KR102574819B1 - Gene editing system for simultaneous gene editing of P34 and its homolog genes and uses thereof - Google Patents

Abstract

The present invention constructed a CRISPR/Cas system that simultaneously corrects the GmP34 gene ( Glyma08g116300 ) and its homolog Glyma08g116400 gene, which express allergens in soybean, and reduces allergens through gene editing-based technology using the same. Soybean transformants were prepared. The CRISPR/Cas-based soybean gene editing system or vector according to the present invention, and the soybean gene editing composition comprising the same, simultaneously express the P34 protein present in soybean and its homologous protein (88.6% homology). have an inhibitory effect.
The present invention can provide a new plant with reduced allergens by simultaneously correcting the GmP34 gene ( Glyma08g116300 ) and its homolog Glyma08g116400 gene in soybean plants, and by cultivating beans with reduced allergens for edible and The usability and value of soybeans for feed can be improved. This not only contributes greatly to enhancing the safety and usability of soybeans, which are important resources as food, but also can be used in various ways as raw materials for foods, feeds, and processed foods that use soybeans as raw materials, so the business potential and marketability are quite large.

Description

Gene editing system for simultaneous gene editing of P34 and its homolog genes and uses thereof {Gene editing system for simultaneous gene editing of P34 and its homolog genes and uses thereof}

The present invention relates to a gene editing system for simultaneously correcting the P34 gene and its homolog gene for developing a soybean transformant in which the P34 protein known as a major allergen in soybean has been removed. Specifically, a gene editing system that simultaneously corrects the P34 ( Glyma08g116300 ) gene and its homolog Glyma08g116400 gene in soybean, a composition for soybean gene correction comprising the same, a soybean gene correction method using the same, and a P34 protein, which is a cause of allergy, removed It relates to soybean transformants and seeds thereof.

Beans are rich in protein, fat, and dietary fiber, and contain various health functional substances such as isoflavones, anthocyanins, lecithin, lutein, and saponin. It is also a great crop.

However, soybean seeds contain allergen-inducing proteins, which can cause allergies when consuming soybean processed foods such as bean sprouts, tofu, and soymilk, and children under the age of 10 are particularly sensitive to soybean allergies. Since more than 65% of soy allergy is known to be caused by P34 protein, P34 protein is a representative anti-nutritional factor that needs to be removed to expand the availability of various foods using soy protein. For this reason, the development of soybeans with P34 protein removed has great economic value by improving the usability and value of soybeans for food and feed.

It can cause introduction, deletion or mutation of genes in various organisms such as bacteria, animals and plants including humans, and through this, it can be used as a research method to reveal the function of genes or as a tool for treatment of diseases and development of new plant varieties. Gene editing technology using the CRISPR/Cas system has been actively researched and used. Recently, gene editing research using the CRISPR/Cas system has been applied in various ways in plants.

The CRISPR/Cas system forms a complex between a Cas endonuclease protein and CRISPR RNA (crRNA) that recognizes a target gene sequence and a transactivating CRISPR RNA (tracrRNA) that binds to the Cas endonuclease while binding to crRNA. it did Here, crRNA and tracrRNA are mainly used in the form of single-stranded guide RNA (sgRNA) connected by a linker, and this single-stranded guide RNA (sgRNA) is the target gene to be cut by Cas endonuclease. Guided by double-stranded DNA sequencing. Cas endonuclease located at the target gene site recognizes a protospacer adjacent motif (PAM) adjacent to the target gene sequence, and then internal or external nucleotide sequences (base pair, base pair) of the target nucleic acid sequence. bp), DNA double strand breaks (DSB) occur (Jinek et al., 2012).

The target nucleic acid cleaved by the CRISPR/Cas system is subjected to homology directed repair (HDR) or non-homologous end joining (non-homology) during error-prone DSB repair. Repair occurs through the DNA repair mechanism of homologous end joining (NHEJ). Through the DNA repair mechanism of non-homologous end joining (NHEJ), insertion and deletion (indel) mutations of random bases between the cut DNA sites occur. As a result, a frameshift mutation or a premature mutation occurs in the coding region of the gene, resulting in knock-out of the target gene. On the other hand, the DNA repair mechanism of homologous recombination (HDR) requires donor DNA to recover the cut DNA. , gene editing is completed.

Gene editing is a technology that freely edits the genetic information of living organisms. It can change the genetic information of animals, plants, and microorganisms, including humans, and dramatically expand its application range. Currently, many researchers are promoting gene editing research, and are conducting research related to basic technologies such as securing original patents for gene editing, developing gene editing vectors, and establishing a gene editing internet platform. The CRISPR-based gene editing market is growing rapidly every year, and is expected to increase to about 20 times the current size within the next 10 years (Inkwood Research, 2019).

However, research on crop improvement using gene editing technology using the genetic scissors (CRISPR/Cas) system is in its infancy. In particular, soybean takes a long time to produce transformants compared to other crops and has relatively low efficiency, so transformation is difficult. In the case of difficult soybeans, the development of soybean breeding materials based on gene editing technology is limited to some laboratories.

Currently, various studies are being conducted to develop soybean transformants and seeds thereof with reduced phytic acid content in order to improve the usability and value of soybeans for food and feed and to preserve the agricultural environment.

However, despite continuous efforts and research to develop soybean transformants and seeds thereof with reduced allergens by introducing a genetic scissors (CRISPR/Cas) system, the results are insignificant. Accordingly, there is an urgent need for an optimal and effective genetic scissors (CRISPR/Cas) system for producing soybean transformants and seeds with reduced allergenic protein content.

KR 10-2015-0016588 A US 2020/0190494 A1 KR 10-2021-0039306 A KR 10-2018-0117785 A

Jinek, M. et al., A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity, Science, Vol. 337, 816-821 (2012) Inkwood Research, GLOBAL CRISPR MARKET FORECAST 2019-2027(2019) Jacobs, TB. et al., Targeted genome modifications in soybean with CRISPR/Cas9, BMC Biotechnology, Vol. 15, no. 16, p. 1-10 (2015)

The present invention is to solve the above needs and overcome the problems of the prior art, in order to develop a genetically modified soybean transformant in which the P34 protein, an allergen, is removed, the P34 ( Glyma08g116300 ) gene and its The purpose is to provide a CRISPR/Cas-based soybean gene editing system that simultaneously corrects the homolog Glyma08g116400 gene (88.6% homology).

Another object of the present invention is to provide a vector comprising the soybean genome editing system, a soybean genome editing composition, and a soybean genome editing method using the same.

In addition, the present invention is a soybean transformant in which the P34 ( Glyma08g116300 ) gene and its homolog Glyma08g116400 gene, which is an allergen in soybean seeds, have been transformed by the soybean gene editing system or the soybean genome editing composition. And it aims to provide its seeds.

Other objects of the present application and technical problems to be achieved by the present invention are not limited to the above-mentioned objects, and will become more clear by the following description of the invention together with the claims and drawings described in this specification. In addition, the technical problems to be achieved by the present invention, which are not described in the present specification, include those that can be clearly understood and inferred by those skilled in the art (hereinafter referred to as 'ordinary technicians').

In order to solve the above problems, the following invention is provided.

The present invention is a CRISPR/Cas-based method for simultaneously deleting the P34 ( Glyma08g116300 ) gene and its homolog, the Glyma08g116400 gene, in order to develop a transgenic soybean transformant in which the P34 protein, an allergen, is removed from soybean. Soybean gene editing system is provided.

Here, the CRISPR/Cas-based soybean gene editing system includes a Cas endonuclease; and gRNA1 and gRNA2 simultaneously targeting the soybean P34 ( Glyma08g116300 ) gene and its homolog Glyma08g116400 gene.

In one embodiment of the present invention, in the CRISPR/Cas-based soybean gene editing system, the nucleic acid sequences encoding the gRNA1 and gRNA2 may be the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2, respectively.

In another embodiment of the present invention, the Cas endonuclease is a plant codon-optimized Cas9 nucleic acid (pcoCas9), an Arabidopsis codon-optimized Cas9 nucleic acid (acoCas9) or a high GC content plant codon-optimized It may include Cas9 nucleic acid (pcohCas9), and may specifically include any one of the nucleotide sequences of SEQ ID NO: 5 to SEQ ID NO: 7.

In another embodiment of the present invention, the soybean gene editing system is a ribonucleoprotein (RNP), and the nucleic acid sequences encoding the gRNA1 and gRNA2 are the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2, respectively. may be

In addition, the present invention provides a codon-optimized Cas9 nucleic acid; and nucleic acids encoding gRNA1 and gRNA2 that simultaneously target the soybean P34 ( Glyma08g116300 ) gene and its homolog Glyma08g116400 gene; a vector for soybean genetic editing operatively linked thereto is provided.

In one embodiment of the present invention, the vector may further include one or more operably linked promoters, plant selectable markers, and one or more nuclear localization sequences (NLS).

In another embodiment of the present invention, the nucleic acid sequences encoding gRNA1 and gRNA2 in the vector may be the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2, respectively. In addition, the codon-optimized Cas9 nucleic acid includes plant codon-optimized Cas9 nucleic acid (pcoCas9), Arabidopsis codon-optimized Cas9 nucleic acid (acoCas9) or plant codon-optimized Cas9 nucleic acid with high GC content (pcohCas9). It may be, and may specifically include the nucleotide sequence of any one of SEQ ID NO: 5 to SEQ ID NO: 7.

In addition, the present invention provides a soybean genome editing composition comprising the CRISPR/Cas-based soybean genome editing system or the vector.

In one embodiment of the present invention, in the soybean gene editing composition, the nucleic acid sequences encoding gRNA1 and gRNA2 are the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and the soybean P34 ( Glyma08g116300 ) gene and It can be characterized by simultaneously correcting its homolog Glyma08g116400 gene.

In addition, the present invention includes the step of transforming soybean using the CRISPR/Cas-based soybean gene editing system or the vector, and simultaneously deleting the soybean P34 ( Glyma08g116300 ) gene and its homolog Glyma08g116400 gene. Characterized in that, it provides a soybean gene editing method.

In one embodiment of the present invention, the soybean gene editing method comprises the steps of correcting a target gene by introducing the CRISPR/Cas-based soybean gene correction system or the vector into soybean plant cells, and soybean plant cells in which the target gene is corrected It may include the step of regenerating soybean plants from.

The present invention also provides a soybean transformant in which the P34 ( Glyma08g116300 ) gene and its homolog Glyma08g116400 gene, which express an allergen in soybean, transformed by the soybean gene editing method have been corrected.

In addition, the present invention is a soybean transformant transformed by the soybean gene editing method, and the P34 ( Glyma08g116300 ) gene and its homolog Glyma08g116400 gene that expresses an allergen in soybean are corrected soybean seeds to provide.

The CRISPR/Cas-based soybean gene editing system or vector according to the present invention, and the soybean gene editing composition comprising the same, simultaneously express the P34 protein present in soybean and its homologous protein (88.6% homology). have an inhibitory effect. In addition, the soybean transformants and their seeds prepared using the soybean gene editing system according to the present invention are allergen-free by correcting the P34 ( Glyma08g116300 ) gene and its homolog Glyma08g116400 gene, which express allergens in soybean. It has the advantage of producing soybeans with reduced triggers.

Therefore, the present invention can provide a new plant with reduced anti-nutritional factors by correcting the target gene in soybean plants, and by cultivating soybeans with reduced allergens in soybean seeds, it will contribute to enhancing the usability and value of soybeans for food and feed. You will be able to.

1 is a diagram showing target sites in target genes of gRNA1 and gRNA2 capable of simultaneously targeting the GmP34 ( Glyma08g116300 ) gene and its homolog Glyma08g116400 gene according to the present invention. (A) shows the final selected target sequence and the chromosomal locations of gRNA1 and gRNA2 binding thereto, respectively. (B) is a target sequence (left) in Exon 1 and a target sequence in Exon 1 or intron among the target sequences of the GmP34 ( Glyma08g116300 ) gene and its homolog Glyma08g116400 gene ( right); and gRNA1 and gRNA2 binding thereto, respectively.
Figure 2 shows a schematic diagram of an exemplary construction of the pMDC123_35S _pro vector.
Figure 3 is an exemplary embodiment comprising selected gRNA1 and gRNA2 and codon-optimized Cas9 genes according to the present invention. A schematic diagram of a vector is shown.
Figure 4 is the result of verifying the transformants in which soybean transformants were prepared and genetically corrected using Agrobacterium. (A) shows the manufacturing process of the soybean transformant according to the present invention, and (B) is PCR amplification for the selectable marker and gRNA expression cassette of the vector used in the present invention for the soybean transformant according to the present invention. It shows the result of confirming transformation. (C) shows a soybean transformant according to the present invention.
Figure 5 shows the results of confirming whether the GmP34 ( Glyma08g116300 ) gene and its homolog Glyma08g116400 gene were corrected in the soybean transformant according to the present invention. (A) is a result of confirming whether the GmP34 ( Glyma08g116300 ) gene was deleted in the soybean transformant using InDel PCR, and (B) is a result confirming whether the homolog Glyma08g116400 gene was deleted.
Figure 6 is a result showing the target sequence region deletion of the GmP34 ( Glyma08g116300 ) gene and protein in the soybean transformant according to the present invention. (A) GmP34 ( Glyma08g116300 ) The target sequence region of the gene shows a 414 bp deletion (deletion), (B) is P34 It represents a 138 amino acid deletion or premature termination mutation in the target sequence region of the protein.
7 is a soybean transformant according to the present invention This is a result showing the deletion of the target sequence region of the GmP34 homolog Glyma08g116400 gene and protein in . (A) shows a 414 bp or 302 bp deletion of the target sequence region of the GmP34 homolog Glyma08g116400 gene, and (B) shows a P34 It represents a 43 amino acid deletion premature termination mutation in the target sequence region of the homologous protein.

The present invention uses the CRISPR/Cas9 (Clustered Regularly-Interspaced Short Palindromic Repeats/CRISPR associated protein 9) system to simultaneously indel the GmP34 ( Glyma08g116300 ) gene and its homolog Glyma08g116400 gene, which express allergens in soybeans. (Indel) mutation, and as a result, soybean transformants and seeds thereof in which expression of soybean P34 and homologous protein (88.6% homology) were simultaneously deleted were prepared. The soybean transformant and its seeds confirmed that the GmP34 gene ( Glyma08g116300 ) and its homolog Glyma08g116400 gene were all deleted through targeted deep sequencing, thereby completing the present invention.

Hereinafter, the present invention will be described in detail through explanation of terms, specific examples and examples. It should be noted that the above specific examples and embodiments include some embodiments of the invention and do not include all embodiments. It is obvious that the content of the invention disclosed by this specification is not limited to the specific embodiments described herein, and includes that a person skilled in the art can implement it in various ways in the technical field to which the present invention belongs. Therefore, it should be understood that the content of the invention disclosed in this specification is not limited to the specific embodiments described herein, and modifications and other embodiments thereof are also included within the scope of the claims.

Definitions of terms used in the present invention are as follows.

[Explanation of terms]

CRISPR/Cas system

As used herein, the term “CRISPR/Cas system” refers to a complex that includes a Cas endonuclease and a guide molecule corresponding to the nuclease, and is capable of binding to a target gene or target nucleic acid. it means. Here, the guide molecule may be a targeting nucleic acid represented by guide RNA (gRNA), but is not limited thereto.

The CRISPR/Cas system can be of various types depending on the Cas endonuclease and the guide RNA (gRNA), and the Cas endonuclease uses the corresponding guide molecule to target nucleic acid. Alternatively, it binds specifically to a target gene to perform gene editing such as DNA double-strand breakage.

Gene editing technology using the CRISPR/Cas system can introduce mutations into the genes of various entities such as humans, animals and plants, and through this, research to reveal the function of genes, production of transformants with new traits, and gene editing It is used as a treatment method for related diseases. The CRISPR/Cas system does not require the production of a custom protein to target a specific sequence, and various repertoires of the CRISPR/Cas system can be produced by changing a spacer sequence, which is a target site binding sequence in a guide molecule.

Cas endonuclease

As used herein, the term “Cas endonuclease” refers to DNA or RNA, which is a target nucleic acid, or a protospacer adjacent motif (PAM) in a target gene, and then converts the sequence of the target nucleic acid. Refers to a nucleolytic enzyme capable of editing DNA double strand breaks (DSB) at an internal or external base pair (bp) Cas endonuclease in this specification is CRISPR/ Refers to the effector proteins constituting the Cas system, wherein the effector protein is a CRISPR protein, a nucleolytic protein capable of binding to a guide RNA (gRNA), or an oligo capable of binding to a target nucleic acid or target gene. It may be a peptide fragment capable of binding to a nucleic acid. Specifically, it may be Cas9 or Cpf1, or a modified nucleolytic protein, but is not limited thereto. In addition, the meaning that those skilled in the art can recognize in the field of CRISPR / Cas technology All inclusive.

Cas9 protein

The "Cas9 protein" according to the present invention recognizes a protospacer adjacent motif (PAM) near the target site sequence in a target nucleic acid or target gene, and then converts the internal or external nucleotide sequence (base pair, bp bp) of the target nucleic acid sequence. ), DNA double strand breaks (DSB) and at least one selected from among all Cas9s capable of causing insertion and/or deletion (Indel) mutation in the target gene. Specifically, The Cas9 protein may be a Cas9 protein derived from the genus Streptococcus, the genus Campylobacter, the genus Neisseria, the genus Pasteurella, the genus Francisella, etc., and more Specifically, Cas9 proteins derived from Streptococcus thermophiles, Streptococcus aureus or Streptococcus pyogenes; Cas9 proteins derived from Campylobacter jejuni Cas9 protein derived from Neisseria meningitidis; Cas9 protein derived from Pasteurella multocida; Cas9 protein derived from Francisella novicida, etc., but are limited thereto. In addition, it includes all meanings that can be recognized by a person skilled in the art in the field of CRISPR/Cas technology The Cas9 protein or gene information can be obtained from a known database such as GenBank of the National Center for Biotechnology Information (NCBI) can

Guide RNA (guide RNA)

The term "guide RNA" according to the present invention is capable of forming a complex with Cas endonuclease, capable of hybridizing with a target nucleic acid sequence, and sequence-specific of the complex to the target nucleic acid sequence. RNA comprising a guide sequence having sufficient complementarity with a target nucleic acid sequence to allow for target binding. Guide molecules or guide RNAs are used interchangeably herein.

Guide RNA herein includes a CRISPR RNA (crRNA) that recognizes a target gene sequence and a transactivating CRISPR RNA (tracrRNA) that binds to the Cas endonuclease while binding to the crRNA. The guide RNA may be in a single-stranded form in which crRNA and tracrRNA are linked by a linker, or a double-stranded form in which crRNA and tracrRNA are complementaryly bonded. A guide RNA herein may be an RNA-based molecule having one or more chemical modifications, such as modifications by chemical linkage of two ribonucleotides or by replacement of one or more ribonucleotides with one or more deoxyribonucleotides. .

Since the specificity of guide RNA according to Cas endonuclease depends on the scaffold, which is a direct repeat sequence, the optimal guide RNA for Cas endonuclease RNA), the direct repeat sequence can be designed according to the origin of Cas. The guide RNA according to the present invention is a guide RNA found in nature or a modification of its length or sequence in order to specifically show the maximum activity for Cas endonuclease. may have In addition, it includes all meanings that can be recognized by a person skilled in the art in the field of CRISPR/Cas technology.

Scaffold area

The term "Scafold region" according to the present invention refers to a part of a guide RNA that can interact with a nucleolytic protein, excluding a spacer among parts of a guide RNA found in nature. You can refer to the rest. The scaffold region includes parts of tracrRNA and crRNA, and does not necessarily refer to one molecule of RNA.

spacer sequence

The term "spacer sequence" according to the present invention means a polynucleotide that hybridizes with a portion of a target sequence in the CRISPR/Cas system according to the present invention. The spacer sequence refers to about 20 consecutive nucleotides near the 3'-end of the crRNA of the guide RNA in the gene scissors system according to the present invention, and is also referred to as "gRNA" in the present invention. .

The "gRNA" according to the present invention, that is, the spacer sequence is designed to correspond to a target sequence of a target nucleic acid or target gene to be gene corrected using the gene scissors system. That is, the spacer may have various sequences depending on the target sequence of the target nucleic acid. In the present specification, the spacer sequence refers to a spacer sequence in the crRNA included in the gene scissors system, and is an important part for targeting a target site as a site that binds to a target sequence in a guide RNA. Unless otherwise specified, It is expressed as "gRNA" as a concept distinct from guide RNA. In addition, it includes all meanings that can be recognized by a person skilled in the art in the field of CRISPR/Cas technology.

CRISPR RNA (crRNA)

The term "CRISPR RNA (crRNA)" according to the present invention is an important component constituting the guide RNA in the CRISPR/Cas system according to the present invention. In the present specification, the crRNA includes a spacer sequence that recognizes a target gene sequence, and is part of a guide RNA that binds to a target gene or target nucleic acid.

transactivating CRISPR RNA (tracrRNA)

"tracrRNA" according to the present invention is an important component constituting the guide RNA of the CRISPR/Cas system together with crRNA. In the present specification, at least a portion of the tracrRNA is a guide RNA portion capable of complementary binding to at least a portion of the crRNA to form a double strand and binding to Cas endonuclease. More specifically, some sequences of the tracrRNA have a sequence complementary to all or part of the CRISPR RNA repeat sequence included in the crRNA.

Single guide RNA (sgRNA)

"Single guide RNA (sgRNA)" according to the present invention is a single-stranded RNA molecule in which the tracrRNA and the crRNA are connected by a linker to facilitate the formation of guide RNA. The guide RNA provided herein may be a single guide RNA, and in this case, the single guide RNA is one molecule of RNA including both the tracrRNA and the crRNA including the spacer sequence.

Target gene

The term "target gene" used in the present invention refers to a gene or nucleic acid in a cell that is a target of gene editing by the CRISPR/Cas system according to the present invention. Target nucleic acids or target genes may be used interchangeably and may refer to the same target. Unless otherwise specified, the target gene may be an intrinsic gene or nucleic acid of the target cell, a gene or nucleic acid derived from the outside, or an artificially synthesized nucleic acid or gene, single-stranded DNA, double-stranded DNA and/or RNA. can mean anything The target gene or target nucleic acid is not particularly limited as long as it can be a target of gene editing by the CRISPR/Cas system.

Target region or target sequence

"Target region" or "target sequence" according to the present invention is a sequence present in a target nucleic acid or target gene, and the CRISPR / Cas system according to the present invention cuts the target gene or target nucleic acid. refers to a specific sequence that recognizes The target site or target sequence may be appropriately selected depending on the purpose. Specifically, the target site or target sequence refers to a sequence complementary to a spacer sequence included in the single-stranded guide RNA (sgRNA) of the present invention. The spacer sequence is determined considering the sequence of the target gene or target nucleic acid and the PAM sequence recognized by the effector protein of the CRISPR/Cas system. The target site or target sequence may refer to only a specific strand that complementarily binds to the guide RNA of the gene scissors system, or may refer to the entire target double strand including the specific strand, which may be appropriately interpreted depending on the context. do. In addition, the term includes all meanings that can be recognized by those skilled in the art, and may be appropriately interpreted depending on the context.

homolog

The term "homolog" used in the present invention refers to the preservation of certain traits during the course of evolution, which may mean conservation of morphological traits, molecular traits, or gene base sequences. If evolution is a kind of remodeling process, it means that we can predict that there will be conserved characteristics through the process. Here, the preservation of homology does not mean an exact match, but means that something derived from a common ancestral trait remains without being lost, allowing an evolutionary relationship to be inferred.

In addition, based on the nucleotide sequence of a gene or the amino acid sequence of a protein, the homologue is more likely to be conserved as the species is evolutionarily closer, and the more essential elements are essential for the basic function of organisms, the more likely it is to be conserved. Homologues by reference include functional equivalents. Functional equivalent refers to both nucleotide and nucleic acid sequences of altered mutant sequences that have substantially the same or similar function, with one or more substitutions, deletions or additions from the reference sequence, but without various functional dissimilarities.

transformation

As used herein, the term “transformation” refers to a change in the genetic characteristics of a cell, that is, a phenomenon in which a genetic character is changed. It means that a cell is genetically modified from its natural state by introducing a new foreign DNA or RNA, and is also referred to as transformation, transformation or transformation. After transfection or transduction, the foreign new DNA or RNA can be recombined with that of the cell by being physiologically integrated into the chromosome of the cell, or it can be temporarily maintained as an episomal element without replication, or it can be independent as a plasmid. can be replicated as

Vector

"Vector" according to the present invention collectively refers to all materials capable of delivering genetic material into cells, unless otherwise specified. For example, a vector may be, but is not limited to, a DNA molecule comprising a genetic material of interest, such as a nucleic acid encoding an effector protein of the CRISPR/Cas system and/or a nucleic acid encoding a guide RNA. The term "vector" includes all meanings that can be recognized by a person skilled in the art, and may be appropriately interpreted depending on the context.

In addition, in the present invention, "vector" may be "expression vector" containing essential regulatory elements operably linked so that the inserted gene is normally expressed, and includes plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. can do. Specifically, Ti plasmids pGA3383, pCAMBIA3301, pCAMBIA3300, pGA3426, pGA3780, pGWB12, pGWB14, animal viruses such as retroviruses, adenoviruses or vaccinia viruses, insect viruses such as baculoviruses, or plant viruses may be used, pPZP, Binary vectors such as the pGA, pCAMBIA, pMDC123, pECO100, pECO200 and pECO300 families can be used.

In addition, since the purpose of the vector is to express each component of the CRISPR / Cas system in a cell, the sequence of the vector must necessarily include at least one of the nucleic acid sequences encoding each component of the CRISPR / Cas system . Specifically, the sequence of the vector includes a guide RNA included in the CRISPR/Cas system to be expressed, and/or a nucleic acid sequence encoding the Cas protein. In this case, the sequence of the vector may include a nucleic acid sequence encoding a wild-type guide RNA and a wild-type Cas protein, as well as a nucleic acid sequence encoding a codon-optimized Cas protein.

The vectors may also be plasmids, linear PCR amplicons or viral vectors. Here, the viral vector is a retrovirus vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus (AAV) vector, and a vaccinia virus vector. It may be at least one viral vector selected from the group consisting of a vaccinia virus vector, a poxvirus vector, and a herpes simplex virus vector.

In addition, the vector may be an expression vector, and may be designed in the form of a linear or circular vector. It is functionally linked to an expression control sequence so that the Cas9 gene can be expressed. For example, vectors include expression control elements such as promoters, operators, initiation codons, stop codons, polyadenylation signals, and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and can be prepared in various ways depending on the purpose. . In addition, vectors can include selectable markers, and vectors can replicate autonomously or integrate into host DNA.

The vector of the present invention can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cutting and ligation using enzymes generally known in the art.

promoter

As used herein, the term "promoter" means that an RNA transcription factor can be activated in a cell by being operably linked to a sequence encoding each component for expressing a target of a vector in a cell. The promoter sequence may be designed differently depending on the corresponding RNA transcription factor or expression environment, and is not limited as long as it can appropriately express the components of the CRISPR/Cas system in cells.

Since the vector of the present invention is applied to plant cells, the promoter of the present invention may include a promoter used for gene introduction into plants. For example, SP6 promoter, T7 promoter, T3 promoter, PM promoter, maize ubiquitin promoter (Ubi), cauliflower mosaic virus (CaMV) 35S promoter, nopaline synthase (nos) promoter, pigwort mosaic virus 35S promoter, Suga crane bacilliform virus promoter, Kommelina yellow mottle virus promoter, ribulose-1,5-bis-phosphate carboxylase small subunit (ssRUBISCO) photoinducible promoter, rice cytosol triosephosphate isomerase (TPI) promoter, Arabidopsis adenine phosphoribosyltransferase (APRT) promoter and octopine synthase promoter. The promoter of the present invention may be characterized in that it is specifically a constitutive promoter, and more specifically a 35S promoter.

In addition, the vector may further include a promoter for a nucleic acid encoding the engineered guide RNA. Specifically, the promoter may be AtU6 promoter, H1 promoter or 7SK promoter.

selection marker

The term "selectable marker" of the present invention refers to a nucleic acid sequence having characteristics that can be selected by chemical methods, and includes all genes capable of distinguishing transformed cells from non-transformed cells. The selectable marker may be included in the vector according to the present invention. Specifically, the selectable marker is a herbicide resistance gene such as glyphosate, glufosinate ammonium or phosphinothricin, kanamycin, G418, bleomycin, hygro There are antibiotic resistance genes such as hygromycin and chloramphenicol, but are not limited thereto. Herbicide resistance genes such as glyphosate, glufosinate ammonium or phosphinothricin, ampicillin, kanamycin, G418, bleomycin, hygro It may be an antibiotic resistance gene such as hygromycin or chloramphenicol, but is not limited thereto.

operably linked

As used herein, the term "operably linked" means that, in gene expression technology, a specific component is connected to another component so that the specific component can function in an intended manner. For example, when a promoter sequence is said to be operably linked to a coding sequence, it means that the promoter is linked to affect transcription and/or expression of the coding sequence in a cell. In addition, the term includes all meanings that can be recognized by those skilled in the art, and may be appropriately interpreted depending on the context.

Nuclear Localization Signal (NLS)

The term "nuclear localization signal (NLS)" according to the present invention refers to a schedule that acts as a kind of "tag" attached to a protein to be transported when a substance outside the cell nucleus is transported into the nucleus by nuclear transport. A peptide of any length or its sequence. NLS can be linked to Cas endonuclease for application of the CRISPR/Cas system according to the present invention in plant cells. Here, the NLS may be the NLS of the SV40 virus large T-antigen, the NLS from nucleoplasmin, or the c-myc NLS. Also hRNPA1 M9 NLS sequence; the NLS sequence of the IBB domain from importin-alpha, the NLS sequence of myoma T protein and the NLS sequence of human p53, the NLS sequence of mouse c-abl IV; NLS derived from the NLS sequence of influenza virus NS1, the NLS sequence of hepatitis virus delta antigen, the NLS sequence of mouse Mx1 protein, the NLS sequence of human poly(ADP-ribose) polymerase or the NLS sequence of steroid hormone receptor (human) glucocorticoid It may be a sequence, but is not limited thereto.

For example, the NLS sequence included in the vector according to the present invention is the NLS sequence of any one or more SV40 virus large T-antigens of SEQ ID NOs: 8 to 10 and/or any one or more nucleoplasmin of SEQ ID NOs: 11 to 12. It may be an NLS sequence from (nucleoplasmin).

approximately

As used herein, the term “about” refers to a reference amount, level, value, number, frequency, percentage, dimension, size, weight or length of 30, 25, 20, 15, 10, 9, 8, 7, 6, means an amount, level, value, number, frequency, percentage, dimension, size, weight or length that varies by 5, 4, 3, 2 or 1%.

All technical terms used in the present invention, unless defined otherwise, are used with the same meaning as commonly understood by one of ordinary skill in the art related to the present invention. In addition, although preferred methods or samples are described in this specification, those similar or equivalent thereto are also included in the scope of the present invention.

Hereinafter, the present invention will be described in detail.

[Materials and methods]

1. High-efficiency gRNA selection for gene editing of target genes

For all gRNAs to be used in the present invention, candidate gRNAs were selected using the gene editing web tool and used for soybean transformation.

First, a target gene to be subjected to gene editing is selected. Then, CRISPR-P2.0 ( http://crispr.hzau.edu.cn/ CRISPR2/ ), RGEN ( http://www.rgenome.net/ ), and Phytozome were used to design gRNAs for soybean gene editing. ( https://phytozome.jgi.doe.gov/pz/portal.html ) and other web sites and the 'off-target effect' of correcting off-target genes using the JBrowse tool in Phytozome gRNAs predicted to have low and high gene editing efficiency are selected.

CRISPR-P is a web site that can search for guide RNA with a target sequence, and candidate gRNAs are ranked according to efficiency, and the type of off-target and the location of off-target on the genome ( exon, intron, intergenic, promoter) can also be identified. However, in CRISPR-P2.0, there is a disadvantage of showing off target in a limited way.

RGEN selects the PAM sequence recognized by the CRISPR/Cas9 system among the nucleotide sequences of the target gene, and then generates a 20bp gRNA with a perfect match or 1bp mismatch or 2bp mismatch from the PAM. present it In addition, RGEN provides an out-of-frame score to predict the possibility of frame shift mutation, so if a high score is selected, there is a high possibility of base mutation occurring later. However, although RGEN provides a large number of off-targets, it is necessary to directly check the detailed information of off-targets because it provides only the location of each off-target on the genome.

Therefore, in the present invention, in order to design a high-efficiency gRNA that specifically binds to a target gene in soybean, CRISPR-P2.0 and RGEN tool are used together to compensate for the disadvantages of each tool, thereby providing a high-efficiency gRNA for soybean gene editing. can design In addition, by selecting a combination of two gRNAs specifically recognizing two target sites within one target gene, it was useful for selecting gene-edited lines using only InDel PCR without NGS when developing future generations.

In addition, the soybean genome is duplicated, and homologs with more than 90% homology exist. When designing a gRNA of a specific gene by avoiding highly homologous homologs, it is highly likely that a gRNA ranked below will be selected. In addition, even if the function of the target gene is lost through genome editing, the desired trait is unlikely to appear because a highly homologous homolog gene can replace the function of the target gene due to functional redundancy.

Therefore, considering the characteristics of soybean, first, a gRNA that simultaneously edits the target gene and homolog was designed, and second, two gRNAs were introduced simultaneously to edit at the two target sites simultaneously designed to fly. gRNAs that specifically recognize the two targets are useful for selecting gene-edited lines by InDel PCR alone without NGS when advancing future generations.

Specifically, the gene information of the target plant is searched in 'phytozome', and the genomic DNA sequence of the target gene is obtained. By searching for protein homologs of the target gene, nucleotide sequence information of genes with high similarity in the same plant is also obtained. This is to search for and avoid off-targets that may occur in future homology genes. At this time, depending on the homology between the target gene and the homolog, the gRNA is designed by selecting the base sequence part with high similarity between the two genes, or the case where the homolog is off-target while designing the gRNA by selecting the base sequence of the target gene is ignored. You can choose one of these strategies.

Next, after acquiring the genomic DNA sequence within 1000 bp considering the appropriateness of the gRNA, enter it into 'CRISPR-P' to search for the gRNA within the sequence. Among the gRNA lists found in 'CRISPR-P', the on-target score is high, the thymine (T) base does not repeat 4 or more, and the off-target location of the gRNA is in the exon of another gene. The candidate group with the fewest corresponding cases is selected. After selecting candidate gRNAs, the distance between 2 gRNAs to be applied to the target gene can only be up to 300 bp due to the technical limit of sequencing when identifying indels by targeted deep sequencing after obtaining transformants. Select pairs located within 250 bp. The gRNA sequence that satisfies the above conditions is searched for off-targets that may occur due to the gRNA once more using Cas-OFFinder of 'RGEN'.

'CRISPR-P' is easy to select highly efficient gRNAs due to on-score information, but off-target information is limited to a maximum of 20, so it is cross-validated once more in 'RGEN' to achieve high efficiency for soybean gene editing. of gRNAs are selected. Using the off-target information located on the genome searched in 'RGEN', search where the off-target occurs in the gRNA selected in 'JBrowse'. 'JBrowse' shows the entire genome information of chromosome n, so it is possible to determine whether the site where off-target occurs is an exon or another part, and through this, the appropriateness of the gRNA can be confirmed. By repeating the above process, a gRNA with a high on-target score and the lowest off-target was finally selected and verified.

2. Preparation of CRISPR/Cas9 vectors containing selected high-efficiency gRNAs

Depending on the purpose, a CRISPR/Cas9 vector can be constructed by final selection of gRNAs targeting a target gene. In the present invention, pMDC123_pcoCas9_g1g2 (plant codon-optimized Cas9), pMDC123_acoCas9_g1g2 (Arabidopsis codon-optimized Cas9), pMDC123_pcohCas9_g1g2 (plant codon-optimized Cas9 with high GC content) and pECO201 vector distributed by Professor Kim Sang-kyu of KAIST were used for soybean gene editing. . These vectors have the Bar gene as a selection marker to select transformed plants, and are gateway vectors having attR1 and attR2 sequences for recombination.

The pECO201 vector is a vector modified by Golden gate assembly of a pGRNA vector and a plant binary vector (Oh Y. et al., Plant Methods, Vol. 16, No.37 (2020)), Arabidopsis codon optimized SpCas9 and 35S promoter. In pMDC123_Cas9_g1g2, the bar gene is controlled by the enhanced 35S CaMV promoter, whereas in pECO201, it is controlled by the nos promoter (nopalin synthase promoter) derived from Agrobacterium .

Since the pMDC123 vector does not have a promoter controlling the expression of the target gene, the pMDC123_35S _pro vector was constructed by excising the 2X 35S CaMV promoter from the pMDC32 vector and linking it to the HindIII/Asc I site of the pMDC123 vector. The constructed pMDC123_35S _pro vector It was used for the final Multisite Gateway recombination. Figure 2 shows a schematic diagram of the construction of the pMDC123_35S _pro vector.

To construct a vector having the selected gRNA1 and gRNA2 and the codon-optimized Cas9 gene (codon-optimized SpCas9), two Golden Gate assembly and one Multisite Gateway recombination were performed. First, the gRNA is operatively linked to the AtU6 promoter. After digesting pYPQ131A and pYPQ132A vectors, which are golden gate entry vectors having an AtU6 promoter, with restriction enzyme ESP3I (BsmBI) , gRNA1 and gRNA2 subjected to oligo annealing were ligated to pYPQ131A and pYPQ132A vectors, respectively, by the Golden Gate assembly method. Next, gRNA1 (pYPQ131A_gRNA1) and gRNA2 (pYPQ132A_gRNA2) linked to each AtU6 promoter were ligated to pYPQ142 vector (Golden Gate recipient and gateway entry vector) by Golden Gate assembly method using restriction enzyme BsaI . Then, each Cas9 gene and a gateway entry vector having a nos terminator (pYPQ150 (plant codon optimized), pYPQ154 (Arabidopsis codon optimized), pYPQ167 (plant codon optimized: high GC content at 5' region)) and in the second process Using the pYPQ 142 vector having both gRNA1 and gRNA2 created and pMDC123_35S _pro , a vector for plant expression, the multisite gateway assembly method is used for final linkage. The pYPQ131A, pYPQ132A, pYPQ142, pYPQ150, pYPQ154, and pYPQ167 plasmid DNAs used in this experiment can be purchased from Addgene.

After preparing and annealing sense and antisense primers for the nucleotide sequences of the gRNAs finally selected in the present invention, they are operably cloned behind the AtU6 promoter (Arabidopsis U6 promter) in a plant expression vector. The expression cassettes of each gRNA are cloned into one vector using the Golden gateway method. Finally, a multiplex CRISPR/Cas9 vector for plant transformation with a selectable marker can be constructed by recombining the SpCas9 gene derived from Streptococcus pyogenes , the gRNA expression cassette, and the T-DNA vector having the 35S promoter and ccdB recombination system.

In this case, in the multiplex CRISPR/Cas9 vector, the SpCas9 gene may be a codon-optimized nucleic acid, the selectable marker gene may be a Bar gene, and the vector may include one or more NLS sequences. A multiplex vector containing two gRNAs per one target gene was prepared to facilitate the selection of transformants for gene editing (GE) using Indel PCR.

3. Soybean transformation and selection of transformants

The binary vector for plant transformation to be used for soybean transformation was transformed into Agrobacterium tumefaciens strain EHA105 by electroporation. One colony with confirmed transformation was cultured at 28 ^° C for two days in LB liquid medium (LB, Ripampicin 25 mg/L, kanamycin 50 mg/L or spetinomycin 50 mg/L) supplemented with antibiotics, and then glycerol ( final 30%) was added and stored at -70 ^o C. Two days before transformation, a part of the cell stock stored at -70 ^° C was inoculated into LB liquid medium supplemented with antibiotics and cultured at 28 ^° C for 30 hours.

400ul of well-grown Agrobacterium cells were mixed with antibiotics in AB solid medium (glucose 0.5g per 100㎖, agar 1.5g/90ml water, 5㎖ 20x AB buffer (K2HPO4 60g/L, NaH2PO4 20g/L), 5㎖ 20x Stock of AB salts (NH4Cl 20g/L, MgSO4.7H2O 6g/L, KCl 3g/L, CaCl 0.2g/L, FeSO4.7H2O 50mg/L), Ripampicin 25mg/L, kanamycin 50mg/L) and cultured. On the day of transformation, Agrobacterium cells that were plated on AB solid medium and grown well were cultured in LCM medium (liquid CCM medium, B5 medium 3.2g per liter, Sucrose 30g, Sodium thiosulfate 158mg, DTT 154mg, L-cysteine 580mg, MES 4.26g, pH 5.4 with 1M KOH) to scrape from the surface of AB medium. Some of them were diluted to an OD ₆₀₀ of 0.6 (low concentration), some were diluted to an OD ₆₀₀ of 2 (high concentration), and acetosyringon (final 200uM) was added and cultured at 28 ^° C until Agrobacterium infection.

In addition, transformation of plants with the vector in the present invention may be performed by a transformation technique known to those skilled in the art. Specifically, in addition to transformation methods using Agrobacterium, microprojectile bombardment, electroporation, PEG-mediated fusion, microinjection, and liposome-mediated methods (liposome-mediated method), in-planta transformation, vacuum infiltration method, floral meristem dipping method, and Agrobacterium spraying method It can be used, and more specifically, a transformation method using Agrobacterium or an Agrobacterium spraying method can be used.

Next, after washing the soybean seeds in running tap water for 30 minutes, seeds with normal seed coats are selected. Prepared soybean seeds were sterilized in 70% ethanol for 30 seconds and washed three times with sterile water. Then, sterilize in 1% sodium hypochlorite solution while shaking for 30 minutes, and wash 5 times at 5 minute intervals. Soybean seeds with excess water removed are germinated with the navel side down (Germination medium (GM), B5 medium 3.2g per liter, Sucrose 20g, MES 0.5g, agar (sigma) 8g, pH5.7 with 1M KOH) After sowing, germinate in a dark condition for 20 hours in a 26 ^o C incubator.

Soybean seeds germinated in GM medium for 20 hours were split in half along the length of the seed using a scalpel (#11 blade). For transformation, cothyledon with an embryonic axis was selected, and the embryonic axis tip and axial bud were removed. Apply high concentration (OD ₆₀₀ = 2) Agrobacterium solution prepared in advance to the tip of a knife and injure the area where the axial bud was removed 6-8 times. Injured half seeds were immersed in a low-concentration Agrobacterium solution (OD ₆₀₀ =0.6), treated with sonication for 20 seconds and vacuum for 3 minutes, and then inoculated for 30 minutes. The explants from which the Agrobacterium solution was removed are placed on CCM medium with sterile filter paper on top (7 explants) and co-cultured (23 ^o C, 16h light, 8h dark) for 4 days.

The explants co-cultured for 4 days were washed three times for 5 minutes each with sterile water (250mg/L cefotaxim, 100mg/L Timetin, 50mg/L vancomycin) to which antibiotics were added to remove Agrobacterium , and the same antibiotics were added in order ( shoot) Induction medium (SIMIP0 medium, B5 medium 3.2g per liter, Sucrose 30g, Sodium thiosulfate 158mg, DTT 154mg, MES 0.6g agar (sigma) 8g, pH5.7 with 1M KOH) washed three times for 15 minutes did After washing, excess water was removed from the explants, and the hypocotyl portion was placed at an angle of about 45 degrees in SIMIP0 medium without the addition of antibiotics. After 2 weeks, in families preparing half seeds, large shoots caused by axial buds not being removed properly are completely removed, and antibiotics are added to the remaining small shoots by removing only the upper part of the shoots to facilitate selection of antibiotics. were subcultured in SIMP10 medium.

After 2 weeks, the browned shoots by PPT were removed using scissors, and the large callus mass formed on the hypocotyl part was trimmed and placed in SEMP5 medium (shoot elogtion medium_PPT5, MS B5 medium 4.4 g per 1 liter, Sucrose 30 g, Sodium thiosulfate 158 mg, DTT 154 ㎎, MES 0.6g, Asparagin 50mg, Pyroglutamic acid 100mg, agar(sigma) 8g, pH5.7 with 1M KOH, Zeatin-ribose 1mgL, GA3 1mg, IAA 0.1mg, Cefotaxim 125mg, Timetin 50mg, Vancomycin 50mg). .

When subcultured to the same SEMP5 medium every 2 weeks and the stem elongated more than 4 cm, the stem part of the soybean transformant was cut and antibiotic-free root induction medium (RIMP0, MS B5 medium 4.4 g per 1 liter, Sucrose 30g, Sodium thiosulfate 158mg, DTT 154mg, MES 0.6g, Asparagin 50mg, Pyroglutamic acid 100mg, agar(sigma) 8g, pH5.7 with 1M KOH, IBA 1mg, Cefotaxim 50mg, Timetin 50mg, Vancomycin 25mg) induced. Transformants that developed roots on the cut surface of the stem were transferred to small pots, acclimatized in a plastic container for 2 weeks, and then transferred to large pots and grown in a greenhouse.

After purifying the regenerated soybean plants, they were transplanted and grown in pots. In order to confirm the T-DNA introduction of the regenerated plants, genomic DNA was extracted for each individual and introduced through genomic PCR. Introduction of the selected marker gene and gRNA expression cassette check. In addition, the expression of the selection marker gene ( Bar ) was confirmed in the regenerated plants using Bar-strip.

In addition, it was confirmed whether the deletion occurred through InDel PCR using the genomic DNA extracted from the T ₀ plant in which T-DNA introduction was confirmed as a template. Afterwards, the location and pattern of deletion and genome-editing efficiency of each transformed line were confirmed through targeted deep sequencing.

[Example]

Hereinafter, the present invention will be described in more detail through examples. The examples of the present invention are only for exemplifying the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. High-efficiency gRNA selection

Since P34 is an allergen in soybean, the GmP34 gene was selected as a target gene to be corrected using the CRISPR/Cas9 system in order to prepare a soybean transformant in which the allergen was removed through gene editing.

As a result of searching the soybean genome database, two genes with high homology as genes encoding the P34 protein, the GmP34 ( Glyma08g116300 ) gene and its homolog, the Glyma08g116400 gene (sequence homology of 88.6% at the amino acid level) ) was confirmed to exist, and it was decided to simultaneously delete these two genes.

Accordingly, it was attempted to select gRNAs capable of simultaneously targeting the GmP34 ( Glyma08g116300 ) gene and its homolog, the Glyma08g116400 gene. Here, "gRNA" is a different concept from guide RNA, and refers to a spacer sequence corresponding to about 20 nucleotides (20 nt) that binds to a target sequence in a target nucleic acid or gene in guide RNA. do.

First, in order to select a highly efficient combination of gRNA1 and gRNA2 for the soybean gene editing CRISPR/Cas9 system, the nucleotide sequence information of the target gene for gene editing was obtained from the Phytozome database. 'CRISPR-P' or 'CRISPR RGEN' GmP34 ( Glyma08g116300 ) gene and its homolog predicted to have low off-target effect and high gene editing efficiency by using gene editing web tools such as 'Tools' A target sequence within the Glyma08g116400 gene was selected, and a highly efficient combination of gRNA1 and gRNA2 binding to the target sequence was selected. To this end, the target sequence in the target gene was identified based on the nucleotide sequence of the Willimas 82 cultivar used as a transformation model.

At this time, since RGEN presents more off-target gene candidates than CRISPR-P, the GmP34 ( Glyma08g116300 ) gene and its homologues (which are less likely to be off-target) using both programs A target sequence within the homolog) Glyma08g116400 gene was selected.

In addition, when the off-target gene candidates are finally selected, the gRNA binding position in the off-target gene is finally confirmed using the Jbrowse site, resulting in high gene editing efficiency and off-target gene candidates. - A target sequence within a target gene with low off-target potential was selected.

As a result, two gRNA combinations capable of simultaneously targeting two highly homologous genes, the GmP34 ( Glyma08g116300 ) gene and its homolog, the Glyma08g116400 gene, as genes encoding the P34 protein, were finally selected. The target sequences targeted by the finally selected two gRNAs and their chromosomal locations are shown in FIG. 1 . In addition, in the target sequence (target sequence), the front target sequence (Target 1) in exon (Exon) 1; and the target sequence (Target 2) of the rear (in the case of Glyma08g116300 ) or intron region (in the case of Glyma08g116400 ); respectively, the gRNAs that bind to were named gRNA1 and gRNA2, respectively, and the nucleic acid sequences encoding gRNA1 and gRNA2, respectively, are shown in FIG. shown

In addition, nucleic acid sequences encoding gRNA1 and gRNA2 are shown in Table 1 below, respectively.

Name Sequence (5'→3') sequence number gRNA1 5′-ACTAGAAGAGAGACCTAAGA-3′ One gRNA2 5′-GCCCCCTTGGTACTTTACTT-3′ 2

In addition, in the case of the target sequence (Target 2), the GmP34 ( Glyma08g116300 ) gene and its homolog Glyma08g116400 gene differed by one base from each other, but the gRNA2 binding thereto was the GmP34 gene ( Glyma08g116300 ) It was selected to match the target sequence of and designed to bind to both genes.

Example 2. Codon-optimized CRISPR/Cas9 vector construction

Next, the present invention relates to the GmP34 ( Glyma08g116300 ) gene and its homolog Glyma08g116400 gene, which are target genes of soybean genome editing, and gRNA1 and gRNA1 that bind to two target sequences selected for each target gene, respectively. It was decided to prepare a multiplex vector in which the nucleic acid encoding gRNA2 was introduced into one vector.

This is by cleaving two target sequence sites located in a wide range within one target gene, causing a clear indel mutation of the target gene, securing a transformant, and then using Indel PCR. The purpose is to manufacture a system having the advantage of being able to more efficiently select transformants undergoing gene correction.

A multiplex vector containing gRNA1 and gRNA2 specifically binding to the two target sites selected above and codon-optimized Cas9 gene in soybean was prepared. At this time, when using the AtU6 promoter for the purpose of gRNA expression, since the nucleic acid encoding gRNA is more efficient to start with 'G', SEQ ID NO: 1 (5'-ACTAGAAGAGAGACCTAAGA-3), which are nucleic acids encoding gRNA1 and gRNA2, respectively ') and SEQ ID NO: 2 (5'-GCCCCCTTGGTACTTTACTT-3') by adding a base 'G' in front of the 5'-terminus, which was used when preparing the multiplex vector. That is, gRNA1 contained in a vector having an AtU6 promoter. and nucleic acids encoding gRNA2 include SEQ ID NO: 3 (5'- G ACTAGAAGAGAGACCTAAGA-3') and SEQ ID NO: 4 (5'- G GCCCCCTTGGTACTTTACTT-3'), respectively.

The basic vector used for soybean transformation is the pMDC123 vector or the pECO201 vector. These vectors have the Bar gene as a selection marker and are gateway vectors having attR1 and attR2 sequences for recombination.

Nucleic acids encoding gRNA1 and gRNA2 binding to the two target sequences selected according to the present invention were cloned into the pECO201 vector to prepare a multiplex vector. The pECO201 vector is a golden gateway entry vector for simultaneous introduction of two or more gRNAs, has a Bar gene as a selection marker, and is designed to express Arabidopsis codon optimized SpCas9 under the control of the 35S promoter. It is done. Two gRNAs were combined into the pECO201 vector by two steps. After the first oligo annealing process, gRNA1 and gRNA2 were ligated with pECO400_1,2 and pECO404_2,e cut with Bsa I, respectively, to obtain pGRNA1 and pGRNA2e plasmids. Second, the pECO201_g1_g2 vector was finally constructed through golden gateway assembly of pGRNA1 and pGRNA2e into which each gRNA was introduced and the pECO201 vector.

Exemplary nucleic acids encoding the selected gRNA1 and gRNA2 according to the present invention and the codon-optimized Cas9 gene, respectively. A schematic diagram of the vector is shown in FIG. 3 .

Example 3. Verification of gene editing of soybean transformants

After transformation of the CRISPR/Cas9 vector for soybean gene editing containing the nucleic acid codon-optimized Cas9 genes encoding the selected gRNA1 and gRNA2, respectively, prepared in Example 2 into Agrobacterium EHA105, Williams 82 cultivar Transformation was performed by co-culture with soybean explants. After acclimating the regenerated soybean plants, they were transplanted and grown in pots. In order to confirm the introduction of T-DNA in the redifferentiated plants, genomic DNA was extracted for each individual, and introduction of the introduced selectable marker gene and gRNA expression cassette was confirmed through genomic PCR. In addition, the expression of the selection marker gene ( Bar gene ) was confirmed in the regenerated plants using Bar-strip.

As a result, as shown in FIG. 4, 16 T ₀ lines expressing gRNA1 and gRNA2 simultaneously targeting the GmP34 ( Glyma08g116300 ) gene and its homolog Glyma08g116400 gene were secured (FIG. 4C). In order to confirm the T-DNA introduction of the obtained transformant T ₀ lineage plants, extract genomic DNA for each individual and perform genomic PCR to confirm the introduction of the introduced selection marker gene ( Bar gene) and gRNA expression cassette (Fig. 4B).

In addition, transformants in which the base sequence between the two gRNA1 and gRNA2 targets was deleted were identified through InDel PCR using plants that showed a positive reaction in PCR for the Bar resistance gene. Deletion of the target sequence site in the target gene was confirmed in both the GmP34 ( Glyma08g116300 ) gene and its homolog Glyma08g116400 gene (FIG. 5).

The obtained soybean transformant had a 414 bp deletion or 302 bp deletion of the target sequence region in the target gene in the GmP34 ( Glyma08g116300 ) gene and its homolog Glyma08g116400 gene, respectively. It was confirmed through targeted deep sequencing that the soybean transformant according to the present invention expressed early termination mutants, 138 amino acid deletion mutants or 43 amino acid deletion mutants of GmP34 protein and GmP34 homologue protein. These results are shown in Figures 6 and 7.

This means that the soybean transformant prepared in the present invention cannot express wild-type or normal GmP34 protein and GmP34 homologue protein.

Through the above results, using the CRISPR/Cas-based soybean gene editing system according to the present invention, the present invention efficiently targets the GmP34 ( Glyma08g116300 ) gene and its homolog Glyma08g116400 gene at the same time. In addition, the present invention prepared soybean transformants in which the soybean GmP34 ( Glyma08g116300 ) gene and its homolog Glyma08g116400 gene were simultaneously deleted, and seeds thereof were obtained. In addition, soybean transformants in which the soybean GmP34 ( Glyma08g116300 ) gene and its homolog Glyma08g116400 gene according to the present invention have been corrected and their seeds can be used to produce plants having various combinations of genetically corrected forms in the next generation. there is.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> Gene editing system for simultaneous gene editing of P34 and its homolog genes and uses its <130> PN21233 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence encoding gRNA1 <400> 1 actagaagag agacctaaga 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence encoding gRNA2 <400> 2 gcccccttgg tactttactt 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence encoding gRNA1 <400> 3 gactagaaga gagacctaag a 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence encoding gRNA2 <400> 4 ggcccccttg gtacttact t 21 <210> 5 <211> 4293 <212> DNA <213> Artificial Sequence <220 > <223> pcoCas9 <400> 5 atggataaga agtactctat cggacttgac atcggaacca actctgttgg atgggctgtt 60 atcaccgatg agtacaaggt tccatctaag aagttcaagg ttcttggaaa caccgataga 120 cactctatca agaagaacct tatcggtgct cttcttttc g attctggaga gaccgctgag 180 gctaccagat tgaagagaac cgctagaaga agatacacca gaagaaagaa cagaatctgc 240 taccttcagg aaatcttctc taacgagatg gctaaggttg atgattcttt cttccacaga 300 cttgaggagt ctttccttgt tgaggaggat aagaag cacg agagacaccc aatcttcgga 360 aacatcgttg atgaggttgc ttaccacgag aagtacccaa ccatctacca ccttagaaag 420 aagttggttg attctaccga taaggctgat cttagactta tctaccttgc tcttgctcac 480 atgatcaagt tcagaggaca cttccttatc gagggagacc ttaacccaga taactct gat 540 gttgataagt tgttcatcca gcttgttcag acctacaacc agcttttcga ggagaaccca 600 atcaacgctt ctggagttga tgctaaggct atcctttctg ctagactttc taagtctcgt 660 agacttgaga accttatcgc tcagcttcca ggagagaaga agaacgg act tttcggaaac 720 cttatcgctc tttctcttgg acttacccca aacttcaagt ctaacttcga tcttgctgag 780 gatgctaagt tgcagctttc taaggatacc tacgatgatg atcttgataa ccttcttgct 840 cagatcggag atcagtacgc tgatcttttc cttgctgcta agaacctttc tgatgctatc 900 cttctttctg acatccttag agttaacacc gagatcacca aggctccact ttctgcttct 960 atgatcaaga gatacgatga gcaccaccag gatcttaccc ttttgaaggc tcttgttaga 1020 cagcagcttc cagagaagta caaggaaatc ttcttcgatc agtctaagaa cggatacgct 1080 ggatacatcg atggaggagc ttctcaggag gagt tctaca agttcatcaa gccaatcctt 1140 gagaagatgg atggaaccga ggagcttctt gttaagttga acagagagga tcttcttaga 1200 aagcagagaa ccttcgataa cggatctatc ccacaccaga tccaccttgg agagcttcac 1260 gctatccttc gtagacagga ggatttctac ccattcttga aggataacag agagaagatc 1320 gagaagatcc ttaccttcag aatcccatac tacgttggac cacttgctag aggaaactct 1380 cgtttc gctt ggatgaccag aaagtctgag gagaccatca ccccttggaa cttcgaggag 1440 gtaagtttct gcttctacct ttgatatata tataataatt atcattaatt agtagtaata 1500 taatatttca aatatttttt tcaaaataaa agaatgtagt atatagcaat tgcttttctg 1560 tagtttataa gtgtgtatat tttaatttat aacttttcta atatatgacc aaaatttgtt 1620 gatgtgcagg ttgttgataa gggagcttct gctcagtctt tcatcgagag aatgaccaac 1680 ttcgataaga accttccaaa cgagaaggtt cttccaaagc actctcttct ttacgagtac 1740 ttcaccgttt acaacgagct taccaaggtt aagtacgtta ccgagggaat gagaaagcca 1800 gctttcc ttt ctggagagca gaagaaggct atcgttgatc ttcttttcaa gaccaacaga 1860 aaggttaccg ttaagcagtt gaaggaggat tacttcaaga agatcgagtg cttcgattct 1920 gttgaaatct ctggagttga ggatagattc aacgcttctc ttggaaccta ccacgat ctt 1980 ttgaagatca tcaaggataa ggatttcctt gataacgagg agaacgagga catccttgag 2040 gacatcgttc ttacccttac ccttttcgag gatagagaga tgatcgagga gagactcaag 2100 acctacgctc accttttcga tgataaggtt atgaagcagt tgaagagaag aagatacacc 2160 ggatggggta gactttctcg taagttgatc aacggaatca gagataagca gtctggaaag 2220 accatccttg atttcttgaa gt ctgatgga ttcgctaaca gaaacttcat gcagcttatc 2280 cacgatgatt ctcttacctt caaggaggac atccagaagg ctcaggtttc tggacaggga 2340 gattctcttc acgagcacat cgctaacctt gctggatctc cagctatcaa gaagggaatc 2400 cttcagaccg ttaaggttgt tgatgagctt gttaaggtta tgggtagaca caagccagag 2460 aacatcgtta tcgagatggc tagagagaac cagaccaccc agaagggaca gaagaactct 2520 cgtgagagaa tgaagagaat cgaggaggga atcaaggagc ttggatctca aatcttgaag 2580 gagcacccag ttgagaacac ccagcttcag aacgagaagt tgtaccttta ctaccttcag 2640 aacggaagag atatgtacgt tgatcaggag ctt gacatca acagactttc tgattacgat 2700 gttgatcaca tcgttccaca gtctttcttg aaggatgatt ctatcgataa caaggttctt 2760 acccgttctg ataagaacag aggaaagtct gataacgttc catctgagga ggttgttaag 2820 aagatgaaga actggag acagcttct t aacgctaagt tgatcaccca gagaaagttc 2880 gataacctta ccaaggctga gagaggagga ctttctgagc ttgataaggc tggattcatc 2940 aagagacagc ttgttgagac cagacagatc accaagcacg ttgctcagat ccttgattct 3000 cgtatgaaca ccaagtacga tgagaacgat aagttgatca gagaggttaa ggttatcacc 3060 ttgaagtcta agttggtttc tgatttcaga aa gtctga gcag gagatcggaa aggctaccgc taagtacttc 3300 ttctactcta acatcatgaa cttcttcaag accgagatca cccttgctaa cggagagatc 3480 accgaggttc agaccggagg attctctaag gagtctatcc ttccaaagag a aactctgat 3540 aagttgatcg ctagaaagaa ggattgggac ccaaagaagt acggaggatt cgattctcca 3600 accgttgctt actctgttct tgttgttgct aaggttgaga agggaaagtc taagaagttg 3660 aagtctgtta aggagcttct tggaatcacc atcatggagc gttct tcttt cgagaagaac 3720 ccaatcgatt tccttgaggc taagggatac aaggaggtta agaaggatct tatcatcaag 3780 ttgccaaagt actctctttt cgagcttgag aacggaagaa agagaatgct tgcttctgct 3840 ggagagcttc agaagggaaa cgagcttgct cttccatcta agtacgttaa cttcctttac 3900 cttgcttctc actacgagaa gttgaaggga tctccagagg ataac gagca gaagcagctt 3960 ttcgttgagc agcacaagca ctaccttgat gagatcatcg agcaaatctc tgagttctct 4020 aagagagtta tccttgctga tgctaacctt gataaggttc tttctgctta caacaagcac 4080 agagataagc caatcagaga gcagg ctgag aacatcatcc accttttcac cctaccaac 4140 cttggtgctc cagctgcttt caagtacttc gataccacca tcgatagaaa aagatacacc 4200 tctaccaagg aggttcttga tgctaccctt atccaccagt ctatcaccgg actttacgag 4260 accagaatcg atctttctca gcttggagga gat 4293 <210> 6 <211> 4116 <212> DNA <213> Artificial Sequence <220> <223> acoCas9 <400> 6 atggataaga agtactct at cggactcgat atcggaacta actctgtggg atgggctgtg 60 atcaccgatg agtacaaggt gccatctaag aagttcaagg ttctcggaaa caccgatagg 120 cactctatca agaaaaacct tatcggtgct ctcctcttcg attctggtga aactgctgag 180 gctaccagac tcaagagaac cgctagaaga aggtacacca gaagaaagaa caggatctgc 240 tacctccaag agatcttctc taacgagatg g ctaaagtgg atgattcatt cttccacagg 300 ctcgaagagt cattcctcgt ggaagaagat aagaagcacg agaggcaccc tatcttcgga 360 aacatcgttg atgaggtggc ataccacgag aagtacccta ctatctacca cctcagaaag 420 aagctcgttg attctactga taaggctgat ctca ggctca tctacctcgc tctcgctcac 480 atgatcaagt tcagaggaca cttcctcatc gagggtgatc tcaaccctga taactctgat 540 gtggataagt tgttcatcca gctcgtgcag acctacaacc agcttttcga agagaaccct 600 atcaacgctt caggtgtgga tgctaaggct atcctctctg ctaggctctc taagtcaaga 660 aggcttgaga acctcattgc tcagctccct ggtgagaaga agaac ggact tttcggaaac 720 ttgatcgctc tctctctcgg actcacccct aacttcaagt ctaacttcga tctcgctgag 780 gatgcaaagc tccagctctc aaaggatacc tacgatgatg atctcgataa cctcctcgct 840 cagatcggag atcagtacgc tgatttgtt c ctcgctgcta agaacctctc tgatgctatc 900 ctcctcagtg atatcctcag agtgaacacc gagatcacca aggctccact ctcagcttct 960 atgatcaaga gatacgatga gcaccaccag gatctcacac ttctcaaggc tcttgttaga 1020 cagcagctcc cagagaagta caaagagatt ttcttcgatc agtctaagaa cggatacgct 1080 ggttacatcg atggtggtgc atctcaagaa gagttctaca agttcatcaa gcc tatcctc 1140 gagaagatgg atggaaccga ggaactcctc gtgaagctca atagagagga tcttctcaga 1200 aagcagagga ccttcgataa cggatctatc cctcatcaga tccacctcgg agagttgcac 1260 gctatcctta gaaggcaaga ggatttctac ccattcctca aggataacag gga aaagatt 1320 gagaagattc tcaccttcag aatcccttac tacgtgggac ctctcgctag aggaaactca 1380 agattcgctt ggatgaccag aaagtctgag gaaaccatca ccccttggaa cttcgaagag 1440 gtggtggata agggtgctag tgctcagtct ttcatcgaga ggatgaccaa cttcgataag 1500 aaccttccaa acgagaaggt gctccctaag cactctttgc tctacgagta cttcaccgtg 1560 tacaacgagt tgaccaaggt taagtacgtg accgagggaa tgaggaagcc tgcttttttg 1620 tcaggtgagc aaaagaaggc tatcgttgat ctcttgttca agaccaacag aaaggtgacc 1680 gtgaagcagc tcaaagagga ttacttcaag aaaatcgagt gcttcgattc agttgagatt 1740 tctggtgttg aggataggtt caacgcatct ctcggaacct accacgatct cctcaagatc 1800 attaaggata aggatttctt ggataacgag gaaaacgagg atatcttgga ggatatcgtt 1860 cttaccctca ccctctttga agatagagag atgattgaag aaaggctcaa gacctacgct 1920 catctcttcg atgataaggt gatgaagcag ttgaagagaa gaagatacac tggttgggga 1980 agg ctctcaa gaaagctcat taacggaatc aggtaagc agtctggaaa gacaatcctt 2040 gatttcctca agtctgatgg attcgctaac agaaacttca tgcagctcat ccacgatgat 2100 tctctcacct ttaaagagga tatccagaag gctcaggttt caggacaggg tgatagtctc 2160 catgagcata tcgctaacct cgctggatct cctgcaatca agaagggaat cctccagact 2220 gtgaaggttg tggatgagtt ggtgaaggtg atgggaaggc ataagcctga gaacatcgtg 2280 atcgaaatgg ctagagagaa ccagaccact cagaagggac agaagaactc tagggaaagg 2340 atgaagagga tcgaggaagg tatcaaagag cttggatctc agatcctcaa agagcaccct 2400 gttgagaaca ctcag ctcca gaatgagaag ctctacctct actacctcca gaacggaagg 2460 gatatgtatg tggatcaaga gttggatatc aacaggctct ctgattacga tgttgatcat 2520 atcgtgccac agtcattctt gaaggatgat tctatcgata acaaggtgct caccaggtct 2580 gataagaaca gg ggtaagag tgataacgtg ccaagtgaag aggttgtgaa gaaaatgaag 2640 aactattgga ggcagctcct caacgctaag ctcatcactc agagaaagtt cgataacttg 2700 actaaggctg agaggggagg actctctgaa ttggataagg caggattcat caagaggcag 2760 cttgtggaaa ccaggcagat cactaagcac gttgcacaga tcctcgattc taggatgaac 2820 accaagtacg atgagaacga taag ttgatc agggaagtga aggttatcac cctcaagtca 2880 aagctcgtgt ctgatttcag aaaggatttc caattctaca aggtgaggga aatcaacaac 2940 taccaccacg ctcacgatgc ttaccttaac gctgttgttg gaaccgctct catcaagaag 3000 tatcctaagc tcgagtca ga gttcgtgtac ggtgattaca aggtgtacga tgtgaggaag 3060 atgatcgcta agtctgagca agagatcgga aaggctaccg ctaagtattt cttctactct 3120 aacatcatga atttcttcaa gaccgagatt accctcgcta acggtgagat cagaaagagg 3180 ccactcatcg agacaaacgg tgaaacaggt gagatcgtgt gggataaggg aagggatttc 3240 gctaccgtta gaaaggtgct ctctatgcca caggt gaaca tcgttaagaa aaccgaggtg 3300 cagaccggtg gattctctaa agagtctatc ctccctaaga ggaactctga taagctcatt 3360 gctaggaaga aggattggga ccctaagaaa tacggtggtt tcgattctcc taccgtggct 3420 tactctgttc tcgttgtggc taaggttgag aa gggaaaga gtaagaagct caagtctgtt 3480 aaggaacttc tcggaatcac tatcatgggaa aggtcatctt tcgagaagaa cccaatcgat 3540 ttcctcgagg ctaagggata caaagaggtt aagaaggatc tcatcatcaa gctcccaaag 3600 tactcactct tcgaactcga gaacggtaga aagaggatgc tcgcttctgc tggtgagctt 3660 caaaagggaa acgagcttgc tctcccat cg ataaggtg ttgtctgctt acaacaagca cagagataag 3900 cctatcaggg aacaggcaga gaacatcatc catctcttca cccttaccaa cctcggtgct 21 0> 7 <211> 4104 <212> DNA <213> Artificial Sequence <220> <223> pcohCas9 <400> 7 atggacaaga agtactccat cggcctcgac atcggcacca acagcgtcgg ctgggcggtg 60 atcaccgacg agtacaaggt cccgtccaag aagttcaagg tcctgggcaa caccgaccgc 120 cactccatca agaagaacct catcggcgcc ctcctcttcg actccggcga gacggcggag 180 gcgacccgcc tca agcgcac cgcccgccgc cgctacaccc gccgcaagaa ccgcatctgc 240 tacctccagg agatcttctc caacgagatg gcgaaggtcg acgactcctt cttccaccgc 300 ctcgaggagt ccttcctcgt ggaggaggac aagaagcacg agcgccaccc catcttcggc 36 0 aacatcgtcg acgaggtcgc ctaccacgag aagtacccca ctatctacca ccttcgtaag 420 aagcttgttg actctactga taaggctgat cttcgtctca tctaccttgc tctcgctcac 480 atgatcaagt tccgtggtca cttccttatc gagggtgacc ttaaccctga taactccgac 540 gtggacaagc tcttcatcca gctcgtccag acctacaacc agctcttcga ggagaaccct 600 atcaacgctt ccggtgtcga cgctaaggcg atcctttccg ctaggctctc caagtccagg 660 cgtctcgaga acctcatcgc ccagctccct ggtgagaaga agaacggtct tttcggtaac 720 ctcatcgctc tctccctcgg tctgacccct aacttcaagt ccaacttcga cctcgctgag 780 gacgcta agc ttcagctctc caaggatacc tacgacgatg atctcgacaa cctcctcgct 840 cagattggag atcagtacgc tgatctcttc cttgctgcta agaacctctc cgatgctatc 900 ctcctttcgg atatccttag ggttaacact gagatcacta aggctcctct ttctgcttcc 960 atgatcaagc gctacgacga gcaccaccag gacctcaccc tcctcaaggc tcttgttcgt 1020 cagcagctcc ccgagaagta caaggagat c ttcttcgacc agtccaagaa cggctacgcc 1080 ggttacattg acggtggagc tagccaggag gagttctaca agttcatcaa gccaatcctt 1140 gagaagatgg atggtactga ggagcttctc gttaagctta accgtgagga cctccttagg 1200 aagcagagga ctttcgataa cggctctat c cctcaccaga tccaccttgg tgagcttcac 1260 gccatccttc gtaggcagga ggacttctac cctttcctca aggacaaccg tgagaagatc 1320 gagaagatcc ttactttccg tattccttac tacgttggtc ctcttgctcg tggtaactcc 1380 cgtttcgctt ggatgactag gaagtccgag gagactatca ccccttggaa cttcgaggag 1440 gttgttgaca agggtgcttc cgcccagtcc ttcatcgagc gcatgaccaa cttcgacaag 1500 aacctcccca acgagaaggt cctccccaag cactccctcc tctacgagta cttcacggtc 1560 tacaacgagc tcaccaaggt caagtacgtc accgagggta tgcgcaagcc tgccttcctc 1620 tccggcgagc a gaagaaggc tatcgttgac ctcctcttca agaccaaccg caaggtcacc 1680 gtcaagcagc tcaaggagga ctacttcaag aagatcgagt gcttcgactc cgtcgagatc 1740 agcggcgttg aggaccgttt caacgcttct ctcggtacct accacgatct cctcaagatc 1800 atcaaggaca aggacttcct cgacaacgag gagaacgagg acatcctcga ggacatcgtc 1860 ctcactctta ctctcttcga ggatagggag atgatcgagg agaggctcaa gactt acgct 1920 catctcttcg atgacaaggt tatgaagcag ctcaagcgtc gccgttacac cggttggggt 1980 aggctctccc gcaagctcat caacggtatc aggtataagc agagcggcaa gactatcctc 2040 gacttcctca agtctgatgg tttcgctaac aggaacttca tgca gctcat ccacgatgac 2100 tctcttacct tcaaggagga tattcagaag gctcaggtgt ccggtcaggg cgactctctc 2160 cacgagcaca ttgctaacct tgctggttcc cctgctatca agaagggcat ccttcagact 2220 gttaaggttg tcgatgagct tgtcaaggtt atgggtcgtc acaagcctga gaacatcgtc 2280 atcgagatgg ctcgtgagaa ccagactacc cagaagggtc agaagaactc gaggg agcgc 2340 atgaagagga ttgaggaggg tatcaaggag cttggttctc agatccttaa ggagcaccct 2400 gtcgagaaca cccagctcca gaacgagaag ctctacctct actacctcca gaacggtagg 2460 gatatgtacg ttgaccagga gctcgacatc aacaggcttt ctgactacga cgtcgaccac 2520 attgttcctc agtctttcct taaggatgac tccatcgaca acaaggtcct cacgaggtcc 2580 gacaagaaca ggggtaagtc ggacaacgtc ccttccgagg aggttgtcaa gaagatgaag 2640 aactactgga ggcagcttct caacgctaag ctcattaccc agaggaagtt cgacaacctc 2700 acgaaggctg agaggggtgg cctttccgag cttgacaagg ctggtttcat caagaggcag 27 60 cttgttgaga cgaggcagat taccaagcac gttgctcaga tcctcgattc taggatgaac 2820 accaagtacg acgagaacga caagctcatc cgcgaggtca aggtgatcac cctcaagtcc 2880 aagctcgtct ccgacttccg caaggacttc cagtctaca aggtccgcga gat caacaac 2940 taccaccacg ctcacgatgc ttaccttaac gctgtcgttg gtaccgctct tatcaagaag 3000 taccctaagc ttgagtccga gttcgtctac ggtgactaca aggtctacga cgttcgtaag 3060 atgatcgcca agtccgagca ggagatcggc aaggccacg ccaagtactt cttctactcc 3120 aacatcatga acttcttcaa gaccgagatc accctcgcca acggcgagat ccgcaagcgc 3180 cctctta tcg agacgaacgg tgagactggt gagatcgttt gggacaaggg tcgcgacttc 3240 gctactgttc gcaaggtcct ttctatgcct caggttaaca tcgtcaagaa gaccgaggtc 3300 cagaccggtg gcttctccaa ggaggtctatc cttccaaaga gaaactcgga caagct catc 3360 gctaggaaga aggattggga ccctaagaag tacggtggtt tcgactcccc tactgtcgcc 3420 tactccgtcc tcgtggtcgc caaggtggag aagggtaagt cgaagaagct caagtccgtc 3480 aaggagctcc tcggcatcac catcatggag cgctcctcct tcgagaagaa cccgatcgac 3540 ttcctcgagg ccaagggcta caaggaggtc aagaaggacc tcatcatcaa gctccccaag 3600 tactctcttt tcgagct cga gaacggtcgt aagaggatgc tggcttccgc tggtgagctc 3660 cagaagggta acgagcttgc tcttccttcc aagtacgtga acttcctcta cctcgcctcc 3720 cactacgaga agctcaaggg ttcccctgag gataacgagc agaagcagct cttcgtggag 3780 cagcacaagc actacctcga cgagatcatc gagcagatct ccgagttctc caagcgcgtc 3840 atcctcgctg acgctaacct cgacaaggtc ctctccgcct acaacaagca ccgcgacaag 3900 cccatccgcg agcaggccga gaacatcatc cacctcttca cgctcacgaa cctcggcgcc 3960 cctgctgctt tcaagtactt cgacaccacc atcgacagga agcgttacac gtccaccaag 4020 gaggttctcg acgctactct catccacca g tccatcaccg gtctttacga gactcgtatc 4080 gacctttccc agcttggtgg tgat 4104 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223 > SV40 NLS <400> 8 ccaaagaaga agagaaaggt t 21 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SV40 NLS <400> 9 cctaagaaga agaggaaggt t 21 <210> 10 <211 > 21 <212> DNA <213> Artificial Sequence <220> <223> SV40 NLS <400> 10 cctaagaaga agcggaaggt t 21 <210> 11 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> nucleoplasmin NLS <400> 11 aagagaccag ctgctaccaa gaaggctgga caggctaaga agaagaa 47 <210> 12 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> nucleoplasmin NLS<400> 12 aagcgtcctg ctgccaccaa aaaggccgga caggctaaga aaaagaag 48

Claims (12)

delete delete delete delete codon-optimized Cas9 nucleic acids; And nucleic acids encoding gRNA1 and gRNA2, respectively, that simultaneously target the soybean GmP34 gene ( Glyma08g116300 ) and its homolog Glyma08g116400 gene;
The codon-optimized Cas9 nucleic acid consists of the nucleotide sequence of SEQ ID NO: 6, and the nucleic acids encoding gRNA1 and gRNA2 consist of the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2, respectively; Or consisting of the nucleotide sequence of SEQ ID NO: 3 and the nucleotide sequence of SEQ ID NO: 4; Characterized in that, a vector for soybean genetic correction.
According to claim 5,
The vector for soybean gene editing, characterized in that it further comprises one or more operably linked promoters, plant selectable markers and nuclear localization sequences (NLS).
delete Comprising the vector of claim 5, soybean gene editing composition.
According to claim 8,
The nucleic acids encoding gRNA1 and gRNA2 are composed of the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and the soybean GmP34 gene ( Glyma08g116300 ) and its homolog Glyma08g116400 gene are simultaneously corrected. , Soybean gene editing composition.
Comprising the step of transforming soybean using the vector of claim 5, soybean GmP34 gene ( Glyma08g116300 ) and its homolog (homolog) Glyma08g116400 gene are simultaneously corrected, soybean gene editing method.
A soybean transformant in which the GmP34 gene ( Glyma08g116300 ) and its homolog Glyma08g116400 gene, which are allergens in soybean, transformed by the soybean gene editing method of claim 10 have been corrected.
A seed of a soybean transformant transformed by the method of claim 10, wherein the allergen GmP34 gene ( Glyma08g116300 ) and its homolog Glyma08g116400 gene are corrected.