U.S. patent application number 16/471443 was filed with the patent office on 2020-07-02 for oleic acid-enriched plant body having genetically modified fad2 and production method thereof.
The applicant listed for this patent is TOOLGEN INCORPORATED. Invention is credited to Min Hee JUNG, Seok Joong KIM, Ye Seul KIM, Ok Jae KOO.
Application Number | 20200208166 16/471443 |
Document ID | / |
Family ID | 62626791 |
Filed Date | 2020-07-02 |
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United States Patent
Application |
20200208166 |
Kind Code |
A1 |
KIM; Seok Joong ; et
al. |
July 2, 2020 |
Oleic Acid-Enriched Plant Body Having Genetically Modified FAD2 And
Production Method Thereof
Abstract
The present invention relates to an artificially manipulated
unsaturated fatty acid biosynthesis-associated factor and use
thereof to increase the content of a specific unsaturated fatty
acid of a plant body. More particularly, the present invention
relates to a system capable of artificially controlling unsaturated
fatty acid biosynthesis and a plant body produced thereby, which
include an artificially manipulated unsaturated fatty acid
biosynthesis-associated factor to control unsaturated fatty acid
biosynthesis and a composition capable of artificially manipulating
the factor. In a specific aspect, the present invention relates to
artificially manipulated unsaturated fatty acid
biosynthesis-associated factors such as FAD2, FAD3, FADE, FAD7 and
FAD8 and/or an unsaturated fatty acid biosynthesis controlling
system by an expression product thereof.
Inventors: |
KIM; Seok Joong; (Seoul,
KR) ; KOO; Ok Jae; (Gyeonggi-do, KR) ; JUNG;
Min Hee; (Seoul, KR) ; KIM; Ye Seul; (Seoul,
KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TOOLGEN INCORPORATED |
Seoul |
|
KR |
|
|
Family ID: |
62626791 |
Appl. No.: |
16/471443 |
Filed: |
September 26, 2017 |
PCT Filed: |
September 26, 2017 |
PCT NO: |
PCT/KR2017/010576 |
371 Date: |
February 4, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62438018 |
Dec 22, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/20 20170501;
C12N 15/10 20130101; C12N 15/11 20130101; C12N 15/82 20130101; C12N
9/22 20130101; C12N 15/8247 20130101; C12N 2800/80 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C12N 15/11 20060101 C12N015/11; C12N 9/22 20060101
C12N009/22 |
Claims
1-25. (canceled)
26. A plant comprising an artificially manipulated genome, wherein
the artificially manipulated genome includes one or more
modifications selected from the group consisting of (i) a deletion
of 1 to 30 nucleotides; (ii) an insertion of one or more foreign
nucleotides; (iii) a deletion of 1 to 30 nucleotides and an
insertion of one or more foreign nucleotides; and (iv) a
substitution with one or more nucleotides different from a
wild-type gene, in a continuous 1 to 50 nucleotides sequence
including a PAM sequence in a nucleic acid sequence constituting a
FAD2 gene, wherein the PAM sequence is one or more sequences
selected from 5'-NGG-3'; 5'-NNNNRYAC-3'; 5'-NNAGAAW-3';
5'-NNNNGATT-3'; 5'-NNGRR(T)-3'; and 5'-TTN-3', wherein the each N
is independently A, T, C or G, the each R is independently A or G,
the Y is C or T, and the W is A or T, wherein the plant comprising
an artificially manipulated genome has at least one of the
phenotype selected from the group consisting of, as compared with a
wild type plant: (a) a decreased expression of a FAD2 protein; (b)
a decreased RNA transcripts of a FAD2 gene; (c) an increased
expression of a mutated FAD2 protein; (d) an increased content of
C8.about.24:D1 unsaturated fatty acid; and (e) a decreased content
of C8.about.24:D2 unsaturated fatty acid.
27. The plant of claim 26, wherein the continuous 1 to 50
nucleotides sequence including a PAM sequence in a nucleic acid
sequence constituting a FAD2 gene comprises at least one of the
nucleotide sequence selected from SEQ ID NO: 1 to 30.
28. The plant of claim 26, wherein the C8.about.24:D1 unsaturated
fatty acid is a C16.about.22:D1 unsaturated fatty acid.
29. The plant of claim 26, wherein the C8.about.24:D1 unsaturated
fatty acid is a C18:D1 unsaturated fatty acid.
30. The plant of claim 26, wherein the C8.about.24:D2 unsaturated
fatty acid is a C16.about.22:D2 unsaturated fatty acid.
31. The plant of claim 26, wherein the C8.about.24:D2 unsaturated
fatty acid is a C18:D2 unsaturated fatty acid.
32. The plant of claim 26, wherein the plant is a soybean.
33. A composition for gene manipulation, which is used to produce a
plant including an artificially manipulated genome, comprising: a
guide nucleic acid capable of targeting a FAD2 gene, or a nucleic
acid sequence encoding the same; and an editor protein, or a
nucleic acid sequence encoding the same, wherein the guide nucleic
acid is a RNA sequence capable of binding a target region in a
nucleic acid sequence constituting a FAD2 gene, wherein the target
region gene comprises at least one of the nucleotide sequence
selected from SEQ ID NO: 1 to 30, and 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 Staphylococcus aureus-derived Cas9 protein, a
Neisseria meningitidis-derived Cas9 protein, and a Cpf1
protein.
34. The composition of claim 33, wherein the composition for gene
manipulation is formed in an agrobacterium vector system.
35. The composition of claim 33, wherein the composition for gene
manipulation is formed in a viral vector system.
36. The composition of claim 35, wherein the viral vector includes
one or more selected from a mosaic virus, a retrovirus, a
lentivirus, an adenovirus, an adeno-associated virus (AAV), a
vaccinia virus, a poxvirus and a herpes simplex virus.
37. A method for producing a plant including an artificially
manipulated genome, comprising an introducing (administering) a
composition to a subject, wherein the subject is a plant cell, a
seed, a part of a plant body or whole plant body, wherein the
composition comprising: a guide nucleic acid capable of targeting a
FAD2 gene, or a nucleic acid sequence encoding the same; and an
editor protein, or a nucleic acid sequence encoding the same,
wherein the guide nucleic acid is a RNA sequence capable of binding
a target region in a nucleic acid sequence constituting a FAD2
gene, wherein the target region gene comprises at least one of the
nucleotide sequence selected from SEQ ID NO: 1 to 30, 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 Staphylococcus aureus-derived
Cas9 protein, a Neisseria meningitidis-derived Cas9 protein, and a
Cpf1 protein, wherein the artificially manipulated genome includes
one or more modifications selected from the group consisting of (i)
a deletion of 1 to 30 nucleotides; (ii) an insertion of one or more
foreign nucleotides; (iii) a deletion of 1 to 30 nucleotides and an
insertion of one or more foreign nucleotides; and (iv) a
substitution with one or more nucleotides different from a
wild-type gene, in a continuous 1 to 50 nucleotides sequence
including a PAM sequence in a nucleic acid sequence constituting a
FAD2 gene, wherein the PAM sequence is one or more sequences
selected from 5'-NGG-3'; 5'-NNNNRYAC-3'; 5'-NNAGAAW-3';
5'-NNNNGATT-3'; 5'-NNGRR(T)-3'; and 5'-TTN-3', wherein the each N
is independently A, T, C or G, the each R is independently A or G,
the Y is C or T, and the W is A or T, wherein the plant comprising
an artificially manipulated genome has at least one of the
phenotype selected from the group consisting of, as compared with a
wild type plant: (a) a decreased expression of a FAD2 protein; (b)
a decreased RNA transcripts of a FAD2 gene; (c) an increased
expression of a mutated FAD2 protein; (d) an increased content of
C8.about.24:D1 unsaturated fatty acid; and (e) a decreased content
of C8.about.24:D2 unsaturated fatty acid.
38. The method of claim 37, wherein the composition is formed in an
agrobacterium vector system.
39. The method of claim 37, wherein the composition is formed in a
viral vector system.
40. The method of claim 39, wherein the viral vector includes one
or more selected from a mosaic virus, a retrovirus, a lentivirus,
an adenovirus, an adeno-associated virus (AAV), a vaccinia virus, a
poxvirus and a herpes simplex virus.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a national phase application of PCT
Application No. PCT/KR2017/010576, filed on Sep. 26, 2017, which
claims the benefit of and priority to U.S. Provisional Patent
Application No. 62/438,018, filed Dec. 22, 2016. The entire
disclosures of the applications identified in this paragraph are
incorporated herein by references.
FIELD
[0002] The present invention relates to the manipulation or
modification of a FAD2 gene using a CRISPR-Cas system to increase
the content of oleic acid in a plant body, and more particularly,
to a plant body increased in oleic acid content by modifying a FAD2
gene using a CRISPR-Cas system capable of targeting a corresponding
gene, a manipulation composition capable of manipulating a FAD2
gene, and a method using the same.
BACKGROUND
[0003] Soybean oil is the second most consumed edible oil in the
world due to being rich in essential fatty acids and the high
utilization of soybean oil meal as a by-product, and 45 million
tons thereof are produced annually. About 62% of the fatty acids
constituting soybean oil are polyunsaturated fatty acids (PUFAs),
and 54% of the fatty acids are linoleic acid, and 8% of the fatty
acids are linolenic acid. Since the fatty acids have two or more
double bonds, oxidation easily occurs and the oil easily becomes
rancid, such that it is difficult to be stored and distributed. (It
has a low storage and difficulty in distributions.) Therefore, to
manufacture soybean oil with a stable quality to be used in food
processing or cooking, pretreatment and purification processes are
required. Most soybean oil manufacturers maintain a certain level
of quality by preventing rancidity by treating partial
hydrogenation for adding a hydrogen to an unsaturated double bond
where oxidation easily occurs during a manufacturing process.
[0004] However, partial hydrogenation has a disadvantage of
producing trans-fatty acids having a risk in a process of
saturating the double bond of unsaturated fatty acids with
hydrogens. While, in the natural state, the production of cis-fatty
acids is dominant in oxidation, however, since trans-forms are
thermodynamically stable, trans-fatty acids, which are geometric
isomers that do not naturally occur, are produced in hydrogenation
or processing.
[0005] Due to the controversy over the risk of trans fat, in 2015,
the US FDA decided to eliminate that partially-hydrogenated oil
that is widely used in the process of manufacturing processed food
from the Generally Recognized as Safe (GRAS) list. Accordingly, US
food manufacturers are looking for different edible oils whereby
they can replace, and stop using partially-hydrogenated oils by
2018, and the related industry is expected to spend 6 billion US
dollars to establish an alternative edible oil or a new
manufacturing process. Partially-hydrogenated soybean oil is
expected to be decreased in demand by 9 million tons annually only
in the US according to the FDA action.
[0006] In addition, many countries such as Europe, Korea and Japan
as well as the US are well aware of the risk of trans fatty acids,
and with the trend to encourage people not to eat as much as
possible, globally, regulations on partially hydrogenated oil seem
to be more strengthened in the future. Therefore, there is a need
of developing edible oil products that do not contain trans fatty
acids that can replace soybean oil produced by partial
hydrogenation.
SUMMARY
Technical Problems
[0007] To solve the above-described problems, the present invention
relates to an artificially manipulated unsaturated fatty acid
controlling system, which has an effect of increasing the content
of a specific unsaturated fatty acid. More particularly, the
present invention relates to an artificially manipulated
unsaturated fatty acid biosynthesis-associated factor, and a system
for controlling an unsaturated fatty acid, which artificially
modifies the content of a specific unsaturated fatty acid.
[0008] The present invention is directed to providing a plant body
increased in the content of a specific unsaturated fatty acid due
to an artificially manipulated unsaturated fatty acid
biosynthesis-associated factor.
[0009] The present invention is directed to providing a plant body
decreased in the content of a specific unsaturated fatty acid by an
artificially manipulated unsaturated fatty acid
biosynthesis-associated factor.
[0010] As an exemplary embodiment of the present invention, the
present invention provides an artificially manipulated unsaturated
fatty acid biosynthesis-associated factor.
[0011] As an exemplary embodiment of the present invention, the
present invention provides an artificially manipulated unsaturated
fatty acid controlling system.
[0012] As an exemplary embodiment of the present invention, the
present invention provides an artificially manipulated unsaturated
fatty acid biosynthesis-associated factor and an expression product
thereof.
[0013] As an exemplary embodiment of the present invention, the
present invention provides a composition for manipulating a gene to
manipulate an unsaturated fatty acid biosynthesis-associated factor
and a method using the same.
[0014] As an exemplary embodiment of the present invention, the
present invention provides a method of controlling the biosynthesis
of an unsaturated fatty acid.
[0015] As an exemplary embodiment of the present invention, the
present invention provides a method of controlling the type of an
unsaturated fatty acid and the content thereof.
[0016] As an exemplary embodiment of the present invention, the
present invention provides a composition for controlling an
unsaturated fatty acid to control the biosynthesis of an
unsaturated fatty acid and/or the content of the unsaturated fatty
acid, and various uses thereof.
[0017] As an exemplary embodiment of the present invention, the
present invention provides an artificially manipulated unsaturated
fatty acid biosynthesis-associated factor such as FAD2, FAD3, FAD4,
FAD6, FAD7 or FAD8 and/or an expression product thereof.
[0018] As an exemplary embodiment of the present invention, the
present invention provides a composition for manipulating a gene to
artificially manipulate an unsaturated fatty acid
biosynthesis-associated factor such as FAD2, FAD3, FAD4, FAD6, FAD7
or FAD8.
[0019] As an exemplary embodiment of the present invention, the
present invention provides an artificially manipulated unsaturated
fatty acid biosynthesis-associated factor such as FAD2, FAD3, FAD4,
FADE, FAD7 or FAD8 and/or various uses of the composition for
manipulating a gene for artificial manipulation.
[0020] As an exemplary embodiment of the present invention, the
present invention provides a plant body increased or decreased in
the content of a specific unsaturated fatty acid and a processed
product using the same.
Technical Solutions
[0021] To solve these problems, the present invention provides a
system capable of artificially controlling the biosynthesis of an
unsaturated fatty acid and/or the content of the fatty acids, which
includes an artificially manipulated unsaturated fatty acid
biosynthesis-associated factor and/or a composition capable of
artificially manipulating the unsaturated fatty acid
biosynthesis-associated factor, for controlling the content of a
specific unsaturated fatty acid.
[0022] In one exemplary embodiment, the present invention provides
a plant body increased in the content of a specific unsaturated
fatty acid by an artificially manipulated unsaturated fatty acid
biosynthesis-associated factor.
[0023] In another exemplary embodiment, the present invention
provides a specific unsaturated fatty acid obtained from a plant
body by using an artificially manipulated unsaturated fatty acid
biosynthesis-associated factor.
[0024] The term "specific unsaturated fatty acid" used herein
refers to one or more unsaturated fatty acids selected from various
types of known unsaturated fatty acids, it may be one or more
unsaturated fatty acids selected from the classification system
represented by the number of carbons (C) and the number of double
bonds (D), which are included in an unsaturated fatty acid among
various types of unsaturated fatty acids. The term "CN:DM
unsaturated fatty acid" used herein refers to an unsaturated fatty
acid consisting of N number of carbons (C) and including M number
of double bonds (D). Here, N may be an integer of 4 to 36, and M
may be an integer of 1 to 35.
[0025] The specific unsaturated fatty acid may be a C8.about.24:D1
unsaturated fatty acid.
[0026] The specific unsaturated fatty acid may be a C16.about.22:D1
unsaturated fatty acid.
[0027] The specific unsaturated fatty acid may be a C18:D1
unsaturated fatty acid.
[0028] The specific unsaturated fatty acid may be oleic acid,
elaidic acid or vaccenic acid.
[0029] In addition, the specific unsaturated fatty acid may be a
C8.about.24:D2 unsaturated fatty acid.
[0030] The specific unsaturated fatty acid may be a C16.about.22:D2
unsaturated fatty acid.
[0031] The specific unsaturated fatty acid may be a C18:D2
unsaturated fatty acid.
[0032] The specific unsaturated fatty acid may be linoleic acid or
linoelaidic acid.
[0033] In one exemplary embodiment of the present invention, the
present invention provides an artificially manipulated unsaturated
fatty acid biosynthesis-associated factor.
[0034] The term "unsaturated fatty acid biosynthesis-associated
factor" used herein refers to all factors directly participating in
or indirectly affecting the biosynthesis of an unsaturated fatty
acid. Here, the factor may be DNA, RNA, a gene, a peptide, a
polypeptide or a protein. The factor includes various materials
capable of controlling the biosynthesis of an unsaturated fatty
acid, which are non-natural, that is, artificially manipulated. For
example, the factor may be a genetically manipulated or modified
gene or protein, which is expressed in a plant.
[0035] The unsaturated fatty acid biosynthesis-associated factor
may increase the content of a specific unsaturated fatty acid
included in a plant.
[0036] The unsaturated fatty acid biosynthesis-associated factor
may decrease the content of a specific unsaturated fatty acid
included in a plant.
[0037] The unsaturated fatty acid biosynthesis-associated factor
may affect a direct/indirect mechanism for controlling the content
of a specific unsaturated fatty acid included in a plant.
[0038] In one exemplary embodiment of the present invention, the
unsaturated fatty acid biosynthesis-associated factor may be, for
example, an artificially manipulated a FAD2 gene, a FAD3 gene, a
FAD4 gene, a FAD6 gene, a FAD7 gene or a FAD8 gene, preferably a
FAD2 gene or a FAD3 gene.
[0039] In one exemplary embodiment of the present invention, the
unsaturated fatty acid biosynthesis-associated factor may include
two or more artificially manipulated genes. For example, two or
more genes selected from the group consisting of a FAD2 gene, a
FAD3 gene, a FAD4 gene, a FAD6 gene, a FAD7 gene and a FAD8 gene
may be artificially manipulated.
[0040] Therefore, in an exemplary embodiment of the present
invention, one or more artificially manipulated unsaturated fatty
acid biosynthesis-associated factors selected from the group
consisting of a FAD2 gene, a FAD3 gene, a FAD4 gene, a FAD6 gene, a
FAD7 gene and a FAD8 gene, which have undergone modification in a
nucleic acid sequence, are provided.
[0041] The modification in a nucleic acid sequence may be
non-limitedly, artificially manipulated by a guide nucleic
acid-editor protein complex.
[0042] 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.
[0043] The guide nucleic acid-editor protein complex may serve to
modify a subject.
[0044] The subject may be a target nucleic acid, a gene, a
chromosome or a protein.
[0045] For example, the gene may be an unsaturated fatty acid
biosynthesis-associated factor, artificially manipulated by a guide
nucleic acid-editor protein complex,
[0046] wherein the unsaturated fatty acid biosynthesis-associated
factor artificially manipulated includes one or more modifications
of nucleic acids which is
[0047] 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 unsaturated fatty acid
biosynthesis-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
[0048] a chemical modification of one or more nucleotides in a
nucleic acid sequence constituting the unsaturated fatty acid
biosynthesis-associated factor.
[0049] The modification of nucleic acids may occur in a promoter
region of the gene.
[0050] 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.
[0051] The modification of nucleic acids may occur in an intron
region of the gene.
[0052] The modification of nucleic acids may occur in an enhancer
region of the gene.
[0053] The PAM sequence may be, for example, one or more of the
following sequences (described in the 5' to 3' direction):
[0054] NGG (N is A, T, C or G);
[0055] NNNNRYAC (each of N is independently A, T, C or G, R is A or
G, and Y is C or T);
[0056] NNAGAAW (each of N is independently A, T, C or G, and W is A
or T);
[0057] NNNNGATT (each of N is independently A, T, C or G);
[0058] NNGRR(T) (each of N is independently A, T, C or G, and R is
A or G); and
[0059] TTN (N is A, T, C or G).
[0060] The editor protein may be derived from Streptococcus
pyogenes, Streptococcus thermophilus, Streptococcus sp.,
Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces
pristinaespiralis, Streptomyces viridochromogenes, Streptomyces
viridochromogenes, Streptosporangium roseum, 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.
[0061] 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
Staphylococcus 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.
[0062] 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 30, for example, SEQ ID NOs:7 or 30.
[0063] The guide nucleic acid may form a complementary bond with a
part of nucleic acid sequences of a FAD2 gene. It may create 0 to
5, 0 to 4, 0 to 3, or 0 to 2 mismatches. As a preferable 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
30, for example, SEQ ID NOs: 7 or 30, respectively.
[0064] 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.
[0065] In addition, the present invention provides a composition
for gene manipulation, which may be employed in artificial
manipulation of an unsaturated fatty acid biosynthesis-associated
factor for a specific purpose.
[0066] The composition for gene manipulation may include a guide
nucleic acid-editor protein complex or a nucleic acid sequence
encoding the same.
[0067] The composition for gene manipulation may include:
[0068] (a) a guide nucleic acid capable of forming a complementary
bond with respect to each of target sequences of one or more genes
selected from the group consisting of a FAD2 gene, a FAD3 gene, a
FAD4 gene, a FAD6 gene, a FAD7 gene and a FAD8 gene, respectively
or a nucleic acid sequence encoding the guide nucleic acid;
[0069] (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
Staphylococcus aureus-derived Cas9 protein, a Neisseria
meningitidis-derived Cas9 protein, and a Cpf1 protein or a nucleic
acid sequence encoding the same.
[0070] 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: 1 to 30,
respectively.
[0071] For example, the guide nucleic acid may be a nucleic acid
sequence which forms a complementary bond with the target sequence
of SEQ ID NOs: 7 or 30.
[0072] In one exemplary embodiment, the composition for gene
manipulation may be a viral vector system.
[0073] The viral vector may be an agrobacterium vector system using
an agrobacteria.
[0074] In one exemplary embodiment, the composition for gene
manipulation may be a viral vector system.
[0075] The viral vector may include one or more selected from the
group consisting of a mosaic virus, a retrovirus, a lentivirus, an
adenovirus, an adeno-associated virus (AAV), a vaccinia virus, a
poxvirus and a herpes simplex virus.
[0076] In an exemplary embodiment, the present invention provides a
method for artificially manipulating cells, which includes:
introducing (a) a guide nucleic acid which is capable of forming a
complementary bond with respect to the target sequences of one or
more genes selected from the group consisting of a FAD2 gene, a
FAD3 gene, a FAD4 gene, a FAD6 gene, a FAD7 gene and a FAD8 gene,
respectively, or a nucleic acid sequence encoding the same; and
[0077] (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
Staphylococcus 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.
[0078] 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.
[0079] The introduction may be performed in vivo or ex vivo of a
plant.
[0080] The introduction may be performed by one or more methods
selected from a gene gun, an electroporation, liposomes, plasmids,
agrobacterium vector system, viral vectors, nanoparticles and a
protein translocation domain (PTD) fusion protein method.
[0081] The viral vector may include one or more selected from the
group consisting of a mosaic virus, a retrovirus, a lentivirus, an
adenovirus, an adeno-associated virus (AAV), a vaccinia virus, a
poxvirus and a herpes simplex virus.
[0082] In addition, the present invention provides a composition
for controlling an unsaturated fatty acid to control the
biosynthesis of an unsaturated fatty acid and/or the content of the
unsaturated fatty acid of a plant.
[0083] The composition for controlling an unsaturated fatty acid
may include a composition for gene manipulation, which may be
employed in artificial manipulation of an unsaturated fatty acid
biosynthesis-associated factor.
[0084] The formulation of the composition for gene manipulation is
the same as described above.
[0085] In an exemplary embodiment, the present invention provides a
processed product using a plant body increased or decreased in the
content of a specific unsaturated fatty acid.
[0086] The plant body may include an artificially manipulated
unsaturated fatty acid biosynthesis-associated factor.
[0087] The processed product may be a food which can be ingested by
humans and/or animals.
[0088] In an exemplary embodiment, the present invention provides a
kit for gene manipulation to control the content of a specific
unsaturated fatty acid.
[0089] The kit may include a composition for gene manipulation,
which may be employed in artificial manipulation of an unsaturated
fatty acid biosynthesis-associated factor.
[0090] The gene of interest may be artificially manipulated using
such a kit.
Advantageous Effects
[0091] A plant body increased in the content of a specific
unsaturated fatty acid which is good for human health or decreased
in the content of a specific unsaturated fatty acid which is
harmful for human health, and/or a processed product using the same
can be manufactured by using an artificially manipulated
unsaturated fatty acid biosynthesis-associated factor and a system
for controlling an unsaturated fatty acid, which is artificially
modified thereby.
[0092] For example, one or more genes selected from a FAD2 gene, a
FAD3 gene, a FAD4 gene, a FAD6 gene, a FAD7 gene and a FAD8 gene
can be used.
BRIEF DESCRIPTION OF DRAWINGS
[0093] FIG. 1 is a schematic diagram of CRISPR-Cas9 vectors,
pPZP-FAD2-7 and pPZP-FAD2-30, for modifying a FAD2 gene of
soybeans.
[0094] FIGS. 2A and 2B illustrates the growth processes of soybean
transgenic plant bodies prepared by the knockout of a FAD2 gene
using pPZP-FAD2-7(a) and pPZP-FAD2-30(b).
[0095] FIG. 3 shows T0 transformants of pPZP-FAD2-7 and
pPZP-FAD2-30.
[0096] FIG. 4 shows the PCR results for confirming insert genes of
the T0 transformants of pPZP-FAD2-7 and pPZP-FAD2-30. Here, NT is
Glycine max L. Kwangan (wild type), and #1, #2, #3, #5, #8, #9, #19
and #21 are T0 transformants.
[0097] FIG. 5 shows the contents of oleic acid in T.sub.1 seeds of
pPZP-FAD2-7 and pPZP-FAD2-30.
[0098] FIG. 6 shows the indel frequency of an FAD2 gene targeted by
CRISPR-Cas9 (the target sequence of FAD2A gene is SEQ ID NO: 7, and
the target sequence of FAD2B is SEQ ID NO: 7).
[0099] FIG. 7 shows the sequencing results for an FAD2 gene of
soybeans transformed using CRISPR-Cas9 (a target sequence including
PAM shown with an underline). For targeting FAD2, the target site
(Type WT) located in the chromosome #10 is SEQ ID NO: 71, and the
target site (Type WT) located in the chromosome #20 is SEQ ID NO:
72. Herein, the target site includes the target sequence. The
sequencing results for the FAD2 gene located in the chromosome #10
are shown in SEQ ID NO: 75 to 83 (Type -3 to +1 order). The
sequencing results for the FAD2 gene located in the chromosome #20
are shown in SEQ ID NO: 84 to 92 (Type -4 to -1 order).
[0100] FIGS. 8A and 8B shows the results of target site screening
and indel frequency of an FAD2 gene manipulated in T.sub.1
transformants, shows the results of target site screening and indel
frequency for of an FAD2 gene in (a) Chromosome #10 (chr10), and
(b) Chromosome #20 (chr20).
[0101] FIGS. 9A and 9B shows the target site sequencing results of
an FAD2 gene manipulated in T.sub.1 transformants, shows the target
site sequencing results of an FAD2 gene in (a) Chromosome #10
(chr10), and (b) Chromosome #20 (chr20). For targeting FAD2, the
target site located in the chromosome #10 is SEQ ID NO: 73, and the
target site located in the chromosome #20 is SEQ ID NO: 74. The
target site sequencing results of the FAD2 located in the
chromosome #10 are shown in the following SEQ ID NOs according to
samples: SEQ ID NO: 73 (FAD2-7#1-1 having no indel); SEQ ID NO:93,
94 (FAD2-30#2-4); SEQ ID NO: 73 (FAD2-30#3-1 having no indel); SEQ
ID NO: 95, 96 (FAD2-30#3-2); SEQ ID NO: 97 (FAD2-30#8-1); SEQ ID
NO: 98 to 100 (FAD2-30#8-2); SEQ ID NO: 101 (FAD2-30#9-1), SEQ ID
NO: 73 (FAD2-30#9-1 having no indel); SEQ ID NO: 102, 103
(FAD2-30#19-1); SEQ ID NO: 104 (FAD2-30#21-2); SEQ ID NO: 105
(FAD2-30#21-5); SEQ ID NO: 106 (FAD2-30#22-5); SEQ ID NO: 107
(FAD2-30#22-6). The target site sequencing results of the FAD2
located in the chromosome #20 are shown in the following SEQ ID NOs
according to samples: SEQ ID NO: 108, 109 (FAD2-7#1-1); SEQ ID NO:
74 (FAD2-30#2-4 having no indel), SEQ ID NO: 110 (FAD2-30#2-4); SEQ
ID NO: 74 (FAD2-30#3-1 having no indel); SEQ ID NO: 111
(FAD2-30#3-2); SEQ ID NO: 112 (FAD2-30#8-1); SEQ ID NO: 113
(FAD2-30#8-2); SEQ ID NO: 114 (FAD2-30#9-1); SEQ ID NO: 115
(FAD2-30#19-1); SEQ ID NO: 116, 117 (FAD2-30#21-2); SEQ ID NO: 118
(FAD2-30#21-5); SEQ ID NO: 119 (FAD2-30#22-5); SEQ ID NO: 120
(FAD2-30#22-6).
[0102] FIG. 10 shows T.sub.1 transformants of pPZP-FAD2-7 and
pPZP-FAD2-30.
[0103] FIG. 11 shows the analysis results of the removal of a gene
from T.sub.1 transformants of pPZP-FAD2-7 and pPZP-FAD2-30 using
PCR.
DETAILED DESCRIPTION
[0104] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by those
of ordinary skill in the art to which the present invention
belongs. Although methods and materials similar to or the same as
described in the specification can be used in the implementation or
experiments of the present invention, suitable methods and
materials will be described below. All publications, patent
applications, patents and other references mentioned herein are
incorporated by reference in their entirety. In addition, the
materials, methods and embodiments are merely illustrative, and not
intended to be limitative.
[0105] One aspect of the present invention relates to a transgenic
plant body increased in the content of a C8.about.24:D1 unsaturated
fatty acid.
[0106] Specifically, the present invention relates to a transgenic
plant body increased in the content of a specific unsaturated fatty
acid by artificially manipulating an unsaturated fatty acid
biosynthesis-associated factor, the present invention includes an
unsaturated fatty acid biosynthesis-associated factor in which a
function is artificially changed, an artificial manipulation
composition therefor, a method of preparing the same, and a plant
body including the same.
[0107] Another aspect of the present invention relates to a
transgenic plant body decreased in the content of a C8.about.24:D2
unsaturated fatty acid.
[0108] Specifically, the present invention relates to a transgenic
plant body decreased in the content of a specific unsaturated fatty
acid by artificially manipulating an unsaturated fatty acid
biosynthesis-associated factor, the present invention includes an
unsaturated fatty acid biosynthesis-associated factor in which a
function is artificially changed, an artificial manipulation
composition therefor, a method of preparing the same, and a plant
body including the same.
[0109] Unsaturated Fatty Acid
[0110] One aspect of the present invention is a system for changing
the content of fatty acids.
[0111] In one example, a system for changing the content of a
specific saturated fatty acid in a plant body may be provided.
[0112] In another example, a system for changing the content of a
specific unsaturated fatty acid in a plant body may be
provided.
[0113] The term "fatty acid" used herein refers to a carboxylic
acid having an aliphatic chain, and most fatty acids produced in a
natural state have an even number of carbons ranging from about 4
to 36, which forms a carbon chain. Fatty acids are largely
classified into saturated fatty acids and unsaturated fatty acids
according to the type of a carbon bond.
[0114] The term "saturated fatty acid" used herein refers to fatty
acids formed with a single bond.
[0115] Fatty acids include propionic acid, butyric acid, valeric
acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid,
capric acid, lauric acid, myristic acid, palmitic acid, stearic
acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid
and the like.
[0116] The term "unsaturated fatty acids" used herein refers to
fatty acids having one or more carbon-carbon double bonds. The
unsaturated fatty acids include all of cis-unsaturated fatty acids
and trans-unsaturated fatty acids. The cis-unsaturated fatty acids
refer to unsaturated fatty acids in which two hydrogens
respectively binding to two carbons participating in a double bond
are structurally placed in the same direction. On the other hand,
the trans-unsaturated fatty acids refer to unsaturated fatty acids
in which two hydrogens respectively binding to two carbons
participating in a double bond are structurally placed in different
directions.
[0117] The unsaturated fatty acids may be classified into Omega-3,
6, 7 and 9 according to the position of a carbon participating in a
double bond.
[0118] The unsaturated fatty acids include Omega-3 (.omega.-3)
fatty acids.
[0119] Here, the "omega-3 (.omega.-3) fatty acids" refers to an
unsaturated fatty acid in which a double bond starts from the third
carbon at the end of the carbon chain, and includes alpha-linolenic
acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid
(DHA).
[0120] The unsaturated fatty acids include omega-6 (.omega.-6)
fatty acids.
[0121] Here, the "omega-6 (.omega.-6) fatty acids" refers to
unsaturated fatty acids in which a double bond starts from the
sixth carbon at the end of a carbon chain, and includes linoleic
acid (LA), gamma-linolenic acid (GLA), dihomo-gamma-linolenic acid
(DGLA) and arachidonic acid (AA).
[0122] The unsaturated fatty acids include omega-7 (.omega.-7)
fatty acids.
[0123] Here, the "omega-7 (.omega.-7) fatty acids" refers to
unsaturated fatty acids in which a double bond starts from the
seventh carbon at the end of a carbon chain, and includes paullinic
acid, palmitoleic acid or vaccenic acid.
[0124] The unsaturated fatty acids include omega-9 (.omega.-9)
fatty acids.
[0125] Here, the "omega-9 (.omega.-9) fatty acids" refers to
unsaturated fatty acids in which a double bond starts from the
ninth carbon at the end of a carbon chain, and includes oleic acid,
elaidic acid, eicosenoic acid, erucic acid and nervonic acid.
[0126] In one exemplary embodiment, the unsaturated fatty acid may
be an omega-6 (.omega.-6) fatty acid.
[0127] In another exemplary embodiment, the unsaturated fatty acid
may be an omega-9 (.omega.-9) fatty acid.
[0128] In addition, the unsaturated fatty acid may be classified as
a CN:DN unsaturated fatty acid by representing the number of
carbons and the number of double bonds.
[0129] The term "CN:DM unsaturated fatty acid" refers to an
unsaturated fatty acid consisting of N number of carbons (C) and
including M number of double bonds (D). Here, N may be an integer
of 4 to 36, and M may be an integer of 1 to 35.
[0130] For example, the unsaturated fatty acid consisting of 18
carbons and including 2 double bonds may be classified by being
represented as a C18:D2 unsaturated fatty acid.
[0131] In one exemplary embodiment, the unsaturated fatty acid
includes a CN:D1 unsaturated fatty acid.
[0132] Here, N may be an integer of 4 to 36.
[0133] Preferably, the CN:D1 unsaturated fatty acid may be a C8:D1
unsaturated fatty acid, a C10:D1 unsaturated fatty acid, a C12:D1
unsaturated fatty acid, a C14:D1 unsaturated fatty acid, a C16:D1
unsaturated fatty acid, a C18:D1 unsaturated fatty acid, a C20:D1
unsaturated fatty acid, a C22:D1 unsaturated fatty acid or a C24:D1
unsaturated fatty acid.
[0134] In another exemplary embodiment, the unsaturated fatty acid
includes a CN:D2 unsaturated fatty acid.
[0135] Here, N may be an integer of 4 to 36.
[0136] Preferably, the CN:D2 unsaturated fatty acid may be a C8:D2
unsaturated fatty acid, a C10:D2 unsaturated fatty acid, a C12:D2
unsaturated fatty acid, a C14:D2 unsaturated fatty acid, a C16:D2
unsaturated fatty acid, a C18:D2 unsaturated fatty acid, a C20:D2
unsaturated fatty acid, a C22:D2 unsaturated fatty acid or a C24:D2
unsaturated fatty acid.
[0137] In still another exemplary embodiment, the unsaturated fatty
acid includes a CN:D3 unsaturated fatty acid.
[0138] Here, N may be an integer of 4 to 36.
[0139] Preferably, the CN:D3 unsaturated fatty acid may be a C8:D3
unsaturated fatty acid, a C10:D3 unsaturated fatty acid, a C12:D3
unsaturated fatty acid, a C14:D3 unsaturated fatty acid, a C16:D3
unsaturated fatty acid, a C18:D3 unsaturated fatty acid, a C20:D3
unsaturated fatty acid, a C22:D3 unsaturated fatty acid or a C24:D3
unsaturated fatty acid.
[0140] In yet another exemplary embodiment, the unsaturated fatty
acid includes a CN:D4 unsaturated fatty acid.
[0141] Here, N may be an integer of 4 to 36.
[0142] Preferably, the CN:D4 unsaturated fatty acid may be a C8:D4
unsaturated fatty acid, a C10:D4 unsaturated fatty acid, a C12:D4
unsaturated fatty acid, a C14:D4 unsaturated fatty acid, a C16:D4
unsaturated fatty acid, a C18:D4 unsaturated fatty acid, a C20:D4
unsaturated fatty acid, a C22:D4 unsaturated fatty acid or a C24:D4
unsaturated fatty acid.
[0143] In yet another exemplary embodiment, the unsaturated fatty
acid includes a CN:D5 unsaturated fatty acid.
[0144] Here, N may be an integer of 4 to 36.
[0145] Preferably, the CN:D5 unsaturated fatty acid may be a C8:D5
unsaturated fatty acid, a C10:D5 unsaturated fatty acid, a C12:D5
unsaturated fatty acid, a C14:D5 unsaturated fatty acid, a C16:D5
unsaturated fatty acid, a C18:D5 unsaturated fatty acid, a C20:D5
unsaturated fatty acid, a C22:D5 unsaturated fatty acid or a C24:D5
unsaturated fatty acid.
[0146] In yet another exemplary embodiment, the unsaturated fatty
acid includes a CN:D6 unsaturated fatty acid.
[0147] Here, N may be an integer of 4 to 36.
[0148] Preferably, the CN:D6 unsaturated fatty acid may be a C8:D6
unsaturated fatty acid, a C10:D6 unsaturated fatty acid, a C12:D6
unsaturated fatty acid, a C14:D6 unsaturated fatty acid, a C16:D6
unsaturated fatty acid, a C18:D6 unsaturated fatty acid, a C20:D6
unsaturated fatty acid, a C22:D6 unsaturated fatty acid or a C24:D6
unsaturated fatty acid.
[0149] In yet another exemplary embodiment, the unsaturated fatty
acid includes a CN:DK unsaturated fatty acid.
[0150] Here, N may be an integer of 4 to 36, and K may be an
integer of 7 to 35.
[0151] In one exemplary embodiment, the unsaturated fatty acid may
be a C8 to 24:D1 unsaturated fatty acid.
[0152] Preferably, the unsaturated fatty acid may be selected from
the group consisting of a C16:D1 unsaturated fatty acid, a C18:D1
unsaturated fatty acid, a C20:D1 unsaturated fatty acid and a
C22:D1 unsaturated fatty acid.
[0153] Most preferably, the unsaturated fatty acid may be a C18:D1
unsaturated fatty acid or a C20:D1 unsaturated fatty acid.
[0154] In another exemplary embodiment, the unsaturated fatty acid
may be a C8 to 24:D2 unsaturated fatty acid.
[0155] Preferably, the unsaturated fatty acid may be selected from
the group consisting of a C16:D2 unsaturated fatty acid, a C18:D2
unsaturated fatty acid, a C20:D2 unsaturated fatty acid and a
C22:D2 unsaturated fatty acid.
[0156] Most preferably, the unsaturated fatty acid may be a C18:D2
unsaturated fatty acid or a C20:D2 unsaturated fatty acid.
[0157] Unsaturated Fatty Acid Biosynthesis-Associated Factor
[0158] Unsaturated Fatty Acid Biosynthesis-Associated Factor
[0159] Another aspect of the present invention is an artificially
manipulated or modified unsaturated fatty acid
biosynthesis-associated factor.
[0160] The term "unsaturated fatty acid biosynthesis-associated
factor" used herein refers to all factors directly participating in
or indirectly affecting the biosynthesis of an unsaturated fatty
acid. Here, the factor may be DNA, RNA, a gene, a peptide, a
polypeptide or a protein.
[0161] In an exemplary embodiment, the unsaturated fatty acid
biosynthesis-associated factor includes various materials capable
of controlling the biosynthesis of an unsaturated fatty acid, which
are non-natural, that is, artificially manipulated. For example,
the unsaturated fatty acid biosynthesis-associated factor may be a
genetically manipulated or modified gene or protein, which is
expressed in a plant.
[0162] The term "artificially manipulated" means an artificially
modified state, which is not a naturally occurring state.
[0163] The term "genetically manipulated" means that a genetic
modification is artificially introduced to plant-derived substances
cited in the present invention, and may be, for example, genes and
gene products (polypeptides, proteins, etc.) in which their genomes
are artificially modified for a specific purpose.
[0164] As an preferable example, the present invention provides a
unsaturated fatty acid biosynthesis-associated factor which is
genetically manipulated or modified for a specific purpose.
[0165] Genes or proteins having the functions listed below may have
multiple types of functions, not only one type of unsaturated fatty
acid biosynthesis-associated function. In addition, as needed, two
or more unsaturated fatty acid biosynthesis-associated functions
and factors may be provided.
[0166] An unsaturated fatty acid biosynthesis-associated factor may
produce an unsaturated fatty acid by forming one or more double
bonds in a saturated fatty acid.
[0167] The unsaturated fatty acid biosynthesis-associated factor
may form new one or more double bonds in an unsaturated fatty
acid.
[0168] The unsaturated fatty acid biosynthesis-associated factor
may change a position of one or more double bonds included in an
unsaturated fatty acid.
[0169] The unsaturated fatty acid biosynthesis-associated factor
may remove one or more double bonds of an unsaturated fatty acid
having two or more double bonds.
[0170] The unsaturated fatty acid biosynthesis-associated factor
may change a cis-unsaturated fatty acid into a trans-unsaturated
fatty acid.
[0171] The unsaturated fatty acid biosynthesis-associated factor
may change a trans-unsaturated fatty acid into a cis-unsaturated
fatty acid.
[0172] The unsaturated fatty acid biosynthesis-associated factor
may control the content of an unsaturated fatty acid included in a
plant.
[0173] The unsaturated fatty acid biosynthesis-associated factor
may increase the content of a specific unsaturated fatty acid
included in a plant.
[0174] The unsaturated fatty acid biosynthesis-associated factor
may decrease the content of a specific unsaturated fatty acid
included in a plant.
[0175] In an Exemplary Embodiment, the Unsaturated Fatty Acid
Biosynthesis-Associated Factor May be an Unsaturated Fatty Acid
Biosynthesis-Associated Factor of a Plant.
[0176] Preferably, the unsaturated fatty acid
biosynthesis-associated factor may be a FAD gene or FAD
protein.
[0177] Most preferably, the unsaturated fatty acid
biosynthesis-associated factor may be one or more selected from the
group consisting of FAD2, FAD3, FADE, FAD7 and FAD8.
[0178] In any exemplary embodiment, the unsaturated fatty acid
biosynthesis-associated factor may be FAD2.
[0179] A FAD2 (omega-6 fatty acid desaturase) gene refers to a gene
(full-length DNA, cDNA or mRNA) encoding the FAD2 protein also
referred to as FAD2-1, FAD2-1B or GMFAD2-1B. In one example, the
FAD2 gene may be one or more genes selected from the group
consisting of the following genes, but the present invention is not
limited thereto: genes encoding plant, for example, soybean
(Glycine max) FAD2 (e.g., NCBI Accession No. NP_001341865.1,
XP_006605883.1, XP_006605882.1, XP_006605885.1, XP_006605884.1, or
XP_014627765.1), for example, FAD2 genes represented by NCBI
Accession No. NM_001354936.1, XM_006605820.2, XM_006605819.2,
XM_006605822.2, XM_006605821.2, or XM_014772279.1.
[0180] In any exemplary embodiment, the unsaturated fatty acid
biosynthesis-associated factor may be FAD3.
[0181] A FAD3 (microsomal omega-3 fatty acid desaturase) gene
refers to a gene (full-length DNA, cDNA or mRNA) encoding a FAD3
protein also referred to as Fanx. In one example, the FAD3 gene may
be one or more genes selected from the group consisting of the
following genes, but the present invention is not limited thereto:
a gene encoding plant, for example, soybean (Glycine max) FAD3
(e.g., NCBI Accession No. NP_001237507.1), for example, an FAD3
gene represented by NCBI Accession No. NM_001250578.1.
[0182] In any exemplary embodiment, the unsaturated fatty acid
biosynthesis-associated factor may be FAD6.
[0183] A FAD6 (fatty acid desaturase 6) gene refers to a gene
(full-length DNA, cDNA or mRNA) encoding a FAD6 protein also
referred to as FADC or SFD4. In one example, the FAD6 gene may be
one or more genes selected from the group consisting of the
following genes, but the present invention is not limited thereto:
a gene encoding a plant, for example, Arabidopsis thaliana FAD6
(e.g., NCBI Accession No. NP_194824.1), for example, a FAD6 gene
represented by NCBI Accession No. NM_119243.4.
[0184] In any exemplary embodiment, the unsaturated fatty acid
biosynthesis-associated factor may be FAD7.
[0185] A FAD7 (chloroplast omega 3 fatty acid desaturase isoform 2)
gene refers to a gene (full-length DNA, cDNA or mRNA) encoding a
FAD7 protein. In one example, the FAD7 gene may be one or more
genes selected from the group consisting of the following genes,
but the present invention is not limited thereto: a gene encoding a
plant, for example, soybean (Glycine max) FAD7 (e.g., NCBI
Accession No. NP_001237361.1), for example, a FAD7 gene represented
by NCBI Accession No. NM_001250432.1.
[0186] In any exemplary embodiment, the unsaturated fatty acid
biosynthesis-associated factor may be FAD8.
[0187] A FAD8 (omega-3 fatty acid desaturase, chloroplastic-like)
gene refers to a gene (full-length DNA, cDNA or mRNA) encoding a
FAD8 protein. In one example, the FAD8 gene may be one or more
genes selected from the group consisting of the following genes,
but the present invention is not limited thereto: a gene encoding a
plant, for example, soybean (Glycine max) FAD8 (e.g., NCBI
Accession No. NP_001239777.1), for example, a FAD8 gene represented
by NCBI Accession No. NM_001252848.1.
[0188] The unsaturated fatty acid biosynthesis-associated factor
may be derived from a plant such as soybean, Arabidopsis thaliana,
sesame, corn and the like, etc.
[0189] Information about the genes may be obtained from a known
database such as GeneBank of the National Center for Biotechnology
Information (NCBI).
[0190] In one exemplary embodiment of the present invention, the
unsaturated fatty acid biosynthesis-associated factor, for example,
FAD2, FAD3, FADE, FAD7 or FAD8, may be artificially manipulated
unsaturated fatty acid biosynthesis-associated factor.
[0191] In a certain embodiment, the artificially manipulated
unsaturated fatty acid biosynthesis-associated factor may be
genetically manipulated.
[0192] 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.
[0193] 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. 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.
[0194] 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.
[0195] The manipulated unsaturated fatty acid
biosynthesis-associated factor may produce an unsaturated fatty
acid by forming one or more double bonds in a saturated fatty
acid.
[0196] The manipulated unsaturated fatty acid
biosynthesis-associated factor may form new one or more double
bonds in an unsaturated fatty acid.
[0197] The manipulated unsaturated fatty acid
biosynthesis-associated factor may change positions of one or more
double bonds included in an unsaturated fatty acid.
[0198] The manipulated unsaturated fatty acid
biosynthesis-associated factor may remove one or more double bonds
of an unsaturated fatty acid having two or more double bonds.
[0199] The manipulated unsaturated fatty acid
biosynthesis-associated factor may change a cis-unsaturated fatty
acid into a trans-unsaturated fatty acid.
[0200] The manipulated unsaturated fatty acid
biosynthesis-associated factor may change a trans-unsaturated fatty
acid into a cis-unsaturated fatty acid.
[0201] The manipulated unsaturated fatty acid
biosynthesis-associated factor may control the content of an
unsaturated fatty acid included in a plant.
[0202] The manipulated unsaturated fatty acid
biosynthesis-associated factor may increase the content of a
specific unsaturated fatty acid included in a plant.
[0203] The manipulated unsaturated fatty acid
biosynthesis-associated factor may decrease the content of a
specific unsaturated fatty acid included in a plant.
[0204] The manipulation includes all types of structural or
functional modifications of the unsaturated fatty acid
biosynthesis-associated factor.
[0205] The structural modification of the unsaturated fatty acid
biosynthesis-associated factor includes all types of modifications,
which are not the same as those of a wild type existing in a
natural state.
[0206] For example, when the unsaturated fatty acid
biosynthesis-associated factor is DNA, RNA or a gene, the
structural modification may be the loss of one or more
nucleotides.
[0207] The structural modification may be the insertion of one or
more nucleotides.
[0208] Here, the inserted nucleotides include all of a subject
including an unsaturated fatty acid biosynthesis-associated factor
and nucleotides entering from the outside of the subject.
[0209] The structural modification may be the substitution of one
or more nucleotides.
[0210] The structural modification may include the chemical
modification of one or more nucleotides.
[0211] Here, the chemical modification includes all of the
addition, removal and substitution of chemical functional
groups.
[0212] As another example, when the unsaturated fatty acid
biosynthesis-associated factor is a peptide, a polypeptide or a
protein, the structural modification may be the loss of one or more
amino acids.
[0213] The structural modification may be the insertion of one or
more amino acids.
[0214] Here, the inserted amino acids include all of a subject
including an unsaturated fatty acid biosynthesis-associated factor
and amino acids entering from the outside of the subject.
[0215] The structural modification may be the substitution of one
or more amino acids.
[0216] The structural modification may include the chemical
modification of one or more amino acids.
[0217] Here, the chemical modification includes all of the
addition, removal and substitution of chemical functional
groups.
[0218] The structural modification may be the partial or entire
attachment of a different peptide, polypeptide or protein.
[0219] Here, the different peptide, polypeptide or protein may be
an unsaturated fatty acid biosynthesis-associated factor, or a
peptide, polypeptide or protein having a different function.
[0220] The functional modification of the unsaturated fatty acid
biosynthesis-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.
[0221] For example, when the unsaturated fatty acid
biosynthesis-associated factor is a peptide, polypeptide or
protein, the functional modification may be a mutation of the
unsaturated fatty acid biosynthesis-associated factor.
[0222] Here, the mutation may be a mutation that enhances or
suppresses a function of the unsaturated fatty acid
biosynthesis-associated factor.
[0223] The functional modification may have an additional function
of the unsaturated fatty acid biosynthesis-associated factor.
[0224] Here, the additional function may be the same or a different
function. In addition, the unsaturated fatty acid
biosynthesis-associated factor having the additional function may
be fused with a different peptide, polypeptide or protein.
[0225] The functional modification may be the enhancement in
functionality due to increased expression of the unsaturated fatty
acid biosynthesis-associated factor.
[0226] The functional modification may be the degradation in
functionality due to decreased expression of the unsaturated fatty
acid biosynthesis-associated factor.
[0227] In an exemplary embodiment, the manipulated unsaturated
fatty acid biosynthesis-associated factor may be induced by one or
more of the following mutations:
[0228] all or partial deletions of the unsaturated fatty acid
biosynthesis-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,
[0229] 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
[0230] 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.
[0231] 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.
[0232] System for Controlling Unsaturated Fatty Acids
[0233] One aspect of the present invention relates to a system for
controlling an unsaturated fatty acid, which controls the
biosynthesis of an unsaturated fatty acid by artificially
manipulating an unsaturated fatty acid biosynthesis-associated
factor.
[0234] The term "system for controlling an unsaturated fatty acid"
used herein includes all phenomena affecting the promotion or
inhibition of the biosynthesis of an unsaturated fatty acid, and/or
the increase or inhibition of the production of unsaturated fatty
acids by changing functions of the artificially manipulated
unsaturated fatty acid biosynthesis-associated factor, and includes
all materials, compositions, methods and uses directly or
indirectly involved in the system of controlling the biosynthesis
of an unsaturated fatty acid.
[0235] Each factor constituting the system for controlling the
biosynthesis of an unsaturated fatty acid is also referred to as an
"unsaturated fatty acid controlling factor."
[0236] The system of the present invention includes a modified
mechanism in a plant body, which is associated with an artificially
manipulated unsaturated fatty acid biosynthesis-associated factor.
By the artificially manipulated unsaturated fatty acid
biosynthesis-associated factor,
[0237] in any exemplary embodiment, the biosynthesis of a C8 to
24:D1 unsaturated fatty acid may be controlled,
[0238] in any exemplary embodiment, the biosynthesis of a C8 to
24:D2 unsaturated fatty acid may be controlled,
[0239] in any exemplary embodiment, the production amount of a C8
to 24:D1 unsaturated fatty acid may be controlled,
[0240] in any exemplary embodiment, the production amount of a C8
to 24:D2 unsaturated fatty acid may be controlled,
[0241] in any exemplary embodiment, the content of a C8 to 24:D1
unsaturated fatty acid in a plant body may be controlled,
[0242] in any exemplary embodiment, the content of a C8 to 24:D2
unsaturated fatty acid in a plant body may be controlled,
[0243] in any exemplary embodiment, the content ratio of the C8 to
24:D1 unsaturated fatty acid and the C8 to 24:D2 unsaturated fatty
acid in a plant body may be controlled,
[0244] in any exemplary embodiment, a double bond of the C8 to
24:D1 unsaturated fatty acid may be added or removed, and
[0245] in any exemplary embodiment, a double bond of the C8 to
24:D2 unsaturated fatty acid may be added or removed.
[0246] In another exemplary embodiment, the system for controlling
an unsaturated fatty acid of the present invention includes a
composition for manipulating an unsaturated fatty acid
biosynthesis-associated factor.
[0247] The composition for manipulation may be a composition
capable of artificially manipulating an unsaturated fatty acid
biosynthesis-associated factor, and preferably, a composition for
gene manipulation.
[0248] Hereinafter, the composition for gene manipulation will be
described.
[0249] Composition for Manipulating Unsaturated Fatty Acid
Biosynthesis-Associated Factor
[0250] Manipulation or modification of substances involved in the
unsaturated fatty acid biosynthesis-associated factor and the
system for controlling an unsaturated fatty acid of the present
invention is preferably accomplished by genetic manipulation.
[0251] In one aspect, composition and method for manipulating a
gene by targeting a partial or entire non-coding or coding region
of the unsaturated fatty acid biosynthesis-associated factor may be
provided.
[0252] In an exemplary embodiment, the composition and method may
be used in manipulation or modification of one or more unsaturated
fatty acid biosynthesis-associated genes involved in the formation
of a desired system for controlling an unsaturated fatty acid. The
manipulation or modification may be performed by modification of
nucleic acids constituting a gene. As a result of the manipulation,
all of knock down, knock out, and knock in are included.
[0253] 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, an initial coding region
may be targeted for the modification of expression and
knockout.
[0254] 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.
[0255] In an exemplary embodiment, for the knockout of one or more
unsaturated fatty acid biosynthesis-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 unsaturated fatty acid
biosynthesis-associated genes contain a deletion or mutation.
[0256] In an exemplary embodiment, the knockdown of a gene may be
used to decrease the expression of undesired alleles or
transcriptomes.
[0257] 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 an unsaturated fatty
acid biosynthesis-associated gene affecting an unsaturated fatty
acid biosynthesis function.
[0258] In an exemplary embodiment, the activity of an unsaturated
fatty acid biosynthesis-associated gene may be regulated, for
example, activated or inactivated by the modification of nucleic
acids of the gene.
[0259] 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.
[0260] 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).
[0261] 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.
[0262] In another aspect, the present invention provides a
composition for manipulating an unsaturated fatty acid
biosynthesis-associated factor.
[0263] The composition for manipulation is a composition that is
able to artificially manipulate an unsaturated fatty acid
biosynthesis-associated factor, and preferably, a composition for
gene manipulation.
[0264] The composition may be employed in gene manipulation for one
or more unsaturated fatty acid biosynthesis-associated factors
involved in formation of a desired system for controlling an
unsaturated fatty acid.
[0265] The gene manipulation may be performed in consideration of a
gene expression regulating process.
[0266] 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.
[0267] 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.
[0268] The gene manipulation may be performed by modification of
nucleic acids constituting an unsaturated fatty acid
biosynthesis-associated factor. As manipulation results, all of
knockdown, knockout, and knockin are included.
[0269] 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.
[0270] In a certain embodiment, for knockout of one or more
unsaturated fatty acid biosynthesis-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 unsaturated fatty acid biosynthesis-associated factors contain
a deletion or mutation.
[0271] In a certain embodiment, knockdown of the unsaturated fatty
acid biosynthesis-associated factor may be used to decrease
expression of undesired alleles or transcriptomes.
[0272] 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 unsaturated fatty
acid biosynthesis-associated factors, or a gene having a different
function.
[0273] 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.
[0274] 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.
[0275] 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.
[0276] 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.
[0277] In another aspect, the present invention may provide the
gene manipulation site.
[0278] 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 an unsaturated fatty acid biosynthesis-associated
gene product.
[0279] For example, the site may be in an initial coding
region,
[0280] a promoter sequence,
[0281] an enhancer sequence,
[0282] a specific intron sequence, or
[0283] a specific exon sequence.
[0284] In an exemplary embodiment, the composition for manipulating
an unsaturated fatty acid biosynthesis-associated factor may
target
[0285] an unsaturated fatty acid biosynthesis-associated factor
affecting the regulation of biosynthesis of unsaturated fatty acid,
such as an FAD gene, preferably an FAD2 gene, an FAD3 gene, an FAD6
gene, an FAD7 gene, or an FAD8 gene, as a manipulation subject.
Most preferably, the composition for manipulating an unsaturated
fatty acid biosynthesis-associated factor may target an FAD2 gene
as a manipulation subject.
[0286] Examples of target regions of the FAD2 gene, that is, target
sequences for regions in which gene manipulation occurs or which
are recognized for gene manipulation are summarized in Table 1.
[0287] The target sequence may target one or more genes.
[0288] The target sequence may simultaneously target two or more
genes. Here, the two or more genes may be homologous genes or
heterologous genes.
[0289] The gene may contain one or more target sequences.
[0290] The gene may be simultaneously targeted at two or more
target sequences.
[0291] The gene may be changed in the site and number of gene
manipulations according to the number of target sequences.
[0292] The gene manipulation may be designed in various forms
depending on the number and positions of the target sequences.
[0293] 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.
[0294] 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.
[0295] Hereinafter, examples of target sequences which are able to
be used in embodiments of the present invention are shown in the
following tables:
TABLE-US-00001 TABLE 1 Target sequences of FAD2 gene No. Target
sequence(including PAM) 1 ATAGATTGGCCATGCAATGAGGG (SEQ ID NO: 1) 2
AATAGATTGGCCATGCAATGAGG (SEQ ID NO: 2) 3 CCTTGGAGAACCCAATAGATTGG
(SEQ ID NO: 3) 4 TGGGTGATTGCTCACGAGTGTGG (SEQ ID NO: 4) 5
TTTTAGTCCCTTATTTCTCATGG (SEQ ID NO: 5) 6 AAACACTTCATCACGGTCAAGGG
(SEQ ID NO: 6) 7 GTGTTTGGAACCCTTGAGAGAGG (SEQ ID NO: 7) 8
GTGAATGGTGGCTTTGTGTTTGG (SEQ ID NO: 8) 9 ACAAAGCCACCATTCACTGTTGG
(SEQ ID NO: 9) 10 AGTTGGCCAACAGTGAATGGTGG (SEQ ID NO: 10) 11
TTGAGTTGGCCAACAGTGAATGG (SEQ ID NO: 11) 12 TGAAAGGTCATAAACAACATAGG
(SEQ ID NO: 12) 13 CAAACACTTCATCACGGTCAAGG (SEQ ID NO: 13) 14
AACCAAAATCCAAAGTTGCATGG (SEQ ID NO: 14) 15 TGGGAGCATAAGGGTGGTAGTGG
(SEQ ID NO: 15) 16 AATATATGGGAGCATAAGGGTGG (SEQ ID NO: 16) 17
GTTTGGCTGCTATGTGTTTATGG (SEQ ID NO: 17) 18 TTTGGCTGCTATGTGTTTATGGG
(SEQ ID NO: 18) 19 TTGGCTGCTATGTGTTTATGGGG (SEQ ID NO: 19) 20
GCAACTATGGACAGAGATTATGG (SEQ ID NO: 20) 21 CACCATTTTACAAGGCACTGTGG
(SEQ ID NO: 21) 22 CTTCATCTGGCTCCACATAGAGG (SEQ ID NO: 22) 23
CTCTATGTGGAGCCAGATGAAGG (SEQ ID NO: 23) 24 TTCTCGGATGTTCCTTCATCTGG
(SEQ ID NO: 24) 25 AGATGAAGGAACATCCGAGAAGG (SEQ ID NO: 25) 26
GATGAAGGAACATCCGAGAAGGG (SEQ ID NO: 26) 27 CATCCGAGAAGGGCGTGTATTGG
(SEQ ID NO: 27) 28 GTACCAATACACGCCCTTCTCGG (SEQ ID NO: 28) 29
AGAAGGGCGTGTATTGGTACAGG (SEQ ID NO: 29) 30 TTGGGACAAACACTTCATCACGG
(SEQ ID NO: 30)
[0296] Composition for Manipulation-Gene Scissors System
[0297] The system for controlling an unsaturated fatty acid of the
present invention may include a guide nucleic acid-editor protein
complex as a composition for manipulating an unsaturated fatty acid
biosynthesis-associated factor.
[0298] Guide Nucleic Acid-Editor Protein Complex
[0299] 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.
[0300] The term "guide nucleic acid" refers to a nucleic acid
capable of recognizing a target nucleic acid, gene, chromosome or
protein.
[0301] 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.
[0302] The guide nucleic acid may include one or more domains.
[0303] 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.
[0304] The guide nucleic acid may include two or more domains,
which may be the same domain repeats, or different domains.
[0305] The guide nucleic acid may have one continuous nucleic acid
sequence.
[0306] 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.
[0307] The guide nucleic acid may have two or more continuous
nucleic acid sequences.
[0308] 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.
[0309] 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.
[0310] The editor protein may be an enzyme.
[0311] The editor protein may be a fusion protein.
[0312] Here, the "fusion protein" refers to a protein that is
produced by fusing an enzyme with an additional domain, peptide,
polypeptide or protein.
[0313] The term "enzyme" refers to a protein that contains a domain
capable of cleaving a nucleic acid, gene, chromosome or
protein.
[0314] 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.
[0315] 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.
[0316] 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.
[0317] The guide nucleic acid-editor protein complex may serve to
modify a subject.
[0318] The subject may be a target nucleic acid, gene, chromosome
or protein.
[0319] 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.
[0320] Here, the guide nucleic acid-editor protein complex may act
at a DNA, RNA, gene or chromosome level.
[0321] The guide nucleic acid-editor protein complex may act in
gene transcription and translation stages.
[0322] The guide nucleic acid-editor protein complex may act at a
protein level.
[0323] 1. Guide Nucleic Acids
[0324] 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.
[0325] 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.
[0326] 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.
[0327] The guide nucleic acid may be present in a linear or
circular shape.
[0328] The guide nucleic acid may be one continuous nucleic acid
sequence.
[0329] For example, the one continuous nucleic acid sequence may be
(N)m, where N is A, T, C or G, or A, U, C or G, and m is an integer
of 1 to 150.
[0330] The guide nucleic acid may be two or more continuous nucleic
acid sequences.
[0331] 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.
[0332] The guide nucleic acid may include one or more domains.
[0333] 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.
[0334] The guide nucleic acid may include two or more domains,
which may be the same domain repeats, or different domains.
[0335] The domains will be described below.
[0336] i) Guide Domain
[0337] 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.
[0338] 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%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95% or more complementarity or complete complementarity.
[0339] The guide domain may be a sequence of 5 to 50 bases.
[0340] 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.
[0341] 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.
[0342] The guide domain may have a guide sequence.
[0343] 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.
[0344] 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%, 95%
or more complementarity or complete complementarity.
[0345] The guide sequence may be a 5 to 50 bases sequence.
[0346] 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.
[0347] 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.
[0348] In addition, the guide domain may include a guide sequence
and an additional base sequence.
[0349] The additional base sequence may be utilized to improve or
degrade the function of the guide domain.
[0350] The additional base sequence may be utilized to improve or
degrade the function of the guide sequence.
[0351] The additional base sequence may be a 1 to 35-base
sequence.
[0352] 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.
[0353] 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.
[0354] The additional base sequence may be located at the 5' end of
the guide sequence.
[0355] The additional base sequence may be located at the 3' end of
the guide sequence.
[0356] ii) First Complementary Domain
[0357] 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.
[0358] The first complementary domain may be a 5 to 35-base
sequence.
[0359] 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.
[0360] 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.
[0361] iii) Linker Domain
[0362] 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.
[0363] The linker domain may be a 1 to 30-base sequence.
[0364] 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.
[0365] 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.
[0366] iv) Second Complementary Domain
[0367] 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.
[0368] 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.
[0369] The second complementary domain may have a 5 to 35-base
sequence.
[0370] 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.
[0371] 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.
[0372] v) Proximal Domain
[0373] The term "proximal domain" is a nucleic acid sequence
located adjacent to the second complementary domain.
[0374] The proximal domain may have a complementary base sequence
therein, and may be formed in a double strand due to a
complementary base sequence.
[0375] The proximal domain may be a 1 to 20-base sequence.
[0376] In one example, the proximal domain may be a 1 to 20, 5 to
20, 10 to 20 or 15 to 20-base sequence.
[0377] In another example, the proximal domain may be a 1 to 5, 5
to 10, 10 to 15 or 15 to 20-base sequence.
[0378] vi) Tail Domain
[0379] The term "tail domain" is a nucleic acid sequence located at
one or more ends of the both ends of the guide nucleic acid.
[0380] The tail domain may have a complementary base sequence
therein, and may be formed in a double strand due to a
complementary base sequence.
[0381] The tail domain may be a 1 to 50-base sequence.
[0382] 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.
[0383] 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.
[0384] 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.
[0385] 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).
[0386] The guide nucleic acid includes one or more domains.
[0387] The guide nucleic acid may include a guide domain.
[0388] The guide nucleic acid may include a first complementary
domain.
[0389] The guide nucleic acid may include a linker domain.
[0390] The guide nucleic acid may include a second complementary
domain.
[0391] The guide nucleic acid may include a proximal domain.
[0392] The guide nucleic acid may include a tail domain.
[0393] Here, there may be 1, 2, 3, 4, 5, 6 or more domains.
[0394] The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more
guide domains.
[0395] The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more
first complementary domains.
[0396] The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more
linker domains.
[0397] The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more
second complementary domains.
[0398] The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more
proximal domains.
[0399] The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more
tail domains.
[0400] Here, in the guide nucleic acid, one type of domain may be
duplicated.
[0401] The guide nucleic acid may include several domains with or
without duplication.
[0402] 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.
[0403] 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.
[0404] 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.
[0405] 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.
[0406] 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.
[0407] 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.
[0408] 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.
[0409] As a specific example of the guide nucleic acid of the
present invention, gRNA will be described below.
[0410] gRNA
[0411] 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.
[0412] 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.
[0413] The gRNA may be called single-stranded gRNA (single RNA
molecule); or double-stranded gRNA (including more than one,
generally, two discrete RNA molecules).
[0414] 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.
[0415] 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.
[0416] 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.
[0417] 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.
[0418] i) Guide Domain
[0419] 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.
[0420] The guide domain may be a 5 to 50-base sequence.
[0421] As an exemplary embodiment, the guide domain may be a 16,
17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
[0422] As an exemplary embodiment, the guide domain may include a
16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
[0423] Here, the guide domain may include a guide sequence.
[0424] 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.
[0425] 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.
[0426] 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 unsaturated fatty acid biosynthesis-associated
factor such as an FAD gene, preferably an FAD2 gene, an FAD3 gene,
an FAD6 gene, an FAD7 or an FAD8 gene, which has, for example, at
least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or
complete complementarity.
[0427] The guide sequence may be a 5 to 50-base sequence.
[0428] In an exemplary embodiment, the guide sequence may be a 16,
17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
[0429] In one exemplary embodiment, the guide sequence may include
a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
[0430] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target sequence of the
FAD2 gene, which is a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base
sequence.
[0431] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target sequence of the
FAD3 gene, which is a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base
sequence.
[0432] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target sequence of the
FAD6 gene, which is a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base
sequence.
[0433] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target sequence of the
FAD7 gene, which is a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base
sequence.
[0434] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target sequence of the
FAD8 gene, which is a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base
sequence.
[0435] Here, target sequences of the target genes, that is, the
unsaturated fatty acid biosynthesis-associated factors such as the
FAD2 gene for the guide sequence are listed above in Table 1, but
the present invention is not limited thereto.
[0436] Here, the guide domain may include a guide sequence and an
additional base sequence.
[0437] The additional base sequence may be a 1 to 35-base
sequence.
[0438] In one exemplary embodiment, the additional base sequence
may be a 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10-base sequence.
[0439] For example, the additional base sequence may be a single
base sequence, guanine (G), or a sequence of two bases, GG.
[0440] The additional base sequence may be located at the 5' end of
the guide sequence.
[0441] The additional base sequence may be located at the 3' end of
the guide sequence.
[0442] 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.
[0443] ii) First Complementary Domain
[0444] 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.
[0445] 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.
[0446] 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.
[0447] 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.
[0448] 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.
[0449] 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,
Staphylococcus aureus or Neisseria meningitides, or a first
complementary domain derived therefrom.
[0450] 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'(SEQ ID NO: 42) or a base sequence
having partial, that is, at least 50% or more, or complete homology
with 5'-GUUUUAGAGCUA-3'(SEQ ID NO: 42). Here, the first
complementary domain may further include (X)n, resulting in
5'-GUUUUAGAGCUA(X)n-3'(SEQ ID NO: 42). 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.
[0451] 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'(SEQ ID
NO: 43), or a base sequence having partial, that is, at least 50%
or more, or complete homology with
5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3'(SEQ ID NO: 43). Here, the first
complementary domain may further include (X)n, resulting in
5'-GUUUUAGUCCCUUUUUAAAUUUCUU(X)n-3'(SEQ ID NO: 43). 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.
[0452] 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.
[0453] 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.
[0454] 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.
[0455] iii) Linker Domain
[0456] 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 by covalent bonding or non-covalent bonding, or may connect
two or more domains by covalent or non-covalent bonding.
[0457] The linker domain may be a nucleic acid sequence connecting
a first complementary domain with a second complementary domain to
produce single-stranded gRNA.
[0458] The linker domain may be connected with the first
complementary domain and the second complementary domain by
covalent or non-covalent bonding.
[0459] The linker domain may connect the first complementary domain
with the second complementary domain by covalent or non-covalent
bonding
[0460] The linker domain may be a 1 to 30-base sequence. The linker
domain may include a 1 to 30-base sequence.
[0461] 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.
[0462] 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.
[0463] 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.
[0464] The linker domain may have homology with a natural sequence,
for example, a partial sequence of tracrRNA, or may be derived
therefrom.
[0465] 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.
[0466] iv) Second Complementary Domain
[0467] 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.
[0468] 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.
[0469] 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.
[0470] 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.
[0471] 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.
[0472] 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,
Staphylococcus aureus or Neisseria meningitides, or a second
complementary domain derived therefrom.
[0473] 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'(SEQ ID NO: 44), or a base
sequence having partial, that is, at least 50% or more homology
with 5'-UAGCAAGUUAAAAU-3'(SEQ ID NO: 44) (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'(SEQ ID NO:
44). 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.
[0474] 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'(SEQ ID NO: 45), or a
base sequence having partial, that is, at least 50% or more
homology with 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3'(SEQ ID NO: 45) (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'(SEQ ID NO: 45). 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.
[0475] 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.
[0476] 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(SEQ ID NO: 46)-3', or a base sequence
having partial, that is, at least 50% or more homology with
5'-AAAUUUCUACU-3'(SEQ ID NO: 46) (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'(SEQ ID NO: 46). 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.
[0477] 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.
[0478] v) Proximal Domain
[0479] 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.
[0480] In one exemplary embodiment, the proximal domain may be a 5,
6, 7, 8, 8, 9, 10, 11, 12, 13, 14 or 15-base sequence.
[0481] In another embodiment, the proximal domain may include a 5,
6, 7, 8, 8, 9, 10, 11, 12, 13, 14 or 15-base sequence.
[0482] 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.
[0483] 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, Staphylococcus aureus or Neisseria
meningitides, or a proximal domain derived therefrom.
[0484] 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'(SEQ ID NO: 47), or a
base sequence having partial, that is, at least 50% or more
homology with 5'-AAGGCUAGUCCG-3'(SEQ ID NO: 47). Here, the proximal
domain may further include (X)n, resulting in
5'-AAGGCUAGUCCG(X)n-3'(SEQ ID NO: 47). 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.
[0485] 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'(SEQ
ID NO: 48), or a base sequence having at least 50% or more homology
with 5'-AAAGAGUUUGC-3'(SEQ ID NO: 48). Here, the proximal domain
may further include (X)n, resulting in 5'-AAAGAGUUUGC(X)n-3'(SEQ ID
NO: 48). 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.
[0486] 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.
[0487] vi) Tail Domain
[0488] 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.
[0489] 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.
[0490] 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.
[0491] 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.
[0492] 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, Staphylococcus aureus or Neisseria
meningitides or a tail domain derived therefrom.
[0493] 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'(SEQ ID NO:
49), or a base sequence having partial, that is, at least 50% or
more homology with 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3'(SEQ ID
NO: 49). Here, the tail domain may further include (X)n, resulting
in 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(X)n-3'(SEQ ID NO: 49). 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 such as A, T, U and G.
[0494] 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'(SEQ ID
NO: 50), or a base sequence having partial, that is, at least 50%
or more homology with
5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3'(SEQ ID NO: 50). Here,
the tail' domain may further include (X)n, resulting in
5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU(X)n-3'(SEQ ID NO: 50).
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.
[0495] 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.
[0496] 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.
[0497] 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.
[0498] 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.
[0499] 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).
[0500] Double-Stranded gRNA
[0501] The double-stranded gRNA consists of a first strand and a
second strand.
[0502] Here, the first strand may consist of
[0503] 5'-[guide domain]-[first complementary domain]-3', and
[0504] the second strand may consist of
[0505] 5'-[second complementary domain]-[proximal domain]-3' or
[0506] 5'-[second complementary domain]-[proximal domain]-[tail
domain]-3'.
[0507] Here, the first strand may be referred to as crRNA, and the
second strand may be referred to as tracrRNA.
[0508] First Strand
[0509] Guide Domain
[0510] 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.
[0511] 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.
[0512] In addition, the guide domain may include a guide
sequence.
[0513] 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.
[0514] 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 unsaturated fatty acid biosynthesis-associated
factor such as an FAD gene, preferably an FAD2 gene, an FAD3 gene,
an FAD6 gene, an FAD7 gene or an FAD8 gene, which has, for example,
at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or
complete complementarity.
[0515] 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.
[0516] In one exemplary embodiment, the guide sequence is a nucleic
acid sequence complementary to a target sequence of the FAD2 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.
[0517] In one exemplary embodiment, the guide sequence is a nucleic
acid sequence complementary to a target sequence of the FAD3 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.
[0518] In one exemplary embodiment, the guide sequence is a nucleic
acid sequence complementary to a target sequence of the FAD6 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.
[0519] In one exemplary embodiment, the guide sequence is a nucleic
acid sequence complementary to a target sequence of the FAD7 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.
[0520] In one exemplary embodiment, the guide sequence is a nucleic
acid sequence complementary to a target sequence of the FAD8 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.
[0521] Here, for the guide sequence, target sequences of a target
gene, that is, unsaturated fatty acid biosynthesis-associated
factors such as an FAD2 gene are listed above in Table 1, but the
present invention is not limited thereto.
[0522] Selectively, the guide domain may include a guide sequence
and an additional base sequence.
[0523] 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.
[0524] In one exemplary embodiment, the additional base sequence
may include one base, guanine (G), or two bases, GG.
[0525] 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.
[0526] The additional base sequence may be located at the 3' end of
the guide domain, or at the 3' end of the guide sequence.
[0527] First Complementary Domain
[0528] 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.
[0529] 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.
[0530] 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.
[0531] 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,
Staphylococcus aureus or Neisseria meningitides, or a first
complementary domain derived therefrom.
[0532] 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.
[0533] 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.
[0534] 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.
[0535] Therefore, the first strand may consist of 5'-[guide
domain]-[first complementary domain]-3' as described above.
[0536] In addition, the first strand may optionally include an
additional base sequence.
[0537] In one example, the first strand may be
[0538] 5'-(N.sub.target)-(Q).sub.m-3'; or
[0539]
5'-(X).sub.a-(N.sub.target)-(X).sub.b-(Q).sub.m-(X).sub.c-3'.
[0540] 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.
[0541] 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 an unsaturated fatty acid
biosynthesis-associated factor such as an FAD gene, preferably an
FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene or an FAD8
gene.
[0542] 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.
[0543] 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'(SEQ ID NO:
42), or a base sequence having at least 50% or more homology with
5'-GUUUUAGAGCUA-3'(SEQ ID NO: 42).
[0544] 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'(SEQ ID NO: 43), or a base sequence
having at least 50% or more homology with
5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3'(SEQ ID NO: 43).
[0545] 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'(SEQ ID NO: 51), or a base sequence
having at least 50% or more homology with
5'-GUUUUAGAGCUGUGUUGUUUCG-3'(SEQ ID NO: 51).
[0546] 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.
[0547] Second Strand
[0548] The second strand may consist of a second complementary
domain and a proximal domain, and selectively include a tail
domain.
[0549] Second Complementary Domain
[0550] 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.
[0551] 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.
[0552] 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.
[0553] 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,
Staphylococcus aureus or Neisseria meningitides, or a second
complementary domain derived therefrom.
[0554] 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.
[0555] 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.
[0556] Proximal Domain
[0557] 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, 8, 9, 10, 11, 12, 13, 14 or
15 bases.
[0558] Here, the proximal domain may have a double strand bond
between complementary base sequences therein.
[0559] 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.
[0560] 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, Staphylococcus aureus or Neisseria
meningitides, or a proximal domain derived therefrom.
[0561] Tail Domain
[0562] 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.
[0563] Here, the tail domain may have a double strand bond between
complementary base sequences therein.
[0564] 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.
[0565] 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, Staphylococcus aureus or Neisseria
meningitides, or a tail domain derived therefrom.
[0566] 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.
[0567] 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.
[0568] 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.
[0569] 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.
[0570] In addition, the second strand may selectively include an
additional base sequence.
[0571] 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'.
[0572] 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'.
[0573] 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.
[0574] 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'(SEQ ID NO: 44), or
a base sequence having at least 50% or more homology with
5'-UAGCAAGUUAAAAU-3'(SEQ ID NO: 44).
[0575] 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'(SEQ ID
NO: 45), or a base sequence having at least 50% or more homology
with 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3'(SEQ ID NO: 45).
[0576] 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'(SEQ ID NO: 52), or a base sequence
having at least 50% or more homology with
5'-CGAAACAACACAGCGAGUUAAAAU-3'(SEQ ID NO: 52).
[0577] 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.
[0578] 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'(SEQ ID NO: 47), or a base sequence having at
least 50% or more homology with 5'-AAGGCUAGUCCG-3'(SEQ ID NO:
47).
[0579] 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'(SEQ ID NO: 48), or a base sequence having at
least 50% or more homology with 5'-AAAGAGUUUGC-3'(SEQ ID NO:
48).
[0580] 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'(SEQ ID NO: 53), or a base
sequence having at least 50% or more homology with
5'-AAGGCUUAGUCCG-3'(SEQ ID NO: 53).
[0581] 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.
[0582] 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'(SEQ ID NO: 49), or a base
sequence having at least 50% or more homology with
5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3'(SEQ ID NO: 49).
[0583] 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'(SEQ ID NO: 50), or a
base sequence having at least 50% or more homology with
5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3'(SEQ ID NO: 50).
[0584] 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'(SEQ ID NO: 54), or a
base sequence having at least 50% or more homology with
5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3'(SEQ ID NO: 54).
[0585] 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.
[0586] 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.
[0587] 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.
[0588] Single-Stranded gRNA
[0589] Single-stranded gRNA may be classified into two types.
[0590] i) Single-Stranded gRNA
[0591] 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'.
[0592] Specifically, the single-stranded gRNA may consist of
[0593] 5'-[guide domain]-[first complementary domain]-[linker
domain]-[second complementary domain]-[proximal domain]-3' or
[0594] 5'-[guide domain]-[first complementary domain]-[linker
domain]-[second complementary domain]-[proximal domain]-[tail
domain]-3'.
[0595] 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.
[0596] Linker Domain
[0597] 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 by
covalent bonding or non-covalent bonding, or connect the first
complementary domain with the second complementary domain by
covalent or non-covalent bonding.
[0598] 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.
[0599] 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.
[0600] The linker domain may have homology with a natural sequence,
for example, a partial sequence of tracrRNA, or may be derived
therefrom.
[0601] 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.
[0602] 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.
[0603] In addition, the single-stranded gRNA may selectively
include an additional base sequence.
[0604] In one exemplary embodiment, the single-stranded gRNA may
be
[0605] 5-(N.sub.target)-(Q).sub.m-(L).sub.j-(Z).sub.h-(P).sub.k-3';
or
[0606]
5'-(N.sub.target)-(Q).sub.m-(L).sub.j-(Z).sub.h-(P).sub.k-(F).sub.i-
-3'.
[0607] In another embodiment, the single-stranded gRNA may be
[0608]
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
[0609]
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'.
[0610] 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 capable of being changed
according to a target sequence on a target gene or nucleic
acid.
[0611] 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 an unsaturated fatty acid
biosynthesis-associated factor such as an FAD gene, preferably an
FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene, or an FAD8
gene.
[0612] 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.
[0613] 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'(SEQ ID NO: 42), or a
base sequence having at least 50% or more homology with
5'-GUUUUAGAGCUA-3'(SEQ ID NO: 42).
[0614] 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'(SEQ ID
NO: 43), or a base sequence having at least 50% or more homology
with 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3'(SEQ ID NO: 43).
[0615] 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'(SEQ ID NO: 51), or a base sequence
having at least 50% or more homology with
5'-GUUUUAGAGCUGUGUUGUUUCG-3'(SEQ ID NO: 51).
[0616] 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.
[0617] 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.
[0618] 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'(SEQ ID NO: 44), or
a base sequence having at least 50% or more homology with
5'-UAGCAAGUUAAAAU-3'(SEQ ID NO: 44).
[0619] 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'(SEQ ID
NO: 45), or a base sequence having at least 50% or more homology
with 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3'(SEQ ID NO: 45).
[0620] 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'(SEQ ID NO: 52), or a base sequence
having at least 50% or more homology with
5'-CGAAACAACACAGCGAGUUAAAAU-3'(SEQ ID NO: 52).
[0621] 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.
[0622] 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'(SEQ ID NO: 47), or a base sequence having at
least 50% or more homology with 5'-AAGGCUAGUCCG-3'(SEQ ID NO:
47).
[0623] 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'(SEQ ID NO: 48), or a base sequence having at
least 50% or more homology with 5'-AAAGAGUUUGC-3'(SEQ ID NO:
48).
[0624] 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'(SEQ ID NO: 53), or a base
sequence having at least 50% or more homology with
5'-AAGGCUUAGUCCG-3'(SEQ ID NO: 53).
[0625] 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.
[0626] 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'(SEQ ID NO: 49), or a base
sequence having at least 50% or more homology with
5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3'(SEQ ID NO: 49).
[0627] 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'(SEQ ID NO: 50), or a
base sequence having at least 50% or more homology with
5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3'(SEQ ID NO: 50).
[0628] 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'(SEQ ID NO: 54), or a
base sequence having at least 50% or more homology with
5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3'(SEQ ID NO: 54).
[0629] 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.
[0630] 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.
[0631] 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.
[0632] ii) Single-Stranded gRNA
[0633] 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:
[0634] 5'-[second complementary domain]-[first complementary
domain]-[guide domain]-3'; or
[0635] 5'-[second complementary domain]-[linker domain]-[first
complementary domain]-[guide domain]-3'.
[0636] Guide Domain
[0637] 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 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.
[0638] 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.
[0639] In addition, the guide domain may include a guide
sequence.
[0640] 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.
[0641] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target gene, that is, a
target sequence of an unsaturated fatty acid
biosynthesis-associated factor such as an FAD gene, preferably an
FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene or an FAD8
gene, which has, for example, at least 70%, 75%, 80%, 85%, 90% or
95% or more complementarity or complete complementarity.
[0642] 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.
[0643] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target sequence of the
FAD2 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.
[0644] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target sequence of the
FAD3 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.
[0645] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target sequence of the
FAD6 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.
[0646] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target sequence of the
FAD7 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.
[0647] In one exemplary embodiment, the guide sequence may be a
nucleic acid sequence complementary to a target sequence of the
FAD8 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.
[0648] Here, target sequences of the target genes, that is, the
unsaturated fatty acid biosynthesis-associated factor such as the
FAD2 gene for the guide sequence are listed above in Table 1, but
the present invention is not limited thereto.
[0649] Selectively, the guide domain may include a guide sequence
and an additional base sequence.
[0650] 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.
[0651] In one exemplary embodiment, the additional base sequence
may be a single base sequence, guanine (G), or a sequence of two
bases, GG.
[0652] 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.
[0653] The additional base sequence may be located at the 3' end of
the guide domain, or at the 3' end of the guide sequence.
[0654] First Complementary Domain
[0655] 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.
[0656] 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.
[0657] 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.
[0658] 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.
[0659] Selectively, the first complementary domain may include an
additional base sequence which does not undergo complementary
bonding with the second complementary domain.
[0660] 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.
[0661] Second Complementary Domain
[0662] 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.
[0663] 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.
[0664] 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.
[0665] 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.
[0666] Selectively, the second complementary domain may include an
additional base sequence which does not undergo complementary
bonding with the first complementary domain.
[0667] 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.
[0668] Linker Domain
[0669] 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 by covalent or non-covalent bonding, or may
connect the first and second complementary domains by covalent or
non-covalent bonding.
[0670] 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.
[0671] 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.
[0672] 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.
[0673] In addition, the single-stranded gRNA may selectively
include an additional base sequence.
[0674] In one exemplary embodiment, the single-stranded gRNA may
be
[0675] 5'-(Z).sub.h-(Q).sub.m-(N.sub.target)-3'; or
[0676]
5'-(X).sub.a-(Z).sub.h-(X).sub.b-(Q).sub.m-(X).sub.c-(N.sub.target)-
-3'.
[0677] In another embodiment, the single-stranded gRNA may be
[0678] 5'-(Z).sub.h-(L).sub.j-(Q).sub.m-(N.sub.target)--'; or
[0679]
5'-(X).sub.a-(Z).sub.h-(L).sub.j-(Q).sub.m-(X).sub.c-(N.sub.target)-
-3'.
[0680] 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.
[0681] 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 an unsaturated fatty acid
biosynthesis-associated factor such as an FAD gene, preferably an
FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene or an FAD8
gene.
[0682] 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.
[0683] 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'.
[0684] 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.
[0685] 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'(SEQ ID NO:
46), or a base sequence having at least 50% or more homology with
5'-AAAUUUCUACU-3'(SEQ ID NO: 46).
[0686] 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.
[0687] 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.
[0688] 2. Editor Protein
[0689] 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.
[0690] The nucleic acid may be a nucleic acid contained in a target
nucleic acid, gene or chromosome.
[0691] The nucleic acid may be a guide nucleic acid.
[0692] The editor protein may be an enzyme.
[0693] The editor protein may be a fusion protein.
[0694] Here, the fusion protein refers to a protein produced by
fusing an enzyme with an additional domain, peptide, polypeptide or
protein.
[0695] The enzyme refers to a protein including a domain which is
able to cleave a nucleic acid, gene, chromosome or protein.
[0696] The enzyme may be a nuclease, protease or restriction
enzyme.
[0697] 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.
[0698] 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-term inus of the enzyme or
the proximity thereof; the middle region of an enzyme; and a
combination thereof.
[0699] 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-term inus of the enzyme or the
proximity thereof; the middle region of an enzyme; and a
combination thereof.
[0700] 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.
[0701] The functional domain, peptide, polypeptide or protein may
be a deaminase.
[0702] 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,
p-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.
[0703] 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).
[0704] The NLS may be NLS of SV40 virus large T-antigen with an
amino acid sequence PKKKRKV(SEQ ID NO: 55); NLS derived from
nucleoplasmin (e.g., nucleoplasmin bipartite NLS with a sequence
KRPAATKKAGQAKKKK(SEQ ID NO: 56)); c-myc NLS with an amino acid
sequence PAAKRVKLD(SEQ ID NO: 57) or RQRRNELKRSP(SEQ ID NO: 58);
hRNPA1 M9 NLS with a sequence
NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY(SEQ ID NO: 59); an
importin-.alpha.-derived IBB domain sequence
RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV(SEQ ID NO: 60); myoma T
protein sequences VSRKRPRP(SEQ ID NO: 61) and PPKKARED(SEQ ID NO:
62); human p53 sequence PQPKKKPL(SEQ ID NO: 63); a mouse c-abl IV
sequence SALIKKKKKMAP(SEQ ID NO: 64); influenza virus NS1 sequences
DRLRR and PKQKKRK(SEQ ID NO: 66); a hepatitis virus-.delta. antigen
sequence RKLKKKIKKL(SEQ ID NO: 67); a mouse Mx1 protein sequence
REKKKFLKRR(SEQ ID NO: 68); a human poly(ADP-ribose) polymerase
sequence KRKGDEVDGVDEVAKKKSKK(SEQ ID NO: 69); or steroid hormone
receptor (human) glucocorticoid sequence RKCLQAGMNLEARKTKK(SEQ ID
NO: 70), but the present invention is not limited thereto.
[0705] The editor protein may include a complete active enzyme.
[0706] 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.
[0707] 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.
[0708] The editor protein may include an incomplete or partially
active enzyme.
[0709] 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.
[0710] The editor protein may include an inactive enzyme.
[0711] 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.
[0712] The editor protein may be a natural enzyme or fusion
protein.
[0713] The editor protein may be present in the form of a partially
modified natural enzyme or fusion protein.
[0714] The editor protein may be an artificially produced enzyme or
fusion protein, which does not exist in nature.
[0715] The editor protein may be present in the form of a partially
modified artificial enzyme or fusion protein, which does not exist
in nature.
[0716] Here, the modification may be substitution, removal,
addition of amino acids contained in the editor protein, or a
combination thereof.
[0717] In addition, the modification may be substitution, removal,
addition of some bases in the base sequence encoding the editor
protein, or a combination thereof.
[0718] As one exemplary embodiment of the editor protein of the
present invention, a CRISPR enzyme will be described below.
[0719] CRISPR Enzyme
[0720] 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.
[0721] 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.
[0722] 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, Streptomyces viridochromogenes,
Streptosporangium roseum, 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.
[0723] 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.
[0724] 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.
[0725] 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.
[0726] 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.
[0727] Type II CRISPR Enzyme
[0728] 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).
[0729] The type II CRISPR enzyme includes two lobes, that is,
recognition (REC) and nuclease (NUC) lobes, and each lobe includes
several domains.
[0730] The REC lobe includes an arginine-rich bridge helix (BH)
domain, an REC1 domain and an REC2 domain.
[0731] 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.
[0732] 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.
[0733] 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 ofamino acids 1 to
59, 718 to 769 and 909 to 1098 of SpCas9, respectively.
[0734] 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.
[0735] 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.
[0736] 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.
[0737] Type V CRISPR Enzyme
[0738] 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.
[0739] 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.
[0740] 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.
[0741] In one example, the PAM sequence recognized by Cpf1 may be
5'-TTN-3' (N is A, T, C or G).
[0742] CRISPR Enzyme Activity
[0743] 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.
[0744] 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.
[0745] Incompletely or Partially Active Enzyme
[0746] A CRISPR enzyme modified to change enzyme activity, thereby
exhibiting incomplete or partial activity is called a nickase.
[0747] 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.
[0748] 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.
[0749] In one example, provided that the CRISPR enzyme is the type
II CRISPR enzyme, in the case of SpCas9, 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.
[0750] In another exemplary embodiment, in the case of CjCas9, 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.
[0751] 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.
[0752] In one example, provided that the CRISPR enzyme is the type
II CRISPR enzyme, in one exemplary embodiment, in the case of
SpCas9, 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.
[0753] In another exemplary embodiment, in the case of CjCas9, 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.
[0754] Inactive Enzyme
[0755] A CRISPR enzyme which is modified to make enzyme activity
completely inactive is called an inactive CRISPR enzyme.
[0756] 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.
[0757] 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.
[0758] In one example, provided that the CRISPR enzyme is the type
II CRISPR enzyme, 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,
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.
[0759] In another exemplary embodiment, in the case of CjCas9, 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.
[0760] Other Activities
[0761] 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.
[0762] In addition, the CRISPR enzyme may be modified to
completely, incompletely, or partially activate the endonuclease
activity, exonuclease activity or helicase activity.
[0763] Targeting of CRISPR Enzyme
[0764] 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.
[0765] 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.
[0766] In one example, provided that the CRISPR enzyme is the type
II CRISPR enzyme,
[0767] in the case of SpCas9, the PAM sequence may be 5'-NGG-3',
5'-NAG-3' and/or 5'-NGA-3',
[0768] in the case of StCas9, the PAM sequence may be 5'-NGGNG-3'
and/or 5'-NNAGAAW-3' (W=A or T),
[0769] in the case of NmCas9, the PAM sequence may be
5'-NNNNGATT-3' and/or 5'-NNNGCTT-3',
[0770] 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),
[0771] 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
[0772] 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).
[0773] In another example, provided that the CRISPR enzyme is the
type V CRISPR enzyme,
[0774] in the case of Cpf1, the PAM sequence may be 5'-TTN-3'.
[0775] Here, the N may be A, T, G or C; or A, U, G or C.
[0776] 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.
[0777] CRISPR Enzyme Mutant
[0778] 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.
[0779] 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.
[0780] 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.
[0781] 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.
[0782] In the CRISPR enzyme mutant,
[0783] 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.
[0784] 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.
[0785] 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.
[0786] 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.
[0787] 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.
[0788] 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.
[0789] 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.
[0790] 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.
[0791] 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.
[0792] 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.
[0793] 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.
[0794] 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.
[0795] 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.
[0796] 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.
[0797] 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.
[0798] 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.
[0799] 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.
[0800] 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.
[0801] 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.
[0802] The modification may be one or two or more of the amino
acids present in the PI domain of the CRISPR enzyme.
[0803] 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.
[0804] 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.
[0805] 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, 1601, M763, D965 and F1038 amino acids
contained in the REC and RuvC domains of SpCas9.
[0806] 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.
[0807] 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, 1601 and K890 amino acids contained in the
REC and HNH domains of SpCas9.
[0808] 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.
[0809] 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, 1601, T1102 and D1127 amino acids contained
in the REC and PI domains of SpCas9.
[0810] 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.
[0811] 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, 1601, M763, K890, D965 and F1038 amino
acids contained in the REC, RuvC and HNH domains of SpCas9.
[0812] 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.
[0813] 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, 1601, M763, D965, F1038, T1102 and D1127
amino acids contained in the REC, RuvC and PI domains of
SpCas9.
[0814] 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.
[0815] 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, 1601, K890, T1102 and D1127 amino acids
contained in the REC, HNH and PI domains of SpCas9.
[0816] 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.
[0817] 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.
[0818] 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.
[0819] 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, 1601, M763, K890, D965, F1038, T1102 and
D1127 amino acids contained in the REC, RuvC, HNH and PI domains of
SpCas9.
[0820] In addition, in the CRISPR enzyme mutant, 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.
[0821] 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.
[0822] 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.
[0823] 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.
[0824] 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.
[0825] 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.
[0826] 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.
[0827] 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.
[0828] 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 gRNA.
[0829] 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.
[0830] 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.
[0831] 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.
[0832] 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.
[0833] 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.
[0834] 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.
[0835] The functional domain, peptide, polypeptide or protein may
be a deaminase.
[0836] 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.
[0837] 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.
[0838] In addition, the functional domain may be a nuclear
localization sequence or signal (NLS) or a nuclear export sequence
or signal (NES).
[0839] 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(SEQ ID NO: 55);
NLS from nucleoplasmin (e.g., nucleoplasmin bipartite NLS having
the sequence KRPAATKKAGQAKKKK(SEQ ID NO: 56)); c-myc NLS having the
amino acid sequence PAAKRVKLD(SEQ ID NO: 57) or RQRRNELKRSP(SEQ ID
NO: 58); hRNPA1 M9 NLS having the sequence
NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY(SEQ ID NO: 59); the sequence
RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV(SEQ ID NO: 60) of the
IBB domain from importin-.alpha.; the sequences VSRKRPRP(SEQ ID NO:
61) and PPKKARED(SEQ ID NO: 62) of a myoma T protein; the sequence
PQPKKKPL(SEQ ID NO: 63) of human p53; the sequence SALIKKKKKMAP(SEQ
ID NO: 64) of mouse c-abl IV; the sequences DRLRR(SEQ ID NO: 65)
and PKQKKRK(SEQ ID NO: 66) of influenza virus NS1; the sequence
RKLKKKIKKL(SEQ ID NO: 67) of a hepatitis delta virus antigen; the
sequence REKKKFLKRR(SEQ ID NO: 68) of a mouse Mx1 protein; the
sequence KRKGDEVDGVDEVAKKKSKK(SEQ ID NO: 69) of a human poly
(ADP-ribose) polymerase; or the NLS sequence RKCLQAGMNLEARKTKK(SEQ
ID NO: 70), derived from a sequence of a steroid hormone receptor
(human) glucocorticoid.
[0840] 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.
[0841] Here, the split-type CRISPR enzyme may be a completely,
incompletely or partially active enzyme or inactive enzyme.
[0842] For example, the SpCas9 may be divided into two parts
between the residue 656, tyrosine, and the residue 657, threonine,
thereby generating split SpCas9.
[0843] In addition, the split-type CRISPR enzyme may selectively
include an additional domain, peptide, polypeptide or protein for
reconstitution.
[0844] 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.
[0845] 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.
[0846] 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.
[0847] 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.
[0848] 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.
[0849] 3. Target Sequence
[0850] 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.
[0851] 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.
[0852] The target sequence may be a 5 to 50-base sequence.
[0853] In an embodiment, the target sequence may be a 16, 17, 18,
19, 20, 21, 22, 23, 24 or 25-base sequence.
[0854] 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.
[0855] 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.
[0856] 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.
[0857] 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.
[0858] In one exemplary embodiment, target sequences for a
gRNA-CRISPR enzyme complex will be described below.
[0859] 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.
[0860] 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.
[0861] 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.
[0862] 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.
[0863] 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.
[0864] 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.
[0865] 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.
[0866] 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.
[0867] 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.
[0868] 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.
[0869] 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 an FAD2 gene, an
FAD3 gene, an FAD6 gene, an FAD7 gene, and an FAD8 gene.
[0870] The target sequence may be a nucleic acid sequence contained
in the FAD2 gene.
[0871] The target sequence may be a nucleic acid sequence contained
in the FAD3 gene.
[0872] The target sequence may be a nucleic acid sequence contained
in the FAD6 gene.
[0873] The target sequence may be a nucleic acid sequence contained
in the FAD7 gene.
[0874] The target sequence may be a nucleic acid sequence contained
in the FAD8 gene.
[0875] Alternatively, the target sequence may be a partial nucleic
acid sequence of one or more genes selected from the group
consisting of an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7
gene, and an FAD8 gene.
[0876] The target sequence may be a partial nucleic acid sequence
of the FAD2 gene.
[0877] The target sequence may be a partial nucleic acid sequence
of the FAD3 gene.
[0878] The target sequence may be a partial nucleic acid sequence
of the FAD6 gene.
[0879] The target sequence may be a partial nucleic acid sequence
of the FAD7 gene.
[0880] The target sequence may be a partial nucleic acid sequence
of the FAD8 gene.
[0881] 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 an FAD2
gene, an FAD3 gene, an FAD6 gene, an FAD7 gene, and an FAD8
gene.
[0882] The target sequence may be a nucleic acid sequence of the
coding or non-coding region or a mixture thereof of the FAD2
gene.
[0883] The target sequence may be a nucleic acid sequence of the
coding or non-coding region or a mixture thereof of the FAD3
gene.
[0884] The target sequence may be a nucleic acid sequence of the
coding or non-coding region or a mixture thereof of the FAD6
gene.
[0885] The target sequence may be a nucleic acid sequence of the
coding or non-coding region or a mixture thereof of the FAD7
gene.
[0886] The target sequence may be a nucleic acid sequence of the
coding or non-coding region or a mixture thereof of the FAD8
gene.
[0887] 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 an FAD2 gene, an FAD3 gene, an FAD6 gene, an
FAD7 gene, and an FAD8 gene.
[0888] 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 FAD2 gene.
[0889] 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 FAD3 gene.
[0890] 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 FAD6 gene.
[0891] 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 FAD7 gene.
[0892] 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 FAD8 gene.
[0893] 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 an FAD2 gene, an FAD3
gene, an FAD6 gene, an FAD7 gene, and an FAD8 gene.
[0894] The target sequence may be a nucleic acid sequence of an
exon, an intron or a mixture thereof of the FAD2 gene.
[0895] The target sequence may be a nucleic acid sequence of an
exon, an intron or a mixture thereof of the FAD3 gene.
[0896] The target sequence may be a nucleic acid sequence of an
exon, an intron or a mixture thereof of the FAD6 gene.
[0897] The target sequence may be a nucleic acid sequence of an
exon, an intron or a mixture thereof of the FAD7 gene.
[0898] The target sequence may be a nucleic acid sequence of an
exon, an intron or a mixture thereof of the FAD8 gene.
[0899] 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 an FAD2 gene, an FAD3 gene, an FAD6 gene,
an FAD7 gene, and an FAD8 gene.
[0900] The target sequence may be a nucleic acid sequence including
or adjacent to a mutated region of the FAD2 gene.
[0901] The target sequence may be a nucleic acid sequence including
or adjacent to a mutated region of the FAD3 gene.
[0902] The target sequence may be a nucleic acid sequence including
or adjacent to a mutated region of the FAD6 gene.
[0903] The target sequence may be a nucleic acid sequence including
or adjacent to a mutated region of the FAD7 gene.
[0904] The target sequence may be a nucleic acid sequence including
or adjacent to a mutated region of the FAD8 gene.
[0905] 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 an FAD2 gene, an FAD3 gene, an FAD6 gene, an
FAD7 gene, and an FAD8 gene.
[0906] The target sequence may be a continuous 5 to 50-nucleic acid
sequence of the FAD2 gene.
[0907] The target sequence may be a continuous 5 to 50-nucleic acid
sequence of the FAD3 gene.
[0908] The target sequence may be a continuous 5 to 50-nucleic acid
sequence of the FAD6 gene.
[0909] The target sequence may be a continuous 5 to 50-nucleic acid
sequence of the FAD7 gene.
[0910] The target sequence may be a continuous 5 to 50-nucleic acid
sequence of the FAD8 gene.
[0911] As one exemplary embodiment of the present invention, the
above target sequences of the FAD2 gene are summarized in Table
1.
[0912] Unsaturated Fatty Acid Biosynthesis-Associated
Factor-Manipulated Product
[0913] 4. Guide Nucleic Acid-Editor Protein Complex and Use
Thereof
[0914] A guide nucleic acid-editor protein complex may modify a
target.
[0915] 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.
[0916] Here, the guide nucleic acid-editor protein complex may act
at a DNA, RNA, gene or chromosomal level.
[0917] 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.
[0918] 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.
[0919] 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.
[0920] 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.
[0921] The guide nucleic acid-editor protein complex may act at
gene transcription and translation stages.
[0922] 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.
[0923] 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.
[0924] The guide nucleic acid-editor protein complex may act at a
protein level.
[0925] 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.
[0926] In one exemplary embodiment, the present invention provides
a guide nucleic acid-editor protein complex used to manipulate a
unsaturated fatty acid biosynthesis-associated factor, for example,
an FAD gene, preferably an FAD2 gene, an FAD3 gene, an FAD6 gene,
an FAD7 gene, and/or an FAD8 gene. Preferably, a gRNA-CRISPR enzyme
complex is provided.
[0927] 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.
[0928] 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 an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7
gene, and/or an FAD8 gene.
[0929] 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.
[0930] The gRNA may target, for example, two or more of the FAD2
gene, the FAD3 gene, the FAD6 gene, the FAD7 gene, and/or the FAD8
gene, or
[0931] two or more regions in each of the FAD2 gene, the FAD3 gene,
the FAD6 gene, the FAD7 gene, and/or the FAD8 gene, and
[0932] may independently induce the cleavage of a double strand
and/or a single strand of the FAD2 gene, the FAD3 gene, the FAD6
gene, the FAD7 gene, and/or the FAD8 gene, or
[0933] may induce the insertion of one foreign nucleotide into a
cleavage site of the FAD2 gene, the FAD3 gene, the FAD6 gene, the
FAD7 gene, and/or the FAD8 gene.
[0934] In addition, in another exemplary embodiment of the present
invention, a nucleic acid constituting the guide nucleic
acid-editor protein complex may include:
[0935] (a) a sequence encoding a guide nucleic acid including a
guide domain, which is complementary to a target sequence of the
FAD2 gene as described herein; and
[0936] (b) a sequence encoding an editor protein.
[0937] 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.
[0938] 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 FAD2 gene.
[0939] 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
FAD2 gene, the FAD3 gene, the FAD6 gene, the FAD7 gene, and/or the
FAD8 gene.
[0940] Use of Guide Nucleic Acid-Editor Protein Complex
[0941] 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.
[0942] Gene Manipulation
[0943] 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.
[0944] i) Cleavage or Damage of Target Gene or Nucleic Acid
[0945] 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.
[0946] 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.
[0947] 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.
[0948] 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.
[0949] 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 gRNS may be cleaved by the SpCas9 nickase (H840A),
and the cleavages may take place sequentially or
simultaneously.
[0950] 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.
[0951] 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.
[0952] 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.
[0953] 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.
[0954] 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.
[0955] 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).
[0956] 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).
[0957] 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).
[0958] 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).
[0959] ii) Repair or Restoration of Damaged Target Gene or Nucleic
Acid
[0960] The target gene or nucleic acid cleaved or damaged by the
CRISPR complex may be repaired or restored through non-homologous
end joining (NHEJ) and homology-directed repairing (HDR).
[0961] Non-Homologous End Joining (NHEJ)
[0962] 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.
[0963] 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 mutation in a significant functional domain
is probably less tolerated than mutations in a non-significant
region of a protein.
[0964] 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.
[0965] 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.
[0966] 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.
[0967] Homology Directed Repairing (HDR)
[0968] 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.
[0969] 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.
[0970] 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.
[0971] 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.
[0972] 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.
[0973] 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.
[0974] Other than the NHEJ and HDR, there are methods of repairing
or restoring broken DNA.
[0975] Single-Strand Annealing (SSA)
[0976] 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.
[0977] 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.
[0978] Single-Strand Break Repair (SSBA)
[0979] 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.
[0980] Mismatch Repair (MMR)
[0981] 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.
[0982] Base Excision Repair (BER)
[0983] 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.
[0984] Nucleotide Excision Repair (NER)
[0985] 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.
[0986] Gene Manipulation Effects
[0987] Manipulation or correction of a target gene or nucleic acid
may largely lead to effects of knockout, knockdown, and
knockin.
[0988] Knockout
[0989] 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.
[0990] 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.
[0991] Knockdown
[0992] 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.
[0993] 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.
[0994] Knockin
[0995] 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.
[0996] In addition, the knockin may further need a donor.
[0997] 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.
[0998] 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.
[0999] The donor may be a double- or single-stranded nucleic
acid.
[1000] The donor may be present in a linear or circular shape.
[1001] The donor may include a nucleic acid sequence having
homology with a target gene or nucleic acid.
[1002] 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.
[1003] 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.
[1004] 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).
[1005] 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
unsaturated fatty acid biosynthesis-associated factor may have the
following constitution.
[1006] In one exemplary embodiment, when the unsaturated fatty acid
biosynthesis-associated factor is a gene,
[1007] the constitution of the artificially manipulated unsaturated
fatty acid biosynthesis-associated factor by the gRNA-CRISPR enzyme
complex may include modification of one or more nucleic acids
among
[1008] a deletion or insertion of one or more nucleotides;
[1009] a substitution with one or more nucleotides different from a
wild-type gene; and
[1010] an insertion of one or more foreign nucleotides
[1011] 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 unsaturated fatty acid biosynthesis-associated factor or
adjacent to a 5' end and/or 3' end thereof.
[1012] In addition, a chemical modification of one or more
nucleotides may be included in the nucleic acid sequence
constituting the unsaturated fatty acid biosynthesis-associated
factor.
[1013] Here, the "foreign nucleotide" is the concept including all
exogeneous, for example, heterologous or artificially-synthesized
nucleotides, other than nucleotides innately included in the
unsaturated fatty acid biosynthesis-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.
[1014] 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.
[1015] To form a desired unsaturated fatty acid biosynthesis
controlling system, artificial modification using a gRNA-CRISPR
enzyme complex may be applied to the nucleic acid constituting the
unsaturated fatty acid biosynthesis-associated factor.
[1016] A region including the nucleic acid modification of the
unsaturated fatty acid biosynthesis-associated factor may be a
target region or target sequence.
[1017] 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.
[1018] Such nucleic acid modification includes the "cleavage" of a
nucleic acid.
[1019] 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.
[1020] 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 unsaturated fatty acid
biosynthesis-associated factor without the cleavage process.
Chemical modification of one or more nucleotides in the nucleic
acid sequence of the unsaturated fatty acid biosynthesis-associated
factor may be included according to such a specific function.
[1021] 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.
[1022] 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 unsaturated
fatty acid biosynthesis-associated factor as described above.
[1023] The artificially manipulated unsaturated fatty acid
biosynthesis-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 system for controlling
unsaturated fatty acids biosynthesis, for example, exhibition of an
effect of promoting or suppressing a specific unsaturated fatty
acid.
[1024] For example, a specific protein may be expressed and its
activity may be stimulated by the artificially manipulated
unsaturated fatty acid biosynthesis-associated factor.
[1025] A specific protein may be inactivated by the artificially
manipulated unsaturated fatty acid biosynthesis-associated
factor.
[1026] In one example, a specific target region of each unsaturated
fatty acid biosynthesis-associated factor of the genome, for
example, reverse regulatory genes such as an FAD2 gene, an FAD3
gene, an FAD6 gene, an FAD7 gene, and/or an FAD8 gene may be
cleaved, resulting in knockdown or knockout of the gene.
[1027] 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 an FAD2 gene, an FAD3 gene, an FAD6
gene, an FAD7 gene, and/or an FAD8 gene.
[1028] A production of unsaturated fatty acids may be regulated by
the artificially manipulated unsaturated fatty acid
biosynthesis-associated factor.
[1029] A plant body increased or decreased in the content of a
specific unsaturated fatty acid or a processed product using the
plant body may be produced by the artificially manipulated
unsaturated fatty acid biosynthesis-associated factor.
[1030] In one exemplary embodiment of the present invention, the
artificially manipulated unsaturated fatty acid
biosynthesis-associated factor may provide various artificially
manipulated unsaturated fatty acid biosynthesis-associated factors
according to the constitutional characteristic of the gRNA-CRISPR
enzyme complex (e.g., included in a target region of the
unsaturated fatty acid biosynthesis-associated factor or different
in the adjacent major PAM sequence).
[1031] Hereinafter, while representative examples of CRISPR enzymes
and an unsaturated fatty acid biosynthesis-associated gene have
been illustrated, they are merely specific examples, and thus the
present invention is not limited thereto.
[1032] 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.
[1033] The present invention may provide an artificially
manipulated unsaturated fatty acid biosynthesis-associated factor,
for example, an artificially manipulated FAD2 gene, FAD3 gene, FAD6
gene, FAD7 gene, and/or FAD8 gene, which is prepared by
[1034] 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,
[1035] 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,
[1036] 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
[1037] d) a combination of two or more selected from a) through
c)
[1038] in the nucleic acid sequence of the unsaturated fatty acid
biosynthesis-associated factor.
[1039] 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.
[1040] The present invention may provide an artificially
manipulated unsaturated fatty acid biosynthesis-associated factor,
for example, an artificially manipulated FAD2 gene, FAD3 gene, FAD6
gene, FAD7 gene, and/or FAD8 gene, which is prepared by
[1041] 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),
[1042] 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,
[1043] 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
[1044] d') a combination of two or more selected from a') through
c') in the nucleic acid sequence of the unsaturated fatty acid
biosynthesis-associated factor.
[1045] 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.
[1046] The present invention may provide an artificially
manipulated unsaturated fatty acid biosynthesis-associated factor,
for example, an artificially manipulated FAD2 gene, FAD3 gene, FAD6
gene, FAD7 gene, and/or FAD8 gene, which is prepared by
[1047] 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 of the 5'-NNAGAAW-3' sequence (each
N is independently A, T, C or G, and W is A or T),
[1048] 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,
[1049] 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
[1050] d'') a combination of two or more selected from a'') through
c'') in the nucleic acid sequence of the unsaturated fatty acid
biosynthesis-associated factor.
[1051] 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.
[1052] The present invention may provide an artificially
manipulated unsaturated fatty acid biosynthesis-associated factor,
for example, an artificially manipulated FAD2 gene, FAD3 gene, FAD6
gene, FAD7 gene, and/or FAD8 gene, which is prepared by
[1053] 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),
[1054] 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,
[1055] 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
[1056] d''') a combination of two or more selected from a''')
through c''') in the nucleic acid sequence of the unsaturated fatty
acid biosynthesis-associated factor.
[1057] 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.
[1058] The present invention may provide an artificially
manipulated unsaturated fatty acid biosynthesis-associated factor,
for example, an artificially manipulated FAD2 gene, FAD3 gene, FAD6
gene, FAD7 gene, and/or FAD8 gene, which is prepared by
[1059] 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),
[1060] 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,
[1061] 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
[1062] d'''') a combination of two or more selected from a'''')
through c'''') in the nucleic acid sequence of the unsaturated
fatty acid biosynthesis-associated factor.
[1063] 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.
[1064] 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.
[1065] The present invention may provide an artificially
manipulated unsaturated fatty acid biosynthesis-associated factor,
for example, an artificially manipulated FAD2 gene, FAD3 gene, FAD6
gene, FAD7 gene, and/or FAD8 gene, which is prepared by
[1066] 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),
[1067] 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,
[1068] 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
[1069] d''''') a combination of two or more selected from a''''')
through c''''') in the nucleic acid sequence of the unsaturated
fatty acid biosynthesis-associated factor.
[1070] In another exemplary embodiment, when the unsaturated fatty
acid biosynthesis-associated factor is a protein,
[1071] the artificially manipulated protein includes all proteins
involved in formation of new or modified unsaturated fatty acid
biosynthesis by a direct or indirect action of the gRNA-CRISPR
enzyme complex.
[1072] For example, the artificially manipulated protein may be a
protein expressed by an unsaturated fatty acid
biosynthesis-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.
[1073] The artificially manipulated unsaturated fatty acid
biosynthesis-associated factor (protein) may have an amino acid
composition and activity corresponding to the composition of the
artificially manipulated unsaturated fatty acid
biosynthesis-associated factor (gene).
[1074] As an embodiment, an (i) artificially manipulated protein
which is changed in expression characteristics may be provided.
[1075] For example, protein modification may have one or more
characteristics:
[1076] 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 unsaturated fatty acid
biosynthesis-associated factor or adjacent to the 5' end and/or the
3' end thereof;
[1077] a decrease or increase in expression level according to the
substitution with one or more nucleotides different from those of a
wild-type gene;
[1078] 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
[1079] a decrease or increase in expression level of a third
protein influenced by expression characteristics of the
above-described proteins.
[1080] An (ii) artificially manipulated protein which is changed in
structural characteristics may be provided.
[1081] For example, protein modification may have one or more
characteristics:
[1082] 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 unsaturated
fatty acid biosynthesis-associated factor or adjacent to the 5' end
and/or the 3' end thereof;
[1083] 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;
[1084] 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
[1085] a change in codons, amino acids, and three-dimensional
structure of a third protein influenced by the above-described
protein changed in structural characteristic.
[1086] An (iii) artificially manipulated protein changed in
functional characteristics may be provided.
[1087] For example, protein modification may have one or more
characteristics:
[1088] the activation or inactivation of a specific 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 unsaturated
fatty acid biosynthesis-associated factor or adjacent to the 5' end
and/or the 3' end thereof;
[1089] 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;
[1090] 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
[1091] the change in the function of a third protein influenced by
the above-described protein changed in functional
characteristics.
[1092] In addition, a protein artificially manipulated by the
chemical modification of one or more nucleotides in the nucleic
acid sequence constituting the unsaturated fatty acid
biosynthesis-associated factor may be included.
[1093] 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.
[1094] 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.
[1095] 5. Other Additional Components
[1096] 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.
[1097] The additional component may be selectively used to improve
the efficiency of the guide nucleic acid-editor protein
complex.
[1098] Activator
[1099] 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.
[1100] 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.
[1101] The activator may be a double-stranded nucleic acid or
single-stranded nucleic acid.
[1102] The activator may be linear or circular.
[1103] 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.
[1104] 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.
[1105] 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.
[1106] 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.
[1107] 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.
[1108] The homologous nucleic acid sequence may have at least
50%.sup., 55%.sup., 60%.sup., 65%, 70%, 75%, 80%, 85%, 90% or 95% A
or more homology, or complete homology.
[1109] In addition, the additional component may be selectively
used to improve the repair efficiency of the damaged gene or
nucleic acid.
[1110] Assistor
[1111] The additional component may be used as an assistor to
improve the repair efficiency of the damaged gene or nucleic
acid.
[1112] 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.
[1113] The assistor may be a double-stranded nucleic acid or
single-stranded nucleic acid.
[1114] The assistor may be present in a linear or circular
shape.
[1115] 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.
[1116] The NHEJ assistor may participate in a repair process or
improve the repair efficiency of the damaged gene or nucleic acid
using NHEJ.
[1117] 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.
[1118] The HDR assistor may participate in the repair process or
improve repair efficiency of the damaged gene or nucleic acid using
HDR.
[1119] 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.
[1120] 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.
[1121] 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.
[1122] 6. Subject
[1123] 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.
[1124] The subject may be an organism including a target nucleic
acid, gene, chromosome or protein of the guide nucleic acid-editor
protein complex.
[1125] The organism may be cells, tissue, or a plant.
[1126] The cells may be eukaryotic cells.
[1127] The eukaryotic cells may be plant cells.
[1128] The tissue may be tissue of a plant such as a leaf, stem,
root, flower, fruit or callus, and the like, etc.
[1129] The plant may be a plant in various periods from a seed to a
mature body.
[1130] The plant may be a plant body including an unsaturated fatty
acid.
[1131] In addition, 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.
[1132] The specimen or sample may be obtained from a plant
body.
[1133] 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.
[1134] Here, the target gene may be an unsaturated fatty acid
biosynthesis-associated factor, for example, an FAD2 gene, an FAD3
gene, an FAD6 gene, an FAD7 gene, and/or an FAD8 gene.
[1135] The target gene may be a wild type, or a modified form in
the wild-type.
[1136] 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.
[1137] Here, the manipulated gene may be an unsaturated fatty acid
biosynthesis-associated factor, for example, an FAD2 gene, an FAD3
gene, an FAD6 gene, an FAD7 gene, and/or an FAD8 gene.
[1138] Here, the guide nucleic acid may target an unsaturated fatty
acid biosynthesis-associated factor, for example, an FAD2 gene, an
FAD3 gene, an FAD6 gene, an FAD7 gene, and/or an FAD8 gene.
[1139] The guide nucleic acid may be a nucleic acid sequence
complementary to a target sequence of the FAD2 gene, FAD3 gene,
FAD6 gene, FAD7 gene, and/or FAD8 gene.
[1140] The guide nucleic acid may target one or more genes.
[1141] The guide nucleic acid may simultaneously target two or more
genes. Here, the two or more genes may be homologous or
heterologous genes.
[1142] The guide nucleic acid may target one or more target
sequences.
[1143] The guide nucleic acid may be designed in various forms
according to the number or locations of the target sequences.
[1144] 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.
[1145] In a certain embodiment, for artificial manipulation of the
FAD2 gene, a guide nucleic acid sequence corresponding to any one
of the target sequences of SEQ ID NOs: 1 to 30.
[1146] In a certain embodiment, for artificial manipulation of the
FAD2 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 30, for
example, SEQ ID NOs: 7 or 30, is provided.
[1147] 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 30, for example, SEQ
ID NOs: 7 or 30, and an expression product thereof are
provided.
[1148] 7. Delivery
[1149] 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.
[1150] The guide nucleic acid may be delivered or introduced into a
subject in the form of DNA, RNA or a mixed form.
[1151] 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.
[1152] 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.
[1153] 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.
[1154] 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.
[1155] 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.
[1156] i) Delivery in Form of DNA, RNA or Mixture Thereof
[1157] 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.
[1158] 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.
[1159] The vector may be a viral or non-viral vector (e.g., a
plasmid).
[1160] The non-vector may be naked DNA, a DNA complex or mRNA.
[1161] Vector-Based Introduction
[1162] The nucleic acid sequence encoding the guide nucleic acid
and/or editor protein may be delivered or introduced into a subject
by a vector.
[1163] The vector may include a nucleic acid sequence encoding a
guide nucleic acid and/or editor protein.
[1164] For example, the vector may simultaneously include nucleic
acid sequences, which encode the guide nucleic acid and the editor
protein, respectively.
[1165] For example, the vector may include the nucleic acid
sequence encoding the guide nucleic acid.
[1166] 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.
[1167] For example, the vector may include the nucleic acid
sequence encoding the editor protein.
[1168] 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.
[1169] The vector may include one or more regulatory/control
components.
[1170] 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.
[1171] The promoter may be a promoter recognized by RNA polymerase
II.
[1172] The promoter may be a promoter recognized by RNA polymerase
III.
[1173] The promoter may be an inducible promoter.
[1174] The promoter may be a subject-specific promoter.
[1175] The promoter may be a viral or non-viral promoter.
[1176] 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).
[1177] 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.
[1178] For example, a promoter useful for the guide nucleic acid
and/or the editor protein may be a root specific expression
promoter, a seed specific promoter, whole-body expression inducible
promoter or a leaf or the others tissue specific promoter.
[1179] The vector may be a viral vector or recombinant viral
vector.
[1180] The virus may be a DNA virus or an RNA virus.
[1181] Here, the DNA virus may be a double-stranded DNA (dsDNA)
virus or single-stranded DNA (ssDNA) virus.
[1182] Here, the RNA virus may be a single-stranded RNA (ssRNA)
virus.
[1183] The virus may be a mosaic virus, 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.
[1184] 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).
[1185] 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.
[1186] In one example, a nucleic acid sequence encoding a guide
nucleic acid and/or editor protein may be delivered or introduced
by a recombinant mosaic virus.
[1187] 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.
[1188] 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.
[1189] 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.
[1190] In addition, a vector may be included in a bacterium and
introduced into a subject.
[1191] Here, the vector may be included in an agrobacterium and
introduced into a subject, but the present invention is not limited
thereto.
[1192] Generally, a method of transferring a desired genetic
material to a subject using agrobacteria is most widely used, and
in the case of a plant, to genetically modify the plant, the DNA of
the agrobacteria may be inserted into the chromosome of a plant
body in a form of a nucleic acid protein called a plasmid. This may
serve to transfer a genetic material into cells of the plant body,
and the transferred genetic material is fused in the cells. The
above-described method may be widely used to produce an
agrobacterium gene-modified crop, and other than this, it can also
be used as a system for studying the reaction of cells for genetic
transformation.
[1193] Non-Vector-Based Introduction
[1194] 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.
[1195] The non-vector may include a nucleic acid sequence encoding
a guide nucleic acid and/or editor protein.
[1196] The non-vector may be naked DNA, a DNA complex, mRNA, or a
mixture thereof.
[1197] 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.
[1198] 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.
[1199] 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.).
[1200] In a certain embodiment, the non-vector may be delivered
using a lipid shell.
[1201] 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.
[1202] 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.
[1203] Other additives may be included.
[1204] ii) Delivery in Form of Peptide, Polypeptide or Protein
[1205] 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.
[1206] 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.
[1207] The peptide, polypeptide or protein may be delivered with a
nucleic acid sequence encoding a guide nucleic acid.
[1208] 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.
[1209] iii) Delivery in Form of Nucleic Acid-Protein Mixture
[1210] 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.
[1211] For example, the guide nucleic acid may be DNA, RNA or a
mixture thereof. The editor protein may be a peptide, polypeptide
or protein.
[1212] 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).
[1213] 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.
[1214] 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.
[1215] The vector may include the nucleic acid sequence encoding
the gRNA and/or CRISPR enzyme.
[1216] For example, the vector may simultaneously include the
nucleic acid sequences encoding the gRNA and the CRISPR enzyme.
[1217] For example, the vector may include the nucleic acid
sequence encoding the gRNA.
[1218] In one example, domains contained in the gRNA may be
contained in one vector, or may be divided and then contained in
different vectors.
[1219] For example, the vector may include the nucleic acid
sequence encoding the CRISPR enzyme.
[1220] 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.
[1221] The vector may include one or more regulatory/control
components.
[1222] 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.
[1223] The promoter may be a promoter recognized by RNA polymerase
II.
[1224] The promoter may be a promoter recognized by RNA polymerase
III.
[1225] The promoter may be an inducible promoter.
[1226] The promoter may be a subject-specific promoter.
[1227] The promoter may be a viral or non-viral promoter.
[1228] 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).
[1229] 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.
[1230] For example, a promoter useful for the guide nucleic acid
and/or the editor protein may be a root specific expression
promoter, a seed specific promoter, whole-body expression inducible
promoter or a leaf or the others tissue specific promoter.
[1231] The vector may be a viral vector or recombinant viral
vector.
[1232] The virus may be a DNA virus or an RNA virus.
[1233] Here, the DNA virus may be a double-stranded DNA (dsDNA)
virus or single-stranded DNA (ssDNA) virus.
[1234] Here, the RNA virus may be a single-stranded RNA (ssRNA)
virus.
[1235] The virus may be a mosaic virus, 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.
[1236] 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).
[1237] 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.
[1238] In one example, a nucleic acid sequence encoding gRNA and/or
a CRISPR enzyme may be delivered or introduced by a recombinant
mosaic virus.
[1239] In another example, a nucleic acid sequence encoding gRNA
and/or a CRISPR enzyme may be delivered or introduced by a
recombinant adenovirus.
[1240] In still another example, a nucleic acid sequence encoding
gRNA and/or a CRISPR enzyme may be delivered or introduced by
recombinant AAV.
[1241] 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.
[1242] The vector may be included in a bacterium and introduced
into a subject.
[1243] Here, the vector may be included in an agrobacterium and
introduced into a subject, but the present invention is not limited
thereto.
[1244] As an example, a vector including a nucleic acid sequence(s)
encoding gRNA and/or a CRISPR enzyme may be included in an
agrobacterium and introduced into a plant body.
[1245] In one exemplary embodiment of the present invention, the
gRNA-CRISPR enzyme complex may be delivered or introduced into a
subject.
[1246] 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.
[1247] 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).
[1248] 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.
[1249] 8. Transformant
[1250] 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.
[1251] 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.
[1252] 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.
[1253] 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.
[1254] 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.
[1255] 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.
[1256] 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.
[1257] The transformant may be an organism including a target
nucleic acid, gene, chromosome or protein of the guide nucleic
acid-editor protein complex.
[1258] The organism may be cells, tissue or a plant.
[1259] The cells may be prokaryotic cells or eukaryotic cells.
[1260] The eukaryotic cells may be plant cells, but the present
invention is not limited thereto.
[1261] The tissue may be tissue of a plant body such as a root, a
stem, a leaf, a flower, a fruit or a callus, and the like, etc.
[1262] The transformant may be a plant body into 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 plant body.
[1263] The specimen or sample may be a root, a stem, a leaf, a
flower, a fruit, a callus or cells thereof.
[1264] Use
[1265] One exemplary embodiment of the present invention releates
to a use for producing a composition for artificially manipulating
an unsaturated fatty acid biosynthesis-associated factor of a
subject such as a plant, a plant body in which the content of a
specific unsaturated fatty acid is controlled by an artificially
manipulated unsaturated fatty acid biosynthesis-associated factor,
or a processed product using the same.
[1266] Specific Unsaturated Fatty Acids
[1267] Soybean (Glycine max L.)
[1268] Soybeans are the most widely cultivated crop in the
worldwide, and provide the highest quality vegetable oil and
proteins in terms of production and use. Transformation technology
is widely used to improve genetic characteristics of various
effective genes in soybeans. A transgenic soybean plant body has
been developed using a transformation system using an agrobacterium
based on a cotyledonary-node (CN) method (Hinchee et al., 1988,
Nat. Biotechnol., Vol. 6, 915-922), and recently, a system for
producing a stable transformant was improved using half-see
explants (Paz et al., 2006, Plant Cell Rep., Vol. 25, 206-213).
Moreover, the application of a wound in a target site using a mixed
use of a thiol compound, an agrobacterium concentrate and
ultrasound degradation resulted in a positive improvement in
transformation efficiency (Meurer et al., 1998, Plant Cell Rep.,
Vol. 18, 180-186; Olhoft et al., 2003, Planta, Vol. 216, 723-735;
Kim et al., 2013, Plant Biotechnol Rep., Vol. 7, 425-433; Kim et
al., 2016, Plant Biotechnol Rep., Vol. 10, 257-267).
[1269] Soybean contains about 20% fat in the total composition, and
the fat consists of fatty acids. The fatty acids consist of
saturated fatty acids and unsaturated fatty acids. The unsaturated
fatty acids consist of oleic acid, linoleic acid and
.alpha.-linolenic acid. Among these, .alpha.-linolenic acid is a
vegetable omega-3 fatty acid, which has been known to inhibit
cancer cell growth, prevent a cardiovascular disease, inhibit
inflammation and blood clotting, and degrade fat. Linoleic acid is
an omega-6 fatty acid, and has been known to promote cancer cell
growth, drop blood pressure, produce inflammation and thrombi, and
accumulate fat, unlike .alpha.-linolenic acid.
[1270] It has been reported that a low ratio of omega-6/omega-3 in
fats has an effect of inhibiting the above-mentioned diseases. In
some examples, when the ratio is 4:1, prevention of a
cardiovascular disease may be excellent and blood circulation may
be improved, when the ratio is 2-3:1, the inflammation of
rheumatoid arthritis may be inhibited, and therefore the most ideal
ratio is known to be 2-1:1. The soybean oil contains 54% of the
omega-6 fatty acid and 8% of the omega-3 fatty acid, and a ratio of
the two fatty acids is significantly high at 6-7:1.
[1271] In the unsaturated fatty acid metabolism of soybean, it has
been known that a FAD2 gene serves to change oleic acid to linoleic
acid, and a FAD3 gene serves to change linoleic acid to
.alpha.-linolenic acid. In the fatty acid metabolism, variations in
the contents of oleic acid and linoleic acid by the mutation of the
FAD2 gene has been reported. When there is a mutation in the FAD2
gene, the oleic acid content is increased, and the linoleic acid
content is decreased, such that the ratio of the linoleic acid to
the .alpha.-linolenic acid was adjusted, but there is a limitation
in finding a satisfactory ratio of 4:1 or less. Therefore, it is
necessary to control the contents of oleic acid and linoleic
acid.
[1272] Specific Unsaturated Fatty Acids
[1273] One exemplary embodiment of the present invention may
provide a plant body increased in the content of a specific
unsaturated fatty acid or a processed product using the same.
[1274] Here, the specific unsaturated fatty acid may be a C8 to
24:D1 unsaturated fatty acid.
[1275] The specific unsaturated fatty acid may be a C16 to 22:D1
unsaturated fatty acid.
[1276] The specific unsaturated fatty acid may be a C18:D1
unsaturated fatty acid.
[1277] The specific unsaturated fatty acid may be oleic acid.
[1278] Alternatively,
[1279] the specific unsaturated fatty acid may be an unsaturated
fatty acid produced by removing one double bond from a C8 to 24:D2
unsaturated fatty acid,
[1280] the specific unsaturated fatty acid may be an unsaturated
fatty acid produced by removing one double bond from a C16 to 22:D2
unsaturated fatty acid,
[1281] the specific unsaturated fatty acid may be an unsaturated
fatty acid produced by removing one double bond from a C18:D2
unsaturated fatty acid, and
[1282] the specific unsaturated fatty acid may be an unsaturated
fatty acid produced by removing one double bond from linoleic
acid.
[1283] Another exemplary embodiment of the present invention
relates to a plant body decreased in the content of a specific
unsaturated fatty acid or a processed product produced using the
same.
[1284] Here, the specific unsaturated fatty acid may be a C8 to
24:D2 unsaturated fatty acid.
[1285] The specific unsaturated fatty acid may be a C16 to 22:D2
unsaturated fatty acid.
[1286] The specific unsaturated fatty acid may be a C18:D2
unsaturated fatty acid.
[1287] The specific unsaturated fatty acid may be linoleic
acid.
[1288] Alternatively,
[1289] the specific unsaturated fatty acid may be an unsaturated
fatty acid produced by forming one double bond in a C8 to 24:D1
unsaturated fatty acid,
[1290] the specific unsaturated fatty acid may be an unsaturated
fatty acid produced by forming one double bond in a C16 to 22:D1
unsaturated fatty acid,
[1291] the specific unsaturated fatty acid may be an unsaturated
fatty acid produced by forming one double bond in a C18:D1
unsaturated fatty acid, and
[1292] the specific unsaturated fatty acid may be an unsaturated
fatty acid produced by forming one double bond in oleic acid.
[1293] Alternatively,
[1294] the specific unsaturated fatty acid may be an unsaturated
fatty acid produced by removing one double bond from a C8 to 24:D3
unsaturated fatty acid,
[1295] the specific unsaturated fatty acid may be an unsaturated
fatty acid produced by removing one double bond from a C16 to 22:D3
unsaturated fatty acid,
[1296] the specific unsaturated fatty acid may be an unsaturated
fatty acid produced by removing one double bond from a C18:D3
unsaturated fatty acid, and
[1297] the specific unsaturated fatty acid may be an unsaturated
fatty acid produced by removing one double bond from
.alpha.-linolenic acid.
[1298] In one embodiment, the specific unsaturated fatty acid may
be a C18:D1 unsaturated fatty acid or a C18:D2 unsaturated fatty
acid.
[1299] In one embodiment, the specific unsaturated fatty acid may
be oleic acid or linoleic acid.
[1300] In another exemplary embodiment, the present invention may
provide a use of a system for controlling an additional third
mechanism in a body, which is involved in various functions of a
specific factor whose function is artificially modified (e.g., a
gene known as an unsaturated fatty acid biosynthesis-associated
factor).
[1301] For example, the specific factor whose function is
artificially modified may be one or more genes selected from a FAD2
gene, a FAD3 gene, a FAD6 gene, a FAD7 gene and a FAD8 gene.
[1302] The third mechanism may be a mechanism in a plant body,
other than the biosynthesis of an unsaturated fatty acid, involved
in these genes.
[1303] Compositions for Controlling Unsaturated Fatty Acid
[1304] One exemplary embodiment of the present invention relates to
a composition used to control the content of an unsaturated fatty
acid of a plant using an artificially manipulated unsaturated fatty
acid biosynthesis-associated factor.
[1305] The composition may include an artificially manipulated
unsaturated fatty acid biosynthesis-associated factor or a
manipulation composition that can artificially manipulate an
unsaturated fatty acid biosynthesis-associated.
[1306] In one exemplary embodiment, the composition may include an
artificially manipulated unsaturated fatty acid
biosynthesis-associated factor, that is, a gene and/or a
protein.
[1307] In one exemplary embodiment, the composition may include a
manipulation composition that can artificially manipulate an
unsaturated fatty acid biosynthesis-associated factor.
[1308] The manipulation composition may include a guide nucleic
acid-editor protein complex.
[1309] The manipulation composition may include a guide nucleic
acid and/or editor protein.
[1310] The manipulation composition may include a nucleic acid
encoding the guide nucleic acid and/or editor protein.
[1311] The manipulation composition may include a virus comprising
a nucleic acid encoding the guide nucleic acid and/or editor
protein.
[1312] In another exemplary embodiment, the composition may further
include an additional element.
[1313] The additional factor may include a suitable carrier for
transferring it into a plant body of a subject.
[1314] In an exemplary embodiment, the composition may include an
expression product of an unsaturated fatty acid
biosynthesis-associated factor which is manipulated to an amount
sufficient to increase or decrease the content of a specific
unsaturated fatty acid.
[1315] The "amount sufficient to increase or decrease the content
of a specific unsaturated fatty acid" means an effective amount
required to increase or decrease the content of a specific
unsaturated fatty acid.
[1316] In one exemplary embodiment, the present invention may
provide compositions for controlling an unsaturated fatty acid as
follows:
[1317] A composition for controlling the content of a specific
unsaturated fatty acid, which includes a guide nucleic acid capable
of forming a complementary bond independently with one or more
target sequences in the nucleic acid sequence of one or more genes
selected from the group consisting of a FAD2 gene, a FAD3 gene, a
FAD6 gene, a FAD7 gene and a FAD8 gene, or a nucleic acid sequence
encoding the same, and
[1318] an editor protein or a nucleic acid sequence encoding the
same;
[1319] a composition for controlling the content of a specific
unsaturated fatty acid, which includes a guide nucleic acid capable
of forming a complementary bond independently with a target
sequence of one or more genes selected from the group consisting of
a FAD2 gene, a FAD3 gene, a FAD6 gene, a FAD7 gene and a FAD8 gene,
or a nucleic acid sequence encoding the same; and
[1320] an editor protein or a nucleic acid sequence encoding the
same; and
[1321] a composition for controlling the content of a specific
unsaturated fatty acid, which includes a complex formed of a guide
nucleic acid capable of forming a complementary bond independently
with a target sequence of one or more genes selected from the group
consisting of a FAD2 gene, a FAD3 gene, a FAD6 gene, a FAD7 gene
and a FAD8 gene, or a nucleic acid sequence encoding the same, and
an editor protein.
[1322] Here, a guide nucleic acid or a 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. They may
be present in the form of a homologous or heterologous vector.
[1323] Method of Controlling Unsaturated Fatty Acid
[1324] In another exemplary embodiment of the present invention,
the present invention provides a method of controlling the content
of an unsaturated fatty acid, which includes producing the
above-described composition and administering an effective amount
of the composition to a target plant body.
[1325] Gene Manipulation
[1326] A method of controlling the content of an unsaturated fatty
acid by manipulating the gene of a body may be used. Such a
controlling method may consist of introducing the composition for
genetic manipulation for manipulating the gene of a plant body into
the plant body.
[1327] The composition for genetic manipulation may include a guide
nucleic acid-editor protein complex.
[1328] The composition for genetic manipulation may be injected
into a specific plant type.
[1329] Here, the specific plant type may be a seed, but the present
invention is not limited thereto.
[1330] In one aspect, the present invention may provide a method of
modifying a target polynucleotide in plant cells.
[1331] In one exemplary embodiment, the method includes obtaining
cells or a cell population from a plant as a sample, and modifying
the cells or cell population. The culturing may be performed at any
step outside a plant body. The cell or cells may be re-introduced
into a plant.
[1332] In addition, in another exemplary embodiment, the present
invention may provide a method of artificially manipulating cells,
which includes:
[1333] introducing (a) a guide nucleic acid capable of forming a
complementary bond with each of target sequences of one or more
genes selected from the group consisting of a FAD2 gene, a FAD3
gene, a FAD6 gene, a FAD7 gene and a FAD8 gene, or a nucleic acid
sequence encoding the same; and
[1334] (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
Staphylococcus aureus-derived Cas9 protein, a Neisseria
meningitidis-derived Cas9 protein and a Cpf1 protein, or a nucleic
acid sequence encoding the same to plant cells.
[1335] 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.
[1336] A technique of the above-described "7. Delivery" section may
be referenced before the introduction step.
[1337] For example, the introduction stage may be achieved by one
or more methods selected from a gene gun, electroporation,
liposomes, plasmids, viral vectors, nanoparticles, and a protein
translocation domain (PTD) fusion protein method.
[1338] For example, the viral vector may be one or more selected
from the group consisting of a mosaic virus, a retrovirus, a
lentivirus, an adenovirus, adeno-associated virus (AAV), vaccinia
virus, a poxvirus and a herpes simplex virus.
[1339] For example, a vector may be included in agrobacteria and
introduced.
[1340] When an unsaturated fatty acid biosynthesis-associated
factor is artificially manipulated using the methods and
compositions according to some exemplary embodiments of the present
invention, it is possible to control the type and/or content of an
unsaturated fatty acid, for example, an increase or decrease of a
specific unsaturated fatty acid, and/or a change in the content of
a specific unsaturated fatty acid, and therefore, a plant in which
an unsaturated fatty acid advantageous for human health is
increased or a harmful unsaturated fatty acid is decreased, and/or
a processed product (food, etc.) thereof may be obtained.
[1341] Additional Uses
[1342] In any exemplary embodiment, the present invention may
provide a kit for preparing a composition for controlling the
content of an unsaturated fatty acid, which includes the
composition.
[1343] The kit may be prepared by a conventional preparation method
known in the art.
[1344] 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.
[1345] In any exemplary embodiment, the present invention may
provide a method of screening a material capable of controlling an
expression level of one or more genes selected from a FAD2 gene, a
FAD3 gene, a FAD6 gene, a FAD7 gene and a FAD8 gene, which are
artificially manipulated.
[1346] In any exemplary embodiment, the present invention may
provide a method of providing information on the sequence at an
artificially manipulated target position to a subject by analyzing
the sequence of one or more genes selected from the group
consisting of a FAD2 gene, a FAD3 gene, a FAD6 gene, a FAD7 gene
and a FAD8 gene.
[1347] In addition, the present invention provides a method of
constructing a library using the provided information.
[1348] Here, a known database may be used.
[1349] In specific exemplary embodiments, the present invention may
provide a plant or cells that can be used for research using the
method of the present invention.
[1350] A plant or cells that include a chromosome editing in one or
more nucleic acid sequences associated with the biosynthesis of an
unsaturated fatty acid may be produced using the method of the
present invention. The nucleic acid sequence may be a sequence that
can encode a protein sequence associated with the biosynthesis of
an unsaturated fatty acid, or a reference sequence associated with
the biosynthesis of an unsaturated fatty acid.
[1351] In one exemplary embodiment, the effect of mutation and the
mechanism of the biosynthesis of an unsaturated fatty acid may be
studied in a plant or cells using measurement conventionally used
in a study related to the biosynthesis of an unsaturated fatty acid
using the plant or cells manufactured by the method of the present
invention. Alternatively, the effect of an active compound in the
biosynthesis of an unsaturated fatty acid may be studied using the
plant or cells.
[1352] In another exemplary embodiment, the effect of an available
gene manipulation strategy may be evaluated using the plant or
cells manufactured by the method of the present invention. In other
words, by modifying a chromosomal sequence encoding a protein
related to the biosynthesis of an unsaturated fatty acid, the
biosynthesis of the corresponding unsaturated fatty acid may be
promoted or inhibited. Particularly, this method includes forming a
modified protein by editing a chromosomal sequence encoding a
protein related to the biosynthesis of an unsaturated fatty acid,
resulting in modification of the plant or cells. Therefore, in some
exemplary embodiments, an effect of the genetically modified plant
may be evaluated by comparing a mechanism of the biosynthesis of an
unsaturated fatty acid with that of a wild-type.
[1353] The genetically modified plant may be used to produce a
plant body which is increased or decrease in specific unsaturated
fatty acids by an artificially manipulated unsaturated fatty acid
biosynthesis-associated factor and a system for controlling an
unsaturated fatty acid, which is artificially modified in function
by the same. Through the control of various mechanisms involved in
a variety of unsaturated fatty acid biosynthesis-associated
factors, a system for controlling an unsaturated fatty acid may be
improved.
Examples
[1354] Hereinafter, the present invention will be described in
further detail with reference to examples.
[1355] These 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.
[1356] Experimental Methods
[1357] 1. gRNA Design
[1358] A CRISPR/Cas9 target site of a FAD2 gene of soybean was
selected using CRISPR RGEN tools (Institute for Basic Science,
Korea). The target site of each gene may be different according to
the type of a CRISPR enzyme, and a target sequence of the gene for
SpCas9 was summarized in Table 1 described above.
[1359] 2. Construction of Vector for Soybean Transformation
[1360] In a soybean transformation test, a pPZP vector including
gRNA of FAD2-7 or FAD2-30 for targeting a FAD2 gene was used, and
the vector also includes Cas9. An Agrobacterium tumefaciens strain,
EHA105, was transformed with the constructed pPZP-FAD2-7 and
pPZP-FAD2-30 vectors (FIG. 1).
[1361] 3. Soybean Transformants and Production of T.sub.1 Seeds
[1362] 1) Sterilization and Soaking of Seeds
[1363] Seeds were sterilized with hydrochloric acid gas generated
by mixing chlorine bleach (100 mL of 12% sodium hypochlorite) with
strong hydrochloric acid (12N HCl, 5 mL) for 20 hours,
suspension-cultured in 1% sodium hypochlorite for 10 minutes for
secondary sterilization, and washed with sterile water three times
at an interval of 10 minutes. Each of the sterile seeds was put
into a 50 mL conical tube, and then sterile water was poured into
the conical tube to perform soaking at room temperature for 20
hours.
[1364] 2) Preparation of Inoculum (Agrobacterium tumefaciens)
[1365] In a soybean transformation test, the pPZP-FAD2-7 and
pPZP-FAD2-30 vectors constructed in the Agrobacterium tumefaciens
strain EHA105 were used, and include PPTR. A bacterial strain
containing the vector was streaked on a solid YEP medium [75 mg/L
of spectinomycin, 25 mg/L of rifampicin, 10 g/L of peptone, 5 g/L
of NaCl, 5 g/L of an yeast extract, 1.5% (w/v) agar (pH 7.0)] and
cultured at 28.degree. C., thereby obtaining a single colony. The
colony was suspended in 10 mL of a liquid YEP medium containing the
same antibiotic included in the solid YEP medium, and stirred at
220 rpm at 28.degree. C. until OD650 reached 0.6 to 0.8. 10 mL of a
30% glycerol stock was added to and mixed with the fully-grown
bacterial cells, 1 mL of the cell suspension was dispensed to each
1.5 mL tube, rapidly cooled with liquid nitrogen, and stored at
-70.degree. C. One day before inoculation, 1 mL of the
Agrobacterium tumefaciens stock that had been stored at -70.degree.
C. was added to 200 mL of a liquid YEP medium containing an
antibiotic, and shake-cultured in an incubator at 250 rpm at
25.degree. C. until OD650 reached 0.6 to 0.8. On the day of
inoculation, 200 mL of a liquid YEP medium was divided into 50 mL,
and centrifuged at 20.degree. C. and 3,270 g for 10 minutes. 15 mL
of a liquid co-cultivation medium (CCM; 0.32 g/L of B5 salt, 1.67
mg/L of BA, 20 mM MES, 0.25 mg/L of GA3, 0.2 mM acetosyringone, 3.3
mM L-Cysteine, 1.0 mM sodium thiosulfate, 1.0 mM DTT, 3% sucrose,
pH 5.4) was added to the Agrobacterium tumefaciens pellet in each
tube.
[1366] 3) Inoculation and Cocultivation
[1367] After the soaked seed was vertically cut to the hypocotyl by
a scalpel inserted between the both cotyledons, a seed coat was
removed. The embryonic axis was cut at about 1 cm beneath the
cytoledon, and one side to which the embryonic axis was attached
was wounded 7 to 8 times with a scalpel (#11 blade). Here, the
scalpel was coated with a 15 mL concentrate, and a wound was made
in a target site. About 50 explants were put into 15 mL
co-culture/Agrobacterium tumefaciens and sonicated for 20 seconds,
followed by inoculation for 30 minutes. After each explant was
taken out of the tube and put on a filter paper to remove moisture,
a sheet of filter paper was put on solid CCM (the same as liquid
CCM, agar (0.7%)), and then 10 explants were put thereon (to place
the adaxial side down). The plate was sealed with a micropore tape,
and co-cultured photoperiodically for 18 hours at 25.degree. C. for
5 days.
[1368] 4) Washing and Shoot Induction
[1369] Five days after the co-culture, the explants were briefly
washed with a liquid 1/2 shoot induction medium (SIM) for 10
minutes for sterilization. The explants were placed on a filter
paper to remove moisture, and then six explants per plate were
embeded in plates containing selectable antibiotic-free SI-{circle
around (1)} (shoot induction medium; 3.2 g/L of B5 salt, 1.67 mg/L
of BA, 3 mM MES, 0.8% agar, 3% sucrose, 250 mg/L of cefotaxime, 50
mg/L of vancomycin, 100 mg/L of ticarcillin, pH 5.6), and a
regeneration part of the explant was positioned with side up at an
angle of about 30.degree.. Each plate was sealed with a micropore
tape and cultured photoperiodically at 25.degree. C. for 18
hours.
[1370] After two weeks, shooting explants were embedded in 10 mg/L
of selectable antibiotic PPT-containing SI-{circle around (2)} (the
same as SI-{circle around (1)}, 10 mg/L of DL-phosphinothricin was
added, pH 5.6), and the other part excluding a shoot was removed
and embedded so that the adaxial part faced downward.
[1371] 5) Shoot Elongation
[1372] After two weeks, a browning shoot/shoot pad was excised with
a scalpel (#15 blade) and embedded in a 5 mg/L selectable
antibiotic PPT-containing shoot elongation medium (SEM; 4.4 g/L of
MS salt, 3 mM MES, 0.5 mg/L GA3, 50 mg/L asparagine, 100 mg/L
pyroglutamic acid, 0.1 mg/L IAA, 1 mg/L zeatin, 3% sucrose, 0.6%
agar, 250 mg/L cefotaxime, 50 mg/L vancomycin, 100 mg/L
ticarcillin, 5 mg/L DL-phosphinothricin, pH 5.6). Every two weeks,
the shoot/shoot pad was transferred to fresh SEM, and the browning
part of the shoot was removed by using the upper side of a scalpel
(#15 blade), and the shoot pad was shaved off gradually so that the
medium was well absorbed. When the shoot was grown up to the lid of
the petri dish, two petri dishes (100 mm.times.40 mm) were stacked
so that the shoot was grown to about 8 cm. Each plate was sealed
with a micropore tape, and incubated photoperiodically at
25.degree. C. for 18 hours.
[1373] 6) Rooting, Acclimatization and PPT Leaf Painting
[1374] When the shoot elongated through selection on SEM was 4 cm
or more, the shoot was excised with a scalpel (#11 blade) and
transferred to a rooting medium (RM; 4.4 g/L of MS salt, 3 mM MES,
3% sucrose, 0.8% agar, 50 mg/L cefotaxime, 50 mg/L vancomycin, 50
mg/L ticarcillin, 25 mg/L asparagine, 25 mg/L pyroglutamic acid, pH
5.6). Here, the lower part of the elongated shoot separated by
cutting was dipped in 1 mg/mL IBA for three minutes, and then put
into a test tube containing RM.
[1375] When the root was sufficiently grown, the medium was washed
off from the root with tertiary distilled water. The grown root was
transplanted into a small pot (6 cm.times.6 cm.times.5.6 cm)
containing a mixture of bed soil (Bio Plug No. 2, Heungnong
seeding) and vermiculite (2:1) and placed in a Magenda box. About
10 days later, a leaf surface was painted with 100 mg/L
DL-phosphinothricin.
[1376] 7) Production of T.sub.1 Seeds
[1377] When the plant body was sufficiently grown, the plant body
was transplanted into a larger pot, and covered with a transparent
plastic lid having about 10 pores. After 10 days, the plant body
was treated with Basta.RTM. (BAYER, 53 mg/L). As a result,
non-transformants (Glycine max L. Kwangan, NT) sensitively reacted
and thus failed, but transformants did not show any change and
exhibited resistance. Nine soybean transformants (eight pPZP-FAD2-7
and one pPZP-FAD2-30) exhibiting resistance were transferred to a
green house, thereby obtaining T.sub.1 seeds.
[1378] 8) Removal of T.sub.1 Transformant-Introduced Gene and
Production of T2 Seeds
[1379] To confirm that a transgenic T.sub.1 plant body-induced gene
was removed, T.sub.1 seeds were sowed and the plant body was grown
15 cm or more over a small port and had 9 or more leaves, followed
by performing leaf painting with phosphinothricin. The selected
transgenic plant body was transferred to a larger pot (20 cm
(diameter).times.25 cm (height)) and grown in a greenhouse to
obtain a T.sub.1 sample and obtain T2 seeds.
[1380] 4. Gene Transfer Analysis
[1381] 1 g of leaves of transient transformants were quantified and
frozen with liquid nitrogen, and the frozen leaves were well
grinded in a mortar, followed by extracting genomic DNA using a
CTAB method. To determine whether a gene was introduced, PCR was
performed using sequences of gRNA FAD2-7 and FAD2-30, selectable
gene Bar and a Cas9 gene. To confirm the introduction of FAD2-7 and
FAD2-30 genes, the following primers were used.
[1382] For the FAD2-7 gene, promoter AtU6p forward primer
(5'-GAATGATTAGGCATCGAACC-3'(SEQ ID NO: 31) and FAD2-7 reverse
primer (5'-AAACTCCTCAAGGGTTCCAAACAC-3'(SEQ ID NO: 31)) were used.
For the FAD2-30 gene, promoter AtU6p forward primer
(5'-GAATGATTAGGCATCGAACC-3'(SEQ ID NO: 31)) and FAD2-7 reverse
primer (5'-AAACTCCTCAAGGGTTCCAAACACC-3'(SEQ ID NO: 33)) were used.
To confirm the introduction of the Bar gene, Bar forward primer
(5'-TCCGTACCGAGCCGCAGGAA-3'(SEQ ID NO: 34)) and Bar reverse primer
(5'-CCGGCAGGCTGAAGTCCAGC-3'(SEQ ID NO: 35)) were used.
[1383] In addition, to confirm the introduction of Cas9, PCR was
performed using three primer sets as follows: a set of Cas9-{circle
around (1)} forward primer (5'-ATGGACAAGAAGTACAGCATCGGC-3'(SEQ ID
NO: 36)) and Cas9-{circle around (1)} reverse primer
(5'-AACTTGTAGAACTCCTCCTGGCTG-3'(SEQ ID NO: 37)); a set of
Cas9-{circle around (2)} forward primer
(5'-TTCAGGAAGTCCAGGATGGTCTTG-3'(SEQ ID NO: 38)) and Cas9-{circle
around (2)} reverse primer (5'-AGAACTGGAAGTCCTTGCGGAAGT-3'(SEQ ID
NO: 39)); and a set of Cas9-{circle around (3)} forward primer
(5'-CTGAGCGAGCTGGACAAGGCCGG-3'(SEQ ID NO: 40)) and Cas9-{circle
around (3)} reverse primer (5'-TTAGGCGTAGTCGGGCACGTCGTA-3'(SEQ ID
NO: 41)) were used.
[1384] 5. Gene Removal Analysis
[1385] 1 g of leaves of a transgenic T.sub.1 plant body were
quantified and frozen with liquid nitrogen, and the frozen leaves
were well grinded in a mortar. 0.5 g of the grinded leaves were put
into a 2 mL tube, 1 mL of a solution prepared by mixing
cetyltrimethylammonium (cTAB) buffer and .beta.-mercaptoethanol
(2-ME) in a ratio of 20:1 was added and treated in a 65.degree. C.
heat-block for 1 hour, and then 10 .mu.L of RNase (1.5 mg/150 .mu.L
H2O) was added to allow incubation at 37.degree. C. for 1 hour.
After one hour, chloroform:isoamyl alcohol (24:1) were mixed in the
same volume and well mixed to prepare a reagent, and then
centrifuged at 4.degree. C. and 12,000 rpm for 10 minutes. A
supernatant was transferred to a new tube, and treated with a
chloroform:isoamylalchol (24:1) reagent once again. After a
supernatant was put into the same volume of isopropanol and mixed
by being inverted 3 to 4 times, placed in a -20.degree. C.
refrigerator for 1 hour, and centrifuged at 4.degree. C. and 12,000
rpm for 10 minutes. After a supernatant was discarded, 1 mL of 70%
ethanol was added thereto, and the resulting solution was
centrifuged again for 2 minutes, thereby obtaining a pellet. After
a supernatant was discarded, the pellet was dried at room
temperature for 20 minutes and then suspended in 30 .mu.L of
distilled water to extract genomic DNA. The extracted genomic DNA
was diluted 20-fold, and used as a template.
[1386] The base sequences of the introduced gene and the selectable
antibiotic gene BAR were subjected to 35 cycles of PCR consisting
of pre-denaturation at 95.degree. C. for 10 minutes, denaturation
at 95.degree. C. for 30 seconds, annealing at 55 to 65.degree. C.
for 30 seconds and extension at 72.degree. C. for 30 seconds to 1.5
minutes, and additionally subjected to extension at 72.degree. C.
for 10 minutes, and the result was determined using a PCR kit
(Prime Taq Premix, GENETBIO, Korea).
[1387] 6. Analysis of Oleic Acid Content
[1388] For fatty acid analysis, three soybean seeds obtained from
each transformant was individually analyzed. Each seed was put into
a paper bag, crushed using a hammer, and fatty acid was extracted
using 5 mL of an extraction solvent (chloroform:hexane:methanol,
8:5:2) at room temperature for 12 hours. 150 .mu.L of the extracted
fatty acid was transferred to a vial, 75 .mu.L of a methylation
reagent (0.25M methanolic sodium methoxide:petroleum ether:ethyl
ether, 1:5:2) was added, and then hexane was added to reach 1 mL. 1
.mu.L of a sample was injected and analyzed using a gas
chromatography apparatus (GC, Aglient Technologies, USA), and
analysis conditions are shown in Table 2 below. A fatty acid ratio
was calculated with an area of each fatty acid with respect to the
total area of the fatty acids.
TABLE-US-00002 TABLE 2 GC conditions for analyzing fatty acid in
soybean Item Condition Instrument Agilent 7890A Column 0.25 .mu.m
i.d. .times. 30 m DB-FFAP capillary column Detector Flame
ionization detector Oven temperature 230.degree. C. Injection
temperature 210.degree. C. Detector temperature 250.degree. C.
Carrier gas N.sub.2 (1.5 mL/min) Injection volume 1 .mu.L
[1389] 7. Analysis of FAD2 Gene Sequence
[1390] PCR-amplification was performed for a target region to have
a size of 200 to 300 bp using Hipi Plus DNA polymerase (Elpisbio).
The PCR product obtained by the above-described method was
subjected to sequencing using a MiSeq system (IIlumina), and then
analyzed using a Cas analyzer, CRISPR RGEN tool
(www.rgenome.net)).
Example 1. Transformant Vector and Production of Transformant
[1391] To knock out the FAD2 gene, as shown in Table 1, guide RNA
targeting the FAD2 gene was designed, and cloned with a Cas9
protein in a pPZP vector, thereby constructing a vector for soybean
transformation (FIG. 1). As shown in FIG. 2, through the
regeneration process of a plant, a total of 9 transformants (T0)
(eight pPZP-FAD2-7 8 and one pPZP-FAD2-30) were produced (FIG. 3).
T.sub.1 seeds were collected from the transformants T0, thereby
producing transformants T.sub.1 (FIG. 10).
Example 2. PCR Analysis for Determining Gene Transfer into
Transformant
[1392] DNA was isolated from the FAD2-7 and FAD2-30 T0
transformants and subjected to PCR according to the above-described
method, confirming all of the introduction of introduced guide RNA,
FAD2-7 and FAD2-30, and selectable gene Bar and Cas9 (FIG. 4).
Example 3. Analysis of Oleic Acid Content in Transgenic Soybean
[1393] The contents of oleic acid were analyzed from the FAD2-7 and
FAD2-30 T.sub.1 seeds according to the above-described method. The
contents of oleic acid in the FAD2-7 and FAD2-30 T.sub.1 seeds were
significantly higher than those of wild-type seeds such as Glycine
max L. Pungsan, Glycine max L. Kwangan and Glycine max L. Hosim
(FIG. 5).
Example 4. Analysis of FAD2 Gene Sequence in Transgenic Soybean
[1394] Indel frequencies (FIG. 6) and sequences (FIG. 7) were
analyzed to confirm whether a mutation was induced in FAD2 genes of
the FAD2-7 and FAD2-30 T.sub.1 seeds according to the above
described method. It was confirmed that a mutation was induced in
the FAD2-7 T.sub.1 seed.
[1395] In addition, T.sub.1 transformants were analyzed through
deep sequencing to confirm whether mutation was induced in a FAD2
gene. As a result, it was confirmed that a mutation was induced
into a target site of a FAD2 gene in chromosome #10 (chr10) and
chromosome #20 (chr20) of other T.sub.1 transformants except
FAD2-7#1-1 and FAD2-30#2-4, #9-1 and #3-1 (FIGS. 8 and 9).
Example 5. Analysis of Removal of Introduced Gene of Transgenic
Soybean
[1396] To confirm gene removal from selected T.sub.1 transformants,
PCR was performed for a selectable gene BAR and an introduced gene
of an introduced vector. As a result, it was confirmed that, from
one FAD2-7 T.sub.1 transformant, both of the introduced gene and
the BAR gene were removed, and among fifteen FAD2-30 T.sub.1
transformants, individuals (9-1, 19-1, 21-1, 21-5) from which genes
were not partially removed were confirmed (FIG. 11).
INDUSTRIAL APPLICABILITY
[1397] A a processed product may be manufactured using plant body
increased in the content of a specific unsaturated fatty acid which
is good for human health or decreased in the content of a specific
unsaturated fatty acid which is harmful for human health by using
an artificially manipulated unsaturated fatty acid
biosynthesis-associated factor and a system for controlling an
unsaturated fatty acid, which is artificially modified thereby, and
thus can be used for food.
[1398] 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
70123DNAGlycine max 1atagattggc catgcaatga ggg 23223DNAGlycine max
2aatagattgg ccatgcaatg agg 23323DNAGlycine max 3ccttggagaa
cccaatagat tgg 23423DNAGlycine max 4tgggtgattg ctcacgagtg tgg
23523DNAGlycine max 5ttttagtccc ttatttctca tgg 23623DNAGlycine max
6aaacacttca tcacggtcaa ggg 23723DNAGlycine max 7gtgtttggaa
cccttgagag agg 23823DNAGlycine max 8gtgaatggtg gctttgtgtt tgg
23923DNAGlycine max 9acaaagccac cattcactgt tgg 231023DNAGlycine max
10agttggccaa cagtgaatgg tgg 231123DNAGlycine max 11ttgagttggc
caacagtgaa tgg 231223DNAGlycine max 12tgaaaggtca taaacaacat agg
231323DNAGlycine max 13caaacacttc atcacggtca agg 231423DNAGlycine
max 14aaccaaaatc caaagttgca tgg 231523DNAGlycine max 15tgggagcata
agggtggtag tgg 231623DNAGlycine max 16aatatatggg agcataaggg tgg
231723DNAGlycine max 17gtttggctgc tatgtgttta tgg 231823DNAGlycine
max 18tttggctgct atgtgtttat ggg 231923DNAGlycine max 19ttggctgcta
tgtgtttatg ggg 232023DNAGlycine max 20gcaactatgg acagagatta tgg
232123DNAGlycine max 21caccatttta caaggcactg tgg 232223DNAGlycine
max 22cttcatctgg ctccacatag agg 232323DNAGlycine max 23ctctatgtgg
agccagatga agg 232423DNAGlycine max 24ttctcggatg ttccttcatc tgg
232523DNAGlycine max 25agatgaagga acatccgaga agg 232623DNAGlycine
max 26gatgaaggaa catccgagaa ggg 232723DNAGlycine max 27catccgagaa
gggcgtgtat tgg 232823DNAGlycine max 28gtaccaatac acgcccttct cgg
232923DNAGlycine max 29agaagggcgt gtattggtac agg 233023DNAGlycine
max 30ttgggacaaa cacttcatca cgg 233120DNAArtificial Sequenceprimer
31gaatgattag gcatcgaacc 203224DNAArtificial Sequenceprimer
32aaactcctca agggttccaa acac 243325DNAArtificial Sequenceprimer
33aaactcctca agggttccaa acacc 253420DNAArtificial Sequenceprimer
34tccgtaccga gccgcaggaa 203520DNAArtificial Sequenceprimer
35ccggcaggct gaagtccagc 203624DNAArtificial Sequenceprimer
36atggacaaga agtacagcat cggc 243724DNAArtificial Sequenceprimer
37aacttgtaga actcctcctg gctg 243824DNAArtificial Sequenceprimer
38ttcaggaagt ccaggatggt cttg 243924DNAArtificial Sequenceprimer
39agaactggaa gtccttgcgg aagt 244023DNAArtificial Sequenceprimer
40ctgagcgagc tggacaaggc cgg 234124DNAArtificial Sequenceprimer
41ttaggcgtag tcgggcacgt cgta 244212RNAStreptococcus pyogenes
42guuuuagagc ua 124325RNACampylobacter jejuni 43guuuuagucc
cuuuuuaaau uucuu 254414RNAStreptococcus pyogenes 44uagcaaguua aaau
144525RNACampylobacter jejuni 45aagaaauuua aaaagggacu aaaau
254611RNAUnknownParcubacteria bacterium 46aaauuucuac u
114712RNAStreptococcus pyogenes 47aaggcuaguc cg
124811RNACampylobacter jejuni 48aaagaguuug c 114934RNAStreptococcus
pyogenes 49uuaucaacuu gaaaaagugg caccgagucg gugc
345038RNACampylobacter jejuni 50gggacucugc gggguuacaa uccccuaaaa
ccgcuuuu 385122RNAStreptococcus thermophilus 51guuuuagagc
uguguuguuu cg 225224RNAStreptococcus thermophilus 52cgaaacaaca
cagcgaguua aaau 245313RNAStreptococcus thermophilus 53aaggcuuagu
ccg 135438RNAStreptococcus thermophilus 54uacucaacuu gaaaaggugg
caccgauucg guguuuuu 38557PRTArtificial Sequencenuclear localization
sequence 55Pro Lys Lys Lys Arg Lys Val1 55616PRTArtificial
Sequencenuclear localization sequence 56Lys Arg Pro Ala Ala Thr Lys
Lys Ala Gly Gln Ala Lys Lys Lys Lys1 5 10 15579PRTArtificial
Sequencenuclear localization sequence 57Pro Ala Ala Lys Arg Val Lys
Leu Asp1 55811PRTArtificial Sequencenuclear localization sequence
58Arg Gln Arg Arg Asn Glu Leu Lys Arg Ser Pro1 5
105938PRTArtificial Sequencenuclear localization sequence 59Asn Gln
Ser Ser Asn Phe Gly Pro Met Lys Gly Gly Asn Phe Gly Gly1 5 10 15Arg
Ser Ser Gly Pro Tyr Gly Gly Gly Gly Gln Tyr Phe Ala Lys Pro 20 25
30Arg Asn Gln Gly Gly Tyr 356042PRTArtificial Sequencenuclear
localization sequence 60Arg Met Arg Ile Glx Phe Lys Asn Lys Gly Lys
Asp Thr Ala Glu Leu1 5 10 15Arg Arg Arg Arg Val Glu Val Ser Val Glu
Leu Arg Lys Ala Lys Lys 20 25 30Asp Glu Gln Ile Leu Lys Arg Arg Asn
Val 35 40618PRTArtificial Sequencenuclear localization sequence
61Val Ser Arg Lys Arg Pro Arg Pro1 5628PRTArtificial
Sequencenuclear localization sequence 62Pro Pro Lys Lys Ala Arg Glu
Asp1 5638PRTArtificial Sequencenuclear localization sequence 63Pro
Gln Pro Lys Lys Lys Pro Leu1 56412PRTArtificial Sequencenuclear
localization sequence 64Ser Ala Leu Ile Lys Lys Lys Lys Lys Met Ala
Pro1 5 10655PRTArtificial Sequencenuclear localization sequence
65Asp Arg Leu Arg Arg1 5667PRTArtificial Sequencenuclear
localization sequence 66Pro Lys Gln Lys Lys Arg Lys1
56710PRTArtificial Sequencenuclear localization sequence 67Arg Lys
Leu Lys Lys Lys Ile Lys Lys Leu1 5 106810PRTArtificial
Sequencenuclear localization sequence 68Arg Glu Lys Lys Lys Phe Leu
Lys Arg Arg1 5 106920PRTArtificial Sequencenuclear localization
sequence 69Lys Arg Lys Gly Asp Glu Val Asp Gly Val Asp Glu Val Ala
Lys Lys1 5 10 15Lys Ser Lys Lys 207017PRTArtificial Sequencenuclear
localization sequence 70Arg Lys Cys Leu Gln Ala Gly Met Asn Leu Glu
Ala Arg Lys Thr Lys1 5 10 15Lys
* * * * *