1- 322
CATGGAAGAGAGAGGGGTGC (SEQ ID NO: 966) Sp322 EPAS1- 323
CAGCACCCCTCTCTCTTCCA (SEQ ID NO: 967) Sp323 EPAS1- 324
AGCACCCCTCTCTCTTCCAT (SEQ ID NO: 968) Sp324 EPAS1- 325
GCACCCCTCTCTCTTCCATG (SEQ ID NO: 969) Sp325 EPAS1- 326
CACCCCTCTCTCTTCCATGG (SEQ ID NO: 970) Sp326 EPAS1- 327
ACCCCTCTCTCTTCCATGGG (SEQ ID NO: 971) Sp327 EPAS1- 328
GCCCCCCATGGAAGAGAGAG (SEQ ID NO: 972) Sp328 EPAS1- 329
TGCCCCCCATGGAAGAGAGA (SEQ ID NO: 973) Sp329 EPAS1- 330
CTGCCCCCCATGGAAGAGAG (SEQ ID NO: 974) Sp330 EPAS1- 331
GGTATTGGATCTGCCCCCCA (SEQ ID NO: 975) Sp331 EPAS1- 332
GGGGCAGATCCAATACCCAG (SEQ ID NO: 976) Sp332 EPAS1- 333
ATCTGGGGGCCACTGGGTAT (SEQ ID NO: 977) Sp333
EPAS1- 334 TGGTGGATCTGGGGGCCACT (SEQ ID NO: 978) Sp334 EPAS1- 335
ATGGTGGATCTGGGGGCCAC (SEQ ID NO: 979) Sp335 EPAS1- 336
AAATGTAATGGTGGATCTGG (SEQ ID NO: 980) Sp336 EPAS1- 337
AAAATGTAATGGTGGATCTG (SEQ ID NO: 981) Sp337 EPAS1- 338
CAAAATGTAATGGTGGATCT (SEQ ID NO: 982) Sp338 EPAS1- 339
CCAGATCCACCATTACATTT (SEQ ID NO: 983) Sp339 EPAS1- 340
CCAAAATGTAATGGTGGATC (SEQ ID NO: 984) Sp340 EPAS1- 341
CAGATCCACCATTACATTTT (SEQ ID NO: 985) Sp341 EPAS1- 342
GTGGGCCCAAAATGTAATGG (SEQ ID NO: 986) Sp342 EPAS1- 343
TTTGTGGGCCCAAAATGTAA (SEQ ID NO: 987) Sp343 EPAS1- 344
TACATTTTGGGCCCACAAAG (SEQ ID NO: 988) Sp344 EPAS1- 345
ACATTTTGGGCCCACAAAGT (SEQ ID NO: 989) Sp345 EPAS1- 346
GGGCCCACAAAGTGGGCCGT (SEQ ID NO: 990) Sp346 EPAS1- 347
GGCCCACAAAGTGGGCCGTC (SEQ ID NO: 991) Sp347 EPAS1- 348
GCCCACAAAGTGGGCCGTCG (SEQ ID NO: 992) Sp348 EPAS1- 349
TCCCCGACGGCCCACTTTGT (SEQ ID NO: 993) Sp349 EPAS1- 350
ATCCCCGACGGCCCACTTTG (SEQ ID NO: 994) Sp350 EPAS1- 351
CTCTGTGCGCTGATCCCCGA (SEQ ID NO: 995) Sp351 EPAS1- 352
GGATCAGCGCACAGAGTTCT (SEQ ID NO: 996) Sp352 EPAS1- 353
GATCAGCGCACAGAGTTCTT (SEQ ID NO: 997) Sp353 EPAS1- 354
GTTCTTGGGAGCAGCGCCGT (SEQ ID NO: 998) Sp354 EPAS1- 355
TTCTTGGGAGCAGCGCCGTT (SEQ ID NO: 999) Sp355 EPAS1- 356
TCTTGGGAGCAGCGCCGTTG (SEQ ID NO: 1000) Sp356 EPAS1- 357
GGAGAGACAGGGGGCCCCAA (SEQ ID NO: 1001) Sp357 EPAS1- 358
ACATGGGGTGGAGAGACAGG (SEQ ID NO: 1002) Sp358 EPAS1- 359
GACATGGGGTGGAGAGACAG (SEQ ID NO: 1003) Sp359 EPAS1- 360
AGACATGGGGTGGAGAGACA (SEQ ID NO: 1004) Sp360 EPAS1- 361
GAGACATGGGGTGGAGAGAC (SEQ ID NO: 1005) Sp361 EPAS1- 362
TTGAAGGTGGAGACATGGGG (SEQ ID NO: 1006) Sp362 EPAS1- 363
GTCTTGAAGGTGGAGACATG (SEQ ID NO: 1007) Sp363 EPAS1- 364
TGTCTTGAAGGTGGAGACAT (SEQ ID NO: 1008) Sp364 EPAS1- 365
TTGTCTTGAAGGTGGAGACA (SEQ ID NO: 1009) Sp365 EPAS1- 366
ATGTCTCCACCTTCAAGACA (SEQ ID NO: 1010) Sp366 EPAS1- 367
TGCCACTTACCTTGTCTTGA (SEQ ID NO: 1011) Sp367 EPAS1- 368
CGGGCTTGGCAGGTCTGCAA (SEQ ID NO: 1012) Sp368 EPAS1- 369
GGGCTTGGCAGGTCTGCAAA (SEQ ID NO: 1013) Sp369 EPAS1- 370
GGCAGGTCTGCAAAGGGTTT (SEQ ID NO: 1014) Sp370 EPAS1- 371
GCAGGTCTGCAAAGGGTTTT (SEQ ID NO: 1015) Sp371 EPAS1- 373
CAGGTCTGCAAAGGGTTTTG (SEQ ID NO: 1016) Sp372 EPAS1- 374
GCAAAGGGTTTTGGGGCTCG (SEQ ID NO: 1017) Sp373 EPAS1- 374
AGGCCCAGACGTGCTGAGTC (SEQ ID NO: 1018) Sp374 EPAS1- 375
TGGCCGGACTCAGCACGTCT (SEQ ID NO: 1019) Sp375 EPAS1- 376
ATGGCCGGACTCAGCACGTC (SEQ ID NO: 1020) Sp376 EPAS1- 377
AGACGTGCTGAGTCCGGCCA (SEQ ID NO: 1021) Sp377 EPAS1- 378
TTGGAGAGGGCTACCATGGC (SEQ ID NO: 1022) Sp378 EPAS1- 379
CTTGTTGGAGAGGGCTACCA (SEQ ID NO: 1023) Sp379 EPAS1- 380
CAGCTTCAGCTTGTTGGAGA (SEQ ID NO: 1024) Sp380 EPAS1- 381
TCAGCTTCAGCTTGTTGGAG (SEQ ID NO: 1025) Sp381 EPAS1- 382
TCGCTTCAGCTTCAGCTTGT (SEQ ID NO: 1026) Sp382 EPAS1- 383
GCTGAAGCTGAAGCGACAGC (SEQ ID NO: 1027) Sp383 EPAS1- 384
GTATGAAGAGCAAGCCTTCC (SEQ ID NO: 1028) Sp384 EPAS1- 385
CAAGCCTTCCAGGACCTGAG (SEQ ID NO: 1029) Sp385 EPAS1- 386
AAGCCTTCCAGGACCTGAGC (SEQ ID NO: 1030) Sp386 EPAS1- 387
AGCCTTCCAGGACCTGAGCG (SEQ ID NO: 1031) Sp387 EPAS1- 388
CACCCCGCTCAGGTCCTGGA (SEQ ID NO: 1032) Sp388 EPAS1- 389
GACTCACCCCGCTCAGGTCC (SEQ ID NO: 1033) Sp389 EPAS1- 390
GGGGATGACTCACCCCGCTC (SEQ ID NO: 1034) Sp390 EPAS1- 391
TACTCCCAGGGGGACCCACC (SEQ ID NO: 1035) Sp391 EPAS1- 392
TCCCAGGGGGACCCACCTGG (SEQ ID NO: 1036) Sp392 EPAS1- 393
GCCACCAGGTGGGTCCCCCT (SEQ ID NO: 1037) Sp393 EPAS1- 394
TGCCACCAGGTGGGTCCCCC (SEQ ID NO: 1038) Sp394 EPAS1- 395
GTGAGGTGCTGCCACCAGGT (SEQ ID NO: 1039) Sp395 EPAS1- 396
TGTGAGGTGCTGCCACCAGG (SEQ ID NO: 1040) Sp396 EPAS1- 397
AAATGTGAGGTGCTGCCACC (SEQ ID NO: 1041) Sp397 EPAS1- 398
GCAGCACCTCACATTTGATG (SEQ ID NO: 1042) Sp398 EPAS1- 399
CCTCACATTTGATGTGGAAA (SEQ ID NO: 1043) Sp399 EPAS1- 400
CCGTTTCCACATCAAATGTG (SEQ ID NO: 1044) Sp400 EPAS1- 401
GGAAACGGATGAAGAACCTC (SEQ ID NO: 1045) Sp401 EPAS1- 402
GAAACGGATGAAGAACCTCA (SEQ ID NO: 1046) Sp402 EPAS1- 403
AAACGGATGAAGAACCTCAG (SEQ ID NO: 1047) Sp403 EPAS1- 404
CGGATGAAGAACCTCAGGGG (SEQ ID NO: 1048) Sp404 EPAS1- 405
GGATGAAGAACCTCAGGGGT (SEQ ID NO: 1049) Sp405 EPAS1- 406
AAGGGCAGCTCCCACCCCTG (SEQ ID NO: 1050) Sp406 EPAS1- 407
TGGGAGCTGCCCTTTGATGC (SEQ ID NO: 1051) Sp407 EPAS1- 408
GTGGCTTGTCCGGCATCAAA (SEQ ID NO: 1052) Sp408 EPAS1- 409
AGTGGCTTGTCCGGCATCAA (SEQ ID NO: 1053) Sp409 EPAS1- 410
TTTGCGCTCAGTGGCTTGTC (SEQ ID NO: 1054) Sp410 EPAS1- 411
TTGGGTACATTTGCGCTCAG (SEQ ID NO: 1055) Sp411 EPAS1- 412
CTGAGCGCAAATGTACCCAA (SEQ ID NO: 1056) Sp412 EPAS1- 413
TGTGGCCGCTGCTCACCATT (SEQ ID NO: 1057) Sp413 EPAS1- 414
CTGTGGCCGCTGCTCACCAT (SEQ ID NO: 1058) Sp414 EPAS1- 415
AGTTCACCCAAAACCCCATG (SEQ ID NO: 1059) Sp415 EPAS1- 416
GTTCACCCAAAACCCCATGA (SEQ ID NO: 1060) Sp416 EPAS1- 417
TTCACCCAAAACCCCATGAG (SEQ ID NO: 1061)
Sp417 EPAS1- 418 CAGGCCCCTCATGGGGTTTT (SEQ ID NO: 1062) Sp418
EPAS1- 419 CCAAAACCCCATGAGGGGCC (SEQ ID NO: 1063) Sp419 EPAS1- 420
CCAGGCCCCTCATGGGGTTT (SEQ ID NO: 1064) Sp420 EPAS1- 421
CAAAACCCCATGAGGGGCCT (SEQ ID NO: 1065) Sp421 EPAS1- 422
GATGGCCCAGGCCCCTCATG (SEQ ID NO: 1066) Sp422 EPAS1- 423
GGATGGCCCAGGCCCCTCAT (SEQ ID NO: 1067) Sp423 EPAS1- 424
GGGATGGCCCAGGCCCCTCA (SEQ ID NO: 1068) Sp424 EPAS1- 425
GATGTCTCAGGGGATGGCCC (SEQ ID NO: 1069) Sp425 EPAS1- 426
GCGGCAGATGTCTCAGGGGA (SEQ ID NO: 1070) Sp426 EPAS1- 427
GGCAGCGGCAGATGTCTCAG (SEQ ID NO: 1071) Sp427 EPAS1- 428
TGGCAGCGGCAGATGTCTCA (SEQ ID NO: 1072) Sp428 EPAS1- 429
GTGGCAGCGGCAGATGTCTC (SEQ ID NO: 1073) Sp429 EPAS1- 430
GCAGATGGAGGCTGTGGCAG (SEQ ID NO: 1074) Sp430 EPAS1- 431
CTGATGGCAGATGGAGGCTG (SEQ ID NO: 1075) Sp431 EPAS1- 432
CCTCCATCTGCCATCAGTCC (SEQ ID NO: 1076) Sp432 EPAS1- 433
CCGGGACTGATGGCAGATGG (SEQ ID NO: 1077) Sp433 EPAS1- 434
CTCCATCTGCCATCAGTCCC (SEQ ID NO: 1078) Sp434 EPAS1- 435
TCCATCTGCCATCAGTCCCG (SEQ ID NO: 1079) Sp435 EPAS1- 436
TCCCCGGGACTGATGGCAGA (SEQ ID NO: 1080) Sp436 EPAS1- 437
GCTGTTCTCCCCGGGACTGA (SEQ ID NO: 1081) Sp437 EPAS1- 438
CTGCTCTTGCTGTTCTCCCC (SEQ ID NO: 1082) Sp438 EPAS1- 439
CCGGGGAGAACAGCAAGAGC (SEQ ID NO: 1083) Sp439 EPAS1- 440
CCTGCTCTTGCTGTTCTCCC (SEQ ID NO: 1084) Sp440 EPAS1- 442
GGGTGGCGTAGCACTGTGGG (SEQ ID NO: 1085) Sp441 EPAS1- 442
TGGGTGGCGTAGCACTGTGG (SEQ ID NO: 1086) Sp442 EPAS1- 413
CTGGGTGGCGTAGCACTGTG (SEQ ID NO: 1087) Sp443 EPAS1- 444
ACTGGGTGGCGTAGCACTGT (SEQ ID NO: 1088) Sp444 EPAS1- 445
TACTGGGTGGCGTAGCACTG (SEQ ID NO: 1089) Sp445 EPAS1- 446
GTGCTACGCCACCCAGTACC (SEQ ID NO: 1090) Sp446 EPAS1- 447
GCTGTAGTCCTGGTACTGGG (SEQ ID NO: 1091) Sp447 EPAS1- 448
CAGGCTGTAGTCCTGGTACT (SEQ ID NO: 1092) Sp448 EPAS1- 449
ACAGGCTGTAGTCCTGGTAC (SEQ ID NO: 1093) Sp449 EPAS1- 450
CTGACGACAGGCTGTAGTCC (SEQ ID NO: 1094) Sp450 EPAS1- 451
CAGCCTGTCGTCAGCCCACA (SEQ ID NO: 1095) Sp451 EPAS1- 452
ACACCTTGTGGGCTGACGAC (SEQ ID NO: 1096) Sp452 EPAS1- 453
TCGTCAGCCCACAAGGTGTC (SEQ ID NO: 1097) Sp453 EPAS1- 454
TCAGCCCACAAGGTGTCAGG (SEQ ID NO: 1098) Sp454 EPAS1- 455
CAGCCCACAAGGTGTCAGGT (SEQ ID NO: 1099) Sp455 EPAS1- 456
ACACCTACCTGACACCTTGT (SEQ ID NO: 1100) Sp456 EPAS1- 457
CACACCCACCTGACACCTTG (SEQ ID NO: 1101) Sp457 EPAS1- 458
ACCAACCCTTCTTTCAGGCA (SEQ ID NO: 1102) Sp458 EPAS1- 459
TTCTTTCAGGCATGGCAAGC (SEQ ID NO: 1103) Sp459 EPAS1- 460
GGCATGGCAAGCCGGCTGCT (SEQ ID NO: 1104) Sp460 EPAS1- 461
GCATGGCAAGCCGGCTGCTC (SEQ ID NO: 1105) Sp461 EPAS1- 462
CAAATGAGGGCCCGAGCAGC (SEQ ID NO: 1106) Sp462 EPAS1- 463
AGCAGGTAGGACTCAAATGA (SEQ ID NO: 1107) Sp463 EPAS1- 464
CAGCAGGTAGGACTCAAATG (SEQ ID NO: 1108) Sp464 EPAS1- 465
GGTCAGTTCGGGCAGCAGGT (SEQ ID NO: 1109) Sp465 EPAS1- 466
ATCTGGTCAGTTCGGGCAGC (SEQ ID NO: 1110) Sp466 EPAS1- 467
CAGTCATATCTGGTCAGTTC (SEQ ID NO: 1111) Sp467 EPAS1- 468
ACAGTCATATCTGGTCAGTT (SEQ ID NO: 1112) Sp468 EPAS1- 469
ACTGACCAGATATGACTGTS (SEQ ID NO: 1113) Sp469 EPAS1- 470
GTTCACCTCACAGTCATATC (SEQ ID NO: 1114) Sp470 EPAS1- 471
TGAGGTGAACGTGCCCGTGC (SEQ ID NO: 1115) Sp471 EPAS1- 472
GAGGTGAACGTGCCCGTGCT (SEQ ID NO: 1116) Sp472 EPAS1- 473
AGCGTGGAGCTTCCCAGCAC (SEQ ID NO: 1117) Sp473 EPAS1- 474
GAGCGTGGAGCTTCCCAGCA (SEQ ID NO: 1118) Sp474 EPAS1- 475
GGAAGCTCCACGCTCCTGCA (SEQ ID NO: 1119) Sp475 EPAS1- 476
AGCTCCACGCTCCTGCAAGG (SEQ ID NO: 1120) Sp476 EPAS1- 477
GCTCCACGCTCCTGCAAGGA (SEQ ID NO: 1121) Sp477 EPAS1- 478
CTCCACGCTCCTGCAAGGAG (SEQ ID NO: 1122) Sp478 EPAS1- 479
GTCCCCTCCTTGCAGGAGCG (SEQ ID NO: 1123) Sp479 EPAS1- 480
TGAGGAGGTCCCCTCCTTGC (SEQ ID NO: 1124) Sp480 EPAS1- 481
AGGGGACCTCCTCAGAGCCC (SEQ ID NO: 1125) Sp481 EPAS1- 482
CCTCCTCAGAGCCCTGGACC (SEQ ID NO: 1126) Sp482 EPaS1- 483
CCTGGTCCAGGGCTCTGAGG (SEQ ID NO: 1127) Sp483 EPAS1- 484
TGGCCTGGTCCAGGGCTCTG (SEQ ID NO: 1128) Sp484 EPAS1- 485
GGCTCAGGTGGCCTGGTCCA (SEQ ID NO: 1129) Sp485 EPAS1- 486
TGGCTCAGGTGGCCTGGTCC (SEQ ID NO: 1130) Sp486 EPAS1- 487
TGGACCAGGCCACCTGAGCC (SEQ ID NO: 1131) Sp487 EPAS1- 488
AAGGCCTGGCTCAGGTGGCC (SEQ ID NO: 1132) Sp488 EPAS1- 489
GGTAGAAGGCCTGGCTCAGG (SEQ ID NO: 1133) Sp489
TABLE-US-00013 TABLE 13 Target sequences of ANGPT2 gene for SpCas9
Gene No. Target sequence ANGPT2- 1 TCTGAGCTGTGATCTTGTCT (SEQ ID NO:
1134) Sp1 ANGPT2- 2 CCGCAGCCTATAACAACTTT (SEQ ID NO: 1135) Sp2
ANGPT2- 3 CCGAAAGTTGTTATAGGCTG (SEQ ID NO: 1136) Sp3 ANGPT2- 4
GCTCTTCCGAAAGTTGTTAT (SEQ ID NO: 1137) Sp4 ANGPT2- 5
TAACAACTTTCGGAAGAGCA (SEQ ID NO: 1138) Sp5 ANGPT2- 6
CGGAAGAGCATGGACAGCAT (SEQ ID NO: 1139) Sp6 ANGPT2- 7
CATAGGAAAGAAGCAATATC (SEQ ID NO: 1140) Sp7 ANGPT2- 8
AAGCAATATCAGGTCCAGCA (SEQ ID NO: 1141) Sp8 ANGPT2- 9
AGCAATATCAGGTCCAGCAT (SEQ ID NO: 1142) Sp9 ANGPT2- 10
TGTAGCTGCAGGACCCATGC (SEQ ID NO: 1143) Sp10 ANGPT2- 11
CAGGAGGAAAGTGTAGCTGC (SEQ ID NO: 1144) Sp11 ANGPT2- 12
CACTTTCCTCCTGCCAGAGA (SEQ ID NO: 1145) Sp12 ANGPT2- 13
AGTTGTCCATCTCTGGCAGG (SEQ ID NO: 1146) Sp13 ANGPT2- 14
GGCAGTTGTCCATCTCTGGC (SEQ ID NO: 1147) Sp14 ANGPT2- 15
GAGCGGCAGTTGTCCATCTC (SEQ ID NO: 1148) Sp15 ANGPT2- 16
CGTAGGGGCTGGAGGAAGAG (SEQ ID NO: 1149) Sp16 ANGPT2- 17
ATTGGACACGTAGGGGCTGG (SEQ ID NO: 1150) Sp17 ANGPT2- 18
AGCATTGGACACGTAGGGGC (SEQ ID NO: 1151) Sp18 ANGPT2- 19
GCACAGCATTGGACACGTAG (SEQ ID NO: 1152) Sp19 ANGPT2- 20
TGCACAGCATTGGACACGTA (SEQ ID NO: 1153) Sp20 ANGPT2- 21
CTGCACAGCATTGGACACGT (SEQ ID NO: 1154) Sp21 ANGPT2- 22
ACGTGTCCAATGCTGTGCAG (SEQ ID NO: 1155) Sp22 ANGPT2- 23
CGTGTCCAATGCTGTGCAGA (SEQ ID NO: 1156) Sp23 ANGPT2- 24
CGCGTCCCTCTGCACAGCAT (SEQ ID NO: 1157) Sp24 ANGPT2- 25
GCCGCTCGAATACGATGACT (SEQ ID NO: 1158) Sp25 ANGPT2- 26
ACCGAGTCATCGTATTCGAG (SEQ ID NO: 1159) Sp26 ANGPT2- 27
AATACGATGACTCGGTGCAG (SEQ ID NO: 1160) Sp27 ANGPT2- 28
GGTGCAGAGGCTGCAAGTGC (SEQ ID NO: 1161) Sp28 ANGPT2- 29
GCAAGTGCTGGAGAACATCA (SEQ ID NO: 1162) Sp29 ANGPT2- 30
TCATGGAAAACAACACTCAG (SEQ ID NO: 1163) Sp30 ANGPT2- 31
CAACACTCAGTGGCTAATGA (SEQ ID NO: 1164) Sp31 ANGPT2- 32
ACTCAGTGGCTAATGAAGGT (SEQ ID NO: 1165) Sp32 ANGPT2- 33
CTAGCTTGAGAATTATATCC (SEQ ID NO: 1166) Sp33 ANGPT2- 34
TTTCTTTCTTCATGTTGTCC (SEQ ID NO: 1167) Sp34 ANGPT2- 35
GGACAACATGAAGAAAGAAA (SEQ ID NO: 1168) Sp35 ANGPT2- 36
GAATGCAGTACAGAACCAGA (SEQ ID NO: 1169) Sp36 ANGPT2- 37
TTTCTATCATCACAGCCGTC (SEQ ID NO: 1170) Sp37 ANGPT2- 38
ACGGCTGTGATGATAGAAAT (SEQ ID NO: 1171) Sp38 ANGPT2- 39
CGGCTGTGATGATAGAAATA (SEQ ID NO: 1172) Sp39 ANGPT2- 40
AAACCTGTTGAACCAAACAG (SEQ ID NO: 1173) Sp40 ANGPT2- 41
GCTCCGCTGTTTGGTTCAAC (SEQ ID NO: 1174) Sp41 ANGPT2- 42
ACCAAACAGCGGAGCAAACG (SEQ ID NO: 1175) Sp42 ANGPT2- 43
TCCGCGTTTGCTCCGCTGTT (SEQ ID NO: 1176) Sp43 ANGPT2- 44
AACGCGGAAGTTAACTGATG (SEQ ID NO: 1177) Sp44 ANGPT2- 45
CTAGCTTGAGAATTATATCC (SEQ ID NO: 1178) Sp45 ANGPT2- 46
TTTCTTTCTTCATGTTGTCC (SEQ ID NO: 1179) Sp46 ANGPT2- 47
GGACAACATGAAGAAAGAAA (SEQ ID NO: 1180) Sp47 ANGPT2- 48
GAATGCAGTACAGAACCAGA (SEQ ID NO: 1181) Sp48 ANGPT2- 49
TTTCTATCATCACAGCCGTC (SEQ ID NO: 1182) Sp49 ANGPT2- 50
ACGGCTGTGATGATAGAAAT (SEQ ID NO: 1183) Sp50 ANGPT2- 51
CGGCTGTGATGATAGAAATA (SEQ ID NO: 1184) Sp51 ANGPT2- 52
AAACCTGTTGAACCAAACAG (SEQ ID NO: 1185) Sp52 ANGPT2- 53
GCTCCGCTGTTTGGTTCAAC (SEQ ID NO: 1186) Sp53 ANGPT2- 54
ACCAAACAGCGGAGCAAACG (SEQ ID NO: 1187) Sp54 ANGPT2- 55
TCCGCGTTTGCTCCGCTGTT (SEQ ID NO: 1188) Sp55 ANGPT2- 56
AACGCGGAAGTTAACTGATG (SEQ ID NO: 1189) Sp56 ANGPT2- 57
TACAAGTTTCCTAGAAAAGA (SEQ ID NO: 1190) Sp57 ANGPT2- 58
TAGCTAGCACCTTCTTTTCT (SEQ ID NO: 1191) Sp58 ANGPT2- 59
AGAAAAGAAGGTGCTAGCTA (SEQ ID NO: 1192) Sp59 ANGPT2- 60
CTTCTTTTTATTGACTGTAGT (SEQ ID NO: 1193) Sp60 ANGPT2- 61
AGAAGAGAAAGATCAGCTAC (SEQ ID NO: 1194) Sp61 ANGPT2- 62
TTCAATGATGGAATTTTGCT (SEQ ID NO: 1195) Sp62 ANGPT2- 63
TTTTTCTAGTTCTTTCAATGA (SEQ ID NO: 1196) Sp63 ANGPT2- 64
AAAAAAAATAGTGACTGCCA (SEQ ID NO: 1197) Sp64 ANGPT2- 65
AAGAACTGAATTATTCACCG (SEQ ID NO: 1198) Sp65 ANGPT2- 66
GAAGCAGCAACATGATCTCA (SEQ ID NO: 1199) Sp66 ANGPT2- 67
TGTAAACTTACAGTTTGATG (SEQ ID NO: 1200) Sp67 ANGPT2- 68
CTATTTTTTAAAAGCAGCTA (SEQ ID NO: 1201) Sp68 ANGPT2- 69
GTTCTTCTTTAGCAACAGTG (SEQ ID NO: 1202) Sp69 ANGPT2- 70
TGTTCTTCTTTAGCAACAGT (SEQ ID NO: 1203) Sp70 ANGPT2- 71
TTGTTCTTCTTTAGCAACAG (SEQ ID NO: 1204) Sp71 ANGPT2- 72
TGTGCTGAAGTATTCAAATC (SEQ ID NO: 1205) Sp72 ANGPT2- 73
AAATCAGGACACACCACGAA (SEQ ID NO: 1206) Sp73 ANGPT2- 74
TAACGTGTAGATGCCATTCG (SEQ ID NO: 1207) Sp74 ANGPT2- 75
TGATCTCTTCTGTAGAATTA (SEQ ID NO: 1208) Sp75 ANGPT2- 76
TTGATCTCTTCTGTAGAATT (SEQ ID NO: 1209) Sp76 ANGPT2- 77
TAATTCTACAGAAGAGATCA (SEQ ID NO: 1210) Sp77 ANGPT2- 78
CTACAGAAGAGATCAAGGTG (SEQ ID NO: 1211) Sp78 ANGPT2- 79
TTTGCAGGCCTACTGTGACA (SEQ ID NO: 1212) Sp79 ANGPT2- 80
GCCTACTGTGACATGGAAGC (SEQ ID NO: 1213) Sp80 ANGPT2- 81
TCCAGCTTCCATGTCACAGT (SEQ ID NO: 1214) Sp81 ANGPT2- 82
TACTGTGACATGGAAGCTGG (SEQ ID NO: 1215) Sp82
ANGPT2- 83 TGTGACATGGAAGCTGGAGG (SEQ ID NO: 1216) Sp83 ANGPT2- 84
GACATGGAAGCTGGAGGAGG (SEQ ID NO: 1217) Sp84 ANGPT2- 85
ACATGGAAGCTGGAGGAGGC (SEQ ID NO: 1218) Sp85 ANGPT2- 86
TGGAAGCTGGAGGAGGCGGG (SEQ ID NO: 1219) Sp86 ANGPT2- 87
GACAATTATTCAGCGACGTG (SEQ ID NO: 1220) Sp87 ANGPT2- 88
ATTATTCAGCGACGTGAGGA (SEQ ID NO: 1221) Sp88 ANGPT2- 89
ATGGCAGCGTTGATTTTCAG (SEQ ID NO: 1222) Sp89 ANGPT2- 90
GCGTTGATTTTCAGAGGACT (SEQ ID NO: 1223) Sp90 ANGPT2- 91
GACTTGGAAAGAATATAAAG (SEQ ID NO: 1224) Sp91 ANGPT2- 92
GGAAAGAATATAAAGTGGTA (SEQ ID NO: 1225) Sp92 ANGPT2- 93
CAGGGATTTGGTAACCCTTC (SEQ ID NO: 1226) Sp93 ANGPT2- 94
GTAACCCTTCAGGAGAATAT (SEQ ID NO: 1227) Sp94 ANGPT2- 95
CCCTTCAGGAGAATATTGGC (SEQ ID NO: 1228) Sp95 ANGPT2- 96
CCAGCCAATATTCTCCTGAA (SEQ ID NO: 1229) Sp56 ANGPT2- 97
CCTTCAGGAGAATATTGGCT (SEQ ID NO: 1230) Sp97 ANGPT2- 98
CCCAGCCAATATTCTCCTGA (SEQ ID NO: 1231) Sp98 ANGPT2- 99
TTAAAATACACCTTAAAGAC (SEQ ID NO: 1232) Sp99 ANGPT2- 100
TAAAATACACCTTAAAGACT (SEQ ID NO: 1233) Sp100 ANGPT2- 101
ATACACCTTAAAGACTGGGA (SEQ ID NO: 1234) Sp101 ANGPT2- 102
TACACCTTAAAGACTGGGAA (SEQ ID NO: 1235) Sp102 ANGPT2- 103
CATTCCCTTCCCAGTCTTTA (SEQ ID NO: 1236) Sp103 ANGPT2- 104
TAAAGACTGGGAAGGGAATG (SEQ ID NO: 1237) Sp104 ANGP12- 105
CAAGTGAAGAACTCAATTAT (SEQ ID NO: 1238) Sp105 ANGPT2- 106
GCTTACAGGATTCACCTTAA (SEQ ID NO: 1239) Sp106 ANGPT2- 107
ATTCACCTTAAAGGACTTAC (SEQ ID NO: 1240) Sp107 ANGPT2- 108
TTCACCTTAAAGGACTTACA (SEQ ID NO: 1241) Sp108 ANGPT2- 109
CTGTCCCTGTAAGTCCTTTA (SEQ ID NO: 1242) Sp109 ANGPT2- 110
AAAGGACTTACAGGGACAGC (SEQ ID NO: 1243) Sp110 ANGPT2- 111
GCTGATGCTGCTTATTTTGC (SEQ ID NO: 1244) Sp111 ANGPT2- 112
ATAAGCAGCATCAGCCAACC (SEQ ID NO: 1245) Sp112 ANGPT2- 113
TGCTAAAATCATTTCCTGGT (SEQ ID NO: 1246) Sp113 ANGPT2- 114
TTTGTGCTAAAATCATTTCC (SEQ ID NO: 1247) Sp114 ANGPT2- 115
AGGAAATGATTTTAGCACAA (SEQ ID NO: 1248) Sp115 ANGPT2- 116
AATGATTTTAGCACAAAGGA (SEQ ID NO: 1249) Sp116 ANGPT2- 117
AAATGTTCACAAATGCTAAC (SEQ ID NO: 1250) Sp117 ANGPT2- 118
TGTTCACAAATGCTAACAGG (SEQ ID NO: 1251) Sp118 ANGPT2- 119
CACAAATGCTAACAGGAGGT (SEQ ID NO: 1252) Sp119 ANGPT2- 120
ACAAATGCTAACAGGAGGTA (SEQ ID NO: 1253) Sp120 ANGPT2- 121
GGCTGGTGGTTTGATGCATG (SEQ ID NO: 1254) Sp121 ANGPT2- 122
TGTGGTCCTTCCAACTTGAA (SEQ ID NO: 1255) Sp122 ANGPT2- 123
TACATTCCGTTCAAGTTTGGA (SEQ ID NO: 1256) Sp123 ANGPT2- 124
ATAGTACATTCCGTTCAAGT (SEQ ID NO: 1257) Sp124 ANGPT2- 125
ACGGAATGTACTATCCACAG (SEQ ID NO: 1258) Sp125 ANGPT2- 126
TTATTTGTGTTCTGCCTCTG (SEQ ID NO: 1259) Sp126 ANGPT2- 127
CAGAACACAAATAAGTTCAA (SEQ ID NO: 1260) Sp127 ANGPT2- 128
ATAAGTTCAACGGCATTAAA (SEQ ID NO: 1261) Sp128 ANGPT2- 129
ACGGCATTAAATGGTACTAC (SEQ ID NO: 1262) Sp129 ANGPT2- 130
ATTAAATGGTACTACTGGAA (SEQ ID NO: 1263) Sp130 ANGPT2- 131
TGGTACTACTGGAAAGGCTC (SEQ ID NO: 1264) Sp131 ANGPT2- 132
AGGCTCAGGCTATTCGCTCA (SEQ ID NO: 1265) Sp132 ANGPT2- 133
TGGTCGGATCATCATGGTTG (SEQ ID NO: 1266) Sp133 ANGPT2- 134
ATCTGCTGGTCGGATCATCA (SEQ ID NO: 1267) Sp134 ANGPT2- 135
ATGTTTAGAAATCTGCTGGT (SEQ ID NO: 1268) Sp135 ANGPT2- 136
TGGGATGTTTAGAAATCTGC (SEQ ID NO: 1269) Sp136
TABLE-US-00014 TABLE 14 Target sequences of ANGPTL4 gene for SpCas9
Gene No. Target sequence ANGPTL4-Sp1 1 AGGCTACCTAAGAGGATGAG (SEQ ID
NO: 1270) ANGPTL4-Sp2 2 GAGGATGAGCGGTGCTCCGA (SEQ ID NO: 1271)
ANGPTL4-Sp3 3 ATGAGCGGTGCTCCGACGGC (SEQ ID NO: 1272) ANGPTL4-Sp4 4
TGAGCGGTGCTCCGACGGCC (SEQ ID NO: 1273) ANGPTL4-Sp5 5
GAGCGGTGCTCCGACGGCCG (SEQ ID NO: 1274) ANGPTL4-Sp6 6
ATCAGGGCTGCCCCGGCCGT (SEQ ID NO: 1275) ANGPTL4-Sp7 7
GCAGAGCATCAGGGCTGCCC (SEQ ID NO: 1276) ANGPTL4-Sp8 8
GGTGGCGGCGCAGAGCATCA (SEQ ID NO: 1277) ANGPTL4-Sp9 9
CGGTGGCGGCGCAGAGCATC (SEQ ID NO: 1278) ANGPTL4-Sp10 10
GCTCAGTAGCACGGCGGTGG (SEQ ID NO: 1279) ANGPTL4-Sp11 11
AGCGCTCAGTAGCACGGCGG (SEQ ID NO: 1280) ANGPTL4-Sp12 12
CTGAGCGCTCAGTAGCACGG (SEQ ID NO: 1281) ANGPTL4-Sp13 13
CGCCGTGCTACTGAGCGCTC (SEQ ID NO: 1282) ANGPTL4-Sp14 14
GCCGTGCTACTGAGCGCTCA (SEQ ID NO: 1283) ANGPTL4-Sp15 15
GCCCTGAGCGCTCAGTAGCA (SEQ ID NO: 1284) ANGPTL4-Sp16 16
GTGCTACTGAGCGCTCAGGG (SEQ ID NO: 1285) ANGPTL4-Sp17 17
CGCGGCGACTTGGACTGCAC (SEQ ID NO: 1286) ANGPTL4-Sp18 18
GCGCGGCGACTTGGACTGCA (SEQ ID NO: 1287) ANGPTL4-Sp19 19
GGACGCAAAGCGCGGCGACT (SEQ ID NO: 1288) ANGPTL4-Sp20 20
AGTCGCCGCGCTTTGCGTCC (SEQ ID NO: 1289) ANGPTL4-Sp21 21
GTCGCCGCGCTTTGCGTCCT (SEQ ID NO: 1290) ANGPTL4-Sp22 22
TCGTCCCAGGACGCAAAGCG (SEQ ID NO: 1291) ANGPTL4-Sp23 23
CAGGACATTCATCTCGTCCC (SEQ ID NO: 1292) ANGPTL4-Sp24 24
CTGGGACGAGATGAATGTCC (SEQ ID NO: 1293) ANGPTL4-Sp25 25
GAGATGAATGTCCTGGCGCA (SEQ ID NO: 1294) ANGPTL4-Sp26 26
GCTGCAGGAGTCCGTGCGCC (SEQ ID NO: 1295) ANGPTL4-Sp27 27
GCGCACGGACTCCTGCAGCT (SEQ ID NO: 1296) ANGPTL4-Sp28 28
CGGACTCCTGCAGCTCGGCC (SEQ ID NO: 1297) ANGPTL4-Sp28 29
GGACTCCTGCAGCTCGGCCA (SEQ ID NO: 1298) ANGPTL4-Sp30 30
GACTCCTGCAGCTCGGCCAG (SEQ ID NO: 1299) ANGPTL4-Sp31 31
GCAGCCCCTGGCCGAGCTGC (SEQ ID NO: 1300) ANGPTL4-Sp32 32
CCAGGGGCTGCGCGAACACG (SEQ ID NO: 1301) ANGPTL4-Sp33 33
CCGCGTGTTCGCGCAGCCCC (SEQ ID NO: 1302) ANGPTL4-Sp34 34
CAGCGCGCTCAGCTGACTGC (SEQ ID NO: 1303) ANGPTL4-Sp35 35
CCGCAGTCAGCTGAGCGCGC (SEQ ID NO: 1304) ANGPTL4-Sp36 36
CCAGCGCGCTCAGCTGACTG (SEQ ID NO: 1305) ANGPTL4-Sp37 37
GTCAGCTGAGCGCGCTGGAG (SEQ ID NO: 1306) ANGPTL4-Sp38 38
GAGCGGCGCCTGAGCGCGTG (SEQ ID NO: 1307) ANGPTL4-Sp39 39
AGCGGCGCCTGAGCGCGTGC (SEQ ID NO: 1308) ANGPTL4-Sp40 40
AGGCGGACCCGCACGCGCTC (SEQ ID NO: 1309) ANGPTL4-Sp41 41
CGCGTGCGGGTCCGCCTGTC (SEQ ID NO: 1310) ANGPTL4-Sp42 42
GCGTGCGGGTCCGCCTGTCA (SEQ ID NO: 1311) ANGPTL4-Sp43 43
GTCCGCCTGTCAGGGAACCG (SEQ ID NO: 1312) ANGPTL4-Sp44 44
TCCGCCTGTCAGGGAACCGA (SEQ ID NO: 1313) ANGPTL4-Sp45 45
CCGCCTGTCAGGGAACCGAG (SEQ ID NO: 1314) ANGPTL4-Sp46 46
CCCCTCGGTTCCCTGACAGG (SEQ ID NO: 1315) ANGPTL4-Sp47 47
GGACCCCTCGGTTCCCTGAC (SEQ ID NO: 1316) ANGPTL4-Sp48 48
CGGGAGGTCGGTGGACCCCT (SEQ ID NO: 1317) ANGPTL4-Sp49 49
AGGGGCTAACGGGAGGTCGG (SEQ ID NO: 1318) ANGPTL4-Sp50 50
CTCAGGGGCTAACGGGAGGT (SEQ ID NO: 1319) ANGPTL4-Sp51 51
GGCTCTCAGGGGCTAACGGG (SEQ ID NO: 1320) ANGPTL4-Sp52 52
TCCCGTTAGCCCCTGAGAGC (SEQ ID NO: 1321) ANGPTL4-Sp53 53
CCCGTTAGCCCCTGAGAGCC (SEQ ID NO: 1322) ANGPTL4-Sp54 54
CCCGGCTCTCAGGGGCTAAC (SEQ ID NO: 1323) ANGPTL4-Sp55 55
ACCCGGCTCTCAGGGGCTAA (SEQ ID NO: 1324) ANGPTL4-Sp56 56
GTTAGCCCCTGAGAGCCGGG (SEQ ID NO: 1325) ANGPTL4-Sp57 57
AGGGTCCACCCGGCTCTCAG (SEQ ID NO: 1326) ANGPTL4-Sp58 58
CAGGGTCCACCCGGCTCTCA (SEQ ID NO: 1327) ANGPTL4-Sp59 59
TCAGGGTCCACCCGGCTCTC (SEQ ID NO: 1328) ANGPTL4-Sp60 60
TGAGAGCCGGGTGGACCCTG (SEQ ID NO: 1329) ANGPTL4-Sp61 61
GAAGGACCTCAGGGTCCACC (SEQ ID NO: 1330) ANGPTL4-Sp62 62
GCAGGCTGTGAAGGACCTCA (SEQ ID NO: 1331) ANGPTL4-Sp63 63
TGCAGGCTGTGAAGGACCTC (SEQ ID NO: 1332) ANGPTL4-Sp64 64
TGAGGTCCTTCACAGCCTGC (SEQ ID NO: 1333) ANGPTL4-Sp65 65
CACGTACCTGCAGGCTGTGA (SEQ ID NO: 1334) ANGPTL4-Sp66 66
CCCTGGGGACACGTACCTGC (SEQ ID NO: 1335) ANGPTL4-Sp67 67
CCCTCCCCAGACACAACTCA (SEQ ID NO: 1336) ANGPTL4-Sp68 68
TGAGCCTTGAGTTGTGTCTG (SEQ ID NO: 1337) ANGPTL4-Sp69 69
CTGAGCCTTGAGTTGTGTCT (SEQ ID NO: 1338) ANGPTL4-Sp70 70
TCTGAGCCTTGAGTTGTGTC (SEQ ID NO: 1339) ANGPTL4-Sp71 71
AACTCAAGGCTCAGAACAGC (SEQ ID NO: 1340) ANGPTL4-Sp72 72
GATCCAGCAACTCTTCCACA (SEQ ID NO: 1341) ANGPTL4-Sp73 73
CCAGCAACTCTTCCACAAGG (SEQ ID NO: 1342) ANGPTL4-Sp74 74
CCACCTTGTGGAAGAGTTGC (SEQ ID NO: 1343) ANGPTL4-Sp75 75
GCTGCTGCTGGGCCACCTTG (SEQ ID NO: 1344) ANGPTL4-Sp76 76
ACAAGGTGGCCCAGCAGCAG (SEQ ID NO: 1345) ANGPTL4-Sp77 77
GGCCCAGCAGCAGCGGCACC ANGPTL4-Sp78 78 CTCCAGGTGCCGCTGCTGCT (SEQ ID
NO: 1347) ANGPTL4-Sp79 79 TCTCCAGGTGCCGCTGCTGC (SEQ ID NO: 1348)
ANGPTL4-Sp80 80 TTCGCAGGTGCTGCTTCTCC (SEQ ID NO: 1349) ANGPTL4-Sp81
81 TTTGCAGATGCTGAATTCGC (SEQ ID NO: 1350) ANGPTL4-Sp82 82
AATTCAGCATCTGCAAAGCC (SEQ ID NO: 1351) ANGPTL4-Sp83 83
CCCTTGATCCTAGGGTTACC
(SEQ ID NO: 1352) ANGPTL4-Sp84 84 CCCATCCTAGTTTGGCCTCC (SEQ ID NO:
1353) ANGPTL4-Sp85 85 GTGGTCCAGGAGGCCAAACT (SEQ ID NO: 1354)
ANGPTL4-Sp86 86 CTAGGTGCTTGTGGTCCAGG (SEQ ID NO: 1355) ANGPTL4-Sp87
87 GGTCTAGGTGCTTGTGGTCC (SEQ ID NO: 1356) ANGPTL4-Sp88 88
CCACAAGCACCTAGACCATG (SEQ ID NO: 1357) ANGPTL4-Sp89 89
CCTCATGGTCTAGGTGCTTG (SEQ ID NO: 1358) ANGPTL4-Sp90 90
CAAGCACCTAGACCATGAGG (SEQ ID NO: 1359) ANGPTL4-Sp91 91
GCTTGGCCACCTCATGGTCT (SEQ ID NO: 1360) ANGPTL4-Sp92 92
GGGCAGGCTTGGCCACCTCA (SEQ ID NO: 1361) ANGPTL4-Sp93 93
CCAAGCCTGCCCGAAGAAAG (SEQ ID NO: 1362) ANGPTL4-Sp94 94
CCTCTTTCTTCGGGCAGGCT (SEQ ID NO: 1363) ANGPTL4-Sp95 95
GGCAGCCTCTTTCTTCGGGC (SEQ ID NO: 1364) ANGPTL4-Sp96 96
CTCGGGCAGCCTCTTTCTTC (SEQ ID NO: 1365) ANGPTL4-Sp97 97
TCTCGGGCAGCCTCTTTCTT (SEQ ID NO: 1366) ANGPTL4-Sp98 98
AAGAAAGAGGCTGCCCGAGA (SEQ ID NO: 1367) ANGPTL4-Sp99 99
TCAACTGGCTGGGCCATCTC (SEQ ID NO: 1368) ANGPTL4-Sp100 100
GTCAACTGGCTGGGCCATCT (SEQ ID NO: 1369) ANGPTL4-Sp101 101
GATGGCCCAGCCAGTTGACC (SEQ ID NO: 1370) ANGPTL4-Sp102 102
GTGAGCCGGGTCAACTGGCT (SEQ ID NO: 1371) ANGPTL4-Sp103 103
TGTGAGCCGGGTCAACTGGC (SEQ ID NO: 1372) ANGPTL4-Sp104 104
ACATTGTGAGCCGGGTCAAC (SEQ ID NO: 1373) ANGPTL4-Sp105 105
GGCGGCTGACATTGTGAGCC (SEQ ID NO: 1374) ANGPTL4-Sp106 106
AGGCGGCTGACATTGTGAGC (SEQ ID NO: 1375) ANGPTL4-Sp107 107
GCAGACACTCACGGTGCAGG (SEQ ID NO: 1376) ANGPTL4-Sp108 108
GGGGCAGACACTCACGGTGC (SEQ ID NO: 1377) ANGPTL4-Sp109 109
ATCTCCCTTCAGGGCTGCCC (SEQ ID NO: 1378) ANGPTL4-Sp110 110
TCTCCCTTCAGGGCTGCCCA (SEQ ID NO: 1379) ANGPTL4-Sp111 111
AGGGCTGCCCAGGGATTGCC (SEQ ID NO: 1380) ANGPTL4-Sp112 112
AACAGCTCCTGGCAATCCCT (SEQ ID NO: 1381) ANGPTL4-Sp113 113
GAACAGCTCCTGGCAATCCC (SEQ ID NO: 1382) ANGPTL4-Sp114 114
GGATTGCCAGGAGCTGTTCC (SEQ ID NO: 1383) ANGPTL4-Sp115 115
TGCCAGGAGCTGTTCCAGGT (SEQ ID NO: 1384) ANGPTL4-Sp116 116
CCAGGAGCTGTTCCAGGTTG (SEQ ID NO: 1385) ANGPTL4-Sp117 117
CCCCAACCTGGAACAGCTCC (SEQ ID NO: 1386) ANGPTL4-Sp118 118
AGCTGTTCCAGGTTGGGGAG (SEQ ID NO: 1387) ANGPTL4-Sp119 119
CACTCTGCCTCTCCCCAACC (SEQ ID NO: 1388) ANGPTL4-Sp120 120
CAGGTTGGGGAGAGGCACAG (SEQ ID NO: 1389) ANGPTL4-Sp121 121
ACTATTTGAAATCCAGCCTC (SEQ ID NO: 1390) ANGPTL4-Sp122 122
CTATTTGAAATCCAGCCTCA (SEQ ID NO: 1391) ANGPTL4-Sp123 123
TATTTGAAATCCAGCCTCAG (SEQ ID NO: 1392) ANGPTL4-Sp124 124
ATGGCGGAGACCCCTGAGGC (SEQ ID NO: 1393) ANGPTL4-Sp125 125
AAAAATGGCGGAGACCCCTG (SEQ ID NO: 1394) ANGPTL4-Sp126 126
TCAGGGGTCTCCGCCATTTT (SEQ ID NO: 1395) ANGPTL4-Sp127 127
TTGCAGTTCACCAAAAATGG (SEQ ID NO: 1396) ANGPTL4-Sp128 128
ATCTTGCAGTTCACCAAAAA (SEQ ID NO: 1397) ANGPTL4-Sp129 129
GTGAACTGCAAGATGACCTC (SEQ ID NO: 1398) ANGPTL4-Sp130 130
ACTGCAAGATGACCTCAGGT (SEQ ID NO: 1399) ANGPTL4-Sp131 131
CTGCAAGATGACCTCAGGTA (SEQ ID NO: 1400) ANGPTL4-Sp132 132
GGACTAACACACCCTACCTG (SEQ ID NO: 1401) ANGPTL4-Sp133 133
GTACCTTTCTGGGCAGATGG (SEQ ID NO: 1402) ANGPTL4-Sp134 134
CTTTCTGGGCAGATGGAGGC (SEQ ID NO: 1403) ANGPTL4-Sp135 135
GAGGCTGGACAGTAATTCAG (SEQ ID NO: 1404) ANGPTL4-Sp136 136
GTAATTCAGAGGCGCCACGA (SEQ ID NO: 1405) ANGPTL4-Sp137 137
GAGGCGCCACGATGGCTCAG (SEQ ID NO: 1406) ANGPTL4-Sp138 138
TGAAGTCCACTGAGCCATCG (SEQ ID NO: 1407) ANGPTL4-Sp139 139
ATGGCTCAGTGGACTTCAAC (SEQ ID NO: 1408) ANGPTL4-Sp140 140
CAGTGGACTTCAACCGGCCC (SEQ ID NO: 1409) ANGPTL4-Sp141 141
AGTGGACTTCAACCGGCCCT (SEQ ID NO: 1410) ANGPTL4-Sp142 142
CCGGCCCTGGGAAGCCTACA (SEQ ID NO: 1411) ANGPTL4-Sp143 143
CCTTGTAGGCTTCCCAGGGC (SEQ ID NO: 1412) ANGPTL4-Sp144 144
GCCCTGGGAAGCCTACAAGG (SEQ ID NO: 1413) ANGPTL4-Sp145 145
CCCTGGGAAGCCTACAAGGC (SEQ ID NO: 1414) ANGPTL4-Sp146 146
CCCGCCTTGTAGGCTTCCCA (SEQ ID NO: 1415) ANGPTL4-Sp147 147
CCTGGGAAGCCTACAAGGCG (SEQ ID NO: 1416) ANGPTL4-Sp148 148
CCCCGCCTTGTAGGCTTCCC (SEQ ID NO: 1417) ANGPTL4-Sp149 149
GAAGCCTACAAGGCGGGGTT (SEQ ID NO: 1418) ANGPTL4-Sp150 150
AAGCCTACAAGGCGGGGTTT (SEQ ID NO: 1419) ANGPTL4-Sp151 151
AGCCTACAAGGCGGGGTTTG (SEQ ID NO: 1420) ANGPTL4-Sp152 152
ATCCCCAAACCCCGCCTTGT (SEQ ID NO: 1421) ANGPTL4-Sp153 153
GCGGGGTTTGGGGATCCCCA (SEQ ID NO: 1422) ANGPTL4-Sp154 154
GGTTTGGGGATCCCCACGGT (SEQ ID NO: 1423) ANGPTL4-Sp155 155
CACTAGAAACACCTACCGTG (SEQ ID NO: 1424) ANGPTL4-Sp156 156
CCACTAGAAACACCTACCGT (SEQ ID NO: 1425) ANGPTL4-Sp157 157
CTCCCACTCCAGGCGAGTTC (SEQ ID NO: 1426) ANGPTL4-Sp158 158
CACTCCAGGCGAGTTCTGGC (SEQ ID NO: 1427) ANGPTL4-Sp159 159
ACTCCAGGCGAGTTCTGGCT (SEQ ID NO: 1428) ANGPTL4-Sp160 160
ACACCCAGCCAGAACTCGCC (SEQ ID NO: 1429) ANGPTL4-Sp161 161
AGGCGAGTTCTGGCTGGGTC (SEQ ID NO: 1430) ANGPTL4-Sp162 162
GTTCTGGCTGGGTCTGGAGA (SEQ ID NO: 1431) ANGPTL4-Sp163 163
GGAGAAGGTGCATAGCATCA (SEQ ID NO: 1432) ANGPTL4-Sp164 164
GAGAAGGTGCATAGCATCAC (SEQ ID NO: 1433) ANGPTL4-Sp165 165
AGAAGGTGCATAGCATCACG (SEQ ID NO: 1434) ANGPTL4-Sp166 166
GAAGGTGCATAGCATCACGG (SEQ ID NO: 1435)
ANGPTL4-Sp167 167 GGGGGACCGCAACAGCCGCC (SEQ ID NO: 1436)
ANGPTL4-Sp168 168 GCACGGCCAGGCGGCTGTTG (SEQ ID NO: 1437)
ANGPTL4-Sp169 169 GCCGCCTGGCCGTGCAGCTG (SEQ ID NO: 1438)
ANGPTL4-Sp170 170 CCGCCTGGCCGTGCAGCTGC (SEQ ID NO: 1439)
ANGPTL4-Sp171 171 CCCGCAGCTGCACGGCCAGG (SEQ ID NO: 1440)
ANGPTL4-Sp172 172 AGTCCCGCAGCTGCACGGCC (SEQ ID NO: 1441)
ANGPTL4-Sp173 173 TGGCCGTGCAGCTGCGGGAC (SEQ ID NO: 1442)
ANGPTL4-Sp174 174 GGCCGTGCAGCTGCGGGACT (SEQ ID NO: 1443)
ANGPTL4-Sp175 175 ATCCCAGTCCCGCAGCTGCA (SEQ ID NO: 1444)
ANGPTL4-Sp176 176 GTGCAGCTGCGGGACTGGGA (SEQ ID NO: 1445)
ANGPTL4-Sp177 177 CACGGAGAACTGCAGCCCCT (SEQ ID NO: 1446)
ANGPTL4-Sp178 178 GCTGCAGTTCTCCGTGCACC (SEQ ID NO: 1447)
ANGPTL4-Sp179 179 CTGCAGTTCTCCGTGCACCT (SEQ ID NO: 1448)
ANGPTL4-Sp180 180 CAGTTCTCCGTGCACCTGGG (SEQ ID NO: 1449)
ANGPTL4-Sp181 181 CTCCGTGCACCTGGGTGGCG (SEQ ID NO: 1450)
ANGPTL4-Sp182 182 GTCCTCGCCACCCAGGTGCA (SEQ ID NO: 1451)
ANGPTL4-Sp183 183 GCACCTGGGTGGCGAGGACA (SEQ ID NO: 1452)
ANGPTL4-Sp184 184 AGGCCGTGTCCTCGCCACCC (SEQ ID NO: 1453)
ANGPTL4-Sp185 185 TGCAGTGAGCTGCAGGCTAT (SEQ ID NO: 1454)
ANGPTL4-Sp186 186 CCTGCAGCTCACTGCACCCG (SEQ ID NO: 1455)
ANGPTL4-Sp187 187 CCACGGGTGCAGTGAGCTGC (SEQ ID NO: 1456)
ANGPTL4-Sp188 188 CAGCTCACTGCACCCGTGGC (SEQ ID NO: 1457)
ANGPTL4-Sp189 189 TGCACCCGTGGCCGGCCAGC (SEQ ID NO: 1458)
ANGPTL4-Sp190 190 GCACCCGTGGCCGGCCAGCT (SEQ ID NO: 1459)
ANGPTL4-Sp191 191 GCGCCCAGCTGGCCGGCCAC (SEQ ID NO: 1460)
ANGPTL4-Sp192 192 GGCGCCCAGCTGGCCGGCCA (SEQ ID NO: 1461)
ANGPTL4-Sp193 193 GGTGGTGGCGCCCAGCTGGC (SEQ ID NO: 1462)
ANGPTL4-Sp194 194 GGACGGTGGTGGCGCCCAGC (SEQ ID NO: 1463)
ANGPTL4-Sp195 195 GCCACCACCGTCCCACCCAG (SEQ ID NO: 1464)
ANGPTL4-Sp196 196 GCCGCTGGGTGGGACGGTGG (SEQ ID NO: 1465)
ANGPTL4-Sp197 197 GAGGCCGCTGGGTGGGACGG (SEQ ID NO: 1466)
ANGPTL4-Sp198 198 GGAGAGGCCGCTGGGTGGGA (SEQ ID NO: 1467)
ANGPTL4-Sp199 199 GTACGGAGAGGCCGCTGGGT (SEQ ID NO: 1468)
ANGPTL4-Sp200 200 GGTACGGAGAGGCCGCTGGG (SEQ ID NO: 1469)
ANGPTL4-Sp201 201 AAGGGTACGGAGAGGCCGCT (SEQ ID NO: 1470)
ANGPTL4-Sp202 202 GAAGGGTACGGAGAGGCCGC (SEQ ID NO: 1471)
ANGPTL4-Sp203 203 AAGTGGAGAAGGGTACGGAG (SEQ ID NO: 1472)
ANGPTL4-Sp204 204 TCTCCGTACCCTTCTCCACT (SEQ ID NO: 1473)
ANGPTL4-Sp205 205 CTCCGTACCCTTCTCCACTT (SEQ ID NO: 1474)
ANGPTL4-Sp206 206 GTCCCAAGTGGAGAAGGGTA (SEQ ID NO: 1475)
ANGPTL4-Sp207 207 ACCCTTCTCCACTTGGGACC (SEQ ID NO: 1476)
ANGPTL4-Sp208 208 TCCTGGTCCCAAGTGGAGAA (SEQ ID NO: 1477)
ANGPTL4-Sp209 209 ATCCTGGTCCCAAGTGGAGA (SEQ ID NO: 1478)
ANGPTL4-Sp210 210 GTCGTGATCCTGGTCCCAAG (SEQ ID NO: 1479)
ANGPTL4-Sp211 211 ACCAGGATCACGACCTCCGC (SEQ ID NO: 1480)
ANGPTL4-Sp212 212 CCAGGATCACGACCTCCGCA (SEQ ID NO: 1481)
ANGPTL4-Sp213 213 CCCTGCGGAGGTCGTGATCC (SEQ ID NO: 1482)
ANGPTL4-Sp214 214 CGCAGTTCTTGTCCCTGCGG (SEQ ID NO: 1483)
ANGPTL4-Sp215 215 TGGCGCAGTTCTTGTCCCTG (SEQ ID NO: 1484)
ANGPTL4-Sp216 216 AACTGCGCCAAGAGCCTCTC (SEQ ID NO: 1485)
ANGPTL4-Sp217 217 CTGCTCACCAGAGAGGCTCT (SEQ ID NO: 1486)
ANGPTL4-Sp218 218 GCAGGGCCTGCTCACCAGAG (SEQ ID NO: 1487)
ANGPTL4-Sp219 219 CCCTGACCCCGGCAGGAGGC (SEQ ID NO: 1488)
ANGPTL4-Sp220 220 TGACCCCGGCAGGAGGCTGG (SEQ ID NO: 1489)
ANGPTL4-Sp221 221 CCGGCAGGAGGCTGGTGGTT (SEQ ID NO: 1490)
ANGPTL4-Sp222 222 GTTGAGGTTGGAATGGCTGC (SEQ ID NO: 1491)
ANGPTL4-Sp223 223 TGCAGCCATTCCAACCTCAA (SEQ ID NO: 1492)
ANGPTL4-Sp224 224 ACTGGCCGTTGAGGTTGGAA (SEQ ID NO: 1493)
ANGPTL4-Sp225 225 GAAGTACTGGCCGTTGAGGT (SEQ ID NO: 1494)
ANGPTL4-Sp226 226 GACGGAAGTACTGGCCGTTG (SEQ ID NO: 1495)
ANGPTL4-Sp227 227 GTGGGATGGAGCGGAAGTAC (SEQ ID NO: 1496)
ANGPTL4-Sp228 228 TCCGCTCCATCCCACAGCAG (SEQ ID NO: 1497)
ANGPTL4-Sp229 229 GCCGCTGCTGTGGGATGGAG (SEQ ID NO: 1498)
ANGPTL4-Sp230 230 CTTCTGCCGCTGCTGTGGGA (SEQ ID NO: 1499)
ANGPTL4-Sp231 231 TAAGCTTCTGCCGCTGCTGT (SEQ ID NO: 1500)
ANGPTL4-Sp232 232 TTAAGCTTCTGCCGCTGCTG (SEQ ID NO: 1501)
ANGPTL4-Sp233 233 GCAGCGGCAGAAGCTTAAGA (SEQ ID NO: 1502)
ANGPTL4-Sp234 234 CAGCGGCAGAAGCTTAAGAA (SEQ ID NO: 1503)
ANGPTL4-Sp235 235 AGCTTAAGAAGGGAATCTTC (SEQ ID NO: 1504)
ANGPTL4-Sp236 236 AGGGAATCTTCTGGAAGACC (SEQ ID NO: 1505)
ANGPTL4-Sp237 237 GAATCTTCTGGAAGACCTGG (SEQ ID NO: 1506)
ANGPTL4-Sp238 238 AATCTTCTGGAAGACCTGGC (SEQ ID NO: 1507)
ANGPTL4-Sp239 239 ATCTTCTGGAAGACCTGGCG (SEQ ID NO: 1508)
ANGPTL4-Sp240 240 CGGGTAGTAGCGGCCCCGCC (SEQ ID NO: 1509)
ANGPTL4-Sp241 241 GGGCCGCTACTACCCGCTGC (SEQ ID NO: 1510)
ANGPTL4-Sp242 242 TGGCCTGCAGCGGGTAGTAG (SEQ ID NO: 1511)
ANGPTL4-Sp243 243 ACATGGTGGTGGCCTGCAGC (SEQ ID NO: 1512)
ANGPTL4-Sp244 244 AACATGGTGGTGGCCTGCAG (SEQ ID NO: 1513)
ANGPTL4-Sp245 245 GGGCTGGATCAACATGGTGG (SEQ ID NO: 1514)
ANGPTL4-Sp246 246 CATGGGCTGGATCAACATGG ANGPTL4-Sp247 247
CACCATGTTGATCCAGCCCA (SEQ ID NO: 1516) ANGPTL4-Sp248 248
TGCCATGGGCTGGATCAACA (SEQ ID NO: 1517) ANGPTL4-Sp249 249
GATCCAGCCCATGGCAGCAG (SEQ ID NO: 1518) ANGPTL4-Sp250 250
CTGCCTCTGCTGCCATGGGC (SEQ ID NO: 1519) ANGPTL4-Sp251 251
GAGGCTGCCTCTGCTGCCAT
(SEQ ID NO: 1520) ANGPTL4-Sp252 252 GGAGGCTGCCTCTGCTGCCA (SEQ ID
NO: 1521) ANGPTL4-Sp252 253 GGCCCAGCCAGGACGCTAGG (SEQ ID NO:
1522)
[1288] 2. Construction of CjCas9 and sgRNA Plasmids
[1289] A sequence encoding human codon-optimized CjCas9 (derived
from Campylobacter jejuni subsp. Jejuni NCTC 11168) was synthesized
to have a nuclear localization signal (NLS) and an HA epitope at
the C-terminus (GeneArt.TM. Gene Synthesis, Thermo Fisher
Scientific), and the synthesized nucleic acid sequence was
replicated using a p3s plasmid described in previous research (Cho,
S. W., Kim, S., Kim, J. M. & Kim, J. S. Targeted genome
engineering in human cells with the Cas9 RNA-guided endonuclease.
Nature Biotechnology 31, 230-232 (2013)).
[1290] A tracrRNA (transactivating crRNA) sequence and a pre-crRNA
(precursor CRISPR RNA) sequence were connected using a GAAA or TGAA
linker, thereby producing sgRNA. The sgRNA regulated transcription
with an U6 promoter.
[1291] 3. PAM Characterization Using Cell-Based Reporter
Analysis
[1292] An AAVS1 target site (AAVS1-TS1) having a variable PAM
sequence (5'-NNNNXCAC-3', 5'-NNNNAXAC-3', 5'-NNNNACXC-3', and
5'-NNNNACAX-3') including a random sequence at the X site was
synthesized (Macrogen, Inc.), and the synthesized nucleic acid
sequence may be replicated using a surrogate reporter plasmid
encoding RFP and GFP.
[1293] To determine a suitable PAM sequence, the constructed
reporter plasmid (100 ng), and plasmids encoding CjCas9 (225 ng)
and sgRNA (675 ng) were co-transfected into HEK293 cells
(1.times.10.sup.5) using lipofectamine 2000 (Invitrogen). Two days
after the transfection, a fractionation of the GFP and RFP-positive
cells was measured by flow cytometry (BD Accuri.TM. C6, BD).
[1294] 4. Cell Culture and Mutation Analysis
[1295] HEK293 (ATCC, CRL-1573) cells and mouse NIH 3T3 (ATCC,
CRL-1658) cells were cultured in a Dulbecco's modified Eagle's
medium (DMEM) supplemented with 100 units/mL penicillin, 100 mg/mL
streptomycin, and 10% fetal bovine serum (FBS).
[1296] A sgRNA plasmid (750 ng) and a CjCas9 plasmid (250 ng) were
transfected into cells (0.5.about.1.times.10.sup.5) using
lipofectamine 2000 (Invitrogen). 48 hours after the transfection,
genome DNA was separated using a DNeasy blood & tissue kit
(Qiagen), and an on-target or off-target site was amplified for
targeted deep sequencing. Deep sequencing libraries were generated
by PCR. TruSeq HT Dual Index primers were used to label respective
samples. Mixed libraries were subjected to paired-end sequencing
(LAS, Inc.), and indel frequencies were calculated.
[1297] 5. Construction of AAV Vectors Encoding CjCas9 and sgRNA
Sequences
[1298] An AAV inverted terminal repeat (ITR)-based vector plasmid
containing an sgRNA sequence and a CjCas9 gene having NLS and HA
tags at the C-terminus was constructed. sgRNA transcription was
induced by an U6 promoter, and CjCas9 expression was regulated by
an EFS promoter in C2C12 myoblasts, or by a Spc512 promoter in the
TA muscle of C57BL/6 mice.
[1299] For retinal delivery, an AAV vector encoding the U6
promoter-induced sgRNA and CjCas9 under the control of the EFS
promoter, specific to the Vegfa gene and Hif1a gene, was
constructed, wherein CjCas9 had eGFP linked to the C-terminus using
a self-cleaved T2A peptide.
[1300] 6. Production, Purification and Characterization of AAV
Vector
[1301] To produce the AAV vector, a pseudotype of AAVDJ or AAV9
capsids was used. The HEK293T cells were transfected with
pAAV-ITR-CjCas9-sgRNA, pAAVED2/9, and a helper plasmid. The HEK293T
cells were cultured in a 2% FBS-containing DMEM. Recombinant AAV
vector stocks were produced using PEI coprecipitation by mixing
polyplus-transfection (PElpro), triple-transfection and the plasmid
in a molar ratio of 1:1:1 in the HEK293T cells. After 72 hours of
culturing, the cells were lysed, and particles were isolated and
purified with iodixanol (Sigma-Aldrich) by step-gradient
ultracentrifugation. The number of vector genomes was quantified
through quantitative PCR.
[1302] 7. AAV Transduction in Mouse Myoblasts
[1303] Mouse myoblasts were infected with various viral amounts of
AAVDJ-CjCas9 (multiplicity of infection (MOI): 1, 5, 10, 50, and
100 (determined by quantitative PCR)), and cultured in 2%
FBS-containing DMEM. For the target deep sequencing, cells were
obtained at different points of time. MOI 1 is considered as
infection by one virus particle among a total of 100 virus
particles determined by quantitative PCR.
[1304] 8. Animals
[1305] Management, use and treatment of all animals used in this
research were performed under the guidance provided by the Seoul
National University Animal Care and Use Committee, ophthalmology
and security research, and the strict agreement according to the
ARVO statement on animal use in veterinary medicine. In this
research, specific male pathogen free (SPF)-6-week old C57BL/6J
mice were used. The mice were maintained in a 12-hour light/dark
cycle.
[1306] Diabetic model mice were induced by injecting streptozotocin
(STZ, Sigma-Aldrich, St. Louis, Mo., USA) intraperitonially once.
As a control, a citrate buffer was injected. 4 days after the STZ
injection, when a blood glucose level of the mouse was 300 mg/dl or
more, diabetes was considered to be induced.
[1307] 9. Injection of AAV into Vitreous Body
[1308] A mixture of tiletamine and zolazepam in a ratio of 1:1
(2.25 mg/kg (body weight) each), and 0.7 mg/kg (body weight) of
xylazine hydrochloride was injected into a vitreous body of a
6-week old mouse for anesthetization. 2 .mu.l (2.times.1010 viral
genome) of AAV9-CjCas9 was injected into the vitreous body using a
Nanofil syringe (World Precision Instruments Inc.) having a 33G
blunt needle under a surgical microscope (Leica Microsystems
Ltd.).
[1309] In the case of diabetic mouse models, the mice were injected
with STZ to induce diabetes, anesthetized after 1 or 7 weeks, and 2
.mu.l of the mixture containing 0.8.times.108 vg/.mu.l of
CjCas9:Vegfa or 1.5.times.109 vg/.mu.l of Rosa26 was injected in
the vitreous body.
[1310] 10. Immunofluorescence Staining and Imaging of Retinal
Tissue
[1311] 42 days after the injection, the sample was fixed with
formalin and embedded in paraffin (n=4). A sample obtained by
cross-section of the sample-embedded paraffin was immunostained
with an anti-HA antibody (Roche, 3F10, 1:1000), an anti-opsin
antibody (Millipore, AB5405, 1:1000), and an Alexa Fluor 488 or 594
antibody (Thermo Fisher Scientific, 1:500). An opsin-positive site
was detected in RPE cells expressing HA-tagged CjCas9 using Image J
software (1.47v, NIH). The distribution of CjCas9 and eGFP was
visualized on RPE flat-mounts using a confocal microscope (LSM 710,
Carl Zeiss).
[1312] 11. Extraction of Genome DNA
[1313] To extract DNA from RPE and the retina, after obtaining
images of RPE and the retina flat-mounts, the tissue samples were
washed with PBS. RPE cells were separated from the choroid/sclera
by vortexing for 30 seconds in a lysis buffer (NucleoSpin Tissue,
Macherey-Nagel). Genome DNA was analyzed to identify complete
isolation of the RPE cells from remaining choroid/sclera tissue.
The genome DNA was analyzed by target deep sequencing.
[1314] 12. Mouse Vegfa ELISA
[1315] 42 days after the injection, a total RPE mixture was
isolated from neural retina tissue, and two kinds of tissue were
frozen for subsequent analysis. The sample tissue was lysed in 120
.mu.l of a cell lysis buffer (CST #9803), and the amount of a Vegfa
protein was measured using a mouse VEGF Quantikine ELISA kit
(MMVOO, R&D Systems).
[1316] 13. Laser-Induced CNV Model
[1317] After anesthetization of a mouse, eye drops containing 0.5%
phenylephrine and 0.5% tropicamide were injected to dilate a pupil.
Laser photocoagulation was performed using an indirect head set
delivery system (Iridex) and a laser system (Ilooda). Parameters of
the laser are a wavelength of 532 nm, a spot size of 200 .mu.m, a
power of 800 mW and exposure time of 70 ms. A laser burn was
induced in the proximity of the optical nerve three to four times.
Burns in which bubbles are generated without bleeding of the
vitreous body were only used for the research. After 7 days, an
eyeball was fixed with 4% paraformaldehyde at room temperature for
one hour. An RPE mixture (RPE/choroid/sclera) was immunostained
overnight at 4.degree. C. using isolectin-B4 (Thermo Fisher
Scientific, cat. no. 121413, 1:100) and an anti-GFP antibody
(Abcam, ab6556, 1:100). The RPE mixture was flat-mounted, and
visualized using a fluorescent microscope (Eclipse 90i, Nikon) or a
confocal microscope (LSM 710, Carl Zeiss) at a 100.times.
magnification. A CNV site was detected using Image J software
(1.47v, NIH). An average of three to four CNV sites per eyeball was
analyzed. Each group consists of 17 to 18 eyeballs.
[1318] 14. Quantitative and Qualitative Analyses for Rupturing of
Retinal Vessels
[1319] To detect vascular leakage, STZ-induced diabetic mouse
models were used. 200 .mu.l of an Evans blue dye (20 mg/ml)
dissolved in PBS was intravenously injected into an anesthetized
mouse. Two hours after perfusion, an eyeball was extracted to be
fixed with 4% paraformaldehyde for 1 hour. The retina was excised
in 2.times.PBS and flat-mounted, and images of the retina were
obtained at 40.times. and 100.times. magnifications using a
fluorescent microscope (Eclipse 90i, Nikon).
[1320] To quantitatively analyze the vascular leakage,
representative four sites of the vascular leakage in the
mid-peripheral retina (0.5 .mu.m.times.0.5 .mu.m) of each mouse
were selected. The mid-peripheral retina was designated as the
middle 1/3 of the retina from the optic nerve head to the ciliary
body, images were modulated according to color threshold values
based on the automatic isodata algorithm using the Image J software
(1.47v, NIH), and regions of interest containing the Evans blue dye
were marked in red. Afterward, the regions marked in red were
detected. The data were normalized with data of control mice, and
represented as vascular leakage (%).
[1321] 15. Data Analysis
[1322] To previously determine a sample size in vitro or in vivo, a
statistical method was not used. For statistical analysis, one-way
ANOVA and Tukey post-hoc tests were used.
Example 1. Confirmation of CjCas9 Expression Through AAV in Mouse
Retinal Tissue
[1323] Since CjCas9 consisting of 984 amino acids has a
considerably smaller size (2.95 kbp) than SpCas9, both CjCas9 gene
and sgRNA are able to be packaged in one AAV vector. Therefore, in
this example, to confirm the possibility of gene manipulation using
CjCas9 as a method for treating AMD, CjCas9-expressing AAV was
used.
[1324] To confirm the expression of CjCas9 through AAV in tissue
such as the retina in a mouse, under the control of U6
promoter-induced sgRNA and an EFS promoter specific to a choroidal
neovascularization (CNV)-associated Vegfa gene and an Hif1a gene, a
CjCas9-coding AAV9 vector was constructed, and here, CjCas9 was
linked to eGFP at the C-terminus using a self-cleaved T2A peptide
(FIGS. 1 and 2). The constructed virus was injected into an eyeball
through injection into the vitreous body, and after 6 weeks, CjCas9
expression in the eyeball was confirmed, an indel frequency was
measured using target deep sequencing, and the amount of Vegfa
protein was measured through ELISA (FIG. 4).
[1325] The expression of the CjCas9-linked eGFP was confirmed in
retinal pigment epithelial (RPE) cells (FIG. 8).
[1326] In addition, indels induced by CjCas9 were observed at
Rosa26, Vegfa, and Hif1a target sites of the RPE cells, indel
frequencies of 14.+-.5%, 22.+-.3%, and 31.+-.2% were confirmed,
respectively (FIG. 3). In addition, noticeable off-target indels
were not induced by Vegfa- or Hif1a-specific sgRNA in the cells
(FIG. 5).
[1327] As expected, the expression level of the Vegfa protein was
decreased in AAV-treated RPE cells encoding Vegfa-specific CjCas9
(AAV-CjCas9: Vegfa), but was not when Hif1a- or Rosa26-specific
CjCas9 (AAV-CjCas9: Hif1a or Rosa26)-coding AAV was treated (FIG.
4). Particularly, since a Hif1a protein is degraded under a
normoxia condition, the expression level of the protein was not
able to be measured.
Example 2. Effect of Vegfa- or Hif1a-Specific AAV-CjCas9 Using
CNV-Induced Mouse
[1328] To induce CNV, an eyeball was injected with AAV, and after 6
weeks, subjected to laser treatment, followed by detecting the CNV
sites one week after the laser treatment (FIG. 6). When AAV-CjCas9:
Vegfa and AAV-CjCas9: Hif1a were injected onto respective eyeballs,
it was confirmed that, compared with an AAV-free negative control,
CNV sites were decreased 24.+-.4% and 20.+-.4%, respectively (FIGS.
6 and 7). In the case of another negative control, that is,
Rosa26-specific CjCas9, no therapeutic effect was confirmed,
either.
Example 3. Effect of AAV-CjCas9 for AMD Treatment
[1329] Since cone dysfunction is caused by conditional knockout of
a Vegfa gene in the mouse RPE cells, the cause of side effects by
the AAV-induced gene knockout in the RPE cells was investigated. To
this end, the size of a cone function-related opsin-positive site
in the retina was measured. As a result, the Vegfa-specific CjCas9
was decreased in size by approximately 30.+-.10% the AAV-free
control (FIGS. 9 and 10). However, as expected, the Hif1a- or
Rosa26-specific CjCas9 did not induce cone dysfunction. Such a
result may suggest that Hif1a inactivation for treatment of AMD
inhibits neovascularization and does not cause cone
dysfunction.
[1330] HIF1A is a hypoxia-inducible transcription factor, and
serves to activate VEGFA transcription. Unlike VEGFA, which is a
primary therapeutic target for AMD treatment and a secretory
protein, HIF1A may not be considered as a drug target, and
generally, a transcription factor such as HIF1A may not be directly
targeted by an antibody or aptamer, or small molecules. In this
research, as the Hif1a gene was effectively inactivated in the RPE
cells using CjCas9 targeting the Hif1a gene on the eyeball of a
mouse, it was confirmed that the CNV sites were reduced in the AMD
mouse models (FIGS. 9 and 10).
Example 4. Effect of AAV-CjCas9 for Treatment of Retinal
Disease
[1331] For extended application to retinal diseases such as
diabetic retinopathy (DR), retinopathy of prematurity, etc., the
constructed virus was injected into the eyeball through injection
into the vitreous body, and 6 weeks later, the in vivo genome
editing effect caused by the indel frequency in retinal tissue was
observed by target deep sequencing, followed by confirming an
expression level of the Vegfa protein through ELISA (FIG. 2). In
the retinal tissue, the indels induced by CjCas9 were observed at
the Rosa26, Vegfa, and Hif1a target sites in the retinal cells,
indel frequencies of 44.+-.8%, 20.+-.2%, and 58.+-.5% were
confirmed, respectively (FIG. 11). As expected, an expression level
of the Vegfa protein was decreased in retinal cells when treated
with AAV encoding Vegfa- or Hif1a-specific CjCas9 (AAV-CjCas9:
Vegfa or Hif1a), but did not when AAV encoding Rosa26-specific
CjCas9 (AAV-CjCas9: Rosa26) was treated (FIG. 12).
Example 5. Effect of AAV-CjCas9 for Treatment of Diabetic
Retinopathy
[1332] Diabetic retinopathy is characterized by the symptoms of
vascular leakage and blood leakage. Therefore, in this example, the
vascular leakage or blood leakage symptom was confirmed using
diabetes-induced mice using STZ, and an improvement or treatment
effect caused by CjCas9 was confirmed. As a result, it was
confirmed that the blood leakage caused by vascular leakage and
rupturing was decreased in the Vegfa-specific CjCas9 (AAV-CjCas9:
Vegfa)-injected retina. Compared with Rosa26-specific CjCas9
(AAV-CjCas9: Rosa26)-injected mice, vascular leakage and blood
leakage in Vegfa-specific CjCas9 (AAV-CjCas9: Vegfa)-injected mice
were decreased and thus recovered to a level similar as in normal
mice of the same age. Such a result was similarly shown in both of
an experiment in which STZ was injected, AAV-CjCas9 was injected
after 7 weeks and observation was performed after 6 weeks (FIG.
13), and an experiment in which STZ was injected, AAV-CjCas9 was
injected after 14 weeks and observation was performed after 7 weeks
(FIGS. 14 and 15). According to the above results, it was confirmed
that Vegfa-specific CjCas9 (AAV-CjCas9: Vegfa) has an effect of
reducing vascular leakage and blood leakage, and thus it can be
expected that the Vegfa-specific CjCas9 (AAV-CjCas9: Vegfa) can
effectively treat diabetic retinopathy.
Example 6. Screening of Target Site of Human
Neovascularization-Associated Factor
[1333] To extend the application of the previously-described
example, in addition to human VEGFA (FIG. 16) and human HIF1A (FIG.
17), human ANGPT2 (FIG. 19), human EPAS1 (FIG. 20) and human
ANGPTL4 (FIG. 21) were selected as potential targets of genome
editing for AMD and DR treatments to screen the target site of each
gene capable of being effectively edited in human cells using a
CjCas9 system. Particularly, the CjCas9 target site of the mouse
Hif1a gene was completely conserved in a human or a different
mammal (FIG. 18). Additionally, the high editing efficiency of the
conserved target site was observed in human cells (FIG. 17, sgRNA
#7). Therefore, it is expected that AAV for Hif1a suggested in this
research or a mutant thereof is able to be used in treatment of a
future human patient.
INDUSTRIAL APPLICABILITY
[1334] An artificially manipulated neovascularization-associated
factor and a neovascularization system artificially modified in
function thereby can be effectively used in treatment of an
angiogenic disease, for example, an angiogenesis-associated ocular
disease.
[1335] Efficiency of the neovascularization system can be improved
by regulating characteristics such as survival, proliferation,
persistency, cytotoxicity, and cytokine-release of various
neovascularization-associated factors.
Original Claims
[1336] 1. An artificially manipulated neovascularization-associated
factor, which is selected from the group consisting of a VEGFA
gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4
gene, which has a modification in a nucleic acid sequence. 2. The
artificially manipulated neovascularization-associated factor of
paragraph 1, wherein the modification in the nucleic acid sequence
is artificially caused by a guide nucleic acid-editor protein
complex. 3. The artificially manipulated
neovascularization-associated factor of paragraph 1, wherein the
neovascularization-associated factor is one or more selected from
the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2
gene, an EPAS1 gene and an ANGPTL4 gene. 4. The artificially
manipulated neovascularization-associated factor of paragraph 1,
wherein the gene is a neovascularization-associated factor
artificially manipulated by a guide nucleic acid-editor protein
complex, wherein the neovascularization-associated factor
artificially manipulated includes one or more modifications of
nucleic acids which is at least one of a deletion or insertion of
one or more nucleotides, a substitution with one or more
nucleotides different from a wild-type gene, and an insertion of
one or more foreign nucleotide, in a proto-spacer-adjacent motif
(PAM) sequence in a nucleic acid sequence constituting the
neovascularization-associated factor or in a continuous 1 bp to 50
bp the base sequence region adjacent to the 5' end and/or 3' end
thereof, or a chemical modification of one or more nucleotides in a
nucleic acid sequence constituting the
neovascularization-associated factor. 5. The artificially
manipulated neovascularization-associated factor of paragraph 1,
wherein the modification of nucleic acids occurs in a promoter
region of the gene. 6. The artificially manipulated
neovascularization-associated factor of paragraph 1, wherein the
modification of nucleic acids occurs in an exon region of the gene.
7. The artificially manipulated neovascularization-associated
factor of paragraph 1, wherein the modification of nucleic acids
occurs in an intron region of the gene. 8. The artificially
manipulated neovascularization-associated factor of paragraph 1,
wherein the modification of nucleic acids occurs in an enhancer
region of the gene. 9. The artificially manipulated
neovascularization-associated factor of paragraph 4, wherein the
PAM sequence is, one or more of the following sequences (described
in the 5' to 3' direction): NGG (N is A, T, C or G); NNNNRYAC (each
N is independently A, T, C or G, R is A or G, and Y is C or T);
NNAGAAW (each N is independently A, T, C or G, and W is A or T);
NNNNGATT (each N is independently A, T, C or G); NNGRR(T) (each N
is independently A, T, C or G, R is A or G); and TTN (N is A, T, C
or G). 10. The artificially manipulated
neovascularization-associated factor of paragraph 2, wherein the
editor protein includes one or more selected from the group
consisting of a Streptococcus pyogenes-derived Cas9 protein, a
Campylobacter jejuni-derived Cas9 protein, a Streptococcus
thermophilus-derived Cas9 protein, a Streptococcus aureus-derived
Cas9 protein, a Neisseria meningitidis-derived Cas9 protein, and a
Cpf1 protein. 11. A guide nucleic acid, which is capable of forming
complementary bonds with respect to the target sequences of SEQ ID
NOs: 1 to 79 in the nucleic acid sequences of one or more genes
selected from the group consisting of a VEGFA gene, an HIF1A gene,
an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene, respectively.
12. The guide nucleic acid of paragraph 11, which includes one or
more guide nucleic acids selected from the group consisting of:
guide nucleic acids capable of forming complementary bonds with
respect to the target sequences of SEQ ID NOs: 3, 4, 7, 9, 10 and
11 in the nucleic acid sequence of the VEGFA gene, respectively;
guide nucleic acids capable of forming complementary bonds with
respect to the target sequences of SEQ ID NOs: 14, 18, 19, 20, 26,
29 and 31 in the nucleic acid sequence of the HIF1A gene,
respectively; guide nucleic acids capable of forming complementary
bonds with respect to the target sequences of SEQ ID NOs: 33, 34,
37, 38, 39 and 43 in the nucleic acid sequence of the ANGPT2 gene,
respectively; guide nucleic acids capable of forming complementary
bonds with respect to the target sequences of SEQ ID NOs: 47, 48,
49, 50, 53, 54 and 55 in the nucleic acid sequence of the EPAS1
gene, respectively; and guide nucleic acids capable of forming
complementary bonds with respect to the target sequences of SEQ ID
NOs: 64, 66, 67, 73, 76 and 79 in the nucleic acid sequence of the
ANGPTL4 gene, respectively. 13. The guide nucleic acid of paragraph
11, wherein the guide nucleic acid is nucleotide molecule of 18 to
23 bp. 14. A composition for gene manipulation, comprising: a guide
nucleic acid capable of forming a complementary bond with respect
to the target sequences of SEQ ID NOs: 1 to 79 in nucleic acid
sequences of one or more genes selected from the group consisting
of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and
an ANGPTL4 gene, respectively, or a nucleic acid sequence encoding
the same; and an editor protein or a nucleic acid sequence encoding
the same. 15. The composition for gene manipulation of paragraph
14, wherein the editor protein includes one or more proteins
selected from the group consisting of a Streptococcus
pyogenes-derived Cas9 protein, a Campylobacter jejuni-derived Cas9
protein, a Streptococcus thermophilus-derived Cas9 protein, a
Streptococcus aureus-derived Cas9 protein, a Neisseria
meningitidis-derived Cas9 protein, and a Cpf1 protein. 16. The
composition for gene manipulation of paragraph 14, wherein the gene
manipulation includes one or more modifications of nucleic acids
which is at least one of a deletion or insertion of one or more
nucleotides, a substitution with one or more nucleotides different
from a wild-type gene, and an insertion of one or more foreign
nucleotide, in a proto-spacer-adjacent motif (PAM) sequence in a
nucleic acid sequence constituting the
neovascularization-associated factor or in a continuous 1 bp to 50
bp the base sequence region adjacent to the 5' end and/or 3' end
thereof, or a chemical modification of one or more nucleotides in a
nucleic acid sequence constituting the
neovascularization-associated factor. 17. The composition for gene
manipulation of paragraph 16, wherein the PAM sequence includes one
or more of the following sequences (described in the 5' to 3'
direction): NGG (N is A, T, C or G); NNNNRYAC (each N is
independently A, T, C or G, R is A or G, and Y is C or T); NNAGAAW
(each N is independently A, T, C or G, and W is A or T); NNNNGATT
(each N is independently A, T, C or G); NNGRR(T) (each N is
independently A, T, C or G, R is A or G); and TTN (N is A, T, C or
G). 18. The composition for gene manipulation of paragraph 14,
wherein the composition for gene manipulation is formed in a viral
vector system. 19. The composition for gene manipulation of
paragraph 18, wherein the viral vector includes one or more
selected from a retrovirus, a lentivirus, an adenovirus,
adeno-associated virus (AAV), vaccinia virus, a poxvirus and a
herpes simplex virus. 20. A method for providing information on a
sequence of an artificially manipulatable target site in a subject
by analyzing sequences of one or more genes selected from the group
consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1
gene and an ANGPTL4 gene. 21. A method for constructing a library
using the information provided by the method of claim 20. 22. A kit
for gene manipulation, comprising: (a) a guide nucleic acid capable
of forming complementary bonds with respect to each of the target
sequences of SEQ ID NOs: 1 to 79, in the nucleic acid sequences of
one or more genes selected from the group consisting of a VEGFA
gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4
gene, respectively, or a nucleic acid sequence encoding the same;
and (b) an editor protein including one or more proteins selected
from the group consisting of a Streptococcus pyogenes-derived Cas9
protein, a Campylobacter jejuni-derived Cas9 protein, a
Streptococcus thermophilus-derived Cas9 protein, a Streptococcus
aureus-derived Cas9 protein, a Neisseria meningitidis-derived Cas9
protein, and a Cpf1 protein, respectively, or a nucleic acid
sequence encoding the same. 23. A composition for treating an
angiovascular disorder, comprising: a guide nucleic acid capable of
forming complementary bonds with respect to each of one or more
target sequences in the nucleic acid sequences of one or more genes
selected from the group consisting of a VEGFA gene, an HIF1A gene,
an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene, respectively, or
a nucleic acid sequence encoding the same; and an editor protein or
a nucleic acid sequence encoding the same. 24. The composition for
treating of paragraph 23, wherein the target sequence includes one
or more of target sequences of SEQ ID NOs: 1 to 79. 25. The
composition for treating of paragraph 23, wherein the editor
protein is a Campylobacter jejuni-derived Cas9 protein. 26. The
composition for treating of paragraph 23, wherein the angiovascular
disorder is ischemic retinopathy or retinopathy of prematurity.
SEQUENCE LISTING
[1337] This application contains references to amino acid sequences
and/or nucleic acid sequences which have been submitted herewith as
the sequence listing text file. The aforementioned sequence listing
is hereby incorporated by reference in its entirety pursuant to 37
C.F.R. .sctn. 1.52(e).
Sequence CWU 1
1
1522122DNAhomo sapiens 1gtagagcagc aaggcaaggc tc 22222DNAhomo
sapiens 2ctttctgtcc tcagtggtcc ca 22322DNAhomo sapiens 3gagaccctgg
tggacatctt cc 22422DNAhomo sapiens 4ttccaggagt accctgatga ga
22522DNAhomo sapiens 5ttgaagatgt actcgatctc at 22622DNAhomo sapiens
6aggggcacac aggatggctt ga 22722DNAhomo sapiens 7agcagccccc
gcatcgcatc ag 22822DNAhomo sapiens 8gcagcagccc ccgcatcgca tc
22922DNAhomo sapiens 9gtgatgttgg actcctcagt gg 221022DNAhomo
sapiens 10tggtgatgtt ggactcctca gt 221122DNAhomo sapiens
11catggtgatg ttggactcct ca 221222DNAhomo sapiens 12atgcggatca
aacctcacca ag 221322DNAhomo sapiens 13cacataggag agatgagctt cc
221422DNAhomo sapiens 14actcaccagc atccagaagt tt 221522DNAhomo
sapiens 15atttggatat tgaagatgac at 221622DNAhomo sapiens
16atttacattt ctgataatgt ga 221722DNAhomo sapiens 17atgtgtttac
agtttgaact aa 221822DNAhomo sapiens 18ctgtgtccag ttagttcaaa ct
221922DNAhomo sapiens 19atggtcacat ggatgagtaa aa 222022DNAhomo
sapiens 20catgaggaaa tgagagaaat gc 222122DNAhomo sapiens
21cccagtgaga aaagggaaag aa 222222DNAhomo sapiens 22ttgtgaaaaa
gggtaaagaa ca 222322DNAhomo sapiens 23atagttcttc ctcggctagt ta
222422DNAhomo sapiens 24tcatagttct tcctcggcta gt 222522DNAhomo
sapiens 25tgttcttcat acacaggtat tg 222622DNAhomo sapiens
26tacgtgaatg tggcctgtgc ag 222722DNAhomo sapiens 27ctgcacaggc
cacattcacg ta 222822DNAhomo sapiens 28ctgaggttgg ttactgttgg ta
222922DNAhomo sapiens 29caggtcatag gtggtttctt at 223022DNAhomo
sapiens 30accaagcagg tcataggtgg tt 223122DNAhomo sapiens
31ttagatagca agactttcct ca 223222DNAhomo sapiens 32tcaggtccag
catgggtcct gc 223322DNAhomo sapiens 33cggcgcgtcc ctctgcacag ca
223422DNAhomo sapiens 34gctgtgcaga gggacgcgcc gc 223522DNAhomo
sapiens 35atcgtattcg agcggcgcgt cc 223622DNAhomo sapiens
36gatgttctcc agcacttgca gc 223722DNAhomo sapiens 37agtgctggag
aacatcatgg aa 223822DNAhomo sapiens 38acaacatgaa gaaagaaatg gt
223922DNAhomo sapiens 39aaatggtaga gatacagcag aa 224022DNAhomo
sapiens 40ttctatcatc acagccgtct gg 224122DNAhomo sapiens
41aagttcaagt ctcgtggtct ga 224222DNAhomo sapiens 42acgagacttg
aacttcagct ct 224322DNAhomo sapiens 43aagaaggtgc tagctatgga ag
224422DNAhomo sapiens 44gatgatgtgc ttgtcttcca ta 224522DNAhomo
sapiens 45aacacctccg tctccttgct cc 224622DNAhomo sapiens
46gaagctgacc agcagatgga ca 224722DNAhomo sapiens 47gcaatgaaac
cctccaaggc tt 224822DNAhomo sapiens 48aaaacatcag caagttcatg gg
224922DNAhomo sapiens 49gcaagttcat gggacttaca ca 225022DNAhomo
sapiens 50ggtcgcaggg atgagtgaag tc 225122DNAhomo sapiens
51gcgggacttc ttcatgagga tg 225222DNAhomo sapiens 52gaagtgcacg
gtcaccaaca ga 225322DNAhomo sapiens 53acagtacggc ctctgttggt ga
225422DNAhomo sapiens 54tccaggtggc tgacttgagg tt 225522DNAhomo
sapiens 55caggacagca ggggctcctt gt 225622DNAhomo sapiens
56tagcccccat gctttgcgag ca 225722DNAhomo sapiens 57gcatcagggc
tgccccggcc gt 225822DNAhomo sapiens 58gcatcagggc tgccccggcc gt
225922DNAhomo sapiens 59ggacgcaaag cgcggcgact tg 226022DNAhomo
sapiens 60tcctgggacg agatgaatgt cc 226122DNAhomo sapiens
61ctgcagctcg gccaggggct gc 226222DNAhomo sapiens 62ccaggggctg
cgcgaacacg cg 226322DNAhomo sapiens 63ccctcggttc cctgacaggc gg
226422DNAhomo sapiens 64accctgaggt ccttcacagc ct 226522DNAhomo
sapiens 65ttccacaagg tggcccagca gc 226622DNAhomo sapiens
66cagcagcagc ggcacctgga ga 226722DNAhomo sapiens 67tcctagtttg
gcctcctgga cc 226822DNAhomo sapiens 68gacccggctc acaatgtcag cc
226922DNAhomo sapiens 69gctgttgcgg tcccccgtga tg 227022DNAhomo
sapiens 70ggcgttgcca tcccagtccc gc 227122DNAhomo sapiens
71aacgccgagt tgctgcagtt ct 227222DNAhomo sapiens 72ataggccgtg
tcctcgccac cc 227322DNAhomo sapiens 73gttctccgtg cacctgggtg gc
227422DNAhomo sapiens 74acacggccta tagcctgcag ct 227522DNAhomo
sapiens 75ccaccgtccc acccagcggc ct 227622DNAhomo sapiens
76gtgatcctgg tcccaagtgg ag 227722DNAhomo sapiens 77gaccccggca
ggaggctggt gg 227822DNAhomo sapiens 78tgcagccatt ccaacctcaa cg
227922DNAhomo sapiens 79tgccgctgct gtgggatgga gc 228022DNAhomo
sapiens 80aaatcccggt ataagtcctg ga 228122DNAhomo sapiens
81gcaccaacgt acacgctcca gg 228222DNAhomo sapiens 82cattagacag
cagcgggcac ca 228322DNAhomo sapiens 83ggcattagac agcagcgggc ac
228422DNAhomo sapiens 84ggctccaggg cattagacag ca 228522DNAhomo
sapiens 85gctcagagcg gagaaagcat tt 228622DNAhomo sapiens
86ggaacattta cacgtctgcg ga 228722DNAhomo sapiens 87gcagacgtgt
aaatgttcct gc 228822DNAhomo sapiens 88gagtctgtgt ttttgcagga ac
228922DNAhomo sapiens 89gcaccaacgt acacgctcca gg 229022DNAhomo
sapiens 90actaaaggac aagtcaccac ag 229122DNAhomo sapiens
91tatccacctc ttttggcaag ca 229222DNAhomo sapiens 92tgaaactcaa
gcaactgtca ta 229322DNAhomo sapiens 93ctcacaacgt aattcacaca ta
229422DNAhomo sapiens 94tacttacctc acaacgtaat tc 229522DNAhomo
sapiens 95aacttactta cctcacaacg ta 229622DNAhomo sapiens
96tcttgttttg acagtggtat ta 229722DNAhomo sapiens 97gggagaaaat
caagtcgtgc tg 229822DNAhomo sapiens 98tatctgaaga ttcaaccggt tt
229922DNAhomo sapiens 99gctattcacc aaagttgaat ca 2210022DNAhomo
sapiens 100aactttgctg gccccagccg ct 2210122DNAhomo sapiens
101aaactgatga ccagcaactt ga 2210222DNAhomo sapiens 102ggggagcatt
acatcattat at 2210322DNAhomo sapiens 103agccacttcg aagtagtgct ga
2210422DNAhomo sapiens 104caacttcttg attgagtgca gg 2210522DNAhomo
sapiens 105ttaccatgcc ccagattcag ga 2210622DNAhomo sapiens
106tcagacacct agtccttccg at 2210722DNAhomo sapiens 107attggtagaa
aaactttttg ct 2210822DNAhomo sapiens 108aactcatgta tttgctgttt ta
2210922DNAhomo sapiens 109aagccctgaa agcgcaagtc ct 2211022DNAhomo
sapiens 110cagttacagt attccagcag ac 2211122DNAhomo sapiens
111aggttcttgt atttgagtct gc 2211222DNAhomo sapiens 112atgcaatcaa
tattttaatg tc 2211322DNAhomo sapiens 113tgattgcatc tccatctcct ac
2211422DNAhomo sapiens 114tagtgccaca tcatcaccat at 2211522DNAhomo
sapiens 115gagtatctct atatggtgat ga 2211622DNAhomo sapiens
116atacctttga ctcaaagcga ca 2211722DNAhomo sapiens 117ttcctgagga
agaactaaat cc 2211822DNAhomo sapiens 118tctgttcact agatttgcat cc
2211922DNAhomo sapiens 119gaatggagca aaagacaatt at 2212022DNAhomo
sapiens 120gttatgattg tgaagttaat gc 2212122DNAhomo sapiens
121aacacctccg tctccttgct cc 2212222DNAhomo sapiens 122tggaggcctt
gtccagatgg ga 2212322DNAhomo sapiens 123tgcgactggc aatcagcttc ct
2212422DNAhomo sapiens 124cgactggcaa tcagcttcct gc 2212522DNAhomo
sapiens 125gaagctgacc agcagatgga ca 2212622DNAhomo sapiens
126gcaatgaaac cctccaaggc tt 2212722DNAhomo sapiens 127aaaacatcag
caagttcatg gg 2212822DNAhomo sapiens 128gcaagttcat gggacttaca ca
2212922DNAhomo sapiens 129ggtcgcaggg atgagtgaag tc 2213022DNAhomo
sapiens 130gcgggacttc ttcatgagga tg 2213122DNAhomo sapiens
131gaagtgcacg gtcaccaaca ga 2213222DNAhomo sapiens 132acagtacggc
ctctgttggt ga 2213322DNAhomo sapiens 133tccaggtggc tgacttgagg tt
2213422DNAhomo sapiens 134tccacgcctg tctcaggtct tg 2213522DNAhomo
sapiens 135caggacagca ggggctcctt gt 2213622DNAhomo sapiens
136ctcatcatca tgtgtgaacc aa 2213722DNAhomo sapiens 137atgtgggatg
ggtgctggat tg 2213822DNAhomo sapiens 138gccacttact acctgaccct tg
2213922DNAhomo sapiens 139tggccactta ctacctgacc ct 2214022DNAhomo
sapiens 140accaagggtc aggtagtaag tg 2214122DNAhomo sapiens
141tagcccccat gctttgcgag ca 2214222DNAhomo sapiens 142gatgaccgtc
ccctgggtct cc 2214322DNAhomo sapiens 143ctcaggacgt agttgacaca ca
2214422DNAhomo sapiens 144catgcttacc tcaggacgta gt 2214522DNAhomo
sapiens 145cacatgctta cctcaggacg ta 2214622DNAhomo sapiens
146cagggattca gtctggtcca tg 2214722DNAhomo sapiens 147ggtgaatagg
aagttactct tc 2214822DNAhomo sapiens 148atgggccacg gagttgagga gc
2214922DNAhomo sapiens 149ctagcccaat agccctgaag ac 2215022DNAhomo
sapiens 150agtgattgag aagctcttcg cc 2215122DNAhomo sapiens
151ggacacagag gccaaggacc aa 2215222DNAhomo sapiens 152cctgatctcc
acagccatct ac 2215322DNAhomo sapiens 153cggatttcaa tgagctggac tt
2215422DNAhomo sapiens 154tcaatgagct ggacttggag ac 2215522DNAhomo
sapiens 155gcggagaacc cacagtccac cc 2215622DNAhomo sapiens
156ccagtggctg gaagatgttt gt 2215722DNAhomo sapiens 157ttccagccac
tggcccctgt ag 2215822DNAhomo sapiens 158ctggagagca agaagacaga gc
2215922DNAhomo sapiens 159gagagagggg tgctggcctg gc 2216022DNAhomo
sapiens 160cctgccaccg tgctgtggcc ag 2216122DNAhomo sapiens
161tctctcttcc atggggggca ga 2216222DNAhomo sapiens 162cacaaagtgg
gccgtcgggg at 2216322DNAhomo sapiens 163ggagagggct accatggccg ga
2216422DNAhomo sapiens 164ctcaggtcct ggaaggcttg ct 2216522DNAhomo
sapiens 165ctcccagggg gacccacctg gt 2216622DNAhomo sapiens
166tgccggacaa gccactgagc gc 2216722DNAhomo sapiens 167ttccccccac
agtgctacgc ca 2216822DNAhomo sapiens 168ctgtagtcct ggtactgggt gg
2216922DNAhomo sapiens 169tgggctgacg acaggctgta gt 2217022DNAhomo
sapiens 170tccttgcagg agcgtggagc tt 2217122DNAhomo sapiens
171gactgtgctg aagtattcaa at 2217222DNAhomo sapiens 172ctgtgctgaa
gtattcaaat ca 2217322DNAhomo sapiens 173tggtgtgtcc tgatttgaat ac
2217422DNAhomo sapiens 174gccattcgtg gtgtgtcctg at 2217522DNAhomo
sapiens 175atcaggacac accacgaatg gc 2217622DNAhomo sapiens
176ctaatcagca acgctatgtg ct 2217722DNAhomo sapiens 177aatcagcaac
gctatgtgct ta 2217822DNAhomo sapiens 178ttcccagtct ttaaggtgta tt
2217922DNAhomo sapiens 179tcttcacttg agagatagaa at 2218022DNAhomo
sapiens 180catcagccaa ccaggaaatg at 2218122DNAhomo sapiens
181ctgttagcat ttgtgaacat tt 2218222DNAhomo sapiens 182tgtggtcctt
ccaacttgaa cg 2218322DNAhomo sapiens 183cggaatgtac tatccacaga gg
2218422DNAhomo sapiens 184ttatttgtgt tctgcctctg tg 2218522DNAhomo
sapiens 185acaaataagt tcaacggcat ta 2218622DNAhomo sapiens
186agcgaatagc ctgagccttt cc 2218720DNAhomo sapiens 187cagctgacca
gtcgcgctga 2018820DNAhomo sapiens 188gtggtagctg gggctggggg
2018920DNAhomo sapiens 189gaggtggtag ctggggctgg
2019020DNAhomo sapiens 190ggaggtggta gctggggctg 2019120DNAhomo
sapiens 191aggaggtggt agctggggct 2019220DNAhomo sapiens
192gaggaggtgg tagctggggc 2019320DNAhomo sapiens 193cggggaggag
gtggtagctg 2019420DNAhomo sapiens 194cccagctacc acctcctccc
2019520DNAhomo sapiens 195ccggggagga ggtggtagct 2019620DNAhomo
sapiens 196gccggggagg aggtggtagc 2019720DNAhomo sapiens
197gctaccacct cctccccggc 2019820DNAhomo sapiens 198accacctcct
ccccggccgg 2019920DNAhomo sapiens 199gccgccggcc ggggaggagg
2020020DNAhomo sapiens 200acctcctccc cggccggcgg 2020120DNAhomo
sapiens 201tccgccgccg gccggggagg 2020220DNAhomo sapiens
202ctgtccgccg ccggccgggg 2020320DNAhomo sapiens 203ccccggccgg
cggcggacag 2020420DNAhomo sapiens 204ccactgtccg ccgccggccg
2020520DNAhomo sapiens 205tccactgtcc gccgccggcc 2020620DNAhomo
sapiens 206gtccactgtc cgccgccggc 2020720DNAhomo sapiens
207ccggcggcgg acagtggacg 2020820DNAhomo sapiens 208ccgcgtccac
tgtccgccgc 2020920DNAhomo sapiens 209gcggcggaca gtggacgcgg
2021020DNAhomo sapiens 210gtggacgcgg cggcgagccg 2021120DNAhomo
sapiens 211tggacgcggc ggcgagccgc 2021220DNAhomo sapiens
212cgcggcggcg agccgcgggc 2021320DNAhomo sapiens 213gcggcggcga
gccgcgggca 2021420DNAhomo sapiens 214cggcggcgag ccgcgggcag
2021520DNAhomo sapiens 215ggcgagccgc gggcaggggc 2021620DNAhomo
sapiens 216cgggctccgg cccctgcccg 2021720DNAhomo sapiens
217caggggccgg agcccgcgcc 2021820DNAhomo sapiens 218gggccggagc
ccgcgcccgg 2021920DNAhomo sapiens 219ccggagcccg cgcccggagg
2022020DNAhomo sapiens 220ccgcctccgg gcgcgggctc 2022120DNAhomo
sapiens 221cggagcccgc gcccggaggc 2022220DNAhomo sapiens
222ggagcccgcg cccggaggcg 2022320DNAhomo sapiens 223gcccgcgccc
ggaggcgggg 2022420DNAhomo sapiens 224tccaccccgc ctccgggcgc
2022520DNAhomo sapiens 225ctccaccccg cctccgggcg 2022620DNAhomo
sapiens 226cgcgcccgga ggcggggtgg 2022720DNAhomo sapiens
227gcgcccggag gcggggtgga 2022820DNAhomo sapiens 228cgcccggagg
cggggtggag 2022920DNAhomo sapiens 229gcccggaggc ggggtggagg
2023020DNAhomo sapiens 230accccctcca ccccgcctcc 2023120DNAhomo
sapiens 231gaccccctcc accccgcctc 2023220DNAhomo sapiens
232ggaggcgggg tggagggggt 2023320DNAhomo sapiens 233gaggcggggt
ggagggggtc 2023420DNAhomo sapiens 234aggcggggtg gagggggtcg
2023520DNAhomo sapiens 235gtggaggggg tcggggctcg 2023620DNAhomo
sapiens 236gaaacttttc gtccaacttc 2023720DNAhomo sapiens
237aaacttttcg tccaacttct 2023820DNAhomo sapiens 238agcgagaaca
gcccagaagt 2023920DNAhomo sapiens 239cttctgggct gttctcgctt
2024020DNAhomo sapiens 240ctgggctgtt ctcgcttcgg 2024120DNAhomo
sapiens 241ttctcgcttc ggaggagccg 2024220DNAhomo sapiens
242cggaggagcc gtggtccgcg 2024320DNAhomo sapiens 243ggaggagccg
tggtccgcgc 2024420DNAhomo sapiens 244gaggagccgt ggtccgcgcg
2024520DNAhomo sapiens 245aggagccgtg gtccgcgcgg 2024620DNAhomo
sapiens 246ggcttccccc gcgcggacca 2024720DNAhomo sapiens
247tcggctcggc ttcccccgcg 2024820DNAhomo sapiens 248gcgggggaag
ccgagccgag 2024920DNAhomo sapiens 249tctcgcggct ccgctcggct
2025020DNAhomo sapiens 250gcacttctcg cggctccgct 2025120DNAhomo
sapiens 251agccgcgaga agtgctagct 2025220DNAhomo sapiens
252gccgcgagaa gtgctagctc 2025320DNAhomo sapiens 253gcccgagcta
gcacttctcg 2025420DNAhomo sapiens 254cgagaagtgc tagctcgggc
2025520DNAhomo sapiens 255gagaagtgct agctcgggcc 2025620DNAhomo
sapiens 256aagtgctagc tcgggccggg 2025720DNAhomo sapiens
257gggccgggag gagccgcagc 2025820DNAhomo sapiens 258ccgggaggag
ccgcagccgg 2025920DNAhomo sapiens 259cctccggctg cggctcctcc
2026020DNAhomo sapiens 260ggaggagccg cagccggagg 2026120DNAhomo
sapiens 261gaggagccgc agccggagga 2026220DNAhomo sapiens
262aggagccgca gccggaggag 2026320DNAhomo sapiens 263ggagccgcag
ccggaggagg 2026420DNAhomo sapiens 264gccgcagccg gaggaggggg
2026520DNAhomo sapiens 265tcctccccct cctccggctg 2026620DNAhomo
sapiens 266gcagccggag gagggggagg 2026720DNAhomo sapiens
267tcttcctcct ccccctcctc 2026820DNAhomo sapiens 268gaagagaagg
aagaggagag 2026920DNAhomo sapiens 269aagagaagga agaggagagg
2027020DNAhomo sapiens 270aagaggagag ggggccgcag 2027120DNAhomo
sapiens 271agggggccgc agtggcgact 2027220DNAhomo sapiens
272cgagcgccga gtcgccactg 2027320DNAhomo sapiens 273cgcagtggcg
actcggcgct 2027420DNAhomo sapiens 274gcgactcggc gctcggaagc
2027520DNAhomo sapiens 275cgactcggcg ctcggaagcc 2027620DNAhomo
sapiens 276gcgctcggaa gccgggctca 2027720DNAhomo sapiens
277tcggaagccg ggctcatgga 2027820DNAhomo sapiens 278cggaagccgg
gctcatggac 2027920DNAhomo sapiens 279gccgggctca tggacgggtg
2028020DNAhomo sapiens 280gcctcacccg tccatgagcc 2028120DNAhomo
sapiens 281gggctcatgg acgggtgagg 2028220DNAhomo sapiens
282ctcatggacg ggtgaggcgg 2028320DNAhomo sapiens 283tccagccgcg
cgcgctcccc 2028420DNAhomo sapiens 284gcctggggag cgcgcgcggc
2028520DNAhomo sapiens 285cagggcctgg ggagcgcgcg 2028620DNAhomo
sapiens 286cgcgcgcgct ccccaggccc 2028720DNAhomo sapiens
287gcgctcccca ggccctggcc 2028820DNAhomo sapiens 288cgctccccag
gccctggccc 2028920DNAhomo sapiens 289gaggcccggg ccagggcctg
2029020DNAhomo sapiens 290cgaggcccgg gccagggcct 2029120DNAhomo
sapiens 291ccaggccctg gcccgggcct 2029220DNAhomo sapiens
292ccgaggcccg ggccagggcc 2029320DNAhomo sapiens 293caggccctgg
cccgggcctc 2029420DNAhomo sapiens 294ccctggcccg ggcctcgggc
2029520DNAhomo sapiens 295ccggcccgag gcccgggcca 2029620DNAhomo
sapiens 296cctggcccgg gcctcgggcc 2029720DNAhomo sapiens
297cccggcccga ggcccgggcc 2029820DNAhomo sapiens 298ctggcccggg
cctcgggccg 2029920DNAhomo sapiens 299gcccgggcct cgggccgggg
2030020DNAhomo sapiens 300tcctccccgg cccgaggccc 2030120DNAhomo
sapiens 301ttcctccccg gcccgaggcc 2030220DNAhomo sapiens
302tactcttcct ccccggcccg 2030320DNAhomo sapiens 303ggcgagctac
tcttcctccc 2030420DNAhomo sapiens 304ggaggaagag tagctcgccg
2030520DNAhomo sapiens 305agtagctcgc cgaggcgccg 2030620DNAhomo
sapiens 306cgccgaggcg ccgaggagag 2030720DNAhomo sapiens
307gccgaggcgc cgaggagagc 2030820DNAhomo sapiens 308gcccgctctc
ctcggcgcct 2030920DNAhomo sapiens 309gtggggcggc ccgctctcct
2031020DNAhomo sapiens 310ggccgcccca cagcccgagc 2031120DNAhomo
sapiens 311ctccggctcg ggctgtgggg 2031220DNAhomo sapiens
312ccccacagcc cgagccggag 2031320DNAhomo sapiens 313cctctccggc
tcgggctgtg 2031420DNAhomo sapiens 314cccacagccc gagccggaga
2031520DNAhomo sapiens 315ccctctccgg ctcgggctgt 2031620DNAhomo
sapiens 316tccctctccg gctcgggctg 2031720DNAhomo sapiens
317tcgcgctccc tctccggctc 2031820DNAhomo sapiens 318ctcgcgctcc
ctctccggct 2031920DNAhomo sapiens 319cgcggctcgc gctccctctc
2032020DNAhomo sapiens 320agagggagcg cgagccgcgc 2032120DNAhomo
sapiens 321cgagccgcgc cggccccggt 2032220DNAhomo sapiens
322gagccgcgcc ggccccggtc 2032320DNAhomo sapiens 323aggcccgacc
ggggccggcg 2032420DNAhomo sapiens 324ttcggaggcc cgaccggggc
2032520DNAhomo sapiens 325tggtttcgga ggcccgaccg 2032620DNAhomo
sapiens 326atggtttcgg aggcccgacc 2032720DNAhomo sapiens
327catggtttcg gaggcccgac 2032820DNAhomo sapiens 328cagaaagttc
atggtttcgg 2032920DNAhomo sapiens 329cagcagaaag ttcatggttt
2033020DNAhomo sapiens 330ccatgaactt tctgctgtct 2033120DNAhomo
sapiens 331ccaagacagc agaaagttca 2033220DNAhomo sapiens
332catgaacttt ctgctgtctt 2033320DNAhomo sapiens 333ttctgctgtc
ttgggtgcat 2033420DNAhomo sapiens 334ggaggtagag cagcaaggca
2033520DNAhomo sapiens 335atggtggagg tagagcagca 2033620DNAhomo
sapiens 336gctctacctc caccatgcca 2033720DNAhomo sapiens
337cgcttacctt ggcatggtgg 2033820DNAhomo sapiens 338gaccgcttac
cttggcatgg 2033920DNAhomo sapiens 339cacgaccgct taccttggca
2034020DNAhomo sapiens 340tttctgtcct cagtggtccc 2034120DNAhomo
sapiens 341ggtgcagcct gggaccactg 2034220DNAhomo sapiens
342gtggtcccag gctgcaccca 2034320DNAhomo sapiens 343ttctgccatg
ggtgcagcct 2034420DNAhomo sapiens 344cttctgccat gggtgcagcc
2034520DNAhomo sapiens 345caggctgcac ccatggcaga 2034620DNAhomo
sapiens 346gctgcaccca tggcagaagg 2034720DNAhomo sapiens
347gcacccatgg cagaaggagg 2034820DNAhomo sapiens 348cacccatggc
agaaggagga 2034920DNAhomo sapiens 349tgccctcctc cttctgccat
2035020DNAhomo sapiens 350ctgccctcct ccttctgcca 2035120DNAhomo
sapiens 351ggagggcaga atcatcacga 2035220DNAhomo sapiens
352tcatgcagtg gtgaagttca 2035320DNAhomo sapiens 353ctgccatcca
atcgagaccc 2035420DNAhomo sapiens 354ccatccaatc gagaccctgg
2035520DNAhomo sapiens 355ccaccagggt ctcgattgga 2035620DNAhomo
sapiens 356atgtccacca gggtctcgat 2035720DNAhomo sapiens
357gaccctggtg gacatcttcc 2035820DNAhomo sapiens 358ctcctggaag
atgtccacca 2035920DNAhomo sapiens 359actcctggaa gatgtccacc
2036020DNAhomo sapiens 360cgatctcatc agggtactcc 2036120DNAhomo
sapiens 361agatgtactc gatctcatca 2036220DNAhomo sapiens
362aagatgtact cgatctcatc 2036320DNAhomo sapiens 363cgcatcaggg
gcacacagga 2036420DNAhomo sapiens 364gcatcgcatc aggggcacac
2036520DNAhomo sapiens 365tgtgtgcccc tgatgcgatg 2036620DNAhomo
sapiens 366gtgtgcccct gatgcgatgc 2036720DNAhomo sapiens
367tgtgcccctg atgcgatgcg 2036820DNAhomo sapiens 368gtgcccctga
tgcgatgcgg 2036920DNAhomo sapiens 369cagcccccgc atcgcatcag
2037020DNAhomo sapiens 370gcagcccccg catcgcatca 2037120DNAhomo
sapiens 371agcagccccc gcatcgcatc 2037220DNAhomo sapiens
372cgggggctgc tgcaatgacg 2037320DNAhomo sapiens 373gggggctgct
gcaatgacga 2037420DNAhomo sapiens 374ctgctgcaat gacgagggcc
2037520DNAhomo sapiens 375cctggagtgt gtgcccactg 2037620DNAhomo
sapiens 376cctcagtggg cacacactcc 2037720DNAhomo sapiens
377gtgatgttgg actcctcagt
2037820DNAhomo sapiens 378ggtgatgttg gactcctcag 2037920DNAhomo
sapiens 379ggagtccaac atcaccatgc 2038020DNAhomo sapiens
380gtccaacatc accatgcagg 2038120DNAhomo sapiens 381tccaacatca
ccatgcaggt 2038220DNAhomo sapiens 382gcccacctgc atggtgatgt
2038320DNAhomo sapiens 383cccaaagatg cccacctgca 2038420DNAhomo
sapiens 384tccttccttt ccagattatg 2038520DNAhomo sapiens
385gaggtttgat ccgcataatc 2038620DNAhomo sapiens 386atgcggatca
aacctcacca 2038720DNAhomo sapiens 387cctcaccaag gccagcacat
2038820DNAhomo sapiens 388cctatgtgct ggccttggtg 2038920DNAhomo
sapiens 389tctctcctat gtgctggcct 2039020DNAhomo sapiens
390agctcatctc tcctatgtgc 2039120DNAhomo sapiens 391attcacattt
gttgtgctgt 2039220DNAhomo sapiens 392agcacaacaa atgtgaatgc
2039320DNAhomo sapiens 393aacaaatgtg aatgcaggtg 2039420DNAhomo
sapiens 394tgtcttgctc tatctttctt 2039520DNAhomo sapiens
395ttttccagaa aatcagttcg 2039620DNAhomo sapiens 396ctttcctcga
actgattttc 2039720DNAhomo sapiens 397cagaaaatca gttcgaggaa
2039820DNAhomo sapiens 398agaaaatcag ttcgaggaaa 2039920DNAhomo
sapiens 399atcagttcga ggaaagggaa 2040020DNAhomo sapiens
400tcagttcgag gaaagggaaa 2040120DNAhomo sapiens 401cagttcgagg
aaagggaaag 2040220DNAhomo sapiens 402aacgaaagcg caagaaatcc
2040320DNAhomo sapiens 403agaaatcccg gtataagtcc 2040420DNAhomo
sapiens 404cacgctccag gacttatacc 2040520DNAhomo sapiens
405acacgctcca ggacttatac 2040620DNAhomo sapiens 406aagtcctgga
gcgtgtacgt 2040720DNAhomo sapiens 407ggcaccaacg tacacgctcc
2040820DNAhomo sapiens 408cccgctgctg tctaatgccc 2040920DNAhomo
sapiens 409ccagggcatt agacagcagc 2041020DNAhomo sapiens
410tccagggcat tagacagcag 2041120DNAhomo sapiens 411ctaatgccct
ggagcctccc 2041220DNAhomo sapiens 412tgggggccag ggaggctcca
2041320DNAhomo sapiens 413ctgggggcca gggaggctcc 2041420DNAhomo
sapiens 414ggttgtactg ggggccaggg 2041520DNAhomo sapiens
415ggaggttgta ctgggggcca 2041620DNAhomo sapiens 416cggaggttgt
actgggggcc 2041720DNAhomo sapiens 417gcaggcggag gttgtactgg
2041820DNAhomo sapiens 418ttgccttttt gcagtccctg 2041920DNAhomo
sapiens 419tgcctttttg cagtccctgt 2042020DNAhomo sapiens
420cgctctgagc aaggcccaca 2042120DNAhomo sapiens 421cctgtgggcc
ttgctcagag 2042220DNAhomo sapiens 422ccgctctgag caaggcccac
2042320DNAhomo sapiens 423tgctttctcc gctctgagca 2042420DNAhomo
sapiens 424caggaacatt tacacgtctg 2042520DNAhomo sapiens
425acgcgagtct gtgtttttgc 2042620DNAhomo sapiens 426aaacacagac
tcgcgttgca 2042720DNAhomo sapiens 427cagactcgcg ttgcaaggcg
2042820DNAhomo sapiens 428agttaaacga acgtacttgc 2042920DNAhomo
sapiens 429aaacgaacgt acttgcaggt 2043020DNAhomo sapiens
430ccctcagatg tgacaagccg 2043120DNAhomo sapiens 431gcctcggctt
gtcacatctg 2043220DNAhomo sapiens 432tcagatgtga caagccgagg
2043320DNAhomo sapiens 433acaagccgag gcggtgagcc 2043420DNAhomo
sapiens 434gccgaggcgg tgagccgggc 2043520DNAhomo sapiens
435tcctgcccgg ctcaccgcct 2043620DNAhomo sapiens 436tttaaatgag
ctcccaatgt 2043720DNAhomo sapiens 437gagctcccaa tgtcggagtt
2043820DNAhomo sapiens 438gttttccaaa ctccgacatt 2043920DNAhomo
sapiens 439tgttttccaa actccgacat 2044020DNAhomo sapiens
440aaatttgtct ttttaaaaga 2044120DNAhomo sapiens 441gtctttttaa
aagaaggtct 2044220DNAhomo sapiens 442aaactcaaaa cctgaagaat
2044320DNAhomo sapiens 443ctgatttctt ccaattcttc 2044420DNAhomo
sapiens 444gaagaaatca gaatagaaaa 2044520DNAhomo sapiens
445aagaaatcag aatagaaaat 2044620DNAhomo sapiens 446atcagaatag
aaaatgggta 2044720DNAhomo sapiens 447ctcgagatgc agccagatct
2044820DNAhomo sapiens 448ttctttactt cgccgagatc 2044920DNAhomo
sapiens 449gaactcacat tatgtggaag 2045020DNAhomo sapiens
450agatgcgaac tcacattatg 2045120DNAhomo sapiens 451tgtgagttcg
catcttgata 2045220DNAhomo sapiens 452ttgataaggc ctctgtgatg
2045320DNAhomo sapiens 453gatggtaagc ctcatcacag 2045420DNAhomo
sapiens 454ccatcagcta tttgcgtgtg 2045520DNAhomo sapiens
455cctcacacgc aaatagctga 2045620DNAhomo sapiens 456tttgcgtgtg
aggaaacttc 2045720DNAhomo sapiens 457gtgaggaaac ttctggatgc
2045820DNAhomo sapiens 458tgtgcccttt ttaggtgatt 2045920DNAhomo
sapiens 459ttgcttttat ttgaaagcct 2046020DNAhomo sapiens
460ttttatttga aagccttgga 2046120DNAhomo sapiens 461agccttggat
ggttttgtta 2046220DNAhomo sapiens 462aaccataaca aaaccatcca
2046320DNAhomo sapiens 463gttatggttc tcacagatga 2046420DNAhomo
sapiens 464tgataatgtg aacaaataca 2046520DNAhomo sapiens
465gataatgtga acaaatacat 2046620DNAhomo sapiens 466caaatacatg
ggattaactc 2046720DNAhomo sapiens 467tgtttacagt ttgaactaac
2046820DNAhomo sapiens 468tactcatcca tgtgaccatg 2046920DNAhomo
sapiens 469ctcatttcct catggtcaca 2047020DNAhomo sapiens
470gcatttctct catttcctca 2047120DNAhomo sapiens 471gaaatgctta
cacacagaaa 2047220DNAhomo sapiens 472ttcattaggc cttgtgaaaa
2047320DNAhomo sapiens 473tcattaggcc ttgtgaaaaa 2047420DNAhomo
sapiens 474gttctttacc ctttttcaca 2047520DNAhomo sapiens
475aagtgtaccc taactagccg 2047620DNAhomo sapiens 476agttcttcct
cggctagtta 2047720DNAhomo sapiens 477tagttcttcc tcggctagtt
2047820DNAhomo sapiens 478ttatgttcat agttcttcct 2047920DNAhomo
sapiens 479tgaacataaa gtctgcaaca 2048020DNAhomo sapiens
480cataaagtct gcaacatgga 2048120DNAhomo sapiens 481acacaggtat
tgcactgcac 2048220DNAhomo sapiens 482tggtatcata tacgtgaatg
2048320DNAhomo sapiens 483acactgaggt tggttactgt 2048420DNAhomo
sapiens 484aacagtaacc aacctcagtg 2048520DNAhomo sapiens
485acagtaacca acctcagtgt 2048620DNAhomo sapiens 486tcttataccc
acactgaggt 2048720DNAhomo sapiens 487ggtttcttat acccacactg
2048820DNAhomo sapiens 488gaaaccacct atgacctgct 2048920DNAhomo
sapiens 489agcaccaagc aggtcatagg 2049020DNAhomo sapiens
490atcagcacca agcaggtcat 2049120DNAhomo sapiens 491ttcacaaatc
agcaccaagc 2049220DNAhomo sapiens 492atatttgatg ggtgaggaat
2049320DNAhomo sapiens 493aatatttgat gggtgaggaa 2049420DNAhomo
sapiens 494atttcaatat ttgatgggtg 2049520DNAhomo sapiens
495aaggaatttc aatatttgat 2049620DNAhomo sapiens 496aaaggaattt
caatatttga 2049720DNAhomo sapiens 497aggaaagtct tgctatctaa
2049820DNAhomo sapiens 498tttcctcagt cgacacagcc 2049920DNAhomo
sapiens 499tatccaggct gtgtcgactg 2050020DNAhomo sapiens
500aataagaaaa tttcatatcc 2050120DNAhomo sapiens 501aattttctta
ttgtgatgaa 2050220DNAhomo sapiens 502taacagaatt accgaattga
2050320DNAhomo sapiens 503aacagaatta ccgaattgat 2050420DNAhomo
sapiens 504tggctcatat cccatcaatt 2050520DNAhomo sapiens
505tatgagccag aagaactttt 2050620DNAhomo sapiens 506gagcggccta
aaagttcttc 2050720DNAhomo sapiens 507gataatattc ataaattgag
2050820DNAhomo sapiens 508ttatgaatat tatcatgctt 2050920DNAhomo
sapiens 509cttactatca tgatgagttt 2051020DNAhomo sapiens
510tcccccctag tgtttactaa 2051120DNAhomo sapiens 511ttgtccttta
gtaaacacta 2051220DNAhomo sapiens 512cttgtccttt agtaaacact
2051320DNAhomo sapiens 513actaaaggac aagtcaccac 2051420DNAhomo
sapiens 514aagtcaccac aggacagtac 2051520DNAhomo sapiens
515aagcatcctg tactgtcctg 2051620DNAhomo sapiens 516tacaggatgc
ttgccaaaag 2051720DNAhomo sapiens 517aggatgcttg ccaaaagagg
2051820DNAhomo sapiens 518ccaaaagagg tggatatgtc 2051920DNAhomo
sapiens 519ccagacatat ccacctcttt 2052020DNAhomo sapiens
520caaaagaggt ggatatgtct 2052120DNAhomo sapiens 521gcactgtggt
tgagaattct 2052220DNAhomo sapiens 522ttcacacata caatgcactg
2052320DNAhomo sapiens 523tatgtgtgaa ttacgttgtg 2052420DNAhomo
sapiens 524gacacattct gtttgttgaa 2052520DNAhomo sapiens
525ggacacattc tgtttgttga 2052620DNAhomo sapiens 526aacagaatgt
gtccttaaac 2052720DNAhomo sapiens 527ctgaagattc aaccggttta
2052820DNAhomo sapiens 528ttcatatctg aagattcaac 2052920DNAhomo
sapiens 529tgtatcttct gattcaactt 2053020DNAhomo sapiens
530cctctttgac aaacttaaga 2053120DNAhomo sapiens 531ccttcttaag
tttgtcaaag 2053220DNAhomo sapiens 532acctgatgct ttaactttgc
2053320DNAhomo sapiens 533gccagcaaag ttaaagcatc 2053420DNAhomo
sapiens 534actttgctgg ccccagccgc 2053520DNAhomo sapiens
535gattgtgtct ccagcggctg 2053620DNAhomo sapiens 536tgattgtgtc
tccagcggct 2053720DNAhomo sapiens 537atgattgtgt ctccagcggc
2053820DNAhomo sapiens 538agatatgatt gtgtctccag 2053920DNAhomo
sapiens 539acaatcatat ctttagattt 2054020DNAhomo sapiens
540tctttagatt ttggcagcaa 2054120DNAhomo sapiens 541gtcatcagtt
tctgtgtctg 2054220DNAhomo sapiens 542aactgatgac cagcaacttg
2054320DNAhomo sapiens 543atggtacttc ctcaagttgc 2054420DNAhomo
sapiens 544agcattacat cattatataa 2054520DNAhomo sapiens
545gtaatttttc gttgggtgag 2054620DNAhomo sapiens 546tgtaattttt
cgttgggtga 2054720DNAhomo sapiens 547ctgtaatttt tcgttgggtg
2054820DNAhomo sapiens 548atattctgta atttttcgtt 2054920DNAhomo
sapiens 549tatattctgt aatttttcgt 2055020DNAhomo sapiens
550aaaattacag aatataaatt 2055120DNAhomo sapiens 551ggcgtttcag
cggtgggtaa 2055220DNAhomo sapiens 552ggctttggcg tttcagcggt
2055320DNAhomo sapiens 553tggctttggc gtttcagcgg 2055420DNAhomo
sapiens 554aagtggcttt ggcgtttcag 2055520DNAhomo sapiens
555gcactacttc gaagtggctt 2055620DNAhomo sapiens 556gggtcagcac
tacttcgaag 2055720DNAhomo sapiens 557caacttcttg attgagtgca
2055820DNAhomo sapiens 558gcaacttctt gattgagtgc 2055920DNAhomo
sapiens 559agaaccaaat ccagagtcac 2056020DNAhomo sapiens
560agttccagtg actctggatt 2056120DNAhomo sapiens 561aaagaaagtt
ccagtgactc 2056220DNAhomo sapiens 562ttttaccatg ccccagattc
2056320DNAhomo sapiens 563ctgatcctga atctggggca 2056420DNAhomo
sapiens 564ggtgtctgat cctgaatctg 2056520DNAhomo sapiens
565aggtgtctga tcctgaatct 2056620DNAhomo sapiens
566taggtgtctg atcctgaatc 2056720DNAhomo sapiens 567cagacaccta
gtccttccga 2056820DNAhomo sapiens 568gtgcttccat cggaaggact
2056920DNAhomo sapiens 569tgtctagtgc ttccatcgga 2057020DNAhomo
sapiens 570actttgtcta gtgcttccat 2057120DNAhomo sapiens
571cactagacaa agttcacctg 2057220DNAhomo sapiens 572agacaaagtt
cacctgaggt 2057320DNAhomo sapiens 573tatatcatga cacctacctc
2057420DNAhomo sapiens 574caatattcac tgggactatt 2057520DNAhomo
sapiens 575acataaaaac aatattcact 2057620DNAhomo sapiens
576cacataaaaa caatattcac 2057720DNAhomo sapiens 577cagtgaatat
tgtttttatg 2057820DNAhomo sapiens 578tttttatgtg gatagtgata
2057920DNAhomo sapiens 579tatggtcaat gaattcaagt 2058020DNAhomo
sapiens 580caatgaattc aagttggaat 2058120DNAhomo sapiens
581aaagaaccca ttttctactc 2058220DNAhomo sapiens 582catatacctg
agtagaaaat 2058320DNAhomo sapiens 583tcatatacct gagtagaaaa
2058420DNAhomo sapiens 584aaaggacaca gatttagact 2058520DNAhomo
sapiens 585gttagctccc tatatcccaa 2058620DNAhomo sapiens
586tcatcatcca ttgggatata 2058720DNAhomo sapiens 587gtcatcatcc
attgggatat 2058820DNAhomo sapiens 588actggaagtc atcatccatt
2058920DNAhomo sapiens 589aactggaagt catcatccat 2059020DNAhomo
sapiens 590actgatcgaa ggaacgtaac 2059120DNAhomo sapiens
591taatggtgac aactgatcga 2059220DNAhomo sapiens 592cttgcggaac
tgctttctaa 2059320DNAhomo sapiens 593acttgcgctt tcagggcttg
2059420DNAhomo sapiens 594tttgaggact tgcgctttca 2059520DNAhomo
sapiens 595ctttgaggac ttgcgctttc 2059620DNAhomo sapiens
596aatactgtaa ctgtgctttg 2059720DNAhomo sapiens 597gttcttgtat
ttgagtctgc 2059820DNAhomo sapiens 598gtagtggtgg cattagcagt
2059920DNAhomo sapiens 599agtggtggca gtggtagtgg 2060020DNAhomo
sapiens 600atcagtggtg gcagtggtag 2060120DNAhomo sapiens
601taattcatca gtggtggcag 2060220DNAhomo sapiens 602tgtttttaat
tcatcagtgg 2060320DNAhomo sapiens 603cactgttttt aattcatcag
2060420DNAhomo sapiens 604aacagtgaca aaagaccgta 2060520DNAhomo
sapiens 605atattttaat gtcttccata 2060620DNAhomo sapiens
606ttatgtatgt gggtaggaga 2060720DNAhomo sapiens 607gtttctttat
gtatgtgggt 2060820DNAhomo sapiens 608agtagtttct ttatgtatgt
2060920DNAhomo sapiens 609tagtagtttc tttatgtatg 2061020DNAhomo
sapiens 610atctctatat ggtgatgatg 2061120DNAhomo sapiens
611cgactttgag tatctctata 2061220DNAhomo sapiens 612catatagaga
tactcaaagt 2061320DNAhomo sapiens 613acagcctcac caaacagagc
2061420DNAhomo sapiens 614ttttcctgct ctgtttggtg 2061520DNAhomo
sapiens 615tcaccaaaca gagcaggaaa 2061620DNAhomo sapiens
616actccttttc ctgctctgtt 2061720DNAhomo sapiens 617gataacacgt
tagggcttct 2061820DNAhomo sapiens 618aagcgacaga taacacgtta
2061920DNAhomo sapiens 619aaagcgacag ataacacgtt 2062020DNAhomo
sapiens 620tatctgtcgc tttgagtcaa 2062120DNAhomo sapiens
621tttcagaact acagttcctg 2062220DNAhomo sapiens 622tttggattta
gttcttcctc 2062320DNAhomo sapiens 623ttctgcaaag ctagtatctt
2062420DNAhomo sapiens 624tgctcagaga aagcgaaaaa 2062520DNAhomo
sapiens 625aagcgaaaaa tggaacatga 2062620DNAhomo sapiens
626gtagtagctg catgatcgtc 2062720DNAhomo sapiens 627cagctactac
atcactttct 2062820DNAhomo sapiens 628ctttcttgga aacgtgtaaa
2062920DNAhomo sapiens 629acaattattt taataccctc 2063020DNAhomo
sapiens 630aaaagaataa actaaccaga 2063120DNAhomo sapiens
631aaaaagaata aactaaccag 2063220DNAhomo sapiens 632agatttagca
tgtagactgc 2063320DNAhomo sapiens 633gatttagcat gtagactgct
2063420DNAhomo sapiens 634atttagcatg tagactgctg 2063520DNAhomo
sapiens 635tagactgctg gggcaatcaa 2063620DNAhomo sapiens
636gggcaatcaa tggatgaaag 2063720DNAhomo sapiens 637caatcataac
tggtcagctg 2063820DNAhomo sapiens 638attaacttca caatcataac
2063920DNAhomo sapiens 639gaagttaatg ctcctataca 2064020DNAhomo
sapiens 640aggtttctgc tgccttgtat 2064120DNAhomo sapiens
641aggcagcaga aacctactgc 2064220DNAhomo sapiens 642ggcagcagaa
acctactgca 2064320DNAhomo sapiens 643gtaattcttc accctgcagt
2064420DNAhomo sapiens 644tgaagaatta ctcagagctt 2064520DNAhomo
sapiens 645agcgacaatg acagctgaca 2064620DNAhomo sapiens
646cagctgacaa ggagaagaaa 2064720DNAhomo sapiens 647ttctccactt
aggagtagct 2064820DNAhomo sapiens 648cacttaggag tagctcggag
2064920DNAhomo sapiens 649ttaggagtag ctcggagagg 2065020DNAhomo
sapiens 650gagtagctcg gagaggagga 2065120DNAhomo sapiens
651agaggaggaa ggagaagtcc 2065220DNAhomo sapiens 652gaggaggaag
gagaagtccc 2065320DNAhomo sapiens 653agaagtcccg ggatgctgcg
2065420DNAhomo sapiens 654cccgggatgc tgcgcggtgc 2065520DNAhomo
sapiens 655ccggcaccgc gcagcatccc 2065620DNAhomo sapiens
656gccggcaccg cgcagcatcc 2065720DNAhomo sapiens 657gggatgctgc
gcggtgccgg 2065820DNAhomo sapiens 658tgcgcggtgc cggcggagca
2065920DNAhomo sapiens 659gtgccggcgg agcaaggaga 2066020DNAhomo
sapiens 660ccggcggagc aaggagacgg 2066120DNAhomo sapiens
661cctccgtctc cttgctccgc 2066220DNAhomo sapiens 662gacggaggtg
ttctatgagc 2066320DNAhomo sapiens 663gtggggcaga ggcagctcat
2066420DNAhomo sapiens 664tgtggggcag aggcagctca 2066520DNAhomo
sapiens 665gagctcacac tgtggggcag 2066620DNAhomo sapiens
666agatgggagc tcacactgtg 2066720DNAhomo sapiens 667cagatgggag
ctcacactgt 2066820DNAhomo sapiens 668ccacagtgtg agctcccatc
2066920DNAhomo sapiens 669ccagatggga gctcacactg 2067020DNAhomo
sapiens 670tgtgagctcc catctggaca 2067120DNAhomo sapiens
671gatggaggcc ttgtccagat 2067220DNAhomo sapiens 672tgatggaggc
cttgtccaga 2067320DNAhomo sapiens 673caaggcctcc atcatgcgac
2067420DNAhomo sapiens 674gattgccagt cgcatgatgg 2067520DNAhomo
sapiens 675gctgattgcc agtcgcatga 2067620DNAhomo sapiens
676agaggagctt gtgtgttcgc 2067720DNAhomo sapiens 677acacacaagc
tcctctcctc 2067820DNAhomo sapiens 678caagctcctc tcctcaggta
2067920DNAhomo sapiens 679tgctggcctt acctgaggag 2068020DNAhomo
sapiens 680gagcctgctg gccttacctg 2068120DNAhomo sapiens
681gactcgtttt cagagcaaac 2068220DNAhomo sapiens 682ctgctggtca
gcttcggctt 2068320DNAhomo sapiens 683agccgaagct gaccagcaga
2068420DNAhomo sapiens 684gtccatctgc tggtcagctt 2068520DNAhomo
sapiens 685ggtacaagtt gtccatctgc 2068620DNAhomo sapiens
686caacttgtac ctgaaagcct 2068720DNAhomo sapiens 687cttgtacctg
aaagccttgg 2068820DNAhomo sapiens 688ttgtacctga aagccttgga
2068920DNAhomo sapiens 689tgaaaccctc caaggctttc 2069020DNAhomo
sapiens 690cacggcaatg aaaccctcca 2069120DNAhomo sapiens
691cttggagggt ttcattgccg 2069220DNAhomo sapiens 692attgccgtgg
tgacccaaga 2069320DNAhomo sapiens 693gtcgccatct tgggtcacca
2069420DNAhomo sapiens 694aaagatcatg tcgccatctt 2069520DNAhomo
sapiens 695gaaagatcat gtcgccatct 2069620DNAhomo sapiens
696agaaaacatc agcaagttca 2069720DNAhomo sapiens 697gaaaacatca
gcaagttcat 2069820DNAhomo sapiens 698caagttcatg ggacttacac
2069920DNAhomo sapiens 699ttgaaacagg tggagctaac 2070020DNAhomo
sapiens 700cactcatccc tgcgaccatg 2070120DNAhomo sapiens
701cgaatctcct catggtcgca 2070220DNAhomo sapiens 702acgaatctcc
tcatggtcgc 2070320DNAhomo sapiens 703ggttctcacg aatctcctca
2070420DNAhomo sapiens 704gagaacctga gtctcaaaaa 2070520DNAhomo
sapiens 705ggataccatt tttgagactc 2070620DNAhomo sapiens
706atccttccac atccaggctc 2070720DNAhomo sapiens 707ccacatccag
gctctggttt 2070820DNAhomo sapiens 708cacatccagg ctctggtttt
2070920DNAhomo sapiens 709tttttcccaa aaccagagcc 2071020DNAhomo
sapiens 710gcaaagacat gtccacagag 2071120DNAhomo sapiens
711caaagacatg tccacagagc 2071220DNAhomo sapiens 712catgaagaag
tcccgctctg 2071320DNAhomo sapiens 713cagagcggga cttcttcatg
2071420DNAhomo sapiens 714cttcatgagg atgaagtgca 2071520DNAhomo
sapiens 715aagtgcacgg tcaccaacag 2071620DNAhomo sapiens
716gttgacagta cggcctctgt 2071720DNAhomo sapiens 717ctgacttgag
gttgacagta 2071820DNAhomo sapiens 718tcaacctcaa gtcagccacc
2071920DNAhomo sapiens 719cctcaagtca gccacctgga 2072020DNAhomo
sapiens 720ccttccaggt ggctgacttg 2072120DNAhomo sapiens
721aagtcagcca cctggaaggt 2072220DNAhomo sapiens 722agtcagccac
ctggaaggta 2072320DNAhomo sapiens 723atgttgccct accttccagg
2072420DNAhomo sapiens 724ctgatgttgc cctaccttcc 2072520DNAhomo
sapiens 725gtctcaggtc ttgcactgca 2072620DNAhomo sapiens
726tctcaggtct tgcactgcac 2072720DNAhomo sapiens 727ggtcttgcac
tgcacgggcc 2072820DNAhomo sapiens 728agttgttgta gactttcacc
2072920DNAhomo sapiens 729cacacagact attgtgagga 2073020DNAhomo
sapiens 730cctcctcaca atagtctgtg 2073120DNAhomo sapiens
731ccacacagac tattgtgagg 2073220DNAhomo sapiens 732tagccacaca
gactattgtg 2073320DNAhomo sapiens 733caatagtctg tgtggctaca
2073420DNAhomo sapiens 734atgatgaggc aggacagcag 2073520DNAhomo
sapiens 735gatgatgagg caggacagca 2073620DNAhomo sapiens
736tgatgatgag gcaggacagc 2073720DNAhomo sapiens 737ttcacacatg
atgatgaggc 2073820DNAhomo sapiens 738ttggttcaca catgatgatg
2073920DNAhomo sapiens 739atgtgggatg ggtgctggat 2074020DNAhomo
sapiens 740aatccagcac ccatcccaca 2074120DNAhomo sapiens
741tgtccatgtg ggatgggtgc 2074220DNAhomo sapiens 742gggggatgtc
catgtgggat 2074320DNAhomo sapiens 743agggggatgt ccatgtggga
2074420DNAhomo sapiens 744atcccacatg gacatccccc 2074520DNAhomo
sapiens 745atccaggggg atgtccatgt 2074620DNAhomo sapiens
746tatccagggg gatgtccatg 2074720DNAhomo sapiens 747ggaaggtctt
gctatccagg 2074820DNAhomo sapiens 748aggaaggtct tgctatccag
2074920DNAhomo sapiens 749caggaaggtc ttgctatcca 2075020DNAhomo
sapiens 750tcaggaaggt cttgctatcc 2075120DNAhomo sapiens
751catgctgtgg cggctcagga 2075220DNAhomo sapiens 752cttcctgagc
cgccacagca 2075320DNAhomo sapiens 753tgtccatgct gtggcggctc
2075420DNAhomo sapiens 754acttcatgtc catgctgtgg
2075520DNAhomo sapiens 755tgaacttcat gtccatgctg 2075620DNAhomo
sapiens 756agttcaccta ctgtgatgac 2075720DNAhomo sapiens
757cacctactgt gatgacaggt 2075820DNAhomo sapiens 758acctactgtg
atgacaggta 2075920DNAhomo sapiens 759cccctacctg tcatcacagt
2076020DNAhomo sapiens 760ctcagaatca cagaactgat 2076120DNAhomo
sapiens 761actgattggt taccaccctg 2076220DNAhomo sapiens
762taccaccctg aggagctgct 2076320DNAhomo sapiens 763ggccaagcag
ctcctcaggg 2076420DNAhomo sapiens 764agcggccaag cagctcctca
2076520DNAhomo sapiens 765gagcggccaa gcagctcctc 2076620DNAhomo
sapiens 766ggtagaattc ataggctgag 2076720DNAhomo sapiens
767tagcgcatgg tagaattcat 2076820DNAhomo sapiens 768tgttctcgga
gtctagcgca 2076920DNAhomo sapiens 769gtgactcttg gtcatgttct
2077020DNAhomo sapiens 770ctcacagttc tggtgactct 2077120DNAhomo
sapiens 771actcctggaa ctcacagttc 2077220DNAhomo sapiens
772tcctccccta gtgtgcacca 2077320DNAhomo sapiens 773cctcccctag
tgtgcaccaa 2077420DNAhomo sapiens 774ctgacccttg gtgcacacta
2077520DNAhomo sapiens 775cctagtgtgc accaagggtc 2077620DNAhomo
sapiens 776cctgaccctt ggtgcacact 2077720DNAhomo sapiens
777accaagggtc aggtagtaag 2077820DNAhomo sapiens 778gccacttact
acctgaccct 2077920DNAhomo sapiens 779aggtagtaag tggccagtac
2078020DNAhomo sapiens 780gctttgcgag catccggtac 2078120DNAhomo
sapiens 781taccggatgc tcgcaaagca 2078220DNAhomo sapiens
782accggatgct cgcaaagcat 2078320DNAhomo sapiens 783ccggatgctc
gcaaagcatg 2078420DNAhomo sapiens 784ccccatgctt tgcgagcatc
2078520DNAhomo sapiens 785cggatgctcg caaagcatgg 2078620DNAhomo
sapiens 786caaagcatgg gggctacgtg 2078720DNAhomo sapiens
787gcatgggggc tacgtgtggc 2078820DNAhomo sapiens 788ctacgtgtgg
ctggagaccc 2078920DNAhomo sapiens 789tacgtgtggc tggagaccca
2079020DNAhomo sapiens 790acgtgtggct ggagacccag 2079120DNAhomo
sapiens 791gtggctggag acccagggga 2079220DNAhomo sapiens
792gttgtagatg accgtcccct 2079320DNAhomo sapiens 793ggttgtagat
gaccgtcccc 2079420DNAhomo sapiens 794actggggctg caggttgcga
2079520DNAhomo sapiens 795cactggggct gcaggttgcg 2079620DNAhomo
sapiens 796acatgatgca ctggggctgc 2079720DNAhomo sapiens
797ttgacacaca tgatgcactg 2079820DNAhomo sapiens 798gttgacacac
atgatgcact 2079920DNAhomo sapiens 799agttgacaca catgatgcac
2080020DNAhomo sapiens 800tgtgtgtcaa ctacgtcctg 2080120DNAhomo
sapiens 801agccctcaca tgcttacctc 2080220DNAhomo sapiens
802tgagattgag aagaatgacg 2080320DNAhomo sapiens 803gaatgacgtg
gtgttctcca 2080420DNAhomo sapiens 804cagggattca gtctggtcca
2080520DNAhomo sapiens 805gcttgaacag ggattcagtc 2080620DNAhomo
sapiens 806catcaggtgg ggcttgaaca 2080720DNAhomo sapiens
807cctgttcaag ccccacctga 2080820DNAhomo sapiens 808ccatcaggtg
gggcttgaac 2080920DNAhomo sapiens 809ctgttcatgg ccatcaggtg
2081020DNAhomo sapiens 810gctgttcatg gccatcaggt 2081120DNAhomo
sapiens 811tgctgttcat ggccatcagg 2081220DNAhomo sapiens
812agatgctgtt catggccatc 2081320DNAhomo sapiens 813gctatcaaag
atgctgttca 2081420DNAhomo sapiens 814aacagcatct ttgatagcag
2081520DNAhomo sapiens 815catctttgat agcagtggca 2081620DNAhomo
sapiens 816atctttgata gcagtggcaa 2081720DNAhomo sapiens
817tctttgatag cagtggcaag 2081820DNAhomo sapiens 818ctttgatagc
agtggcaagg 2081920DNAhomo sapiens 819cttcctattc accaagctaa
2082020DNAhomo sapiens 820cctattcacc aagctaaagg 2082120DNAhomo
sapiens 821cctcctttag cttggtgaat 2082220DNAhomo sapiens
822ctcgggctcc tcctttagct 2082320DNAhomo sapiens 823caagctaaag
gaggagcccg 2082420DNAhomo sapiens 824aaaggaggag cccgaggagc
2082520DNAhomo sapiens 825gcccgaggag ctggcccagc 2082620DNAhomo
sapiens 826gccagctggg ccagctcctc 2082720DNAhomo sapiens
827agccagctgg gccagctcct 2082820DNAhomo sapiens 828gcccagctgg
ctcccacccc 2082920DNAhomo sapiens 829tcctggggtg ggagccagct
2083020DNAhomo sapiens 830ctcctggggt gggagccagc 2083120DNAhomo
sapiens 831atgatggcgt ctcctggggt 2083220DNAhomo sapiens
832gatgatggcg tctcctgggg 2083320DNAhomo sapiens 833agagatgatg
gcgtctcctg 2083420DNAhomo sapiens 834gagagatgat ggcgtctcct
2083520DNAhomo sapiens 835agagagatga tggcgtctcc 2083620DNAhomo
sapiens 836aggagacgcc atcatctctc 2083720DNAhomo sapiens
837gccatcatct ctctggattt 2083820DNAhomo sapiens 838accgaaatcc
agagagatga 2083920DNAhomo sapiens 839atcatctctc tggatttcgg
2084020DNAhomo sapiens 840tcatctctct ggatttcggt 2084120DNAhomo
sapiens 841ctcgaagttc tgattccctg 2084220DNAhomo sapiens
842cacagggaat cagaacttcg 2084320DNAhomo sapiens 843ttcgaggagt
cctcagccta 2084420DNAhomo sapiens 844ggagtcctca gcctatggca
2084520DNAhomo sapiens 845gatggccttg ccataggctg 2084620DNAhomo
sapiens 846gggcaggatg gccttgccat 2084720DNAhomo sapiens
847tggctggctc gggggcagga 2084820DNAhomo sapiens 848tcctgccccc
gagccagcca 2084920DNAhomo sapiens 849cctgcccccg agccagccat
2085020DNAhomo sapiens 850cccatggctg gctcgggggc 2085120DNAhomo
sapiens 851gtggcccatg gctggctcgg 2085220DNAhomo sapiens
852cgtggcccat ggctggctcg 2085320DNAhomo sapiens 853cccgagccag
ccatgggcca 2085420DNAhomo sapiens 854ccgtggccca tggctggctc
2085520DNAhomo sapiens 855tccgtggccc atggctggct 2085620DNAhomo
sapiens 856tcaactccgt ggcccatggc 2085720DNAhomo sapiens
857agccatgggc cacggagttg 2085820DNAhomo sapiens 858ctcctcaact
ccgtggccca 2085920DNAhomo sapiens 859gctgtggctc ctcaactccg
2086020DNAhomo sapiens 860gagccacagc acccagagcg 2086120DNAhomo
sapiens 861cagcctcgct ctgggtgctg 2086220DNAhomo sapiens
862cacagcaccc agagcgaggc 2086320DNAhomo sapiens 863acagcaccca
gagcgaggct 2086420DNAhomo sapiens 864caggctccca gcctcgctct
2086520DNAhomo sapiens 865gcaggctccc agcctcgctc 2086620DNAhomo
sapiens 866ggggcacggt gaaggcaggc 2086720DNAhomo sapiens
867gcctgccttc accgtgcccc 2086820DNAhomo sapiens 868gcctggggca
cggtgaaggc 2086920DNAhomo sapiens 869agctgcctgg ggcacggtga
2087020DNAhomo sapiens 870cggggcagct gcctggggca 2087120DNAhomo
sapiens 871cgtgccccag gcagctgccc 2087220DNAhomo sapiens
872gtgccccagg cagctgcccc 2087320DNAhomo sapiens 873ctgcccgggg
cagctgcctg 2087420DNAhomo sapiens 874gctgcccggg gcagctgcct
2087520DNAhomo sapiens 875tgctgcccgg ggcagctgcc 2087620DNAhomo
sapiens 876actgggggtg gtgctgcccg 2087720DNAhomo sapiens
877cactgggggt ggtgctgccc 2087820DNAhomo sapiens 878gcactggggg
tggtgctgcc 2087920DNAhomo sapiens 879gctgctggtg gcactggggg
2088020DNAhomo sapiens 880gctgctgctg gtggcactgg 2088120DNAhomo
sapiens 881tgctgctgct ggtggcactg 2088220DNAhomo sapiens
882ctgctgctgc tggtggcact 2088320DNAhomo sapiens 883gctgctgctg
ctggtggcac 2088420DNAhomo sapiens 884gcagctgctg ctgctgctgg
2088520DNAhomo sapiens 885cagcagcagc agctgctcca 2088620DNAhomo
sapiens 886tcttcagggc tattgggcta 2088720DNAhomo sapiens
887gtcttcaggg ctattgggct 2088820DNAhomo sapiens 888taatagtctt
cagggctatt 2088920DNAhomo sapiens 889gtaatagtct tcagggctat
2089020DNAhomo sapiens 890aagatgtgta atagtcttca 2089120DNAhomo
sapiens 891aaagatgtgt aatagtcttc 2089220DNAhomo sapiens
892tgaagactat tacacatctt 2089320DNAhomo sapiens 893tctcaatcac
ttcaatcttc 2089420DNAhomo sapiens 894gattgagaag ctcttcgcca
2089520DNAhomo sapiens 895gctcttcgcc atggacacag 2089620DNAhomo
sapiens 896cgccatggac acagaggcca 2089720DNAhomo sapiens
897gtccttggcc tctgtgtcca 2089820DNAhomo sapiens 898ctgggtactg
cattggtcct 2089920DNAhomo sapiens 899caaggaccaa tgcagtaccc
2090020DNAhomo sapiens 900catctacctg ggtactgcat 2090120DNAhomo
sapiens 901agctcattga aatccgtctg 2090220DNAhomo sapiens
902tcagacggat ttcaatgagc 2090320DNAhomo sapiens 903ggatttcaat
gagctggact 2090420DNAhomo sapiens 904tgagctggac ttggagacac
2090520DNAhomo sapiens 905actggcaccc tatatcccca 2090620DNAhomo
sapiens 906gcaccctata tccccatgga 2090720DNAhomo sapiens
907caccctatat ccccatggac 2090820DNAhomo sapiens 908accctatatc
cccatggacg 2090920DNAhomo sapiens 909tccccgtcca tggggatata
2091020DNAhomo sapiens 910ttccccgtcc atggggatat 2091120DNAhomo
sapiens 911ggaagtcttc cccgtccatg 2091220DNAhomo sapiens
912tggaagtctt ccccgtccat 2091320DNAhomo sapiens 913ctggaagtct
tccccgtcca 2091420DNAhomo sapiens 914cggggcagat ggggcttagc
2091520DNAhomo sapiens 915gctaagcccc atctgccccg 2091620DNAhomo
sapiens 916gccccatctg ccccgaggag 2091720DNAhomo sapiens
917gccgctcctc ggggcagatg 2091820DNAhomo sapiens 918agccgctcct
cggggcagat 2091920DNAhomo sapiens 919gagccgctcc tcggggcaga
2092020DNAhomo sapiens 920ctgccccgag gagcggctct 2092120DNAhomo
sapiens 921ccccgaggag cggctcttgg 2092220DNAhomo sapiens
922ccgccaagag ccgctcctcg 2092320DNAhomo sapiens 923tccgccaaga
gccgctcctc 2092420DNAhomo sapiens 924ctccgccaag agccgctcct
2092520DNAhomo sapiens 925agtgctgggg ggtggactgt 2092620DNAhomo
sapiens 926cagtgctggg gggtggactg 2092720DNAhomo sapiens
927actgaagcag tgctgggggg 2092820DNAhomo sapiens 928ggcactgaag
cagtgctggg 2092920DNAhomo sapiens 929tggcactgaa gcagtgctgg
2093020DNAhomo sapiens 930atggcactga agcagtgctg 2093120DNAhomo
sapiens 931catggcactg aagcagtgct 2093220DNAhomo sapiens
932tcatggcact gaagcagtgc 2093320DNAhomo sapiens 933tggctggaag
atgtttgtca 2093420DNAhomo sapiens 934gacaaacatc ttccagccac
2093520DNAhomo sapiens 935gggctacagg ggccagtggc 2093620DNAhomo
sapiens 936tgcggggcta caggggccag 2093720DNAhomo sapiens
937gggactgtgc ggggctacag 2093820DNAhomo sapiens 938agggactgtg
cggggctaca 2093920DNAhomo sapiens 939aagggactgt gcggggctac
2094020DNAhomo sapiens 940caggaggaag ggactgtgcg 2094120DNAhomo
sapiens 941cccgcacagt cccttcctcc 2094220DNAhomo sapiens
942ccaggaggaa gggactgtgc
2094320DNAhomo sapiens 943tccaggagga agggactgtg 2094420DNAhomo
sapiens 944tgaaacttgt ccaggaggaa 2094520DNAhomo sapiens
945ctgaaacttg tccaggagga 2094620DNAhomo sapiens 946gctgctgaaa
cttgtccagg 2094720DNAhomo sapiens 947gctgctgctg aaacttgtcc
2094820DNAhomo sapiens 948ggacaagttt cagcagcagc 2094920DNAhomo
sapiens 949agaagacaga gcccgagcac 2095020DNAhomo sapiens
950gaggacatgg gccggtgctc 2095120DNAhomo sapiens 951ggaggacatg
ggccggtgct 2095220DNAhomo sapiens 952agaagatgga ggacatgggc
2095320DNAhomo sapiens 953tcaaagaaga tggaggacat 2095420DNAhomo
sapiens 954atcaaagaag atggaggaca 2095520DNAhomo sapiens
955tcctccatct tctttgatgc 2095620DNAhomo sapiens 956tccggcatca
aagaagatgg 2095720DNAhomo sapiens 957gcttccggca tcaaagaaga
2095820DNAhomo sapiens 958tggcagggat gctttgcttc 2095920DNAhomo
sapiens 959gcatccctgc caccgtgctg 2096020DNAhomo sapiens
960ctggccacag cacggtggca 2096120DNAhomo sapiens 961cctgccaccg
tgctgtggcc 2096220DNAhomo sapiens 962cctggccaca gcacggtggc
2096320DNAhomo sapiens 963ctggcctggc cacagcacgg 2096420DNAhomo
sapiens 964gtgctggcct ggccacagca 2096520DNAhomo sapiens
965aagagagagg ggtgctggcc 2096620DNAhomo sapiens 966catggaagag
agaggggtgc 2096720DNAhomo sapiens 967cagcacccct ctctcttcca
2096820DNAhomo sapiens 968agcacccctc tctcttccat 2096920DNAhomo
sapiens 969gcacccctct ctcttccatg 2097020DNAhomo sapiens
970cacccctctc tcttccatgg 2097120DNAhomo sapiens 971acccctctct
cttccatggg 2097220DNAhomo sapiens 972gccccccatg gaagagagag
2097320DNAhomo sapiens 973tgccccccat ggaagagaga 2097420DNAhomo
sapiens 974ctgcccccca tggaagagag 2097520DNAhomo sapiens
975ggtattggat ctgcccccca 2097620DNAhomo sapiens 976ggggcagatc
caatacccag 2097720DNAhomo sapiens 977atctgggggc cactgggtat
2097820DNAhomo sapiens 978tggtggatct gggggccact 2097920DNAhomo
sapiens 979atggtggatc tgggggccac 2098020DNAhomo sapiens
980aaatgtaatg gtggatctgg 2098120DNAhomo sapiens 981aaaatgtaat
ggtggatctg 2098220DNAhomo sapiens 982caaaatgtaa tggtggatct
2098320DNAhomo sapiens 983ccagatccac cattacattt 2098420DNAhomo
sapiens 984ccaaaatgta atggtggatc 2098520DNAhomo sapiens
985cagatccacc attacatttt 2098620DNAhomo sapiens 986gtgggcccaa
aatgtaatgg 2098720DNAhomo sapiens 987tttgtgggcc caaaatgtaa
2098820DNAhomo sapiens 988tacattttgg gcccacaaag 2098920DNAhomo
sapiens 989acattttggg cccacaaagt 2099020DNAhomo sapiens
990gggcccacaa agtgggccgt 2099120DNAhomo sapiens 991ggcccacaaa
gtgggccgtc 2099220DNAhomo sapiens 992gcccacaaag tgggccgtcg
2099320DNAhomo sapiens 993tccccgacgg cccactttgt 2099420DNAhomo
sapiens 994atccccgacg gcccactttg 2099520DNAhomo sapiens
995ctctgtgcgc tgatccccga 2099620DNAhomo sapiens 996ggatcagcgc
acagagttct 2099720DNAhomo sapiens 997gatcagcgca cagagttctt
2099820DNAhomo sapiens 998gttcttggga gcagcgccgt 2099920DNAhomo
sapiens 999ttcttgggag cagcgccgtt 20100020DNAhomo sapiens
1000tcttgggagc agcgccgttg 20100120DNAhomo sapiens 1001ggagagacag
ggggccccaa 20100220DNAhomo sapiens 1002acatggggtg gagagacagg
20100320DNAhomo sapiens 1003gacatggggt ggagagacag 20100420DNAhomo
sapiens 1004agacatgggg tggagagaca 20100520DNAhomo sapiens
1005gagacatggg gtggagagac 20100620DNAhomo sapiens 1006ttgaaggtgg
agacatgggg 20100720DNAhomo sapiens 1007gtcttgaagg tggagacatg
20100820DNAhomo sapiens 1008tgtcttgaag gtggagacat 20100920DNAhomo
sapiens 1009ttgtcttgaa ggtggagaca 20101020DNAhomo sapiens
1010atgtctccac cttcaagaca 20101120DNAhomo sapiens 1011tgccacttac
cttgtcttga 20101220DNAhomo sapiens 1012cgggcttggc aggtctgcaa
20101320DNAhomo sapiens 1013gggcttggca ggtctgcaaa 20101420DNAhomo
sapiens 1014ggcaggtctg caaagggttt 20101520DNAhomo sapiens
1015gcaggtctgc aaagggtttt 20101620DNAhomo sapiens 1016caggtctgca
aagggttttg 20101720DNAhomo sapiens 1017gcaaagggtt ttggggctcg
20101820DNAhomo sapiens 1018aggcccagac gtgctgagtc 20101920DNAhomo
sapiens 1019tggccggact cagcacgtct 20102020DNAhomo sapiens
1020atggccggac tcagcacgtc 20102120DNAhomo sapiens 1021agacgtgctg
agtccggcca 20102220DNAhomo sapiens 1022ttggagaggg ctaccatggc
20102320DNAhomo sapiens 1023cttgttggag agggctacca 20102420DNAhomo
sapiens 1024cagcttcagc ttgttggaga 20102520DNAhomo sapiens
1025tcagcttcag cttgttggag 20102620DNAhomo sapiens 1026tcgcttcagc
ttcagcttgt 20102720DNAhomo sapiens 1027gctgaagctg aagcgacagc
20102820DNAhomo sapiens 1028gtatgaagag caagccttcc 20102920DNAhomo
sapiens 1029caagccttcc aggacctgag 20103020DNAhomo sapiens
1030aagccttcca ggacctgagc 20103120DNAhomo sapiens 1031agccttccag
gacctgagcg 20103220DNAhomo sapiens 1032caccccgctc aggtcctgga
20103320DNAhomo sapiens 1033gactcacccc gctcaggtcc 20103420DNAhomo
sapiens 1034ggggatgact caccccgctc 20103520DNAhomo sapiens
1035tactcccagg gggacccacc 20103620DNAhomo sapiens 1036tcccaggggg
acccacctgg 20103720DNAhomo sapiens 1037gccaccaggt gggtccccct
20103820DNAhomo sapiens 1038tgccaccagg tgggtccccc 20103920DNAhomo
sapiens 1039gtgaggtgct gccaccaggt 20104020DNAhomo sapiens
1040tgtgaggtgc tgccaccagg 20104120DNAhomo sapiens 1041aaatgtgagg
tgctgccacc 20104220DNAhomo sapiens 1042gcagcacctc acatttgatg
20104320DNAhomo sapiens 1043cctcacattt gatgtggaaa 20104420DNAhomo
sapiens 1044ccgtttccac atcaaatgtg 20104520DNAhomo sapiens
1045ggaaacggat gaagaacctc 20104620DNAhomo sapiens 1046gaaacggatg
aagaacctca 20104720DNAhomo sapiens 1047aaacggatga agaacctcag
20104820DNAhomo sapiens 1048cggatgaaga acctcagggg 20104920DNAhomo
sapiens 1049ggatgaagaa cctcaggggt 20105020DNAhomo sapiens
1050aagggcagct cccacccctg 20105120DNAhomo sapiens 1051tgggagctgc
cctttgatgc 20105220DNAhomo sapiens 1052gtggcttgtc cggcatcaaa
20105320DNAhomo sapiens 1053agtggcttgt ccggcatcaa 20105420DNAhomo
sapiens 1054tttgcgctca gtggcttgtc 20105520DNAhomo sapiens
1055ttgggtacat ttgcgctcag 20105620DNAhomo sapiens 1056ctgagcgcaa
atgtacccaa 20105720DNAhomo sapiens 1057tgtggccgct gctcaccatt
20105820DNAhomo sapiens 1058ctgtggccgc tgctcaccat 20105920DNAhomo
sapiens 1059agttcaccca aaaccccatg 20106020DNAhomo sapiens
1060gttcacccaa aaccccatga 20106120DNAhomo sapiens 1061ttcacccaaa
accccatgag 20106220DNAhomo sapiens 1062caggcccctc atggggtttt
20106320DNAhomo sapiens 1063ccaaaacccc atgaggggcc 20106420DNAhomo
sapiens 1064ccaggcccct catggggttt 20106520DNAhomo sapiens
1065caaaacccca tgaggggcct 20106620DNAhomo sapiens 1066gatggcccag
gcccctcatg 20106720DNAhomo sapiens 1067ggatggccca ggcccctcat
20106820DNAhomo sapiens 1068gggatggccc aggcccctca 20106920DNAhomo
sapiens 1069gatgtctcag gggatggccc 20107020DNAhomo sapiens
1070gcggcagatg tctcagggga 20107120DNAhomo sapiens 1071ggcagcggca
gatgtctcag 20107220DNAhomo sapiens 1072tggcagcggc agatgtctca
20107320DNAhomo sapiens 1073gtggcagcgg cagatgtctc 20107420DNAhomo
sapiens 1074gcagatggag gctgtggcag 20107520DNAhomo sapiens
1075ctgatggcag atggaggctg 20107620DNAhomo sapiens 1076cctccatctg
ccatcagtcc 20107720DNAhomo sapiens 1077ccgggactga tggcagatgg
20107820DNAhomo sapiens 1078ctccatctgc catcagtccc 20107920DNAhomo
sapiens 1079tccatctgcc atcagtcccg 20108020DNAhomo sapiens
1080tccccgggac tgatggcaga 20108120DNAhomo sapiens 1081gctgttctcc
ccgggactga 20108220DNAhomo sapiens 1082ctgctcttgc tgttctcccc
20108320DNAhomo sapiens 1083ccggggagaa cagcaagagc 20108420DNAhomo
sapiens 1084cctgctcttg ctgttctccc 20108520DNAhomo sapiens
1085gggtggcgta gcactgtggg 20108620DNAhomo sapiens 1086tgggtggcgt
agcactgtgg 20108720DNAhomo sapiens 1087ctgggtggcg tagcactgtg
20108820DNAhomo sapiens 1088actgggtggc gtagcactgt 20108920DNAhomo
sapiens 1089tactgggtgg cgtagcactg 20109020DNAhomo sapiens
1090gtgctacgcc acccagtacc 20109120DNAhomo sapiens 1091gctgtagtcc
tggtactggg 20109220DNAhomo sapiens 1092caggctgtag tcctggtact
20109320DNAhomo sapiens 1093acaggctgta gtcctggtac 20109420DNAhomo
sapiens 1094ctgacgacag gctgtagtcc 20109520DNAhomo sapiens
1095cagcctgtcg tcagcccaca 20109620DNAhomo sapiens 1096acaccttgtg
ggctgacgac 20109720DNAhomo sapiens 1097tcgtcagccc acaaggtgtc
20109820DNAhomo sapiens 1098tcagcccaca aggtgtcagg 20109920DNAhomo
sapiens 1099cagcccacaa ggtgtcaggt 20110020DNAhomo sapiens
1100acacccacct gacaccttgt 20110120DNAhomo sapiens 1101cacacccacc
tgacaccttg 20110220DNAhomo sapiens 1102accaaccctt ctttcaggca
20110320DNAhomo sapiens 1103ttctttcagg catggcaagc 20110420DNAhomo
sapiens 1104ggcatggcaa gccggctgct 20110520DNAhomo sapiens
1105gcatggcaag ccggctgctc 20110620DNAhomo sapiens 1106caaatgaggg
cccgagcagc 20110720DNAhomo sapiens 1107agcaggtagg actcaaatga
20110820DNAhomo sapiens 1108cagcaggtag gactcaaatg 20110920DNAhomo
sapiens 1109ggtcagttcg ggcagcaggt 20111020DNAhomo sapiens
1110atctggtcag ttcgggcagc 20111120DNAhomo sapiens 1111cagtcatatc
tggtcagttc 20111220DNAhomo sapiens 1112acagtcatat ctggtcagtt
20111320DNAhomo sapiens 1113actgaccaga tatgactgtg 20111420DNAhomo
sapiens 1114gttcacctca cagtcatatc 20111520DNAhomo sapiens
1115tgaggtgaac gtgcccgtgc 20111620DNAhomo sapiens 1116gaggtgaacg
tgcccgtgct 20111720DNAhomo sapiens 1117agcgtggagc ttcccagcac
20111820DNAhomo sapiens 1118gagcgtggag cttcccagca 20111920DNAhomo
sapiens 1119ggaagctcca cgctcctgca 20112020DNAhomo sapiens
1120agctccacgc tcctgcaagg 20112120DNAhomo sapiens 1121gctccacgct
cctgcaagga 20112220DNAhomo sapiens 1122ctccacgctc ctgcaaggag
20112320DNAhomo sapiens 1123gtcccctcct tgcaggagcg 20112420DNAhomo
sapiens 1124tgaggaggtc ccctccttgc 20112520DNAhomo sapiens
1125aggggacctc ctcagagccc 20112620DNAhomo sapiens 1126cctcctcaga
gccctggacc 20112720DNAhomo sapiens 1127cctggtccag ggctctgagg
20112820DNAhomo sapiens 1128tggcctggtc cagggctctg 20112920DNAhomo
sapiens 1129ggctcaggtg gcctggtcca 20113020DNAhomo sapiens
1130tggctcaggt ggcctggtcc
20113120DNAhomo sapiens 1131tggaccaggc cacctgagcc 20113220DNAhomo
sapiens 1132aaggcctggc tcaggtggcc 20113320DNAhomo sapiens
1133ggtagaaggc ctggctcagg 20113420DNAhomo sapiens 1134tctgagctgt
gatcttgtct 20113520DNAhomo sapiens 1135ccgcagccta taacaacttt
20113620DNAhomo sapiens 1136ccgaaagttg ttataggctg 20113720DNAhomo
sapiens 1137gctcttccga aagttgttat 20113820DNAhomo sapiens
1138taacaacttt cggaagagca 20113920DNAhomo sapiens 1139cggaagagca
tggacagcat 20114020DNAhomo sapiens 1140cataggaaag aagcaatatc
20114120DNAhomo sapiens 1141aagcaatatc aggtccagca 20114220DNAhomo
sapiens 1142agcaatatca ggtccagcat 20114320DNAhomo sapiens
1143tgtagctgca ggacccatgc 20114420DNAhomo sapiens 1144caggaggaaa
gtgtagctgc 20114520DNAhomo sapiens 1145cactttcctc ctgccagaga
20114620DNAhomo sapiens 1146agttgtccat ctctggcagg 20114720DNAhomo
sapiens 1147ggcagttgtc catctctggc 20114820DNAhomo sapiens
1148gagcggcagt tgtccatctc 20114920DNAhomo sapiens 1149cgtaggggct
ggaggaagag 20115020DNAhomo sapiens 1150attggacacg taggggctgg
20115120DNAhomo sapiens 1151agcattggac acgtaggggc 20115220DNAhomo
sapiens 1152gcacagcatt ggacacgtag 20115320DNAhomo sapiens
1153tgcacagcat tggacacgta 20115420DNAhomo sapiens 1154ctgcacagca
ttggacacgt 20115520DNAhomo sapiens 1155acgtgtccaa tgctgtgcag
20115620DNAhomo sapiens 1156cgtgtccaat gctgtgcaga 20115720DNAhomo
sapiens 1157cgcgtccctc tgcacagcat 20115820DNAhomo sapiens
1158gccgctcgaa tacgatgact 20115920DNAhomo sapiens 1159accgagtcat
cgtattcgag 20116020DNAhomo sapiens 1160aatacgatga ctcggtgcag
20116120DNAhomo sapiens 1161ggtgcagagg ctgcaagtgc 20116220DNAhomo
sapiens 1162gcaagtgctg gagaacatca 20116320DNAhomo sapiens
1163tcatggaaaa caacactcag 20116420DNAhomo sapiens 1164caacactcag
tggctaatga 20116520DNAhomo sapiens 1165actcagtggc taatgaaggt
20116620DNAhomo sapiens 1166ctagcttgag aattatatcc 20116720DNAhomo
sapiens 1167tttctttctt catgttgtcc 20116820DNAhomo sapiens
1168ggacaacatg aagaaagaaa 20116920DNAhomo sapiens 1169gaatgcagta
cagaaccaga 20117020DNAhomo sapiens 1170tttctatcat cacagccgtc
20117120DNAhomo sapiens 1171acggctgtga tgatagaaat 20117220DNAhomo
sapiens 1172cggctgtgat gatagaaata 20117320DNAhomo sapiens
1173aaacctgttg aaccaaacag 20117420DNAhomo sapiens 1174gctccgctgt
ttggttcaac 20117520DNAhomo sapiens 1175accaaacagc ggagcaaacg
20117620DNAhomo sapiens 1176tccgcgtttg ctccgctgtt 20117720DNAhomo
sapiens 1177aacgcggaag ttaactgatg 20117820DNAhomo sapiens
1178ctagcttgag aattatatcc 20117920DNAhomo sapiens 1179tttctttctt
catgttgtcc 20118020DNAhomo sapiens 1180ggacaacatg aagaaagaaa
20118120DNAhomo sapiens 1181gaatgcagta cagaaccaga 20118220DNAhomo
sapiens 1182tttctatcat cacagccgtc 20118320DNAhomo sapiens
1183acggctgtga tgatagaaat 20118420DNAhomo sapiens 1184cggctgtgat
gatagaaata 20118520DNAhomo sapiens 1185aaacctgttg aaccaaacag
20118620DNAhomo sapiens 1186gctccgctgt ttggttcaac 20118720DNAhomo
sapiens 1187accaaacagc ggagcaaacg 20118820DNAhomo sapiens
1188tccgcgtttg ctccgctgtt 20118920DNAhomo sapiens 1189aacgcggaag
ttaactgatg 20119020DNAhomo sapiens 1190tacaagtttc ctagaaaaga
20119120DNAhomo sapiens 1191tagctagcac cttcttttct 20119220DNAhomo
sapiens 1192agaaaagaag gtgctagcta 20119320DNAhomo sapiens
1193cttcttttat tgactgtagt 20119420DNAhomo sapiens 1194agaagagaaa
gatcagctac 20119520DNAhomo sapiens 1195ttcaatgatg gaattttgct
20119620DNAhomo sapiens 1196tttttctagt tcttcaatga 20119720DNAhomo
sapiens 1197aaaaaaaata gtgactgcca 20119820DNAhomo sapiens
1198aagaactgaa ttattcaccg 20119920DNAhomo sapiens 1199gaagcagcaa
catgatctca 20120020DNAhomo sapiens 1200tgtaaactta cagtttgatg
20120120DNAhomo sapiens 1201ctatttttta aaagcagcta 20120220DNAhomo
sapiens 1202gttcttcttt agcaacagtg 20120320DNAhomo sapiens
1203tgttcttctt tagcaacagt 20120420DNAhomo sapiens 1204ttgttcttct
ttagcaacag 20120520DNAhomo sapiens 1205tgtgctgaag tattcaaatc
20120620DNAhomo sapiens 1206aaatcaggac acaccacgaa 20120720DNAhomo
sapiens 1207taacgtgtag atgccattcg 20120820DNAhomo sapiens
1208tgatctcttc tgtagaatta 20120920DNAhomo sapiens 1209ttgatctctt
ctgtagaatt 20121020DNAhomo sapiens 1210taattctaca gaagagatca
20121120DNAhomo sapiens 1211ctacagaaga gatcaaggtg 20121220DNAhomo
sapiens 1212tttgcaggcc tactgtgaca 20121320DNAhomo sapiens
1213gcctactgtg acatggaagc 20121420DNAhomo sapiens 1214tccagcttcc
atgtcacagt 20121520DNAhomo sapiens 1215tactgtgaca tggaagctgg
20121620DNAhomo sapiens 1216tgtgacatgg aagctggagg 20121720DNAhomo
sapiens 1217gacatggaag ctggaggagg 20121820DNAhomo sapiens
1218acatggaagc tggaggaggc 20121920DNAhomo sapiens 1219tggaagctgg
aggaggcggg 20122020DNAhomo sapiens 1220gacaattatt cagcgacgtg
20122120DNAhomo sapiens 1221attattcagc gacgtgagga 20122220DNAhomo
sapiens 1222atggcagcgt tgattttcag 20122320DNAhomo sapiens
1223gcgttgattt tcagaggact 20122420DNAhomo sapiens 1224gacttggaaa
gaatataaag 20122520DNAhomo sapiens 1225ggaaagaata taaagtggta
20122620DNAhomo sapiens 1226cagggatttg gtaacccttc 20122720DNAhomo
sapiens 1227gtaacccttc aggagaatat 20122820DNAhomo sapiens
1228cccttcagga gaatattggc 20122920DNAhomo sapiens 1229ccagccaata
ttctcctgaa 20123020DNAhomo sapiens 1230ccttcaggag aatattggct
20123120DNAhomo sapiens 1231cccagccaat attctcctga 20123220DNAhomo
sapiens 1232ttaaaataca ccttaaagac 20123320DNAhomo sapiens
1233taaaatacac cttaaagact 20123420DNAhomo sapiens 1234atacacctta
aagactggga 20123520DNAhomo sapiens 1235tacaccttaa agactgggaa
20123620DNAhomo sapiens 1236cattcccttc ccagtcttta 20123720DNAhomo
sapiens 1237taaagactgg gaagggaatg 20123820DNAhomo sapiens
1238caagtgaaga actcaattat 20123920DNAhomo sapiens 1239gcttacagga
ttcaccttaa 20124020DNAhomo sapiens 1240attcacctta aaggacttac
20124120DNAhomo sapiens 1241ttcaccttaa aggacttaca 20124220DNAhomo
sapiens 1242ctgtccctgt aagtccttta 20124320DNAhomo sapiens
1243aaaggactta cagggacagc 20124420DNAhomo sapiens 1244gctgatgctg
cttattttgc 20124520DNAhomo sapiens 1245ataagcagca tcagccaacc
20124620DNAhomo sapiens 1246tgctaaaatc atttcctggt 20124720DNAhomo
sapiens 1247tttgtgctaa aatcatttcc 20124820DNAhomo sapiens
1248aggaaatgat tttagcacaa 20124920DNAhomo sapiens 1249aatgatttta
gcacaaagga 20125020DNAhomo sapiens 1250aaatgttcac aaatgctaac
20125120DNAhomo sapiens 1251tgttcacaaa tgctaacagg 20125220DNAhomo
sapiens 1252cacaaatgct aacaggaggt 20125320DNAhomo sapiens
1253acaaatgcta acaggaggta 20125420DNAhomo sapiens 1254ggctggtggt
ttgatgcatg 20125520DNAhomo sapiens 1255tgtggtcctt ccaacttgaa
20125620DNAhomo sapiens 1256tacattccgt tcaagttgga 20125720DNAhomo
sapiens 1257atagtacatt ccgttcaagt 20125820DNAhomo sapiens
1258acggaatgta ctatccacag 20125920DNAhomo sapiens 1259ttatttgtgt
tctgcctctg 20126020DNAhomo sapiens 1260cagaacacaa ataagttcaa
20126120DNAhomo sapiens 1261ataagttcaa cggcattaaa 20126220DNAhomo
sapiens 1262acggcattaa atggtactac 20126320DNAhomo sapiens
1263attaaatggt actactggaa 20126420DNAhomo sapiens 1264tggtactact
ggaaaggctc 20126520DNAhomo sapiens 1265aggctcaggc tattcgctca
20126620DNAhomo sapiens 1266tggtcggatc atcatggttg 20126720DNAhomo
sapiens 1267atctgctggt cggatcatca 20126820DNAhomo sapiens
1268atgtttagaa atctgctggt 20126920DNAhomo sapiens 1269tgggatgttt
agaaatctgc 20127020DNAhomo sapiens 1270aggctaccta agaggatgag
20127120DNAhomo sapiens 1271gaggatgagc ggtgctccga 20127220DNAhomo
sapiens 1272atgagcggtg ctccgacggc 20127320DNAhomo sapiens
1273tgagcggtgc tccgacggcc 20127420DNAhomo sapiens 1274gagcggtgct
ccgacggccg 20127520DNAhomo sapiens 1275atcagggctg ccccggccgt
20127620DNAhomo sapiens 1276gcagagcatc agggctgccc 20127720DNAhomo
sapiens 1277ggtggcggcg cagagcatca 20127820DNAhomo sapiens
1278cggtggcggc gcagagcatc 20127920DNAhomo sapiens 1279gctcagtagc
acggcggtgg 20128020DNAhomo sapiens 1280agcgctcagt agcacggcgg
20128120DNAhomo sapiens 1281ctgagcgctc agtagcacgg 20128220DNAhomo
sapiens 1282cgccgtgcta ctgagcgctc 20128320DNAhomo sapiens
1283gccgtgctac tgagcgctca 20128420DNAhomo sapiens 1284gccctgagcg
ctcagtagca 20128520DNAhomo sapiens 1285gtgctactga gcgctcaggg
20128620DNAhomo sapiens 1286cgcggcgact tggactgcac 20128720DNAhomo
sapiens 1287gcgcggcgac ttggactgca 20128820DNAhomo sapiens
1288ggacgcaaag cgcggcgact 20128920DNAhomo sapiens 1289agtcgccgcg
ctttgcgtcc 20129020DNAhomo sapiens 1290gtcgccgcgc tttgcgtcct
20129120DNAhomo sapiens 1291tcgtcccagg acgcaaagcg 20129220DNAhomo
sapiens 1292caggacattc atctcgtccc 20129320DNAhomo sapiens
1293ctgggacgag atgaatgtcc 20129420DNAhomo sapiens 1294gagatgaatg
tcctggcgca 20129520DNAhomo sapiens 1295gctgcaggag tccgtgcgcc
20129620DNAhomo sapiens 1296gcgcacggac tcctgcagct 20129720DNAhomo
sapiens 1297cggactcctg cagctcggcc 20129820DNAhomo sapiens
1298ggactcctgc agctcggcca 20129920DNAhomo sapiens 1299gactcctgca
gctcggccag 20130020DNAhomo sapiens 1300gcagcccctg gccgagctgc
20130120DNAhomo sapiens 1301ccaggggctg cgcgaacacg 20130220DNAhomo
sapiens 1302ccgcgtgttc gcgcagcccc 20130320DNAhomo sapiens
1303cagcgcgctc agctgactgc 20130420DNAhomo sapiens 1304ccgcagtcag
ctgagcgcgc 20130520DNAhomo sapiens 1305ccagcgcgct cagctgactg
20130620DNAhomo sapiens 1306gtcagctgag cgcgctggag 20130720DNAhomo
sapiens 1307gagcggcgcc tgagcgcgtg 20130820DNAhomo sapiens
1308agcggcgcct gagcgcgtgc 20130920DNAhomo sapiens 1309aggcggaccc
gcacgcgctc 20131020DNAhomo sapiens 1310cgcgtgcggg tccgcctgtc
20131120DNAhomo sapiens 1311gcgtgcgggt ccgcctgtca 20131220DNAhomo
sapiens 1312gtccgcctgt cagggaaccg 20131320DNAhomo sapiens
1313tccgcctgtc agggaaccga 20131420DNAhomo sapiens 1314ccgcctgtca
gggaaccgag 20131520DNAhomo sapiens 1315cccctcggtt ccctgacagg
20131620DNAhomo sapiens 1316ggacccctcg gttccctgac 20131720DNAhomo
sapiens 1317cgggaggtcg gtggacccct 20131820DNAhomo sapiens
1318aggggctaac gggaggtcgg 20131920DNAhomo sapiens
1319ctcaggggct aacgggaggt 20132020DNAhomo sapiens 1320ggctctcagg
ggctaacggg 20132120DNAhomo sapiens 1321tcccgttagc ccctgagagc
20132220DNAhomo sapiens 1322cccgttagcc cctgagagcc 20132320DNAhomo
sapiens 1323cccggctctc aggggctaac 20132420DNAhomo sapiens
1324acccggctct caggggctaa 20132520DNAhomo sapiens 1325gttagcccct
gagagccggg 20132620DNAhomo sapiens 1326agggtccacc cggctctcag
20132720DNAhomo sapiens 1327cagggtccac ccggctctca 20132820DNAhomo
sapiens 1328tcagggtcca cccggctctc 20132920DNAhomo sapiens
1329tgagagccgg gtggaccctg 20133020DNAhomo sapiens 1330gaaggacctc
agggtccacc 20133120DNAhomo sapiens 1331gcaggctgtg aaggacctca
20133220DNAhomo sapiens 1332tgcaggctgt gaaggacctc 20133320DNAhomo
sapiens 1333tgaggtcctt cacagcctgc 20133420DNAhomo sapiens
1334cacgtacctg caggctgtga 20133520DNAhomo sapiens 1335ccctggggac
acgtacctgc 20133620DNAhomo sapiens 1336ccctccccag acacaactca
20133720DNAhomo sapiens 1337tgagccttga gttgtgtctg 20133820DNAhomo
sapiens 1338ctgagccttg agttgtgtct 20133920DNAhomo sapiens
1339tctgagcctt gagttgtgtc 20134020DNAhomo sapiens 1340aactcaaggc
tcagaacagc 20134120DNAhomo sapiens 1341gatccagcaa ctcttccaca
20134220DNAhomo sapiens 1342ccagcaactc ttccacaagg 20134320DNAhomo
sapiens 1343ccaccttgtg gaagagttgc 20134420DNAhomo sapiens
1344gctgctgctg ggccaccttg 20134520DNAhomo sapiens 1345acaaggtggc
ccagcagcag 20134620DNAhomo sapiens 1346ggcccagcag cagcggcacc
20134720DNAhomo sapiens 1347ctccaggtgc cgctgctgct 20134820DNAhomo
sapiens 1348tctccaggtg ccgctgctgc 20134920DNAhomo sapiens
1349ttcgcaggtg ctgcttctcc 20135020DNAhomo sapiens 1350tttgcagatg
ctgaattcgc 20135120DNAhomo sapiens 1351aattcagcat ctgcaaagcc
20135220DNAhomo sapiens 1352cccttgatcc tagggttacc 20135320DNAhomo
sapiens 1353cccatcctag tttggcctcc 20135420DNAhomo sapiens
1354gtggtccagg aggccaaact 20135520DNAhomo sapiens 1355ctaggtgctt
gtggtccagg 20135620DNAhomo sapiens 1356ggtctaggtg cttgtggtcc
20135720DNAhomo sapiens 1357ccacaagcac ctagaccatg 20135820DNAhomo
sapiens 1358cctcatggtc taggtgcttg 20135920DNAhomo sapiens
1359caagcaccta gaccatgagg 20136020DNAhomo sapiens 1360gcttggccac
ctcatggtct 20136120DNAhomo sapiens 1361gggcaggctt ggccacctca
20136220DNAhomo sapiens 1362ccaagcctgc ccgaagaaag 20136320DNAhomo
sapiens 1363cctctttctt cgggcaggct 20136420DNAhomo sapiens
1364ggcagcctct ttcttcgggc 20136520DNAhomo sapiens 1365ctcgggcagc
ctctttcttc 20136620DNAhomo sapiens 1366tctcgggcag cctctttctt
20136720DNAhomo sapiens 1367aagaaagagg ctgcccgaga 20136820DNAhomo
sapiens 1368tcaactggct gggccatctc 20136920DNAhomo sapiens
1369gtcaactggc tgggccatct 20137020DNAhomo sapiens 1370gatggcccag
ccagttgacc 20137120DNAhomo sapiens 1371gtgagccggg tcaactggct
20137220DNAhomo sapiens 1372tgtgagccgg gtcaactggc 20137320DNAhomo
sapiens 1373acattgtgag ccgggtcaac 20137420DNAhomo sapiens
1374ggcggctgac attgtgagcc 20137520DNAhomo sapiens 1375aggcggctga
cattgtgagc 20137620DNAhomo sapiens 1376gcagacactc acggtgcagg
20137720DNAhomo sapiens 1377ggggcagaca ctcacggtgc 20137820DNAhomo
sapiens 1378atctcccttc agggctgccc 20137920DNAhomo sapiens
1379tctcccttca gggctgccca 20138020DNAhomo sapiens 1380agggctgccc
agggattgcc 20138120DNAhomo sapiens 1381aacagctcct ggcaatccct
20138220DNAhomo sapiens 1382gaacagctcc tggcaatccc 20138320DNAhomo
sapiens 1383ggattgccag gagctgttcc 20138420DNAhomo sapiens
1384tgccaggagc tgttccaggt 20138520DNAhomo sapiens 1385ccaggagctg
ttccaggttg 20138620DNAhomo sapiens 1386ccccaacctg gaacagctcc
20138720DNAhomo sapiens 1387agctgttcca ggttggggag 20138820DNAhomo
sapiens 1388cactctgcct ctccccaacc 20138920DNAhomo sapiens
1389caggttgggg agaggcagag 20139020DNAhomo sapiens 1390actatttgaa
atccagcctc 20139120DNAhomo sapiens 1391ctatttgaaa tccagcctca
20139220DNAhomo sapiens 1392tatttgaaat ccagcctcag 20139320DNAhomo
sapiens 1393atggcggaga cccctgaggc 20139420DNAhomo sapiens
1394aaaaatggcg gagacccctg 20139520DNAhomo sapiens 1395tcaggggtct
ccgccatttt 20139620DNAhomo sapiens 1396ttgcagttca ccaaaaatgg
20139720DNAhomo sapiens 1397atcttgcagt tcaccaaaaa 20139820DNAhomo
sapiens 1398gtgaactgca agatgacctc 20139920DNAhomo sapiens
1399actgcaagat gacctcaggt 20140020DNAhomo sapiens 1400ctgcaagatg
acctcaggta 20140120DNAhomo sapiens 1401ggactaacac accctacctg
20140220DNAhomo sapiens 1402gtacctttct gggcagatgg 20140320DNAhomo
sapiens 1403ctttctgggc agatggaggc 20140420DNAhomo sapiens
1404gaggctggac agtaattcag 20140520DNAhomo sapiens 1405gtaattcaga
ggcgccacga 20140620DNAhomo sapiens 1406gaggcgccac gatggctcag
20140720DNAhomo sapiens 1407tgaagtccac tgagccatcg 20140820DNAhomo
sapiens 1408atggctcagt ggacttcaac 20140920DNAhomo sapiens
1409cagtggactt caaccggccc 20141020DNAhomo sapiens 1410agtggacttc
aaccggccct 20141120DNAhomo sapiens 1411ccggccctgg gaagcctaca
20141220DNAhomo sapiens 1412ccttgtaggc ttcccagggc 20141320DNAhomo
sapiens 1413gccctgggaa gcctacaagg 20141420DNAhomo sapiens
1414ccctgggaag cctacaaggc 20141520DNAhomo sapiens 1415cccgccttgt
aggcttccca 20141620DNAhomo sapiens 1416cctgggaagc ctacaaggcg
20141720DNAhomo sapiens 1417ccccgccttg taggcttccc 20141820DNAhomo
sapiens 1418gaagcctaca aggcggggtt 20141920DNAhomo sapiens
1419aagcctacaa ggcggggttt 20142020DNAhomo sapiens 1420agcctacaag
gcggggtttg 20142120DNAhomo sapiens 1421atccccaaac cccgccttgt
20142220DNAhomo sapiens 1422gcggggtttg gggatcccca 20142320DNAhomo
sapiens 1423ggtttgggga tccccacggt 20142420DNAhomo sapiens
1424cactagaaac acctaccgtg 20142520DNAhomo sapiens 1425ccactagaaa
cacctaccgt 20142620DNAhomo sapiens 1426ctcccactcc aggcgagttc
20142720DNAhomo sapiens 1427cactccaggc gagttctggc 20142820DNAhomo
sapiens 1428actccaggcg agttctggct 20142920DNAhomo sapiens
1429agacccagcc agaactcgcc 20143020DNAhomo sapiens 1430aggcgagttc
tggctgggtc 20143120DNAhomo sapiens 1431gttctggctg ggtctggaga
20143220DNAhomo sapiens 1432ggagaaggtg catagcatca 20143320DNAhomo
sapiens 1433gagaaggtgc atagcatcac 20143420DNAhomo sapiens
1434agaaggtgca tagcatcacg 20143520DNAhomo sapiens 1435gaaggtgcat
agcatcacgg 20143620DNAhomo sapiens 1436gggggaccgc aacagccgcc
20143720DNAhomo sapiens 1437gcacggccag gcggctgttg 20143820DNAhomo
sapiens 1438gccgcctggc cgtgcagctg 20143920DNAhomo sapiens
1439ccgcctggcc gtgcagctgc 20144020DNAhomo sapiens 1440cccgcagctg
cacggccagg 20144120DNAhomo sapiens 1441agtcccgcag ctgcacggcc
20144220DNAhomo sapiens 1442tggccgtgca gctgcgggac 20144320DNAhomo
sapiens 1443ggccgtgcag ctgcgggact 20144420DNAhomo sapiens
1444atcccagtcc cgcagctgca 20144520DNAhomo sapiens 1445gtgcagctgc
gggactggga 20144620DNAhomo sapiens 1446cacggagaac tgcagcaact
20144720DNAhomo sapiens 1447gctgcagttc tccgtgcacc 20144820DNAhomo
sapiens 1448ctgcagttct ccgtgcacct 20144920DNAhomo sapiens
1449cagttctccg tgcacctggg 20145020DNAhomo sapiens 1450ctccgtgcac
ctgggtggcg 20145120DNAhomo sapiens 1451gtcctcgcca cccaggtgca
20145220DNAhomo sapiens 1452gcacctgggt ggcgaggaca 20145320DNAhomo
sapiens 1453aggccgtgtc ctcgccaccc 20145420DNAhomo sapiens
1454tgcagtgagc tgcaggctat 20145520DNAhomo sapiens 1455cctgcagctc
actgcacccg 20145620DNAhomo sapiens 1456ccacgggtgc agtgagctgc
20145720DNAhomo sapiens 1457cagctcactg cacccgtggc 20145820DNAhomo
sapiens 1458tgcacccgtg gccggccagc 20145920DNAhomo sapiens
1459gcacccgtgg ccggccagct 20146020DNAhomo sapiens 1460gcgcccagct
ggccggccac 20146120DNAhomo sapiens 1461ggcgcccagc tggccggcca
20146220DNAhomo sapiens 1462ggtggtggcg cccagctggc 20146320DNAhomo
sapiens 1463ggacggtggt ggcgcccagc 20146420DNAhomo sapiens
1464gccaccaccg tcccacccag 20146520DNAhomo sapiens 1465gccgctgggt
gggacggtgg 20146620DNAhomo sapiens 1466gaggccgctg ggtgggacgg
20146720DNAhomo sapiens 1467ggagaggccg ctgggtggga 20146820DNAhomo
sapiens 1468gtacggagag gccgctgggt 20146920DNAhomo sapiens
1469ggtacggaga ggccgctggg 20147020DNAhomo sapiens 1470aagggtacgg
agaggccgct 20147120DNAhomo sapiens 1471gaagggtacg gagaggccgc
20147220DNAhomo sapiens 1472aagtggagaa gggtacggag 20147320DNAhomo
sapiens 1473tctccgtacc cttctccact 20147420DNAhomo sapiens
1474ctccgtaccc ttctccactt 20147520DNAhomo sapiens 1475gtcccaagtg
gagaagggta 20147620DNAhomo sapiens 1476acccttctcc acttgggacc
20147720DNAhomo sapiens 1477tcctggtccc aagtggagaa 20147820DNAhomo
sapiens 1478atcctggtcc caagtggaga 20147920DNAhomo sapiens
1479gtcgtgatcc tggtcccaag 20148020DNAhomo sapiens 1480accaggatca
cgacctccgc 20148120DNAhomo sapiens 1481ccaggatcac gacctccgca
20148220DNAhomo sapiens 1482ccctgcggag gtcgtgatcc 20148320DNAhomo
sapiens 1483cgcagttctt gtccctgcgg 20148420DNAhomo sapiens
1484tggcgcagtt cttgtccctg 20148520DNAhomo sapiens 1485aactgcgcca
agagcctctc 20148620DNAhomo sapiens 1486ctgctcacca gagaggctct
20148720DNAhomo sapiens 1487gcagggcctg ctcaccagag 20148820DNAhomo
sapiens 1488ccctgacccc ggcaggaggc 20148920DNAhomo sapiens
1489tgaccccggc aggaggctgg 20149020DNAhomo sapiens 1490ccggcaggag
gctggtggtt 20149120DNAhomo sapiens 1491gttgaggttg gaatggctgc
20149220DNAhomo sapiens 1492tgcagccatt ccaacctcaa 20149320DNAhomo
sapiens 1493actggccgtt gaggttggaa 20149420DNAhomo sapiens
1494gaagtactgg ccgttgaggt 20149520DNAhomo sapiens 1495agcggaagta
ctggccgttg 20149620DNAhomo sapiens 1496gtgggatgga gcggaagtac
20149720DNAhomo sapiens 1497tccgctccat cccacagcag 20149820DNAhomo
sapiens 1498gccgctgctg tgggatggag 20149920DNAhomo sapiens
1499cttctgccgc tgctgtggga 20150020DNAhomo sapiens 1500taagcttctg
ccgctgctgt 20150120DNAhomo sapiens 1501ttaagcttct gccgctgctg
20150220DNAhomo sapiens 1502gcagcggcag aagcttaaga 20150320DNAhomo
sapiens 1503cagcggcaga agcttaagaa 20150420DNAhomo sapiens
1504agcttaagaa gggaatcttc 20150520DNAhomo sapiens 1505agggaatctt
ctggaagacc 20150620DNAhomo sapiens 1506gaatcttctg gaagacctgg
20150720DNAhomo sapiens 1507aatcttctgg aagacctggc
20150820DNAhomo sapiens 1508atcttctgga agacctggcg 20150920DNAhomo
sapiens 1509cgggtagtag cggccccgcc 20151020DNAhomo sapiens
1510gggccgctac tacccgctgc 20151120DNAhomo sapiens 1511tggcctgcag
cgggtagtag 20151220DNAhomo sapiens 1512acatggtggt ggcctgcagc
20151320DNAhomo sapiens 1513aacatggtgg tggcctgcag 20151420DNAhomo
sapiens 1514gggctggatc aacatggtgg 20151520DNAhomo sapiens
1515catgggctgg atcaacatgg 20151620DNAhomo sapiens 1516caccatgttg
atccagccca 20151720DNAhomo sapiens 1517tgccatgggc tggatcaaca
20151820DNAhomo sapiens 1518gatccagccc atggcagcag 20151920DNAhomo
sapiens 1519ctgcctctgc tgccatgggc 20152020DNAhomo sapiens
1520gaggctgcct ctgctgccat 20152120DNAhomo sapiens 1521ggaggctgcc
tctgctgcca 20152220DNAhomo sapiens 1522ggcccagcca ggacgctagg 20
* * * * *