Base correction of plant organelle DNA

Abstract

The present invention relates to a composition for base correction of plant organelle DNA, and more particularly to a composition and method for correcting adenine to guanine and cytosine to thymine in plant organelle DNA.

Description

[Technical Field]

[0001] The present invention relates to a composition for base correction of plant organelle DNA, and in particular to a composition and method for simultaneously correcting adenine to guanine and cytosine to thymine in plant organelle DNA. [Background technology]

[0002] Fusion proteins linking DNA-binding proteins and deaminase enzymes enable targeted nucleotide substitution or base editing in genes without generating DNA double-strand breaks (DSBs), single-nucleotide transversion in a targeted manner to correct point mutations that cause genetic disorders, or introduce desired single-nucleotide mutations in prokaryotic, human, and other eukaryotic cells.

[0003] Programmed gene correction tools, such as zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeat (CRISPR) systems, CRISPR-associated protein 9 (Cas9) mutants, and base deaminase protein base correction techniques, have been developed for plant genetic research and crop trait improvement through base sequence alterations. However, these tools are not suitable for correcting DNA sequences in plant organelles, including mitochondria and chloroplasts. This is primarily due to the difficulty of transporting RNA into organelles and / or expressing two compounds simultaneously in organelles. Plant organelles encode various essential genes required for photosynthesis and respiration. Methods and tools for correcting genes in these organelles are critically needed for studying the functions of these genes and improving crop productivity and traits. For example, targeted mutations in the mitochondrial atp6 gene can induce male sterility, a trait useful for reproduction, and specific point mutations in the 16S rRNA gene of the chloroplast genome can induce antibiotic resistance.

[0004] The bacterial toxin DddAtox is the enzymatic part of a bacterial toxin derived from Burkholderia cenocepacia, and is capable of deaminating cytosine in double-stranded DNA. As an example of a deaminase, DddAtox is cytotoxic to cells, so to avoid toxicity in host cells, DddAtox is split into two inactive splits, and each half can be linked to a DNA-binding protein designed to bind to DNA to form a functional DdCBE pair.

[0005] Based on this technical background, we have confirmed that AtoG base correction and CtoT base correction occur simultaneously in plant organelle DNA, and have completed the present invention. Summary of the Invention

[0006] An object of the present invention is to provide a composition for base correction of plant organelle DNA, which comprises a DNA-binding protein, cytosine deaminase or an isolated form of cytosine deaminase, and adenine deaminase or a nucleic acid encoding the same.

[0007] It is an object of the present invention to provide a method for correcting plant organelle DNA.

[0008] To achieve the above object, the present invention provides a composition for base correction of plant organelle DNA, comprising a DNA-binding protein or a nucleic acid encoding the same, cytosine deaminase or a first and second segment derived from cytosine deaminase or a nucleic acid encoding the same, and adenine deaminase or a nucleic acid encoding the same.

[0009] The present invention also provides a method for correcting bases in plant organelle DNA, comprising the step of treating plant cells with the composition. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram of DNA cloning for producing transformed plants using Agrobacterium. [Figure 2] Transformant plants targeting the psaA gene. (a) The efficiency and location of base correction in each plant, (b) plant phenotype, and (c) the changes in amino acid base sequence resulting from DNA base correction. [Figure 3] Transformant plants targeting the rbcL gene. (a) The efficiency and location of base correction in each plant, and (b) the changes in amino acid base sequence resulting from DNA base correction. [Figure 4] This shows the results of confirming the efficiency and location of base correction in transformed plants targeting the rrn16S gene. [Figure 5]Base correction efficiency and phenotype of first-generation transformants targeting the psaA, rbcL, and rrn16S genes in Arabidopsis chloroplasts. (a) Base correction efficiency and position for psaA #1, 2, and 3. Not only adenine base correction but also cytosine base correction occurs simultaneously. (b) The phenotype of psaA #3 shows green, chimeric, and pale green traits. (c) Base correction efficiency, position, and phenotype for rbcL #1 and 2. (d) Results of confirming the base correction efficiency of first-generation transformants targeting the rrn16S gene. [Figure 6] The figure shows the base correction efficiency and phenotype of second-generation transformed plants. (a) Phenotype of second-generation transformed plants of psaA #3, (b) base correction efficiency and location. (c) Phenotype of second-generation transformed plants of rrn16S that are resistant to spectinomycin, (d) base correction efficiency and location. (e) PCR was used to confirm the presence or absence of foreign genes introduced into the transformed plants, and it was confirmed that rrn16S #9-1 and #9-3 did not contain foreign genes. [Figure 7] The figure shows the results of confirming the base correction site and efficiency of plants that are resistant or sensitive to spectinomycin among the second generation of rrn16S transformed plants. [Figure 8] Off-target mutations observed through whole genome analysis of chloroplasts of psaA #1 (a), psaA #3 chimeric (b), psaA #3 pale green (c), rrn16s #1 (d), 6 (e), and wild-type Col-0 (f). No obvious off-target mutations were observed in the transformed plants compared to the wild-type. [Figure 9] This shows the efficiency of base correction of the lettuce mitochondrial gene atp6. DETAILED DESCRIPTION OF THE INVENTION

[0011] Detailed Description of the Invention and Preferred Embodiments Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used herein is well known and commonly used in the art.

[0012] The only method available for base correction of organelle DNA in plant cells was to combine DddAtox with TALE to correct cytosine to thymine. The technical objective of this invention is to combine DddAtox and adenine deaminase with TALE to correct bases in organelle DNA. By correcting bases in organelle DNA, which was previously impossible, it is possible to develop functional plants, such as plants with herbicide resistance.

[0013] The inventors of this application linked DddAtox cytosine deaminase and adenine deaminase to a TALE or ZFP protein capable of binding to DNA to create a base editor in which AtoG base correction and CtoT base correction occur simultaneously.

[0014] Based on this, in one aspect, the present invention relates to a composition for base correction of plant organelle DNA, which comprises a DNA-binding protein or a nucleic acid encoding the same, cytosine deaminase or a first and second fragments of cytosine deaminase or a nucleic acid encoding the same, and adenine deaminase or a nucleic acid encoding the same, and is characterized in that the composition simultaneously corrects adenine to guanine and cytosine to thymine in the plant organelle DNA.

[0015] As used herein, "correction" can be used interchangeably with "editing" and refers to a method of altering a nucleic acid sequence by selective deletion of a specific genomic target, including, but not limited to, a chromosomal region, a gene promoter, an open reading frame, or any nucleic acid sequence.

[0016] As used herein, "single base" refers to one and only one nucleotide in a nucleic acid sequence. When used in the context of a single base correction, this refers to the replacement of a base at a specific position in a nucleic acid sequence with a different base. Such a replacement can occur by a variety of mechanisms, including, without limitation, substitution or modification.

[0017] As used herein, "target" or "target site" refers to a pre-defined nucleic acid sequence of any composition and / or length. Such target sites include, but are not limited to, a chromosomal region, a gene, a promoter, an open reading frame, or any nucleic acid sequence.

[0018] As used herein, "on-target" refers to a subsequence of a specific genomic target that can be perfectly complementary to a programmable DNA binding region and / or a single guide RNA sequence.

[0019] As used herein, "off-target" refers to a subsequence of a specific genomic target that may be partially complementary to the programmable DNA-binding region and / or single guide RNA sequence.

[0020] The present invention includes a first segment and a second segment derived from cytosine deaminase or a mutant thereof, and has a form in which the cytosine deaminase binds to a DNA-binding protein and / or the first segment and the second segment each bind to the DNA-binding protein.

[0021] The cytosine deaminase is an amino group-releasing enzyme that can convert cytosine (C) to uridine (U).

[0022] The cytosine deaminase may be a cytosine deaminase. Examples of cytosine deaminase include APOBEC1 (apolipoprotein B editing complex 1) and AID (activation-induced deaminase). However, most DNA deaminases act only on single-stranded DNA and are not suitable for binding to DNA-binding proteins to correct bases. Specifically, the cytosine deaminase may be derived from a deaminase (DddA) that acts on double-stranded DNA or an orthologue thereof. More specifically, the cytosine deaminase may be a double-stranded DNA-specific bacterial cytosine deaminase.

[0023] The cytosine deaminase is in a split form, and the cytosine deaminase comprises a first split body and a second split body, each of which lacks deaminase activity.

[0024] The full-length cytosine deaminase may include the sequence of SEQ ID NO: 1, which corresponds to the tox fragment. The cytosine deaminase includes a first fragment and a second fragment, each of which lacks deaminase activity.

[0025] (SEQ ID NO: 1) GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGC

[0026] In one embodiment, the first or second segment of cytosine deaminase may comprise one or more sequences selected from the group consisting of G33, G44, A54, N68, G82, N98, and G108 at the N-terminus of the sequence of SEQ ID NO: 1. The first or second segment of cytosine deaminase may comprise one or more sequences selected from the group consisting of G34, P45, G55, N69, T83, A99, and A109 at the C-terminus of the sequence of SEQ ID NO: 1.

[0027] Specifically, the cytosine deaminase may include a first split fragment (G1333-N) of SEQ ID NO: 2 and a second split fragment (G1333-C) of SEQ ID NO: 3, and / or a first split fragment (G1397-N) of SEQ ID NO: 4 and a second split fragment (G1397-C) of SEQ ID NO: 5, and / or a first split fragment (G1333-N) of SEQ ID NO: 2 and a second split fragment (G1397-C) of SEQ ID NO: 5, and / or a first split fragment (G1397-N) of SEQ ID NO: 4 and a second split fragment (G1333-C) of SEQ ID NO: 2.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used herein is that which is well known and commonly used in the art.

[0029] The only method available for base correction of organelle DNA in plant cells was to bind DddAtox to a TALE to correct cytosine to thymine. The technical objective of this invention is to bind DddAtox and adenine deaminase to a TALE to correct the base of organelle DNA. By correcting the base of organelle DNA, which was previously impossible, it is possible to develop functional plants, such as plants with herbicide resistance.

[0030] The inventors of this application linked DddAtox cytosine deaminase and adenine deaminase to a TALE or ZFP protein capable of binding to DNA, creating a base editor in which AtoG base correction and CtoT base correction occur simultaneously.

[0031] Based on this, in one aspect, the present invention relates to a composition for base correction of plant organelle DNA, which comprises a DNA-binding protein or a nucleic acid encoding the same, cytosine deaminase or a first and second segment derived from cytosine deaminase or a nucleic acid encoding the same, and adenine deaminase or a nucleic acid encoding the same, and is characterized in that the composition simultaneously corrects adenine to guanine and cytosine to thymine in the plant organelle DNA.

[0032] As used herein, "correction" can be used interchangeably with "editing" and refers to a method of altering a nucleic acid sequence by selective deletion of a specific genomic target, including, but not limited to, a chromosomal region, a gene promoter, an open reading frame, or any nucleic acid sequence.

[0033] As used herein, "single base" refers to one and only one nucleotide in a nucleic acid sequence. When used in the context of a single base correction, this refers to the replacement of a base at a specific position in a nucleic acid sequence with a different base. Such a replacement can occur by a variety of mechanisms, including, without limitation, substitution or modification.

[0034] As used herein, "target" or "target site" refers to a pre-defined nucleic acid sequence of any composition and / or length. Such target sites include, but are not limited to, a chromosomal region, a gene, a promoter, an open reading frame, or any nucleic acid sequence.

[0035] As used herein, "on-target" refers to a subsequence of a specific genomic target that can be perfectly complementary to a programmable DNA binding region and / or a single guide RNA sequence.

[0036] As used herein, "off-target" refers to a subsequence of a specific genomic target that may be partially complementary to the programmable DNA-binding region and / or single guide RNA sequence.

[0037] The present invention includes a first segment and a second segment derived from cytosine deaminase or a mutant thereof, and has a form in which the cytosine deaminase binds to a DNA-binding protein and / or the first segment and the second segment each bind to the DNA-binding protein.

[0038] The cytosine deaminase is an amino group-releasing enzyme that can convert cytosine (C) to uridine (U).

[0039] The cytosine deaminase may be a cytosine deaminase. Examples of cytosine deaminase include APOBEC1 (apolipoprotein B editing complex 1) and AID (activation-induced deaminase). However, most DNA deaminases act only on single-stranded DNA and are not suitable for binding to DNA-binding proteins to correct bases. Specifically, the cytosine deaminase may be derived from a deaminase (DddA) that acts on double-stranded DNA or an orthologue thereof. More specifically, the cytosine deaminase may be a double-stranded DNA-specific bacterial cytosine deaminase.

[0040] The cytosine deaminase is in a split form, and the cytosine deaminase comprises a first split body and a second split body, each of which lacks deaminase activity.

[0041] The full-length cytosine deaminase may include the sequence of SEQ ID NO: 1, which corresponds to the tox fragment. The cytosine deaminase includes a first fragment and a second fragment, each of which lacks deaminase activity.

[0042] (SEQ ID NO: 2) Wild-type DddAtox G1333-N GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGG (SEQ ID NO: 33) GGCTCTGGTTCCTACGCCCTGGGTCCATATCAGATTAGTGCTCCCAACTCCCCGCCTACAACGGTCAGACAGTGGGGACCTTTTACTATGTCAACGACGCCGGGGGATTGGAATCCAAGGTTTTCTCTAGCGGTGGG (SEQ ID NO: 3) Wild-type G1333-C PTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGC (SEQ ID NO: 34) CCAACACCTTATCCTAACTACGCTAACGCCGGGCACGTCGAGGGGCAGTCAGCTCTTTTTATGAGAGATAACGGCATTAGCGAAGGGCTTGTGTTCCATAATAATCCTGAGGGCACCTGTGGCTTCTGTGTAAATATGACC GAAACACTTCTGCTGAGAACGCTAAAATGACTGTCGTACCACCCGAAGGCGCAATCCCAGTTAAACGGGGCGCAACCGGCGAAACCAAAGTATTCACCGGAAACAGCAATAGTCCAAAGTCCCCCACCAAGGGAGGTTGC (SEQ ID NO: 4) Wild-type DddAtox G1397-N GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG (SEQ ID NO: 35) GGTAGCTACGCACTTGGTCCTTACCAGATTAGCGCACCCCAACTCCCCGCCTATAATGGTCAAACCGTCGGGACCTTTTACTACGTAAACGATGCTGGTGGGCTGGAATCCAAAGTATTCTCCTCAGGGGGCCCTACACCCTACCCCAACTACGCCAATGCT GGTCATGTAGAAGGGCAGTCAGCACTGTTTATGCGCGATAATGGTATAAGCGAGGGGTTGGTCTTCCATAACAACCCAGAGGGTACTTGTGGCTTCTGTGTGAATATGACTGAAACCCTTCTGCCCGAAAATGCCAAGATGACTGTCGTCCCACCTGAAGGC

[0043] (SEQ ID NO: 5) Wild-type DddAtox G1397-C AIPVKRGATGETKVFTGNSNSPKSPTKGGC (SEQ ID NO: 36) GCCATACCTGTGAAGCGGGGAGCAACAGGGGAGACAAAGGTGTTCACAGGCAACTCTAACAGTCCAAAGAGCCCCACCAAAGGCGGGTGT

[0044] The G1333N, G1333C, G1397N, and G1397C can be used in combination in a split deaminase, specifically in the form of Left-G1333-N+Right-G1333-C, Left-G1397-N+Right-G1397-C, Left-G1397-N+Right-G1333-C, or Left-G1333-N+Right-G1397-C.

[0045] The DNA binding protein can be, for example, a zinc finger protein, a transcription activator-like effector (TALE) array, or a combination thereof.

[0046] The zinc finger motif of the zinc finger protein has a DNA-binding domain, and the C-terminal portion of the finger specifically recognizes a DNA sequence. DNA-binding proteins containing 3 to 6 zinc finger motifs recognize a DNA sequence.

[0047] In one embodiment, the first cleavage body of cytosine deaminase and the second cleavage body of cytosine deaminase can be bound to the N-terminus or C-terminus of a zinc finger protein, respectively.

[0048] The C-terminus of a zinc finger protein (ZF-Left) binds to the N-terminus of the first cytosine deaminase fragment, and the C-terminus of a zinc finger protein (ZF-Right) binds to the N-terminus of the second cytosine deaminase fragment (CC configuration). The end of the zinc finger protein (ZF-Left) binds to the C-terminus of the first cytosine deaminase fragment, and the C-terminus of the zinc finger protein (ZF-Right) binds to the N-terminus of the second cytosine deaminase fragment (NC configuration). the C-terminus of a zinc finger protein (ZF-Left) is bound to the N-terminus of the first cytosine deaminase fragment, and the N-terminus of a zinc finger protein (ZF-Right) is bound to the C-terminus of the second cytosine deaminase fragment (CN configuration); or

[0049] The N-terminus of a zinc finger protein (ZF-Left) can bind to the C-terminus of the first cytosine deaminase fragment, and the N-terminus of a zinc finger protein (ZF-Right) can bind to the C-terminus of the second cytosine deaminase fragment (NN configuration).

[0050] The ZF-Left may comprise the sequence of SEQ ID NO:6.

[0051] The ZF-Left may comprise the sequence of SEQ ID NO:6. (SEQ ID NO: 6) GIHGVPAAMAERPFQCRICMRNFSDRSNLSRHIRTHTGEKPFACDICGRKFAISSNLNSHTKIHTGSQKPFQCRICMRNFSRSDNLARHIRTHTGEKPFACDICGRKFATSGNLTRHTKIHLR.

[0052] The ZF-Right may comprise the sequence of SEQ ID NO:7.

[0053] (SEQ ID NO: 7) GIHGVPAAMAERPFQCRICMRNFSRSDNLSVHIRTHTGEKPFACDICGRKFAQKINLQVHTKIHTGEKPFQCRICMRNFSRSDVLSEHIRTHTGEKPFACDICGRKFAQRNHRTTHTKIHLR.

[0054] ZFs can vary in sequence depending on the DNA target. ZFs can be customized based on the DNA target sequence. Because ZFs recognize 3 bp of DNA, typically 3-6 ZFs can be combined to create ZF combinations that recognize 9-18 bp of DNA. For example, ZFs can be created using libraries containing modules such as GNNs, TNNs, CNNs, or ANNs.

[0055] In some cases, the zinc finger protein can be linked to the deaminase via a linker, which can be a peptide linker containing 2 to 40 amino acid residues, such as, but not limited to, 2 aa, 5 aa, 10 aa, 16 aa, 24 aa, or 32 aa in length.

[0056] In one embodiment, the linker may comprise:

[0057] 2a.a linker: GS, 5a.a linker: TGEKQ, 10a.a linker: SGAQGSTLDF, 16a.a Linker: SGSETPGTSESATPES, 24a.a linker: SGTPHEVGVYTLSGTPHEVGVYTL, or 32a.a Linker: GSGGSSGGSSGSETPGTSESATPESSGGSSGGS.

[0058] In a specific embodiment of the present invention, the split deaminase and zinc finger protein can be linked via a linker, with the zinc finger protein binding to the N-terminus of the half deaminase containing the first split body and the zinc finger protein binding to the N-terminus of the half deaminase containing the second split body. In this case, a C to T transversion can occur in the spacer between the binding sites of the left and right ZFPs. It was confirmed that when both the left and right ZFPs are linked to the half deaminase containing the first split body and the half deaminase containing the second split body, respectively, via a 24a linker, they exhibit mutual correction efficiency.

[0059] The TAL effector (TALE) is composed of a repeat of a 33-34 amino acid sequence, with approximately nine repeated domains (RVD, Repeated Variant Domain). Each domain can recognize one nucleotide and bind to a specific DNA sequence depending on the 12th-13th amino acid sequence (HD->Cytosine, NI->Adenine, NG->Thymine, NN->Guanine). The de-effector (TALE) recognizes single-stranded DNA within the target site. The distance between target sites can be 12-14 nucleotides.

[0060] The TALE domain refers to a protein domain that binds to nucleotides in a sequence-specific manner through a combination of one or more TALE-Repeat. It includes, but is not limited to, at least one TALE-Repeat, specifically 1 to 30 TALE-Repeat. A TALE-Repeat is a site that recognizes a specific nucleotide sequence within a TALE domain.

[0061] The TALE domain includes a backbone structure containing an N-terminal region of TALE and a C-terminal region of TALE.

[0062] [Table 1]

[0063] Depending on the position at which the TALE domain is bound relative to the cleavage site, a single TALE array or a first TALE array and a second TALE array can be bound, respectively.

[0064] A first TALE (left TALE) can be bound to the first segmen of cytosine deaminase, and a second TALE (right TALE) can be bound to the second segmen of cytosine deaminase, with the structures N'-TALE-first segmen-C' and N'-TALE-second segmen-C', respectively.

[0065] TALE arrays can be customized to suit target DNA sequences. They consist of repeating modules consisting of 33–35 amino acid residues. These modules were derived from the plant pathogen Xanthmonas. Each module recognizes one A, one C, one G, and one T base and binds to DNA. The base specificity of each module is determined by the 12th and 13th amino acid residues, called the RVD (repeat variable di-residue). For example, a module with an RVD of NN recognizes G, NI recognizes A, HD recognizes C, and NG recognizes T. TALE arrays can be designed to consist of a minimum of 14 modules and a maximum of 18 or more modules, and to recognize target DNA sequences of 15–20 bp.

[0066] The Cas protein may be a mutated form, which may mean mutated to lose endonuclease activity that cleaves DNA double strands, for example, one or more of a mutant target-specific nuclease that has lost endonuclease activity but has nicase activity, and a form that has lost both endonuclease activity and nicase activity.

[0067] If the enzyme has nickase activity, a nick can be introduced simultaneously with or sequentially, regardless of order, in the strand where the base conversion occurs (e.g., conversion of cytosine to uridine) by the cytosine deaminase or in the opposite strand (e.g., the opposite strand from the strand where the base conversion occurred) (e.g., a nick can be introduced in the opposite strand from the strand where the PAM is located, at a position corresponding to between the third and fourth nucleotides toward the 5' end of the PAM sequence). Such a mutation (e.g., amino acid substitution, etc.) can occur in the catalytically active domain (e.g., the RuvC catalytic domain in the case of Cas9). In the case of Streptococcus pyogenes Cas9, the mutation can include a substitution of one or more of the catalytic aspartate residues (e.g., aspartic acid at position 10 (D10)), glutamic acid at position 762 (E762), histidine at position 840 (H840), asparagine at position 854 (N854), asparagine at position 863 (N863), aspartic acid at position 986 (D986), etc. In this case, the substituted amino acid can be, but is not limited to, alanine.

[0068] In some cases, one or more of the aspartic acid at position 1135 (D1135), the arginine at position 1335 (R1335), and the threonine at position 1337 (T1337) of the Streptococcus pyogenes Cas9 protein, for example, all three, may be substituted with other amino acids, resulting in a mutation that recognizes NGA (where N is any base selected from A, T, G, and C), which is different from the PAM sequence (NGG) of wild-type Cas9.

[0069] For example, the amino acid sequence of the Cas9 protein derived from Streptococcus pyogenes is (1) D10, H840, or D10 + H840, (2) D1135, R1335, T1337, or D1135 + R1335 + D1337, or (3) Amino acid substitutions can occur at all residues (1) and (2).

[0070] The "other amino acid" refers to an amino acid selected from 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 the amino acids, excluding the amino acid at the original variant position in the wild-type protein. In one example, the "other amino acid" may be alanine, valine, glutamine, or arginine.

[0071] In some cases, a guide RNA may be further included. The guide RNA may be, for example, one or more selected from the group consisting of CRIPSPR RNA (crRNA), trans-activating crRNA (tracrRNA), and single guide RNA (sgRNA), and may specifically be a double-stranded crRNA:tracrRNA complex in which the crRNA and tracrRNA are bound to each other, or a single-stranded guide RNA (sgRNA) in which the crRNA or a portion thereof and the tracrRNA or a portion thereof are linked via an oligonucleotide linker.

[0072] The present invention may include an adenine deaminase. The adenine deaminase may be selected from the group consisting of apolipoprotein B editing complex 1 (APOBEC1), activation-induced deaminase (AID), and tRNA-specific adenosine deaminase (tadA), and may be tRNA-specific adenosine deaminase (tadA). The adenine deaminase may be deoxyadenine deaminase, such as a mutant of Escherichia coli TadA.

[0073] In a specific embodiment of the present invention, the mutant of E. coli TadA is deoxyadenine deaminase, specifically TadA8e or ABE8.0.

[0074] The cytosine deaminase is contained in a split form, and the DNA-binding protein is a zinc finger protein, in which the N-terminus of the zinc finger protein (ZF-Left) is bound to the C-terminus of the first split body of cytosine deaminase, and the C-terminus of the zinc finger protein (ZF-Right) is bound to the N-terminus of the second split body of cytosine deaminase (NC configuration). Adenine deaminase can be bound to the C-terminus of the zinc finger protein (ZF-Left), the N-terminus or C-terminus of the first split body of cytosine deaminase, the N-terminus of the zinc finger protein (ZF-Right), or the N-terminus or C-terminus of the second split body of cytosine deaminase.

[0075] Adenine deaminase is bound to the C-terminus of a zinc finger protein (ZF-Left) at the N-terminus of the first cytosine deaminase fragment and to the C-terminus of a zinc finger protein (ZF-Right) at the N-terminus of the second cytosine deaminase fragment (CC configuration); to the C-terminus of a zinc finger protein (ZF-Left) at the N-terminus of the first cytosine deaminase fragment and to the N-terminus of a zinc finger protein (ZF-Right) at the C-terminus of the second cytosine deaminase fragment (CN configuration); or to the N-terminus of a zinc finger protein (ZF-Left) at the C-terminus of the first cytosine deaminase fragment and to the N-terminus of a zinc finger protein (ZF-Right) at the C-terminus of the second cytosine deaminase fragment (NN configuration). In any of the above configurations, the cytosine deaminase fragment can bind to the C-terminus of the zinc finger protein (ZF-Right), the N-terminus or C-terminus of the first cytosine deaminase fragment, the N-terminus of the zinc finger protein (ZF-Right), or the N-terminus or C-terminus of the second cytosine deaminase fragment.

[0076] When the cytosine deaminase is in a split form and the DNA-binding protein is a TALE, a first TALE can be bound to the first split body of the cytosine deaminase, and a second TALE can be bound to the second split body of the cytosine deaminase, having the structures N'-TALE-first split body DDDA-C' and N'-TALE-second split body DDDA-C', respectively. Adenine deaminase can be bound to the N-terminus or C-terminus of the first split body of the cytosine deaminase, or cytosine deaminase n can be bound to the N-terminus or C-terminus of the second split body.

[0077] The present invention provides a composition for A-to-G base correction of plant organelle DNA (without UGI (uracil DNA-glycosylase inhibitor)), which comprises 1) a DNA-binding protein, 2) a split double-stranded DNA-specific bacterial cytosine deaminase, and 3) deoxyadenine deaminase derived from E. coli TadA, wherein the DNA-binding protein is a zinc finger protein (ZFP) or a transcription activator-like effector (TALE) array, and the split double-stranded DNA-specific bacterial cytosine deaminase is DddAtox derived from Burkholderia cenocepacia.

[0078] The cytosine deaminase is contained in a split form, and the DNA-binding protein is a zinc finger protein, in which the N-terminus of the zinc finger protein (ZF-Left) is bound to the C-terminus of the first split body of cytosine deaminase, and the C-terminus of the zinc finger protein (ZF-Right) is bound to the N-terminus of the second split body of cytosine deaminase (NC configuration). Adenine deaminase can be bound to the C-terminus of the zinc finger protein (ZF-Left), the N-terminus or C-terminus of the first split body of cytosine deaminase, the N-terminus of the zinc finger protein (ZF-Right), or the N-terminus or C-terminus of the second split body of cytosine deaminase.

[0079] Adenine deaminase is bound to the N-terminus of the first cytosine deaminase fragment by the C-terminus of a zinc finger protein (ZF-Left) and to the N-terminus of the second cytosine deaminase fragment by the C-terminus of a zinc finger protein (ZF-Right) (CC configuration); to the N-terminus of the first cytosine deaminase fragment by the C-terminus of a zinc finger protein (ZF-Left) and to the C-terminus of the second cytosine deaminase fragment by the N-terminus of a zinc finger protein (ZF-Right) (CN configuration); or to the C-terminus of the first cytosine deaminase fragment by the C-terminus of a zinc finger protein (ZF-Left) and to the C-terminus of the second cytosine deaminase fragment by the N-terminus of a zinc finger protein (ZF-Right) (NN configuration). In all configurations, the cytosine deaminase fragment can bind to the C-terminus of the zinc finger protein (ZF-Left), the N-terminus or C-terminus of the first cytosine deaminase fragment, the N-terminus of the zinc finger protein (ZF-Right), or the N-terminus or C-terminus of the second cytosine deaminase fragment.

[0080] When the cytosine deaminase is included in a split form and the DNA-binding protein is a TALE, a first TALE can be bound to the first split body of the cytosine deaminase and a second TALE can be bound to the second split body of the cytosine deaminase, having the structures N'-TALE-first split body DDDA-C' and N'-TALE-second split body DDDA-C', respectively. Adenine deaminase can be bound to the N-terminus or C-terminus of the first split body of the cytosine deaminase or the N-terminus or C-terminus of the second split body of the cytosine deaminase.

[0081] The present invention relates to a composition for A-to-G base correction in plant organelles, characterized in that the DNA-binding protein is a zinc finger protein or a TALE array, and the first and second segments derived from cytosine deaminase are derived from bacteria and are specific to double-stranded DNA.

[0082] The present invention relates to a composition for A-to-G base correction in plant organelles, wherein the DNA-binding protein is a zinc finger protein or a TALE array, the first and second segments derived from cytosine deaminase are derived from bacteria and are specific to double-stranded DNA, and the DNA-binding protein is bound to the N-terminus of the first segment, and the DNA-binding protein is bound to the C-terminus of adenine deaminase.

[0083] The present invention provides a composition for plant organelle A-to-G base correction, comprising 1) a DNA-binding protein, 2) a split double-stranded DNA-specific bacterial cytosine deaminase, and 3) deoxyadenine deaminase derived from E. coli TadA, wherein the composition does not contain UGI; the DNA-binding protein is a zinc finger protein (ZFP) or a transcription activator-like effector (TALE) array; and the split double-stranded DNA-specific bacterial cytosine deaminase is DddAtox derived from Burkholderia cenocepacia.

[0084] The present invention relates to a method for A-to-G base correction in plant organelles, which comprises the steps of treating a plant cell with a DNA-binding protein or a nucleic acid encoding the same, a first and second segment derived from cytosine deaminase or a nucleic acid encoding the same, and adenine deaminase or a nucleic acid encoding the same, wherein the DNA-binding protein is a zinc finger protein or a TALE array, and the first and second segment derived from cytosine deaminase are derived from bacteria and are specific to double-stranded DNA.

[0085] The present invention relates to a method for A-to-G base correction in plant organelles, comprising the steps of treating a plant cell with a DNA-binding protein or a nucleic acid encoding the same, a first and second segment derived from cytosine deaminase or a nucleic acid encoding the same, and adenine deaminase or a nucleic acid encoding the same, wherein the DNA-binding protein is a zinc finger protein or a TALE array, the first and second segment derived from cytosine deaminase are derived from bacteria and are specific to double-stranded DNA, and the DNA-binding protein is bound to the N-terminus of the first segment and the DNA-binding protein is bound to the C-terminus of the adenine deaminase.

[0086] The present invention provides a method for plant organelle A-to-G base correction, which comprises the steps of treating plant cells with a DNA-binding protein or a nucleic acid encoding the same, first and second segments derived from cytosine deaminase or nucleic acids encoding them, and adenine deaminase or a nucleic acid encoding the same, wherein the composition for plant organelle A-to-G base correction contains 1) a DNA-binding protein, 2) a segmented double-stranded DNA-specific bacterial cytosine deaminase, and 3) deoxyadenine deaminase derived from E. coli TadA, and does not contain UGI. The DNA-binding protein is a zinc finger protein (ZFP) or a transcription activator-like effector (TALE) array, and is the segmented double-stranded DNA-specific bacterial cytosine deaminase DddAtox derived from Burkholderia cenocepacia.

[0087] Previously, TALEDs that did not contain UGIs did not induce C-to-T editing in animal cell mitochondria, but only A-to-G editing.

[0088] The present invention may additionally include a chloroplast transit peptide or a mitochondrial targeting signal (MTS).

[0089] For example, a chloroplast transit peptide (CTP) or a mitochondrial targeting signal (MTS) binds to the protein and is delivered to the chloroplasts and mitochondria in plant cells. When delivered to the chloroplasts and mitochondria, the remaining portion, excluding the N-terminal CTP or MTS protein, is delivered to the chloroplasts and mitochondria in preprotein form. During the process of entering the chloroplasts and mitochondria, a portion of the delivery protein is shed, allowing it to target the chloroplasts and mitochondria and modify specific portions.

[0090] With reference to nucleic acids, the terms "polynucleotide," "nucleotide," "nucleotide sequence," and "oligonucleotide" are used interchangeably. A polymeric form of nucleotides of any length can contain deoxyribonucleotides or ribonucleotides, or their analogs. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. A polynucleotide can contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure can be made before or after assembly of the polymer.

[0091] The polynucleotide can be an RNA sequence, a DNA sequence, or a combination thereof (combined RNA-DNA sequence).

[0092] As a means for expressing the fusion protein, known expression vectors such as plasmid vectors, cosmid vectors, and bacteriophage vectors can be used, and the vectors can be easily produced by those skilled in the art according to any known method using DNA recombination technology.

[0093] The vector may be a plasmid vector or a viral vector, and the viral vector may specifically be, but is not limited to, an adenovirus, an adeno-associated virus, a lentivirus, or a retrovirus vector.

[0094] A recombinant expression vector can contain a nucleic acid in a form compatible with expression of the nucleic acid in a host cell, meaning that the recombinant expression vector contains one or more regulatory elements that can be selected based on the host cell to be used for expression, i.e., operably linked to the nucleic acid sequence to be expressed.

[0095] Within a recombinant expression vector, "operably linked" means that the nucleotide sequence of interest is linked to regulatory elements in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into a host cell).

[0096] The recombinant expression vector may contain a T7 promoter and may be in a form suitable for messenger RNA synthesis, meaning that it contains one or more regulatory elements that enable messenger RNA to be synthesized in vitro by mRNA synthesis, i.e., by T7 polymerase.

[0097] "Regulatory elements" can include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, e.g., polyadenylation signals, and poly-U sequences). Regulatory elements include elements that direct inducible or constitutive expression of a nucleotide sequence in many types of host cells, as well as elements that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can direct expression primarily in a desired tissue of interest, e.g., muscle, neurons, bone, skin, blood, a particular organ (e.g., liver, pancreas), or a particular cell type (e.g., lymphocytes). Regulatory elements can also direct expression in a temporally-dependent manner, such as in a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue- or cell type-specific.

[0098] In some cases, the vector includes one or more Pol III promoters, one or more Pol II promoters, one or more Pol I promoters, or a combination thereof. Examples of Pol III promoters include, but are not limited to, the U6 and H1 promoters. Examples of Pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (e.g., Boshart et al. (1985) Cell 41: 521-530), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter.

[0099] "Regulatory elements" can include enhancers, such as the WPRE, CMV enhancer, the R-U5' segment of the HTLV-I LTR, the SV40 enhancer, and the intron sequence between exons 2 and 3 of rabbit β-globin. Those skilled in the art will recognize that the design of an expression vector can depend on factors such as the choice of host cell to be transformed and the desired expression level. The vector can be introduced into a host cell to produce transcripts, proteins, or peptides, including fusion proteins or peptides encoded by nucleic acids as described herein (e.g., clustered regularly interspaced short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutants thereof, and fusion proteins thereof). Useful vectors include lentiviruses and adeno-associated viruses, and these vector types can also be selected to target specific cell types.

[0100] The vectors can be delivered in vivo or intracellularly via local injection (e.g., direct injection into a lesion or target site), electroporation, lipofection, viral vectors, nanoparticles, as well as PTD (protein translocation domain) fusion protein methods.

[0101] The nucleic acid can be injected in the form of ribonucleic acid, for example, messenger ribonucleic acid mRNA, to allow gene correction of cells, for example, animal cells or plant cells without restriction.

[0102] The nucleic acid according to the present invention may be in the form of mRNA. When delivered in the form of mRNA, the transcription process into mRNA is unnecessary compared to delivery in the form of a DNA-based vector, and gene correction can be initiated more quickly, and there is a higher possibility of transient protein expression.

[0103] The inventors of the present application confirmed that when a cytosine base editor is injected into plant cells in the form of ribonucleic acid, such as messenger ribonucleic acid, for plant organelle gene correction, off-target effects are reduced compared to when it is delivered via a plasmid. They were the first to demonstrate that when a cytosine base editor is transformed into plant cells in the form of mRNA for plant organelle gene correction, it has advantages in terms of off-target effects compared to plasmids.

[0104] The mRNA can be delivered directly and / or via a carrier. In some cases, the mRNA of the nucleic acid cleaving enzyme and / or cleavage factor can be chemically modified and / or delivered directly in the form of synthetic self-replicative RNA.

[0105] Methods for delivering mRNA molecules to cells in vitro or in vivo can be considered, including methods for delivering mRNA to cells or methods for delivering mRNA to cells in vivo in organisms such as humans and animals. For example, mRNA molecules can be delivered to cells using lipids (e.g., liposomes, micelles, etc.), nanoparticles or nanotubes, and / or cationic compounds (e.g., polyethyleneimine or PEI). In some cases, bolistic methods, such as gene guns or biolistic particle delivery systems, can be used to deliver mRNA to cells.

[0106] The carrier may include, but is not limited to, for example, a cell penetrating peptide (CPP), a nanoparticle, or a polymer.

[0107] The CPPs are short peptides that facilitate the cellular uptake of a variety of molecular cargoes, from nano-sized particles to small chemical molecules and large fragments of DNA.

[0108] Regarding the nanoparticles, the compositions according to the present invention can be delivered via polymeric nanoparticles, metal nanoparticles, metal-inorganic nanoparticles, or lipid nanoparticles. The polymeric nanoparticles can be, for example, DNA nanoclews, or thread-like DNA nanoparticles, synthesized by rolling circle amplification. DNA nanoclews and thread-like DNA nanoparticles can be loaded with mRNA and coated with PEI to enhance their endosomal escape ability. Such complexes can bind to the cell membrane, be internalized, and then transported to the nucleus via endosomal escape and transported.

[0109] In association with the metal nanoparticles, gold particles can be linked and complexed with a cationic endosomal disruptive polymer for delivery to cells, such as polyethylene imine, poly(arginine), poly(lysine), poly(histidine), poly-[2-{(2-aminoethyl)amino}-ethyl-aspartamide] (pAsp(DET)), a block copolymer of poly(ethylene glycol) (PEG) and poly(arginine), a block copolymer of PEG and poly(lysine), or a block copolymer of PEG and poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PEG-pAsp(DET)).

[0110] The mRNA can be encapsulated in association with the metal / inorganic nanoparticles, for example, through ZIF-8 (zeolitic imidazolate framework-8).

[0111] In some cases, the mRNA may be negatively charged and may be bound to a cationic substance to form nanoparticles, which may penetrate cells via receptor-mediated endocytosis or phagocytosis.

[0112] Cationic polymers include polyallylamine (PAH), polyethyleneimine (PEI), poly(L-lysine) (PLL), poly(L-arginine) (PLA), polyvinylamine monomers or copolymers, poly(vinylbenzyl-tri-C1-C4-alkylammonium salts), polymers of aliphatic or aromatic dihalides and aliphatic N,N,N',N'-tetra-C1-C4-alkyl-alkylenediamines, poly(vinylpyridine) or poly(vinylpyridinium salts), poly(N,N-diallyl-N,N-di-C1-C4-alkyl-ammonium halides), homopolymers or copolymers of quaternized di-C1-C4-alkyl-aminoethyl acrylate or methacrylate, and POLYQUAD. TM , polyaminoamides, and the like.

[0113] The cationic lipid can include a cationic liposome formulation, in which the lipid bilayer of the liposome can protect the encapsulated nucleic acid from degradation and prevent specific neutralization by antibodies capable of binding to the nucleic acid. During endosome maturation, fusion of the endosomal membrane with the liposome allows efficient endosomal escape of the cationic lipid-nuclease. Cationic lipids include polyethyleneimine, polyamidoamine (PAMAM)-functionalized resinous bodies, lipopectin (a combination of DOTMA and DOPE), lipopectinase, LIPOFECTAMINE® (e.g., LIPOFECTAMINE® 2000, LIPOFECTAMINE® 3000, LIPOFECTAMINE® RNAiMAX, LIPOFECTAMINE® LTX), SAINT-RED (Synvolux Therapeutics, Groningen, The Netherlands), DOPE, Cytopectin (Gilead Sciences, Foster City, California), and Eupectin (JBL, San Luis Obispo, California). Representative cationic liposomes can be prepared from N-[1-(2,3-dioleoxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoxy)-propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), 3β-[N-(N'-N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2,3-dioleoxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide, or dimethyldioctadecylammonium bromide (DDAB).

[0114] Liposomes can be used in conjunction with lipid nanoparticles as carriers for delivery. Liposomes are spherical vesicular structures composed of a single or multiple lipid bilayers surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomal formulations can contain primarily natural phospholipids and lipids, such as 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, phosphatidylcholine, or monosialogloside. In some cases, cholesterol or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) can be added to the lipid membrane to eliminate instability in plasma. The addition of cholesterol reduces the rapid release of the encapsulated bioactive compound into plasma, and / or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increases stability.

[0115] The composition can be delivered to a plant cell via: Bombardment using a gene gun PEG-mediated protoplast transfection Protoplast transfection (electroporation), or Protoplast injection by microinjection.

[0116] The polynucleotide sequence encoding the fusion protein according to the invention can be an RNA sequence, a DNA sequence, or a combination thereof (combined RNA-DNA sequence).

[0117] The nucleic acid can be delivered to a plant cell via: Transformation using Agrobacterium, such as Agrobacterium tumefaciens, Agrobacterium rhizogene, etc. - binary vector, - Viral vectors: 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. Transfection with viruses, Injection (Bombardment, Gene gun), PEG-mediated protoplast transfection Protoplast transfection (electroporation), or Protoplast injection by microinjection.

[0118] Examples of the virus include viral vectors such as 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).

[0119] Each of the above vectors can be delivered into cells via local injection (e.g., direct injection into a lesion or target site), electroporation, lipofection, viral vectors, nanoparticles, as well as PTD (protein translocation domain) fusion protein methods.

[0120] The present invention relates to a method for correcting bases in plant organelle DNA, which comprises treating plant cells with the composition.

[0121] The present invention relates to atrazine herbicide-resistant plants in which the base of the chloroplast psbA gene containing the GTTGAAAGC sequence has been corrected by the above method.

[0122] The present invention relates to spectinomycin-resistant plants in which the bases of 16s rRNA containing the CGTCATCCTCA sequence have been corrected by the above method.

[0123] The present invention relates to a plant in which the base of the atp6 gene has been corrected by the above method.

[0124] The present invention relates to a plant having an albino phenotype in which the base of the psaA gene containing ATG has been corrected by the above method.

[0125] By correcting the 295th amino acid, phenylalanine, in chloroplast DNA psbA with serine, it is possible to develop plants that are resistant to the herbicide atrazine. By correcting not only chloroplast psbA but also 16s rRNA and psaA, it is possible to develop spectinomycin-resistant plants and albino plants.

[0126] This allows for base correction of the rbcL (Rubisco large subunit) gene, which is involved in photosynthesis, and is expected to regulate photosynthetic efficiency, leading to increased plant production or the creation of plants with high carbon dioxide absorption efficiency.

[0127] The present invention relates to plants in which the bases of the chloroplast rubisco (ribulose bisphosphate carboxylase)-encoding gene have been corrected by the above method. The base correction of the rubisco large unit involved in photosynthesis can be expected to regulate photosynthetic efficiency and increase plant productivity.

[0128] The chloroplast base sequence has a high degree of homology among plants, and therefore, atrazine-resistant plants can be developed not only for the lettuce (Lactuca sativa cv. Cheongchima) used in the present invention, but also for rice, wheat, potato, tomato, and other plants.

[0129] The plasmid was transferred to lettuce protoplasts, and after 7 days, it was confirmed that the adenine base had been corrected at the target in the atp6 gene of mitochondrial DNA.

[0130] [Example] The present invention will be described in more detail below through examples. It will be obvious to those skilled in the art that these examples are intended solely to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0131] [Example 1] A schematic diagram of DNA cloning for generating transgenic plants using Agrobacterium is shown in Figure 1. To clone a TALE pair with repeated base sequences into a single vector, RPS5A promoter-PTP-3xFlag-Left TALE-1397N and RPS5A promoter-PTP-3xFlag-Right TALE-1397C-ABE8.0 were cloned into vectors with different Aat II and Pme I site locations. The two plasmids were then digested with Aat II and Pme I, respectively, and the RPS5A promoter-PTP-3xFlag-Right TALE-1397C-ABE8.0-35S terminator was ligated to the plasmid containing the Left TALE, resulting in a single clone. Figure 1 is merely a schematic diagram; 1397C-ABE8.0 can be cloned into the Left TALE, and 1397N can be cloned into the Right TALE.

[0132] Through Agrobacterium-mediated transformation, we secured 20, 6, and 37 first-generation transformants targeting the psaA, rbcL, and rrn16S genes in Arabidopsis chloroplasts, respectively. Figure 2 shows the transformation plants targeting the psaA gene. (a) The efficiency and location of base correction for each plant, (b) the plant phenotype, and (c) the changes in amino acid base sequence due to DNA base correction. Figure 2(a) confirms that C-12, C-11, and C2 (labeled G2) in the base sequence representation were corrected.

[0133] Transformant plants targeting the rbcL gene. (a) The efficiency and location of base correction in each plant, and (b) the changes in amino acid base sequence due to DNA base correction are shown in Figure 3.

[0134] The efficiency and location of base correction in transformed plants targeting the rrn16S gene are shown in Figure 4.

[0135] Base correction efficiency and phenotype of first-generation transformants targeting the psaA, rbcL, and rrn16S genes in Arabidopsis chloroplasts. (a) The base correction efficiency and position of psaA #1, 2, and 3 are shown. Not only adenine base correction but also cytosine base correction is performed simultaneously. (b) The phenotype of psaA #3 shows green, chimeric, and pale green traits. (c) The base correction efficiency, position, and phenotype of rbcL #1 and 2, and (d) the base correction efficiency of first-generation transformants targeting the rrn16S gene are shown in Figure 5.

[0136] It can be seen that C2 and C-2 in Figure 4 and Figures 5(a) and (d) have been corrected, respectively.

[0137] Figure 6 shows the base correction efficiency and phenotype of the second-generation transformed plants. (a) Phenotype of the second-generation transformed plants of psaA #3, (b) base correction efficiency and location, (c) phenotype of the second-generation transformed plants of rrn16S that are resistant to spectinomycin, (d) base correction efficiency and location, and (e) PCR was used to confirm the presence or absence of foreign genes introduced into the transformed plants, and it was confirmed that rrn16S #9-1 and #9-3 did not contain foreign genes.

[0138] Figure 6 confirms that chlorophyll base correction is transmitted to the next generation, and in the case of (b), it confirms that AtoG and CtoT base corrections are transmitted to the next generation together.

[0139] Based on this, we confirmed that, unlike animals, AtoG base correction and CtoT base correction occur simultaneously in plants.

[0140] Among the second-generation rrn16S transformed plants, the base correction position and efficiency of plants resistant and sensitive to spectinomycin are shown in FIG.

[0141] Figure 8 shows off-target mutations in the chloroplast genomes of psaA #1 (a), psaA #3 chimeric (b), psaA #3 pale green (c), rrn16s #1 (d), 6 (e), and wild-type Col-0 (f). No obvious off-target mutations were observed in the transformed plants compared to the wild-type.

[0142] Figure 9 shows the efficiency of base correction of the lettuce mitochondrial atp6 gene. Lettuce protoplasts were transfected with 15 μg each of PcUBi promoter-MTS-3xFlag-Left TALE-1397N-Pea3A terminator, PcUBi promoter-MTS-3xFlag-Right TALE-1397C-ABE8.0-Pea3A terminator, and PcUBi promoter-MTS-3xFlag-Left TALE-1397C-ABE8.0-Pea3A terminator, for a total of 30 μg. Seven days later, the efficiency of adenine base correction was measured. The base correction efficiencies were 0.99% for A6 and 1.12% for A9.

[0143] The sequences for each configuration used in the examples are as follows:

[0144] CTS (or PTP: Sequence number 16) MDSQLVLSLKLNPSFTPLSPLFPFTPCSSFSPSLRFSSCYSRRLYSPVTVYAAK

[0145] MTS (SEQ ID NO: 17) MFKQASRLLSRSVAAAASSKSVTTRAFSTELPSTLDS

[0146] NTD (TALE N-term SEQ ID NO: 18) DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHERAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLN

[0147] CTD (TALE C-term SEQ ID NO: 19) LTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG

[0148] AD (ABE8.0 or TadA8e: SEQ ID NO: 20) SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN

[0149] Linker 1 (TALE array - Linker 1-DddA tox ) GS

[0150] Linker 2 (DddA tox -Linker 2-AD) SGSETPGTSESATPES

[0151] DddA tox 1397N (SEQ ID NO: 21) GSGSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG

[0152] DddA tox 1397C (SEQ ID NO: 22) GSAIPVKRGATGETKVFTGNSNSPKSPTKGGC

[0153] psbA Left TALE repeat (SEQ ID NO: 23) LTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHG

[0154] psbA Right TALE repeat (SEQ ID NO: 24) LTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHG

[0155] rrn16S Left TALE repeat (SEQ ID NO: 25) LTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHG

[0156] rrn16S Right TALE repeat (SEQ ID NO: 26) LTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHG

[0157] psaA Left TALE repeat (SEQ ID NO: 27) LTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHG

[0158] psaA Right TALE repeat (SEQ ID NO: 28) LTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHG

[0159] rbc L Left TALE repeat (SEQ ID NO: 29) LTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHG

[0160] rbc L Right TALE repeat (SEQ ID NO: 30) LTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHG

[0161] atp6 Left TALE repeat (SEQ ID NO: 31) LTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHG

[0162] atp6 Right TALE repeat (Accession No. 32) LTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHG

[0163] RPS5A promoter (SEQ ID NO: 41)

[0164] 35S terminator (SEQ ID NO: 42) CGGCCATGCTAGAGTCCGCAAAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCTATTTTTCTCCAGAATAATGTGTGAGTAGTTCCCAGATAAGGGAATTAGGGTTCTTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTGACCT

[0165] PcUbi promoter (SEQ ID NO: 43) CTAGCAACGATTGTACAATTGCTTCTTTAAAAAAGGAAGAAAGAAAGAAAGAAAAGAATCAACATCAGCGTTAACAAACGGCCCCGTTACGGCCCAAACGGTCATATAGAGTAACGGCGTTAAGCGTTGAAAGACTCCTATCGAAATACGTAACCGCAAACGTGTCATAGTCAGATCCCCTCTTCCTTCACCGCCTCAAACACAAAAATAATCTTCTACAGCCTATATATACAACCCCCCCTTCTATCTCTCCTTTCTCACAATTCATCATCTTTCTTTCTCTACCCCCAATTTTAAGAAATCCTCTCTTCTCCTCTTCATTTTCAAGGTAAATCTCTCTCTCTCTCTCTCTCTCTGTTATTCCTTGTTTTAATTAGGTATGTATTATTGCTAGTTTGTTAATCTGCTTATCTTATGTATGCCTTATGTGAATATCTTTATCTTGTTCATCTCATCCGTTTAGAAGCTATAAATTTGTTGATTTGACTGTGTATCTACACGTGGTTATGTTTATATCTAATCAGATATGAATTTCTTCATATTGTTGCGTTTGTGTGTACCAATCCGAAATCGTTGATTTTTTTCATTTAATCGTGTAGCTAATTGTACGTATACATATGGATCTACGTATCAATTGTTCATCTGTTTGTGTTTGTATGTATACAGATCTGAAAACATCACTTCTCTCATCTGATTGTGTTGTTACATACATAGATATAGATCTGTTATATCATTTTTTTTATTAATTGTGTATATATATATGTGCATAGATCTGGATTACATGATTGTGATTATTTACATGATTTTGTTATTTACGTATGTATATATGTAGATCTGGACTTTTTGGAGTTGTTGACTTGATTGTATTTGTGTGTGTATATGTGTGTTCTGATCTTGATATGTTATGTATGTGCAGC

[0166] Pea3A terminator (SEQ ID NO: 44) CAGGCCTCCCAGCTTTTCGTCCGTATCATCGGTTTCGACAACGTTCGTCAAGTTCAATGCATCAGTTTCATTGCCCACACACCAGAATCCTACTAAGTTTGAGTATTATGGCATTGGAAAAGCTGTTTTCTTCTATCATTTGTTCTGCTTGTAATTTACTGTGTTCTTTCAGTTTTTGTTTTCGGACATCAAAATGCAAATGGATGGATAAGAGTTAATAAATGATATGGTCCTT TTGTTCATTCTCAAATTATTATTATCTGTTGTTTTTACTTTAATGGGTTGAATTTAAGTAAGAAAGGAACTAACAGTGTGATATTAAGGTGCAATGTTAGACATATAAAACAGTCTTTCACCTCTCTTTGGTTATGTCTTGAATTGGTTTGTTTCTTCACTTATCTGTGTAATCAAGTTTACTATGAGTCTATGATCAAGTAATTATGCAATCAAGTTAAGTACAGTATAGGCTT [Industrial Applicability]

[0167] Previously, base correction of plant organelle DNA was limited to deamination of cytosine to thymine using DddAtox. However, this invention broadens the scope of base correction of organelle DNA by correcting adenine to guanine. AtoG and CtoT base corrections can be performed simultaneously.

[0168] Although the specific details of the present invention have been described above in detail, it will be apparent to those skilled in the art that these specific details are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the true scope of the present invention is to be defined by the appended claims and their equivalents.

[0169] [Sequence]Free Text Electronic file attached.

Claims

1. A composition for base correction of plant organelle DNA, comprising: DNA-binding proteins or nucleic acids encoding the same; Cytosine deaminase, or a first cleavage body and a second cleavage body derived from cytosine deaminase, or a nucleic acid encoding the same; and adenine deaminase or a nucleic acid encoding the same, the first and second division bodies are each bound to the DNA-binding protein; The adenine in the plant organelle DNA is simultaneously corrected to guanine and the cytosine to thymine. composition.

2. The first and second segments each have no cytosine deaminase activity. The composition of claim 1.

3. The cytosine deaminase is derived from a deaminase (DddA) acting on double-stranded DNA or an orthologue thereof. The composition of claim 1.

4. The first segment comprises at least one sequence selected from the group consisting of G33, G44, A54, N68, G82, N98, and G108 at the N-terminus of the sequence of SEQ ID NO: 1; The composition of claim 1.

5. The second segment comprises one or more sequences up to the C-terminus selected from the group consisting of G34, P45, G55, N69, T83, A99, and A109 in the sequence of SEQ ID NO: 1; The composition of claim 1.

6. The DNA binding protein is a zinc-finger protein (ZF protein) or a transcription activator-like effector array (TALE array); The composition of claim 1.

7. The DNA binding protein is linked via a peptide linker containing 2 to 40 amino acid residues. The composition of claim 1.

8. 8. The composition of claim 7, wherein the linker comprises: 2a.a linker: GS, 5a.a linker: TGEKQ, 10a.a linker: SGAQGSTLDF, 16a.a linker: SGSETPGTSESATPES, 24a.a linker: SGTPHEVGVYTLSSGTPHEVGVYTL, or 32a.a Linker: GSGGSSGGSSGSETPGTSESATPESSGGSSGGS.

9. the DNA-binding protein is a zinc finger protein, and the first and second segments are bound to the N-terminus or C-terminus of the zinc finger protein, respectively; The composition of claim 1.

10. The DNA binding protein is a TALE array, and a single TALE array is bound to one end of the segment, or a first TALE array and a second TALE array are bound to the first segment and the second segment, respectively; The composition of claim 1.

11. The adenine deaminase is bound to the N- or C-terminus of a DNA binding protein or a cytosine deaminase. The composition of claim 1.

12. the adenine deaminase is a TadA-derived deoxyadenine deaminase; The composition of claim 1.

13. A chloroplast transit peptide or a nucleic acid encoding the same, The composition of claim 1.

14. Mitochondrial Targeting Signal (MTS) or a nucleic acid encoding the same; The composition of claim 1.

15. Does not contain UGI (uracil DNA-glycosylase inhibitor), The composition of claim 1.

16. The composition of claim 1, which is delivered to a plant cell via: Gene gun injection (Bombardment), PEG-mediated protoplast transfection Protoplast transfection by electroporation Or, Protoplast injection by microinjection.

17. The composition of claim 1, wherein the nucleic acid is delivered to a plant cell via: Agrobacterium-mediated transformation of Agrobacterium tumefaciens or Agrobacterium rhizogenes; Viral transfection, Gene gun injection (Bombardment), PEG-mediated protoplast transfection Protoplast transfection by electroporation Or, Protoplast injection by microinjection.

18. A method for correcting bases in plant organelle DNA, comprising the step of treating plant cells with the composition of any one of claims 1 to 17.

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