Corn plant having herbicide resistance trait through base-editing of epsps gene, and method for producing same

Abstract

The present invention relates to a recombinant vector comprising: a guide RNA expression cassette targeting the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene of corn (Zea mays L.); and a cytosine base editor expression cassette having a cytidine deaminase and a nuclease variant fused therein, and a method for producing a corn plant having a herbicide resistance trait through base-editing of the EPSPS gene using the recombinant vector.

Description

Corn plant having herbicide tolerance trait by EPSPs gene base correction and method for producing same

[0001] The present invention relates to a corn plant having a herbicide tolerance trait by base correction of the EPSPs (5-enolpyruvylshikimate-3-phosphate synthase) gene and a method for producing the same.

[0002]

[0003] This work was carried out with the support of the Rural Development Administration's Next-Generation Crop New Breeding Technology Development (R&D) project (Project Number: PJ014883032021).

[0004] The genetically modified (GM) seed market, which contains herbicide tolerance and pest resistance traits, accounts for approximately one-third (KRW 20 trillion) of the global seed market. Most GM seed development is costly and time-consuming, and is monopolized by multinational corporations, making it difficult for small and medium-sized enterprises (SMEs) to access. Multinational corporations develop GMOs by inserting specific transgenes. Herbicide-resistant seeds have been developed by conferring herbicide resistance to crops through mechanisms that protect herbicide-sensitive amino acid-producing enzymes.

[0005] Attempts to induce herbicide resistance using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, rather than GMOs, require the use of base-editing technology. To preserve the function of enzymes within the amino acid metabolic pathway while maintaining herbicide sensitivity, a small number of bases within the target gene must be mutated to alter the amino acid sequence. Developing seeds using this cutting-edge breeding technology, gene editing, could not only replace GMOs, but also be easily developed by general seed companies or venture companies, rather than multinational corporations.

[0006] The global corn seed market will account for approximately 35% of the global grain and cereal seed market in 2022, and herbicide tolerance, a key trait in corn, is inherent in GM corn varieties. As of 2022, South Korea's imports of GM soybeans and corn reached $4.26 billion (approximately KRW 5.7 trillion), accounting for approximately 25% of the country's total crop production. Due to this massive import volume, South Korea's grain self-sufficiency rate is only 23%. Furthermore, halting imports would have a significant impact on the livestock feed and processed food markets, so imports of GM soybeans and corn from overseas are expected to continue.

[0007] Recently, base editors that can correct the genome without double-strand breaks in DNA have been developed, such as adenine base editors (ABEs) and cytosine base editors (CBEs). Among these, cytosine base editors are constructed by fusing a naturally occurring cytidine deaminase to dCas9 (dead Cas9, a mutant of Cas9 containing D10A and H840A) or nCas9 (nickase Cas9), and can correct cytosine to thymine without gene cleavage or insertion of additional template (donor DNA).

[0008] Meanwhile, Korean Patent Registration No. 2061438 discloses 'a method for converting a monocotyledonous plant genome sequence in which a nucleic acid base in a targeting DNA sequence is specifically converted, and a molecular complex used therefor', and Korean Publication Patent No. 2022-0149325 discloses 'a herbicide-resistant plant and a method for producing the same' using a guide RNA targeting the afb4 or afb5 gene, but 'a corn plant having a herbicide-resistant trait by base correction of the EPSPS gene and a method for producing the same' of the present invention is not described.

[0009] The present invention was derived from the above-mentioned needs, and the inventors of the present invention attempted to develop herbicide-resistant corn using a non-GMO gene editing technology, and to develop a new herbicide-resistant crop by artificially performing base substitution of the EPSPs (5-enolpyruvylshikimate-3-phosphate synthase) gene, which responds to the herbicide glyphosate, using base-editing technology among gene editing technologies.

[0010] To this end, the inventors of the present invention introduced a base correction system targeting the corn EPSPS gene into corn protoplasts and analyzed the C to T base correction efficiency of the EPSPS gene by a specific guide RNA. As a result, excellent base correction efficiency and patterns were confirmed in protoplasts into which a vector expressing dual guide RNA was introduced. Using the transformation vector, several corn T0 plants in which the target amino acid of the corn EPSPS genome was base-corrected were obtained, and F1 seeds were obtained by crossbreeding with the B73 parent line, and F1 plants being cultivated in an LMO greenhouse. F2 seeds were obtained through crossing the F1 plants, and a glyphosate herbicide tolerance test was performed on the F2 seedlings cultivated therefrom. As a result, increased plant tolerance was confirmed compared to the control group, thereby completing the present invention.

[0011] To solve the above problem, the present invention provides a recombinant vector comprising a guide RNA expression cassette targeting the EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene of maize (Zea mays L.); and a cytosine base editor expression cassette fused with a cytidine deaminase and nuclease variant.

[0012] In addition, the present invention provides a method for producing a corn base-corrected plant having a herbicide tolerance trait, comprising the steps of (a) introducing the recombinant vector into a corn plant cell to correct the genome; and (b) regenerating a corn plant from the corn plant cell in which the genome has been corrected.

[0013] In addition, the present invention provides a corn base-corrected plant having a herbicide tolerance trait produced by the above method and a seed having its genome corrected.

[0014] In addition, the present invention provides a base correction composition for increasing herbicide resistance of corn plants, which contains the recombinant vector as an active ingredient.

[0015] The present invention has enabled the acquisition of genetic diversity necessary for corn breeding through Agrobacterium transformation-based base-editing. Furthermore, by utilizing base-editing technology to select herbicide-resistant individuals, it is expected that non-GMO status can be scientifically proven, thereby alleviating public concerns. If commercialized, it is expected to attract attention as an import substitute and reduce GMO imports. Furthermore, utilizing gene editing technology in the development of new varieties can significantly reduce costs compared to GMO development and reduce the time required by conventional breeding methods by more than half.

[0016] Figure 1 shows the base correction sites of the corn EPSPS gene, the locations where glyphosate resistance was reported to be caused by amino acid changes in the EPSPS gene, and the predicted amino acids changed by cytosine base correction.

[0017] Figure 2 is a schematic diagram of a cytosine base editing vector used for corn transformation in the present invention, wherein the optimized binary vector includes two sgRNA expression cassettes and an APOBEC3A expression cassette fused with nCas9-NG. RB: T-DNA right border, OsU6-2: Rice U6-2 Pol Ⅲ promoter, ZmUbi(P): maize Ubiquitin promoter, UTR: 5'-UTR ofOsADH (Oryza sativa alcohol dehydrogenase), NLS: nuclear localization signal, APOBEC3A: Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A, 16aa linker: 16aa residue peptides, nCas9(NG): nickase Cas9(D10A) containing 7 mutations(L111R, D1135V, G1218R, E1219F, A1322R, R1335V, T1337R), UGI: Uracil glycosylase inhibitor, Pea3A(T): pea3A terminator fromPisum sativum. LB: T-DNA left border.

[0018] Figure 3 shows the base correction efficiency of cytosine base correction vectors with single or dual guide RNAs in maize protoplasts. Analysis results confirmed that the pTG5 vector with dual guide RNAs corrected at threonine, alanine, and proline positions.

[0019] Figure 4 shows the results of analyzing the correction efficiency and pattern of two types of cytosine base correction vectors in corn protoplasts.

[0020] Figure 5 shows the appearance of PPT (phosphinothricin) selected transgenic corn callus and the results of analyzing the base correction efficiency in the callus.

[0021] Figure 6 shows the results of analysis of the EPSPS gene base correction pattern of selected transgenic corn calli.

[0022] Figure 7 shows the production of corn T0 transformants and their cultivation in a glass greenhouse.

[0023] Figure 8 shows the results of deep sequencing analysis of the target region of the EPSPS gene in maize T0 regenerated individuals. Maize T0 individuals A26, A32, A39, B3, and C30, which were base-corrected for the EPSPS gene using the pTG4 vector, were derived from the callus line (TG4-1, TG4-8, TG4-9, or TG4-13) of Figure 6, and maize T0 individuals B11, B12, B13, B14, B17, C16, A22, B2, B4, B6, and B37, which were base-corrected for the EPSPS gene using the pTG5 vector, were derived from the callus line (TG5-6 or TG5-13) of Figure 6.

[0024] Figure 9 shows the appearance of seeds of the F1 corn transformant obtained by cross-pollination of the corn T0 transformant and the B73 corn line, in which base correction of the EPSPS gene was confirmed.

[0025] Figure 10 shows the germinated F1 seedlings of base-corrected corn and the artificial cross in the LMO house.

[0026] Figure 11 shows the results of a glyphosate herbicide tolerance test of germinated F2 seedlings of base-corrected corn.

[0027] In order to achieve the object of the present invention, the present invention provides a recombinant vector comprising a guide RNA expression cassette targeting the EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene of corn (Zea mays L.); and a cytosine base editor expression cassette fused with a cytidine deaminase and nuclease variant.

[0028] The term "base editors (BEs)" used in the present invention refers to a single base correction means, and more specifically, are constructed by fusing adenosine deaminase or cytidine deaminase to the amino-terminus of Cas9 nickase (nCas9), and are named adenine base editors (ABEs) and cytosine base editors (CBEs), respectively. The BEs correct adenine to guanine at a specific site and CBEs correct cytosine to thymine at a specific site, without causing double-strand breaks.

[0029] The term "recombinant" as used herein refers to a cell that replicates a heterologous nucleic acid, expresses said nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid. A recombinant cell may express a gene or gene fragment not found in the cell's native form, either in sense or antisense form. Furthermore, a recombinant cell may express a gene found in the cell's native form, but in a modified form that has been reintroduced into the cell by artificial means.

[0030] Additionally, the term "vector" is used to refer to a DNA fragment(s) or nucleic acid molecule that is delivered into a cell. A vector replicates DNA and can reproduce independently in a host cell. The term "vector" is often used interchangeably with "vector."

[0031] The term "expression cassette" as used herein refers to a nucleic acid sequence comprising one or more genes and sequences that regulate their expression, for example, any combination of various cis-acting transcriptional regulatory elements. The expression cassette of the present invention comprises three main elements: i) a promoter; ii) a second polynucleotide, which may be referred to as a "coding polynucleotide" or "coding sequence" (also referred to as a coding gene), which is operably linked to the promoter and whose transcription is directed by the promoter when the expression cassette is introduced into a cell; and iii) a terminator polynucleotide (also referred to as a transcription terminator) that directs the termination of transcription and is located immediately downstream of the second polynucleotide.

[0032] The above promoters are suitable promoters for transformation, and are preferably, but not limited to, the CaMV 35S promoter, the actin promoter, the ubiquitin promoter, the pEMU promoter, the MAS promoter, the histone promoter, or the Clp promoter. The term "promoter" refers to a region of DNA upstream of a structural gene and refers to a DNA molecule to which RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in a plant cell. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental states or cell differentiation, and the use of a constitutive promoter may be preferred in the present invention. Therefore, a constitutive promoter does not limit the possibilities of selection.

[0033] In the recombinant vector of the present invention, conventional terminators can be used, and examples thereof include, but are not limited to, the Pisum sativum3A terminator, the nopaline synthase terminator, the rice α-amylase RAmy1 A terminator, the terminator of the Octopine gene of Agrobacterium tumefaciens, the phaseoline terminator, and the rrnB1 / B2 terminator of Escherichia coli. Regarding the necessity of terminators, it is generally known that terminator regions increase the certainty and efficiency of gene transcription in plant cells. Therefore, the use of terminators is highly preferred in the context of the present invention.

[0034] In the recombinant vector of the present invention, the guide RNA targeting the corn EPSPS gene may target the base sequences of SEQ ID NO: 3 and SEQ ID NO: 4, and two guide RNA expression cassettes are included in the recombinant vector so that each guide RNA can be expressed simultaneously.

[0035] In the present invention, the term "guide RNA" refers to a short single-stranded RNA, which is specific to a target DNA among the base sequences encoding a target gene, and refers to a ribonucleic acid that serves to guide a nuclease protein to the target DNA base sequence by complementarily binding to all or part of the target DNA base sequence. The guide RNA may be a dual RNA comprising two RNAs, namely, crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) as components; or a single-stranded guide RNA (single guide RNA, sgRNA) comprising a first portion including a sequence that is all or partly complementary to a base sequence in a target gene and a second portion including a sequence that interacts with a nuclease (particularly, an RNA-guided nuclease), but may be included in the scope of the present invention without limitation, and may be manufactured and used according to a technique known in the art, taking into account the type of nuclease used together or the microorganism from which the nuclease is derived, etc. The above guide RNA is adjacent to the PAM (protospacer adjacent motif) site and may include a sequence complementary to a 10 to 20 bp base sequence of the DNA to be edited, but is not limited thereto.

[0036] In a recombinant vector according to one embodiment of the present invention, the guide RNA expression cassette may be, but is not limited to, operably linked in the 5' to 3' direction to: a rice U6-2 polymerase III promoter; a guide RNA coding sequence; a guide RNA scaffold coding sequence; and a pea (Pisum sativum) pea3A terminator.

[0037] In the present invention, the base sequences of the rice U6-2 polymerase III promoter, guide RNA scaffold coding sequence, and pea pea3A terminator may be composed of the base sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively, and may all be optimized for corn codons, but are not limited thereto.

[0038] Additionally, in the recombinant vector of the present invention, the cytidine deamination enzyme may be APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like), more preferably APOBEC3A, but is not limited thereto.

[0039] In addition, in the recombinant vector of the present invention, the nuclease variant may preferably be nickase Cas9 (CRISPR associated protein 9) recognizing NGG-PAM (protospacer adjacent motif) or NG-PAM, and more preferably nickase Cas9-NG recognizing NG-PAM, but is not limited thereto.

[0040] In one embodiment of the present invention, the nickase Cas (nCas9) is a Cas9 D10A nickase lacking RuvC-like nuclease domain activity, which is an enzyme protein having an activity of nicking a complementary DNA strand of crRNA. In addition, the Cas9-NG is a mutant protein having seven amino acid mutations (L1111R, D1135V, G1218R, E1219F, A1322R, R1335V, T1337R) in the Cas9 protein (wild type) derived from Streptococcus pyogenes, thereby recognizing the PAM sequence as 5'-NG without dependence on the third G of the 5'-NGG PAM sequence of the wild type Cas9.

[0041] In addition, in the recombinant vector of the present invention, the cytosine base-editing gene scissors expression cassette may additionally include a UGI (uracil DNA-glycosylase inhibitor) peptide coding sequence, and the UGI peptide may be linked directly to the carboxy-terminus of the nuclease variant or via a linker, but is not limited thereto.

[0042] In a recombinant vector according to one embodiment of the present invention, the cytosine base-editing gene scissors expression cassette may be, but is not limited to, operably linked in the 5' to 3' direction to: a corn ubiquitin promoter; a 5'-UTR derived from a rice ADH (Oryza sativaalcohol dehydrogenase) gene; an NLS (nuclear localization signal) coding sequence; an APOBEC3A coding sequence; a linker coding sequence; an nCas9-NG coding sequence; a UGI coding sequence; and a pea pea3A terminator.

[0043] In the present invention, the corn ubiquitin promoter and the 5'-UTR sequence derived from the rice ADH gene may be the base sequences of SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The target gene can be highly expressed in a plant by the enhancer function of the 5'-UTR derived from the rice ADH gene.

[0044] In addition, in the present invention, the APOBEC3A coding sequence, linker coding sequence, nCas9-NG coding sequence, and UGI coding sequence may be composed of the base sequences of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively, and may all be optimized for corn codons, but are not limited thereto.

[0045] Additionally, in the recombinant vector of the present invention, the NLS sequence may be composed of an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15, and its coding sequence may be composed of a base sequence of SEQ ID NO: 16 and SEQ ID NO: 17, respectively, and may be optimized for corn codons, but is not limited thereto.

[0046] In addition, in the recombinant vector of the present invention, the herbicide may preferably be glyphosate, but is not limited thereto. Glyphosate is an aminophosphonic acid (AMPA) analog of the amino acid glycine, and is a broad-spectrum herbicide that kills plants by interfering with the shikimate pathway, which produces aromatic amino acids such as phenyalanine, tyrosine, and tryptophan.

[0047] The present invention also provides a method for producing a corn base-corrected plant having a herbicide tolerance trait, comprising the steps of: (a) introducing a recombinant vector of the present invention into a corn plant cell to correct the genome; and (b) regenerating a corn plant from the corn plant cell in which the genome has been corrected.

[0048] In the method for producing a corn base-corrected plant according to the present invention, the recombinant vector and herbicide are as described above.

[0049] The recombinant vector according to the present invention comprises guide RNAs of SEQ ID NO: 3 and SEQ ID NO: 4 targeting the corn EPSPs gene and cytosine base-editing genetic scissors, so that cytosine can be corrected to thymine at the target site of the corn EPSPs gene. The target site for the guide RNA of the present invention includes a region encoding the 164th threonine, the 165th alanine, and the 168th proline of the corn EPSPs protein (amino acid sequence of SEQ ID NO: 2), and resistance to the herbicide glyphosate is achieved through base-editing of the coding region of the amino acid residues.

[0050] In addition, the method for producing a corn base-corrected plant according to the present invention may additionally include a step of producing F1 seeds by cross-breeding the regenerated corn plant of step (b) with a corn strain with strong water resistance, but is not limited thereto.

[0051] The above-mentioned strong corn strain is not limited to this, but may preferably be B73.

[0052] In the method for producing a corn base-corrected plant according to the present invention, introducing the recombinant vector into a plant cell refers to a transformation method. Transformation of plant species is now commonplace, including both dicotyledonous and monocotyledonous plants. In principle, any transformation method can be used to introduce the recombinant vector according to the present invention into a suitable progenitor cell.

[0053] Methods for transducing the above recombinant vector into plant cells include calcium / polyethylene glycol method for protoplasts (Krens et al., 1982, Nature 296:72-74; Negrutiu et al., 1987, Plant Mol. Biol. 8:363-373), electroporation of protoplasts (Shillito et al., 1985, Bio / Technol. 3:1099-1102), microinjection into plant elements (Crossway et al., 1986, Mol. Gen. Genet. 202:179-185), particle bombardment of various plant elements (DNA or RNA-coated) (Klein et al., 1987, Nature 327:70), and Agrobacterium tumefaciens-mediated gene transfer (incomplete). It can be appropriately selected from infections caused by bacteria, etc.

[0054] In the method for producing a corn base-corrected plant according to the present invention, the "plant cell" into which the recombinant vector is introduced may be any plant cell. The plant cell is a cultured cell, cultured tissue, cultured organ, or whole plant. The "plant tissue" includes differentiated or undifferentiated plant tissues, such as, but not limited to, roots, stems, leaves, pollen, microspores, egg cells, seeds, and various types of cells used for culture, i.e., single cells, protoplasts, shoots, and callus tissues. The plant tissue may be in planta or in an organ culture, tissue culture, or cell culture state. A preferred plant cell according to the present invention is a protoplast.

[0055] In the manufacturing method of the present invention, any method known in the art can be used to regenerate genome-edited plants from genome-edited plant cells. Genome-edited plant cells must be regenerated into whole plants. Techniques for regenerating mature plants from callus or protoplast cultures are well known in the art for numerous and diverse species.

[0056] The present invention also provides a corn base-corrected plant having a herbicide tolerance trait produced by the above method and a seed having its genome corrected.

[0057] The corn base-edited plant having a herbicide tolerance trait according to the present invention is a corn plant in which the EPSPs gene related to glyphosate tolerance, a herbicide, is base-edited using a cytosine base editor system, and cytosine at a specific position of the corn EPSPs gene is corrected to thymine, thereby increasing tolerance to the herbicide glyphosate compared to a corn plant in which base-editing has not occurred.

[0058] The present invention also provides a base correction composition for increasing herbicide resistance of corn plants, which contains the recombinant vector of the present invention as an active ingredient.

[0059] In the base correction composition according to the present invention, the recombinant vector and herbicide are as described above.

[0060]

[0061] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0062]

[0063] Example 1. Construction of a cytosine base correction vector for corn base correction.

[0064] The recombinant vector used in the present invention is constructed with a basic configuration of cytidine deaminase APBEC3A, which includes a wide window of correction efficiency, in a nickase Cas9-NG (PAM sequence NG) expressed by a ZmUbi promoter for corn expression and an sgRNA expression cassette (OsU6-2 promotor) for monocots. nCas9-NG was optimized for plant codons to optimize plant expression, and nickase Cas9-NG, which introduced HindⅢ, XbaI, NdeI, AatⅡ, ApaI, SacI, and EcoRI restriction enzymes for sub-cloning into a transformation vector, and the base sequences of NLS sequence, APOBEC3A sequence, 16aa linker sequence, UGI sequence, and pea 3A terminator were synthesized and recombined to produce a synthetic DNA clone for plant base editing with the basic structure of cytidine deaminase + nCas9-NG + UGI sequence. The final cytidine deaminase base correction vector for corn Agrobacterium-mediated transformation used in the present invention was completed by introducing recombinant synthetic DNA into the HindIII and EcoRI sites of the MCS (multiple cloning site) of the pCAMBIA 3300 binary vector, in which the commonly used PPT (Phosphinothricin) plant selection marker is used, using a restriction enzyme (Fig. 2).

[0065]

[0066] Example 2. Production of corn transformants

[0067] Based on the report by Hong et al. (2019, Korean J. Breed. Sci. 51(4):290-297), the Agrobacterium-mediated transformation process of corn was performed at the National Institute of Agricultural Sciences.

[0068]

[0069] 2-1. Plant materials

[0070] Corn cultivar Hi-ⅡA was obtained from the United States Department of Agriculture (USDA). Corn seeds were soaked in water and germinated at 28°C. Four days after germination, they were transplanted into 50-well pots. Once the leaves reached 15–20 cm in length, they were transplanted to the field for cultivation. Optimal immature embryos were aseptically extracted within 9–15 days after pollination, at a size of 1.5–2.0 mm, and used for transformation.

[0071]

[0072] 2-2. Agrobacterium culture

[0073] For maize transformation, the vectors constructed in Example 1 were introduced into Agrobacterium tumefaciens EHA101 by tri-parental mating, and colonies with confirmed gene introduction were used for maize transformation. Maize transformation was performed based on the method of Ishida et al. (2007, Nat Protoc. 2(7):1614-21) and Lee & Zhang (2016, Curr Protoc Plant Biol. 1(1):121-137) with some modifications to the medium composition. Agrobacterium containing the transformation vector was plated on YEP solid medium containing 50 mg / L spectinomycin and cultured at 28°C for 3 days. The cultured bacteria were plated on AB medium and cultured at 20°C for 3 days. The cultured bacteria were inoculated into 5 ml of inoculation medium (N6 salts, N6 vitamins, 1 g / L casamino and vitamin assay, 1.5 mg / L dicamba, 0.5 mg / L 2,4-D, 68.4 g / L sucrose, 36 g / L glucose, 100 μM acetosyringone, pH 5.2) at an OD 600 After adjusting to 1.0, it was cultured at 24°C at 100 rpm for 4 hours and used as an inoculation solution.

[0074]

[0075] 2-3. Production of transformants

[0076] The optimally immature embryos, 1.5-2.0 mm in size, were soaked in the Agrobacterium inoculum containing the prepared vector for 5 minutes. The inoculated immature embryos were placed on co-culture medium (N6 salts, N6 vitamins, 1.22 mg / L CuSO4, 0.7 mg / L L-proline, 0.5 mg / L MES, 30 g / L sucrose, 10 g / L glucose, 1.5 mg / L dicamba, 0.5 mg / L 2,4-D, 300 mg / L L-cysteine, 1 mM DTT, 0.85 mg / L AgNO3, 100 μM acetosyringone, 3 g / L Gelrite, pH 5.8) with the scutellum tissue facing upward and the hypocotyl touching the medium, and co-cultured at 21°C for 3 days. After co-culture, infected immature embryos were transferred to resting medium (N6 salts, N6 vitamins, 0.7 mg / L L-proline, 0.5 mg / L MES, 30 g / L sucrose, 1.5 mg / L dicamba, 0.5 mg / L 2,4-D, 0.85 mg / L AgNO3, 150 mg / L timentin, 3 g / L Gelrite, pH 5.8) and cultured in the dark at 28°C for 7 days.

[0077] After culture, immature embryos were transferred to the first selection medium (SM1: N6 salts, N6 vitamins, 0.7 mg / L L-proline, 0.5 mg / L MES, 30 g / L sucrose, 1.5 mg / L dicamba, 0.5 mg / L 2,4-D, 1.5 mg / L bialaphos, 0.85 mg / L AgNO3, 150 mg / L timentin, 3 g / L Gelrite, pH 5.8) and cultured in the dark at 25°C for 2 weeks. The generated calli were transferred to the second selection medium (SM2: N6 salts, N6 vitamins, 0.7 mg / L L-proline, 0.5 mg / L MES, 30 g / L sucrose, 1.5 mg / L dicamba, 0.5 mg / L 2,4-D, 3.0 mg / L bialaphos, 0.85 mg / L AgNO3, 150 mg / L timentin, 3 g / L Gelrite, pH 5.8) and subcultured three times at 2-week intervals under dark conditions at 28°C. The vigorously growing embryogenic calli were transferred to the third selection medium (SM3: N6 salts, N6 vitamins, 0.7 mg / L L-proline, 0.5 mg / L MES, 30 g / L sucrose, 1.5 mg / L 2,4-D, 3.0 mg / L bialaphos, 0.85 mg / L AgNO3, 150 mg / L timentin, 3 g / L Gelrite, pH 5.8) and subcultured at 28°C in the dark for 2 months at 2-week intervals. Large and fast-growing calli were split and transferred to new medium.

[0078] Embryo-like calli with Type II characteristics and regeneration potential were transferred to maturation medium (MM: MS salts, MS vitamins, 60 g / L sucrose, 2.0 mg / L glycine, 3.0 mg / L bialaphos, 150 mg / L timentin, 3 g / L Gelrite, pH 5.6) and cultured at 28℃ in the dark for 2-3 weeks to mature the embryos. The matured embryos were transferred to regeneration medium (RM: 2.9 g / L MS salts, MS vitamins, 30 g / L sucrose, 2.0 mg / L glycine, 3 g / L Gelrite, pH 5.6) and cultured at 25℃ in the dark for 16 h until shoots and roots emerged. After this, the small plantlets were transferred to a new regeneration medium and grown for 2 weeks under conditions of 25°C and 16 hours of light. After acclimatizing to soil, they were grown in a greenhouse and self-pollinated to harvest seeds.

[0079]

[0080] Example 3. Comparison of proofreading efficiency of single and dual guide base correction vectors.

[0081] 3-1. Corn protoplast isolation and transformation vector verification

[0082] Corn protoplasts were isolated from the cotyledons of seedlings germinated in vitro, and the final vector DNA was secured at a high concentration and introduced into the protoplasts using the PEG transfection method.

[0083] The method of Yoo et al. (2007, Nat Protoc. 2(7):1565-72) was referenced and modified to isolate maize protoplasts and introduce vectors into the protoplasts using PEG method. More specifically, to isolate maize protoplasts, maize B73 line seeds were sterilized in 70% ethanol, 0.4% hypochlorite solution for 15 min, washed three times with distilled water, germinated on 1 / 2X MS solid medium containing 2% sucrose, and then cultured. The culture was performed in a growth room at 150 μmol / m for 16 h. 2 s), and was performed at 25℃ under 8-hour dark conditions. Corn cob on the 9th day of Magenta box culture was finely chopped and incubated with 15 ml of enzyme solution [2% VCP (Viscozyme, Celluclast, PectinEX 2:1:1), 0.45 M mannitol, 20 mM MES (pH 5.7), CPW solution] at 25℃ in the dark for 4-6 hours with stirring at 40 rpm for digestion, and then diluted with an equal volume of W5 solution. The enzyme-treated mixture was filtered through a 50 μm mesh and centrifuged at 100 g for 5 minutes in a round-bottom tube to recover protoplasts. Protoplasts resuspended in 0.45 M CPW solution were purified by floating on CPW 21S solution (CPW solution containing 21% (w / v) sucrose, pH 5.8) and centrifuged at 150 g for 7 min. The purified protoplasts were washed with W5 solution and centrifuged at 70 g for 5 min to produce a pellet. Finally, the protoplasts were resuspended in W5 solution and counted under a microscope using a hemacytometer.

[0084] 1×10 above 5The maize protoplast mixture was resuspended in 200 μl of MMG solution (0.4 M mannitol, 15 mM MgCl2, 4 mM MES, pH 5.7), and 20–30 μg of the vector DNA constructed in Example 1 was added. The mixture was carefully mixed with 220 μl of freshly prepared PEG solution [40% (w / v) PEG 4000 (Sigma No. 95904), 0.2 M mannitol, 0.1 M CaCl2], and incubated for 10 min in the dark at 25°C. After incubation, 950 μl of W5 solution (2 mM MES (pH 5.7), 154 mM NaCl, 125 mM CaCl2, 5 mM KCl) was slowly added. The solution was then mixed well by inverting the tube. The protoplasts were then pelleted by centrifugation at 100 g for 3 min and carefully resuspended in 1 ml of W5 or WI solution (0.5 M mannitol, 20 mM KCl, and 4 mM MES, pH 5.7). Finally, the protoplasts were transferred to a multi-well plate and incubated at 25°C in the dark for 48 to 72 h.

[0085]

[0086] 3-2. Base correction efficiency analysis

[0087] Maize protoplast genomic DNA was isolated and subjected to tarted deep sequencing using an Illumina Mi-Seq instrument. The raw data was analyzed using the BE-Analyzer program of CRISPR RGRN Tools to analyze the base correction efficiency of guide RNA.

[0088] As shown in Fig. 3, the efficiency of guide RNA (EPSPs #2, EPSPs #3) in maize protoplasts of the four types of ZmEPSPs transformation vectors produced was analyzed by deep-seq to confirm the presence and efficiency of base correction targeting the maize EPSPs gene. Two types of vectors were selected as the final maize transformation vectors: pTG4 vector with a single guide RNA (OsU6-2::EPSPs #3 + ZmUbi P::APOBEC3A_nCas9-NG) and pTG5 vector with dual guides (OsU6-2::EPSPs #2 + ZmUbi P::APOBEC3A_nCas9-NG + OsU6-2::EPSPs #3). At this time, the pTG5 vector showed the efficiency of correcting all four cytosine base target sites.

[0089] To confirm the base correction patterns of the two selected vectors (pTG4, pTG5), the vectors were PEG-transfected into maize protoplasts as in Example 3-1, and genomic DNA was isolated after 72 hours of culture, followed by targeted deep sequencing using an Illumina Mi-Seq instrument. As a result, the C to T correction rate was confirmed to be 3.37% or more under the condition using pTG4 (pTG ZmE #4) and 4.13% or more under the condition using pTG5 (pTG ZmE #5), and various correction patterns were confirmed at the four cytosine base correction target sites (Fig. 4). The final vector was verified by confirming the efficiency within the gene target window and the C to T base correction efficiency.

[0090] Analysis of the base correction efficiency of a total of 34 corn callus lines grown on PPT selection medium after transformation process from corn immature embryos was performed by randomly pooling some callus from the callus grown on the selection medium and confirming it through NGS analysis. As a result, as shown in Fig. 5, the base correction efficiency and base correction pattern of each of the 34 callus lines were confirmed to be diverse. Among the 34 callus lines analyzed, 6 superior callus lines (TG4-1, TG4-8, TG4-9, TG4-13, TG5-6, TG5-13) were selected as shown in Fig. 6 to increase the rate of securing corn base correction bodies, and corn regeneration bodies were induced from the grown callus.

[0091]

[0092] Example 4. Selection of base-editing constructs for the corn EPSPS gene

[0093] As shown in Fig. 7, the production of maize transformants (T0) produced with pTG4 and pTG5 vectors for base correction of the ZmEPSPs gene was performed at the National Institute of Agricultural Sciences through regeneration stages, soil acclimatization, and pot transplantation, and cultivation in a glass greenhouse, and a total of 145 T0 generation plants were secured. At this time, the results of NGS analysis of the transformed maize plants being cultivated in the greenhouse confirmed that 16 of the 145 transformed T0 maize plants were base correction plants, and the analysis of the patterns of these 16 base correction plants confirmed that the T0 maize plants had changed amino acids due to changes in the target base (Fig. 8). At this time, the overall base correction body acquisition efficiency was 11.0% (16 / 145), which was confirmed in 5 out of 70 T0 corn plants (7.1%) produced with the cytosine base correction pTG4 vector with a single guide RNA, and in 11 out of 75 T0 corn plants (14.7%) produced with the cytosine base correction pTG5 vector with dual guide RNAs, indicating that the base correction body acquisition rate was about twice as high in the pTG5 vector with dual guide RNAs (Table 1).

[0094] Through Agrobacterium-mediated transformation EPSPsGenetic base editing corn production efficiency Vector No. of PPT selection of transgenic calli No. of regeneration plantlets (T0) No. of edited plantlets (editing ratio, %) No. of line harvested (T1) (with seed) pTG413705 (7.1) 10 (2) pTG5217511 (14.7) 19 (3) Total 3414516 (11.0) 29 (5)

[0095] T0 corn transformants and base correction plants were subjected to self-pollination or cross-pollination with B73, and among the 16 corn plants in which target region amino acid correction was confirmed, 5 base correction plants were secured by cross-pollination with corn B73 line (paternal line) and F1 seeds were obtained (Fig. 9).

[0096] Corn F1 seeds confirmed by base correction were sown in a growth chamber to induce germination, and the germinated base correction corn seedlings were transplanted into soil in an LMO vinyl house to grow normally, and artificial crossbreeding was performed to secure F2 seeds (Fig. 10).

[0097]

[0098] Example 5. Verification of herbicide tolerance of EPSPS gene base-editing constructs

[0099] The secured corn F1 generation seeds have germinated seedlings and are currently being cultivated in open-field LMO greenhouses. Numerous monoallelic and biallelic F2 generation seeds have been secured through selfing or outcrossing. The secured F2 seeds were tested for herbicide tolerance at various concentrations of glyphosate, and the degree of herbicide tolerance was evaluated during indoor cultivation using commercially available non-selective pesticides containing glyphosate.

[0100] After seed germination in a nursery tray in an indoor greenhouse, commercial herbicide (product name: Geunsami, Farm Hannong) was sprayed once with an electric sprayer at a concentration of 0.2X the recommended dose (original dose 0.4 ppm) and 4 times the recommended dose (200 ml). As a result of observation, all regular corn (control) plants died 9 days after herbicide treatment, but some of the base-corrected corn F2 seedlings (TG5 B4-8) were confirmed to have herbicide resistance (Fig. 11).

Claims

1. A recombinant vector comprising a guide RNA expression cassette targeting the EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene of maize (Zea mays L.); and a cytosine base editor expression cassette fused with a cytidine deaminase and nuclease variant.

2. A recombinant vector according to claim 1, characterized in that the guide RNA targeting the corn EPSPs gene targets the base sequences of sequence number 3 and sequence number 4.

3. A recombinant vector characterized in that the base correction efficiency is increased by overlapping double guide RNA targeting the EPSPS gene of corn in the first paragraph.

4. A recombinant vector according to claim 1, characterized in that the cytidine deamination enzyme is APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like).

5. A recombinant vector according to claim 1, characterized in that the nuclease variant is nickase Cas9 (CRISPR associated protein 9).

6. A recombinant vector according to claim 5, characterized in that the nickase Cas9 recognizes NGG-PAM (protospacer adjacent motif) or NG-PAM.

7. A recombinant vector according to claim 1, characterized in that the cytosine base-editing gene scissors expression cassette additionally includes a UGI (uracil DNA-glycosylase inhibitor) peptide coding sequence.

8. A recombinant vector according to claim 7, characterized in that the UGI peptide is linked to the carboxy-terminus of the nuclease variant.

9. A recombinant vector according to claim 1, characterized in that the herbicide is glyphosate. 10.(a) a step of introducing a recombinant vector of any one of claims 1 to 9 into a corn plant cell to correct the genome; and (b) a method for producing a corn base-corrected plant having a herbicide tolerance trait, comprising the step of re-differentiating a corn plant from a corn plant cell in which the genome has been corrected.

11. In paragraph 10, the re-differentiated corn plant of step (b) is cross-bred with a corn strain with strong water resistance to produce F 1 A manufacturing method characterized by further comprising a step of producing seeds.

12. A manufacturing method according to claim 10, characterized in that the herbicide is glyphosate.

13. A corn base-corrected plant having a herbicide tolerance trait produced by the method of Article 10.

14. Seeds of corn plants whose genomes have been corrected according to Article 13.

15. A composition for base correction to increase herbicide resistance of corn plants, containing the recombinant vector of any one of claims 1 to 9 as an active ingredient.

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