KR102584891B1 - Gene editing system for GmIKP1 gene editing and uses thereof - Google Patents

Abstract

The present invention constructed a CRISPR/Cas system that simultaneously corrects GmIPK1 and its homolog genes that regulate phytic acid biosynthesis in soybean, and developed a gene-edited soybean transformant with low phytic acid content through gene editing-based technology using this system. The CRISPR/Cas-based soybean gene editing system or vector according to the present invention and the soybean gene editing composition containing the same have the effect of simultaneously suppressing the expression of the IPK1 protein present in soybean.
The present invention can provide a new plant that produces soybeans with reduced phytic acid content, an anti-nutritional factor, by specifically correcting the GmIPK1 gene in soybean plants, and by cultivating soybeans with reduced phytic acid content in soybean seeds for use as food and feed. This will improve the usability and value of soybeans and at the same time contribute to preserving a clean agricultural ecosystem.

Description

GmIPK1 gene editing system and uses thereof {Gene editing system for GmIKP1 gene editing and uses thereof}

The present invention relates to a gene editing system for editing the GmIPK1 gene of soybean to develop a gene-edited soybean transformant with a low content of phytic acid, an anti-nutritional factor of soybean. Specifically, it relates to a gene editing system for deleting the GmIPK1 gene in soybean, a soybean gene editing composition containing the same, a soybean gene editing method using the same, and soybean transformants with low phytanic acid content and seeds thereof.

Soybeans are rich in protein, fat, and dietary fiber, and contain various health functional substances such as isoflavones, anthocyanins, lecithin, lutein, and saponin, so they not only have high nutritional value as food, but are also used as industrial feed and processing purposes. It is also a crop of great value.

However, soybeans contain anti-nutritional factors such as phytic acid, indigestible oligosaccharides, and allergens, which hinders the usability of soybeans as a food.

Soybean seeds contain a large amount of phytic acid (phytate), approximately 2% of dry matter. Phytic acid (phytate) combines with minerals such as calcium, iron, and zinc to form sediments, thereby destroying the minerals. It is a representative anti-nutritional factor that interferes with absorption. Phytic acid is known to not only reduce the value of soybeans as food, but also reduce the feed value of soybeans. In particular, livestock manure containing a large amount of phytic acid is known to be a major cause of water pollution due to eutrophication. For this reason, the development of soybeans with reduced phytic acid content has great economic and environmental value, including improving the usability and value of soybeans for food and feed and preserving the agricultural environment.

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

The CRISPR/Cas system forms a complex of the Cas endonuclease protein, CRISPR RNA (crRNA) that recognizes the target gene sequence, and transactivating CRISPR RNA (tracrRNA) that binds to the Cas endonuclease while binding to the crRNA. It was done. 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 used to cut the target gene to be cut by Cas endonuclease. Guide to double-stranded DNA base sequences. Cas endonuclease located at the target gene site recognizes the protospacer adjacent motif (PAM) neighboring the target gene sequence and then changes the internal or external base pair (base pair) sequence of the target nucleic acid sequence. bp), DNA double strand breaks (DSB) occur (Jinek et al., 2012).

Target nucleic acids cut by the CRISPR/Cas system undergo homologous recombination (HDR) or non-homologous end joining (HDR) during error-prone DSB repair. Repair occurs through the DNA repair mechanism of the homologous end joining (NHEJ) process. Through the DNA repair mechanism of non-homologous end joining (NHEJ), insertion and deletion (indel) mutations of random bases occur between the cut DNA regions. As a result, a frameshift mutation or premature mutation occurs in the coding part of the gene, resulting in the knock-out of the target gene. On the other hand, the DNA repair mechanism of homologous recombination (HDR) requires donor DNA to repair cut DNA, and the sequence of the target gene is precisely replaced using the sequence of this donor DNA as a template. , gene editing is completed.

Gene editing is a technology that freely edits the genetic information of living organisms. It can dramatically expand the scope of use by changing the genetic information of animals, plants, and microorganisms, including humans. Currently, many researchers are pursuing gene editing research and are conducting research on basic technologies such as securing gene editing original patents, 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 more than 20 times its current size within the next 10 years (Inkwood Research, 2019).

However, crop improvement research using CRISPR technology is in its early stages. In particular, soybeans require a long period of time to produce transformants compared to other crops and the efficiency is relatively low, so in the case of soybeans that are difficult to transform, soybeans based on gene editing technology are used. The development of breeding materials is limited to only a few research labs.

Currently, various studies are continuously being conducted to develop soybean transformants and seeds 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 with reduced phytanic acid content by introducing the CRISPR/Cas system, the results are minimal. Accordingly, an optimal and effective CRISPR/Cas system for producing soybean transformants and seeds with reduced phytanic acid content is urgently needed.

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, T.B. et al., Targeted genome modifications in soybean with CRISPR/Cas9, BMC Biotechnology, Vol. 15, No. 16, pp. 1-10(2015)

The present invention is intended to solve the above needs and overcome the problems of the prior art. In order to develop a gene-corrected soybean transformant with a low content of phytic acid, an anti-nutritional factor in soybeans, CRISPR/Cas to delete the GmIPK1 gene in soybeans The purpose is to provide a soybean gene editing system.

The present invention also aims to provide a vector containing the soybean gene editing system, a soybean gene editing composition, and a soybean gene editing method using the same.

In addition, the present invention aims to provide soybean transformants and seeds thereof, which are transformed with the soybean gene editing system or soybean gene editing composition and have the GmIPK1 gene, an enzyme that regulates phytic acid biosynthesis in soybean seeds, corrected.

Other objects of this application and technical problems to be achieved by the present invention are not limited to the objects mentioned above, and will become clearer through 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 that are not described in this specification include those that can be clearly understood and inferred by a person with ordinary knowledge in the technical field of the present invention (hereinafter referred to as a “person skilled in the art”).

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

The present invention provides a CRISPR/Cas-based soybean gene editing system that deletes the GmIPK1 gene for the development of gene-edited soybean transformants with low content of phytic acid, an anti-nutritional factor in soybeans. Here, the CRISPR/Cas-based soybean gene editing system includes Cas endonuclease; and gRNA1 and gRNA2 that specifically target the GmIPK1 gene of soybean.

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 plant codon-optimized Cas9 nucleic acid (pcoCas9), Arabidopsis codon-optimized Cas9 nucleic acid (acoCas9), or plant codon-optimized with high GC content. It may contain Cas9 nucleic acid (pcohCas9), and specifically may contain any one of SEQ ID NO: 5 to SEQ ID NO: 7 base sequence.

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 base sequence of SEQ ID NO: 1 and the base sequence of SEQ ID NO: 2, respectively. It may be.

Additionally, the present invention provides codon-optimized Cas9 nucleic acid; and nucleic acids encoding gRNA1 and gRNA2 that specifically target the GmIPK1 gene of soybean. A vector for soybean gene editing operably linked is provided.

In one embodiment of the present invention, the vector may further include one or more operably linked promoters, a plant selection marker, 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 may include 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 specifically may include any one of SEQ ID NO: 5 to SEQ ID NO: 7.

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

In one embodiment of the present invention, in the composition for soybean gene editing, the nucleic acid sequences encoding the gRNA1 and gRNA2 are the base sequence of SEQ ID NO: 1 and the base sequence of SEQ ID NO: 2, respectively, and the GmIPK1 gene of soybean is deleted. It can be characterized.

In addition, the present invention provides a soybean gene editing method comprising the step of transforming soybean using the CRISPR/Cas-based soybean gene editing system or the vector, and deleting the soybean GmIPK1 gene. .

In one embodiment of the present invention, the soybean gene editing method includes the steps of introducing the CRISPR/Cas-based soybean gene editing system or the vector into soybean plant cells to correct the target gene, and the soybean plant cell in which the target gene has been corrected. It may include the step of redifferentiating the soybean plant from.

The present invention also provides a soybean transformant in which the GmIPK1 gene, an enzyme that regulates phytic acid biosynthesis in soybean, has been corrected, transformed by the soybean gene editing method.

In addition, the present invention provides soybean seeds in which the GmIPK1 gene, an enzyme that regulates phytic acid biosynthesis, has been corrected, which are seeds of soybean transformants transformed by the soybean gene editing method.

The CRISPR/Cas-based soybean gene editing system or vector according to the present invention and the soybean gene editing composition containing the same have the effect of suppressing the expression of the IPK1 protein present in soybean. In addition, soybean transformants and their seeds produced using the soybean gene editing system according to the present invention have the advantage of producing soybeans with reduced phytic acid content by correcting the GmIPK1 gene, which regulates phytic acid biosynthesis.

Therefore, the present invention can provide a new plant with reduced anti-nutritional factors by correcting target genes in soybean plants, and improves the usability and value of soybeans for food and feed by cultivating soybeans with reduced phytic acid content in soybean seeds. At the same time, it can contribute to preserving a clean agricultural ecosystem.

Figure 1 is a diagram showing the target site within the target gene of gRNA1 and gRNA2 according to the present invention, which can specifically target the GmIPK1 gene of soybean. (A) shows the final selected target site and the chromosomal locations of gRNA1 and gRNA2 that bind to it, respectively. (B) shows the nucleotide sequences of the target sequence in Exon 2 (left) and the target sequence in Exon 3 (right) among the target regions of the GmIPK1 gene; and base sequences encoding gRNA1 and gRNA2, respectively, binding thereto.
Figure 2 shows an exemplary construction schematic diagram of the pMDC123_35S _pro vector.
3 shows an exemplary plot comprising selected gRNA1 and gRNA2 and codon-optimized Cas9 gene according to the present invention. Shows a schematic diagram of a vector.
Figure 4 shows the results of manufacturing soybean transformants using Agrobacterium and verifying the transformants in which gene editing occurred. (A) shows the manufacturing process of the soybean transformant, and (B) shows the results of confirming transformation of the soybean transformant by PCR amplification reaction for the selection marker and gRNA expression cassette of the vector used in the present invention. .
Figure 5 shows the results of confirming whether the GmIPK1 gene was corrected in the soybean transformant according to the present invention. (A) shows whether the GmIPK1 gene was deleted in the soybean transformant using InDel PCR, and (B) shows the soybean transformant according to the present invention in which the GmIPK1 gene was confirmed to be deleted.
Figure 6 is a result showing deletion of the target sequence region of the GmIPK1 gene and protein in the soybean transformant according to the present invention. (A) shows a 577 bp deletion of the target sequence region of the GmIPK1 gene, and (B) shows the IPK1 It shows a deletion of 95 amino acids in the target sequence region of the protein.

The present invention uses the CRISPR/Cas9 (Clustered Regularly-Interspaced Short Palindromic Repeats/CRISPR associated protein 9) system to specifically cause an indel mutation in the GmIPK1 gene, an enzyme that regulates phytate biosynthesis, known as an anti-nutritional factor in soybeans. As a result, a soybean transformant and its seeds with deletion of soybean IPK1 expression were produced. The present invention was completed by confirming that the soybean transformant and its seeds underwent gene correction resulting in deletion of the GmIPK1 gene through targeted deep sequencing.

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

The definitions of terms used in the invention are as follows.

[Explanation of terms]

CRISPR/Cas system

The term “CRISPR/Cas system” used in the present invention is a complex containing a Cas endonuclease and a guide molecule corresponding to the nucleolytic enzyme, 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 Cas endonuclease and the guide RNA (gRNA), and Cas endonuclease uses the corresponding guide molecule to target nucleic acid. Alternatively, it binds specifically to the target gene and performs gene editing such as DNA double-strand cutting.

Gene editing technology using the CRISPR/Cas system can introduce mutations into the genes of various organisms such as humans, animals, and plants, and through this, research to reveal the function of genes, manufacturing 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 customized protein to target a specific sequence, and a diverse repertoire of CRISPR/Cas systems can be produced by changing the spacer sequence, which is the target site binding sequence within the guide molecule.

Cas endonuclease

The term “Cas endonuclease” used in the present invention refers to the recognition of a protospacer adjacent motif (PAM) within a target nucleic acid, DNA or RNA, or a target gene, and then the formation of the target nucleic acid sequence. It refers to a nucleolytic enzyme that can edit DNA to cause double strand breaks (DSB) in internal or external base pairs (bp). In this specification, Cas endonuclease refers to CRISPR/ Refers to an effector protein that constitutes the Cas system. Here, the effector protein is a CRISPR protein, a nucleic acid decomposition protein that can bind to a guide RNA (gRNA), or an oligonucleotide that can bind 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 a person skilled in the art of CRISPR/Cas technology can recognize is Includes all.

Cas9 protein

“Cas9 protein” according to the present invention recognizes the protospacer adjacent motif (PAM) near the target site sequence within the target nucleic acid or target gene, and then encodes the internal or external base sequence (base pair, bp) of the target nucleic acid sequence. ) may be one or more types selected among all Cas9s that can cause DNA double strand breaks (DSB) and cause insertion and/or deletion (Indel) mutation in the target gene. Specifically, The Cas9 protein may be a Cas9 protein derived from the Streptococcus genus, Campylobacter genus, Neisseria genus, Pasteurella genus, and Francisella genus, etc. Specifically, Cas9 protein from Streptococcus thermophiles, Streptoccus aureus or Streptococcus pyogenes; Cas9 protein from Campylobacter jejuni ; Cas9 protein derived from Neisseria meningitidis; Cas9 protein derived from Pasteurella multocida; Cas9 protein derived from Francisella novicida, etc., but is limited thereto. In addition, it includes all meanings recognized by a person skilled in the art 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). You can.

guide RNA

The term "guide RNA" according to the present invention is capable of forming a complex with Cas endonuclease, hybridizing to a target nucleic acid sequence, and having a sequence-specific effect on the complex to the target nucleic acid sequence. It refers to RNA containing a guide sequence that has sufficient complementarity with the target nucleic acid sequence to bind appropriately. In this specification, guide molecules or guide RNA are used interchangeably.

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. Guide RNA may be in a single-stranded form in which crRNA and tracrRNA are connected by a linker, or it may be in a double-stranded form by complementary binding of crRNA and tracrRNA. A guide RNA herein may be an RNA-based molecule that has one or more chemical modifications, such as by chemical linking of two ribonucleotides or by replacement of one or more ribonucleotides with one or more deoxyribonucleotides. .

Since the specificity of guide RNA for Cas endonuclease is determined by the scaffold, which is a directly repeating sequence, the optimal guide RNA for Cas endonuclease is When designing 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 exhibit maximum activity specifically for Cas endonuclease. It can be something to have. In addition, it includes all meanings that a person skilled in the art can recognize in the field of CRISPR/Cas technology.

Scaffold area

The term “Scaffold region” according to the present invention refers collectively to the portion of guide RNA that can interact with nucleic acid decomposition proteins, and excludes the spacer among the portions of guide RNA found in nature. It can refer to the remaining parts. 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 refers to a polynucleotide that hybridizes with a portion of the 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 expressed as "gRNA" in the present invention. .

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

transactivating CRISPR RNA (tracrRNA)

“TracrRNA” according to the present invention is an important component that constitutes the guide RNA of the CRISPR/Cas system along with crRNA. In the present specification, at least a portion of the tracrRNA is a guide RNA portion that can form a double strand by binding complementary to at least a portion of the crRNA and can bind to Cas endonuclease. More specifically, some sequences of the tracrRNA have complementary sequences 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 with a linker to facilitate the formation of guide RNA. The guide RNA provided herein may be a single guide RNA, in which case the single guide RNA is one molecule of RNA containing both the tracrRNA and the crRNA containing a 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 the target of gene editing by the CRISPR/Cas system according to the present invention. Target nucleic acid or target gene may be used interchangeably and may refer to the same object. Unless otherwise stated, the target gene may be a unique gene or nucleic acid of the target cell, an externally derived gene or nucleic acid, or an artificially synthesized nucleic acid or gene, and may be single-stranded DNA, double-stranded DNA and/or RNA. It can mean everything. The target gene or target nucleic acid is not particularly limited as long as it can be subjected to 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 is used to cleave the target gene or target nucleic acid by the CRISPR/Cas system according to the present invention. refers to a specific sequence that is recognized for 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 that is complementary to the 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 only to a specific strand that binds complementary to the guide RNA of the gene scissors system, or may refer to the entire target double strand including the specific strand portion, which may be interpreted appropriately depending on the context. do. In addition, the above term includes all meanings that can be recognized by a person skilled in the art, and can be appropriately interpreted depending on the context.

homolog

The term “homolog” used in the present invention refers to a trait that is conserved during the process of evolution, which may mean conservation in morphological traits, molecular traits, or gene base sequences. If evolution is a kind of remodeling process, this means that it can be predicted that there will be characteristics that are preserved throughout the process. Here, preserved homology does not mean an exact match, but means that something derived from a common ancestral trait remains without being lost, allowing evolutionary relationships to be inferred.

In addition, based on the base sequence of a gene or the amino acid sequence of a protein, the proportion of conserved sequences is higher in species that are evolutionarily closer to homologs, and the more essential elements for the basic functions of organisms are, the more likely they are to be conserved. Accordingly, in the present invention, Meaningful homologs include functional equivalents. Functional equivalent refers to both nucleotide and nucleic acid sequences of a changed mutant sequence that have one or more substitutions, deletions, or additions from a reference sequence and have substantially the same or similar function without various functional dissimilarities.

transformation

The term “transformation” used in the present invention refers to a change in the genetic characteristics of a cell, that is, a phenomenon that changes the genetic characteristics. It means that a cell is genetically modified from its natural state by introducing new external substances such as DNA or RNA, and is also called transformation, transformation, or type conversion. After transfection or transduction, new external DNA or RNA can be recombined with that of the cell by being physiologically integrated into the cell's chromosome, can be temporarily maintained as an episomal element without a replication process, or can be stored independently as a plasmid. can be copied.

Vector

“Vector” according to the present invention refers collectively to all substances capable of transporting genetic material into a cell, unless otherwise specified. For example, the vector may be a DNA molecule containing target genetic material, for example, a nucleic acid encoding an effector protein of the CRISPR/Cas system and/or a nucleic acid encoding a guide RNA, but is not limited thereto. The above term "vector" includes all meanings that can be recognized by a person skilled in the art, and can be appropriately interpreted depending on the context.

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

In addition, since the purpose of the vector is to express each component of the CRISPR/Cas system within the cell, the sequence of the vector must necessarily include at least one nucleic acid sequence 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 a Cas protein. At this time, the sequence of the vector may include not only a wild-type guide RNA and a nucleic acid sequence encoding a wild-type Cas protein, but also a nucleic acid sequence encoding a codon-optimized Cas protein.

The vector may also be a plasmid, linear PCR amplicon or viral vector. 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.

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

The vector of the present invention can be manufactured using genetic recombination technology well known in the art, and enzymes generally known in the art are used for site-specific DNA cutting and ligation.

promoter

The term “promoter” used in the present invention means that it is operably linked to a sequence encoding each component for expressing the expression target of the vector within a cell, thereby enabling the activation of an RNA transcription factor within the cell. The promoter sequence can be designed differently depending on the corresponding RNA transcription factor or expression environment, and is not limited as long as it can properly express the components of the CRISPR/Cas system within the cell.

As 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, ubiquitin promoter of maize (Ubi), cauliflower mosaic virus (CaMV) 35S promoter, nopaline cintase (nos) promoter, pigwort mosaic virus 35S promoter, Sugacrane bacilliform virus promoter, Commelina yellow mottle virus promoter, light-inducible promoter of ribulose-1,5-bis-phosphate carboxylase small subunit (ssRUBISCO), rice cytosolic triosephosphate isomerase (TPI) promoter, the adenine phosphoribosyltransferase (APRT) promoter of Arabidopsis, and the octopine synthase promoter. The promoter of the present invention can be specifically characterized as a constitutive expression promoter, and more specifically as a 35S promoter.

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

selection marker

The term "selection marker" of the present invention refers to a nucleic acid sequence that has characteristics that can be selected by chemical methods, and includes all genes that can distinguish 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 they are not limited to these. 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 operably linked to a coding sequence, it means that the promoter is linked to affect transcription and/or expression of the coding sequence in the cell. In addition, the above term includes all meanings that can be recognized by a person skilled in the art, and can be appropriately interpreted depending on the context.

Nuclear Localization Signal (NLS)

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

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

approximately

The term “about” according to the present invention means 30, 25, 20, 15, 10, 9, 8, 7, 6, means a quantity, 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 otherwise defined, are used with the same meaning as commonly understood by a person skilled in the art in the field related to the present invention. In addition, preferred methods and samples are described in this specification, but similar or equivalent methods 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 these were used for soybean transformation.

First, select the target gene for gene editing. Afterwards, CRISPR-P2.0 ( http://crispr.hzau.edu.cn/ CRISPR2/ ), RGEN ( http://www.rgenome.net/ ), and Phytozome were used to design gRNA for soybean gene editing. ‘Off-target effect’ that corrects non-target genes using web sites such as ( https://phytozome.jgi. doe.gov/pz/portal.html ) and the JBrowse tool within Phytozome. Select gRNAs that are predicted to have low gene editing efficiency and high gene editing efficiency.

CRISPR-P is a web site where you can search for guide RNA with the target base sequence. Candidate gRNAs are ranked according to efficiency, and the type of off-target and exon, intron, intergenic, and promoter on the genome. You can also check the location of off-targets, such as: However, CRISPR-P2.0 has the disadvantage of showing limited off targets.

In addition, RGEN selects the PAM sequence recognized by the CRISPR/Cas9 system among the base sequences of the target gene and then extracts a 20bp gRNA that is a perfect match, 1bp mismatch, or 2bp mismatch from the PAM. presents. And in RGEN, an out-of-frame score is provided to predict the possibility of frame shift mutation occurring, so if you select one with a high score, there is a high possibility that a mutation will occur in the base in the future. However, although RGEN provides a large number of off-targets, it only provides the location of each off-target on the genome, so it is necessary to directly check the detailed information of the off-target.

Therefore, in the present invention, in order to design a highly efficient gRNA that specifically binds to a target gene in soybean, CRISPR-P2.0 and RGEN tools are used together to compensate for the shortcomings of each tool, thereby creating a highly efficient gRNA for soybean gene editing. can be designed. In addition, by selecting a combination of two gRNAs that specifically recognize two target sites within one target gene, it was useful to select lines that were gene edited using only InDel PCR without NGS when advancing future generations.

In the present invention, the genetic information of the target plant was specifically searched in 'phytozome' and the genomic DNA base sequence of the target gene was obtained. Considering the target specificity and efficiency of gRNA, genomic DNA base sequence within 1000 bp was acquired and entered into 'CRISPR-P' to search for gRNA within the corresponding base sequence.

Among the list of gRNAs searched in 'CRISPR-P', the on-target score is high, the Thymine (T) base is not repeated more than 4 times, and the off-target location of the gRNA is in the exon of another gene. Those with the fewest applicable cases were selected as the candidate group. After selecting candidate gRNAs, the distance between the two gRNAs to be applied to the target gene can only be up to 300bp due to technical limitations of sequencing when confirming indels through targeted deep sequencing after obtaining transformants. Therefore, in order to match the distance between the two targets as much as possible, Pairs located within approximately 250 bp were selected. The gRNA base sequence that satisfied the above conditions was searched again for off-targets that may occur due to the gRNA using Cas-OFFinder of 'RGEN'.

'CRISPR-P' is easy to select gRNAs with high efficiency due to on-score information, but shows limited off-target information (maximum 20), so soybean gene editing is performed through cross-validation once more in 'RGEN'. Highly efficient gRNA was selected for this purpose. Using the off-target information located on the genome retrieved from 'RGEN', we searched for the part where the off-target of the gRNA selected from 'JBrowse' occurs.

'JBrowse' shows the entire genome information of chromosome n, so you can determine whether the off-target site is an exon or another part, and through this, you can check whether the gRNA is appropriate. By repeating the above process, the gRNA with the highest on-target score and the least off-target was finally selected and verified.

2. Preparation of CRISPR/Cas9 vector containing selected high-efficiency gRNA

Depending on the purpose, the CRISPR/Cas9 vector can be produced by final selection of gRNAs targeting the target gene. In the present invention, PMDC123_PCOCAS9_G1G2 (Plant Codon-Optimized Cas9), PMDC123_ACOCAS9_G1G2 (Arabidopsis Codon-Optimized Cas9), PMDC123_PCO HCAS9_G1G2 (Plant Codon-optimized Cas9 with High GC Content) and PECO201 Vector sold by Professor KAIST Kim Sang-gyu . The above vectors are gateway vectors that have the Bar gene as a selection marker to select transformed plants and have 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)), and Arabidopsis codon optimized SpCas9 and 35S promoter. In pMDC123_Cas9_g1g2, the bar gene is controlled by the enhanced 35S CaMV promoter, while in pECO201, it is controlled by the nos promoter (nopalin synthase promoter) from Agrobacterium .

Since the pMDC123 vector does not have a promoter that controls the expression of the gene of interest, ₂ The produced pMDC123_35S _pro vector is It was used for final Multisite Gateway recombination. Figure 2 shows a schematic diagram of the production of the pMDC123_35S _pro vector.

To construct a vector containing the selected gRNA1 and gRNA2 and the codon-optimized Cas9 gene (codon-optimized SpCas9), two Golden Gate assembly and one Multisite Gateway recombination processes were performed. First, it is a process of linking gRNA to the AtU6 promoter so that it can be activated. After chopping the pYPQ131A and pYPQ132A vectors, which are golden gate entry vectors with the AtU6 promoter, with the restriction enzyme ESP3I (BsmBI) , gRNA1 and gRNA2 that had gone through the oligo annealing process were linked to pYPQ131A and pYPQ132A vectors, respectively, using the Golden Gate assembly method. Next, gRNA1 (pYPQ131A_gRNA1) and gRNA2 (pYPQ132A_gRNA2) linked to each AtU6 promoter were linked to the pYPQ142 vector (Golden Gate recipient and Gateway entry vector) using the restriction enzyme BsaI using the Golden Gate assembly method. Afterwards, in the second process, Gateway entry vectors (pYPQ150 (plant codon optimized), pYPQ154 (Arabidopsis codon optimized), pYPQ167 (plant codon optimized: high GC content at 5' region)) with each Cas9 gene and nos Terminator The pYPQ 142 vector, which contains both gRNA1 and gRNA2, and pMDC123_35S _pro , a plant expression vector, are used to final connect using the multisite gateway assembly method. The pYPQ131A, pYPQ132A, pYPQ142, pYPQ150, pYPQ154, and pYPQ167 plasmid DNA used in this experiment can be purchased from Addgene.

In the present invention, sense and antisense primers for the base sequences of the finally selected gRNAs are prepared and annealed, and then operably cloned behind the AtU6 promoter (Arabidopsis U6 promter) in a plant expression vector. Using the golden gateway method, the expression cassettes of each gRNA are cloned into one vector. By recombining the SpCas9 gene derived from Streptococcus pyogenes , a gRNA expression cassette, and a T-DNA vector with a 35S promoter and ccdB recombination system, a multiplex CRISPR/Cas9 vector for plant transformation with a selection marker can be produced.

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

3. Soybean transformation and transformant selection

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

400ul of well-grown Agrobacterium cells were cultured in AB solid medium supplemented with antibiotics (0.5g glucose per 100ml, 1.5g agar/90ml water, 5ml 20x AB buffer (K2HPO4 60g/L, NaH2PO4 20g/L), 5ml 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) were plated and cultured. Agrobacterium cells that grew well by spreading on AB solid medium on the day of transformation were cultured in LCM medium (liquid CCM medium, 3.2g of B5 medium per liter, 30g of Sucrose, 158mg of Sodium thiosulfate, 154mg of DTT, 580mg of L-cysteine, 4.26g of MES, pH 5.4). with 1M KOH) to scrape from the surface of AB medium. Some of this was diluted to an OD ₆₀₀ of 0.6 (low concentration), and some of it was diluted to an OD ₆₀₀ of 2 (high concentration), then Acetosyringon (final 200uM) was added and cultured at 28 ^o C until Agrobacterium infection.

Additionally, in the present invention, transformation of plants with the vector can be performed by transformation techniques 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, wash the soybean seeds under running tap water for 30 minutes and then select seeds with normal seed coats. Prepared soybean seeds were sterilized in 70% ethanol for 30 seconds and washed three times with sterilized water. Afterwards, sterilize by shaking in 1% sodium hypochlorite solution for 30 minutes and then wash 5 times at 5-minute intervals. Germination medium (GM), 3.2g B5 medium per 1 liter, 20g Sucrose, 0.5g MES, 8g agar (sigma), pH5.7 with 1M KOH), with excess water removed, soybean seeds facing down. After sowing, germinate in the dark for 20 hours in an incubator at 26 ^o C.

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

The explants co-cultured for 4 days were washed three times for 5 minutes each with sterile water containing antibiotics (250 mg/L cefotaxim, 100 mg/L Timetin, 50 mg/L vancomycin) to remove Agrobacterium , and then washed in liquid containing the same antibiotics ( shoot) Wash three more times for 15 minutes in induction medium (SIMIP0 medium, 3.2g of B5 medium per 1L, 30g of Sucrose, 158mg of Sodium thiosulfate, 154mg of DTT, 8g of MES 0.6g agar (sigma), pH5.7 with 1M KOH) did. After washing, excess water was removed from the explant, and the hypocotyl portion was placed at an angle of about 45 degrees in SIMIP0 medium without antibiotics. Two weeks later, in households preparing half seed, large shoots that occurred due to axial buds not being removed properly are completely removed, and only the upper part of the shoot is removed and antibiotics are added to the remaining small shoots to facilitate antibiotic selection. Subcultured in SIMP10 medium.

After 2 weeks, the shoots that had browned due to PPT were removed using scissors, and the large callus mass formed in the hypocotyl area was trimmed and grown on SEMP5 medium (shoot elogtion medium_PPT5, 4.4g of MS B5 medium per 1 liter, 30g of Sucrose, 158 mg of Sodium thiosulfate, 154 mg of DTT). ㎎, 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) .

Subculture with the same SEMP5 medium every two weeks, and when the stem grows more than 4cm, cut the stem part of the soybean transformant and add antibiotic-free root induction medium (RIMP0, 4.4g of MS B5 medium 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). was derived. Transformants that developed roots on the cut side of the stem were transferred to small pots and acclimatized in a plastic container for two weeks, then transferred to large pots and grown in a greenhouse.

After purifying the redifferentiated soybean plants, transplant them into pots and grow them. To confirm the introduction of T-DNA into the redifferentiated plants, genomic DNA was extracted for each individual and introduction of the selection marker gene and gRNA expression cassette introduced through genomic PCR. Confirmed. In addition, the expression of the selection marker gene ( Bar ) in redifferentiated plants was confirmed using Bar-strip.

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

[Example]

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

Example 1. Highly efficient gRNA selection

In plants, phytic acid (Ins(1,2,3,4,5,6)P ₆ ) biosynthesis is a process involving many enzymes, and IPK1 (inositol 1,3,5,6-pentakisphosphate) is involved in this biosynthetic pathway. 2-kinase) enzyme is known to catalyze the final step of the phytic acid biosynthesis pathway, so the GmIPK1 gene was selected as a target gene to be deleted using the CRISPR/Cas9 system to produce low phytate soybeans through gene editing.

Accordingly, we attempted to select a gRNA that could specifically target the target sequence for the GmIPK1 (Glyma14g072200) gene. Here, “gRNA” is a different concept from guide RNA and refers to a spacer sequence corresponding to about 20 nucleotides (20nt) that binds to the target nucleic acid or target sequence within the gene in the guide RNA. do.

First, in order to select a highly efficient combination of gRNA1 and gRNA2 for the soybean gene editing CRISPR/Cas9 system, base sequence information on target genes for gene editing was obtained from the Phytozome database.

Within the GmIPK1 gene, which is predicted to have low off-target effects and high gene editing efficiency by correcting non-target genes using gene editing web tools such as 'CRISPR-P' or 'CRISPR RGEN Tools' A target sequence was selected, and a highly efficient combination of gRNA1 and gRNA2 that binds to this target sequence was selected. For this purpose, the target sequence within the target gene was identified based on the base sequence of the Willimas 82 variety used as a transgenic model.

At this time, because RGEN presents more off-target gene candidates than CRISPR-P, both programs were used to select a target sequence within the GmIPK1 gene with a low probability of being off-target. did. In addition, when the off-target gene candidates are finally selected, the gRNA binding site within the off-target gene is finally confirmed using the Jbrowse site to ensure high gene editing efficiency and -Target sequences within target genes with a low probability of being off-target were selected.

As a result, a combination of two gRNAs capable of specifically targeting the GmIPK1 gene encoding the IPK1 protein was finally selected. The target sequences targeted by the two finally selected gRNAs and their locations on the chromosome are shown in Figure 1. In addition, among the target sequences, the gRNAs that bind to the target sequence (Target 1) in Exon 2 and the target sequence (Target 2) in Exon 3 are named gRNA1 and gRNA2, respectively, The nucleic acid sequences encoding gRNA1 and gRNA2, respectively, are shown in Figure 1.

Additionally, Table 1 below shows the nucleic acid sequences encoding gRNA1 and gRNA2, respectively.

Name Sequence (5'→3') sequence number gRNA1 5'-GAAAGTGGTCCGCATACGTA-3' One gRNA2 5'-ATCACAATGTGCATCAACCC-3' 2

Example 2. Codon-optimized CRISPR/Cas9 vector construction

Next, the present invention introduces nucleic acids encoding gRNA1 and gRNA2, which each bind to two target sequences selected for each target gene, into one vector for the GmIPK1 gene, which is a target gene for soybean gene editing. It was decided to prepare a multiplex vector. This causes a definite indel mutation in the target gene by cutting two target sequence regions located in a wide range within one target gene, and after securing the transformant, Indel PCR is used to The purpose is to manufacture a system that has the advantage of more efficiently selecting transformants in which gene correction has occurred.

A multiplex vector containing gRNA1 and gRNA2 that specifically binds to the two selected target sites and a codon-optimized Cas9 gene from soybean was prepared. At this time, when using the AtU6 promoter for the purpose of expressing gRNA, it is more efficient for the nucleic acid encoding the gRNA to start with 'G', so SEQ ID NO: 1 (5'-GAAAGTGGTCCGCATACGTA-3), which is the nucleic acid encoding gRNA1 and gRNA2, respectively ') and SEQ ID NO: 2 (5'-ATCACAATGTGCATCAACCC-3'), base 'G' was added in front of the 5'-end of each, and this was used to prepare the multiplex vector. That is, gRNA1 included in the vector with the AtU6 promoter. and the nucleic acid encoding gRNA2 includes SEQ ID NO: 3 (5'- G GAAAGTGGTCCGCATACGTA-3') and SEQ ID NO: 4 (5'- G ATCACAATGTGCATCAACCC-3'), respectively.

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

A multiplex vector was prepared by cloning nucleic acids encoding gRNA1 and gRNA2 that bind to the two target sequences selected according to the present invention into the pECO201 vector. The pECO201 vector is a golden gateway entry vector for simultaneously introducing two or more gRNAs, has the Bar gene as a selection marker, and was designed to express Arabidopsis codon-optimized SpCas9 ( Arabidopsis codon optimized SpCas9) under the control of the 35S promoter. It is done. Two gRNAs were combined into the pECO201 vector through two processes. gRNA1 and gRNA2, which had undergone the first oligo annealing process, were ligated with pECO400_1,2 and pECO404_2,e, respectively, cut with Bsa I to obtain pGRNA1 and pGRNA2e plasmids. Second, pECO201_g1_g2 vector was finally created through golden gateway assembly of pGRNA1 and pGRNA2e and pECO201 vectors into which each gRNA was introduced.

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

Example 3. Verification of gene editing of soybean transformants

After transforming the CRISPR/Cas9 vector for soybean gene editing containing the nucleic acid encoding the selected gRNA1 and gRNA2, respectively, and the codon-optimized Cas9 gene produced in Example 2, into Agrobacterium EHA105, Willimas 82 It was transformed by co-culturing with explants of various soybean varieties.

After purifying the redifferentiated soybean plants, they were transplanted into pots and grown. To confirm the introduction of T-DNA into redifferentiated plants, genomic DNA was extracted for each individual and the introduction of the introduced selection marker gene and gRNA expression cassette was confirmed through genomic PCR (Figure 4).

As shown in Figure 4, a T ₀ line expressing gRNA1 and gRNA2 specifically targeting two target sites in the GmIPK1 gene was obtained (Bar PCR positive).

In addition, as shown in Figure 5, a transformant with a deletion of the nucleotide sequence between the two gRNA1 and gRNA2 targets was confirmed through InDel PCR using plants that showed a positive reaction in PCR for the Bar resistance gene.

It was confirmed through targeted deep sequencing that the obtained soybean transformant had an indel mutation at both target sequence sites in the GmIPK1 gene, resulting in a 577 bp deletion (Figure 6A). It is shown in Figure 6B that the obtained soybean transformant has 95 amino acids deleted from the IPK1 protein and thus cannot express the wild-type or normally functioning IPK1 protein.

Through the above results, it can be confirmed that gene editing targeting two target sites within the GmIPK1 gene simultaneously occurred efficiently in soybean plants using the CRISPR/Cas-based soybean gene editing system according to the present invention. there was. In addition, the present invention produced a soybean transformant with a deletion of the soybean GmIPK1 gene and secured its seeds. In addition, the soybean transformant and its seeds in which the soybean GmIPK1 gene has been corrected according to the present invention can be used to produce plants with various combinations of gene correction forms in the next generation.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> Gene editing system for GmIPK1 gene editing and uses its <130> PN21249 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA < 213> Artificial Sequence <220> <223> nucleotide sequence encoding gRNA1 <400> 1 gaaagtggtc cgcatacgta 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence encoding gRNA2 <400 > 2 atcacaatgt gcatcaaccc 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence encoding gRNA1 <400> 3 ggaaagtggt ccgcatacgt a 21 <210> 4 <211> 21 <212 > DNA <213> Artificial Sequence <220> <223> nucleotide sequence encoding gRNA2 <400> 4 gatcacaatg tgcatcaacc c 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 cttcttttcg attctggaga gaccgct gag 180 gctaccagat tgaagagaac cgctagaaga agatacacca gaagaaagaa cagaatctgc 240 taccttcagg aaatcttctc taacgagatg gctaaggttg atgattcttt cttccacaga 300 cttgaggagt ctttccttgt tgaggaggat aagaagcacg agagaacaccc aatcttc gga 360 aacatcgttg atgaggttgc ttaccacgag aagtacccaa ccatctacca ccttagaaag 420 aagttggttg attctaccga taaggctgat cttagactta tctaccttgc tcttgctcac 480 atgatcaagt tcagaggaca cttccttatc gagggagacc ttaacccaga taactctgat 540 gttgataagt tgttcatcca gcttgttcag acctacaacc agctt ttcga ggagaaccca 600 atcaacgctt ctggagttga tgctaaggct atcctttctg ctagactttc taagtctcgt 660 agacttgaga accttatcgc tcagcttcca ggagagaaga agaacggact 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 tct tgttaga 1020 cagcagcttc cagagaagta caaggaaatc ttcttcgatc agtctaagaa cggatacgct 1080 ggatacatcg atggaggagc ttctcaggag gagttctaca agttcatcaa gccaatcctt 1140 gagaagatgg atggaaccga ggagcttctt gttaagttga acagaga gga tcttcttaga 1200 aagcagagaa ccttcgataa cggatctatc ccacaccaga tccaccttgg agagcttcac 1260 gctatccttc gtagacagga ggatttctac ccattcttga aggataacag agagaagatc 1320 gagaagatcc ttaccttcag aatcccatac tacgttggac cacttgctag aggaaactct 1380 cgtttcgctt ggatgaccag aaagtctgag gagaccatca ccccttggaa cttcgaggag 14 40 gtaagtttct gcttctacct ttgatatata tataataatt atcattaatt agtagtaata 1500 taatatttca aatattttt tcaaaataaa agaatgtagt atatagcaat tgcttttctg 1560 tagtttataa gtgtgtatat tttaatttat aacttttcta atatatgacc aaaatttgtt 1620 g atgtgcagg ttgttgataa gggagcttct gctcagtctt tcatcgagag aatgaccaac 1680 ttcgataaga accttccaaa cgagaaggtt cttccaaagc actctcttct ttacgagtac 1740 ttcaccgttt acaacgagct taccaaggtt aagtacgtta ccgagggaat gagaaagcca 1800 gctttccttt ctggagagca gaagaaggct atcgttgatc ttcttttcaa gaccaacaga 1 860 aaggttaccg ttaagcagtt gaaggaggat tacttcaaga agatcgagtg cttcgattct 1920 gttgaaatct ctggagttga ggatagattc aacgcttctc ttggaaccta ccacgatctt 1980 ttgaagatca tcaaggataa ggatttcctt gataacgagg agaacgagga catcc ttgag 2040 gacatcgttc ttacccttac ccttttcgag gatagagaga tgatcgagga gagactcaag 2100 acctacgctc accttttcga tgataaggtt atgaagcagt tgaagagaag aagatacacc 2160 ggatggggta gactttctcg taagttgatc aacggaatca gagataagca gtctggaaag 2220 accatccttg atttcttgaa gtctgatgga ttcgctaaca gaaacttcat gcagcttatc 2280 cacgatgatt ctcttacctt caaggaggac atccagaagg ctcaggtttc tggacaggga 2340 gattctcttc acgagcacat cgctaacctt gctggatctc cagctatcaa gaaggggaatc 2400 cttcagaccg ttaaggttgt tgatgagctt gttaaggtta tgggtagaca caagccagag 2460 aa catcgtta tcgagatggc tagagagaac cagaccaccc agaagggaca gaagaactct 2520 cgtgagagaa tgaagagaat cgaggaggga atcaaggagc ttggatctca aatcttgaag 2580 gagcacccag ttgagaacac ccagcttcag aacgagaagt tgtaccttta ctaccttcag 2640 aacggaagag atatgtacgt tgatcaggag cttgacatca acagactttc tgattacgat 2700 gttgatcaca tcgttccac a gtctttcttg aaggatgatt ctatcgataa caaggttctt 2760 acccgttctg ataagaacag aggaaagtct gataacgttc catctgagga ggttgttaag 2820 aagatgaaga actactggag acagcttctt 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 aaggatttcc agttctacaa ggttagagag 3120 atcaacaact accaccacgc tcacgat gct taccttaacg ctgttgttgg aaccgctctt 3180 atcaagaagt acccaaagtt ggagtctgag ttcgtttacg gagattacaa ggtttacgat 3240 gttagaaaga tgatcgctaa gtctgagcag gagatcggaa aggctaccgc taagtacttc 3300 ttctactcta acatcatga a cttcttcaag accgagatca cccttgctaa cggagagatc 3360 agaaagagac cacttatcga gaccaacgga gagaccggag agatcgtttg ggataaggga 3420 agagatttcg ctaccgttag aaaggttctt tctatgccac aggttaacat cgttaagaaa 3480 accgaggttc agaccggagg attctctaag gagtctatcc ttccaaagag aaactctgat 3540 aagttgatcg ctagaaagaa ggattgggac ccaaagaagt acgg aggatt cgattctcca 3600 accgttgctt actctgttct tgttgttgct aaggttgaga agggaaagtc taagaagttg 3660 aagtctgtta aggagcttct tggaatcacc atcatggagc gttcttcttt cgagaagaac 3720 ccaatcgatt tccttgaggc taagggatac aag gaggtta agaaggatct tatcatcaag 3780 ttgccaaagt actctctttt cgagcttgag aacggaagaa agagaatgct tgcttctgct 3840 ggagagcttc agaagggaaa cgagcttgct cttccatcta agtacgttaa cttcctttac 3900 cttgcttctc actacgagaa gttgaaggga tctccagagg ataacgagca gaagcagctt 3960 ttcgttgagc agcacaagca ctaccttgat gagatcatcg a gcaaatctc tgagttctct 4020 aagagagtta tccttgctga tgctaacctt gataaggttc tttctgctta caacaagcac 4080 agagataagc caatcagaga gcaggctgag aacatcatcc accttttcac ccttaccaac 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 agtactctat cggactcgat atcggaacta actctgtggg atgggctgtg 60 atcacc gatg agtacaaggt gccatctaag aagttcaagg ttctcggaaa caccgatagg 120 cactctatca agaaaaacct tatcggtgct ctcctcttcg attctggtga aactgctgag 180 gctaccagac tcaagagaac cgctagaaga aggtacacca gaagaaagaa caggatctgc 240 tacctccaag agatcttctc taacgagatg gctaaagtgg atgattcatt cttccacagg 300 ctcgaagagt cattcctcgt ggaagaagat aaga agcacg agaggcaccc tatcttcgga 360 aacatcgttg atgaggtggc ataccacgag aagtacccta ctatctacca cctcagaaag 420 aagctcgttg attctactga taaggctgat ctcaggctca 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 agaacggact tttcggaaac 720 ttgatcgctc tctctctcgg actcacccct aacttca agt ctaacttcga tctcgctgag 780 gatgcaaagc tccagctctc aaaggatacc tacgatgatg atctcgataa cctcctcgct 840 cagatcggag atcagtacgc tgatttgttc ctcgctgcta agaacctctc tgatgctatc 900 ctcctcagtg atatcctcag ag tgaacacc gagatcacca aggctccact ctcagcttct 960 atgatcaaga gatacgatga gcaccaccag gatctcacac ttctcaaggc tcttgttaga 1020 cagcagctcc cagagaagta caaagagatt ttcttcgatc agtctaagaa cggatacgct 1080 ggttacatcg atggtggtgc atctcaagaa gagttctaca agttcatcaa gcctatcctc 1140 gagaagatgg atggaaccga ggaactcctc gtgaagctca atagagagg a tcttctcaga 1200 aagcagagga ccttcgataa cggatctatc cctcatcaga tccacctcgg agagttgcac 1260 gctatcctta gaaggcaaga ggatttctac ccattcctca aggataacag ggaaaagatt 1320 gagaagattc tcaccttcag aatcccttac tacgtgggac ctctcg ctag 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 tgagg aagcc tgcttttttg 1620 tcaggtgagc aaaagaaggc tatcgttgat ctcttgttca agaccaacag aaaggtgacc 1680 gtgaagcagc tcaaagagga ttacttcaag aaaatcgagt gcttcgattc agttgagatt 1740 tctggtgttg aggataggtt caac gcatct ctcgggaacct accacgatct cctcaagatc 1800 attaaggata aggatttctt ggataacgag gaaaacgagg atatcttgga ggatatcgtt 1860 cttaccctca ccctctttga agatagagag atgattgaag aaaggctcaa gacctacgct 1920 catctcttcg atgataaggt gatgaagcag ttgaagagaa gaagatacac tggttgggga 1980 aggctctcaa gaaagctcat taacggaatc agggataagc agtctggaaa gacaatcctt 20 40 gatttcctca agtctgatgg attcgctaac agaaacttca tgcagctcat ccacgatgat 2100 tctctcacct ttaaagagga tatccagaag gctcaggttt caggacaggg tgatagtctc 2160 catgagcata tcgctaacct cgctggatct cctgcaatca agaagggaat cctccagact 22 20 gtgaaggttg tggatgagtt ggtgaaggtg atgggaaggc ataagcctga gaacatcgtg 2280 atcgaaatgg ctagagagaa ccagaccact cagaagggac agaagaactc tagggaaagg 2340 atgaagagga tcgaggaagg tatcaaagag cttggatctc agatcctcaa agagcaccct 2400 gttgagaaca ctcagctcca gaatgagaag ctctacctct actacctcca gaacggaagg 2460 gatatgtatg t ggatcaaga gttggatatc aacaggctct ctgattacga tgttgatcat 2520 atcgtgccac agtcattctt gaaggatgat tctatcgata acaaggtgct caccaggtct 2580 gataagaaca ggggtaagag tgataacgtg ccaagtgaag aggttgtgaa gaaaatgaag 2640 a actattgga ggcagctcct caacgctaag ctcatcactc agagaaagtt cgataacttg 2700 actaaggctg agaggggagg actctctgaa ttggataagg caggattcat caagaggcag 2760 cttgtggaaa ccaggcagat cactaagcac gttgcacaga tcctcgattc taggatgaac 2820 accaagtacg atgagaacga taagttgatc agggaagtga aggttatcac cctcaagtca 2880 aagctcgtgt ctgatt tcag aaaggatttc caattctaca aggtgaggga aatcaacaac 2940 taccaccacg ctcacgatgc ttaccttaac gctgttgttg gaaccgctct catcaagaag 3000 tatcctaagc tcgagtcaga gttcgtgtac ggtgattaca aggtgtacga tgtgaggaag 3060 atga tcgcta agtctgagca agagatcgga aaggctaccg ctaagtattt cttctactct 3120 aacatcatga atttcttcaa gaccgagatt accctcgcta acggtgagat cagaaagagg 3180 ccactcatcg agacaaacgg tgaaacaggt gagatcgtgt gggataaggg aagggatttc 3240 gctaccgtta gaaaggtgct ctctatgcca caggtgaaca tcgttaagaa aaccgaggtg 3300 cagaccggtg gattctctaa aga gtctatc ctccctaaga ggaactctga taagctcatt 3360 gctaggaaga aggattggga ccctaagaaa tacggtggtt tcgattctcc taccgtggct 3420 tactctgttc tcgttgtggc taaggttgag aagggaaaga gtaagaagct caagtctgtt 3480 aaggaacttc tcggaatcac tatcatggaa aggtcatctt tcgagaagaa cccaatcgat 3540 ttcctcgagg ctaagggata caaagaggtt aagaaggatc tcatcatcaa gctcccaaag 3600 tactcactct tcgaactcga gaacggtaga aagaggatgc tcgcttctgc tggtgagctt 3660 caaaagggaa acgagcttgc tctcccatct aagtacgtta actttcttta cctcgcttct 3720 cactacgaga agttgaaggg atctccagaa gataacgag c agaagcaact tttcgttgag 3780 cagcacaagc actacttgga tgagatcatc gagcagatct ctgagttctc taaaagggtg 3840 atcctcgctg atgcaaacct cgataaggtg ttgtctgctt acaacaagca cagagataag 3900 cctatcaggg aacaggcaga gaacatcatc catctcttca cccttaccaa ccctcggtgct 3960 cctgctgctt tcaagtactt cgatacaacc atcgatagga agagatacac ctctaccaaa 4020 gaagtgctcg atgctaccct catccatcag tctatcactg gactctacga gactaggatc 4080 gatctctcac agctcggtgg tgattcaagg gctgat 4116 <210> 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 tcaagcgcac cgcccgccgc cgctacaccc gccgcaagaa ccgcatctgc 240 tacctccagg agatcttctc caacgagatg gcgaaggtcg acgactcctt cttccaccgc 300 ctcgaggagt ccttcctcgt ggaggaggac aagaagcacg agcgccaccc catcttcggc 360 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 a cctcatcgc ccagctccct ggtgagaaga agaacggtct tttcggtaac 720 ctcatcgctc tctccctcgg tctgacccct aacttcaagt ccaacttcga cctcgctgag 780 gacgctaagc ttcagctctc caaggatacc tacgacgatg atctcgacaa cctcctcgct 840 cagatt ggag atcagtacgc tgatctcttc cttgctgcta agaacctctc cgatgctatc 900 ctcctttcgg atatccttag ggttaacact gagatcacta aggctcctct ttctgcttcc 960 atgatcaagc gctacgacga gcaccacag gacctcaccc tcctcaaggc tcttgttcgt 1020 cagcagctcc ccgagaagta caaggagatc ttcttcgacc agtccaagaa cggctacgcc 1080 ggttacattg acggtggagc tagccaggag gagttctaca agttcatcaa gccaatcctt 1140 gagaagatgg atggtactga ggagcttctc gttaagctta accgtgagga cctccttagg 1200 aagcagagga ctttcgataa cggctctatc cctcaccaga tccaccttgg tgagcttcac 1260 gccatccttc gtaggca gga 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 aacctccccca acgagaaggt cctcccccaag cactccctcc tctacgagta cttcacggtc 1560 tacaacgagc tcaccaaggt caagtacgtc accgagggta tgcgcaagcc tgccttcctc 1620 tccggcgagc agaagaaggc tatcgttgac ctcctcttca agaccaaccg caaggtcacc 1680 gtca agcagc 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 gacttacgct 1920 catctcttcg atgacaaggt tatgaagcag ctcaagcgtc gccgtta cac cggttggggt 1980 aggctctccc gcaagctcat caacggtatc agggataagc agagcggcaa gactatcctc 2040 gacttcctca agtctgatgg tttcgctaac aggaacttca tgcagctcat ccacgatgac 2100 tctcttacct tcaaggagga tattcagaag gctcaggtg t ccggtcaggg cgactctctc 2160 cacgagcaca ttgctaacct tgctggttcc cctgctatca agaagggcat ccttcagact 2220 gttaaggttg tcgatgagct tgtcaaggtt atgggtcgtc acaagcctga gaacatcgtc 2280 atcgagatgg ctcgtgagaa ccagactacc cagaagggtc agaagaactc gagggagcgc 2340 atgaagagga ttgaggaggg tatcaaggag cttggttctc a gatccttaa 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 2760 cttgttgaga cgaggcagat taccaagcac gttgctcaga tcctcgattc taggat gaac 2820 accaagtacg acgagaacga caagctcatc cgcgaggtca aggtgatcac cctcaagtcc 2880 aagctcgtct ccgacttccg caaggacttc cagttctaca aggtccgcga gatcaacaac 2940 taccaccacg ctcacgatgc ttaccttaac gctgtcgttg gta ccgctct tatcaagaag 3000 taccctaagc ttgagtccga gttcgtctac ggtgactaca aggtctacga cgttcgtaag 3060 atgatcgcca agtccgagca ggagatcggc aaggccaccg ccaagtactt cttctactcc 3120 aacatcatga acttcttcaa gaccgagatc accctcgcca acggcgagat ccgcaagcgc 3180 cctcttatcg agacgaacgg tgagactggt gagatcgttt gggacaaggg tcgcgactt c 3240 gctactgttc gcaaggtcct ttctatgcct caggttaaca tcgtcaagaa gaccgaggtc 3300 cagaccggtg gcttctccaa ggagtctatc cttccaaaga gaaactcgga caagctcatc 3360 gctaggaaga aggattggga ccctaagaag tacggtggtt tcgactcccc tact gtcgcc 3420 tactccgtcc tcgtggtcgc caaggtggag aagggtaagt cgaagaagct caagtccgtc 3480 aaggagctcc tcggcatcac catcatggag cgctcctcct tcgagaagaa cccgatcgac 3540 ttcctcgagg ccaagggcta caaggaggtc aagaaggacc tcatcatcaa gctccccaag 3600 tactctcttt tcgagctcga gaacggtcgt aagaggatgc tggcttccgc tggtgagctc 3660 cagaagggta ac gagcttgc tcttccttcc aagtacgtga acttcctcta cctcgcctcc 3720 cactacgaga agctcaaggg ttcccctgag gataacgagc agaagcagct cttcgtggag 3780 cagcacaagc actacctcga cgagatcatc gagcagatct ccgagttctc caagcgcgtc 384 0 atcctcgctg acgctaacct cgacaaggtc ctctccgcct acaacaagca ccgcgacaag 3900 cccatccgcg agcaggccga gaacatcatc cacctcttca cgctcacgaa cctcggcgcc 3960 cctgctgctt tcaagtactt cgacaccacc atcgacagga agcgttacac gtccaccaag 4020 gaggttctcg acgctactct catccaccag tccatcaccg gtctttacga gactcgtatc 4080 gacctttccc agcttggtgg t gat 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 acid; and nucleic acids encoding gRNA1 and gRNA2, respectively targeting two target sites in the GmIPK1 gene of soybean; A vector for soybean gene editing operably linked, comprising:
The codon-optimized Cas9 nucleic acid consists of the base sequence of SEQ ID NO: 6, and the nucleic acids encoding gRNA1 and gRNA2 consist of the base sequence of SEQ ID NO: 1 and SEQ ID NO: 2, respectively; Or a vector for soybean gene editing, characterized in that it consists of the base sequence of SEQ ID NO: 3 and the base sequence of SEQ ID NO: 4.
According to clause 5,
The vector is a vector for soybean gene editing, characterized in that it further contains one or more operably linked promoters, a plant selection marker, and a nuclear localization signal (NLS).
delete A composition for soybean gene editing, comprising the vector of claim 5.
According to clause 8,
A composition for soybean gene editing, wherein the nucleic acids encoding gRNA1 and gRNA2 are composed of the base sequence of SEQ ID NO: 1 and the base sequence of SEQ ID NO: 2, respectively, and delete the soybean GmIPK1 gene.
A soybean gene correction method comprising the step of transforming soybean using the vector of claim 5, and deleting the soybean GmIPK1 gene.
A soybean transformant in which the GmIPK1 gene, an enzyme that regulates phytic acid biosynthesis in soybeans, was transformed by the soybean gene editing method of claim 10.
A soybean transformant transformed by the method of claim 10, in which the GmIPK1 gene, an enzyme that regulates phytic acid biosynthesis, has been corrected.