KR20210077585A - Manufacturing method of non-functional mutant in first filial generation using CRISPR/Cas system - Google Patents
Manufacturing method of non-functional mutant in first filial generation using CRISPR/Cas system Download PDFInfo
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Abstract
Description
본 발명은 CRISPR/Cas 시스템을 이용한 표적 유전자에 대한 돌연변이체 제조 방법; 웅성불임 유지친의 제조 방법; 및 Non-GMO 식물체의 제조 방법에 관한 것이다. The present invention provides a method for producing a mutant for a target gene using the CRISPR / Cas system; Method for producing male infertility eujichin; And it relates to a method for producing a Non-GMO plant.
자연 돌연변이 및 유도 돌연변이로 인한 웅성불임(male sterile) 돌연변이는 여러 종의 고등식물에서 보고되어 왔다. 상기 돌연변이는 여러 가지 다른 표현형들과 연관되어 있으며, 이는 구조적 변이들, 예컨대 짧은 화사(filaments), 열개(dehiscence)의 결여 또는 편평한 표면을 가지고, 또한 배우자 형성, 특히 감수분열과 연관된 기능적 결함을 가진 꽃가루를 포함한다. 이러한 돌연변이들 모두 비기능성 꽃가루를 생성한다. Male sterile mutations due to natural and induced mutations have been reported in several species of higher plants. These mutations have been associated with several different phenotypes, which include structural variations, such as short filaments, lack of dehiscence or flat surfaces, and functional defects associated with gamete formation, particularly meiosis. contains pollen. Both of these mutations produce non-functional pollen.
웅성불임은 유전양식에 따라 핵유전형(genic male sterility)과 세포질유전형(cytoplasmic male sterility)으로 나눌 수 있다. 핵유전형 웅성불임은 생존에 불리하게 작용하기 때문에 발견되는 것으로서, 주로 열성유전을 하며, 열성동형(homozygous recessive)인 경우에 불임성을 띠게 되기 때문에 불임계의 유지 및 증식에 많은 노력이 소요되고, 작물에 따라서는 꽃이 작아 임성(fertility)의 판별이 힘들고, 꽃이 필 때까지 시간이 많이 소요되는 작물에서는 적용이 힘든 단점이 있다.Male infertility can be divided into nuclear genotype (genic male sterility) and cytoplasmic genotype (cytoplasmic male sterility) according to the genetic pattern. Nuclear genotype male infertility is found because it adversely affects survival. It is mainly recessive, and in the case of homozygous recessive, it takes sterility, so it takes a lot of effort to maintain and propagate sterility. Depending on the flower, it is difficult to determine fertility due to small flowers, and it is difficult to apply to crops that take a lot of time to bloom.
세포질 웅성불임은 어느 가임계를 교배해도 100% 불임주가 나오기 때문에, 불임주에 여러 가지 가임주를 교배하여 50% 혹은 100% 불임주를 낼 수 있는 개체 및 계통, 즉 유지계통(maintainer, 유지친)을 찾아내서 심어주어야 하기 때문에 불편한 점은 있으나, 불임활용의 소기의 목적을 달성할 수 있다. 하지만, 일대잡종 육종을 위해 웅성불임계 획득을 위한 방사선 및 화학 약품을 통한 인위적 웅성불임 유기도 시도된 바가 있으나, 부작용을 해소해야 하는 문제점을 가지고 있다. Because cytoplasmic male sterility produces 100% sterile strains by crossing any fertile strain, individuals and strains that can produce 50% or 100% sterility by crossing various fertile strains to sterile strains, that is, maintainers (maintainers) ) is found and planted, so it is inconvenient, but the intended purpose of infertility can be achieved. However, for one-to-one hybrid breeding, artificial male infertility through radiation and chemical agents for obtaining male sterility has also been attempted, but there is a problem in that side effects must be resolved.
일대잡종(F1) 종자를 생산하기 위한 웅성불임 식물의 개발 및 연구에 많은 투자를 하고 있지만, 현재까지 웅성불임 식물이 가지고 있는 문제점으로 인해 이에 관한 연구 성과는 매우 미비한 실정이다. 또한, 유전공학 기법을 이용한 여러 웅성불임 식물 개발 방법들은 일대잡종 교배체가 제초제 저항성 유전자 등 외래 유전자를 포함한 경우가 대부분이며, 이들은 다양한 GMO 작물 규제로 인하여 사용이 매우 제한되어 있다. 이러한 웅성불임계 식물이 가지고 있는 단점들을 극복, 즉 안정성 있는 웅성불임계의 확보 및 다른 유지계통이 필요 없이 불임계를 유지할 수만 있다면, 이를 이용한 손쉬운 신품종 개발 및 고부가가치의 산업생산에 유용하게 이용할 수 있을 것이다. 따라서, 안정성 있는 웅성불임계를 확보할 수 있고, 웅성불임계를 유지할 있는 신규한 방법 또는 신규한 유전자에 대한 연구가 필요한 실정이다.Although a lot of investment is being made in the development and research of male sterile plants for producing F1 seeds, the research results on this are very insignificant due to problems with male sterile plants so far. In addition, in many male sterile plant development methods using genetic engineering techniques, most of the cases in which one-to-one hybrids include foreign genes such as herbicide resistance genes, and their use is very limited due to regulations on various GMO crops. If you can overcome the disadvantages of these male sterility plants, that is, if you can secure a stable male sterility and maintain sterility without the need for other maintenance systems, it can be usefully used for easy new variety development and high value-added industrial production. There will be. Therefore, it is possible to secure stable male sterility, and there is a need for research on novel methods or novel genes for maintaining male sterility.
본 발명의 목적은 (a) 표적 유전자를 인식할 수 있는 sgRNA(single guide RNA)를 코딩하는 폴리뉴클레오티드 및 Cas(CRISPR-associated nuclease) 단백질을 코딩하는 폴리뉴클레오티드가 삽입된 재조합 벡터로 형질전환시켜 형질전환 식물체를 제조하는 단계; 및 (b) 상기 형질전환 식물체와 야생형 식물체를 교배하는 단계를 포함하는 일대잡종(F1)에서 표적 유전자에 대한 동형접합 돌연변이체(homozygous mutant) 또는 이대립인자성 돌연변이체(biallelic mutant)의 제조 방법을 제공하는 것이다. It is an object of the present invention to transform with a recombinant vector into which (a) a polynucleotide encoding a single guide RNA (sgRNA) capable of recognizing a target gene and a polynucleotide encoding a CRISPR-associated nuclease (Cas) protein are inserted. preparing a converted plant; And (b) a method for producing a homozygous mutant or biallelic mutant for the target gene in a one-to-one hybrid (F1) comprising the step of crossing the transgenic plant and the wild-type plant is to provide
본 발명의 또 다른 목적은 (a) 표적 유전자를 인식할 수 있는 sgRNA를 코딩하는 폴리뉴클레오티드, Cas 단백질을 코딩하는 폴리뉴클레오티드 및 형광표지 단백질을 코딩하는 폴리뉴클레오티드가 삽입된 재조합 벡터로 형질전환시켜 형질전환 식물체를 제조하는 단계; (b) 상기 형질전환 식물체와 야생형 식물체를 교배하는 단계; 및 (c) 형광표지 단백질을 발현하는 식물체를 선별하는 단계를 포함하는 웅성불임 유지친(male sterile maintainer)의 제조 방법을 제공하는 것이다. Another object of the present invention is to transform with a recombinant vector into which (a) a polynucleotide encoding an sgRNA capable of recognizing a target gene, a polynucleotide encoding a Cas protein, and a polynucleotide encoding a fluorescent marker protein are inserted. preparing a converted plant; (b) crossing the transgenic plant and the wild-type plant; And (c) to provide a method for producing a male sterile maintainer comprising the step of selecting a plant expressing the fluorescent marker protein.
본 발명의 또 다른 목적은 (a) 표적 유전자를 인식할 수 있는 sgRNA를 코딩하는 폴리뉴클레오티드, Cas 단백질을 코딩하는 폴리뉴클레오티드 및 형광표지 단백질을 코딩하는 폴리뉴클레오티드가 삽입된 재조합 벡터로 형질전환시켜 형질전환 식물체를 제조하는 단계; (b) 상기 형질전환 식물체와 야생형 식물체를 교배하는 단계; 및 (c) 형광표지 단백질을 발현하지 않는 식물체를 선별하는 단계를 포함하는 Non-GMO(Non-Genetically Modified Organism) 식물체의 제조 방법을 제공하는 것이다. Another object of the present invention is to transform with a recombinant vector into which (a) a polynucleotide encoding an sgRNA capable of recognizing a target gene, a polynucleotide encoding a Cas protein, and a polynucleotide encoding a fluorescent marker protein are inserted. preparing a converted plant; (b) crossing the transgenic plant and the wild-type plant; And (c) to provide a method for producing a Non-Genetically Modified Organism (Non-GMO) plant comprising the step of selecting a plant that does not express the fluorescent marker protein.
본 발명의 또 다른 목적은 표적 유전자를 인식할 수 있는 sgRNA를 코딩하는 폴리뉴클레오티드, Cas 단백질을 코딩하는 폴리뉴클레오티드 및 형광표지 단백질을 코딩하는 폴리뉴클레오티드를 포함하는 재조합 벡터로서, 상기 sgRNA를 코딩하는 폴리뉴클레오티드는 서열번호 3, 서열번호 6 및 서열번호 9의 염기서열로 이루어진 것으로부터 선택되는 어느 하나 이상인 것을 특징으로 하는 재조합 벡터를 제공하는 것이다. Another object of the present invention is a recombinant vector comprising a polynucleotide encoding an sgRNA capable of recognizing a target gene, a polynucleotide encoding a Cas protein, and a polynucleotide encoding a fluorescent label protein, the polynucleotide encoding the sgRNA Nucleotides are to provide a recombinant vector, characterized in that at least one selected from the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 9.
본 발명의 또 다른 목적은 상기 재조합 백터로 형질전환된 형질전환 식물체를 제공하는 것이다.Another object of the present invention is to provide a transgenic plant transformed with the recombinant vector.
본 발명의 또 다른 목적은 상기 형질전환 식물체의 종자를 제공하는 것이다.Another object of the present invention is to provide a seed of the transgenic plant.
상기 목적을 달성하기 위하여, 본 발명은 (a) 표적 유전자를 인식할 수 있는 sgRNA(single guide RNA)를 코딩하는 폴리뉴클레오티드 및 Cas(CRISPR-associated nuclease) 단백질을 코딩하는 폴리뉴클레오티드가 삽입된 재조합 벡터로 형질전환시켜 형질전환 식물체를 제조하는 단계; 및 (b) 상기 형질전환 식물체와 야생형 식물체를 교배하는 단계를 포함하는 일대잡종(F1)에서 표적 유전자에 대한 동형접합 돌연변이체(homozygous mutant) 또는 이대립인자성 돌연변이체(biallelic mutant)의 제조 방법을 제공한다. In order to achieve the above object, the present invention provides a recombinant vector into which (a) a polynucleotide encoding a single guide RNA (sgRNA) capable of recognizing a target gene and a polynucleotide encoding a Cas (CRISPR-associated nuclease) protein are inserted Transforming to prepare a transgenic plant; And (b) a method for producing a homozygous mutant or biallelic mutant for the target gene in a one-to-one hybrid (F1) comprising the step of crossing the transgenic plant and the wild-type plant provides
본 발명의 일 실시예에 있어서, 상기 sgRNA를 코딩하는 폴리뉴클레오티드는 서열번호 3, 서열번호 6, 서열번호 9, 서열번호 11 및 서열번호 13의 염기서열로 이루어진 것으로부터 선택되는 어느 하나 이상인 것일 수 있다. In one embodiment of the present invention, the polynucleotide encoding the sgRNA may be any one or more selected from the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13 have.
본 발명의 일 실시예에 있어서, 상기 표적 유전자는 웅성불임과 관련된 유전자인 것일 수 있으나, 이에 한정되는 것은 아니다. In one embodiment of the present invention, the target gene may be a gene related to male infertility, but is not limited thereto.
본 발명의 일 실시예에 있어서, 상기 표적 유전자는 Os12BGlu38 유전자, OsHXK5 유전자 및 OsPLGG1 유전자로 이루어진 군에서 선택되는 어느 하나 이상의 유전자인 것일 수 있으나, 이에 한정되는 것은 아니다. In one embodiment of the present invention, the target gene may be any one or more genes selected from the group consisting of Os12BGlu38 gene, OsHXK5 gene and OsPLGG1 gene, but is not limited thereto.
본 발명의 일 실시예에 있어서, 상기 Os12BGlu38 유전자는 서열번호 1의 염기서열로 이루어진 것이고, 상기 OsHXK5 유전자는 서열번호 4의 염기서열로 이루어진 것이고, 상기 OsPLGG1 유전자는 서열번호 7의 염기서열로 이루어진 것일 수 있다. In one embodiment of the present invention, the Os12BGlu38 gene consists of the nucleotide sequence of SEQ ID NO: 1, the OsHXK5 gene consists of the nucleotide sequence of SEQ ID NO: 4, and the OsPLGG1 gene consists of the nucleotide sequence of SEQ ID NO: 7 can
본 발명의 일 실시예에 있어서, 상기 (a) 단계의 형질전환 식물체는 동형접합 돌연변이체 또는 이대립인자성 돌연변이체인 것일 수 있다. In one embodiment of the present invention, the transgenic plant of step (a) may be a homozygous mutant or a biallelic mutant.
또한, 본 발명은 (a) 표적 유전자를 인식할 수 있는 sgRNA를 코딩하는 폴리뉴클레오티드, Cas 단백질을 코딩하는 폴리뉴클레오티드 및 형광표지 단백질을 코딩하는 폴리뉴클레오티드가 삽입된 재조합 벡터로 형질전환시켜 형질전환 식물체를 제조하는 단계; (b) 상기 형질전환 식물체와 야생형 식물체를 교배하는 단계; 및 (c) 형광표지 단백질을 발현하는 식물체를 선별하는 단계를 포함하는 웅성불임 유지친(male sterile maintainer)의 제조 방법을 제공한다. In addition, the present invention is a transgenic plant by transforming a recombinant vector into which (a) a polynucleotide encoding an sgRNA capable of recognizing a target gene, a polynucleotide encoding a Cas protein, and a polynucleotide encoding a fluorescent marker protein are inserted. preparing a; (b) crossing the transgenic plant and the wild-type plant; And (c) provides a method for producing a male sterile maintainer comprising the step of selecting a plant expressing the fluorescent marker protein.
본 발명의 일 실시예에 있어서, 상기 형광표지 단백질을 발현하는 식물체는 동형접합 돌연변이체 또는 이대립인자성 돌연변이체인 것일 수 있다. In one embodiment of the present invention, the plant expressing the fluorescent marker protein may be a homozygous mutant or a biallelic mutant.
또한, 본 발명은 (a) 표적 유전자를 인식할 수 있는 sgRNA를 코딩하는 폴리뉴클레오티드, Cas 단백질을 코딩하는 폴리뉴클레오티드 및 형광표지 단백질을 코딩하는 폴리뉴클레오티드가 삽입된 재조합 벡터로 형질전환시켜 형질전환 식물체를 제조하는 단계; (b) 상기 형질전환 식물체와 야생형 식물체를 교배하는 단계; 및 (c) 형광표지 단백질을 발현하지 않는 식물체를 선별하는 단계를 포함하는 Non-GMO (Non-Genetically Modified Organism) 식물체의 제조 방법을 제공한다. In addition, the present invention is a transgenic plant by transforming a recombinant vector into which (a) a polynucleotide encoding an sgRNA capable of recognizing a target gene, a polynucleotide encoding a Cas protein, and a polynucleotide encoding a fluorescent marker protein are inserted. preparing a; (b) crossing the transgenic plant and the wild-type plant; And (c) provides a method for producing a Non-GMO (Non-Genetically Modified Organism) plant comprising the step of selecting a plant that does not express the fluorescent marker protein.
본 발명의 일 실시예에 있어서, 상기 형광표지 단백질을 발현하지 않는 식물체는 이형접합 돌연변이체인 것일 수 있다. In one embodiment of the present invention, the plant that does not express the fluorescent marker protein may be a heterozygous mutant.
또한, 본 발명은 표적 유전자를 인식할 수 있는 sgRNA를 코딩하는 폴리뉴클레오티드, Cas 단백질을 코딩하는 폴리뉴클레오티드 및 형광표지 단백질을 코딩하는 폴리뉴클레오티드를 포함하는 재조합 벡터로서, 상기 sgRNA를 코딩하는 폴리뉴클레오티드는 서열번호 3, 서열번호 6, 서열번호 9, 서열번호 11 및 서열번호 13의 염기서열로 이루어진 것으로부터 선택되는 어느 하나 이상인 것을 특징으로 하는 재조합 벡터를 제공한다. In addition, the present invention is a recombinant vector comprising a polynucleotide encoding an sgRNA capable of recognizing a target gene, a polynucleotide encoding a Cas protein, and a polynucleotide encoding a fluorescent marker protein, wherein the polynucleotide encoding the sgRNA comprises: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 11 and provides a recombinant vector, characterized in that any one or more selected from the nucleotide sequence consisting of SEQ ID NO: 13.
본 발명의 일 실시예에 있어서, 상기 sgRNA를 코딩하는 폴리뉴클레오티드는 서열번호 3, 서열번호 11 및 서열번호 13의 염기서열로 이루어진 것에서 선택되는 어느 하나 이상인 것일 수 있다. In one embodiment of the present invention, the polynucleotide encoding the sgRNA may be at least one selected from the group consisting of the nucleotide sequences of SEQ ID NO: 3, SEQ ID NO: 11, and SEQ ID NO: 13.
본 발명의 일 실시예에 있어서, 상기 표적 유전자는 Os12BGlu38 유전자, OsHXK5 유전자 및 OsPLGG1 유전자로 이루어진 군에서 선택되는 어느 하나 이상의 유전자인 것일 수 있다. In one embodiment of the present invention, the target gene may be any one or more genes selected from the group consisting of Os12BGlu38 gene, OsHXK5 gene and OsPLGG1 gene.
본 발명의 일 실시예에 있어서, 상기 형광표지 단백질은 녹색형광단백질(GFP), 황색형광단백질(YFP), 적색형광단백질(RFP) 및 시안형광단백질(CFP)로 이루어진 군에서 선택되는 것일 수 있으나, 이에 한정되는 것은 아니다. In one embodiment of the present invention, the fluorescent label protein may be one selected from the group consisting of green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP) and cyan fluorescent protein (CFP). , but is not limited thereto.
또한, 본 발명은 상기 재조합 백터로 형질전환된 형질전환 식물체를 제공한다. In addition, the present invention provides a transgenic plant transformed with the recombinant vector.
본 발명의 일 실시예에 있어서, 상기 식물체는 표적 유전자에 대한 동형접합 돌연변이체 또는 이대립인자성 돌연변이체인 것일 수 있다. In one embodiment of the present invention, the plant may be a homozygous mutant or a biallelic mutant for the target gene.
또한, 본 발명은 상기 형질전환 식물체의 종자를 제공한다. In addition, the present invention provides a seed of the transgenic plant.
본 발명에 따른 방법은 CRISPR/Cas 시스템을 이용하여 표적 유전자에 대한 돌연변이를 유도한 후, 야생형 식물체를 교배하여 일대잡종(F1)에서 표적 유전자에 대한 동형접합 돌연변이체 또는 이대립인자성 돌연변이체를 제조할 수 있으며, 재조합 벡터에 형광표지 단백질을 추가하여 형광을 통해 웅성불임 유지친 또는 Non-GMO 식물체를 쉽게 분류할 수 있어, 농업분야에서 다양한 활용이 가능하다.The method according to the present invention induces a mutation for a target gene using the CRISPR/Cas system, and then crosses a wild-type plant to generate a homozygous mutant or a biallelic mutant for the target gene in a one-to-one hybrid (F1). It can be manufactured, and by adding a fluorescent labeling protein to the recombinant vector, it is possible to easily classify male sterile oleaginous or Non-GMO plants through fluorescence, so that various applications are possible in the agricultural field.
도 1은 CRISPR/Cas 시스템을 이용하여 Os12BGlu38 유전자의 돌연변이를 유도하기 위한 삽입 유전자의 모식도를 나타낸 것이다.
도 2는 CRISPR/Cas9을 이용하여 제작한 T0 식물체에서 Os12BGlu38 유전자 표적 부위의 염기서열을 분석하여 유전형을 확인한 결과이다.
도 3은 일대잡종(F1)에서 CRISPR/Cas9 운반체의 삽입 여부를 확인한 결과이다.
도 4는 일대잡종(F1)에서 CRISPR/Cas9 운반체가 삽입되지 않은 개체의 유전형을 확인한 결과이다.
도 5는 일대잡종(F1)에서 CRISPR/Cas9 운반체가 삽입된 개체의 유전형을 확인한 결과이다.
도 6는 CRISPR/Cas 시스템을 이용하여 OsHXK5 유전자의 돌연변이를 유도하기 위한 삽입 유전자의 모식도를 나타낸 것이다.
도 7은 CRISPR/Cas9을 이용하여 제작한 T0 식물체에서 OsHXK5 유전자의 염기서열을 분석하여 유전형을 확인한 결과이다.
도 8A은 야생형을 모본으로 사용한 일대잡종(F1)에서 CRISPR/Cas9 운반체가 삽입된 개체에서 OsHXK5 유전자 표적 부위의 염기서열을 분석하여 유전형을 분석한 결과이고, 8B는 야생형을 부본으로 사용한 일대잡종(F1)에서 CRISPR/Cas9 운반체가 삽입된 개체에서 OsHXK5 유전자 표적 부위의 염기서열을 분석하여 유전형을 분석한 결과이다.
도 9는 CRISPR/Cas 시스템을 이용하여 OsPLGG1 유전자의 돌연변이를 유도하기 위한 삽입 유전자의 모식도를 나타낸 것이다.
도 10A은 야생형을 모본으로 사용한 일대잡종(F1)에서 CRISPR/Cas9 운반체가 삽입된 개체에서 OsPLGG1 유전자 표적 부위의 염기서열을 분석하여 유전형을 분석한 결과이고, 10B는 야생형을 부본으로 사용한 일대잡종(F1)에서 CRISPR/Cas9 운반체가 삽입된 개체에서 OsPLGG1 유전자 표적 부위의 염기서열을 분석하여 유전형을 분석한 결과이다.
도 11은 적용되는 운반체(재조합 벡터)의 모식도를 나타낸 것이다.
도 12는 CRISPR/Cas 시스템을 이용하여 웅성불임 유지친(maintainer line) 및 Non-GM(Transgene free) 잡종 개발 모식도를 나타낸 것이다. 1 shows a schematic diagram of an inserted gene for inducing mutation of the Os12BGlu38 gene using the CRISPR/Cas system.
2 is a result of confirming the genotype by analyzing the nucleotide sequence of the Os12BGlu38 gene target site in the T0 plant produced using CRISPR/Cas9.
3 is a result of confirming whether the CRISPR/Cas9 transporter is inserted in a one-to-one hybrid (F1).
4 is a result of confirming the genotype of an individual in which the CRISPR/Cas9 transporter is not inserted in a one-to-one hybrid (F1).
5 is a result of confirming the genotype of the CRISPR/Cas9 transporter inserted in the one-to-one hybrid (F1).
6 shows a schematic diagram of an inserted gene for inducing mutation of the OsHXK5 gene using the CRISPR/Cas system.
7 is a result of confirming the genotype by analyzing the nucleotide sequence of the OsHXK5 gene in the T0 plant produced using CRISPR/Cas9.
Figure 8A is a genotype analysis result by analyzing the nucleotide sequence of the OsHXK5 gene target site in an individual having a CRISPR/Cas9 transporter inserted in a one-to-one hybrid (F1) using a wild type as a model, and 8B is a one-to-one hybrid using a wild type as a copy ( This is the result of genotype analysis by analyzing the nucleotide sequence of the OsHXK5 gene target site in the CRISPR/Cas9 transporter inserted in F1).
9 shows a schematic diagram of an inserted gene for inducing mutation of the OsPLGG1 gene using the CRISPR/Cas system.
Figure 10A is a genotype analysis result by analyzing the nucleotide sequence of the OsPLGG1 gene target site in an individual having a CRISPR/Cas9 transporter inserted in a one-to-one hybrid (F1) using a wild type as a model, and 10B is a one-to-one hybrid using a wild type as a copy ( In F1), the genotype analysis result by analyzing the nucleotide sequence of the OsPLGG1 gene target site in the CRISPR/Cas9 transporter inserted.
11 shows a schematic diagram of an applied carrier (recombinant vector).
Figure 12 shows a schematic diagram of male infertility maintenance parent (maintainer line) and Non-GM (Transgene free) hybrid development using the CRISPR / Cas system.
본원 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함" 한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.Throughout this specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.
본원 명세서 전체에서 사용되는 용어, "~(하는) 단계" 또는 "~의 단계" 는 "~를 위한 단계"를 의미하지 않는다.As used throughout this specification, the terms “step of” or “step of” do not mean “step for”.
본 발명은 (a) 표적 유전자를 인식할 수 있는 sgRNA(single guide RNA)을 코딩하는 폴리뉴클레오티드 및 Cas 단백질을 코딩하는 폴리뉴클레오티드가 삽입된 재조합 벡터로 형질전환시켜 형질전환 식물체를 제조하는 단계; 및 (b) 상기 형질전환 식물체와 야생형 식물체를 교배하는 단계를 포함하는 일대잡종(F1)에서 표적 유전자에 대한 동형접합 돌연변이체(homozygous mutant) 또는 이대립인자성 돌연변이체(biallelic mutant)의 제조 방법을 제공한다. The present invention comprises the steps of (a) transforming a polynucleotide encoding a single guide RNA (sgRNA) capable of recognizing a target gene and a polynucleotide encoding a Cas protein into a recombinant vector to prepare a transgenic plant; And (b) a method for producing a homozygous mutant or biallelic mutant for the target gene in a one-to-one hybrid (F1) comprising the step of crossing the transgenic plant and the wild-type plant provides
상기에서, "gRNA (guide RNA) 또는 sgRNA (single guide RNA)"는 Cas 단백질에 결합할 수 있고, Cas 단백질을 표적 유전자 내의 특정 위치를 인식하여 유전자 편집(gene editing)에 관여하는 RNA 분자를 말하며, gRNA는 Cas9(CRISPR-associated nuclease) 단백질에 특이적으로 표적하는 crRNA(CRISPR RNA) 및 pre-crRNA 와 부분적인 상보적 결합을 형성하는 tracrRNA(trans-activating crRNA)를 포함한다. In the above, "gRNA (guide RNA) or sgRNA (single guide RNA)" refers to an RNA molecule that can bind to a Cas protein and is involved in gene editing by recognizing a specific location within a target gene of the Cas protein. , gRNA includes crRNA (CRISPR RNA) that specifically targets Cas9 (CRISPR-associated nuclease) protein and tracrRNA (trans-activating crRNA) that forms a partial complementary bond with pre-crRNA.
본 발명에서, 상기 sgRNA를 코딩하는 폴리뉴클레오티드는 서열번호 3, 서열번호 6 및 서열번호 9의 염기서열로 이루어진 것으로부터 선택되는 어느 하나 이상인 것일 수 있다. In the present invention, the polynucleotide encoding the sgRNA may be any one or more selected from the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 9.
상기에서, "Cas(CRISPR-associated nuclease)"는 CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)과 관련된 RNA-guided DNA endonuclease enzyme으로서, 유전자 편집(gene editing)에 사용되고 있다.In the above, "Cas (CRISPR-associated nuclease)" is an RNA-guided DNA endonuclease enzyme related to CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), and is used for gene editing.
본 발명에서 표적 유전자는 웅성불임과 관련된 유전자, 즉, 웅성불임 유전자인 것일 수 있고, 웅성불임 유전자가 아닌 일반 유전자인 것일 수 있다. 바람직하게는 웅성불임 유전자인 Os12BGlu38 유전자 또는 OsHXK5 유전자인 것일 수 있고, 일반 유전자인 OsPLGG1 유전자인 것일 수 있으나, 이에 한정되는 것은 아니다. In the present invention, the target gene may be a gene related to male infertility, that is, a male infertility gene, or a general gene other than a male infertility gene. Preferably, it may be a male infertility gene, Os12BGlu38 gene or OsHXK5 gene, and may be a general gene, OsPLGG1 gene, but is not limited thereto.
상기에서, "웅성불임(male sterile)"은 웅성기관의 형태적 또는 기능적 이상 때문에 수분, 수정, 종자형성이 이루어지지 않는 현상을 의미하며, 환경적인 일시적 변이로서 발현하는 경우와 유전형질로서 발현하는 경우가 있으나, 본 발명에서의 웅성불임은 유전형질로서 유도되는 것일 수 있다.As used above, "male sterile" refers to a phenomenon in which pollination, fertilization, and seed formation are not made due to morphological or functional abnormalities of the male organ, and when expressed as a temporary environmental mutation or expressed as a genetic trait In some cases, male infertility in the present invention may be induced as a genetic trait.
상기 Os12BGlu38 유전자는 서열번호 1의 염기서열로 이루어진 것일 수 있고, OsHXK5 유전자는 서열번호 4의 염기서열로 이루어진 것일 수 있으며, OsPLGG1 유전자는 서열번호 7의 염기서열로 이루어진 것일 수 있으나, 이에 한정되는 것은 아니다. The Os12BGlu38 gene may consist of the nucleotide sequence of SEQ ID NO: 1, the OsHXK5 gene may consist of the nucleotide sequence of SEQ ID NO: 4, and the OsPLGG1 gene may consist of the nucleotide sequence of SEQ ID NO: 7, but is limited thereto no.
상기에서, "재조합 벡터"란 적당한 숙주세포에서 표적 단백질 또는 표적 RNA을 발현할 수 있는 벡터로서, 유전자 삽입물이 발현되도록 작동가능하게 연결된 필수적인 조절 요소를 포함하는 유전자 작제물을 말한다. 본 발명의 용어, "작동가능하게 연결된(operably linked)"은 일반적 기능을 수행하도록 핵산 발현조절 서열과 표적하는 단백질 또는 RNA를 코딩하는 핵산서열(염기서열)이 기능적으로 연결(functional linkage)되어 있는 것을 말한다. 예를 들어, 프로모터와 단백질 또는 RNA를 코딩하는 핵산서열이 작동가능하게 연결되어 코딩하는 핵산서열의 발현에 영향을 미칠 수 있다. 재조합 벡터와의 작동적 연결은 당해 기술분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용한다.As used herein, the term "recombinant vector" refers to a vector capable of expressing a target protein or target RNA in a suitable host cell, and refers to a genetic construct comprising essential regulatory elements operably linked to express a gene insert. As used herein, the term “operably linked” means that a nucleic acid expression control sequence and a nucleic acid sequence (nucleotide sequence) encoding a target protein or RNA are functionally linked to perform a general function. say that For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect expression of the encoding nucleic acid sequence. The operable linkage with the recombinant vector can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation using enzymes generally known in the art.
본 발명의 재조합 벡터는 표적 유전자를 인식할 수 있는 sgRNA를 코딩하는 폴리뉴클레오티드 및 Cas 단백질을 코딩하는 폴리뉴클레오티드를 포함하는 것일 수 있고, 형광표지 단백질을 코딩하는 폴리뉴클레오티드를 추가적으로 더 포함하는 것일 수 있다. 또한, 1개의 표적 유전자를 인식할 수 있는 2개 이상의 sgRNA를 코딩하는 폴리뉴클레오티드를 포함하는 것일 수 있고, 2개 이상의 표적 유전자를 각각 인식할 수 있는 2개 이상의 sgRNA를 코딩하는 폴리뉴클레오티드를 포함하는 것일 수 있다. The recombinant vector of the present invention may include a polynucleotide encoding an sgRNA capable of recognizing a target gene and a polynucleotide encoding a Cas protein, and may further include a polynucleotide encoding a fluorescent label protein. . In addition, it may include a polynucleotide encoding two or more sgRNAs capable of recognizing one target gene, and a polynucleotide encoding two or more sgRNAs capable of recognizing two or more target genes, respectively. it could be
예를 들어, Os12BGlu38 유전자를 인식할 수 있는 서열번호 3, 서열번호 11 및 서열번호 13의 염기서열로 이루어진 sgRNA를 코딩하는 폴리뉴클레오티드 중에서 서로 다른 두 개 또는 세 개의 폴리뉴클레오티드를 삽입한 것일 수 있고, 바람직하게는 서열번호 3의 염기서열로 이루어진 gRNA를 코딩하는 폴리뉴클레오티드를 삽인 한 것일 수 있다.For example, two or three different polynucleotides may be inserted from among polynucleotides encoding sgRNA consisting of the nucleotide sequences of SEQ ID NO: 3, SEQ ID NO: 11 and SEQ ID NO: 13 that can recognize the Os12BGlu38 gene, Preferably, the polynucleotide encoding the gRNA consisting of the nucleotide sequence of SEQ ID NO: 3 may be inserted.
또한, Os12BGlu38 유전자를 인식할 수 있는 서열번호 3, 서열번호 11 및 서열번호 13의 염기서열로 이루어진 sgRNA를 코딩하는 폴리뉴클레오티드 중 어느 하나의 폴리뉴클레오티드; OsHXK5 유전자를 인식할 수 있는 서열번호 6의 염기서열로 이루어진 sgRNA를 코딩하는 폴리뉴클레오티드; 및 OsPLGG1 유전자를 인식할 수 있는 서열번호 9의 염기서열로 이루어진 sgRNA를 코딩하는 폴리뉴클레오티드 중에서 서로 다른 두 개 또는 세 개의 폴리뉴클레오티드를 삽입한 것일 수 있다. In addition, any one of the polynucleotides encoding the sgRNA consisting of the base sequence of SEQ ID NO: 3, SEQ ID NO: 11 and SEQ ID NO: 13 capable of recognizing the Os12BGlu38 gene; a polynucleotide encoding an sgRNA consisting of the nucleotide sequence of SEQ ID NO: 6 capable of recognizing the OsHXK5 gene; and two or three different polynucleotides from among polynucleotides encoding sgRNA consisting of the nucleotide sequence of SEQ ID NO: 9 capable of recognizing the OsPLGG1 gene may be inserted.
상기에서, "형질전환"은 상기 재조합 벡터 등의 DNA 분자 등이 숙주세포로 주입되어 숙주세포의 DNA와 결합하여 세포의 유전형질이 변화되는 것을 말한다. 형질전환 방법은 미세주입법, 칼슘포스페이트 침전법, 전기천공법, 리포좀-매개 형질감염법 및 DEAE-덱스트란 처리법 및 아그로박테리움 매개 형질감염법 등 당해 기술분야에서 잘 알려진 기술을 이용할 수 있다. As used herein, "transformation" refers to a change in the genetic characteristics of a cell by injecting a DNA molecule such as the recombinant vector into a host cell and binding it with the DNA of the host cell. As the transformation method, techniques well known in the art such as microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection and DEAE-dextran treatment and Agrobacterium mediated transfection may be used.
상기에서, "교배"는 여교배(back cross) 또는 상호교배(reciprocal cross)를 통해 이루어지는 것일 수 있다. In the above, "crossing" may be made through back cross (back cross) or reciprocal cross (reciprocal cross).
상기에서, "동형접합 돌연변이체(homozygous mutant)"는 두 개의 대립유전자(allele)에 동일한 돌연변이가 나타난 식물체를 말하고, "이대립인자성 돌연변이체(biallelic mutant)"는 두 개의 대립유전자에 동일하지는 않지만 모두 돌연변이가 나타난 식물체를 말하며, "이형접합 돌연변이체(heterozygous mutant)"는 두 개의 대립유전자 중 하나의 대립유전자에만 돌연변이가 나타난 식물체를 말한다. In the above, "homozygous mutant" refers to a plant in which the same mutation appears in two alleles, and "biallelic mutant" refers to a plant that is identical to two alleles. However, it refers to a plant in which all mutations appear, and "heterozygous mutant" refers to a plant in which only one of the two alleles has a mutation.
본 발명에서는 재조합 벡터로 형질전환된 형질전환 식물체가 동형접합 돌연변이 또는 이대립인자성 돌연변이로 나타났고, 상기 돌연변이된 형질전환 식물체와 야생형 식물체를 여교배(back cross) 혹은 상호교배(reciprocal cross)한 경우 일대잡종(F1)에서 멘델의 법칙에 따라 이형접합 돌연변이만 나타난 것이 아니라, 일대잡종(F1)에서 재조합 벡터의 포함 여부에 따라 동형접합 돌연변이 또는 이대립인자성 돌연변이 또한 나타남을 확인하였다. 즉, 일대잡종(F1)에서 동형접합 돌연변이 또는 이대립인자성 돌연변이인 유전형을 제조할 수 있다. In the present invention, a transgenic plant transformed with a recombinant vector was shown as a homozygous mutation or a biallelic mutation, and the mutated transgenic plant and a wild-type plant were backcrossed or crossed. In the case of one-to-one hybrids (F1), it was confirmed that not only heterozygous mutations appeared according to Mendel's law, but also homozygous mutations or biallelic mutations appeared in one-to-one hybrids (F1) depending on whether the recombinant vector was included. That is, a genotype that is a homozygous mutation or a biallelic mutation in a one-to-one hybrid (F1) can be prepared.
본 발명의 식물체는 벼, 보리, 밀, 호밀, 옥수수, 사탕수수, 귀리 및 양파 등의 단자엽 식물, 목화, 토마토, 애기장대, 가지, 담배, 고추, 우엉, 쑥갓, 상추, 도라지, 시금치, 근대, 고구마, 샐러리, 당근, 미나리, 파슬리, 배추, 양배추, 갓무, 수박, 참외, 오이, 호박, 박, 딸기, 대두, 녹두, 강낭콩 및 완두 등의 쌍자엽 식물인 것일 수 있으나, 이에 한정되는 것은 아니다. Plants of the present invention include monocotyledonous plants such as rice, barley, wheat, rye, corn, sugar cane, oats and onions, cotton, tomatoes, Arabidopsis thaliana, eggplant, tobacco, red pepper, burdock, sagebrush, lettuce, bellflower, spinach, chard , sweet potato, celery, carrot, water parsley, parsley, Chinese cabbage, cabbage, radish, watermelon, melon, cucumber, pumpkin, gourd, strawberry, soybean, mung bean, kidney bean and pea, etc. may be dicotyledonous plants, but is not limited thereto. .
또한, 본 발명은 (a) 표적 유전자를 인식할 수 있는 sgRNA를 코딩하는 폴리뉴클레오티드, Cas 단백질을 코딩하는 폴리뉴클레오티드 및 형광표지 단백질을 코딩하는 폴리뉴클레오티드가 삽입된 재조합 벡터로 형질전환시켜 형질전환 식물체를 제조하는 단계; (b) 상기 형질전환 식물체와 야생형 식물체를 교배하는 단계; 및 (c) 형광표지 단백질을 발현하는 식물체를 선별하는 단계를 포함하는 웅성불임 유지친(male sterile maintainer line)의 제조 방법을 제공한다.In addition, the present invention is a transgenic plant by transforming a recombinant vector into which (a) a polynucleotide encoding an sgRNA capable of recognizing a target gene, a polynucleotide encoding a Cas protein, and a polynucleotide encoding a fluorescent marker protein are inserted. preparing a; (b) crossing the transgenic plant and the wild-type plant; And (c) provides a method for producing a male sterile maintainer line comprising the step of selecting a plant expressing the fluorescent marker protein.
상기에서, "웅성불임 유지친(male sterile maintainer line)"은 웅성불임을 유지할 수 있는 친 또는 계통을 말한다. 본 발명에서, 형광표지 단백질을 발현하는 식물체는 CRISPR/Cas9를 갖는 동형접합 돌연변이체 또는 이대립인자성 돌연변이체로서 웅성불임 유지친이 될 수 있어, 형광으로 쉽게 유지친인 것을 구분할 수 있는 장점이 있다. As used above, "male sterile maintainer line" refers to a parent or line capable of maintaining male sterility. In the present invention, a plant expressing a fluorescent label protein can become a male infertility parent as a homozygous mutant or biallelic mutant having CRISPR/Cas9. have.
또한, 본 발명은 (a) 표적 유전자를 인식할 수 있는 sgRNA를 코딩하는 폴리뉴클레오티드, Cas 단백질을 코딩하는 폴리뉴클레오티드 및 형광표지 단백질을 코딩하는 폴리뉴클레오티드가 삽입된 재조합 벡터로 형질전환시켜 형질전환 식물체를 제조하는 단계; (b) 상기 형질전환 식물체와 야생형 식물체를 교배하는 단계; 및 (c) 형광표지 단백질을 발현하지 않는 식물체를 선별하는 단계를 포함하는 Non-GMO(Non-Genetically Modified Organism) 식물체의 제조 방법을 제공한다. In addition, the present invention is a transgenic plant by transforming a recombinant vector into which (a) a polynucleotide encoding an sgRNA capable of recognizing a target gene, a polynucleotide encoding a Cas protein, and a polynucleotide encoding a fluorescent marker protein are inserted. preparing a; (b) crossing the transgenic plant and the wild-type plant; And (c) provides a method for producing a Non-Genetically Modified Organism (Non-GMO) plant comprising the step of selecting a plant that does not express the fluorescent marker protein.
상기에서, "Non-GMO"는 유전자 변형 생물체가 아닌 것을 말한다. 본 발명에서, 형광표지 단백질을 발현하지 않는 식물체는 CRISPR/Cas9를 갖지 않는 이형접합 돌연변이체인 것으로서, 외래 유전자가 삽입되지 않은 식물체가 될 수 있어, 형광 발현 여부에 따라 Non-GMO 를 쉽게 구분할 수 있는 장점이 있다. In the above, "Non-GMO" refers to non-genetically modified organisms. In the present invention, a plant that does not express the fluorescent marker protein is a heterozygous mutant that does not have CRISPR/Cas9, and can be a plant in which a foreign gene is not inserted. There are advantages.
또한, 본 발명은 표적 유전자를 인식할 수 있는 sgRNA를 코딩하는 폴리뉴클레오티드, Cas 단백질을 코딩하는 폴리뉴클레오티드 및 형광표지 단백질을 코딩하는 폴리뉴클레오티드를 포함하는 재조합 벡터로서, 상기 sgRNA를 코딩하는 폴리뉴클레오티드는 서열번호 3, 서열번호 6 및 서열번호 9의 염기서열로 이루어진 것으로부터 선택되는 어느 하나 이상인 것을 특징으로 하는 재조합 벡터를 제공한다. In addition, the present invention is a recombinant vector comprising a polynucleotide encoding an sgRNA capable of recognizing a target gene, a polynucleotide encoding a Cas protein, and a polynucleotide encoding a fluorescent marker protein, wherein the polynucleotide encoding the sgRNA comprises: It provides a recombinant vector, characterized in that at least one selected from the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 9.
상기 표적 유전자는 Os12BGlu38 유전자, OsHXK5 유전자 및 OsPLGG1 유전자로 이루어진 군에서 선택되는 어느 하나 이상의 유전자인 것일 수 있으나, 이에 한정되는 것은 아니다. 또한, 1개의 표적 유전자를 인식할 수 있는 2개 이상의 sgRNA를 코딩하는 폴리뉴클레오티드를 포함하는 것일 수 있고, 2개 이상의 표적 유전자를 각각 인식할 수 있는 2개 이상의 sgRNA를 코딩하는 폴리뉴클레오티드를 포함하는 것일 수 있다. The target gene may be any one or more genes selected from the group consisting of Os12BGlu38 gene, OsHXK5 gene and OsPLGG1 gene, but is not limited thereto. In addition, it may include a polynucleotide encoding two or more sgRNAs capable of recognizing one target gene, and a polynucleotide encoding two or more sgRNAs capable of recognizing two or more target genes, respectively. it could be
상기 형광표지 단백질은 녹색형광단백질(GFP), 황색형광단백질(YFP), 적색형광단백질(RFP) 및 시안형광단백질(CFP)로 이루어진 군에서 선택되는 것일 수 있으나, 이에 한정되는 것은 아니다. The fluorescent label protein may be selected from the group consisting of green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), and cyan fluorescent protein (CFP), but is not limited thereto.
또한, 본 발명은 상기 재조합 백터로 형질전환된 형질전환 식물체 및 상기 형질전환 식물체의 종자를 제공한다. 상기 식물체 또는 종자는 표적 유전자에 대한 동형접합 돌연변이체 또는 이대립인자성 돌연변이체인 것일 수 있다. In addition, the present invention provides a transgenic plant transformed with the recombinant vector and a seed of the transgenic plant. The plant or seed may be a homozygous mutant or a biallelic mutant for the target gene.
이하, 본 발명을 실시예를 통하여 더욱 상세히 설명하기로 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these examples.
실시예 1. CRISPR/Cas9 시스템을 이용한Example 1. Using the CRISPR/Cas9 system Os12BGlu38 유전자의 돌연변이 유도Mutation of the Os12BGlu38 gene
본 발명자들은 CRISPR/Cas9 시스템을 이용하여 만들어진 다양한 형질전환체 및 그 후대 식물체들의 유전형을 분석한 결과, Cas9 단백질 및 sgRNA(single guide RNA)가 동시에 발현하는 식물체와 야생형 식물체를 교배한 경우 야생형의 염색체로부터 전달된 표적 유전자는 수정 혹은 배발생 초기 시기동안 계속적으로 돌연변이의 발생이 유도되어 표적 유전자의 이형접합 돌연변이체(heterozygous mutant)를 표적 유전자의 동형접합 돌연변이체(homozygous mutant) 혹은 표적 유전자의 이대립인자성 돌연변이체(biallelic mutant)로 만드는 현상을 발견하였다. As a result of analyzing the genotypes of various transformants and progeny plants made using the CRISPR/Cas9 system, the present inventors found that when a plant expressing Cas9 protein and sgRNA (single guide RNA) at the same time was crossed with a wild-type plant, the wild-type chromosome In the target gene delivered from the fertilization or embryogenesis, mutations are continuously induced during the early stage of embryogenesis, so that a heterozygous mutant of the target gene is identified as a homozygous mutant of the target gene or a heterozygous allele of the target gene. The phenomenon of making it a biallelic mutant was discovered.
이에 본 발명자들은 웅성불임(male sterile) 유전자에 적용하여 안정적인 웅성불임 식물체를 제조하고자 위하여, 웅성불임을 유도하는 유전자인 Os12BGlu38 유전자(서열번호 1)의 염기서열 중 TCCATGATCAATACAAGGGA(서열번호 2)를 인식할 수 있는 sgRNA에 해당하는 염기서열(서열번호 3) 및 Cas 단백질을 코딩하는 염기서열을 운반체(재조합 벡터, 도 1)에 삽입하여 벼에 형질전환하였고, 형질전환된 식물체를 “T0”라고 명명하였다.Accordingly, the present inventors applied to the male sterile gene to prepare a stable male infertility plant, and to recognize TCCATGATCAATACAAGGGA (SEQ ID NO: 2) among the nucleotide sequences of the Os12BGlu38 gene (SEQ ID NO: 1), which is a gene inducing male infertility. The nucleotide sequence (SEQ ID NO: 3) corresponding to the capable sgRNA and the nucleotide sequence encoding the Cas protein were inserted into the carrier (recombinant vector, FIG. 1) to transform rice, and the transformed plant was named “T0”. .
이후, 본 발명자들은 CRISPR/Cas9을 이용하여 제작한 형질전환된 3개의 T0 식물체에서 Os12BGlu38 유전자의 염기서열 TCCATGATCAATACAAGGGA 의 분석을 통해 유전형을 확인하는 실험을 수행하였다. 간단히, 대조군으로 사용할 야생형(WT)과 일대잡종(F1) 식물의 잎으로부터 DNA를 추출하여 Os12BGlu38의 표적서열(서열번호 2)을 포함하는 구간을 증폭할 수 있는 24bp의 시발체(primer) 염기서열 한 쌍을 제작하여 PCR(Polymerase Chain Reaction)을 수행하였다. PCR을 통해 증폭된 DNA를 전기영동 하였을 때, 708bp 크기의 밴드 또는 돌연변이 양상에 따라 이보다 크거나 작은 밴드를 확인하였다. 이후 해당 밴드를 이용하여 염기서열 결정을 하였을 때, 표적서열을 포함한 708bp 구간 내에서 야생형과 다른 양상의 돌연변이를 확인하였으며 3개의 T0 식물체는 모두 이대립인자성 돌연변이체인 것으로 확인되었다. (도 2). Then, the present inventors performed an experiment to confirm the genotype through the analysis of the nucleotide sequence TCCATGATCAATACAAGGGA of the Os12BGlu38 gene in three T0 plants transformed using CRISPR/Cas9. Briefly, a 24-bp primer sequence capable of amplifying a section containing the Os12BGlu38 target sequence (SEQ ID NO: 2) by extracting DNA from the leaves of wild-type (WT) and one-to-one hybrid (F1) plants to be used as a control. By making a pair, PCR (Polymerase Chain Reaction) was performed. When the DNA amplified through PCR was electrophoresed, a band with a size of 708bp or a band larger or smaller than this was confirmed depending on the mutation pattern. Then, when the nucleotide sequence was determined using the corresponding band, mutations of a different aspect from the wild type were identified within the 708bp section including the target sequence, and all three T0 plants were confirmed to be biallelic mutants. (Fig. 2).
또한, 본 발명자들은 상기 3 개의 T0 식물체 중 하나의 식물체와 야생형인 동진벼와의 여교배를 실시한 후, 일대잡종(F1)의 유전형을 확인하는 실험을 수행하였다. CRISPR/Cas9 운반체에는 존재하면서 야생형 벼에는 존재하지 않는 24bp의 시발체(primer) 염기서열 한 쌍을 제작하여 PCR(Polymerase Chain Reaction)을 수행하였다. PCR을 통해 증폭된 DNA를 전기영동 하였을 때, 운반체 DNA가 증폭되어 931bp 크기의 밴드가 확인된 경우 CRISPR/Cas9 운반체가 포함된 식물체, 확인되지 않은 경우 운반체가 포함되지 않은 식물체라 할 수 있다. 우선, 일대잡종(F1)에서 CRISPR/Cas9 운반체의 삽입 여부를 확인하였고, #2, #4, #7, #9, #10, #12, #13, #18, #19, #24 및 #25 개체에서 CRISPR/Cas9 운반체가 삽입되어 있음을 확인하였다 (도 3).In addition, the present inventors performed an experiment to confirm the genotype of the one-to-one hybrid (F1) after backcrossing one of the three T0 plants with the wild-type Dongjinbyeo. Polymerase Chain Reaction (PCR) was performed by preparing a pair of 24 bp primer nucleotide sequences that are present in the CRISPR/Cas9 transporter but not in wild-type rice. When the DNA amplified through PCR is electrophoresed, if the carrier DNA is amplified and a band with a size of 931 bp is confirmed, it is a plant containing the CRISPR/Cas9 carrier, and if it is not confirmed, it can be called a plant without the carrier. First, it was confirmed whether the CRISPR/Cas9 transporter was inserted in the one-to-one hybrid (F1), #2, #4, #7, #9, #10, #12, #13, #18, #19, #24 and # It was confirmed that the CRISPR/Cas9 transporter was inserted in 25 subjects ( FIG. 3 ).
상기 결과에 따라, 본 발명자들은 일대잡종(F1)에서 CRISPR/Cas9 운반체가 삽입되지 않은 나머지 개체(#1, #3, #5, #6, #8, #11, #14, #15, #16, #17, #20, #21, #22 및 #23)에서 Os12BGlu38 유전자의 유전형을 확인하였다. 그 결과, 상기 개체 모두는 이형접합 돌연변이임을 확인하였다 (도 4). 또한, 일대잡종(F1)에서 CRISPR/Cas9 운반체가 삽입된 개체(#2, #4, #7, #9, #10, #12, #13, #18, #19, #24 및 #25)에서는 Os12BGlu38 유전자의 유전형이 모두 동형접합 혹은 이대립인자성 돌연변이임을 확인하였다 (도 5). According to the above results, the present inventors found that the remaining individuals (#1, #3, #5, #6, #8, #11, #14, #15, # The genotype of the Os12BGlu38 gene was confirmed in 16, #17, #20, #21, #22 and #23). As a result, it was confirmed that all of the individuals were heterozygous mutants ( FIG. 4 ). In addition, the CRISPR/Cas9 transporter inserted in the co-hybrid (F1) (#2, #4, #7, #9, #10, #12, #13, #18, #19, #24 and #25) It was confirmed that all genotypes of the Os12BGlu38 gene were homozygous or biallelic mutations (FIG. 5).
이로써, CRISPR/Cas 운반체가 삽입된 일대잡종(F1) 식물체 모두는 동형접합 돌연변이체 또는 이대립인자성 돌연변이체이고, 모든 돌연변이체가 웅성불임이 유지되는 반면, CRISPR/Cas 운반체가 삽입되지 않은 일대잡종(F1) 식물체 모두는 이형접합 돌연변이인 것을 확인하였다. Thus, all of the CRISPR/Cas transporter-introduced one-to-hybrid (F1) plants are either homozygous or biallelic mutants, and all mutants maintain male sterility, whereas one-to-one CRISPR/Cas transporters are not inserted. (F1) It was confirmed that all of the plants were heterozygous mutants.
또한, CRISPR/Cas 운반체가 삽입되지 않은 일대잡종을 자가수정했을 경우 그 자손은 모두 웅성불임 표현형을 보이지 않았으며, 유전형의 경우 이형접합체 돌연변이와 야생형의 비율이 1:1로 나타남으로써, 일반적인 돌연변이 분리비를 따르는 것으로 확인하였다. In addition, when the CRISPR/Cas transporter was not inserted into a direct hybrid, all of the offspring did not show the male infertility phenotype, and in the case of the genotype, the ratio of heterozygous mutation to wild type was 1:1, resulting in a general mutation separation ratio. confirmed to follow.
실시예 2. CRISPR/Cas9 시스템을 이용한Example 2. Using the CRISPR/Cas9 system OsHXK5 유전자의 돌연변이 유도Mutation of the OsHXK5 gene
본 발명자들은 다른 웅성불임을 유도하는 유전자인 OsHXK5 유전자를 이용하여 안정적인 웅성불임 식물체를 생산하기 위하여 실시예 1과 동일한 실험을 수행하였다. 우선, 웅성불임을 유도하는 유전자인 OsHXK5 유전자(서열번호 4)의 염기서열 중 GCGGGGCATCTCGGACGCCA(서열번호 5)를 인식할 수 있는 sgRNA에 해당하는 염기서열(서열번호 6) 및 Cas 단백질을 코딩하는 염기서열을 운반체(재조합 벡터, 도 6)에 삽입하여 벼에 형질전환하였다. The present inventors performed the same experiment as in Example 1 to produce stable male infertility plants using the OsHXK5 gene, which is another gene inducing male infertility. First, a nucleotide sequence corresponding to sgRNA that can recognize GCGGGGCATCTCGGACGCCA (SEQ ID NO: 5) among the nucleotide sequences of the OsHXK5 gene (SEQ ID NO: 4), which is a gene inducing male infertility (SEQ ID NO: 6) and a nucleotide sequence encoding a Cas protein was inserted into a carrier (recombinant vector, FIG. 6) to transform the rice.
이후, 형질전환된 10개의 식물체에서 OsHXK5 유전자의 염기서열 GCGGGGCATCTCGGACGCCA 의 분석을 통해 유전형을 확인하는 실험을 수행하였다. 그 결과, 10개의 식물체는 모두 동형접합 또는 이대립인자성 돌연변이체인 것으로 확인되었다 (도 7). Thereafter, an experiment was performed to confirm the genotype through analysis of the base sequence GCGGGGCATCTCGGACGCCA of the OsHXK5 gene in 10 transformed plants. As a result, it was confirmed that all 10 plants were homozygous or biallelic mutants ( FIG. 7 ).
또한, 본 발명자들은 상기 10개의 식물체 중 #2 식물체를 자가 수정하여 얻은 T1 식물들 중 CRISPR/Cas 운반체가 삽입된 식물체와 야생형인 동진벼를 상호교배(reciprocal cross)를 실시한 후, 일대잡종(F1) 식물체에서 OsHXK5 유전자의 염기서열 GCGGGGCATCTCGGACGCCA 의 분석을 통해 유전형을 확인하는 실험을 수행하였다. 그 결과, 야생형을 모본으로 사용한 일대잡종(F1) 식물체 중 CRISPR/Cas9 운반체의 삽입된 개체는 모두 동형접합 혹은 이대립인자성 돌연변이인 것으로 확인되었고 (도 8A), 야생형을 부본으로 사용한 일대잡종(F1) 식물체 중 CRISPR/Cas9 운반체의 삽입된 개체도 모두 동형접합 혹은 이대립인자성 돌연변이인 것으로 확인됨으로써 (도 8B), CRISPR/Cas 운반체가 삽입된 식물체의 경우 모두 동형접합체 또는 이대립인자성 돌연변이인 것을 확인하였다. In addition, the present inventors performed a reciprocal cross between a plant in which the CRISPR/Cas carrier was inserted and a wild-type Dongjinbyeo among T1 plants obtained by self-fertilizing
실시예 3. CRISPR/Cas9 시스템을 이용한Example 3. Using the CRISPR/Cas9 system OsPLGG1 유전자의 돌연변이 유도Induction of mutations in the OsPLGG1 gene
본 발명자들은 웅성불임 유전자가 아닌 일반적인 유전자에도 동일한 결과가 나타나는지 확인하기 위하여 OsPLGG1 유전자를 이용하여 실시예 1과 동일한 실험을 수행하였다. OsPLGG1 기능상실 벼는 광호흡이 제한되어 잎의 엽록소 함량이 감소되는 표현형이 나타남에 착안하여, OsPLGG1 유전자(서열번호 7)의 염기서열 CCTCTTCTACGTCCCTTCCC(서열번호 8)를 인식할 수 있는 sgRNA에 해당하는 염기서열(서열번호 9) 및 Cas 단백질을 코딩하는 염기서열을 운반체(재조합 벡터, 도 9)에 삽입하여 벼에 형질전환하였다. The present inventors performed the same experiment as in Example 1 using the OsPLGG1 gene in order to check whether the same results are shown in general genes other than male infertility genes. OsPLGG1 loss-of-function In rice, photorespiration is restricted and the chlorophyll content of leaves is reduced, paying attention to the phenotype of the OsPLGG1 gene (SEQ ID NO: 7). A nucleotide sequence corresponding to sgRNA that can recognize CCTCTTCTACGTCCCTTCCC (SEQ ID NO: 8) (SEQ ID NO: 9) and a nucleotide sequence encoding a Cas protein were inserted into a carrier (recombinant vector, FIG. 9) to transform rice.
이후, 형질전환된 4개 식물체에서 OsPLGG1 유전자의 염기서열 CCTCTTCTACGTCCCTTCCC 의 분석을 통해 유전형을 확인하는 실험을 수행하였고, 모두 이대립인자성 돌연변이체인 것을 확인하였다. 또한, 자가수정한 T1 식물체 중 T가 삽입되어 CCTCTTCTACGTCCCTTCCC의 염기서열을 가진 식물체와 야생형인 동진벼를 상호교배(reciprocal cross)를 실시한 후, 일대잡종(F1) 식물체에서 유전형을 확인하는 실험을 수행하였다. 그 결과, 야생형을 모본으로 사용한 일대잡종(F1) 식물체 중 CRISPR/Cas9 운반체가 삽입된 41개 개체는 모두 동형접합 혹은 이대립인자성 돌연변이인 것으로 확인되었고 (도 10A), 야생형을 부본으로 사용한 일대잡종(F1) 식물체 중 CRISPR/Cas9 운반체가 삽입된 9개 개체도 모두 동형접합 혹은 이대립인자성 돌연변이인 것으로 확인됨으로써 (도 10B), CRISPR/Cas 운반체가 삽입된 식물체는 모두 동형접합체 또는 이대립인자성 돌연변이인 것을 확인하였다. 또한, 50개의 일대잡종(F1) 식물체는 모두 엽록소 함량이 감소되는 표현형이 나타남을 확인하였다. Thereafter, an experiment was performed to confirm the genotype through analysis of the nucleotide sequence CCTCTTCTACGTCCCTTCCC of the OsPLGG1 gene in the four transformed plants, and it was confirmed that all of them were biallelic mutants. In addition, after performing a reciprocal cross between a plant having the nucleotide sequence of CCTCTTCTACGTCCCTTCCC and a wild-type Dongjinbyeo in which T was inserted among the self-fertilized T1 plants, an experiment was performed to confirm the genotype in a one-to-one hybrid (F1) plant. As a result, it was confirmed that all 41 individuals into which the CRISPR/Cas9 transporter was inserted among cohort hybrid (F1) plants using the wild type as a model were homozygous or biallelic mutations (FIG. 10A), and the cohort using the wild type as a copy. Among hybrid (F1) plants, all 9 individuals into which the CRISPR/Cas9 transporter was inserted were also confirmed to be homozygous or biallelic mutations (FIG. 10B), so that all plants in which the CRISPR/Cas transporter was inserted were homozygous or biallelic. It was confirmed that it was a factor mutation. In addition, it was confirmed that all 50 one-to-one hybrid (F1) plants exhibited a phenotype in which the chlorophyll content was decreased.
실시예 4. CRISPR/Cas9 시스템을 이용한 유지친 및 Non-GM(Non-Genetically Modified Organism) 식물체의 제조Example 4. Preparation of oleaginous and Non-Genetically Modified Organism (GM) plants using the CRISPR/Cas9 system
본 발명자들은 CRISPR/Cas9 운반체에 웅성불임 유전자에 대한 sgRNA를 삽입하여 유전자의 돌연변이를 유도함과 동시에 형광표지 유전자를 함께 삽입하여 형질전환된 종자를 형광으로 쉽게 분리할 수 있도록 운반체(재조합 벡터)를 제작하였다 (도 11A).The present inventors inserted the sgRNA for the male infertility gene into the CRISPR/Cas9 carrier to induce gene mutation and at the same time insert the fluorescent marker gene together to create a carrier (recombinant vector) so that the transformed seed can be easily separated by fluorescence (Fig. 11A).
또한, 수정 후 웅성불임 유전자 돌연변이가 유도될 때 우연히 3개 혹은 3의 배수의 유전자가 삽입/결손되어 야생형 웅성불임 단백질의 활성을 유지하고 있을 경우를 대비하여 CRISPR/Cas9 운반체에 한 가지 웅성불임 유전자에 대한 서로 다른 두 개의 sgRNA를 삽입함으로써 한 개체 내에서 서로 다른 두 개의 돌연변이 혹은 긴 염기서열의 결손을 유도하는 동시에 CRISPR/Cas9 운반체에 형광표지 유전자를 함께 삽입하여 형질전환된 종자를 형광으로 쉽게 분리할 수 있도록 운반체(재조합 벡터)를 제작하였다 (도 11B). In addition, when male infertility gene mutation is induced after fertilization, one male infertility gene is added to the CRISPR/Cas9 carrier in case three or a multiple of 3 genes are accidentally inserted/deleted to maintain the activity of the wild-type male infertility protein. Inducing two different mutations or deletion of long nucleotide sequences in one individual by inserting two different sgRNAs for A carrier (recombinant vector) was constructed so that the
또한, 수정 후 웅성불임 유전자 돌연변이가 유도될 때 우연히 3개 혹은 3의 배수의 유전자가 삽입/결손되어 야생형 웅성불임 단백질의 활성을 유지하고 있을 경우를 대비하여 CRISPR/Cas9 운반체에 두 가지의 서로 다른 웅성불임 유전자에 대한 서로 다른 두 개의 sgRNA를 삽입함으로써 한 개체 내에서 서로 다른 두 개의 돌연변이를 유도하는 동시에 CRISPR/Cas 운반체에 형광표지 유전자를 함께 삽입하여 형질전환된 종자를 형광으로 쉽게 분리할 수 있도록 운반체(재조합 벡터)를 제작하였다(도 11C). In addition, when the male infertility gene mutation is induced after fertilization, two different types of CRISPR/Cas9 transporters are added to the CRISPR/Cas9 transporter in case three or a multiple of three genes are accidentally inserted/deleted to maintain the activity of the wild-type male infertility protein. By inserting two different sgRNAs for the male infertility gene, two different mutations are induced in one individual, and at the same time, a fluorescent marker gene is inserted into the CRISPR/Cas carrier so that the transformed seeds can be easily separated by fluorescence. A vehicle (recombinant vector) was constructed ( FIG. 11C ).
형광표지 유전자는 mRFP (단량체 적색 형광 단백질) 인 mCherry cDNA (서열번호 14)를 이용하여 운반체로 형질전환된 식물체는 적색으로 발생되어 형광분광법으로 선별할 수 있도록 하였다.As the fluorescent marker gene, mCherry cDNA (SEQ ID NO: 14), which is mRFP (monomeric red fluorescent protein), was used to transform a plant transformed with the carrier into a red color, so that it could be selected by fluorescence spectroscopy.
상기 운반체(재조합 벡터)로 형질전환된 식물체 중 동형접합 돌연변이체 또는 이대립인자성 돌연변이체는 야생형과 여교배한 후, 형광표지 단백질의 발현을 이용하여 CRISPR/Cas9 운반체가 삽입된 식물체를 선별하고 유전형을 확인할 수 있다. 이 경우 형광표지 단백질을 발현하는 모든 식물체가 웅성불임인 것으로 확인되면, 수정 중에 야생형으로부터 받은 염색체에도 CRISPR/Cas9 유래 돌연변이가 유도되었다는 의미이므로, 형광표지 단백질이 발현하는 식물체를 웅성불임 유지친(maintainer)으로 사용할 수 있다. 또한, 형광표지 단백질이 발현하지 않는 식물체는 웅성불임 유전자에 대하여 이형접합 돌연변이이며, 동시에 외래 유전자가 삽입되지 않아 Non-GMO(Non-Genetically Modified Organism)이므로, 일대잡종(F1) 종자를 상업적으로 활용할 수 있다.Among the plants transformed with the carrier (recombinant vector), the homozygous mutant or the biallelic mutant is backcrossed with the wild type, and then the plant into which the CRISPR / Cas9 carrier is inserted is selected using the expression of the fluorescent marker protein. genotype can be identified. In this case, if all plants expressing the fluorescent marker protein are confirmed to be male infertile, it means that CRISPR/Cas9-derived mutations were induced even in the chromosomes received from the wild-type during fertilization. ) can be used as In addition, a plant that does not express the fluorescent marker protein is a heterozygous mutation for the male infertility gene and at the same time is a Non-GMO (Non-Genetically Modified Organism) because a foreign gene is not inserted. can
실시예 5. CRISPR/Cas9 시스템을 이용한Example 5. Using the CRISPR/Cas9 system Os12BGlu38 유전자의 돌연변이 식물체의 비교Comparison of Os12BGlu38 gene mutant plants
본 발명자들은 CRISPR/Cas9 시스템을 이용한 형질전환 식물체의 제조 효율을 확인하기 위하여 Os12BGlu38 유전자의 다양한 부위를 타겟으로 하는 sgRNA 서열을 구축하고 이를 이용하여 돌연변이된 다양한 식물제를 제조하였다.The present inventors constructed sgRNA sequences targeting various regions of the Os12BGlu38 gene in order to confirm the production efficiency of transgenic plants using the CRISPR/Cas9 system, and prepared various mutated plant preparations using the sgRNA sequences.
구체적으로 실시예 1에 기재된 Os12BGlu38 유전자(서열번호 1)의 염기서열 중 TCCATGATCAATACAAGGGA(서열번호 2)를 인식할 수 있는 sgRNA 염기서열(서열번호 3)과, GACACCTTCCTGATGCAACC(서열번호 10)를 인식할 수 있는 sgRNA 염기서열(서열번호 11) 및, GGTCTCGCATTTTCCCCAGT(서열번호 12)를 인식할 수 있는 gRNA 염기서열(서열번호 13) 중에 하나를 코딩하고, Cas9 단백질을 코딩하는 운반체(재조합 벡터)를 제조하였다. 제조된 운반체는 벼에 형질전환하하였다.Specifically, the sgRNA nucleotide sequence (SEQ ID NO: 3) capable of recognizing TCCATGATCAATACAAGGGA (SEQ ID NO: 2) among the nucleotide sequences of the Os12BGlu38 gene (SEQ ID NO: 1) described in Example 1, and GACACCTTCCTGATGCAACC (SEQ ID NO: 10) capable of recognizing One of the gRNA sequences (SEQ ID NO: 13) capable of recognizing the sgRNA sequence (SEQ ID NO: 11) and GGTCTCGCATTTTCCCCAGT (SEQ ID NO: 12), and a carrier (recombinant vector) encoding the Cas9 protein was prepared. The prepared vehicle was transformed into rice.
이후, 본 발명자들은 각각의 sgRNA를 이용한 3개의 식물체에서 실시예 1에서 개시된 방법을 이용하여 Os12BGlu38 유전자의 염기서열 분석을 통해 유전형을 확인하여 돌연변이 효율 평가를 수행하였다. 수행 결과 서열번호 3의 sgRNA를 코딩하는 재조합 벡터의 경우는 Os12BGlu38의 다른 부위를 타겟하는 서열번호 11 및 서열번호 13의 sgRNA에 비하여 돌연변이 확률이 더 높은 것으로 나타났다.Then, the present inventors performed mutation efficiency evaluation by confirming the genotype through sequencing of the Os12BGlu38 gene using the method disclosed in Example 1 in three plants using each sgRNA. As a result, the recombinant vector encoding the sgRNA of SEQ ID NO: 3 had a higher mutation probability than the sgRNAs of SEQ ID NO: 11 and SEQ ID NO: 13 targeting other sites of Os12BGlu38.
상기 결과에 따라 sgRNA 타겟 서열의 돌연변이 확률이 상이하였으며, 서열번호 11 및 서열번호 13의 sgRNA는 오프타겟(off-target)으로 효율이 낮은 것으로 나타났으며, 이로써 서열번호 3의 sgRNA 염기서열이 높은 확률로 Os12BGlu38 웅성불임 식물체를 제조할 수 있음을 확인하였다.According to the above results, the mutation probability of the sgRNA target sequence was different, and the sgRNA of SEQ ID NO: 11 and SEQ ID NO: 13 was found to have low efficiency as an off-target, whereby the sgRNA nucleotide sequence of SEQ ID NO: 3 was high. It was confirmed that Os12BGlu38 male sterile plants could be produced with probability.
<110> University-Industry Cooperation Group of Kyung Hee University <120> Manufacturing method of non-functional mutant in first filial generation using CRISPR/Cas system <130> PO2009-042 <150> KR 10-2019-0168711 <151> 2019-12-17 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 3139 <212> DNA <213> Oryza sativa <400> 1 actctcaccg ccgctctctc ttcctctcca acatgaatat gccattgcta ctcctcatcg 60 ccatcgtcgt cgtctccctc tcccatggca acggggagca gaccgacctc acgcgggaga 120 cgttccccgc gggcttcgtc ttcggcaccg cgtcgtcggc gtaccaggtg gaggggaacg 180 ccctccagta tggccgaggg ccctgcatct gggacacctt cctgatgcaa cctggtgaga 240 tcgatgatac atgttcaggc aaaactagca atgtggtatc taaatataca gatcataaac 300 agtttgtcgc atttttcaaa taatggaatt aacaatgtgt tgggacgatc cacctaaaaa 360 gagaatatga caacaacaat ctaaaaagaa ttacatagcc tgatattgaa atgttgttgc 420 attacggcgc gttgcatttt gtacgtctct agtggatgta tgactggttg ttgatttggc 480 tctgattgtt cctttggata ggtgtaactc ctgataattc gaccgcgaat gtgaccgtcg 540 acgagtacca ccgctacatg gtcagtgaaa gtctcatgtg tttcagtgcc tgttctaatg 600 ggtttctgtt cctctgtatg actcttgatt tgtagcttat gtggctcctc tgatgatctt 660 ctttgcagga tgatgtggac aacatggtga gagtgggctt cgacgcgtat cgcttctcga 720 tctcctggtc tcgcattttc cccagtaagt taccggctga aacatggtca ggcacagtga 780 tgagatcatg ggatattcct ttcattgatg gtttttcttc tgcgtgttgt aggtggactt 840 gggaagatta acaaagacgg cgtggattat taccacaggc tcattgatta catgcttgct 900 aacagtacgt tggtcttggc atttcttcta gttcttgccc atttcggatt tcatgatctc 960 gaattaatag gttttttttt ggtttgattt gcacatcaga cattattcca tatgttgtgc 1020 tctaccacta cgaccttcca caggtgctcc atgatcaata caagggatgg ctacacccca 1080 gaattgtgta agtgttcgtg atcaaagcca tctgtttggc tgtaaaactt aaaattaaat 1140 accctttaaa ttgtttgaca aactacagaa gcacctctga agattttgtg ctaaattatg 1200 aaacccatag ctctctaact gcgaaatcct gacagtgaaa gttctgcagg tgtttaacca 1260 aaataggttt cactgtagca tatccgacat acacaaatat caaacccaga caatctgtat 1320 cctccctttc ttacgttcct tactcccttt ttcttgaatt aatgtgtgat tcatgtctgc 1380 gacttcattt tgcaggagag attttgtgag atttgcagac ttctgcttca agacatatgg 1440 tcataaggtg aagaactggt ttaccattaa cgaacctagg atgatggcaa atcatggcta 1500 cggtgacggc ttcttccccc ctggcagatg caccggctgc caacccggtg ggaattccgc 1560 caccgagcct tacatcgcag cccataacct tctcctttca catgccgctg ctgtcaggac 1620 atatcgtgac aagtaccagg ctagtgaatg atatccatgc ccctactatc caattcaaca 1680 tttcttaaaa tctccaatgt atcagctcat gttcatgata ccttgcgatt ttcaggctat 1740 tcagaagggg aagattggca tccttctcga ttttgtatgg tatgagccac tcaccgacaa 1800 agaagaggat cacgcagctg cacatagagc cagggagttt acccttggct ggtgatacat 1860 cttacactgt tcatcaatca ttgctcttac acggtgtccg catgaaggtt gaactgaact 1920 tccacttgaa acttttgcta ggtacctgca cccgattaca tatggtcatt acccagaaac 1980 tatgcagaat gctgttaagg aaaggctgcc caatttcaca cgtgagcagt ctgagatgat 2040 aaaaggatca gcggattata ttgcgatcaa ccattacaca acttattatg tcagtcacca 2100 cgtcaacaag acatccatca gctatctcaa tgattgggat gtgaaaattt catgtatgac 2160 actttcaaat cacacagtat tagaccagaa atatttgtac cttttcaatt ctttgccttc 2220 ctagctaaca cattttggtc ctctattcag atgagcgtaa cggtgtgcca attgggaaac 2280 aggtaagcaa caatttacaa gaaaggttga aaagaatggt agatcatgaa ctaaattctg 2340 gtttttaaac atctatgcag gcgtactcga actggcttta tgttgttcct tgggggatct 2400 acaaagctgt catgcatgtc aaggagaagt acaaggaccc cattataatc atcggagaaa 2460 atggtaaata aagataatac tggagaccct ttcaattttc ggacagtgaa cagtacaact 2520 ataaaagtgc tctgcagaat gaatctagga gcaaaatcta acaagaacct tttgcaggca 2580 ttgaccagcc aggcaatgag accctacctg gtgcactgta tgacttcttc aggatacaat 2640 attttgatca gtacctccat gagcttaaga gggcgatcaa ggatggtgca agggtcactg 2700 ggtattttgc ttggtccctg cttgacaact tcgagtggcg gctcgggttc acttcaaaat 2760 tcggaatcgt ctatgtagac cggagcactt tcacacggta ccctaaagac tcaacacgtt 2820 ggttcaggaa gatgataaaa agtgaggttt gagttggatt attatcactg ttggcagctg 2880 ctggagtgct tttttgttat gctagttttg gttgtatgaa taataagatg tcttgtacta 2940 gttgagaact tcttcagatt ttgtacttct agtattctac tttttgtact tcagattttg 3000 caagcaagga tgattttagt ttaatagatt caggacatct atatatactg gtgataagag 3060 aggcagattt caatttttgg cttgaattgc ccatatttgt gttttccaat gttttttaac 3120 ttcaaatttt aaactttgg 3139 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Os12Bglu38 target site <400> 2 tccatgatca atacaaggga 20 <210> 3 <211> 104 <212> DNA <213> Artificial Sequence <220> <223> Os12Bglu38 sgRNA <400> 3 tccatgatca atacaaggga gttttagagc tatgctgaaa agcatagcaa gttaaaataa 60 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgc 104 <210> 4 <211> 5643 <212> DNA <213> Oryza sativa <400> 4 accggttaaa acgcccaagt cagtggctct cgtcctcctt tctcctacgc ggccggagag 60 aggagggggg aagagaggat ctcgcagagc gccatagcct cggatccaga agcggaattc 120 ggtggaggtc tcgggcacct ggatcgatcg gaggaaggga aggcggagca gcggtgatgg 180 ggaaggcggc ggcggtgggg acggcggtgg tggtggccgc ggcggtcggg gtggcggtgg 240 tgctggcgcg gaggcggagg aggagggacc tggagctggt ggagggagcc gcggcggaga 300 ggaagaggaa ggtggcggcg gtgatcgagg acgtggagca cgcgctgtcg accccgacgg 360 cgctgctgcg gggcatctcg gacgccatgg tcaccgagat ggagcgaggc ctgcgcgggg 420 acagccacgc catggttaag atgctcatca cctacgtcga caacctcccc accgggtaag 480 cgcctcgatt ccgatctgat gtctgattcc tttcgttttt cgtattatgt ttcctttcgt 540 ttttagtatt atgggtttgg aactttgtga tctaaaaagt ttggaattgg gaaaggtctc 600 gtattgatgc gaatcgttcg ggaacaaatc tagtagtgac gcgcgtaacc gtgggaattt 660 atctccgtgc tctgattagg accgactgac cttttgatgg gatggtagga aatgtgcgaa 720 ttctttttat gatgtttgta cgaaaatcag attcccccga aaattgcgcc tagtggtgat 780 ccgaagagaa tggaactata atctcccatt tgatcttcat gtggttttgg tgtctcgtat 840 tcgtgaattg ggattttttt tgtacgttcc gatcgaagct agatcaatcc tagctttgcc 900 aaacaatata agccatttgt tataactgaa ataagttatg taaccacaag gaaacgcttg 960 ctctcggact ctgctgagga gagccaaggc aaattggctc ataccagtga ttgaaggaaa 1020 aatgtggagt cgtgggcatc tgaaattgct tctcatgcac caggacgcag gatttcggtg 1080 atgggggtac agagttacta aagcatcatg caatgagaaa aaaaatgaag tgctagtatg 1140 tgttattaac acaaacataa atcattgtta tagaaaaaaa tataaagtag ccaagagaaa 1200 atgataatac tcttgacaag gatgattttt gtgcttgaaa atgattgttt taatttagct 1260 gattgtgcat acacatacta tacatacatg ttatagatta tagatcacat ttgtttgcta 1320 aaacaatttg cggggtaccg gattacgaac tgtacccctc atgtccttgt gtaaacagga 1380 ttctacatag aactcaaaca agatagataa attggcctat ctaagctagg aaagataggt 1440 ctcccgtcaa catatcctat cgttctcaga tgatcttcgg ttttgtgatc tatagtagat 1500 cgtccatttg ttcctaattt ggctctttag gatttctgat tatgatatgt gattaattaa 1560 tttgatcgtc catttgttcc tactttggct ctttaggatt tctgattatg atatgtgatt 1620 aattaatttc ttgttggttt gatttgttaa ggcttcagtg acaggtaatt aactaatgta 1680 tggcagagag tttctttgca ttgtggtttc cttgttagca atctgaccaa ttagatccat 1740 cagtcgagct tatctcatgg tatagttggt ctggtaataa gtttctctgt aacctccaca 1800 tcgttaagct aaatgtggca ggcacatcct gaaggaagtt tcttaaatat aatgattcat 1860 tcttattcta tcggatcctt ctaccagtct gttagaactc actcacccat aagcatacaa 1920 tgtttcctgc tgccttcttc caaaagtagt aatttcattc tttgatcata atgatttccc 1980 tttgcagaaa tgaacagggg ttgttttatg cattggatct tggaggaacc aacttccgcg 2040 tcctgcgagt ccaactcggt ggcaaggaga aacgtgtcgt ccaacaacag tacgaggaag 2100 tctcaattcc accacatctg atggttggaa cttccatggt aagcatttgc tgcttgattt 2160 taaatttctt ggagctgtac attccatgct tgtcttatgt attttctgac tattaatgca 2220 ggaactgttt gattttattg cttctgcatt gtcaaaattt gttgatactg aaggtgatga 2280 tttccacctc ccagaaggga gacagagaga gctgggcttc accttttcct tcccagtgag 2340 ccagacatca atatcgtcag gaacgctcat caagtggaca aagggtttct ccatcaatga 2400 cgcggtaaaa gtcaaaaata acttttggac ataccctgac tagaacacaa tcctgatcct 2460 tctttaaaca catgaattat gtcacaaggt tccttgataa agcttgattg aatatggcaa 2520 tggagtattt caattagcta tttgctacaa tctccctttg tgtacattgt aatcaactta 2580 actaggttag tgaaaagaaa tatcgacctt gatcaggaag catatttaat ttgaggatct 2640 ttaatagtac tcagtattat gtttgcctgg ttgatcatat ctgtgtgaac tgtcaagcat 2700 ccaataagaa taatttgatt ttgctatatt atctcctgaa ttaggttggc gaagatgttg 2760 tatctgagtt gggcaaggcc atggagaggc agggattaga tatgaaaatt gcagcattgg 2820 taagttaata tatattaatt tcgtcaattt cttacttcca cactatatct atgctcagct 2880 ccgttatctg aggcattgat aatcatttgt atcctgatgc tgcttttggg gtattcacat 2940 ctgatgatgt gatgcaaaca ttgttgacat gatttaccac tgatgttagg ttaatgacac 3000 tgtcggcaca ttggctggtg ggaggtatgc ggataacagt gttgttgctg ctataatatt 3060 gggcactggt acaaatgcag catatgttga gaatgctaat gcaattccta aatggaccgg 3120 tttactgcct aggtccggaa atatggttag tgtgtgaata ccttatgctt gggattatca 3180 ttagttgtct atgtcttata gatgatcaat gccaacactc ttgaatgtta accctactta 3240 aaaaaatatg taaacgtgga attatttggt attctctagg taatcaacac agaatggggg 3300 agctttaaat cagataagct tcctctttca gaattcgata aagcaatgga ttttgaaagc 3360 ttgaatcctg gagagcaggt attctatccc cagtattttc ctctcctttt agttttagcc 3420 ttttaggtgg accgcattaa cttgtatgta ttttccaaca gatatatgaa aagttgattt 3480 ctggcatgta tcttggagag atagtgcgaa gaatcttgct gaagcttgct catgatgcag 3540 ctttgtttgg ggatgttgtt ccatctaagc tagagcaacc gtttgtacta aggtatattt 3600 ctgtaccatc tctcttttga tcttcattcc ttccttttgt catggcaaaa gccgttttac 3660 tttgttgtta actatcatgc tggtcaagtt ttttttttca cgatttctgt gactacccct 3720 tggataagag tgcacatgat ataaagacgt tggcatcgtt tccattataa tattcgcact 3780 gttgataata tacccagata gattatggaa ggggaagttg tttgtatatc ttgaaattat 3840 catgctaaat cagttagcaa atccatggaa ggtgacaaac atacactttg ttgttttatc 3900 cattactact ttcttctggt tctggttgat tctcataatt gtgtgcaact agaagttatc 3960 tagcttggat aattagggtg gtttggtttt tgctcaccag tttttcttta tgcttctaac 4020 ccctcaatct tatgcaactc tggtagaatt ttttataagt atctcaattg ttgtatcaca 4080 ggacaccgga tatgtcagcc atgcatcatg actcgtcaca tgaccttaaa actgttggag 4140 ctaagctaaa ggatatcgtc ggggtatgaa atttcttgac ccaaagtgga tacgcaacat 4200 ccaacactgt tcaatgagta ctggttgctt atttatgatt tgatctttta ttcaggtccc 4260 agatacttcc ctggaagtaa gatacattac cagtcacatt tgtgacatag ttgcagagcg 4320 tgctgcacgc ttggctgctg ctggcatata tggggtccta aagaagctag gtcgggacaa 4380 gatgccaaaa gacggcagta agatgcctag gactgtcatt gccttggatg gtgggctcta 4440 tgaacattac aagaagttca gcagttgctt agaatcaact ctaacagacc ttcttgggga 4500 tgatgtctcg tcttcggtgg ttaccaagct ggccaacgat ggttctggca ttggagctgc 4560 tcttctcgca gcctcgcact cccagtatgc cgagatcgac tagctttaag gatgatcttg 4620 atgaatgatg aatcaaactc cgtttgtagg ttctcatttc ccccttcaaa atccacataa 4680 tactcctggc tccccccttg aaatcttacc atcttttttt ggctattctg agggcaaaca 4740 taagtgcctc tgcagcggga tatagctagt atagcgccaa tgagtttgga ggttttctaa 4800 tggcataaaa cgttggatgg cagtagcaga ctaacaggga aatggaggca caggcaattt 4860 ccattcctgt tctgtcagat tcttttcccc cttaattgat gttgagaacc aagatttttt 4920 tgctctgtat tttctcttcg taataaagaa ggggacataa tctaattgct cttgtttgat 4980 ctcatatatg tatgtgactg cactctcact gtcccaaaat tttatagatg catttacaaa 5040 gttacaatac tcattacatg caaataactg aaggatggcg aatgtatttg tctggtatct 5100 acctgctctc ctaactagcg tgccatctct cctagatggt aaccattgga tcaggttata 5160 cttcttattg accattgttc tcatactatg ctgtccaagc tgccgttgcc gtcgtcgatg 5220 cctttggtgt ccgaaagctg cttccggcgg ctgccacccc tgatcggcag aaacggcgcg 5280 ccgtcgccgt cgtactccgc caccttcccg tccacggtgt tctggagcgc ctgctcgcac 5340 ctcaccagaa tctgcagaac gtccctcatt gtcggcctcg tcgccggctg cgcgccggtg 5400 cagatgatgc cgagcttgaa cacgacctcg gcgtcgtcgc cgtacccgga gtcggtgatg 5460 cacctgtcca ccgcgtcggc gatgctcctg cccgattgca ggtgccgcca cgcccactcc 5520 gccagcgagc cgtgctcgcc gccgtcgtgg gcttccctgc cggtgatcag ctcgaggagc 5580 accaccccga agctgtacac gtcgaccttc tcgttcacct tcctcgtgta cgcgcactct 5640 gca 5643 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OsHXK5 target site <400> 5 gcggggcatc tcggacgcca 20 <210> 6 <211> 104 <212> DNA <213> Artificial Sequence <220> <223> OsHXK5 sgRNA <400> 6 gcggggcatc tcggacgcca gttttagagc tatgctgaaa agcatagcaa gttaaaataa 60 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgc 104 <210> 7 <211> 3312 <212> DNA <213> Oryza sativa <400> 7 ggccaaagga gaaggagaag aagagaagag aagagaagag atggcagcga cctcatcatc 60 atcctccccc tgcatgattg cctcgctacg cagcagccat cgctgccgcc tttcgccctc 120 cgccaccgcc acgtcgccgc cccgcctccg cacgcttcct ccccgccgat gccgacgcaa 180 cccctcctcc tcctcctcct cctcccgcca agcggcggca atctccatgg ctcccgccaa 240 tcctcgccac cgcctcatcg ccccgatcca cgccgccgcc gccgccggag gaggaggagg 300 aggcgcgggc gccacctctc cctcgggcct acccagcgtg agtcgtcaat cagtcaaaat 360 gcacgcagcc tcgtcgcgat ttcgcatgac ctgccgatcg aactgacatg cgtgagctat 420 gtatggattt tatagctcgt cgggatcgcg cacctgctgg tgtcgctggg gatcgtgctg 480 gcctccgaca agttcctgaa gcaggcgttc gcggcggcgt ccatcaagtt ccccagcgcc 540 ctcttcggca tgttctgcgt cttctccgtc ctcgtcgtcc tcgacgcctt cgcgcccgcg 600 ctcgccaagg ggttcatgga cttcttcgag ccggccaccc tcttcatcca gaggtggctg 660 cccctcttct acgtcccttc cctggtcgtc ctgccgctcg ccgtcaggga cgtcccggct 720 gcttccggcc tcaagatctt cctcatcatt tgtatgcaca tcactacgct cctatcgttg 780 catgcattct gctacttctt tcttcctcct gatcatcgtg cttatctgct ccgcctctgc 840 ttcctccgga ttttacattc tactacttcg tatttgatat gctgatcaat taacttgcag 900 taaatattac cacaccaact tgcattgtgg attggcccca ttcataaatc acaacaattt 960 tacttagagg gtgtttagat tcaggggtgt aaagttttac cgtgtcacat cggatattat 1020 atagggtgtc gtaatagggt gtttggatac taataaaaaa aactaattac agaatccggc 1080 agtaaactgc gagacgaatt tattaagtct aattaatccg tcattagcaa atgtttactg 1140 taaaaccaca ttatcaaatc atagagcaat tagacttaaa agattcgtct cgcaaattag 1200 tggcaatctg tgcaattagt tattttttta gcctatattt aatacttcgt gcaagtgttc 1260 aaacgttcga tgtgacaggg tgtaaaattt tagggtggga tctaaacatg cccttggttt 1320 cagattaatt tgcattcaat ctgaaaaaac cgatggtgtg atgttatctg gttttcattt 1380 cccagttggt ggctggtttg cttcactcat ggtggccgga tacacagcac tcaccgtgag 1440 gaagattgtc cagacacagc ttataccagc tgagccgatg agcaagccgt ctccgttcgc 1500 gacgctggaa ttctgggcct ggggtgctgt ctttgtcgca tcgtttgctg ttgcgtatgt 1560 taaccccaca gcgcttggca ccacggcaag aacatgtctt cctttcctcc tcgcttcaac 1620 tgtactggga tacatggttg gttctgggta aatacatatg ctttttgtat acacttaatc 1680 tgtaagatct gacgaagcca ccagctgcct tgtttactcc atttctctaa caacatacat 1740 gggtcttatt gattacttgg caggatacca tctggtgtca agaaagtgct tcacccaatc 1800 atttgttgtg cactctctgc agatttggca gcaatagcat atgggtatct ctcccggtct 1860 ggagttgatg cagtgctagg ttgtaaacta aaccctgatc aatagtgctc taatcaagtt 1920 gtgaattctg aaataagttg gttttaattg ccaggtgatt acctcacaaa ggcgccatct 1980 aatcctggag ctggtgacgt cctgatggga tttcttggtt cagtcatcat atcgtttgca 2040 ttctcaatgt tcaagcagag aaaggtagtg cttgctgctg tttctgcaga attattctca 2100 gttagcatgc atggttatgg tgtgagatga ttcatatata tcctgaggct ctgtgtttgt 2160 tgttgctggt gtgtgttggc taatcaacta ataatgtttt gcggtgggta aaactcagct 2220 tgtgaagagg catgcagcgg aaattttcac atcaattgcc atcgcatcaa cattctcgtt 2280 atactcaact gccatcctag gacgggtgat agggctagag ccttcactga ccatatcaat 2340 attaccgagg tgcataaccg tggcgttggc tctgagcatc gtgtctttct ttgaaggtac 2400 ggacctagta catacttata ttgctggaaa tcaatttctg ttcttgctgg tcgatcagct 2460 agatatataa tatggagtac tcagctagct tattatatat ggtatggatg acttagtttg 2520 tgtgtcccgg gcatgcaggt gtaaattctt cgctaaccgc tgcggtcgtc gtcctcaccg 2580 ggctgatcgg cgcaaatttt gtgcaagcag ccatggacaa gcttggcctc aacgacccca 2640 tcgcgcgagg aatagggacg gcttccaggt acatatatac ctttgagttt tacttttgag 2700 atttgttccg gtccaacaaa aagtatttcg agataccgat acctcacggg ttcacagtat 2760 caaatcgttt ccgatcgtta gatctaacaa tgtccatcct acataactag atccaatggt 2820 cggaaacgat ttagatcaaa tgatcgaaaa tgatttggta taccacgaga tagcaaattt 2880 ctttactttt acctacctgc tggctgcttt ttttagtaag ttaagctgca tatatataca 2940 tacatacagc agtatgctct atgaataatg tttgcattta cgttgcagtg ctcatggact 3000 gggaacagca gcgctgtcgg ccaaggagcc tgaagcgctg cccttctgcg ccatcgctta 3060 cggcctgacg gggatcttcg gttcgctcat ttgctcggtt ccggccgtca ggcagagctt 3120 agtgttcata gctggctgac aacacgcacg gtacatcagt tttgtttttg tttgtttttt 3180 ctttttcact ttcttttggg tcgtcgtgcg tgtatagagc gatgctcgca agattgcaag 3240 aatatatgta tacagacaca ttttttgtga ttatgtctcc gttgtaataa gaagcaagag 3300 agtaattttg ta 3312 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OsPLGG1 target site <400> 8 cctcttctac gtcccttccc 20 <210> 9 <211> 104 <212> DNA <213> Artificial Sequence <220> <223> OsPLGG1 sgRNA <400> 9 cctcttctac gtcccttccc gttttagagc tatgctgaaa agcatagcaa gttaaaataa 60 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgc 104 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Os12Bglu38 target site 2 <400> 10 gacaccttcc tgatgcaacc 20 <210> 11 <211> 104 <212> DNA <213> Artificial Sequence <220> <223> Os12Bglu38 sgRNA <400> 11 gacaccttcc tgatgcaacc gttttagagc tatgctgaaa agcatagcaa gttaaaataa 60 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgc 104 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Os12Bglu38 target site 3 <400> 12 ggtctcgcat tttccccagt 20 <210> 13 <211> 104 <212> DNA <213> Artificial Sequence <220> <223> Os12Bglu38 sgRNA <400> 13 actggggaaa atgcgagacc gttttagagc tatgctgaaa agcatagcaa gttaaaataa 60 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgc 104 <210> 14 <211> 705 <212> DNA <213> Artificial Sequence <220> <223> mCherry cDNA <400> 14 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag 60 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc 120 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc 180 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac 240 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc 300 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac 360 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta 420 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc 480 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct 540 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc 600 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa 660 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtac 705 <110> University-Industry Cooperation Group of Kyung Hee University <120> Manufacturing method of non-functional mutant in first filial generation using CRISPR/Cas system <130> PO2009-042 <150> KR 10-2019-0168711 <151> 2019-12-17 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 3139 <212> DNA <213> Oryza sativa <400> 1 actctcaccg ccgctctctc ttcctctcca acatgaatat gccattgcta ctcctcatcg 60 ccatcgtcgt cgtctccctc tcccatggca acggggagca gaccgacctc acgcgggaga 120 cgttccccgc gggcttcgtc ttcggcaccg cgtcgtcggc gtaccaggtg gaggggaacg 180 ccctccagta tggccgaggg ccctgcatct gggacacctt cctgatgcaa cctggtgaga 240 tcgatgatac atgttcaggc aaaactagca atgtggtatc taaatataca gatcataaac 300 agtttgtcgc atttttcaaa taatggaatt aacaatgtgt tgggacgatc cacctaaaaa 360 gagaatatga caacaacaat ctaaaaagaa ttacatagcc tgatattgaa atgttgttgc 420 attacggcgc gttgcatttt gtacgtctct agtggatgta tgactggttg ttgatttggc 480 tctgattgtt cctttggata ggtgtaactc ctgataattc gaccgcgaat gtgaccgtcg 540 acgagtacca ccgctacatg gtcagtgaaa gtctcatgtg tttcagtgcc tgttctaatg 600 ggtttctgtt cctctgtatg actcttgatt tgtagcttat gtggctcctc tgatgatctt 660 ctttgcagga tgatgtggac aacatggtga gagtgggctt cgacgcgtat cgcttctcga 720 tctcctggtc tcgcattttc cccagtaagt taccggctga aacatggtca ggcacagtga 780 tgagatcatg ggatattcct ttcattgatg gtttttcttc tgcgtgttgt aggtggactt 840 gggaagatta acaaagacgg cgtggattat taccacaggc tcattgatta catgcttgct 900 aacagtacgt tggtcttggc atttcttcta gttcttgccc atttcggatt tcatgatctc 960 gaattaatag gttttttttt ggtttgattt gcacatcaga cattattcca tatgttgtgc 1020 tctaccacta cgaccttcca caggtgctcc atgatcaata caagggatgg ctacacccca 1080 gaattgtgta agtgttcgtg atcaaagcca tctgtttggc tgtaaaactt aaaattaaat 1140 accctttaaa ttgtttgaca aactacagaa gcacctctga agattttgtg ctaaattatg 1200 aaacccatag ctctctaact gcgaaatcct gacagtgaaa gttctgcagg tgtttaacca 1260 aaataggttt cactgtagca tatccgacat acacaaatat caaacccaga caatctgtat 1320 cctccctttc ttacgttcct tactcccttt ttcttgaatt aatgtgtgat tcatgtctgc 1380 gacttcattt tgcaggagag attttgtgag atttgcagac ttctgcttca agacatatgg 1440 tcataaggtg aagaactggt ttaccattaa cgaacctagg atgatggcaa atcatggcta 1500 cggtgacggc ttcttccccc ctggcagatg caccggctgc caacccggtg ggaattccgc 1560 caccgagcct tacatcgcag cccataacct tctcctttca catgccgctg ctgtcaggac 1620 atatcgtgac aagtaccagg ctagtgaatg atatccatgc ccctactatc caattcaaca 1680 tttcttaaaa tctccaatgt atcagctcat gttcatgata ccttgcgatt ttcaggctat 1740 tcagaagggg aagattggca tccttctcga ttttgtatgg tatgagccac tcaccgacaa 1800 agaagaggat cacgcagctg cacatagagc cagggagttt acccttggct ggtgatacat 1860 cttacactgt tcatcaatca ttgctcttac acggtgtccg catgaaggtt gaactgaact 1920 tccacttgaa acttttgcta ggtacctgca cccgattaca tatggtcatt acccagaaac 1980 tatgcagaat gctgttaagg aaaggctgcc caatttcaca cgtgagcagt ctgagatgat 2040 aaaaggatca gcggattata ttgcgatcaa ccattacaca acttattatg tcagtcacca 2100 cgtcaacaag acatccatca gctatctcaa tgattgggat gtgaaaattt catgtatgac 2160 actttcaaat cacacagtat tagaccagaa atatttgtac cttttcaatt ctttgccttc 2220 ctagctaaca cattttggtc ctctattcag atgagcgtaa cggtgtgcca attgggaaac 2280 aggtaagcaa caatttacaa gaaaggttga aaagaatggt agatcatgaa ctaaattctg 2340 gtttttaaac atctatgcag gcgtactcga actggcttta tgttgttcct tgggggatct 2400 acaaagctgt catgcatgtc aaggagaagt acaaggaccc cattataatc atcggagaaa 2460 atggtaaata aagataatac tggagaccct ttcaattttc ggacagtgaa cagtacaact 2520 ataaaagtgc tctgcagaat gaatctagga gcaaaatcta acaagaacct tttgcaggca 2580 ttgaccagcc aggcaatgag accctacctg gtgcactgta tgacttcttc aggatacaat 2640 attttgatca gtacctccat gagcttaaga gggcgatcaa ggatggtgca agggtcactg 2700 ggtattttgc ttggtccctg cttgacaact tcgagtggcg gctcgggttc acttcaaaat 2760 tcggaatcgt ctatgtagac cggagcactt tcacacggta ccctaaagac tcaacacgtt 2820 ggttcaggaa gatgataaaa agtgaggttt gagttggatt attatcactg ttggcagctg 2880 ctggagtgct tttttgttat gctagttttg gttgtatgaa taataagatg tcttgtacta 2940 gttgagaact tcttcagatt ttgtacttct agtattctac tttttgtact tcagattttg 3000 caagcaagga tgattttagt ttaatagatt caggacatct atatatactg gtgataagag 3060 aggcagattt caatttttgg cttgaattgc ccatatttgt gttttccaat gttttttaac 3120 ttcaaatttt aaactttgg 3139 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Os12Bglu38 target site <400> 2 tccatgatca atacaaggga 20 <210> 3 <211> 104 <212> DNA <213> Artificial Sequence <220> <223> Os12Bglu38 sgRNA <400> 3 tccatgatca atacaaggga gttttagagc tatgctgaaa agcatagcaa gttaaaataa 60 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgc 104 <210> 4 <211> 5643 <212> DNA <213> Oryza sativa <400> 4 accggttaaa acgcccaagt cagtggctct cgtcctcctt tctcctacgc ggccggagag 60 aggagggggg aagagaggat ctcgcagagc gccatagcct cggatccaga agcggaattc 120 ggtggaggtc tcgggcacct ggatcgatcg gaggaaggga aggcggagca gcggtgatgg 180 ggaaggcggc ggcggtgggg acggcggtgg tggtggccgc ggcggtcggg gtggcggtgg 240 tgctggcgcg gaggcggagg aggagggacc tggagctggt ggagggagcc gcggcggaga 300 ggaagaggaa ggtggcggcg gtgatcgagg acgtggagca cgcgctgtcg accccgacgg 360 cgctgctgcg gggcatctcg gacgccatgg tcaccgagat ggagcgaggc ctgcgcgggg 420 acagccacgc catggttaag atgctcatca cctacgtcga caacctcccc accgggtaag 480 cgcctcgatt ccgatctgat gtctgattcc tttcgttttt cgtattatgt ttcctttcgt 540 ttttagtatt atgggtttgg aactttgtga tctaaaaagt ttggaattgg gaaaggtctc 600 gtattgatgc gaatcgttcg ggaacaaatc tagtagtgac gcgcgtaacc gtgggaattt 660 atctccgtgc tctgattagg accgactgac cttttgatgg gatggtagga aatgtgcgaa 720 ttctttttat gatgtttgta cgaaaatcag attcccccga aaattgcgcc tagtggtgat 780 ccgaagagaa tggaactata atctcccatt tgatcttcat gtggttttgg tgtctcgtat 840 tcgtgaattg ggattttttt tgtacgttcc gatcgaagct agatcaatcc tagctttgcc 900 aaacaatata agccatttgt tataactgaa ataagttatg taaccacaag gaaacgcttg 960 ctctcggact ctgctgagga gagccaaggc aaattggctc ataccagtga ttgaaggaaa 1020 aatgtggagt cgtgggcatc tgaaattgct tctcatgcac caggacgcag gatttcggtg 1080 atgggggtac agagttacta aagcatcatg caatgagaaa aaaaatgaag tgctagtatg 1140 tgttattaac acaaacataa atcattgtta tagaaaaaaa tataaagtag ccaagagaaa 1200 atgataatac tcttgacaag gatgattttt gtgcttgaaa atgattgttt taatttagct 1260 gattgtgcat acacatacta tacatacatg ttatagatta tagatcacat ttgtttgcta 1320 aaacaatttg cggggtaccg gattacgaac tgtacccctc atgtccttgt gtaaacagga 1380 ttctacatag aactcaaaca agatagataa attggcctat ctaagctagg aaagataggt 1440 ctcccgtcaa catatcctat cgttctcaga tgatcttcgg ttttgtgatc tatagtagat 1500 cgtccatttg ttcctaattt ggctctttag gatttctgat tatgatatgt gattaattaa 1560 tttgatcgtc catttgttcc tactttggct ctttaggatt tctgattatg atatgtgatt 1620 aattaatttc ttgttggttt gatttgttaa ggcttcagtg acaggtaatt aactaatgta 1680 tggcagagag tttctttgca ttgtggtttc cttgttagca atctgaccaa tagatccat 1740 cagtcgagct tatctcatgg tatagttggt ctggtaataa gtttctctgt aacctccaca 1800 tcgttaagct aaatgtggca ggcacatcct gaaggaagtt tcttaaatat aatgattcat 1860 tcttattcta tcggatcctt ctaccagtct gttagaactc actcacccat aagcatacaa 1920 tgtttcctgc tgccttcttc caaaagtagt aatttcattc tttgatcata atgatttccc 1980 tttgcagaaa tgaacagggg ttgttttatg cattggatct tggaggaacc aacttccgcg 2040 tcctgcgagt ccaactcggt ggcaaggaga aacgtgtcgt ccaacaacag tacgaggaag 2100 tctcaattcc accacatctg atggttggaa cttccatggt aagcatttgc tgcttgattt 2160 taaatttctt ggagctgtac attccatgct tgtcttatgt attttctgac tattaatgca 2220 ggaactgttt gattttattg cttctgcatt gtcaaaattt gttgatactg aaggtgatga 2280 tttccacctc ccagaaggga gacagagaga gctgggcttc accttttcct tcccagtgag 2340 ccagacatca atatcgtcag gaacgctcat caagtggaca aagggtttct ccatcaatga 2400 cgcggtaaaa gtcaaaaata acttttggac ataccctgac tagaacacaa tcctgatcct 2460 tctttaaaca catgaattat gtcacaaggt tccttgataa agcttgattg aatatggcaa 2520 tggagtattt caattagcta tttgctacaa tctccctttg tgtacattgt aatcaactta 2580 actaggttag tgaaaagaaa tatcgacctt gatcaggaag catatttaat ttgaggatct 2640 ttaatagtac tcagtattat gtttgcctgg ttgatcatat ctgtgtgaac tgtcaagcat 2700 ccaataagaa taatttgatt ttgctatatt atctcctgaa ttaggttggc gaagatgttg 2760 tatctgagtt gggcaaggcc atggagaggc agggattaga tatgaaaatt gcagcattgg 2820 taagttaata tatattaatt tcgtcaattt cttacttcca cactatatct atgctcagct 2880 ccgttatctg aggcattgat aatcatttgt atcctgatgc tgcttttggg gtattcacat 2940 ctgatgatgt gatgcaaaca ttgttgacat gatttaccac tgatgttagg ttaatgacac 3000 tgtcggcaca ttggctggtg ggaggtatgc ggataacagt gttgttgctg ctataatatt 3060 gggcactggt acaaatgcag catatgttga gaatgctaat gcaattccta aatggaccgg 3120 tttactgcct aggtccggaa atatggttag tgtgtgaata ccttatgctt gggattatca 3180 ttagttgtct atgtcttata gatgatcaat gccaacactc ttgaatgtta accctactta 3240 aaaaaatatg taaacgtgga attatttggt attctctagg taatcaacac agaatggggg 3300 agctttaaat cagataagct tcctctttca gaattcgata aagcaatgga ttttgaaagc 3360 ttgaatcctg gagagcaggt attctatccc cagtattttc ctctcctttt agttttagcc 3420 ttttaggtgg accgcattaa cttgtatgta ttttccaaca gatatatgaa aagttgattt 3480 ctggcatgta tcttggagag atagtgcgaa gaatcttgct gaagcttgct catgatgcag 3540 ctttgtttgg ggatgttgtt ccatctaagc tagagcaacc gtttgtacta aggtatattt 3600 ctgtaccatc tctcttttga tcttcattcc ttccttttgt catggcaaaa gccgttttac 3660 tttgttgtta actatcatgc tggtcaagtt ttttttttca cgatttctgt gactacccct 3720 tggataagag tgcacatgat ataaagacgt tggcatcgtt tccattataa tattcgcact 3780 gttgataata tacccagata gattatggaa ggggaagttg tttgtatatc ttgaaattat 3840 catgctaaat cagttagcaa atccatggaa ggtgacaaac atacactttg ttgttttatc 3900 cattactact ttcttctggt tctggttgat tctcataatt gtgtgcaact agaagttatc 3960 tagcttggat aattagggtg gtttggtttt tgctcaccag tttttcttta tgcttctaac 4020 ccctcaatct tatgcaactc tggtagaatt ttttataagt atctcaattg ttgtatcaca 4080 ggacaccgga tatgtcagcc atgcatcatg actcgtcaca tgaccttaaa actgttggag 4140 ctaagctaaa ggatatcgtc ggggtatgaa atttcttgac ccaaagtgga tacgcaacat 4200 ccaacactgt tcaatgagta ctggttgctt atttatgatt tgatctttta ttcaggtccc 4260 agatacttcc ctggaagtaa gatacattac cagtcacatt tgtgacatag ttgcagagcg 4320 tgctgcacgc ttggctgctg ctggcatata tggggtccta aagaagctag gtcgggacaa 4380 gatgccaaaa gacggcagta agatgcctag gactgtcatt gccttggatg gtgggctcta 4440 tgaacattac aagaagttca gcagttgctt agaatcaact ctaacagacc ttcttgggga 4500 tgatgtctcg tcttcggtgg ttaccaagct ggccaacgat ggttctggca ttggagctgc 4560 tcttctcgca gcctcgcact cccagtatgc cgagatcgac tagctttaag gatgatcttg 4620 atgaatgatg aatcaaactc cgtttgtagg ttctcatttc ccccttcaaa atccacataa 4680 tactcctggc tccccccttg aaatcttacc atcttttttt ggctattctg agggcaaaca 4740 taagtgcctc tgcagcggga tatagctagt atagcgccaa tgagtttgga ggttttctaa 4800 tggcataaaa cgttggatgg cagtagcaga ctaacaggga aatggaggca caggcaattt 4860 ccattcctgt tctgtcagat tcttttcccc cttaattgat gttgagaacc aagatttttt 4920 tgctctgtat tttctcttcg taataaagaa ggggacataa tctaattgct cttgtttgat 4980 ctcatatatg tatgtgactg cactctcact gtcccaaaat tttatagatg catttacaaa 5040 gttacaatac tcattacat caaataactg aaggatggcg aatgtatttg tctggtatct 5100 acctgctctc ctaactagcg tgccatctct cctagatggt aaccattgga tcaggttata 5160 cttcttattg accattgttc tcatactatg ctgtccaagc tgccgttgcc gtcgtcgatg 5220 cctttggtgt ccgaaagctg cttccggcgg ctgccacccc tgatcggcag aaacggcgcg 5280 ccgtcgccgt cgtactccgc caccttcccg tccacggtgt tctggagcgc ctgctcgcac 5340 ctcaccagaa tctgcagaac gtccctcatt gtcggcctcg tcgccggctg cgcgccggtg 5400 cagatgatgc cgagcttgaa cacgacctcg gcgtcgtcgc cgtacccgga gtcggtgatg 5460 cacctgtcca ccgcgtcggc gatgctcctg cccgattgca ggtgccgcca cgcccactcc 5520 gccagcgagc cgtgctcgcc gccgtcgtgg gcttccctgc cggtgatcag ctcgaggagc 5580 accaccccga agctgtacac gtcgaccttc tcgttcacct tcctcgtgta cgcgcactct 5640 gca 5643 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OsHXK5 target site <400> 5 gcggggcatc tcggacgcca 20 <210> 6 <211> 104 <212> DNA <213> Artificial Sequence <220> <223> OsHXK5 sgRNA <400> 6 gcggggcatc tcggacgcca gttttagagc tatgctgaaa agcatagcaa gttaaaataa 60 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgc 104 <210> 7 <211> 3312 <212> DNA <213> Oryza sativa <400> 7 ggccaaagga gaaggagaag aagagaagag aagagaagag atggcagcga cctcatcatc 60 atcctccccc tgcatgattg cctcgctacg cagcagccat cgctgccgcc tttcgccctc 120 cgccaccgcc acgtcgccgc cccgcctccg cacgcttcct ccccgccgat gccgacgcaa 180 cccctcctcc tcctcctcct cctcccgcca agcggcggca atctccatgg ctccccgccaa 240 tcctcgccac cgcctcatcg ccccgatcca cgccgccgcc gccgccggag gaggaggagg 300 aggcgcgggc gccacctctc cctcgggcct acccagcgtg agtcgtcaat cagtcaaaat 360 gcaccgcagcc tcgtcgcgat ttcgcatgac ctgccgatcg aactgacatg cgtgagctat 420 gtatggattt tatagctcgt cgggatcgcg cacctgctgg tgtcgctggg gatcgtgctg 480 gcctccgaca agttcctgaa gcaggcgttc gcggcggcgt ccatcaagtt ccccagcgcc 540 ctcttcggca tgttctgcgt cttctccgtc ctcgtcgtcc tcgacgcctt cgcgcccgcg 600 ctcgccaagg ggttcatgga cttcttcgag ccggccaccc tcttcatcca gaggtggctg 660 cccctcttct acgtcccttc cctggtcgtc ctgccgctcg ccgtcaggga cgtcccggct 720 gcttccggcc tcaagatctt cctcatcatt tgtatgcaca tcactacgct cctatcgttg 780 catgcattct gctacttctt tcttcctcct gatcatcgtg cttatctgct ccgcctctgc 840 ttcctccgga ttttacattc tactacttcg tatttgatat gctgatcaat taacttgcag 900 taaatattac cacaccaact tgcattgtgg attggcccca ttcataaatc acaacaattt 960 tacttagagg gtgtttagat tcaggggtgt aaagttttac cgtgtcacat cggatattat 1020 atagggtgtc gtaatagggt gtttggatac taataaaaaa aactaattac agaatccggc 1080 agtaaactgc gagacgaatt tattaagtct aattaatccg tcattagcaa atgtttactg 1140 taaaaccaca ttatcaaatc atagagcaat tagacttaaa agattcgtct cgcaaattag 1200 tggcaatctg tgcaattagt tattttttta gcctatattt aatacttcgt gcaagtgttc 1260 aaacgttcga tgtgacaggg tgtaaaattt tagggtggga tctaaacatg cccttggttt 1320 cagattaatt tgcattcaat ctgaaaaaac cgatggtgtg atgttatctg gttttcattt 1380 cccagttggt ggctggtttg cttcactcat ggtggccgga tacacagcac tcaccgtgag 1440 gaagattgtc cagacacagc ttataccagc tgagccgatg agcaagccgt ctccgttcgc 1500 gacgctggaa ttctgggcct ggggtgctgt ctttgtcgca tcgtttgctg ttgcgtatgt 1560 taaccccaca gcgcttggca ccacggcaag aacatgtctt cctttcctcc tcgcttcaac 1620 tgtactggga tacatggttg gttctgggta aatacatatg ctttttgtat acacttaatc 1680 tgtaagatct gacgaagcca ccagctgcct tgtttactcc atttctctaa caacatacat 1740 gggtcttatt gattacttgg caggatacca tctggtgtca agaaagtgct tcacccaatc 1800 atttgttgtg cactctctgc agatttggca gcaatagcat atgggtatct ctcccggtct 1860 ggagttgatg cagtgctagg ttgtaaacta aaccctgatc aatagtgctc taatcaagtt 1920 gtgaattctg aaataagttg gttttaattg ccaggtgatt acctcacaaa ggcgccatct 1980 aatcctggag ctggtgacgt cctgatggga tttcttggtt cagtcatcat atcgtttgca 2040 ttctcaatgt tcaagcagag aaaggtagtg cttgctgctg tttctgcaga attattctca 2100 gttagcatgc atggttatgg tgtgagatga ttcatatata tcctgaggct ctgtgtttgt 2160 tgttgctggt gtgtgttggc taatcaacta ataatgtttt gcggtgggta aaactcagct 2220 tgtgaagagg catgcagcgg aaattttcac atcaattgcc atcgcatcaa cattctcgtt 2280 atactcaact gccatcctag gacgggtgat agggctagag ccttcactga ccatatcaat 2340 attaccgagg tgcataaccg tggcgttggc tctgagcatc gtgtctttct ttgaaggtac 2400 ggacctagta catacttata ttgctggaaa tcaatttctg ttcttgctgg tcgatcagct 2460 agatatataa tatggagtac tcagctagct tattatatat ggtatggatg acttagtttg 2520 tgtgtcccgg gcatgcaggt gtaaattctt cgctaaccgc tgcggtcgtc gtcctcaccg 2580 ggctgatcgg cgcaaatttt gtgcaagcag ccatggacaa gcttggcctc aacgacccca 2640 tcgcgcgagg aatagggacg gcttccaggt acatatatac ctttgagttt tacttttgag 2700 atttgttccg gtccaacaaa aagtatttcg agataccgat acctcagggg ttcacagtat 2760 caaatcgttt ccgatcgtta gatctaacaa tgtccatcct acataactag atccaatggt 2820 cggaaacgat tagatcaaa tgatcgaaaa tgatttggta taccacgaga tagcaaattt 2880 ctttactttt acctacctgc tggctgcttt ttttagtaag ttaagctgca tatatata 2940 tacatacagc agtatgctct atgaataatg tttgcattta cgttgcagtg ctcatggact 3000 gggaacagca gcgctgtcgg ccaaggagcc tgaagcgctg cccttctgcg ccatcgctta 3060 cggcctgacg gggatcttcg gttcgctcat ttgctcggtt ccggccgtca ggcagagctt 3120 agtgttcata gctggctgac aacacgcacg gtacatcagt tttgtttttg tttgtttttt 3180 ctttttcact ttcttttggg tcgtcgtgcg tgtatagagc gatgctcgca agattgcaag 3240 aatatatgta tacagacaca ttttttgtga ttatgtctcc gttgtaataa gaagcaagag 3300 agtaattttg ta 3312 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OsPLGG1 target site <400> 8 cctcttctac gtcccttccc 20 <210> 9 <211> 104 <212> DNA <213> Artificial Sequence <220> <223> OsPLGG1 sgRNA <400> 9 cctcttctac gtcccttccc gttttagagc tatgctgaaa agcatagcaa gttaaaataa 60 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgc 104 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Os12Bglu38 target site 2 <400> 10 gacaccttcc tgatgcaacc 20 <210> 11 <211> 104 <212> DNA <213> Artificial Sequence <220> <223> Os12Bglu38 sgRNA <400> 11 gacaccttcc tgatgcaacc gttttagagc tatgctgaaa agcatagcaa gttaaaataa 60 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgc 104 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Os12Bglu38 target site 3 <400> 12 ggtctcgcat tttccccagt 20 <210> 13 <211> 104 <212> DNA <213> Artificial Sequence <220> <223> Os12Bglu38 sgRNA <400> 13 actggggaaa atgcgagacc gttttagagc tatgctgaaa agcatagcaa gttaaaataa 60 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgc 104 <210> 14 <211> 705 <212> DNA <213> Artificial Sequence <220> <223> mCherry cDNA <400> 14 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag 60 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc 120 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc 180 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac 240 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc 300 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac 360 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta 420 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc 480 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct 540 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc 600 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa 660 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtac 705
Claims (19)
(b) 상기 형질전환 식물체와 야생형 식물체를 교배하는 단계를 포함하는 일대잡종(F1)에서 표적 유전자에 대한 동형접합 돌연변이체(homozygous mutant) 또는 이대립인자성 돌연변이체(biallelic mutant)의 제조 방법. (a) Transforming with a recombinant vector into which a polynucleotide encoding a sgRNA (single guide RNA) capable of recognizing a target gene and a polynucleotide encoding a Cas (CRISPR-associated nuclease) protein are inserted to prepare a transgenic plant step; and
(B) a method of producing a homozygous mutant or biallelic mutant for a target gene in a one-to-one hybrid (F1) comprising the step of crossing the transgenic plant and the wild-type plant.
상기 sgRNA를 코딩하는 폴리뉴클레오티드는 서열번호 3, 서열번호 6, 서열번호 9, 서열번호 11 및 서열번호 13의 염기서열로 이루어진 것으로부터 선택되는 어느 하나 이상인 것을 특징으로 하는 방법. The method of claim 1,
The polynucleotide encoding the sgRNA is any one or more selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13.
상기 표적 유전자는 웅성불임과 관련된 유전자인 것을 특징으로 하는 방법. The method of claim 1,
The method, characterized in that the target gene is a gene related to male infertility.
상기 표적 유전자는 Os12BGlu38 유전자, OsHXK5 유전자 및 OsPLGG1 유전자로 이루어진 군에서 선택되는 어느 하나 이상의 유전자인 것을 특징으로 하는 방법. The method of claim 1,
The target gene is any one or more genes selected from the group consisting of Os12BGlu38 gene, OsHXK5 gene and OsPLGG1 gene.
상기 Os12BGlu38 유전자는 서열번호 1의 염기서열로 이루어진 것이고, 상기 OsHXK5 유전자는 서열번호 4의 염기서열로 이루어진 것이고, 상기 OsPLGG1 유전자는 서열번호 7의 염기서열로 이루어진 것을 특징으로 하는 방법. 5. The method of claim 4,
The Os12BGlu38 gene consists of the nucleotide sequence of SEQ ID NO: 1, the OsHXK5 gene consists of the nucleotide sequence of SEQ ID NO: 4, and the OsPLGG1 gene consists of the nucleotide sequence of SEQ ID NO: 7. Method, characterized in that.
상기 (a) 단계의 형질전환 식물체는 동형접합 돌연변이체 또는 이대립인자성 돌연변이체인 것을 특징으로 하는 방법.The method of claim 1,
The transgenic plant of step (a) is a method characterized in that it is a homozygous mutant or a biallelic mutant.
(b) 상기 형질전환 식물체와 야생형 식물체를 교배하는 단계; 및
(c) 형광표지 단백질을 발현하는 식물체를 선별하는 단계를 포함하는 웅성불임 유지친(male sterile maintainer)의 제조 방법. (a) preparing a transgenic plant by transforming it with a recombinant vector into which a polynucleotide encoding an sgRNA capable of recognizing a target gene, a polynucleotide encoding a Cas protein, and a polynucleotide encoding a fluorescent marker protein are inserted;
(b) crossing the transgenic plant and the wild-type plant; and
(c) a method for producing a male sterile maintainer comprising the step of selecting a plant expressing the fluorescent marker protein.
상기 형광표지 단백질을 발현하는 식물체는 동형접합 돌연변이체 또는 이대립인자성 돌연변이체인 것을 특징으로 하는 방법.8. The method of claim 7,
The method, characterized in that the plant expressing the fluorescent marker protein is a homozygous mutant or a biallelic mutant.
(b) 상기 형질전환 식물체와 야생형 식물체를 교배하는 단계; 및
(c) 형광표지 단백질을 발현하지 않는 식물체를 선별하는 단계를 포함하는 Non-GMO(Non-Genetically Modified Organism) 식물체의 제조 방법. (a) preparing a transgenic plant by transforming it with a recombinant vector into which a polynucleotide encoding an sgRNA capable of recognizing a target gene, a polynucleotide encoding a Cas protein, and a polynucleotide encoding a fluorescent marker protein are inserted;
(b) crossing the transgenic plant and the wild-type plant; and
(c) A method for producing a Non-Genetically Modified Organism (Non-GMO) plant comprising the step of selecting a plant that does not express the fluorescent marker protein.
상기 형광표지 단백질을 발현하지 않는 식물체는 이형접합 돌연변이체인 것을 특징으로 하는 방법.10. The method of claim 9,
The method, characterized in that the plant that does not express the fluorescent marker protein is a heterozygous mutant.
상기 형광표지 단백질을 코딩하는 폴리뉴클레오티드는 서열번호 14의 염기서열로 이루어지는 것을 특징으로 하는 방법.10. The method of claim 9,
The method, characterized in that the polynucleotide encoding the fluorescent labeling protein consists of the nucleotide sequence of SEQ ID NO: 14.
상기 sgRNA를 코딩하는 폴리뉴클레오티드는 서열번호 3, 서열번호 6, 서열번호 9, 서열번호 11 및 서열번호 13의 염기서열로 이루어진 것에서 선택되는 어느 하나 이상인 것을 특징으로 하는 재조합 벡터. A recombinant vector comprising a polynucleotide encoding an sgRNA capable of recognizing a target gene, a polynucleotide encoding a Cas protein, and a polynucleotide encoding a fluorescent label protein,
The polynucleotide encoding the sgRNA is a recombinant vector, characterized in that at least one selected from the group consisting of the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13.
상기 sgRNA를 코딩하는 폴리뉴클레오티드는 서열번호 3, 서열번호 11 및 서열번호 13의 염기서열로 이루어진 것에서 선택되는 어느 하나 이상인 것을 특징으로 하는 재조합 벡터.13. The method of claim 12,
The polynucleotide encoding the sgRNA is a recombinant vector, characterized in that at least one selected from the group consisting of nucleotide sequences of SEQ ID NO: 3, SEQ ID NO: 11 and SEQ ID NO: 13.
상기 표적 유전자는 Os12BGlu38 유전자, OsHXK5 유전자 및 OsPLGG1 유전자로 이루어진 군에서 선택되는 어느 하나 이상의 유전자인 것을 특징으로 하는 재조합 벡터. 13. The method of claim 12,
The target gene is a recombinant vector, characterized in that any one or more genes selected from the group consisting of Os12BGlu38 gene, OsHXK5 gene and OsPLGG1 gene.
상기 형광표지 단백질은 녹색형광단백질(GFP), 황색형광단백질(YFP), 적색형광단백질(RFP) 및 시안형광단백질(CFP)로 이루어진 군에서 선택되는 것을 특징으로 하는 재조합 벡터. 13. The method of claim 12,
The fluorescent label protein is a recombinant vector, characterized in that selected from the group consisting of green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP) and cyan fluorescent protein (CFP).
상기 형광표지 단백질을 코딩하는 폴리뉴클레오티드는 서열번호 14의 염기서열로 이루어지는 것을 특징으로 하는 재조합 벡터.13. The method of claim 12,
The polynucleotide encoding the fluorescent label protein is a recombinant vector, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 14.
상기 식물체는 표적 유전자에 대한 동형접합 돌연변이체 또는 이대립인자성 돌연변이체인 것을 특징으로 하는 형질전환 식물체. 18. The method of claim 17,
The plant is a transgenic plant, characterized in that it is a homozygous mutant or a biallelic mutant for the target gene.
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