Method for producing plants having herbicide resistance by base editing of DNA encoding plant chloroplast D1 protein

Abstract

The present invention provides a method for producing a plant resistant to herbicides, the method expressing a base editing composition or base editor in a target plant or part of the plant, and growing or culturing the plant or part of the plant, the base editing composition has an activity of editing an adenine (A) base of a DNA encoding a plant chloroplast D1 protein into a guanine (G) base and / or editing a cytosine (C) base into a thymine (T) base. The herbicide is preferably a triazine herbicide, and more preferably atrazine. A plant with resistance to the herbicide is a plant in which a serine residue of the D1 protein is mutated into glycine or an alanine residue is mutated into valine, the serine residue is a serine residue corresponding to a 264th-site serine residue of the D1 protein of Arabidopsis thaliana, and the alanine residue is a serine residue corresponding to a 264th-site serine residue of the D1 protein of the Arabidopsis thaliana and is a serine residue corresponding to a 264th-site serine residue of the D1 protein of the Arabidopsis thaliana and a valine residue corresponding to the 264th-site serine residue of the D1 protein of the Arabidopsis thaliana. The alanine residue is an alanine residue corresponding to the 251 alanine residue of the D1 protein of the arabidopsis thaliana. The base editing composition or base editor may comprise one or more fusion proteins, the one or more fusion proteins may each independently comprise a DNA binding protein, and the enzyme protein may comprise at least one of a cytosine deaminase, an adenine deaminase, and a nicking enzyme. The plant produced by the method is obtained by editing a DNA sequence of 5 '-AGT-3' of a serine residue (a serine residue corresponding to a 264th-site serine residue of D1 protein of arabidopsis thaliana) of encoding chloroplast D1 protein into 5 '-GGT-3' of encoding glycine, and editing a DNA sequence of 5 '-AGT-3' of encoding chloroplast D1 protein into 5 '-GGT-3' of encoding glycine. Or the DNA sequence encoding the 5 '-GCT-3'of the alanine residue is edited into an initial gene edited plant encoding the 5'-GTT-3 'of valine, or the DNA sequence encoding the 5'-GCT-3 'of the alanine residue is edited into a 5'-GTT-3 'of valine. The invention provides a genetically edited plant or part of a plant, a progeny or seed thus obtained, and a method for controlling weeds by applying herbicides in the vicinity of these plants.

Description

Technical Field

[0001] This invention relates to a base-editing composition or base editor for editing DNA bases encoding the D1 protein in a plant organelle (chloroplast) to make it herbicide resistant; expressing the base-editing composition or base editor in a target plant or a portion of a plant and causing the plant or portion of a plant to grow or be cultured to make it herbicide resistant; a method for producing such herbicide-resistant gene-edited plants or portions of plants; and a method for using such herbicide-resistant gene-edited plants or portions of plants. Background Technology

[0002] Triazine herbicides interfere with electron transport during photosynthesis by binding to the D1 protein (encoded by the psbA gene) in plant chloroplasts, leading to oxidative stress and nutrient deficiency, ultimately causing plant death. First discovered in 1952 by JR Geigy in Switzerland, triazine herbicides are now registered in over 100 countries and used for weed control on more than 50 different crops. Commonly used triazine herbicides include: atrazine, cyprodinil, fenpropathrin, fenpropathrin, fenpropathrin, fenpropathrin, fenpropathrin, simazine, terbufos, etc. Triazine herbicides were among the first widely recognized and reported herbicides that induce weed resistance. Since the discovery of groundsel resistant to simazine in 1968, at least 55 weed species resistant to triazine herbicides have been reported.

[0003] Despite efforts to develop herbicide-resistant plants through genetic engineering, the herbicide-resistant plants developed to date have been achieved by introducing exogenous genes that confer herbicide resistance and then inducing their strong expression. Therefore, they are classified as genetically modified organisms (GMOs). These GMO crops involve the introduction of exogenous genes and are subject to safety regulations.

[0004] A fusion protein linking a DNA-binding protein and a deaminase can replace nucleotides or edit bases in the genome without generating a double-strand break (DSB), or edit point mutations that cause genetic disorders, or achieve DNA editing such as targeted conversion of single nucleotides (to introduce desired single nucleotide variations into prokaryotic and eukaryotic cells, such as humans).

[0005] Programmable genome editing tools, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeat (CRISPR) systems, and base editors composed of CRISPR-associated protein 9 (Cas9) variants lacking nucleic acid degradation efficiency and base deaminase proteins, have the potential to be used for plant genetic research and crop trait improvement by altering base sequences.

[0006] However, these existing genome editing tools are not well-suited for editing the DNA sequences of plant organelles, including mitochondria and chloroplasts. This is primarily because it is impossible to deliver the guide RNA required to run the most widely used CRISPR systems into the organelles, or because it is difficult to simultaneously express two compounds within the organelles. Furthermore, mitochondria and chloroplasts in plants are organelles with DNA of a different morphology than that of animal nuclei or mitochondria. For example, human mitochondrial DNA has 16,569 base pairs, while Arabidopsis thaliana mitochondrial DNA has 366,924 base pairs. On the other hand, Arabidopsis thaliana chloroplast DNA has 154,478 base pairs. As mentioned above, the DNA length and composition of plant chloroplasts and mitochondria differ from those of animal nuclei or mitochondria, and the types of genes they contain are completely different. In particular, plant chloroplasts contain proteins related to DNA replication and repair, which are different from those found in animal nuclei or mitochondria. Therefore, the DNA repair patterns exhibited when changes occur in the chloroplasts and mitochondrial DNA of plants differ from those in the nuclear or mitochondrial DNA of animals. Consequently, methods effective in editing nuclear or mitochondrial DNA in animals may not be effective in editing plant organelle DNA. Plant organelles encode numerous essential genes required for photosynthesis and respiration. Methods or tools for editing these organelle genes are crucial for studying their function and improving crop productivity and traits.

[0007] Existing technical documents Patent documents Patent Document 1: International Patent Application Publication WO2022 / 060185A Patent Document 2: International Patent Application Publication WO2023 / 086953A Summary of the Invention The problem the invention aims to solve The purpose of this invention is to provide a plant that exhibits herbicide resistance through editing chloroplast DNA. Specifically, it provides a method for producing a plant resistant to herbicides by editing DNA bases encoding the plant chloroplast D1 protein, and a plant prepared by this method.

[0008] means for solving problems This invention provides a method for producing herbicide-resistant plants, wherein the method expresses a base-editing composition or base editor in a target plant or a portion of a plant, and causes the plant or portion of a plant to grow or be cultured. The base-editing composition has the activity of editing adenine (A) bases to guanine (G) bases and / or editing cytosine (C) bases to thymine (T) bases in the DNA encoding the plant chloroplast D1 protein. The herbicide is preferably a triazine herbicide, more preferably atrazine. The herbicide-resistant plant is one in which the serine residue of the D1 protein is mutated to glycine, or the alanine residue is mutated to valine (including cases where both are mutated), wherein the serine residue corresponds to the 264th serine residue of the Arabidopsis thaliana D1 protein, and the alanine residue corresponds to the 251st alanine residue of the Arabidopsis thaliana D1 protein. The base editing composition or base editor may contain one or more fusion proteins, each of which may independently contain a DNA-binding protein, and the enzyme protein may contain at least one of cytosine deaminase, adenine deaminase, and nicking enzyme.

[0009] The plant produced by this method is an initial gene-edited plant in which the 5'-AGT-3' DNA sequence encoding a serine residue of the chloroplast D1 protein (corresponding to the serine residue at position 264 of the Arabidopsis D1 protein) is edited to encode glycine (5'-GGT-3'), or the 5'-GCT-3' DNA sequence encoding an alanine residue of the chloroplast D1 protein (corresponding to the alanine residue at position 251 of the Arabidopsis D1 protein) is edited to encode valine (5'-GTT-3'). This invention provides such a gene-edited plant or a portion thereof, the resulting offspring or seeds, and a method for controlling weeds by applying herbicides near these plants.

[0010] Invention Effects The herbicide-resistant plants of this invention are prepared through base editing of chloroplast DNA, thus these plants are resistant to different types of herbicides than plants resistant to herbicides acquired through base editing of genes in the cell nucleus. Furthermore, herbicide-resistant plants developed to date are classified as genetically modified organisms (GMOs) because they acquire resistance by inducing strong expression of exogenous genes after introduction. However, the herbicide-resistant plants of this invention, developed through base editing of plant organelle DNA, are relatively unrestricted under GMO and Living Modified Organisms (LMO) regulations, and therefore have higher applicability.

[0011] Furthermore, by introducing the chloroplast DNA base editing of this invention into plants that are currently widely used as GMO crops and are resistant to herbicides that inhibit amino acid synthesis or promote the generation of reactive oxygen species (ROS), it is possible to prepare plants resistant to two or more herbicides. When a single herbicide is used for a long period in the same location, resistant weeds will develop. Therefore, the use of two or more herbicides is advantageous in making it more difficult for weeds resistant to previously used herbicides to survive. However, it is difficult to provide plants resistant to two or more different herbicides using existing gene recombination technologies. Attached Figure Description

[0012] Figure 1 This is a schematic diagram of different base editors used to edit the DNA sequence encoding the 5'-AGT-3' serine residue at position 264 of the Arabidopsis D1 protein into a 5'-GGT-3' (A-to-G) sequence encoding glycine. CTS represents the chloroplast transit signal, NTD represents the N-terminal domain of the TALE protein, and CTD represents the C-terminal domain of the TALE protein. "Left TALE repeats" and "Right TALE repeats" represent TALE arrays, respectively. Figure 1In the diagram, "Left 1" and "Left 2" represent "AtpsbA S264G Left 1" and "AtpsbA S264G Left 2" as described in the examples, respectively. "Right 1" and "Right 2" represent "AtpsbA S264G Right 1" and "AtpsbA S264G Right 2" as described in the examples, respectively. The single underlined base sequence indicates the base sequence in which the TALE protein (composed of an N-terminal domain, a TALE array, and a C-terminal domain) binds to the target gene (psbA). The underlined "AGT" is the target editing site encoding serine at position 264. UDG represents uracil DNA glycosylation enzyme, and AD represents adenine deaminase. Figure 1 Part A uses DddA tox Splitting cells (1397N and 1397C). Figure 1 Part B and Figure 1 Part C uses full-length DddA tox (GSVG), and respectively using only left TALE repeats or only right TALE repeats.

[0013] Figure 2 The diagram shows the base editing efficiency measured in individuals that survived atrazine treatment in the first generation of transformed plants. Proteins enclosed in "[ ]" refer to proteins that constitute a fusion protein. For example, [Left1-AD-GSVG-UDG] indicates a fusion protein linking Left1 (TALE), AD, GSVG, and UDG (linker omitted). Col-0 is an untransformed wild-type individual. Protein-related abbreviations are as described. Figure 1 As shown.

[0014] Figure 3 The images show second-generation plants transformed with the base-editing fusion protein used in Example 1 and wild-type (Col-0) plants sown in atrazine-containing and atrazine-free media, and photographs taken 14 days later. Figure 3 Part A) and base editing efficiency ( Figure 3 Part B). Abbreviations related to proteins are as described. Figure 1 As shown. Unlike wild-type individuals, the gene-edited plants of this invention did not exhibit growth inhibition even after atrazine treatment, which warrants attention.

[0015] Figure 4 Shown in the case of Figure 3 The second-generation transformed plants (third-generation transformations) constructed using the base editor [Left 1-1397C-AD-UDG] + [Right 2-1397N-UDG#18] showed growth when treated with medium (1 / 2 MS) alone, or further treated with Basta (PPT: glufosinate) or atrazine herbicides. Figure 4 (Part a). #18-2, #18-4, #18-7 and #18-8 did not show growth inhibition even after atrazine treatment, and because they did not contain exogenous genes, they showed sensitivity to styrax. Figure 4 Part b shows the base editing efficiency analysis results for #18-2-1, #18-2-2, #18-4-1, #18-4-2, #18-7-1, #18-7-2, #18-8-1, and #18-8-2, all of which show more than 99% homology. Figure 4 Part c shows the results of PCR using primers for the Procymidone resistance gene and primers for the psbA gene. The results indicate that the foreign gene was detected in #18-1-1, #18-3-1, #18-5-1, and #18-6-1, which have Procymidone resistance, while the foreign gene was not introduced into #18-2-1, #18-4-1, #18-7-1, and #18-8-1, which do not have Procymidone resistance.

[0016] Figure 5 The graph shows the growth of the third-generation transformed plant of this invention (represented by the base editor [Left 1-1397C-AD-UDG] + [Right 2-1397N-UDG]#18-2) after 5 days of treatment with different concentrations of atrazine.

[0017] Figure 6 This shows whether third-generation transformed plants of [Left 1-AD-GSVG-UDG] #25 and [Left 1-AD-GSVG-UDG] #28 grew when treated with medium (1 / 2 MS) alone, or further treated with glufosinate-ammonium (PPT) or atrazine herbicide. Figure 6 (part a). Figure 4Part b shows the base editing efficiency analysis results for #25-1-1, #25-2-1, #25-3-1, #25-4-1, #25-5-1, #25-6-1, #25-7-1, #25-8-1, #28-1-1, #28-2-1, #28-3-1, #28-4-1, #28-5-1, #28-6-1, #28-7-1, and #28-8-1, all showing homogeneity of over 99%.

[0018] Figure 7 The results show the evaluation of the C (cytosine) to T (thymine) editing efficiency in 5'-GCT-3', which is mitochondrial DNA encoding amino acid 251 (alanine) in the D1 protein of lettuce involved in resistance to benzoxazine. Base positions in the region between two TALE protein-binding DNA base sequences (spacer regions) are indicated by subscript numbers, with the first base in the spacer region being the first to be counted. Single-underlined base sequences indicate the base sequences bound to the TALE protein (composed of an N-terminal domain, TALE array, and C-terminal domain) in the target gene (psbA), with the underlined "A8G9T10" being the target editing site encoding alanine at position 251. Figure 7 In the illustration, "Left 1" and "Left 2" represent "LspsbA A251V Left 1" and "LspsbA A251V Left 2" as described in the examples, respectively, and "Right" represents "LspsbA A251V Right" as described in the examples. "FZY2 v1.6 N" and "FZY2 v1.6 C" are the mitotic bodies of the cytosine deaminase used.

[0019] Figure 8 The efficiency of A-to-G base editing in the Arabidopsis chloroplast gene psbA obtained using Nt.BspD6I(C) as a nicking enzyme and with deaminase (TadA8e) is shown, and the results obtained from a single T1 individual obtained from transformed Arabidopsis are presented. Figures 8 to 12 These are experimental results obtained from Example 4, and the composition of the base editor used is described in Example 4. Figure 8 and Figure 9 The base sequence shown is similar to Figure 11The sequences described are identical and represent the bases located in the region (spacer region) between the DNA base sequences bound to the two TALE proteins used ("AtpsbA Left TALE NK" and "AtpsbA Right TALE NK"). The first base in the spacer is counted starting from the first base and indicates its respective position. A6G7T8 is the AGT base associated with herbicide resistance and encoding the base editing target (serine at position 264) of this invention.

[0020] Figure 9 The efficiency of A-to-G base editing in the Arabidopsis chloroplast gene psbA, obtained using Nt-BspD6I(C) as a nicking enzyme and with a deaminase (TadA8e), is shown, along with results obtained from a single T2 individual from transformed Arabidopsis. "AtpsbA Left TALE NK" and "AtpsbA Right TALE NK" were used as TALE proteins as illustrated in the examples. The psbA base editor used produced the following result through A-to-G base editing: the serine residue at position 264 (encoded by 5'-AGT-3') of the D1 protein encoded by the psbA gene was converted to a glycine residue (encoded by 5'-GGT-3'), which confers resistance to the atrazine herbicide. Figure 9 The eight individuals shown are all those that survived even after atrazine treatment.

[0021] Figure 10 Shown in the case of from Figure 9 The T3 individuals derived from seeds of individuals #2-1, #2-3, and #2-4, as well as wild-type individuals, were shown to exhibit growth when treated with culture medium (1 / 2 MS) alone, or further treated with Basta (PPT: glufosinate) or atrazine herbicides. The growth of T3 individuals derived from #2-1, #2-3, and #2-4 was not inhibited even after atrazine treatment. The #2-1 individual, which survived Basta treatment, showed the introduction of a foreign gene.

[0022] Figure 11 Showing the Figure 10 Sequencing analysis of the T3 individuals with atrazine resistance showed that they all exhibited more than 99% homology. Figure 11The base sequences shown represent the bases in the region (spacer region) between the DNA base sequences that bind to the two TALE proteins ("AtpsbA Left TALE NK" and "AtpsbA Right TALE NK"). The AGT base encoding serine at position 264, which is associated with herbicide resistance, is underlined. Left TadA8e indicates that TadA8e is linked to and used with AtpsbA Left TALE NK, and Right Nt.BspD6I(C) indicates that Nt.BspD6I(C) is linked to and used with AtpsbA Right TALE NK.

[0023] Figure 12 It is aimed at Figure 11 The results of PCR analysis on T3 individuals with atrazine resistance, using primers for the Procymidone resistance gene and the psbA gene, showed that... Figure 10 The foreign gene was detected in #2-1-1, which is derived from #2-1 and has Procymidone resistance, while the foreign gene was not introduced into #2-3-1 and #2-4-1, which are derived from #2-3 and #2-4 and do not have Procymidone resistance. Detailed Implementation

[0024] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the terms used in this specification are those well-known and commonly used in the art.

[0025] The terms “correction,” “modification,” and “editing” used in this specification are used interchangeably and refer to methods for altering nucleic acid sequences through selective mutations of specific genomic targets. These specific genomic targets include, but are not limited to, genes, promoters, open reading frames, or any nucleic acid sequence.

[0026] The terms "base editor," "base editing system," and "base correction system" used in this specification are used interchangeably to refer to a substance having the activity of altering nucleic acid sequences through selective mutation of genomic targets, and include a combination of one or more different base editors. The terms "base editor," "base editing system," or "base correction system" used in this specification, depending on the context, can be a polypeptide (which may be a fusion protein) or a polynucleotide or a combination thereof, or a composition containing one or more polypeptides (which may be fusion proteins) or polynucleotides or combinations thereof. Therefore, the terms "base editing system" or "base editing composition" used in this specification can include one base editor or a combination of two or more different base editors, wherein the different base editors can be used simultaneously or individually.

[0027] As used in this specification, the term "fusion protein" refers to a polypeptide composed of two or more different polypeptides linked by peptide bonds. The fusion protein used in this invention independently comprises a DNA-binding protein, and as an enzyme protein, it further comprises at least one of adenine deaminase, cytosine deaminase, and nicking enzyme directly or indirectly linked to the DNA-binding protein. It may also contain additional sequences such as UDG, UGI, NLS, NES, and CTS, and may further contain other sequences for biotechnological methods such as tagging. These individual polypeptides can be directly linked or linked via a linker. A "linker" refers to any molecule that links two different molecules. In the field of biotechnology, any linker known for providing fusion proteins or protein conjugates can be used; for example, peptide linkers containing 1 to 100 amino acid residues can be used.

[0028] In this specification, when referring to the “linkage” of two proteins, it can be a direct link or an indirect link through a linker or other (multiple) proteins.

[0029] As used in this specification, the term "variant" refers to a mutated form of a wild-type gene and its amino acid sequence. This includes naturally occurring variants, such as genes and their amino acid sequences from other species that diverged from a common ancestor (orthologs), or artificially created variants, such as engineered or evolved genes and their amino acid sequences. The term "homolog" includes related genes that diverged through speciation and their resulting protein variants (orthologs), as well as related genes that diverged within a genome through gene replication and their resulting protein variants (paralogs). As used in this specification, "homolog" and "ortholog" are substantially the same concept.

[0030] The design and construction methods of the fusion protein with base editing activity or the polynucleotide encoding it used in the production of gene-edited plants according to the present invention can employ any method known in the art. The polynucleotide can be inserted into a vector, and the vector can be introduced into cells. The single protein constituting the fusion protein with base editing activity used in the production of gene-edited plants according to the present invention is typically cloned into a single polynucleotide and expressed as a single polypeptide (fusion protein). However, one or more of the single proteins can also be cloned into (multiple) separate polynucleotides and expressed as two or more separate polypeptides, and this is also within the scope of the present invention.

[0031] As used in this specification, the terms “target,” “targeting,” “target site,” or “targeting site” refer to a predetermined nucleic acid sequence of any composition and / or length. Such target sites include, but are not limited to, genes, promoters, open reading frames, or any nucleic acid sequence.

[0032] The terms “gene delivery vector,” “delivery composition,” or “vector” used in this specification are used interchangeably to refer to a medium or substance used to deliver or introduce a base editing composition or base editor into a cell, tissue, or organism containing target DNA for base editing, or to express target DNA.

[0033] This invention provides a method for producing herbicide-resistant plants, the method comprising: expressing a base-editing composition or base editor in a target plant or a portion of a plant, and causing the plant or portion of a plant to grow or be cultured, wherein the base-editing composition has the activity of editing adenine (A) bases to guanine (G) bases and / or cytosine (C) bases to thymine (T) bases in DNA encoding plant chloroplast D1 protein. The step of expressing the base-editing composition or base editor in the target plant or a portion of a plant may include: introducing a polynucleotide encoding one or more fusion proteins contained in the base-editing composition or base editor into the plant or a portion of a plant.

[0034] In this invention, the term "plant" can refer not only to a plant at any developmental stage, but also to any part that can be attached to or separated from a whole, undamaged plant. Such a "part" of a plant includes, but is not limited to, plant organs, tissues, and cells, including plant callus, plant clusters, plant protoplasts, and plant cell tissue cultures of regenerable plants. Examples of "parts" of a particular plant include stems, leaves, roots, inflorescences, flowers, florets, fruits, pollen grains, pedicels, stamens, anthers, pistils, stigmas, styles, ovaries, petals, calyxes, carpels, root tips, root caps, root hairs, leaf hairs, carpels, pollen grains, microspores, plumules, ovules, cotyledons, hypocotyls, epicotyls, xylem, phloem, parenchyma, endosperm, companion cells, guard cells, and any other known organs, tissues, and cells of a plant. Additionally, the plant part can be a seed.

[0035] In this invention, "plant" includes both non-GMO plants and GMO plants. A "non-GMO plant" refers to a plant whose genome does not contain recombinant DNA. A "GMO plant" refers to a plant whose genome contains recombinant DNA. Such a GMO plant can be produced by introducing recombinant DNA into the plant's genome; for example, it could be a plant with recombinant DNA that confers herbicide resistance.

[0036] In this invention, the term "herbicide" refers to an active ingredient that causes death of plants (e.g., vegetation) or has an adverse effect on their growth. Preferably, the herbicide in this invention can be a triazine herbicide, such as atrazine, propargite, atrazine, fenpropathrin, simazine, cyclomethrin, methamidophos, terbufenozide, terbufenozide, terbufenozide, diuron, fluroxypyr, linuron, terbufenozide, and benzoxazine, or their salts; more preferably, it can be atrazine or benzoxazine, or their salts. In this invention, the term "resistance" may be used interchangeably with "tolerance," and "plants resistant to herbicides" means plants that, despite treatment with an effective dose of a specific herbicide that causes death or adverse effect on the growth of normal or wild-type plants, do not exhibit the responses (death or adverse effect on growth) shown by normal or wild-type plants under that effective dose of herbicide treatment.

[0037] In this specification, "expressing" a base editing composition or base editor in a plant or a part of a plant can refer to any technique known to those skilled in the art that enables the base editing composition or base editor to function in a plant or a part of a plant. Typically, this can be the introduction of a polynucleotide (which may be a DNA sequence, an RNA sequence, or a combination thereof) encoding the base editing composition or base editor into the plant or a part of a plant. The steps of introducing the polynucleotide into the plant or a part of a plant can be, but are not limited to, integrating DNA into a suitable vector and transforming plant cells or protoplasts, or injecting it into plant cells or protoplasts in the form of mRNA or ribonucleoprotein (RNP). The part of the plant can be plant cells, protoplasts, or callus tissue, but is not limited to these.

[0038] The method of delivering a polynucleotide encoding a base-editing fusion protein into plant cells or protoplasts can be any method known to those skilled in the art, for example, it may include transformation using Agrobacterium (e.g., Agrobacterium tumefaciens, Agrobacterium rhizogene, etc.), transfection using viruses (e.g., Geminivirus, Tobacco rattle virus (TRV), Tomato mosaic virus (ToMV), Foxtail mosaic virus (FoMV), Barley Yellow Striate Mosaic Virus (BYSMV), Sonchus Yellow Net Rhabdovirus (SYNV), etc.), injection using a gene gun, transfection by protoplast fusion, electroporation-based transfection, microinjection-based injection, etc.

[0039] This invention relates to mutating serine residues in the D1 protein of plant chloroplasts to glycine and / or alanine residues to valine residues. The serine residues are those corresponding to the 264th serine residue in the Arabidopsis thaliana D1 protein. Specifically, the base editing composition or base editor used in this invention mutates the sequence to 5'-GGT-3' (glycine) by editing the adenine (A) base in the 5'-AGT-3' sequence encoding the 264th serine residue to a guanine (G) base. Thus, when the serine residue in the D1 protein is edited to glycine, herbicide resistance can be conferred without the introduction of a separate exogenous gene; for example, growth is not impaired even when treated with atrazine. Depending on the plant, the serine residue may be at a different site than the 264th, but this site can be easily determined by sequence alignment by those skilled in the art. In this specification, the expression "corresponding to a serine residue" related to the serine site is intended to refer to a serine residue that functions equivalently to the 264th serine residue in Arabidopsis thaliana, even if the site is not the 264th position of the D1 protein. The alanine residue is the alanine residue corresponding to the 251st alanine residue in the Arabidopsis thaliana D1 protein. Specifically, the base editing composition or base editor used in this invention mutates the sequence to 5'-GTT-3' (valine) by editing the cytosine (C) base in the 5'-GCT-3' sequence encoding the 251st alanine residue to a thymine (T) base. Thus, when editing the alanine residue of the D1 protein to valine, herbicide resistance can be conferred without the introduction of a separate exogenous gene; for example, growth is not weakened even when treated with benzoxazine. Depending on the plant, the alanine residue may be located at other sites besides the 251st position, but this site can be easily determined by sequence alignment by those skilled in the art. In this specification, the phrase "corresponding to an alanine residue" related to the serine site is intended to refer to an alanine residue that functions identically to alanine at position 251 in Arabidopsis thaliana, even if the site is not position 251 of the D1 protein. The phrase "serine residue mutated to glycine, or alanine residue mutated to valine" in this specification includes cases where both mutations occur.

[0040] The plants to which the herbicide-resistant plant production method of the present invention is applicable can be any plant that has developed resistance to triazine herbicides through the D1 protein. For example, these plants can be row crop plants (e.g., corn, soybeans, cotton, rapeseed, beets, alfalfa, sugarcane, rice, and wheat), vegetables (e.g., tomatoes, peppers, chilies, melons, watermelons, cucumbers, eggplants, cauliflower, broccoli, lettuce, spinach, onions, peas, carrots, sweet corn, cabbage, leeks, fennel, pumpkins, squash, or zucchini, radishes, Brussels sprouts, tomatillos, kidney beans, dry beans, or okra), culinary plants (e.g., basil, parsley, coffee, or tea), fruits (e.g., apples, pears, cherries, peaches, plums, apricots, bananas, plantains, table grapes, wine grapes, citrus fruits, avocados, mangoes, or berries), trees, or ornamental plants (e.g., ornamental flowering plants or shrubs, or zoysia grass), but are not limited thereto. Preferably, the invention is applicable to soybeans, zoysia grass, or lettuce. The following shows the serine residues corresponding to the 264th serine residue of the Arabidopsis D1 protein in representative species such as soybean, lettuce, and Zoysia japonica.

[0041]

[0042] The herbicide-resistant gene-edited plants of this invention can be genetically modified or transgenic plants or their offspring that have previously been made herbicide-resistant by inserting recombinant DNA. For example, such genetically modified or transgenic plants or their offspring can be genetically modified to be resistant to herbicides that inhibit amino acid synthesis or promote the generation of reactive oxygen species (ROS). Base editing of such plants according to this invention refers to conferring resistance to triazine herbicides in addition to existing herbicide resistance. This approach is advantageous because it makes it more difficult for weeds resistant to previously used herbicides to survive by combining two or more herbicides.

[0043] Composition of base editing composition or base editor This invention relates to base editing in plants or parts of plants (e.g., plant cells or protoplasts). Base editing compositions or base editors delivered to plants or parts of plants (e.g., plant cells or protoplasts) for base editing can be composed of proteins (such as DNA-binding proteins and enzyme proteins) or polynucleotides encoding said proteins. In this specification, when the base editing compositions or base editors used in this invention are defined from the perspective of proteins(s) that may be contained therein, it should be understood that this description implies that the base editing compositions or base editors used in this invention may contain polynucleotides(s) encoding such proteins(s).

[0044] The herbicide-resistant plants of the present invention are characterized by being produced using a base editing composition or base editor comprising a DNA-binding protein and an enzyme protein, or a polynucleotide encoding said protein. The enzyme protein may comprise one or more of cytosine deaminase, adenine deaminase, and nicking enzyme, wherein the cytosine deaminase may exist in full-length or in two mitotic forms.

[0045] This invention provides a method for producing herbicide-resistant plants using a base editing composition or base editor comprising a DNA-binding protein and an enzyme protein, or a polynucleotide encoding said protein. The method includes: expressing the base editing composition or base editor comprising a DNA-binding protein and an enzyme protein, or a polynucleotide encoding said protein, in a target plant or a portion of a plant, and allowing said plant or a portion of a plant to grow or be cultured. The enzyme protein may comprise one or more of cytosine deaminase, adenine deaminase, and nicking enzyme, wherein the cytosine deaminase may be present in full-length or in two mitotic forms.

[0046] The base editing composition or base editor used in the production of herbicide-resistant plants according to the present invention, or the DNA-binding protein and enzyme protein used in the method of producing herbicide-resistant plants using said composition or base editor, can exist in the form of more than one fusion protein, preferably in the form of two fusion proteins. In this respect, the base editing composition or base editor used in the production of herbicide-resistant plants according to the present invention may contain two polynucleotides encoding the two fusion proteins as described above.

[0047] When the base editing composition or base editor used in the production of herbicide-resistant plants according to the present invention, or the DNA-binding protein and enzyme protein used in the method of producing herbicide-resistant plants using the composition or base editor, exist in the form of two fusion proteins, each of the two fusion proteins independently comprises a DNA-binding protein, and at least one of the two fusion proteins comprises one or more of cytosine deaminase, adenine deaminase, and nicking enzyme. The cytosine deaminase may exist in full-length or in the form of two mitotic figures; when existing in the form of two mitotic figures, the two mitotic figures are contained in different fusion proteins. In this respect, the base editing composition or base editor used in the production of herbicide-resistant plants according to the present invention may comprise two polynucleotides encoding the two fusion proteins as described above.

[0048] For base editing compositions or base editors used to produce herbicide-resistant plants via C-to-T editing, when the DNA-binding protein and the enzyme protein are present in the form of two fusion proteins, the two fusion proteins may each contain a mitochondrial of cytosine deaminase. The mitochondrial of cytosine deaminase cannot individually possess deaminase activity; they only exert deaminase activity when the two mitochondrial cells are adjacent to each other. Alternatively, one of the two fusion proteins may contain a full-length cytosine deaminase, and the other fusion protein may contain a nicking enzyme. In this case, preferably, the cytosine deaminase has deaminase activity against single-stranded DNA. In this respect, according to the invention, base editing compositions or base editors used to produce herbicide-resistant plants via C-to-T editing may contain two polynucleotides encoding the two fusion proteins as described above.

[0049] For base editing compositions or base editors used to produce herbicide-resistant plants through A-to-G editing, when the DNA-binding protein and the enzyme protein are present in the form of two fusion proteins, the two fusion proteins may each contain a mitotic form of cytosine deaminase, and one of the two fusion proteins may contain adenine deaminase. Alternatively, one of the two fusion proteins may contain a nicking enzyme, and the other fusion protein may contain adenine deaminase. In this respect, according to the invention, base editing compositions or base editors used to produce herbicide-resistant plants through A-to-G editing may contain two polynucleotides encoding the two fusion proteins as described above.

[0050] When the base editing composition or base editor used in the production of herbicide-resistant plants according to the present invention, or the DNA-binding protein and enzyme protein used in the method of producing herbicide-resistant plants using said composition or base editor, exist in the form of a fusion protein, said fusion protein comprises a DNA-binding protein and contains one or more of cytosine deaminase, adenine deaminase, and nicking enzyme. In this case, the cytosine deaminase exists in its full-length form. In this respect, the base editing composition or base editor used in the production of herbicide-resistant plants according to the present invention may contain a polynucleotide encoding a fusion protein as described above.

[0051] For base editing compositions or base editors used to produce herbicide-resistant plants via C-to-T editing, when the DNA-binding protein and the enzyme protein are present in the form of a fusion protein, said fusion protein may contain a full-length cytosine deaminase. Alternatively, a fusion protein may contain a full-length cytosine deaminase and a nicking enzyme, wherein, preferably, the cytosine deaminase has deaminase activity against single-stranded DNA. In this respect, according to the invention, base editing compositions or base editors used to produce herbicide-resistant plants via C-to-T editing may contain a polynucleotide encoding a fusion protein as described above.

[0052] For base editing compositions or base editors used to produce herbicide-resistant plants edited from A to G, when the DNA-binding protein and the enzyme protein are present in the form of a fusion protein, said fusion protein may contain a full-length cytosine deaminase and an adenine deaminase. Alternatively, a fusion protein may contain a nicking enzyme and an adenine deaminase. In this respect, according to the invention, base editing compositions or base editors used to produce herbicide-resistant plants edited from A to G may contain a polynucleotide encoding a fusion protein as described above.

[0053] DNA-binding protein The DNA-binding protein used in the base editing composition or base editor for producing herbicide-resistant plants of the present invention is a nucleic acid-programmable DNA-binding protein, which refers to a protein capable of targeting and binding to specific sequences in a DNA molecule. The DNA-binding protein used in the base editing composition or base editor for producing herbicide-resistant plants of the present invention may be selected from zinc finger proteins, transcription activator-like effector (TALE) proteins, or CRISPR-associated nucleases, or combinations thereof, but is not necessarily limited thereto. Regarding the composition of zinc finger proteins, TALE proteins, and CRISPR-associated nucleases, reference can be made to prior art, including all technical content described in International Patent Application Publication WO2022 / 060185, which is incorporated herein by reference.

[0054] When two or more DNA-binding proteins are used in the base editing of the present invention for producing herbicide-resistant plants, the two or more DNA-binding proteins may be of the same or different types. For example, when two DNA-binding proteins are used in the base editing composition or base editor (e.g., when two fusion proteins are used), the two DNA-binding proteins may both be zinc finger proteins, both be TALE proteins, or both be CRISPR-associated nucleases. Alternatively, one of the two DNA-binding proteins may be a zinc finger protein and the other may be a TALE protein or a CRISPR-associated nuclease, or one may be a TALE protein and the other may be a zinc finger protein or a CRISPR-associated nuclease. Preferably, when the DNA-binding protein used in the present invention is a TALE protein and is used in the form of two fusion proteins, the two fusion proteins each contain a TALE protein. In this respect, according to the present invention, the base editing composition or base editor used for producing herbicide-resistant plants by base editing may contain two or more polynucleotides encoding two or more DNA-binding proteins, which may be the same or different as described above.

[0055] In one embodiment of the present invention, the DNA-binding protein may be a zinc finger protein. A “zinc finger protein” (which may be referred to as “ZF” or “ZFP”) refers to a protein that binds to DNA in a sequence-specific manner via one or more zinc finger motifs, or as a DNA-binding portion of a larger protein. A zinc finger protein may contain 3 to 6 zinc finger motifs, but the zinc finger proteins used in the present invention are not necessarily limited to this. A zinc finger motif is a small polypeptide domain consisting of approximately 20 to 40 amino acid residues, with four amino acid residues as cysteine ​​and / or histidine that may be appropriately positioned and coordinated to bind to zinc ions. For example, depending on the type of residues that coordinate to zinc ions, zinc finger motifs may be classified as C2H2 (Cys2-His2), C3H (Cys2-CysHis), C4 (Cys2-Cys2), etc. The zinc finger motifs used in the zinc finger proteins used in the present invention may be naturally occurring zinc fingers or variants thereof found in any eukaryote, including humans, or artificially prepared.

[0056] In one embodiment of the present invention, the DNA-binding protein may be a TALE protein. A TALE protein is a protein that binds to nucleotides in a sequence-specific manner via one or more TALE-repeat modules. The TALE protein comprises at least one TALE-repeat module, preferably 1 to 30 TALE-repeat modules, but is not limited thereto. As used herein, a TALE-repeat module may also be referred to as a "TALE array," and the term "TALE protein" refers to a TALE array flanked by an N-terminal domain and a C-terminal domain (which may include a half domain). Depending on the context, the term "TALE" as used herein may refer to either a "TALE array" or a "TALE protein."

[0057] When two DNA-binding proteins are used in the base editing process of this invention for producing herbicide-resistant plants, one can be designated by the modifiers "first" or "left," and the other by the modifiers "second" or "right." For example, when both DNA-binding proteins are TALE, one TALE protein can be called the "first TALE protein" or "left TALE protein," and the other TALE protein can be called the "second TALE protein" or "right TALE protein."

[0058] When the TALE protein is used as the DNA-binding protein of the base editing composition or base editor for producing herbicide-resistant plants according to the present invention, a single-module TALE array or a multi-module TALE array (e.g., a dual-module TALE array of a first TALE (or left TALE) array and a second TALE (or right TALE) array) may also be used. When the base editing composition or base editor used in the present invention comprises two fusion proteins, the two fusion proteins may each have a first TALE protein and a second TALE protein. In this case, the first TALE protein and / or the second TALE protein each independently comprise one or more enzyme proteins selected from cytosine deaminase, adenine deaminase, and nicking enzymes. For example, one of the first TALE protein and the second TALE protein may be linked to a cytosine deaminase mitochondria, and the other TALE protein may be linked to other cytosine deaminase mitochondria. For example, one of the first TALE protein and the second TALE protein may be linked to a cytosine deaminase mitochondria, and the other TALE protein may be linked to other cytosine deaminase mitochondria and adenine deaminase. For example, one of the first and second TALE proteins may be linked to a cytosine deaminase, and the other TALE protein may be linked to a nicking enzyme. For example, one of the first and second TALE proteins may be linked to an adenine deaminase, and the other TALE protein may be linked to a nicking enzyme. In this respect, according to the present invention, a base editing composition or base editor used for producing herbicide-resistant plants through base editing may comprise two polynucleotides encoding two fusion proteins, as described above, each comprising a first TALE protein and a second TALE protein. When the base editing composition or base editor used in the present invention comprises a fusion protein, the fusion protein may have a monomodal TALE protein. In this case, the TALE protein may be linked to one or more enzyme proteins selected from cytosine deaminase, adenine deaminase, and nicking enzyme. For example, the monomodal TALE protein may be linked to a full-length cytosine deaminase with deaminase activity against double-stranded DNA. For example, the monomodal TALE protein may be linked to both a full-length cytosine deaminase and an adenine deaminase with deaminase activity against double-stranded DNA. For example, a single-module TALE protein can be linked to a full-length cytosine deaminase that has deaminase activity against nicking enzymes and single-stranded DNA. For example, a single-module TALE protein can be linked to nicking enzymes and adenine deaminase.

[0059] In one embodiment of the present invention, the DNA-binding protein may be a CRISPR-associated nuclease. CRISPR-associated nucleases are proteins that recognize specific base sequences in DNA by forming a complex with an RNA component called guide RNA, and possess endonuclease or nicking enzyme activity, thereby performing the function of editing that site; they are also known as "Cas proteins." Currently, more than 40 different Cas protein families have been documented. Information on known Cas genes and proteins can be obtained from the GenBank database of the National Center for Biotechnology Information (NCBI), but is not limited thereto.

[0060] The Cas protein can be any Cas protein, as long as it possesses endonuclease or nickase activity when forming a complex with guide RNA. For example, it can be Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, Cas12j, Cas13a, Cas13b, Cas13c, Cas13d, Cas14, Csy1, Csy2, Csy3, etc. Nucleases of Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, CsMT2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, or Csf4, or variants thereof, but not limited thereto. Preferably, the Cas protein is Cas9 or Cas12a, or variants thereof.

[0061] The term "guide RNA" refers to a DNA that is specific to a target DNA and can form a complex with the Cas protein, specifically an RNA that guides the Cas protein to the target DNA. The guide RNA may consist of two RNAs: CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA), or it may be a single-chain RNA (sgRNA) formed by fusing essential portions of crRNA and tracrRNA. The guide RNA may also be a dual RNA containing crRNA and tracrRNA. Any guide RNA can be used in this invention if it contains essential portions of crRNA and tracrRNA, as well as a portion complementary to the target.

[0062] The base editing composition or base editor used in this invention may contain a polynucleotide encoding a DNA-binding protein as described above.

[0063] Cytosine deaminase The cytosine deaminase used in the base editing composition or base editor for producing herbicide-resistant plants of the present invention refers to an aminohydrolase having the activity of converting cytosine bases into uridine. Enzymes possessing both adenine deaminase and cytosine deaminase activities also correspond to the cytosine deaminase referred to in this specification. Cytosine deaminases can be derived from or mutated (e.g., engineered or evolved) from any organism (e.g., eukaryotes or prokaryotes), wherein said organisms include, but are not limited to, algae, bacteria, fungi, plants, invertebrates, and mammals. For example, it could be: apolipoprotein B editing complex (APOBEC), activation-induced cytidine deaminase (AID), cytosine deaminase derived from or mutated from tRNA-specific adenosine deaminase (TadA) or its orthologs, or cytosine deaminase derived from or mutated from DddA or its orthologs, or its mitogens. The cytosine deaminase with the TadA mutation mentioned above can be, for example, a product in which one or more amino acid residues at positions 6, 26, 27, 28, 46, 48, 49, 61, 74, 76, 77, 82, 96, 107, 108, 112, 114, 115, 119, 122, 127, 142, 143, 151, 154, and 158 in the amino acid sequence of SEQ ID NO: 39 are mutated to other amino acids. For example, it can be a polypeptide in which amino acid position 27 is mutated to lysine, amino acid position 28 is mutated to alanine, amino acid position 61 is mutated to isoleucine, and amino acid position 96 is mutated to asparagine. Regarding the composition of the cytosine deaminase that can be used in this invention, reference can be made to the prior art, including all technical contents recorded in the international patent application publications WO2022 / 060185, WO2023 / 086953, etc., which are incorporated herein by reference.

[0064] The cytosine deaminase that can be used in this invention can be DddA tox (SEQ ID NO: 1) or a variant thereof, or a conserved amino acid substitute thereof, the cytosine deaminase may exist in full-length or in two mitotic forms. In this specification, DddA tox Used interchangeably with DddA.

[0065] DddA tox It originates from Burkholderia neonsis ( Burkholderia cenocepacia The enzymatic portion of bacterial toxins (DddA) can deaminate cytosine in double-stranded DNA. tox To avoid toxicity to host cells, the cytosine deaminase is used in two inactive splits, namely, a first split and a second split, as reported in the literature [Mok, BY et al. A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature 583, 631-637 (2020)]. When the cytosine deaminase used in this invention is used in the form of a first split and a second split, neither the first nor the second split has deaminase activity on its own; deaminase activity is only exerted when the two splits are adjacent to each other. In other words, in order to use the cytosine deaminase in the form of two splits, the full-length sequence of the cytosine deaminase needs to be formed when the sequences of the two splits are merged, and this is well known to those skilled in the art of base editing using cytosine deaminase.

[0066] In one embodiment, the DddA tox Cytosine deaminase can exist in the form of two mitotic figures, wherein one of the two mitotic figures may contain a sequence from the N-terminus of the amino acid sequence of SEQ ID NO: 1 to amino acids 33, 44, 54, 68, 82, 98 or 108, and the other of the two mitotic figures may contain a sequence from amino acids 34, 45, 55, 69, 83, 99 or 109 to the C-terminus of the amino acid sequence of SEQ ID NO: 1.

[0067] In one embodiment, one of the two split bodies may contain the amino acid sequence of SEQ ID NO: 2 (G1333-N) or a conserved amino acid substitute thereof, and the other may contain the amino acid sequence of SEQ ID NO: 3 (G1333-C) or a conserved amino acid substitute thereof.

[0068] In one embodiment, one of the two split bodies may contain the amino acid sequence of SEQ ID NO: 4 (G1397-N) or a conserved amino acid substitute thereof, and the other may contain the amino acid sequence of SEQ ID NO: 5 (G1397-C) or a conserved amino acid substitute thereof.

[0069] In one embodiment, cytosine deaminase can exist in the form of two mitotic forms, wherein one or more amino acids in the two mitotic forms can be replaced by other amino acids. For example, DddA tox The first and second cleavages may respectively contain the amino acid sequences of SEQ ID NO: 2 (G1333-N) and SEQ ID NO: 3 (G1333-C). In this case, one or more amino acids selected from the group consisting of positions 3, 5, 10, 11, 13, 14, 15, 16, 17, 18, 19, 28, 30, and 31 of SEQ ID NO: 2, or one or more amino acids selected from the group consisting of positions 13, 16, 17, 20, 21, 28, 29, 30, 31, 32, 33, 56, 57, 58, and 60 of SEQ ID NO: 3, may be substituted with other amino acids. As another example, DddA tox The first cleavage may contain the amino acid sequences of SEQ ID NO: 4 (G1397-N) and SEQ ID NO: 5 (G1397-C), in which case one or more amino acids selected from the group consisting of positions 87, 88, 91, 92, 95, 100, 101, 102, and 103 of SEQ ID NO: 4, or one or more amino acids selected from the group consisting of positions 13, 14, 15, and 16 of SEQ ID NO: 5, may be substituted with other amino acids. The “other amino acids” refer to amino acids selected from all known variants of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, aspartic acid, cysteine, glutamine, glycine, serine, threonine, tyrosine, aspartic acid, glutamic acid, arginine, histidine, lysine, and all known variants of said amino acids, excluding the amino acids present in the wild-type protein at the original mutation site. When using such a variant, two DddA molecules are respectively linked to the DNA-binding protein. tox When a pair of mitochondria cannot bind to DNA, they cannot function properly, thus enabling highly efficient and precise C-to-T editing without resulting in unwanted off-target C-to-T editing.

[0070] The cytosine deaminase used in this invention can also be used in full-length form, in which case the full-length cytosine deaminase used (e.g., DddA) tox () is made by modifying the amino acid sequence to make it non-toxic or only have low toxicity.

[0071] In DddA tox The C-terminus of DNA specifically enriches positively charged amino acids. Since DNA carries a negative charge, it binds to positively charged amino acids in proteins. By substituting these positively charged amino acids, the binding of DddA can be weakened.tox This reduces or eliminates intracellular toxicity by binding to DNA. In other words, if the toxicity is neutralized by substituting positively charged amino acids, it can be cloned using E. coli, thus ensuring the full-length DddA. tox .

[0072] Wild-type DddA tox Due to its endocytotoxicity, the use of a split approach (divided into two parts) imposes several limitations on experiments. In particular, when using Cas9, orthogonal Cas9 variants that recognize different PAMs are employed. In this case, the presence of protospacer adjacent motifs (PAMs) restricts the precise editing of cytosine bases to thymine at the desired site. Furthermore, since the target window exhibiting the highest activity is a 40 bp region between the binding sites of the two Cas9 variants, unwanted cytosine present in this region may also be edited. However, due to the full-length DddA... tox It does not separate, and therefore is not limited by PAM. In addition, it exhibits the best activity on the TC motif within 10 bp of the target site, thus resulting in higher accuracy.

[0073] Full length DddA tox Not only Cas9 can be used, but also TALE modules and zinc finger proteins can be used to edit cytosine (in the TC motif) at the desired site into thymine. Existing DddA... tox It needs to be used in the form of splits, so it needs to be delivered in pairs, but the full length DddA tox Only the TALE module and one module of zinc finger proteins are needed, so target sites can be selected without restriction.

[0074] Additionally, due to the total length DddA tox Only one DNA-binding protein module is needed, thus offering the advantage of a smaller overall size for the base editor. Existing cytosine base editors (CBEs) can edit cytosine in the R-loop formed by Cas9 binding to the target site into thymine, but the full-length DddA... tox Cytosine outside the R-ring can be edited. Therefore, using existing CBEs, it is possible to edit cytosine at restricted sites into thymine.

[0075] Based on this, the non-toxic full-length cytosine deaminase can be provided by replacing one, two, three, four, or five amino acids in the wild-type amino acid sequence of SEQ ID NO: 1 with other amino acids. The "other amino acids" refer to amino acids selected from all known variants of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, asparagine, cysteine, glutamine, glycine, serine, threonine, tyrosine, aspartic acid, glutamic acid, arginine, histidine, lysine, and all known variants of said amino acids, excluding the amino acids present in the wild-type protein at the original mutation site. For example, the other amino acid could be alanine.

[0076] The non-toxic full-length DddA tox It may contain amino acid sequences selected from the group consisting of SEQ ID NO: 17 to SEQ ID NO: 27.

[0077] Preferably, the full-length cytosine deaminase variant that can be used in the present invention may have one or more amino acid substitutions selected from the group consisting of the group consisting of S substitution at position 37 to G, G substitution at position 59 to S, A substitution at position 109 to V, and S substitution at position 129 to G in the amino acid sequence of SEQ ID NO.1.

[0078] More preferably, the full-length cytosine deaminase variant that can be used in the present invention may have all of the following in the amino acid sequence of SEQ ID NO: 1: S replaced by G at position 37, G replaced by S at position 59, A replaced by V at position 109, and S replaced by G at position 129, in which case its sequence is as shown in SEQ ID NO: 24.

[0079] As yet another example, the full-length cytosine deaminase variant that can be used in this invention may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 25 to SEQ ID NO: 27.

[0080] In one embodiment, the cytosine deaminase usable in this invention may be a variant of DddA of SEQ ID NO: 1, and may be used in full-length or in two-split form. For example, the cytosine deaminase usable in this invention may be a variant evolved from DddA. Such evolved DddA variants may include DddA6 and DddA11, etc., in which regard references can be made to previously known information, including International Patent Application Publication Publication WO2022 / 221337, etc. Additionally, the variant of DddA usable in this invention may be an ortholog of DddA. In one embodiment, the ortholog of DddA usable in this invention may be FZY2 or an engineered or evolved variant thereof, but is not limited thereto. FZY2 is a DddA homolog derived from the Lachnospiraceae bacterium sunii NSJ-8, which has the amino acid sequence of SEQ ID NO: 6.

[0081] In the base editing composition or base editor of the present invention for preparing herbicide-resistant plants, when FZY2 is used as a cytosine deaminase, a variant having the amino acid sequence of SEQ ID NO: 7 (FZY2 v1.6) can be used, and in the amino acid sequence of SEQ ID NO: 7, it can be used as an N-terminal splitting form in the form of a splitting form having the amino acid sequence of SEQ ID NO: 8 (FZY2 v1.6 S100N), and as a C-terminal splitting form in the form of a splitting form having the amino acid sequence of SEQ ID NO: 10 (FZY2 v1.6 S100C).

[0082] In the base editing composition or base editor for producing herbicide-resistant plants of the present invention, when cytosine deaminase is used, it may be contained in a fusion protein comprising a DNA-binding protein, preferably directly or indirectly (e.g., via linkers and / or other protein components) linked to the C-terminus of the DNA-binding protein. When the fusion protein contains other protein components besides cytosine deaminase, the cytosine deaminase may be directly or indirectly (e.g., via linkers and / or other protein components) linked to the N-terminus or C-terminus of said other protein components.

[0083] The base editing composition or base editor used in this invention may contain a polynucleotide encoding a cytosine deaminase as described above.

[0084] adenine deaminase The adenine deaminase that can be used in the base editing composition or base editor for producing herbicide-resistant plants of the present invention refers to any aminohydrolase having the activity of converting adenine bases into hypoxanthine (inosine as a nucleoside). Enzymes having both adenine deaminase and cytosine deaminase activity also correspond to the adenine deaminase referred to in this specification. The adenine deaminase that can be used in the present invention can be derived from or mutated (e.g., engineered or evolved) from any organism (e.g., eukaryotes or prokaryotes), including but not limited to algae, bacteria, fungi, plants, invertebrates, and mammals, for example, it can be derived from or mutated from *Escherichia coli* (E. coli), *Staphylococcus aureus* (… Staphylococcus aureus , S.aureus ), Salmonella typhi Salmonella Typhi , S.Typhi Shewanella putrefactive bacteria ( Shewanella putrefaciens , S.putrefaciens Haemophilus influenzae ( ) Haemophilus influenzae , H. influenzae ) or Crescentella ( Caulobacter crescentus , C. crescentus The adenine deaminase may be, for example, tRNA-specific adenosine deaminase (TadA) or a variant thereof.

[0085] The aforementioned TadA can be, for example, TadA8e (SEQ ID NO: 39) or a truncated form or variant thereof (e.g., a variant modified or evolved in a manner applicable to deoxynucleotides). Variants of the aforementioned TadA8e can be, for example, products of SEQ ID NO: 39 where one or more amino acid residues at positions 26, 28, 30, 36, 46, 48, 49, 51, 70, 72, 73, 76, 77, 82, 84, 96, 106, 108, 110, 111, 123, 134, 146, 147, 152, 154, 155, 156, 157, and 166 are substituted with other amino acids, or conserved amino acid substitutes thereof. For example, variants of TadA8e may contain one or more amino acid substitutions selected from the group consisting of V28Q, V28R, A48W, F84M, V106A, K110S, K110T, K110V, R111F, R111Q, R111S, R111T, and R111Y. For example, variants of TadA8e may contain one or more amino acid substitutions selected from the group consisting of R26, V28, A48, Y73, and H96. It is a common practice in the field of biotechnology to describe specific amino acid substitutions by indicating the amino acid before substitution, the position number of the residue, and the sequence of the amino acid after substitution. Regarding the composition of the adenine deaminase that can be used in this invention, reference can be made to all previously known content, including all technical contents described in international patent application publications WO2022 / 060185 and WO2023 / 086953, which are incorporated herein by reference.

[0086] When adenine deaminase is used in the base editing composition or base editor of the present invention for producing herbicide-resistant plants, it may be contained in a fusion protein comprising a DNA-binding protein, preferably directly or indirectly (e.g., via linkers and / or other protein components) linked to the C-terminus of the DNA-binding protein. When the fusion protein contains other protein components besides adenine deaminase, the adenine deaminase may be directly or indirectly (e.g., via linkers and / or other protein components) linked to the N-terminus or C-terminus of said other protein components.

[0087] The base editing composition or base editor used in this invention may contain a polynucleotide encoding adenine deaminase as described above.

[0088] Cutting enzyme The nicking enzyme that can be used in the base editing composition or base editor for producing herbicide-resistant plants of the present invention refers to any enzyme that produces single-strand breaks (also called "nicks") in double-stranded DNA; in other words, it has the activity of cutting one strand of the DNA double helix but not the other strand. The nicking enzyme can be selected from MutH, BspD6I, FokI (including homodimers or heterodimers), BsaI, BsmBI, BsmAI, BsrDI, CviPII, BspQI, AlwI, and I-TevI, fragments thereof, and variants thereof, or their conserved amino acid substitutions. For example, MutH (SEQ ID NO: 12), Nt.BspD6I(C) (the catalytically active fragment of Nt.BspD6I; SEQ ID NO: 16), or a heterodimer of the catalytically active domain of FokI (SEQ ID NO: 13) can be used as the nicking enzyme. MutH can be used in the form of a variant or fragment thereof, wherein one or more amino acids selected from the group consisting of residues 48, 91, 94, 184, and 212 of the amino acid sequence of SEQ ID NO: 6 are mutated to other amino acids. For example, it may have substitutions of one or more amino acids selected from the group consisting of K48A, E91A, F94A, R184A, and Y212S. FokI can be used in the form of a variant or fragment thereof, wherein one or more amino acids selected from the group consisting of residues 469, 483, 486, 487, 490, 496, 499, 537, and 538 of the amino acid sequence of SEQ ID NO: 14 are mutated to other amino acids, and can be used in monomeric or dimer form, wherein the dimer may be a homodimer or a heterodimer. For example, it can be used in the form of a heterodimer as shown in SEQ ID NO: 13 (a FokI fragment (domain) with amino acid substitutions of E490K and I538K and a FokI fragment (domain) with amino acid substitutions of D450A, Q486E and I499L linked by 40 amino acid linkers). The amino acid substitution positions (numbers) mentioned above are based on SEQ ID NO: 14. It is a common practice in the biotechnology field to describe specific amino acid substitutions by indicating the position number of the amino acid before substitution and the sequence of the amino acid after substitution.

[0089] In the base editing composition or base editor for producing herbicide-resistant plants of the present invention, when a nicking enzyme is used, it can be used as a fusion protein linked to a DNA-binding protein and / or other proteins, or it can be expressed as a single protein. When more than one fusion protein is used, only one of the more than one fusion protein contains a nicking enzyme, or multiple fusion proteins may contain the same or different nicking enzymes.

[0090] When the nicking enzyme is contained in a fusion protein that includes a DNA-binding protein, it is preferably directly or indirectly (e.g., via a linker and / or other protein components) linked to the C-terminus of the DNA-binding protein. When the fusion protein contains other protein components besides the nicking enzyme, the nicking enzyme can be directly or indirectly (e.g., via a linker and / or other protein components) linked to the N-terminus or C-terminus of the other protein components.

[0091] The base editing composition or base editor used in this invention may contain a polynucleotide encoding the nicking enzyme as described above.

[0092] accessory proteins One or more of the fusion proteins used in the base editing compositions or base editors of this invention may contain a nuclear export signal (NES) as part of the fusion protein. When an NES is attached to a base editing protein, base editing can be performed with greater efficiency. The NES sequence can be any signal sequence that confers nuclear export capability (e.g., VDEMTKKFGTLTIHDTEK), and can use natural nuclear export signals (NES) or artificially synthesized NES. For example, it may be derived from mouse parvovirus (MVM), but is not limited thereto. When using an NES, its location can be diverse, for example, it may be directly or indirectly (e.g., through linkers and / or other protein components) linked to the N-terminus of a DNA-binding protein, but is not limited thereto. In this respect, the base editing compositions or base editors used in this invention may contain polynucleotides encoding the NES as described above.

[0093] One or more of the fusion proteins contained in the base editing composition or base editor used in this invention may also contain a chloroplast transit signal (CTS) or a plasmid transit peptide (PTP). Such a base editing composition or base editor can be used to edit chloroplast, chromoplast, or leucoplast DNA in plant cells. The CTS used in this invention can be any signal sequence capable of translocating into chloroplasts, and can be a natural CTS present at the N-terminus of various chloroplast proteins; additionally, artificially synthesized CTSs can also be used. When using a CTS or PTP, its location can be diverse, for example, it can be directly or indirectly (e.g., through linkers and / or other protein components) linked to the N-terminus of a DNA-binding protein or the N-terminus of a NES, but is not limited thereto. In this regard, the base editing composition or base editor used in this invention may contain a polynucleotide encoding a CTS or PTP as described above.

[0094] The base editing composition or base editor used in this invention can be a protein containing uracil DNA glycosylase (UDG). UDG is known to recognize damaged DNA in its native state and has the activity of selectively removing only uracil bases from DNA. During A-to-G base editing, when combined with cytosine deaminases (such as DddA...), toxWhen used together with UDG, cytosine bases are deaminated and converted to uracil. When UDG is expressed simultaneously, the converted uracil bases can be removed, restoring the original DNA sequence and avoiding unwanted cytosine base editing. Therefore, UDG is used for A-to-G base editing via base editing compositions or base editors containing cytosine deaminase, thereby conferring herbicide resistance traits. This technique is particularly useful for selectively editing adenine bases in environments where the natural expression level of UDG is low, especially in plant cell organelles. UDG from any species can be used, such as UDG from Arabidopsis, humans, mice, tobacco, rice, etc., and UDG can have various lengths, such as containing 2, 5, 10, 16, 24, or 32 amino acids. UDG is preferably used as a fusion protein in conjunction with the DNA-binding protein, cytosine deaminase, and adenine deaminase used in this invention, but it can also be delivered to the target DNA in a form separate from the components (protein or polynucleotide). When used as a fusion protein, UDG can be linked to the C-terminus of cytosine deaminase (or its mitochondria) or the C-terminus of adenine deaminase, but is not limited thereto. In this regard, the base editing composition or base editor used in this invention may contain a polynucleotide encoding UDG as described above, which may be linked to a polynucleotide encoding other proteins or exist alone.

[0095] The base editing compositions or base editors used in this invention may further comprise a uracil glycosylase inhibitor (UGI) as an enzyme protein. The UGI is preferably contained in a base editor fusion protein comprising cytosine deaminase, because the UGI can increase C-to-T base editing efficiency by inhibiting the activity of uracil DNA glycosylase (UDG), an enzyme that catalyzes the removal of uracil (U) from DNA to restore mutant DNA. When using the UGI, it can be used as a fusion protein linked to other proteins or expressed as a standalone protein. When used as a fusion protein, the location of the UGI can be varied; preferably, it can be directly or indirectly (e.g., through linkers and / or other protein components) linked to the C-terminus of cytosine deaminase. In this regard, the base editing compositions or base editors used in this invention may comprise a polynucleotide encoding the UGI as described above, which may be linked to a polynucleotide encoding other proteins or exist alone.

[0096] When the protein used in the base editing method of this invention is in the form of a fusion protein(s), the fusion protein comprises a DNA-binding protein and an enzyme protein. In addition to the DNA-binding protein and the enzyme protein, it may contain signal sequences such as NES and CTS, and may contain other sequences for biotechnological methods such as tagging. These individual polypeptides can be directly linked or linked via linkers. A "linker" refers to any molecule that connects two different molecules. In the field of biotechnology, any linker known to be used to provide fusion proteins or protein conjugates can be used; for example, a peptide linker containing 1 to 100 amino acid residues can be used. In this respect, the base editing composition or base editor used in this invention may contain multiple polynucleotides encoding the fusion protein(s) as described above. Such a fusion protein (or the polynucleotide encoding the fusion protein) can be designed and prepared by any method known in the field of biotechnology.

[0097] In the base editor of the present invention, the polypeptide included as part of the fusion protein, in particular, one or more of adenine deaminase, cytosine deaminase and nickase, may be expressed by a polynucleotide that has been codon-optimized for expression in plants.

[0098] Composition of an exemplary base composition The base editing composition or base editor used in this invention and the DNA binding protein used in the base editing method, as a whole or in part, can recognize and bind, in the DNA sequence encoding the chloroplast D1 protein, a nucleotide sequence comprising (1) 5'-TTTTGGCCGATTGATTTTCCAATATGCTAGTTTCAACAATTCTCGTTCTTTACA-3' (SEQ ID NO: 29) or a part thereof, or its complementary sequence; or (2) 5'-GAAGAAGAAACTTATAATATCGTAGCCGCTCATGGTTATTTTGGCCGATTGATCTTCCA-3' (SEQ ID NO: 30) or a part thereof, or its complementary sequence. For example, one or more DNA-binding proteins contained in one or more fusion proteins, as a whole or in part, can recognize and bind to the nucleotide sequence of (3) 5'-TTTTGGCCGATTGATTTTCCAAT-3' (SEQ ID NO: 31) or a part thereof or its complementary sequence in the DNA sequence encoding chloroplast D1 protein; (4) 5'-TGTAAAGAACGAGAATTGTT-3' (SEQ ID NO: 32) or a part thereof or its complementary sequence; (5) 5'-GAAGAAGAAACTTATAATAT-3' (SEQ ID NO: 33) or a part thereof or its complementary sequence; or (6) 5'-TGGAAGATCAATCGGCCAAA-3' (SEQ ID NO: 34) or a part thereof or its complementary sequence. For example, when two DNA-binding proteins are contained in two fusion proteins and used, one DNA-binding protein recognizes and binds to the nucleotide sequence of (3) 5'-TTTTGGCCGATTGATTTTCCAAT-3' (SEQ ID NO: 31) or a portion thereof, or its complementary sequence, and the other DNA-binding protein recognizes and binds to the nucleotide sequence of (4) 5'-TGTAAAGAACGAGAATTGTT-3' (SEQ ID NO: 32) or a portion thereof, or its complementary sequence. For example, when two DNA-binding proteins are contained in two fusion proteins and used, one DNA-binding protein recognizes and binds to the nucleotide sequence of (5) 5'-GAAGAAGAAACTTATAATAT-3' (SEQ ID NO: 33) or a portion thereof, or its complementary sequence, and the other DNA-binding protein recognizes and binds to the nucleotide sequence of (6) 5'-TGGAAGATCAATCGGCCAAA-3' (SEQ ID NO: 34) or a portion thereof, or its complementary sequence.

[0099] The base editing composition or base editor used in this invention may comprise a fusion protein, which may comprise a DNA-binding protein, a cytosine deaminase, and an adenine deaminase. In this case, the DNA-binding protein may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 55 to SEQ ID NO: 58, the cytosine deaminase may comprise the amino acid sequence of SEQ ID NO: 24, and the adenine deaminase may comprise the amino acid sequence of SEQ ID NO: 39. The fusion protein is used to edit an adenine (A) base encoding a plant chloroplast D1 protein to a guanine (G) base, specifically, by means of the A-to-G base editing, mutating a serine residue corresponding to the 264th serine residue of the Arabidopsis D1 protein to glycine. The fusion protein may also comprise UDG, which may comprise the amino acid sequence of SEQ ID NO: 41. The base editing composition or base editor used in this invention may comprise a polynucleotide encoding the fusion protein as described above.

[0100] The base editing composition or base editor used in this invention may comprise two fusion proteins. One of the two fusion proteins may comprise a DNA-binding protein, a first cytosine deaminase mitochondria, and an adenine deaminase. The other of the two fusion proteins may comprise a DNA-binding protein and a second cytosine deaminase mitochondria. In this case, the DNA-binding protein may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 55 to SEQ ID NO: 58, the first cytosine deaminase mitochondria may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 5, the second cytosine deaminase mitochondria may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 5, and the adenine deaminase may comprise the amino acid sequence of SEQ ID NO: 39. The fusion protein is used to edit the adenine (A) bases encoding the plant chloroplast D1 protein to guanine (G) bases. Specifically, through this A-to-G base editing, the serine residue corresponding to the 264th serine residue of the Arabidopsis D1 protein is mutated to glycine. The fusion protein may also contain an UDG, which may contain the amino acid sequence of SEQ ID NO: 41. The base editing composition or base editor used in this invention may contain a polynucleotide encoding the two fusion proteins as described above.

[0101] The base editing composition or base editor used in this invention may comprise two fusion proteins, one of which may comprise a DNA-binding protein and a first cytosine deaminase mitochondrion, and the other of which may comprise a DNA-binding protein and a second cytosine deaminase mitochondrion. In this case, the DNA-binding protein may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 63 to SEQ ID NO: 65, the first or second cytosine deaminase mitochondrion may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 10, and the second cytosine deaminase mitochondrion may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 10. The fusion protein is used to edit the cytosine (C) bases encoding the plant chloroplast D1 protein to thymine (T) bases, and in particular, through the C-to-T base editing, the alanine residue corresponding to the 251st alanine residue of the Arabidopsis D1 protein is mutated to valine. The fusion protein may also contain a UGI, which may contain the amino acid sequence of SEQ ID NO: 43. The base editing composition or base editor used in this invention may contain polynucleotides encoding the two fusion proteins as described above.

[0102] The base editing composition or base editor used in this invention may comprise two fusion proteins. One of the two fusion proteins may comprise a DNA-binding protein and an adenine deaminase, and the other of the two fusion proteins may comprise a nicking enzyme. The DNA-binding protein may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 71 and SEQ ID NO: 72. The adenine deaminase may comprise an amino acid sequence consisting of SEQ ID NO: 39. The nicking enzyme may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 16. The fusion protein is used to edit an adenine (A) base encoding a guanine (G) base into a guanine (G) base. In particular, through the A-to-G base editing, the serine residue corresponding to the 264th serine residue of the Arabidopsis D1 protein is mutated to glycine. The base editing composition or base editor used in this invention may comprise a polynucleotide encoding the two fusion proteins as described above.

[0103] The present invention provides a gene-edited plant or part of a plant, wherein the DNA sequence encoding the 5'-AGT-3' serine residue of the chloroplast D1 protein is edited to encode the 5'-GGT-3' glycine, thereby making the plant resistant to herbicides, wherein the serine residue is the serine residue corresponding to the 264th serine residue of the D1 protein of Arabidopsis thaliana.

[0104] The present invention also provides a gene-edited plant or part of a plant, wherein the DNA sequence encoding the 5'-GCT-3' alanine residue of the chloroplast D1 protein is edited to encode the 5'-GTT-3' valine residue, thereby making the plant resistant to herbicides, wherein the alanine residue is the alanine residue corresponding to the 251st alanine residue of the Arabidopsis thaliana D1 protein.

[0105] In this invention, the term "gene-edited plant" is intended as a concept in contrast to transgenic plants (GMOs) involving the insertion of foreign genes, referring to plants in which the introduced foreign gene has been removed and a single base in the endogenous genome has been mutated in a targeted manner in the absence of foreign gene insertion.

[0106] The gene-edited plant or a portion thereof of the present invention is provided by editing a DNA sequence encoding a 5'-AGT-3' serine residue of chloroplast D1 protein to a 5'-GGT-3' glycine residue or a 5'-GCT-3' a alanine residue to a 5'-GTT-3' valine residue using a base editing composition or base editor, wherein the base editing composition or base editor has the activity of editing adenine (A) bases of DNA encoding plant chloroplast D1 protein to guanine (G) bases and / or editing cytosine (C) bases to thymine (T) bases. Preferably, it can be provided by the gene editing composition or gene editing method described throughout this specification.

[0107] The present invention also provides a plant cell or protoplast that, according to the invention, confers herbicide resistance by base editing in the gene encoding the D1 protein.

[0108] Another aspect of the present invention provides a plant or a portion thereof grown or cultured from plant cells or protoplasts edited from the bases described above, or a progeny or clone of said plant or a portion thereof.

[0109] Another aspect of the invention provides a seed obtained from the plant, its offspring or clone, or a portion thereof as described above.

[0110] Another aspect of the present invention provides a plant or its offspring, or a portion thereof, grown from the seeds described above.

[0111] When the base editing composition or base editor used in this invention is in the form of a polynucleotide, the transformation of plants by such a polynucleotide can be achieved using transformation techniques well known to those skilled in the art of biotechnology. For example, transformation methods using Agrobacterium (e.g., Agrobacterium tumefaciens, Agrobacterium rhizogene, etc.), microprojectile bombardment, electroporation, PEG-mediated fusion, microinjection, liposome-mediated method, in-planta transformation, vacuum infiltration method, floral meristem dipping method, and Agrobacterium spraying method can be used. Regarding Agrobacterium transformation, a binary vector system utilizing two replicons can be used.

[0112] When the base editing composition or base editor used in this invention is in the form of a polynucleotide, a viral vector may be used. For example, such a viral vector may include, but is not limited to, Geminivirus, Tobacco Rattle Virus (TRV), Tomato Mosaic Virus (ToMV), Foxtail Mosaic Virus (FoMV), Barley Yellow Striate Mosaic Virus (BYSMV), and Sonchus Yellow Net Rhabdovirus (SYNV).

[0113] When the base editing composition or base editor used in this invention is in the form of mRNA, the mRNA can be delivered directly or via a carrier. Methods for delivering mRNA molecules into cells, including methods for delivering mRNA into cells in vivo or in vitro, are considered. For example, mRNA molecules can be delivered into cells by comprising lipids (e.g., liposomes, micelles, etc.), nanoparticles or nanotubes, or cationic compounds (e.g., polyethyleneimine or PEI). In some cases, a bolistic method (e.g., a gene gun or a bioballistic particle delivery system) can be used to deliver mRNA into cells. The carrier may comprise, but is not limited to, cell-penetrating peptides (CPPs), nanoparticles, or polymers.

[0114] The plant cells, protoplasts, plants, and seeds of the present invention are characterized in that the adenine (A) bases of the DNA encoding the chloroplast D1 protein are edited to guanine (G) bases and / or the cytosine (C) bases are edited to thymine (T) bases, thereby achieving herbicide resistance. By using the base editing compositions or base editors described in this specification, not only can cytosine bases present in the 5'-TC-3' sequence of the target DNA sequence be edited to thymine, but also cytosine bases present in the 5'-AC-3', 5'-CC-3', or 5'-GC-3' sequences can be edited to thymine. Furthermore, by using a nicking enzyme, even without the use of DddA... tox It can also achieve targeted C-to-T and / or A-to-G base editing.

[0115] In T2 and subsequent generations of individuals transformed using the base editing composition or base editor used in this invention, homologous individuals were successfully obtained, exhibiting base editing only on the desired target bases in the chloroplast DNA. Homologousity refers to the identical copies of DNA contained in chloroplasts or mitochondria, indicating a genetically stable state, which is a crucial aspect for improving plant traits through gene editing.

[0116] In techniques that manipulate plant genes through gene editing to provide improved plants, commercialization is hampered by regulations related to genetically modified plants (GMOs), as plants from which foreign genes introduced for gene editing are not removed face difficulties. It has been confirmed that by using the base editing composition or base editor used in this invention, herbicide-resistant plants can be stably obtained even without the presence of foreign genes conferring herbicide resistance.

[0117] The herbicide-resistant plants of the present invention play a role in methods for controlling weeds. Therefore, the present invention also provides a method for controlling weeds near the herbicide-resistant plants of the present invention. The method includes the step of applying an effective amount of herbicide to the weeds and the herbicide-resistant plants, wherein the plants exhibit enhanced resistance to more than one herbicide, particularly triazine herbicides, compared to wild-type plants. In such methods for controlling weeds, preferably, the herbicide-resistant plants of the present invention include, but are not limited to, crops such as soybeans, lettuce, and zoysia grass.

[0118] The term "weeds" can be understood in the broadest sense as all plants growing in undesirable locations.

[0119] This invention also provides a herbicide-treated turf, wherein the turf is a ground cover plant (such as Zoysia japonica) attached to the soil. The ground cover plant is preferably a plant, which, according to this invention, is genetically modified to be resistant to herbicides, preferably Zoysia japonica. When herbicide treatment is applied starting with the turf, the overall vegetation yield of the plant can be increased, which is therefore beneficial. Since the gene-edited plant of this invention is herbicide-resistant, it can be used in the form of herbicide-treated turf. To date, there are no publicly reported examples of turf as ground cover plants that can use herbicide-treated turf.

[0120] The present invention may be based on the above and may involve, but is not limited to, the production methods of plants or parts thereof that are resistant to herbicides through DNA base editing as described below.

[0121] 1. A method for producing a plant or a portion of a plant resistant to herbicides through DNA base editing, comprising the steps of: expressing a base editing composition in a target plant or a portion of a plant, and causing said plant or the portion of a plant to grow or be cultured, said base editing composition having the activity of editing adenine (A) bases of DNA encoding plant chloroplast D1 protein to guanine (G) bases and / or editing cytosine (C) bases to thymine (T) bases.

[0122] 2. The method according to Embodiment 1, wherein the herbicide is a triazine herbicide.

[0123] 3. The method according to Embodiment 1, wherein the herbicide is selected from the group consisting of propargite, atrazine, pymetrozine, simazine, cyclomethrin, cypermethrin, terbuprofen, diuron, fluroxypyr, linuron, terbutaline, and benzimidone.

[0124] 4. The method according to embodiment 1, wherein the herbicide is atrazine.

[0125] 5. The method according to Embodiment 1, wherein the plant resistant to herbicides through DNA base editing is a plant in which the serine residue of the D1 protein is mutated to glycine or the alanine residue is mutated to valine, wherein the serine residue corresponds to the serine residue at position 264 of the D1 protein of Arabidopsis thaliana, and the alanine residue corresponds to the alanine residue at position 251 of the D1 protein of Arabidopsis thaliana.

[0126] 6. The method according to embodiment 5, wherein the plant resistant to herbicides through DNA base editing is a plant in which the DNA sequence encoding the 5'-AGT-3' of the serine residue is edited to encode the 5'-GGT-3' of glycine, or the DNA sequence encoding the 5'-GCT-3' of the alanine residue is edited to encode the 5'-GTT-3' of valine.

[0127] 7. The method according to Embodiment 1, wherein the target plant is a row crop, fruit, cooking plant, vegetable, forest tree, or ornamental plant.

[0128] 8. The method according to Embodiment 1, wherein the target plant is selected from soybean, zoysia grass and lettuce.

[0129] 9. The method according to embodiment 1, wherein the target plant is a transgenic (GMO) plant or a progeny of a plant that has previously been made herbicide resistant by inserting recombinant DNA.

[0130] 10. The method according to embodiment 9, wherein the transgenic plant previously made resistant to herbicides by inserting recombinant DNA is genetically modified to be resistant to herbicides that control amino acid synthesis or herbicides that promote the generation of reactive oxygen species (ROS).

[0131] 11. The method according to Embodiment 1, wherein the base editing composition comprises a DNA-binding protein and an enzyme protein, or a polynucleotide encoding the protein, wherein the enzyme protein comprises one or more of cytosine deaminase, adenine deaminase, and nicking enzyme, wherein the cytosine deaminase exists in full-length or in two mitotic forms.

[0132] 12. The method according to embodiment 11, wherein the DNA-binding protein and the enzyme protein exist in the form of more than one fusion protein.

[0133] 13. The method according to embodiment 11, wherein the DNA-binding protein is selected from the group consisting of zinc finger protein, transcription activator-like effector (TALE) protein and CRISPR-related nuclease.

[0134] 14. The method according to embodiment 11, wherein, in the DNA sequence encoding the chloroplast D1 protein, the DNA-binding protein recognizes (1) a portion of the nucleotide sequence of 5'-TTTTGGCCGATTGATTTTCCAATATGCTAGTTTCAACAATTCTCGTTCTTTACA-3' (SEQ ID NO: 29) or its complementary sequence; or (2) a portion of the nucleotide sequence of 5'-GAAGAAGAAACTTATAATATCGTAGCCGCTCATGGTTATTTTGGCCGATTGATCTTCCA-3' (SEQ ID NO: 30) or its complementary sequence.

[0135] 15. The method according to embodiment 11, wherein, in the DNA sequence encoding the chloroplast D1 protein, the DNA-binding protein recognizes (3) a portion of the nucleotide sequence of 5'-TTTTGGCCGATTGATTTTCCAAT-3' (SEQ ID NO: 31) or its complementary sequence; (4) a portion of the nucleotide sequence of 5'-TGTAAAGAACGAGAATTGTT-3' (SEQ ID NO: 32) or its complementary sequence; (5) a portion of the nucleotide sequence of 5'-GAAGAAGAAACTTATAATAT-3' (SEQ ID NO: 33) or its complementary sequence; or (6) a portion of the nucleotide sequence of 5'-TGGAAGATCAATCGGCCAAA-3' (SEQ ID NO: 34) or its complementary sequence.

[0136] 16. The method according to embodiment 11, wherein the adenine deaminase comprises the amino acid sequence of SEQ ID NO: 39 or a conserved amino acid substitute thereof.

[0137] 17. The method according to embodiment 11, wherein the adenine deaminase comprises a variant or a conserved amino acid substitute thereof in which one or more amino acids selected from the group consisting of positions 28, 30, 46, 48, 49, 82, 84, 106, 108, 110 and 111 of the amino acid sequence of SEQ ID NO: 39 are substituted with other amino acids.

[0138] 18. The method according to embodiment 11, wherein the cytosine deaminase is an apolipoprotein B editing complex (APOBEC), an activation-induced deaminase (AID), or DddA, or a variant thereof, or a variant of tRNA-specific adenosine deaminase (TadA).

[0139] 19. The method according to embodiment 11, wherein the cytosine deaminase is a DddA having the amino acid sequence of SEQ ID NO: 1. tox It or its variants, or its conserved amino acid substitutes, and exist in full-length or in two cleaved forms.

[0140] 20. The method according to embodiment 11, wherein the cytosine deaminase exists in the form of two mitotic bodies, one of which contains a sequence from the N-terminus of the amino acid sequence of SEQ ID NO: 1 to amino acid positions 33, 44, 54, 68, 82, 98, or 108, and the other of the two mitotic bodies contains a sequence from amino acid positions 34, 45, 55, 69, 83, 99, or 109 of the amino acid sequence of SEQ ID NO: 1 to the C-terminus.

[0141] 21. The method according to embodiment 20, wherein either of the two split bodies contains the amino acid sequence of SEQ ID NO: 2 or a conserved amino acid substitute thereof, and the other contains the amino acid sequence of SEQ ID NO: 3 or a conserved amino acid substitute thereof; or either of the two split bodies contains the amino acid sequence of SEQ ID NO: 4 or a conserved amino acid substitute thereof, and the other contains the amino acid sequence of SEQ ID NO: 5 or a conserved amino acid substitute thereof.

[0142] 22. The method according to embodiment 11, wherein the cytosine deaminase exists in the form of two mitotic figures, one of which is a variant or a conserved substitute thereof in which one or more amino acids selected from the group consisting of positions 3, 5, 10, 11, 13, 14, 15, 16, 17, 18, 19, 28, 30, and 31 of SEQ ID NO: 2 are substituted with other amino acids; and the other is a variant or a conserved substitute thereof in which one or more amino acids selected from the group consisting of positions 13, 16, 17, 20, 21, 28, 29, 30, 31, 32, 33, 56, 57, 58, and 60 of SEQ ID NO: 3 are substituted with other amino acids; or One of the two splits is a variant or a conservative substitute thereof in which one or more amino acid sequences selected from the group consisting of positions 87, 88, 91, 92, 95, 100, 101, 102 and 103 of SEQ ID NO: 4 are replaced by other amino acids, and the other is a variant or a conservative substitute thereof in which one or more amino acids selected from the group consisting of positions 13, 14, 15 and 16 of SEQ ID NO: 5 are replaced by other amino acids.

[0143] 23. The method according to embodiment 11, wherein the cytosine deaminase exists in the form of two mitotic bodies, one of which contains the amino acid sequence of SEQ ID NO: 8 or a conserved amino acid substitute thereof, and the other of which contains the amino acid sequence of SEQ ID NO: 10 or a conserved amino acid substitute thereof.

[0144] 24. The method according to embodiment 11, wherein the cytosine deaminase comprises the amino acid sequence of SEQ ID NO: 24 or a conserved amino acid substitute thereof.

[0145] 25. The method according to embodiment 11, wherein the nicking enzyme is selected from the group consisting of MutH, BspD6I, FokI (including FokI-FokI homodimer or heterodimer), BsaI, BsmBI, BsmAI, BsrDI, CviPII, BspQI, AlwI and I-TevI, fragments thereof and variants thereof.

[0146] 26. The method according to embodiment 12, wherein the base editing composition comprises a fusion protein comprising a DNA-binding protein, a cytosine deaminase, and an adenine deaminase, the DNA-binding protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 55 to SEQ ID NO: 58, the cytosine deaminase comprising the amino acid sequence of SEQ ID NO: 24, and the adenine deaminase comprising the amino acid sequence of SEQ ID NO: 39.

[0147] 27. The method according to embodiment 12, wherein the base editing composition comprises two fusion proteins, one of the two fusion proteins comprising a DNA-binding protein, a first cytosine deaminase mitochondria, and an adenine deaminase, the other of the two fusion proteins comprising a DNA-binding protein and a second cytosine deaminase mitochondria, the DNA-binding protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 55 to SEQ ID NO: 58, the first cytosine deaminase mitochondria comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 5, the second cytosine deaminase mitochondria comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 5, and the adenine deaminase comprising the amino acid sequence of SEQ ID NO: 39.

[0148] 28. The method according to embodiment 26 or 27, wherein the fusion protein further comprises UDG of SEQ ID NO: 41.

[0149] 29. The method of claim 12, wherein the base editing composition comprises two fusion proteins, one of the two fusion proteins comprising a DNA-binding protein and a first cytosine deaminase mitochondria, the other of the two fusion proteins comprising a DNA-binding protein and a second cytosine deaminase mitochondria, the DNA-binding protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 63 to SEQ ID NO: 65, the first cytosine deaminase mitochondria comprising the amino acid sequence of SEQ ID NO: 8, and the second cytosine deaminase mitochondria comprising the amino acid sequence of SEQ ID NO: 10.

[0150] 30. The method according to embodiment 29, wherein the fusion protein further comprises UGI of SEQ ID NO: 43.

[0151] 31. The method according to embodiment 12, wherein the base editing composition comprises two fusion proteins, one of the two fusion proteins comprising a DNA-binding protein and an adenine deaminase, the other of the two fusion proteins comprising a DNA-binding protein and a nicking enzyme, the DNA-binding protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 71 and SEQ ID NO: 72, the adenine deaminase comprising the amino acid sequence of SEQ ID NO: 39, and the nicking enzyme comprising the amino acid sequence of SEQ ID NO: 16.

[0152] 32. The method according to any one of embodiments 1 to 31, wherein the step of expressing the base composition in a target plant or a part of a plant comprises: introducing a polynucleotide encoding one or more fusion proteins contained in the base composition into the plant or a part of a plant.

[0153] 33. The method according to embodiment 32, wherein at least a portion of the polynucleotide is codon-optimized for expression in the plant or a portion of the plant.

[0154] 34. The method according to any one of embodiments 1 to 33, wherein a portion of the plant is a plant cell, protoplast, or callus.

[0155] 35. A gene-edited plant or part of a plant, said plant being a herbicide-resistant plant in which the DNA sequence encoding a serine residue of chloroplast D1 protein at 5'-AGT-3' is edited to 5'-GGT-3', or the DNA sequence encoding an alanine residue at 5'-GCT-3' is edited to 5'-GTT-3', wherein... The serine residue is the serine residue corresponding to the 264th serine residue of the Arabidopsis thaliana D1 protein, and the alanine residue is the alanine residue corresponding to the 251st alanine residue of the Arabidopsis thaliana D1 protein.

[0156] 36. The gene-edited plant or part of a plant according to embodiment 35, wherein the DNA sequence encoding a serine residue of chloroplast D1 protein is edited to 5'-GGT-3' or the DNA sequence encoding an alanine residue of 5'-GCT-3' is edited to 5'-GTT-3' by a base editing composition, said base editing composition having the activity of editing adenine (A) bases of DNA encoding plant chloroplast D1 protein to guanine (G) bases and / or editing cytosine (C) bases to thymine (T) bases.

[0157] 37. A plant or part of a plant, wherein it is produced according to the method described in any one of embodiments 1 to 34.

[0158] 38. A plant or part of a plant, wherein it is a progeny or clone of the plant described in any one of embodiments 35 to 37.

[0159] 39. A seed, wherein the seed is obtained from a plant according to any one of embodiments 35 to 38.

[0160] 40. A plant or a offspring of a plant, or a part of a plant, wherein it is grown from a seed according to embodiment 39.

[0161] 41. A method for controlling weeds near a target plant, comprising: applying an effective amount of herbicide to the weeds and the target plant, wherein the target plant is a plant according to any one of embodiments 35 to 38 and 40.

[0162] 42. A turf comprising plants and soil, wherein the turf is a herbicide-treated turf.

[0163] 43. The turf according to embodiment 42, wherein the plant is the plant according to any one of embodiments 35 to 38 and 40.

[0164] 44. Turf according to embodiment 42 or 43, wherein the plants are ground cover plants.

[0165] 45. Turf according to any one of embodiments 42 to 44, wherein the plant is Zoysia japonica.

[0166] Furthermore, based on the content described above in this specification, the present invention may also relate to, but is not limited to, base editing compositions or base editors for producing herbicide-resistant plants or parts thereof.

[0167] 1. A base editing composition or base editor, wherein adenine (A) bases of DNA encoding a plant chloroplast D1 protein are edited to guanine (G) bases and / or cytosine (C) bases are edited to thymine (T) bases, comprising a DNA-binding protein and an enzyme protein, or a polynucleotide encoding said protein, said enzyme protein comprising one or more of cytosine deaminase, adenine deaminase, and nicking enzyme, said cytosine deaminase being present in full-length or in two mitotic forms.

[0168] 2. The base editing composition or base editor according to Embodiment 1, wherein the herbicide is a triazine herbicide.

[0169] 3. The base editing composition or base editor according to Embodiment 1 or 2, wherein the herbicide is selected from the group consisting of propargite, atrazine, pymetrozine, simazine, cyclomethrin, cypermethrin, terbuprofen, diuron, fluroxypyr, linuron, terbutaline, and benzimidone.

[0170] 4. The base editing composition or base editor according to any one of embodiments 1 to 3, wherein the herbicide is atrazine.

[0171] 5. The base editing composition or base editor according to any one of embodiments 1 to 4, wherein the plant resistant to herbicides through DNA base editing is a plant in which a serine residue of the D1 protein is mutated to glycine or an alanine residue is mutated to valine, wherein the serine residue corresponds to the serine residue at position 264 of the D1 protein of Arabidopsis thaliana, and the alanine residue corresponds to the alanine residue at position 251 of the D1 protein of Arabidopsis thaliana.

[0172] 6. The base editing composition or base editor according to claim 5, wherein the plant resistant to herbicides through DNA base editing is a plant in which the DNA sequence encoding the 5'-AGT-3' of the serine residue is edited to encode the 5'-GGT-3' of glycine, or the DNA sequence encoding the 5'-GCT-3' of the alanine residue is edited to encode the 5'-GTT-3' of valine.

[0173] 7. The base editing composition or base editor according to any one of embodiments 1 to 6, wherein the target plant is a row crop plant, fruit, culinary plant, vegetable, forest tree, or ornamental plant.

[0174] 8. The base editing composition or base editor according to any one of embodiments 1 to 7, wherein the target plant is selected from soybean, zoysia grass and lettuce.

[0175] 9. The base editing composition or base editor according to any one of embodiments 1 to 8, wherein the target plant is a transgenic (GMO) plant or a progeny of a plant that has previously been made herbicide resistant by inserting recombinant DNA.

[0176] 10. The base editing composition or base editor according to Embodiment 9, wherein the transgenic plant previously made herbicide resistant by inserting recombinant DNA has been genetically modified to be resistant to herbicides that inhibit amino acid synthesis or herbicides that promote the generation of reactive oxygen species (ROS).

[0177] 11. The base editing composition or base editor according to any one of embodiments 1 to 10, wherein the DNA binding protein and the enzyme protein are present in the form of more than one fusion protein.

[0178] 12. The base editing composition or base editor according to any one of embodiments 1 to 11, wherein the DNA binding protein is selected from the group consisting of zinc finger protein, transcription activator-like effector (TALE) protein and CRISPR-related nuclease.

[0179] 13. The base editing composition or base editor according to any one of embodiments 11 to 12, wherein, in the DNA sequence encoding the chloroplast D1 protein, the DNA-binding protein recognizes (1) a portion of the nucleotide sequence of 5'-TTTTGGCCGATTGATTTTCCAATATGCTAGTTTCAACAATTCTCGTTCTTTACA-3' (SEQ ID NO: 29) or its complementary sequence; or (2) a portion of the nucleotide sequence of 5'-GAAGAAGAAACTTATAATATCGTAGCCGCTCATGGTTATTTTGGCCGATTGATCTTCCA-3' (SEQ ID NO: 30) or its complementary sequence.

[0180] 14. The base editing composition or base editor according to any one of embodiments 1 to 13, wherein, in the DNA sequence encoding the chloroplast D1 protein, the DNA-binding protein recognizes (3) a portion of the nucleotide sequence of 5'-TTTTGGCCGATTGATTTTCCAAT-3' (SEQ ID NO: 31) or its complementary sequence; (4) a portion of the nucleotide sequence of 5'-TGTAAAGAACGAGAATTGTT-3' (SEQ ID NO: 32) or its complementary sequence; (5) a portion of the nucleotide sequence of 5'-GAAGAAGAAACTTATAATAT-3' (SEQ ID NO: 32) or its complementary sequence; or (6) a portion of the nucleotide sequence of 5'-TGGAAGATCAATCGGCCAAA-3' (SEQ ID NO: 33) or its complementary sequence.

[0181] 15. The base editing composition or base editor according to any one of embodiments 1 to 14, wherein the adenine deaminase comprises the amino acid sequence of SEQ ID NO: 39 or a conserved amino acid substitute thereof.

[0182] 16. The base editing composition or base editor according to any one of embodiments 1 to 15, wherein the adenine deaminase comprises a variant or a conserved amino acid substitute thereof in which one or more amino acids selected from the group consisting of positions 28, 30, 46, 48, 49, 82, 84, 106, 108, 110 and 111 of the amino acid sequence of SEQ ID NO: 39 are substituted with other amino acids.

[0183] 17. The base editing composition or base editor according to any one of embodiments 1 to 16, wherein the cytosine deaminase is an apolipoprotein B editing complex (APOBEC), an activation-induced deaminase (AID), or DddA, or a variant thereof, or a variant of tRNA-specific adenosine deaminase (TadA).

[0184] 18. The base editing composition or base editor according to any one of embodiments 1 to 17, wherein the cytosine deaminase is DddA having the amino acid sequence of SEQ ID NO: 1. tox It may be a variant, homolog, or a conserved amino acid substitute thereof, and may exist in full-length or in two cleaved forms.

[0185] 19. The base editing composition or base editor according to any one of embodiments 1 to 18, wherein the cytosine deaminase exists in the form of two mitotic figures, one of which contains a sequence from the N-terminus of the amino acid sequence of SEQ ID NO: 1 to amino acid positions 33, 44, 54, 68, 82, 98, or 108, and the other of the two mitotic figures contains a sequence from amino acid positions 34, 45, 55, 69, 83, 99, or 109 of the amino acid sequence of SEQ ID NO: 1 to its C-terminus.

[0186] 20. The base editing composition or base editor according to embodiment 19, wherein either of the two splits comprises the amino acid sequence of SEQ ID NO: 2 or a conserved amino acid substitute thereof, and the other comprises the amino acid sequence of SEQ ID NO: 3 or a conserved amino acid substitute thereof; or either of the two splits comprises the amino acid sequence of SEQ ID NO: 4 or a conserved amino acid substitute thereof, and the other comprises the amino acid sequence of SEQ ID NO: 5 or a conserved amino acid substitute thereof.

[0187] 21. A base editing composition or base editor according to any one of embodiments 1 to 20, wherein the cytosine deaminase exists in the form of two mitotic figures, one of which is a variant or a conserved substitute thereof in which one or more amino acids selected from the group consisting of positions 3, 5, 10, 11, 13, 14, 15, 16, 17, 18, 19, 28, 30, and 31 of SEQ ID NO: 2 are substituted with other amino acids, and the other is a variant or a conserved substitute thereof in which one or more amino acids selected from the group consisting of positions 13, 16, 17, 20, 21, 28, 29, 30, 31, 32, 33, 56, 57, 58, and 60 of SEQ ID NO: 3 are substituted with other amino acids; or One of the two splits is a variant or a conservative substitute thereof in which one or more amino acid sequences selected from the group consisting of positions 87, 88, 91, 92, 95, 100, 101, 102 and 103 of SEQ ID NO: 4 are replaced by other amino acids, and the other is a variant or a conservative substitute thereof in which one or more amino acids selected from the group consisting of positions 13, 14, 15 and 16 of SEQ ID NO: 5 are replaced by other amino acids.

[0188] 22. The base editing composition or base editor according to any one of embodiments 1 to 21, wherein the cytosine deaminase is present in the form of two mitotic figures, one of which contains the amino acid sequence of SEQ ID NO: 8 or a conserved amino acid substitute thereof, and the other of which contains the amino acid sequence of SEQ ID NO: 10 or a conserved amino acid substitute thereof.

[0189] 23. The base editing composition or base editor according to any one of embodiments 1 to 18, wherein the cytosine deaminase comprises the amino acid sequence of SEQ ID NO: 24 or a conserved amino acid substitute thereof.

[0190] 24. The base editing composition or base editor according to any one of embodiments 1 to 23, wherein the nicking enzyme is selected from the group consisting of MutH, BspD6I, FokI (including FokI-FokI homodimer or heterodimer), BsaI, BsmBI, BsmAI, BsrDI, CviPII, BspQI, AlwI and I-TevI, fragments thereof and variants thereof.

[0191] 25. The base editing composition or base editor according to any one of embodiments 1 to 24, wherein the base editing composition or base editor comprises a fusion protein comprising a DNA-binding protein, a cytosine deaminase, and an adenine deaminase, the DNA-binding protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 55 to SEQ ID NO: 58, the cytosine deaminase comprising the amino acid sequence of SEQ ID NO: 24, and the adenine deaminase comprising the amino acid sequence of SEQ ID NO: 39.

[0192] 26. A base editing composition or base editor according to any one of embodiments 1 to 24, wherein the base editing composition or base editor comprises two fusion proteins, one of the two fusion proteins comprising a DNA-binding protein, a first cytosine deaminase mitochondria, and an adenine deaminase, the DNA-binding protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 55 to SEQ ID NO: 58, the first cytosine deaminase mitochondria comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 5, the second cytosine deaminase mitochondria comprising an amino acid selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 5, and the adenine deaminase comprising the amino acid sequence of SEQ ID NO: 39.

[0193] 27. The base editing composition or base editor according to embodiment 25 or 26, wherein the fusion protein further comprises UDG of SEQ ID NO: 41.

[0194] 28. A base editing composition or base editor according to any one of embodiments 1 to 24, wherein the base editing composition or base editor comprises two fusion proteins, one of the two fusion proteins comprising a DNA-binding protein and a first cytosine deaminase mitochondria, the other of the two fusion proteins comprising a DNA-binding protein and a second cytosine deaminase mitochondria, the DNA-binding protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 63 to SEQ ID NO: 65, the first cytosine deaminase mitochondria comprising the amino acid sequence of SEQ ID NO: 8, and the second cytosine deaminase mitochondria comprising the amino acid sequence of SEQ ID NO: 10.

[0195] 29. The base editing composition or base editor according to embodiment 28, wherein the fusion protein further comprises the UGI of SEQ ID NO: 43.

[0196] 30. The base editing composition or base editor according to any one of embodiments 1 to 24, wherein the base editing composition or base editor comprises two fusion proteins, one of the two fusion proteins comprising a DNA-binding protein and an adenine deaminase, the other of the two fusion proteins comprising a nicking enzyme, the DNA-binding protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 71 and SEQ ID NO: 72, the adenine deaminase comprising the amino acid sequence of SEQ ID NO: 39, and the nicking enzyme comprising the amino acid sequence of SEQ ID NO: 16.

[0197] 31. The base editing composition or base editor according to any one of embodiments 1 to 30, wherein the base editing composition or base editor comprises a polynucleotide, at least a portion of which has been codon-optimized for expression in the plant or a part of the plant.

[0198] 32. The base editing composition or base editor according to any one of embodiments 1 to 31, wherein a portion of the plant is a plant cell, protoplast, or callus tissue.

[0199] The present invention will now be described in detail through the following embodiments. However, the following embodiments are only for illustrating the present invention, and the present invention is not limited to these embodiments.

[0200] Example 1 To edit the 264th serine base of the psbA gene in Arabidopsis chloroplasts to glycine, a fusion protein was prepared, as shown in Table 4. The DNA sequence bound to the fusion protein is shown in Table 1. To express the fusion protein, the following DNA was cloned, and transformed plants were prepared by transformation using Agrobacterium.

[0201] [Table 1] DNA-binding protein recognition sequence for base editing AtpsbA S264.

[0202]

[0203] Specifically, the gene construct encoding the fusion protein shown in Table 4 was designed to be placed between the RPS5A promoter, which is preferentially induced to be expressed during embryonic development, and the 35S terminator, so that editing could be initiated from early development. Using Gibson assembly, Golden Gate cloning, and restriction endonucleases, it was cloned into a vector suitable for transforming Agrobacterium tumefaciens (a strain that can deliver gene constructs to the Arabidopsis nuclear genome using T-DNA). Subsequently, using Agrobacterium strain GV3101 transformed with this vector, Arabidopsis Columbia ecotype (Col-0) plants were transformed using the known method (Zhang et al., Nat. Protoc. 1, 641-646 (2006)) via the floral dip method.

[0204] Approximately 15,000–20,000 Arabidopsis seeds, each inoculated with the fusion protein via the flower-dip method, were sown in the soil. After 7 and 14 days, the seeds were treated with atrazine (40 g / ha) to ensure the survival of the first generation of transformed plants, and the gene-editing efficiency was analyzed. Figure 2 ).

[0205] First-generation transformed plants were sown on semi-MS medium containing 1 mg / L atrazine. After screening for surviving second-generation transformed plants, gene editing efficiency was analyzed. Figure 3 ).

[0206] Gene editing efficiency analysis was performed by extracting DNA from a portion of the rosette leaf of a plant, using primers listed in Tables 2 and 3 below for PCR, followed by base sequence analysis using Illumina MiSeq (v2, 300 cycles), and gene editing efficiency was analyzed using https: / / github.com / ibs-cge2 / prime_editor_analysis.

[0207] [Table 2] Information on AtpsbA primers used in PCR for base editing analysis.

[0208]

[0209] [Table 3] Primer information used in PCR for base editing analysis.

[0210]

[0211] The composition of the prepared fusion protein is as follows: [Table 4]

[0212] like Figure 3 The results clearly show that the experimental results indicate that, in the AGT base sequence encoding serine at position 264 of the D1 protein, plants transformed with the base editor used in this invention underwent base editing in a highly efficient and position-specific manner, thereby preventing the growth inhibition observed in the wild type when treated with atrazine.

[0213] The analyzed transformed plants survived even after atrazine treatment, thus indicating that gene editing at the target site was achieved. However, in Figure 2 The reason why base editing efficiency cannot be confirmed in some individuals is that chloroplasts that have not undergone base editing were sampled from multiple chloroplasts present in a plant cell. Specifically, it is known that a single Arabidopsis cell contains 80-120 chloroplasts, and the DNA copy number in a single chloroplast is up to 560. The RPS5a promoter used in this invention is a promoter that induces the expression of downstream genes present in the plant embryo, and it is difficult to induce base editing of all chloroplast DNA in the embryo. Therefore, after chloroplast DNA base editing in the plant embryo, when the plant develops into an adult, a so-called rosette leaf exists in a plant containing chloroplasts with edited DNA and unedited chloroplasts. When sampling a portion of the rosette leaf to confirm base editing efficiency, unedited chloroplasts may be sampled, making it impossible to confirm the base editing efficiency. The literature [Nature Plants 8, 1378-1384 (2022)] also reports that during the induction of base editing of the chloroplast gene psaA, when psaA is edited, it is visually identifiable as a light green rosette leaf. However, in the first generation of transformed plants, both light green (from base editing of chloroplast DNA) and normal green rosette leaves were observed simultaneously in one plant. Therefore, even in atrazine-resistant first-generation transformed plants, the base editing efficiency may not be definitively confirmed experimentally. In such cases, if... Figure 3 As shown, base editing efficiency can be confirmed in the second-generation transformed plants.

[0214] Example 2: Evaluating herbicide resistance by editing the 264th serine base of the Arabidopsis chloroplast gene psbA to glycine. Transformed plants obtained by editing the 264th serine base of the psbA gene in the chloroplasts of Arabidopsis thaliana prepared in Example 1 to glycine were further used to evaluate herbicide resistance. The transformed plants from Example 1 were treated with atrazine at concentrations of 1 mg / L, 5 mg / L, 20 mg / L, and 50 mg / L, respectively. Growth inhibition was confirmed after 5 days. The control group consisted of wild-type plants without base editing. Figure 4 As shown, the experimental results indicate that with the increase of atrazine concentration, the control group showed almost no growth, while the transformed plants that underwent base editing according to this invention did not show any growth inhibition.

[0215] Example 3: Base editing via FZY2 import To edit the 251st alanine base of the psbA gene in lettuce chloroplasts to valine, a fusion protein as shown in Table 6 was prepared. To express the fusion protein, DNA was cloned, and transformed plants were prepared using transformation with *Agrobacterium*.

[0216] [Table 5] The recognition base sequence of the DNA-binding protein AtpsbA A251V used for base editing.

[0217]

[0218] [Table 6]

[0219] Agrobacterium tumefaciens strain GV3101 was transformed using a vector cloning a gene construct encoding the fusion proteins shown in Table 6. Arabidopsis thaliana plants were then transformed using a known method (Zhang et al., Nat. Protoc. 1, 641-646 (2006)) via the floral dip method. Arabidopsis T1 seedlings were screened in half-strength MS medium supplemented with 2% sucrose, 20 mg / L glufosinate (PPT), and 250 mg / L cefotaxime.

[0220] To target deep sequencing, total DNA was extracted from actual leaves of the selected plants or transfected lettuce protoplasts using the DNeasy Plant Mini kit (Qiagen). For large-scale analysis, DNA was extracted using 100 μL of cell lysis buffer (20 mM Tris-HCl, pH 8.0, Sigma-Aldrich, 5 mM EDTA, 400 mM NaCl, 0.05% sodium dodecyl sulfate) containing 2 μL of proteinase K (Qiagen). The lysates were incubated at 55°C for 2 hours, followed by incubation at 95°C for 10 minutes. Deep sequencing libraries were prepared by amplifying the target site using TruSeq HT dual-index primers and PrimeSTAR® GXL DNA polymerase (TAKARA) via three PCR cycles (first, second, and third). Sequencing of the libraries was performed using Illumina. MiSeq paired-end sequencing technology is used. Base editing efficiency (frequency) is calculated as the percentage of sequencing reads that present the desired base editing result among all sequencing reads.

[0221] Primers used in PCR for base editing analysis First time: F-GAGCTTAGTTTCCGTCTGGGTATGCG (SEQ ID NO: 104) First time: R-CATTTGTAGATGGAGCTTCGATAGCAGC (SEQ ID NO: 105) Second time: F-ACACTCTTTCCCTACACGACCGCTTCCGATCTGATCAGGGAAACCACAGAAAATGAATC (SEQ ID NO: 106) Second time: R-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGTTGAAAGCCATAGTGCTGATACC (SEQ ID NO: 107) Experimental results show that, Figure 7As shown, in the region encoding resistance to LspsbA benzoxazine, the base editor prepared according to the present invention edits the C (cytosine) to T (thymine) in the chloroplast DNA at amino acid 251 (alanine), thereby replacing alanine with valine, and confirms excellent editing efficiency. In particular, when the same FZY2 mitochondria are included, it was confirmed that the base editing efficiency using LspsbA A251V left 2 is superior to that using LspsbA A251V left 1.

[0222] Example 4: Production of herbicide-resistant plants via a nick enzyme-based base editor (1) Plasmid construction DNA encoding a base editor fusion protein was cloned, which was obtained by base editing of the psbA gene in Arabidopsis thaliana.

[0223] As shown in Table 7 below, the DNA encoding the TALE protein that recognizes the psbA gene sequence of Arabidopsis thaliana (denoted as At) was constructed using the known Golden Gate cloning technique and cloned in a manner that linked the DNA sequences of other proteins that constitute the base editor fusion protein.

[0224] [Table 7]

[0225] Specifically, to transform Arabidopsis thaliana, the TALE protein-coding base sequence was assembled at the desired position in a master vector containing the RPS5A promoter and the CTS and tag sequences using Golden Gate cloning technology. PCR products encoding the desired fusion protein were generated using PrimeSTAR® GXL DNA polymerase (TAKARA), assembled using NEBuilder, and then chemically transformed into E. coli DH5α. Antibiotic-resistant colonies were screened, and surviving colonies were analyzed using Sanger sequencing. Each construct, containing deaminase and cleavage enzyme, was cleaved using AatII and PmeI restriction endonucleases, and then incorporated into the whole construct using T4 DNA ligase. The DNA for Arabidopsis plant transformation was placed between the RPS5A promoter and the 35S terminator.

[0226] (2) Plant transformation and growth conditions The *Agrobacterium tumefaciens* strain GV3101 was transformed using the vector cloned in Example 1, and *Arabidopsis thaliana* plants were transformed using the floral dip method according to known methods (Zhang et al., *Nat. Protoc. 1*, 641-646 (2006)). *Arabidopsis* T1 seedlings were screened in half-strength MS medium supplemented with 2% sucrose, 20 mg / L glufosinate (PPT), and 250 mg / L cefotaxime. To evaluate resistance to atrazine or PPT, T2 or T3 seeds were sown in half-strength MS medium containing 2% sucrose with or without atrazine (1 mg / L) and with or without PPT (20 mg), and allowed to grow for 2 weeks. All transgenic plants were cultivated under long-day conditions at 23°C (16 hours of light / 8 hours of darkness).

[0227] (3) Targeted deep sequencing for analyzing base editing frequencies For targeted deep sequencing, total DNA was extracted from actual leaves of screened plants using the DNeasy Plant Mini kit (Qiagen). For large-scale analysis, DNA was extracted using 100 μL of cell lysis buffer (20 mM Tris-HCl, pH 8.0 (Sigma-Aldrich), 5 mM EDTA (Sigma-Aldrich), 400 mM NaCl, 0.05% sodium dodecyl sulfate (Sigma-Aldrich)) containing 2 μL of proteinase K (Qiagen). The lysates were incubated at 55°C for 2 hours, followed by incubation at 95°C for 10 minutes. Deep sequencing libraries were prepared by amplifying the target sites using TruSeq HT dual-index primers and PrimeSTAR® GXL DNA polymerase (TAKARA) via three PCR amplifications (first, second, and third). The library was sequenced using Illumina MiSeq paired-end sequencing technology. Base editing efficiency (frequency) was calculated as the percentage of sequencing reads that yielded the desired base editing results. The PCR primer sequences used for sequencing are shown in Table 8.

[0228] [Table 8]

[0229] The base editor fusion protein used has the following composition: [Table 9]

[0230] Using the psaA gene of Arabidopsis thaliana as an example, when the base editor of this invention (using Nt-BspD6I(C) as the nicking enzyme and TadA8e as the deaminase) was used, A-to-G base editing at the target site was confirmed. Figure 8 and Figure 9 ).

[0231] The psbA gene encodes a D1 protein, an important subunit of the photosystem II complex. In various plant species, resistance to atrazine herbicides is often associated with the substitution of glycine for serine at position 264 in the D1 protein. DNA encoding the TALE protein was cloned and introduced into Arabidopsis thaliana using a binary vector. T1 plants were screened in half-strength MS medium supplemented with glufosinate (20 mg / L) under long-day conditions (16 hours light / 8 hours dark). Targeted depth sequencing confirmed the A-to-G editing frequency in the DNA base sequence corresponding to amino acid S264. Figure 8 Then, T2 seeds were collected from T1 plants and cultured in half-strength MS medium containing atrazine (1 mg / L) to obtain 8 atrazine-resistant progeny. The base editing frequency was then confirmed by targeted deep sequencing. Figure 9 Next, under the same conditions as T2 seeds, T3 seeds of T2 plants were cultivated, and... Figure 10 The image shows plants exhibiting resistance to phosphinothricin (20 mg / L, also known as Basta) (plant #2-1) and resistance to atrazine (plants #2-3 and #2-4). Targeted deep sequencing of the atrazine-resistant T3 plants showed that editing the 6 bases of the a portion encoding amino acid S264 to G bases resulted in over 99% homogeneity. Figure 11 ).

[0232] (4) Genotype analysis used to confirm the presence of exogenous genes Whole DNA was extracted from actual leaves of T3 plants using the DNeasy Plant Mini kit (Qiagen). The target region was then amplified throughout the DNA using PrimeSTAR® GXL DNA polymerase (TAKARA). The resulting PCR products were analyzed on a 1% agarose gel. The PCR primer sequences used are shown in Table 10.

[0233] To confirm that the atrazine-resistant lines #2-3-1 and #2-4-1 did not contain a foreign gene, genomic PCR was performed using primers specific to the atrazine resistance gene included in the cloning of the foreign gene (base editor). The results confirmed that, unlike #2-1-1, #2-3-1 and #2-4-1 plants did not contain a foreign gene. Figure 12 This result demonstrates that base editing induced by the base editor used in this invention (even if the base editor is not introduced into the gene) can be stably inherited and maintained.

[0234] [Table 10]

[0235] The sequences used in this invention that are not listed in Tables 1 to 10 above are as follows: SEQ ID NO: 1: wild-type DddA tox GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGC.

[0236] SEQ ID NO: 2: wild-type DddA tox G1333-N GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGG.

[0237] SEQ ID NO: 3: wild-type DddA tox G1333-C PTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGC。

[0238] SEQ ID NO: 4: Wild-type DddA tox G1397-N GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG。

[0239] SEQ ID NO: 5: Wild-type DddA tox G1397-C AIPVKRGATGETKVFTGNSNSPKSPTKGGC。

[0240] SEQ ID NO: 6: FZY2 wild type MSLPEYDGTTTHGVLVLDDGTQIGFTSGNGDPRYTNYRNNGHVEQKSALYMRENNISNATVYHNNTNGTCGYCNTMTATFLPEGATLTVVPPENAVANNRAIDYVKTYTGTSNDPKISPRYKGN。

[0241] SEQ ID NO: 7: FZY2 v1.6 Full-length MSLPEYDGTTTHGVLVLDDGTQIGFTSGNGDPRYTNYRNNGHVEQKSALYMRENNISNATVYHNNTNGTCGYCNTMIATFLPEGATLTVVPPENAVANNRAIDYVKTYTGTSNDPKISPRYKGN。

[0242] SEQ ID NO: 8: FZY2 V1.6 S100 N MSLPEYDGTTTHGVLVLDDGTQIGFTSGNGDPRYTNYRNNGHVEQKSALYMRENNISNATVYHNNTNGTCGYCNTMIATFLPEGATLTVVPPENAVANN。

[0243] SEQ ID NO:9:FZY2 V1.6 S100 N ATGTCCCTGCCAGAGTATGATGGGACGACAACGCATGGTGTATTGGTTCTAGATGATGGCACGCAGATCGGGTTCACCTCCGGCAATGGCGACCCTAGATACACAAACTATCGTAACAATGGACACGTCGAACAAAAGTCTGCGCTCTATATGCGAGAGAACAACATTAGCAATGCAACAGTGTATCATAACAATACCAATGGGACCTGCGGATATTGCAATACGATGATTGCAACTTTCCTCCCCGAGGGCGCAACTCTCACTGTTGTTCCGCCGGAAAATGCGGTAGCAAACAAC。

[0244] SEQ ID NO:10:FZY2 V1.6 S100 C RAIDYVKTYTGTSNDPKISPRYKGN。

[0245] SEQ ID NO:11:FZY2 V1.6 S100 C CGAGCCATCGACTACGTTAAAACTTACACGGGAACAAGTAACGATCCGAAAATCAGCCCCCGTTACAAGGGAAAT。

[0246] SEQ ID NO:12:MutH MSQPRPLLSPPETEEQLLAQAQQLSGYTLGELAALAGLVTPENLKRDKGWIGVLLEIWLGASAGSKPEQDFAALGVELKTIPVDSLGRPLETTFVCVAPLTGNSGVTWETSHVRHKLKRVLWIPVEGERSIPLAKRRVGSPLLWSPNEEEDRQLREDWEELMDMIVLGQIERITARHGEYLQIRPKAANAKALTEAIGARGERILTLPRGFYLKKNFTSALLARHFLIQ。

[0247] SEQ ID NO:13:FokI E490K I538K-40aa-FokI D450A Q486E I499L LVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTRAYSGGYNLPIGQADEMQRYVKENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHKTNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFSSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPAGAIYTVGSPIDYGVIVDTRAYSGGYNLPIGQADEMERYVEENQTRNKHLNPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF.

[0248] SEQ ID NO:14:FokI wild type(wild type) KIRTFGWVQNPGKFENLKRVVQVFDRNSKVHNEVKNIKIPTLVKESKIQKELVAIMNQHDLIYTYKELVGTGTSIRSEAPCDAIIQATIADQGNKKGYIDNWSSDGFLRWAHALGFIEYINKSDSFVITDVGLAYSKSADGSAIEKEILIEAISSYPPAIRILTLLEDGQHLTKFDLGKNLGFSGESGFTSLPEGILLDTLANAMPKDKGEIRNNWEGSSDKYARMIGGWLDKLGLVKQGKKEFIIPTLGKPDNKEFISHAFKITGEGLKVLRRAKGSTKFTRVPKRVYWEMLATNLTDKEYVRTRRALILEILIKAGSLKIEQIQDNLKKLGFDEVIETIENDIKGLINTGIFIEIKGRFYQLKDHILQFVIPNRGVTKQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF。

[0249] SEQ ID NO:15:Nt.BspD6I(C) 。

[0250] SEQ ID NO:16:Nt.BspD6I(C) MRQLEEVIDLLEVYHEKKNVIEEKIKARFIANKNTVFEWLTWNGFIILGNALEYKNNFVIDEELQPVTHAAGNQPDMEIIYEDFIVLGEVTTSKGATQFKMESEPVTRHYLN KKKELEKQGVEKELYCLFIAPEINKNTFEEFMKYNIVQNTRIIPLSLKQFNMLLMVQKKLIEKGRRLSSYDIKNLMVSLYRTTIECERKYTQIKAGLEETLNNWVVDKEVRF。

[0251] SEQ ID NO: 17: A1341D KRKKA variant GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYDNAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTAGGC。

[0252] SEQ ID NO: 18: AAAAA variant GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVAAGATGETAVFTGNSNSPASPTAGGC。

[0253] SEQ ID NO: 19: AAAAK variant GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVAAGATGETAVFTGNSNSPASPTKGGC。

[0254] SEQ ID NO: 20: AAKAA variant GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVAAGATGETKVFTGNSNSPASPTAGGC。

[0255] SEQ ID NO: 21: AAKAK variant GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVAAGATGETKVFTGNSNSPASPTKGGC。

[0256] SEQ ID NO: 22: KAAAA variant GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKAGATGETAVFTGNSNSPASPTAGGC。

[0257] SEQ ID NO: 23: E1347A variant GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVAGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGC。

[0258] SEQ ID NO: 24: GSVG variant GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLEGKVFSSGGPTPYPNYANAGHVESQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGVIPVKRGATGETKVFTGNSNGPKSPTKGGC。

[0259] SEQ ID NO: 25: SSVG variant GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVESQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGVIPVKRGATGETKVFTGNSNGPKSPTKGGC。

[0260] SEQ ID NO: 26: GSAG variant GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLEGKVFSSGGPTPYPNYANAGHVESQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNGPKSPTKGGC。

[0261] SEQ ID NO: 27: GSVS variant.

[0262] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLEGKVFSSGGPTPYPNYANAGHVESQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGVIPVKRGATGETKVFTGNSNSPKSPTKGGC.

[0263] SEQ ID NO: 28: GSVG (full-length DddA tox variant).

[0264] GGCAGCTACGCCCTGGGTCCGTATCAGATTAGCGCCCCGCAGCTGCCAGCATACAATGGTCAGACCGTGGGTACCTTCTACTATGTGAACGACGCTGGCGGTCTGGAGGGCAAGGTGTTTAGCAGCGGCGGTCCAACCCCGTACCCAAACTATGCCAATGCCGGTCATGTGGAGAGTCAGAGCGCCCTGTTCATGCGTGATAACGGCATCAGCGAGGGTCTGGTGTTCCACAACAACCCGGAAGGCACCTGCGGTTTTTGCGTGAACATGACCGAGACACTGCTGCCGGAAAACGCGAAAATGACCGTGGTGCCGCCGGAAGGTGTCATTCCAGTGAAGCGCGGCGCTACCGGTGAAACCAAAGTGTTTACCGGTAACAGCAACGGCCCGAAGAGCCCGACCAAAGGCGGTTGC.

[0265] (1) 5'-TTTTGGCCGATTGATTTTCCAATATGCTAGTTTCAACAATTCTCGTTCTTTACA-3' (SEQ ID NO: 29).

[0266] (2) 5'-GAAGAAGAAACTTATAATATCGTAGCCGCTCATGGTTATTTTGGCCGATTGATCTTCCA-3' (SEQ ID NO: 30).

[0267] (3) 5'-TTTTGGCCGATTGATTTTCCAAT-3' (SEQ ID NO: 31).

[0268] (4) 5'-TGTAAAGAACGAGAATTGTT-3' (SEQ ID NO: 32).

[0269] (5) 5'-GAAGAAGAAACTTATAATAT-3' (SEQ ID NO: 33).

[0270] (6) 5'-TGGAAGATCAATCGGCCAAA-3' (SEQ ID NO: 34).

[0271] SEQ ID NO: 35: CTS (Chloroplast Transit Signal) MDSQLVLSLKLNPSFTPLSPLFPFTPCSSFSPSLRFSSCYSRRLYSPVTVYAAK.

[0272] SEQ ID NO: 36: CTS (Chloroplast Transit Signal) ATGGATTCACAGCTAGTCTTGTCTCTGAAGCTGAATCCAAGCTTCACTCCTCTTTCTCCTCTCTTCCCTTTCACTCCATGTTCTTCTTTTTCGCCGTCGCTCCGGTTTTCTTCTTGCTACTCCCGCCGCCTCTATTCTCCGGTTACCGTCTACGCCGCGAAG.

[0273] SEQ ID NO: 37: 3xFlag DYKDHDGDYKDHDIDYKDDDDK.

[0274] SEQ ID NO: 38: 3xFlag GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAG.

[0275] SEQ ID NO: 39: TadA8e SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN.

[0276] SEQ ID NO: 40: TadA8e TCTGAGGTGGAGTTTTCCCACGAGTACTGGATGAGACATGCCCTGACCCTGGCCAAGAGGGCACGGGATGAGAGGGAGGTGCCTGTGGGAGCCGTGCTGGTGCTGAACAATAGAGTGATCGGCGAGGGCTGGAACAGAGCCATCGGCCTGCACGACCCAACAGCCCATGCCGAAATTATGGCCCTGAGACAGGGCGGCCTGGTCATGCAGAACTACAGACTGATTGACGCCACCCTGTACGTGACATTCGAGCCTTGCGTGATGTGCGCCGGCGCCATGATCCACTCTAGGATCGGCCGCGTGGTGTTTGGCGTGAGGAACTCAAAAAGAGGCGCCGCAGGCTCCCTGATGAACGTGCTGAACTACCCCGGCATGAATCACCGCGTCGAAATTACCGAGGGAATCCTGGCAGATGAATGTGCCGCCCTGCTGTGCGATTTCTATCGGATGCCTAGACAGGTGTTCAATGCTCAGAAGAAGGCCCAGAGCTCCATCAAC。

[0277] SEQ ID NO: 41: UDG (Uracil DNA Glycosylase) MASSTPKTLMDFFQPAKRLKASPSSSSFPAVSVAGGSRDLGSVANSPPRVTVTTSVADDSSGLTPEQIARAEFNKFVAKSKRNLAVCSERVTKAKSEGNCYVPLSELLVEESWLKALPGEFHKPYAKSLSDFLEREIITDSKSPLIYPPQHLIFNALNTTPFDRVKTVIIGQDPYHGPGQAMGLSFSVPEGEKLPSSLLNIFKELHKDVGCSIPRHGNLQKWAVQGVLLLNAVLTVRSKQPNSHAKKGWEQFTDAVIQSISQQKEGVVFLLWGRYAQEKSKLIDATKHHILTAAHPSGLSANRGFFDCRHFSRANQLLEEMGIPPIDWQL。

[0278] SEQ ID NO: 42: UDG (Uracil DNA Glycosylase) ATGGCTTCGTCGACACCTAAAACCCTAATGGATTTTTTTCAACCTGCCAAACGCCTCAAAGCTTCCCCTTCTTCCTCCTCTTTCCCCGCCGTCTCCGTCGCCGGTGGTTCCCGTGATTTGGGTTCTGTAGCAAACTCGCCGCCTCGTGTAACCGTTACCACTTCCGTCGCCGATGATTCGTCTGGCCTTACACCTGAACAGATCGCTCGCGCAGAGTTCAACAAGTTCGTCGCCAAGTCCAAGCGTAACCTCGCCGTCTGCTCCGAGAGGGTCACAAAAGCAAAATCTGAAGGAAACTGCTACGTACCATTGAGTGAGCTCTTAGTAGAAGAATCATGGCTTAAAGCTCTTCCTGGGGAATTTCATAAACCCTACGCCAAATCACTTTCTGATTTCCTTGAACGTGAGATCATCACTGACAGTAAAAGCCCTCTGATTTATCCACCGCAGCACTTGATTTTCAATGCTCTTAATACAACTCCTTTTGATCGAGTTAAGACTGTCATTATCGGACAGGATCCTTATCATGGACCTGGTCAAGCTATGGGTTTGTCCTTCTCTGTACCTGAAGGAGAAAAGCTTCCTTCTAGTCTGTTGAACATCTTTAAGGAGCTTCATAAAGATGTTGGCTGTTCCATCCCACGTCACGGTAATCTACAGAAATGGGCTGTGCAGGGTGTGTTACTCCTGAATGCTGTTCTTACAGTAAGGAGTAAACAGCCTAATTCACATGCAAAGAAAGGATGGGAACAATTCACTGATGCTGTTATTCAAAGTATCTCACAGCAGAAGGAAGGTGTTGTTTTTCTTCTCTGGGGAAGATACGCTCAAGAGAAATCCAAGTTGATAGATGCGACTAAACATCATATACTCACAGCAGCTCATCCATCTGGTTTGTCGGCGAATAGAGGCTTCTTCGACTGCAGGCATTTCTCTCGCGCAAACCAGCTACTCGAGGAAATGGGGATTCCTCCCATAGACTGGCAACTT。

[0279] SEQ ID NO:43:UGI TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML。

[0280] SEQ ID NO:44:UGI ACAAACCTGAGTGATATTATTGAAGGAGACGGGTAAACAATTAGTGATTCAAGAAAGTATACTCATGCTACCAGAAAGTGGAGGAAGTCATAGGCAACAAACCGGAATCTGACATACTGTGT TCACACAGCTTATGACGAATCTACAGATGAAAATGTCATGCTGCTAACTTCTGATGCTCCCGAGTACAAGCCCTGGGCTTTTAATTCAAGATTCCAACGGCGAGAATAAAATTAAGATGTTG。

[0281] SEQ ID NO:45:Linker1 GSSGSETPGTSESATPES。

[0282] SEQ ID NO:46:Linker2 LVGS。

[0283] SEQ ID NO:47:Linker3 SGSETPGTSESATPES。

[0284] SEQ ID NO:48:Linker4 GSGS。

[0285] 2aa Linker:GS。

[0286] SEQ ID NO:49:Linker5 SGGS。

[0287] SEQ ID NO:50:Linker1 GGATCCTCAGGAAGCGAAACTCCTGGTACCTCAGAGTCCGCTACTCCCGAATCC。

[0288] SEQ ID NO: 51: Linker 2 CTAGTCGGTTCC.

[0289] SEQ ID NO: 52: Linker 3 TCAGGTTCTGAGACGCCTGGCACAAGTGAATCCGCCACACCGGAGTCC.

[0290] SEQ ID NO: 53: Linker 4 GGATCCGGCAGT.

[0291] 2aa Linker: GGATCC.

[0292] SEQ ID NO: 54: Linker 5 TCTGGGGGAAGC.

[0293] SEQ ID NO: 55: AtpsbA S264G Left (Left) 1 DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG。

[0294] SEQ ID NO: 56: AtpsbA S264G Left 2 DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG。

[0295] SEQ ID NO: 57: AtpsbA S264G Right 1 DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG。

[0296] SEQ ID NO: 58: AtpsbA S264G Right 2 DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG。

[0297] SEQ ID NO: 59: AtpsbA S264G Left 1

[0298] SEQ ID NO: 60: AtpsbA S264G Left 2

[0299] SEQ ID NO: 61: AtpsbA S264G Right 1:

[0300] SEQ ID NO: 62: AtpsbA S264G Right 2

[0301] SEQ ID NO: 63: LspsbA A251V Left 1 DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG。

[0302] SEQ ID NO: 64: LspsbA A251V Left 2 DLRTLGYSQQQQEKIKPKVRSTVAQHHGALVGHGFTHAHIVALSQHPAALGTVAVTYQHIITALPEATHEDIVGVGKRGAGARALEALLTDAGELRGPPLQLDTGQLVKIAKRGGVTAMEAVHASRNALTGAPLNLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG。

[0303] SEQ ID NO: 65: LspsbA A251V Right DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG。

[0304] SEQ ID NO: 66: LspsbA A251V Left 1

[0305] SEQ ID NO: 67: LspsbA A251V Left 2

[0306] SEQ ID NO: 68: LspsbA A251V Right

[0307] SEQ ID NO: 69: AtpsbA Left TALE NK

[0308] SEQ ID NO: 70: AtpsbA Right TALE NK

[0309] SEQ ID NO: 71: AtpsbA Left TALE NK DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG。

[0310] SEQ ID NO: 72: AtpsbA Right TALE NK 。

Claims

1. A method for producing a plant or part of a plant resistant to herbicides through DNA base editing, characterized in that, The method includes the steps of expressing a base editing composition in a target plant or a portion of a plant, and causing said plant or portion of a plant to grow or be cultured, said base editing composition having the activity of editing adenine bases to guanine bases and / or editing cytosine bases to thymine bases in DNA encoding plant chloroplast D1 protein.

2. The method according to claim 1, characterized in that, The herbicide is a triazine herbicide.

3. The method according to claim 1, characterized in that, The herbicides selected are composed of a group consisting of propargite, atrazine, pymetrozine, simazine, cyclomethrin, cypermethrin, terbuprofen, diuron, fluroxypyr, linuron, terbutaline, and benzimidone.

4. The method according to claim 1, characterized in that, The herbicide is atrazine.

5. The method according to claim 1, characterized in that, Plants that develop herbicide resistance through DNA base editing are those in which the serine residues of the D1 protein are mutated to glycine or the alanine residues are mutated to valine. The serine residues correspond to the 264th serine residue of the Arabidopsis thaliana D1 protein, and the alanine residues correspond to the 251st alanine residue of the Arabidopsis thaliana D1 protein.

6. The method according to claim 5, characterized in that, Plants that are resistant to herbicides through DNA base editing are those whose DNA sequence encoding the 5'-AGT-3' of the serine residue is edited to encode the 5'-GGT-3' of glycine, or whose DNA sequence encoding the 5'-GCT-3' of the alanine residue is edited to encode the 5'-GTT-3' of valine.

7. The method according to claim 1, characterized in that, The target plants are row-sown crops, fruits, culinary plants, vegetables, trees, or ornamental plants.

8. The method according to claim 1, characterized in that, The target plants were selected from soybean, zoysia grass and lettuce.

9. The method according to claim 1, characterized in that, The target plant is a transgenic plant that has previously been made herbicide resistant by inserting recombinant DNA, or a progeny of the transgenic plant.

10. The method according to claim 9, characterized in that, Previously, transgenic plants that were made resistant to herbicides by inserting recombinant DNA were genetically modified to be resistant to herbicides that inhibit amino acid synthesis or promote the generation of reactive oxygen species.

11. The method according to claim 1, characterized in that, The base editing composition comprises a DNA-binding protein and an enzyme protein, or a polynucleotide encoding the protein, wherein the enzyme protein comprises one or more of cytosine deaminase, adenine deaminase, and nicking enzyme, wherein the cytosine deaminase exists in full-length or in two mitotic forms.

12. The method according to claim 11, characterized in that, DNA-binding proteins and enzyme proteins exist in the form of more than one fusion protein.

13. The method according to claim 11, characterized in that, DNA-binding proteins were selected from a group consisting of zinc finger proteins, transcription activator-like effector proteins, and CRISPR-related nucleases.

14. The method according to claim 11, characterized in that, In the DNA sequence encoding the chloroplast D1 protein, the DNA-binding protein recognizes (1) the nucleotide sequence of 5'-TTTTGGCCGATTGATTTTCCAATATGCTAGTTTCAACAATTCTCGTTCTTTACA-3' (SEQ ID NO: 29) or a portion thereof, or its complementary sequence; or (2) the nucleotide sequence of 5'-GAAGAAGAAACTTATAATATCGTAGCCGCTCATGGTTATTTTGGCCGATTGATCTTCCA-3' (SEQ ID NO: 30) or a portion thereof, or its complementary sequence.

15. The method according to claim 11, characterized in that, In the DNA sequence encoding the chloroplast D1 protein, the DNA-binding protein recognizes (3) the nucleotide sequence of 5'-TTTTGGCCGATTGATTTTCCAAT-3' (SEQ ID NO: 31) or a portion thereof, or its complementary sequence; (4) the nucleotide sequence of 5'-TGTAAAGAACGAGAATTGTT-3' (SEQ ID NO: 32) or a portion thereof, or its complementary sequence; (5) the nucleotide sequence of 5'-GAAGAAGAAACTTATAATAT-3' (SEQ ID NO: 33) or a portion thereof, or its complementary sequence; or (6) the nucleotide sequence of 5'-TGGAAGATCAATCGGCCAAA-3' (SEQ ID NO: 34) or a portion thereof, or its complementary sequence.

16. The method according to claim 11, characterized in that, Adenine deaminase contains the amino acid sequence of SEQ ID NO: 39 or its conserved amino acid substitutes.

17. The method according to claim 11, characterized in that, Adenine deaminase comprises a variant or a conserved amino acid substitute thereof in which one or more amino acids selected from the group consisting of positions 28, 30, 46, 48, 49, 82, 84, 106, 108, 110 and 111 of the amino acid sequence of SEQ ID NO: 39 are replaced by other amino acids.

18. The method according to claim 11, characterized in that, Cytosine deaminase is a lipoprotein B mRNA editing complex, an activation-induced deaminase or DddA, or a variant thereof, or a variant of tRNA-specific adenosine deaminase.

19. The method according to claim 11, characterized in that, Cytosine deaminase is DddA with the amino acid sequence SEQ ID NO:

1. tox It or its variants, or its conserved amino acid substitutes, and exist in full-length or two-part form.

20. The method according to claim 11, characterized in that, Cytosine deaminase exists in the form of two mitotic figures, one of which contains a sequence from the N-terminus of the amino acid sequence of SEQ ID NO: 1 to amino acid positions 33, 44, 54, 68, 82, 98, or 108, and the other of the two mitotic figures contains a sequence from amino acid positions 34, 45, 55, 69, 83, 99, or 109 of the amino acid sequence of SEQ ID NO: 1 to its C-terminus.

21. The method according to claim 20, characterized in that, One of the two split bodies contains the amino acid sequence of SEQ ID NO: 2 or a conserved amino acid substitute thereof, and the other contains the amino acid sequence of SEQ ID NO: 3 or a conserved amino acid substitute thereof; or one of the two split bodies contains the amino acid sequence of SEQ ID NO: 4 or a conserved amino acid substitute thereof, and the other contains the amino acid sequence of SEQ ID NO: 5 or a conserved amino acid substitute thereof.

22. The method according to claim 11, characterized in that, Cytosine deaminase exists in the form of two mitotic figures, one of which is a variant or a conserved substitute thereof in which one or more amino acids selected from the group consisting of positions 3, 5, 10, 11, 13, 14, 15, 16, 17, 18, 19, 28, 30, and 31 of SEQ ID NO: 2 are substituted with other amino acids; the other is a variant or a conserved substitute thereof in which one or more amino acids selected from the group consisting of positions 13, 16, 17, 20, 21, 28, 29, 30, 31, 32, 33, 56, 57, 58, and 60 of SEQ ID NO: 3 are substituted with other amino acids; or One of the two splits is a variant or a conservative substitute thereof in which one or more amino acid sequences selected from the group consisting of positions 87, 88, 91, 92, 95, 100, 101, 102 and 103 of SEQ ID NO: 4 are replaced by other amino acids, and the other is a variant or a conservative substitute thereof in which one or more amino acids selected from the group consisting of positions 13, 14, 15 and 16 of SEQ ID NO: 5 are replaced by other amino acids.

23. The method according to claim 11, characterized in that, Cytosine deaminase exists in the form of two mitotic figures, one of which contains the amino acid sequence of SEQ ID NO: 8 or a conserved amino acid substitution thereof, and the other of which contains the amino acid sequence of SEQ ID NO: 10 or a conserved amino acid substitution thereof.

24. The method according to claim 11, characterized in that, Cytosine deaminase contains the amino acid sequence of SEQ ID NO: 24 or its conserved amino acid substitutions.

25. The method according to claim 11, characterized in that, The nicking enzymes were selected from MutH, BspD6I, FokI (including FokI-FokI homodimers or heterodimers), BsaI, BsmBI, BsmAI, BsrDI, CviPII, BspQI, AlwI and I-TevI, their fragments and their variants.

26. The method according to claim 12, characterized in that, The base editing composition comprises a fusion protein comprising a DNA-binding protein, a cytosine deaminase, and an adenine deaminase, wherein the DNA-binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 55 to SEQ ID NO: 58, the cytosine deaminase comprises the amino acid sequence of SEQ ID NO: 24, and the adenine deaminase comprises the amino acid sequence of SEQ ID NO:

39.

27. The method according to claim 12, characterized in that, The base editing composition comprises two fusion proteins, one of which comprises a DNA-binding protein, a first cytosine deaminase mitochondria, and an adenine deaminase, and the other of which comprises a DNA-binding protein and a second cytosine deaminase mitochondria. The DNA-binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 55 to SEQ ID NO: 58, the first cytosine deaminase mitochondria comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 5, the second cytosine deaminase mitochondria comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 5, and the adenine deaminase comprises an amino acid sequence consisting of SEQ ID NO:

39.

28. The method according to claim 26 or 27, characterized in that, The fusion proteins also contain UDG of SEQ ID NO:

41.

29. The method according to claim 12, characterized in that, The base editing composition comprises two fusion proteins, one of which comprises a DNA-binding protein and a first cytosine deaminase mitochondria, and the other of which comprises a DNA-binding protein and a second cytosine deaminase mitochondria. The DNA-binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63 to SEQ ID NO: 65, the first cytosine deaminase mitochondria comprises the amino acid sequence of SEQ ID NO: 8, and the second cytosine deaminase mitochondria comprises the amino acid sequence of SEQ ID NO:

10.

30. The method according to claim 29, characterized in that, The fusion proteins also contain UGI of SEQ ID NO:

43.

31. The method according to claim 12, characterized in that, The base editing composition comprises two fusion proteins, one of which comprises a DNA-binding protein and an adenine deaminase, and the other of which comprises a DNA-binding protein and a nicking enzyme. The DNA-binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 71 and SEQ ID NO: 72, the adenine deaminase comprises an amino acid sequence of SEQ ID NO: 39, and the nicking enzyme comprises an amino acid sequence of SEQ ID NO:

16.

32. The method according to any one of claims 1 to 31, characterized in that, The step of expressing the base composition in a target plant or a part of a plant includes the step of introducing a polynucleotide encoding one or more fusion proteins contained in the base composition into the plant or a part of a plant.

33. The method according to claim 32, characterized in that, At least a portion of the polynucleotides has been codon-optimized for expression in the plant or a part of the plant.

34. The method according to any one of claims 1 to 33, characterized in that, A part of a plant is a plant cell, protoplast, or callus.

35. A gene-edited plant or part of a plant, said plant being a herbicide-resistant plant in which the DNA sequence encoding a serine residue of chloroplast D1 protein at 5'-AGT-3' is edited to 5'-GGT-3', or the DNA sequence encoding an alanine residue at 5'-GCT-3' is edited to 5'-GTT-3', characterized in that, The serine residue is the serine residue corresponding to the 264th serine residue of the Arabidopsis thaliana D1 protein, and the alanine residue is the alanine residue corresponding to the 251st alanine residue of the Arabidopsis thaliana D1 protein.

36. The gene-edited plant or part of a plant according to claim 35, characterized in that, Using a base editing composition, the DNA sequence encoding the serine residue 5'-AGT-3' of chloroplast D1 protein is edited to 5'-GGT-3', or the DNA sequence encoding the alanine residue 5'-GCT-3' is edited to 5'-GTT-3', wherein... The base editing composition has the activity of editing adenine bases to guanine bases and / or editing cytosine bases to thymine bases in DNA encoding plant chloroplast D1 protein.

37. A plant or a part of a plant, characterized in that... ， Produced according to the method of any one of claims 1 to 34.

38. A plant or a part of a plant, characterized in that, It is a progeny or clone of the plant according to any one of claims 35 to 37.

39. A seed, characterized in that, Obtained from the plant according to any one of claims 35 to 38.

40. A plant, its offspring, or a part of a plant, characterized in that, It is grown from the seed according to claim 39.

41. A method for controlling weeds near a target plant. include: The step of applying an effective amount of herbicide to the weeds and the target plants. The method is characterized in that, The target plant is the plant described in any one of claims 35 to 38 and 40.

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