Base editing of plant organelle DNA

Abstract

The present invention relates to a composition for editing a base of a plant organelle DNA, and more particularly, to a composition for editing adenine of the plant organelle DNA into guanine and editing cytosine into thymine, and to an editing method.

Description

Technical Field

[0001] The present invention relates to a composition for base editing of plant organelle DNA, and in particular, to a composition and an editing method capable of simultaneously editing adenine into guanine and cytosine into thymine in plant organelle DNA. Background Art

[0002] The fusion protein linked to the DNA binding protein and the deaminase does not generate DNA double-strand breaks (DSBs) and can achieve single nucleotide conversion in a targeted manner to achieve targeted nucleotide substitution or base editing in the genome, edit point mutations that can induce genetic disorders, or introduce desired single nucleotide variations in prokaryotes, humans and other eukaryotic cells.

[0003] Programs (sets) of genome editing tools such as zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeat (CRISPR) system and CRISPR-associated protein 9 (Cas9) variants without nucleic acid degradation efficiency and base editing technology of base deaminase protein are used for plant genetic research and crop trait improvement by changing the base sequence. However, these tools are not suitable for editing DNA sequences of plant organs including mitochondria and chloroplasts, mainly because it is difficult to deliver guide RNA to organelles or to express two compounds simultaneously in organelles. Plant organelles encode a variety of essential genes required for photosynthesis and respiration. Methods or tools for editing the genes of these organelles are crucial for functional studies of these genes or improvement of crop productivity and traits. For example, targeted mutations in the mitochondrial atp6 gene can induce male sterility (a trait useful for reproduction), while specific point mutations in the 16S rRNA gene of the chloroplast genome can induce antibiotic resistance.

[0004] Bacterial toxin DddA tox It is derived from Burkholderia cenocepacia and has the enzyme function of a bacterial toxin that deaminates cytosine in double-stranded DNA. As an example of a deaminase, DddA tox To avoid toxicity to host cells, DddA toxIt is used as two inactive splits, and each split half can be used as a functional DdCBE pair by connecting to a DNA-binding protein designed to bind to DNA.

[0005] In this technical context, it was confirmed that A to G base editing and C to T base editing of plant organelle DNA can occur simultaneously, thereby completing the present invention. Summary of the invention

[0006] Problem that the invention aims to solve

[0007] The object of the present invention is to provide a composition for base editing of plant organelle DNA, wherein the composition comprises a DNA binding protein, cytosine deaminase or an isolated form of cytosine deaminase, adenine deaminase, or nucleic acids encoding them.

[0008] The object of the present invention is to provide a method for editing plant organelle DNA.

[0009] In order to achieve the above objectives, the present invention provides a composition for base editing of plant organelle DNA, the composition comprising: a DNA binding protein or a nucleic acid encoding the same; a cytosine deaminase or a first segment and a second segment derived from a cytosine deaminase or a nucleic acid encoding the same; and an adenine deaminase or a nucleic acid encoding the same.

[0010] In addition, the present invention provides a method for editing bases of plant organelle DNA, the method comprising the step of treating plant cells with the composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Schematic diagram of DNA cloning for producing transformed plants using Agrobacterium.

[0012] Figure 2 The results of transformant plants targeting the psaA gene are shown, where a is the base editing efficiency and position of each plant, b is the phenotype of the plant, and c is the change in the amino acid base sequence through DNA base editing.

[0013] Figure 3 The results of transformant plants targeting the rbcL gene are shown, where a is the base editing efficiency and position of each plant, and b is the change in the amino acid base sequence through DNA base editing.

[0014] Figure 4 The results show the base editing efficiency and confirmed position of the transformed plants targeting the rrn16S gene.

[0015] Figure 5 The base editing efficiency and phenotype of the first-generation transformants targeting psaA, rbcL, and rrn16S genes in the chloroplasts of Arabidopsis thaliana are shown. Among them, a is the base editing efficiency and position of psaA#1, 2, and 3, and not only base editing of adenine but also base editing of cytosine occurs. b shows that the phenotype of psaA#3 can show green, chimeric, and pale green traits. c is the base editing efficiency, position, and phenotype of rbcL#1 and 2. d is the result of confirming the base editing efficiency of the first-generation transformant targeting rrn16S gene.

[0016] Figure 6 The base editing efficiency and phenotype of the second-generation transformed plants are shown. Among them, a is the phenotype of the second-generation transformed psaA#3, b is the base editing efficiency and position, c is the phenotype of the second-generation transformed rrn16S resistant to spectinomycin, d is the base editing efficiency and position, and e is the result of confirming whether the external gene was introduced into the transformed plant by PCR, confirming that rrn16S#9-1, 3 has no external gene.

[0017] Figure 7 The results of confirming the base editing position and efficiency of plants resistant and sensitive to spectinomycin in the second generation of rrn16S-transformed plants are shown.

[0018] Figure 8 The results of confirming off-target base mutations by full-length chloroplast genome analysis of psaA#1 (a), psaA#3 chimeric (b), psaA#3 palegreen (c), rrn16s#1 (d), rrn16s#6 (e), and wild-type Col-0 (f) are shown. No significant off-target base mutations were observed in the transformed plants compared to the wild-type.

[0019] Fig. 9 Shown is the base editing efficiency of the lettuce mitochondrial gene atp6. DETAILED DESCRIPTION

[0020] Unless otherwise defined, all technical terms and scientific terms used in this specification have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention belongs. Generally, the nomenclature used in this specification is a method well known and commonly used in the art.

[0021] The base editing method for organellar DNA in plant cells only involves replacing DddA tox The invention relates to a method for editing cytosine into thymine by combining with TALE. The technical problem of the invention is to convert DddA tox By combining TALE with adenine deaminase, the bases of organelle DNA can be edited. By editing the bases of organelle DNA, which was not possible before, functional plants (such as plants with herbicide resistance) can be developed.

[0022] The inventors of the present invention prepared a base editor that converts DddA tox Cytosine deaminase and adenine deaminase are linked to DNA-binding TALE or ZFP proteins, thereby enabling simultaneous A to G base editing and C to T base editing.

[0023] Based on this, from one point of view, the present invention relates to a composition that can edit adenine of the plant organelle DNA into guanine and cytosine into thymine at the same time. The composition is a base editing composition for plant organelle DNA, characterized in that it comprises: a DNA binding protein or a nucleic acid encoding it; cytosine deaminase or a first segment and a second segment derived from cytosine deaminase (deaminase), or a nucleic acid encoding it; and adenine deaminase or a nucleic acid encoding it.

[0024] "Editing" as used in this specification can be used interchangeably with "correction", which refers to a method of changing a nucleic acid sequence by selective deletion of a specific genomic target. These specific genomic targets include, but are not limited to, chromosomal regions, genes, promoters, open reading frames, or any nucleic acid sequence.

[0025] As used in this specification, "single base" refers to one and only one nucleotide in a nucleic acid sequence. When single base editing is used herein, it means that the base at a specific position in the nucleic acid sequence is replaced by a different base. These substitutions can be achieved by a variety of mechanisms, including substitution or modification, but not limited to this.

[0026] "Target" or "targeting site" as used in this specification refers to a pre-confirmed nucleic acid sequence of any composition and / or length. These targeting sites include but are not limited to chromosomal regions, genes, promoters, open reading frames or any nucleic acid sequence.

[0027] As used herein, "on-target" refers to a subsequence of a specific genomic target that is fully complementary to a programmable DNA binding domain and / or a single guide RNA sequence.

[0028] As used herein, "off-target" refers to a subsequence of a specific genomic target that is partially complementary to a programmable DNA binding domain and / or a single guide RNA sequence.

[0029] In the present invention, the first segment and the second segment derived from cytosine deaminase or a variant thereof are included, and the cytosine deaminase is bound to a DNA binding protein or the first segment and the second segment are respectively bound to the DNA binding protein.

[0030] The cytosine deaminase is a deaminase that converts cytosine (C) to uridine (U).

[0031] It can be the cytosine deaminase. For example, the cytosine deaminase may exist in the form of apolipoprotein B editing complex 1 (apolipoprotein B editing complex 1, APOBEC1) and activation-induced deaminase (activation-induced deaminase, AID), but most DNA deaminases only act on single-stranded DNA, so it may not be suitable for base editing by being connected to DNA binding proteins. Specifically, the cytosine deaminase can be derived from a deaminase (DddA) acting on double-stranded DNA or its orthologue (orthologue). More specifically, the cytosine deaminase can be a double-stranded DNA-specific bacterial cytosine deaminase.

[0032] The cytosine deaminase is in a segmented form, and includes a first segment and a second segment separated from each other, wherein the first segment and the second segment each have no deaminase activity.

[0033] The full-length cytosine deaminase may include the sequence of SEQ ID NO. 1 corresponding to the tox segment. The cytosine deaminase includes a first segment and a second segment, and each of the first segment and the second segment does not have deaminase activity.

[0034] (SEQ ID NO.1)

[0035] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPT PYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETL LPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGC.

[0036] In one embodiment, the first segment or the second segment of the cytosine deaminase may include a sequence from the N-terminus to one or more selected from the group consisting of G33, G44, A54, N68, G82, N98 and G108 in the sequence of SEQ ID NO. 1. The first segment or the second segment of the cytosine deaminase may include a sequence from one or more selected from the group consisting of G34, P45, G55, N69, T83, A99 and A109 to the C-terminus in the sequence of SEQ ID NO. 1.

[0037] Specifically, the cytosine deaminase may include the first segment (G1333-N) of SEQ ID NO.2 and the second segment (G1333-C) of SEQ ID NO.3, or may include the first segment (G1397-N) of SEQ ID NO.4 and the second segment (G1397-C) of SEQ ID NO.5, or may include the first segment (G1333-N) of SEQ ID NO.2 and the second segment (G1397-C) of SEQ ID NO.5, or may include the first segment (G1397-N) of SEQ ID NO.4 and the second segment (G1333-C) of SEQ ID NO.2.

[0038] Unless otherwise defined, all technical terms and scientific terms used in this specification have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention belongs. Generally, the nomenclature used in this specification is a method well known and commonly used in the art.

[0039] The base editing method for organellar DNA in plant cells only involves replacing DddA tox The invention relates to a method for editing cytosine into thymine by combining with TALE. The technical problem of the invention is to convert DddA tox By combining TALE with adenine deaminase, the bases of organelle DNA can be edited. By editing the bases of organelle DNA, which was not possible before, functional plants (such as plants with herbicide resistance) can be developed.

[0040] The inventors of the present invention prepared a base editor that converts DddA tox Cytosine deaminase and adenine deaminase are linked to DNA-binding TALE or ZFP proteins, thereby enabling simultaneous A to G base editing and C to T base editing.

[0041] Based on this, from one point of view, the present invention relates to a composition that can edit adenine of the plant organelle DNA into guanine and cytosine into thymine at the same time. The composition is a base editing composition for plant organelle DNA, characterized in that it comprises: a DNA binding protein or a nucleic acid encoding it; cytosine deaminase or a first segment and a second segment derived from cytosine deaminase (deaminase), or a nucleic acid encoding it; and adenine deaminase or a nucleic acid encoding it.

[0042] "Editing" as used in this specification can be used interchangeably with "correction", which refers to a method of changing a nucleic acid sequence by selective deletion of a specific genomic target. These specific genomic targets include, but are not limited to, chromosomal regions, genes, promoters, open reading frames, or any nucleic acid sequence.

[0043] As used in this specification, "single base" refers to one and only one nucleotide in a nucleic acid sequence. When single base editing is used herein, it means that the base at a specific position in the nucleic acid sequence is replaced by a different base. These substitutions can be achieved by a variety of mechanisms, including substitution or modification, but not limited to this.

[0044] "Target" or "targeting site" as used in this specification refers to a pre-confirmed nucleic acid sequence of any composition and / or length. These targeting sites include but are not limited to chromosomal regions, genes, promoters, open reading frames or any nucleic acid sequence.

[0045] As used herein, "on-target" refers to a subsequence of a specific genomic target that is fully complementary to a programmable DNA binding domain and / or a single guide RNA sequence.

[0046] As used herein, "off-target" refers to a subsequence of a specific genomic target that is partially complementary to a programmable DNA binding domain and / or a single guide RNA sequence.

[0047] In the present invention, the first segment and the second segment derived from cytosine deaminase or a variant thereof are included, and the cytosine deaminase is bound to a DNA binding protein or the first segment and the second segment are respectively bound to the DNA binding protein.

[0048] The cytosine deaminase is a deaminase that converts cytosine (C) to uridine (U).

[0049] It can be the cytosine deaminase. For example, the cytosine deaminase may exist in the form of apolipoprotein B editing complex 1 (apolipoprotein B editing complex 1, APOBEC1) and activation-induced deaminase (activation-induced deaminase, AID), but most DNA deaminases only act on single-stranded DNA, so it may not be suitable for base editing by being connected to DNA binding proteins. Specifically, the cytosine deaminase can be derived from a deaminase (DddA) acting on double-stranded DNA or its orthologue (orthologue). More specifically, the cytosine deaminase can be a double-stranded DNA-specific bacterial cytosine deaminase.

[0050] The cytosine deaminase is in a segmented form, and includes a first segment and a second segment separated from each other, wherein the first segment and the second segment each have no deaminase activity.

[0051] The full-length cytosine deaminase may include the sequence of SEQ ID NO. 1 corresponding to the tox segment. The cytosine deaminase includes a first segment and a second segment, and each of the first segment and the second segment does not have deaminase activity.

[0052] (SEQ ID NO.2)Wild-type DddA tox G1333-N

[0053] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGG.

[0054] (SEQ ID NO.33)

[0055] GGCTCTGGTTCCTACGCCCTGGGTCCATATCAGATTAGTGCTCCCC AACTCCCCGCCTACAACGGTCAGACAGTGGGGACCTTTTACTATGTCA ACGACGCCGGGGGATTGGAATCCAAGGTTTTCTCTAGCGGTGGG.

[0056] (SEQ ID NO.3)Wild-type G1333-C

[0057] PTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVN MTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGC.

[0058] (SEQ ID NO.34)

[0059] CCAACACCTTATCCTAACTACGCTAACGCCGGGCACGTCGAGGGGCAGTCAGCTCTTTTTATGAGAGATAACGGCATTAGCGAAGGGCTTGTGTTCCATAATAATCCTGAGGGCACCTGTGGCTTCTGTGTAAATATGACCGAAACACTTCTGCCTGAGAACGCTAAAATGACTGTCGTACCACCCGAAGGCGCAATCCCAGTTAAACGGGGCGCAACCGGCGAAACCAAAGTATTCACCGGAAACAGCAATAGTCCAAAGTCCCCCACCAAGGGAGGTTGC。

[0060] (SEQ ID NO.4) Wild-type DddA tox G1397-N

[0061] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPT PYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETL LPENAKMTVVPPEG。

[0062] (SEQ ID NO.35)

[0063] GGTAGCTACGCACTTGGTCCTTACCAGATTAGCGCACCCCAACTCCCCGCCTATAATGGTCAAACCGTCGGGACCTTTTACTACGTAAACGATGCTGGTGGGCTGGAATCCAAAGTATTCTCCTCAGGGGGCCCTACACCCTACCCCAACTACGCCAATGCTGGTCATGTAGAAGGGCAGTCAGCACTGTTTATGCGCGATAATGGTATAAGCGAGGGGTTGGTCTTCCATAACAACCCAGAGGGTACTTGTGGCTTCTGTGTGAATATGACTGAAACCCTTCTGCCCGAAAATGCCAAGATGACTGTCGTCCCACCTGAAGGC。

[0064] (SEQ ID NO.5)Wild-type DddA tox G1397-C

[0065] AIPVKRGATGETKVFTGNSSPKSPTKGGC.

[0066] (SEQ ID NO.36)

[0067] GCCATACCTGTGAAGCGGGGAGCAACAGGGGAGACAAAGGTGT TCACAGGCAACTCTAACAGTCCAAAGAGCCCCACCAAAGGCGGGTGT.

[0068] The above-mentioned G1333N, G1333C, G1397N and G1397C can be used as split deaminase by combination. Specifically, they can be used 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.

[0069] For example, the DNA binding protein can be a zinc finger protein, a transcription activator-like effector (TALE) array, or a combination thereof.

[0070] The zinc finger motif of the zinc finger protein has a DNA binding domain, and the c-terminal part of the zinc finger specifically recognizes a DNA sequence. The DNA binding protein including 3 to 6 zinc finger motifs recognizes a DNA sequence.

[0071] In one embodiment, the first segment of cytosine deaminase and the second segment of cytosine deaminase can be bound to the N-terminus or the C-terminus of the zinc finger protein, respectively.

[0072] The C-terminus of the zinc finger protein (ZF-Left) can bind to the N-terminus of the first segment of cytosine deaminase, and the C-terminus of the zinc finger protein (ZF-Right) can bind to the N-terminus of the second segment of cytosine deaminase (CC configuration);

[0073] The N-terminus of the zinc finger protein (ZF-Left) can bind to the C-terminus of the first segment of cytosine deaminase, and the C-terminus of the zinc finger protein (ZF-Right) can bind to the N-terminus of the second segment of cytosine deaminase (NC configuration);

[0074] The C-terminus of the zinc finger protein (ZF-Left) can bind to the N-terminus of the first segment of cytosine deaminase, and the N-terminus of the zinc finger protein (ZF-Right) can bind to the C-terminus of the second segment of cytosine deaminase (CN configuration); or

[0075] The N-terminus of the zinc finger protein (ZF-Left) can bind to the C-terminus of the first segment of CDase, and the N-terminus of the zinc finger protein (ZF-Right) can bind to the C-terminus of the second segment of CDase (NN configuration).

[0076] The ZF-Left may include the following sequence of SEQ ID NO.6:

[0077] [SEQ ID NO.6]

[0078] GIHGVPAAMAERPFQCRICMRNFSDRSNLSRHIRTHTGEKPFACDIC GRKFAISSNLNSHTKIHTGSQKPFQCRICMRNFSRSDNLARHIRTHTGEKP FACDICGRKFATSGNLTRHTKIHLR.

[0079] The ZF-right (Right) may include the following sequence of SEQ ID NO.7:

[0080] [SEQ ID NO.7]

[0081] GIHGVPAAMAERPFQCRICMRNFSRSDNLSVHIRTHTGEKPFACDIC GRKFAQKINLQVHTKIHTGEKPFQCRICMRNFSRSDVLSEHIRTHTGEKPF ACDICGRKFAQRNHRTTHTKIHLR.

[0082] ZFs can have different sequences depending on different DNA targets. ZFs can be customized according to the DNA targeting sequence. Since ZFs recognize 3 bp of DNA, a ZF combination that recognizes 9 to 18 bp of DNA can usually be created by connecting 3 to 6 ZFs. For example, a library including modules such as GNN, TNN, CNN, or ANN can be used to create it.

[0083] Depending on the situation, the zinc finger protein can be connected to the deaminase via a linker. The linker can be a peptide linker comprising 2 to 40 amino acid residues. For example, the linker can be a linker with a length of 2aa, 5aa, 10aa, 16aa, 24aa or 32aa, etc., but is not limited thereto.

[0084] In one embodiment, the connector may include:

[0085] 2a.a Connector: GS;

[0086] 5a.a Linker: TGEKQ (SEQ ID NO. 8);

[0087] 10a.a Linker: SGAQGSTLDF (SEQ ID NO. 9);

[0088] 16a.a Linker: SGSETPGTSESATPES (SEQ ID NO. 10);

[0089] 24a.a Linker: SGTPHEVGVYTLSGTPHEVGVYTL (SEQ ID NO. 45); or

[0090] 32a.a Linker: GSGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO. 11).

[0091] In a specific embodiment of the present invention, the split deaminase and the zinc finger protein can be connected through a linker, the zinc finger protein is bound to the N-terminus of the half-deaminase comprising the first split, and the zinc finger protein is bound to the N-terminus of the half-deaminase comprising the second split. At this time, a base conversion from C to T can occur in the spacer between the left and right ZFP binding sites. When both the left and right ZFPs are connected to the half-deaminase comprising the first split and the half-deaminase comprising the second split respectively through a 24a.a linker, it is confirmed that high editing efficiency is exhibited.

[0092] The TAL effector (TALE) is composed of 33 to 34 repeated amino acid sequences and about 9 domains (repeated variant domains, RVD) are repeated. Each domain can recognize a nucleotide and can bind to a specific DNA sequence according to the 12th to 13th amino acid sequence (HD->Cytosine, NI->Adenine, NG->Thymine, NN->Guanine). The TAL effector (TALE) recognizes single-stranded DNA in the target site. The distance between the target sites can be 12 to 14 nucleotides.

[0093] The TALE domain refers to a protein domain that binds to nucleotides in a sequence-specific manner through a combination of more than one TALE-repeat sequence. At least one TALE-repeat sequence specifically includes but is not limited to 1 to 30 TALE-repeat sequences. The TALE-repeat sequence is a site that recognizes a specific nucleotide sequence in a TALE domain.

[0094] The TALE domain includes the N-terminal inclusive region of TALE and the C-terminal inclusive region of TALE as a backbone structure.

[0095]

[0096]

[0097] Based on the cleavage site, a single TALE array, or a first TALE array and a second TALE array can be bound separately according to the site to which the TALE domain binds.

[0098] The first TALE (left TALE) can bind to the first segment of the cytosine deaminase, and the second TALE (right TALE) can bind to the second segment of the cytosine deaminase. They have the structures of N'-TALE-first segment-C' and N'-TALE-second segment-C' respectively.

[0099] TALE arrays can be customized according to the targeted DNA sequence. The TALE array is composed of a repetitive arrangement of modules consisting of 33 to 35 amino acid residues, which are derived from the plant pathogen Xanthomonas. One module recognizes an A, C, G and T base and binds to DNA. The base specificity of each module is determined by the 12th and 13th amino acid residues, which are called repeat variable di-residues (RVD). For example, the module with RVD NN recognizes G, NI recognizes A, HD recognizes C, and NG recognizes T. The TALE array consists of a minimum of 14 to a maximum of more than 18 modules, which can be designed to recognize targeted DNA sequences of 15bp to 20bp.

[0100] The Cas protein may include a variant form. It may refer to a variant that loses the endonuclease activity of cutting double-stranded DNA, for example, it may be a variant targeted nuclease that loses the endonuclease activity and has nickase activity and a variant that loses both the endonuclease activity and the nickase activity.

[0101] When it has nickase activity, a nick can be introduced into the chain or the opposite chain (e.g., the opposite chain of the chain where the base conversion occurs) simultaneously with the base conversion (e.g., cytosine is converted to uracil) based on the cytosine deaminase or in sequence regardless of the order (e.g., on the opposite chain of the chain where the PAM is located, along the 5' end direction of the PAM sequence, a nick is introduced in the position corresponding to between the third nucleotide and the fourth nucleotide). Such variation (e.g., amino acid substitution, etc.) can occur in the catalytically active domain (e.g., in the case of Cas9, the RuvC catalytic domain). For Cas9 derived from Streptococcus pyogenes, the mutation may include a mutation in which one or more of the group consisting of a catalytic aspartate residue (catalytic aspartate residue; aspartic acid at position 10 (D10), etc.), glutamic acid at position 762 (E762), histidine at position 840 (H840), asparagine at position 854 (N854), asparagine at position 863 (N863), and aspartic acid at position 986 (D986) are replaced by any other amino acid. In this case, any other amino acid substituted may be alanine, but is not limited thereto.

[0102] Depending on the situation, one or more (for example, three) of the aspartic acid (D1135) at position 1135, the arginine (R1335) at position 1335, and the threonine (T1337) at position 1337 of the Cas9 protein derived from Streptococcus pyogenes are replaced by other amino acids, thereby achieving mutation in a manner that recognizes NGA (N is any base selected from A, T, G and C) that is different from the PAM sequence (NGG) of the wild-type Cas9.

[0103] For example, in the amino acid sequence of the Cas9 protein derived from Streptococcus pyogenes, it can be:

[0104] (1) D10, H840 or D10+H840;

[0105] (2) D1135, R1335, T1337 or D1135+R1335+T1337; or

[0106] (3) Amino acid substitutions occur in the residues of (1) and (2).

[0107] The "other amino acids" refer to the following amino acids except the amino acids in the wild-type protein at the original mutation site: alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, aspartic acid, cysteine, glutamine, glycine, serine, threonine, tyrosine, aspartic acid, glutamic acid, arginine, histidine, lysine, all known variants of the amino acids. In one example, the "other amino acids" may refer to alanine, valine, glutamine or arginine.

[0108] Depending on the circumstances, guide RNA may also be included. For example, the guide RNA may be selected from one or more of the group consisting of CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA) and single-stranded guide RNA (sgRNA), specifically, a double-stranded crRNA:tracrRNA complex in which crRNA and tracrRNA are bound to each other, or a single-stranded guide RNA (sgRNA) in which crRNA or a portion thereof is connected to tracrRNA or a portion thereof via an oligonucleotide linker.

[0109] The present invention includes adenine deaminase. For example, the adenine deaminase can be selected from the group consisting of apolipoprotein B editing complex 1 (APOBEC1), activation-induced deaminase (AID) and tadA (tRNA-specific adenosine deaminase), specifically, it can be tRNA-specific adenosine deaminase (tRNA-specific adenosine deaminase, tadA). For example, the adenine deaminase is a variant of Escherichia coli TadA, and can be deoxy-adeninedeaminase.

[0110] In a specific embodiment of the present invention, deoxy-adenine deaminase is used as a variant of Escherichia coli TadA, specifically, TadA8e or ABE 8.0.

[0111] The cytosine deaminase is contained in a split form, and the DNA binding protein is a zinc finger protein. In a structure in which the N-terminus of the zinc finger protein (ZF-Left) is bound to the C-terminus of the first split body of the 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 the 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 the cytosine deaminase, the N-terminus of the zinc finger protein (ZF-Right), and the N-terminus or C-terminus of the second split body of the cytosine deaminase.

[0112] The C-terminus of the zinc finger protein (ZF-Left) binds to the N-terminus of the first segment of cytosine deaminase and the C-terminus of the zinc finger protein (ZF-Right) binds to the N-terminus of the second segment of cytosine deaminase (CC configuration); the C-terminus of the zinc finger protein (ZF-Left) binds to the N-terminus of the first segment of cytosine deaminase and the N-terminus of the zinc finger protein (ZF-Right) binds to the C-terminus of the second segment of cytosine deaminase (CN configuration) ); or when the N-terminus of the zinc finger protein (ZF-Left) is bound to the C-terminus of the first segment of cytosine deaminase and the N-terminus of the zinc finger protein (ZF-Right) is bound to the C-terminus of the second segment of cytosine deaminase (NN 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 segment of cytosine deaminase, the N-terminus of the zinc finger protein (ZF-Right), and the N-terminus or C-terminus of the second segment of cytosine deaminase.

[0113] When the cytosine deaminase is included in a segmented form and the DNA binding protein is a TALE, the first TALE can bind to the first segment of the cytosine deaminase, and the second TALE can bind to the second segment of the cytosine deaminase, and have the structures of N'-TALE-first segment DDDA-C' and N'-TALE-second segment DDDA-C', respectively. Adenine deaminase can bind to the N-terminus or C-terminus of the first segment of the cytosine deaminase or the N-terminus or C-terminus of the second segment of the cytosine deaminase.

[0114] The present invention is a composition, which is a composition for A-to-G base editing of plant organelle DNA (without uracil DNA-glycosylase inhibitor (UGI)) comprising 1) a DNA binding protein, 2) a split double-stranded DNA-specific bacterial cytosine deaminase, and 3) a deoxyadenine deaminase derived from TadA of Escherichia coli (E. coli), 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 DddA derived from Burkholderia cenocepacia. tox .

[0115] The cytosine deaminase is contained in a split form, and the DNA binding protein is a zinc finger protein. In a structure in which the N-terminus of the zinc finger protein (ZF-Left) is bound to the C-terminus of the first split body of the 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 the 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 the cytosine deaminase, the N-terminus of the zinc finger protein (ZF-Right), and the N-terminus or C-terminus of the second split body of the cytosine deaminase.

[0116] The C-terminus of the zinc finger protein (ZF-Left) binds to the N-terminus of the first segment of cytosine deaminase and the C-terminus of the zinc finger protein (ZF-Right) binds to the N-terminus of the second segment of cytosine deaminase (CC configuration); the C-terminus of the zinc finger protein (ZF-Left) binds to the N-terminus of the first segment of cytosine deaminase and the N-terminus of the zinc finger protein (ZF-Right) binds to the C-terminus of the second segment of cytosine deaminase (CN configuration) ); or when the N-terminus of the zinc finger protein (ZF-Left) is bound to the C-terminus of the first segment of cytosine deaminase and the N-terminus of the zinc finger protein (ZF-Right) is bound to the C-terminus of the second segment of cytosine deaminase (NN 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 segment of cytosine deaminase, the N-terminus of the zinc finger protein (ZF-Right), and the N-terminus or C-terminus of the second segment of cytosine deaminase.

[0117] When the cytosine deaminase is included in a segmented form and the DNA binding protein is a TALE, the first TALE can bind to the first segment of the cytosine deaminase, and the second TALE can bind to the second segment of the cytosine deaminase, and have the structures of N'-TALE-first segment DDDA-C' and N'-TALE-second segment DDDA-C', respectively. Adenine deaminase can bind to the N-terminus or C-terminus of the first segment of the cytosine deaminase or the N-terminus or C-terminus of the second segment of the cytosine deaminase.

[0118] The present invention relates to a composition, which is a composition for editing bases from A to G in plant organelles, characterized in that the DNA binding protein is a zinc finger protein or a TALE array, and the first segment and the second segment derived from cytosine deaminase are derived from bacteria and are specific for double-stranded DNA.

[0119] The present invention relates to a composition, which is a composition for editing plant organelle A to G bases, wherein the DNA binding protein is a zinc finger protein or a TALE array, the first segment and the second segment derived from cytosine deaminase are derived from bacteria and are double-stranded DNA specific, and the N-terminus of the first segment is bound to the DNA binding protein, and the C-terminus of the adenine deaminase is bound to the DNA binding protein.

[0120] The present invention relates to a composition for editing plant organelle A to G bases, the composition comprising 1) DNA binding protein, 2) split double-stranded DNA-specific bacterial cytosine deaminase and 3) deoxyadenine deaminase derived from TadA of Escherichia coli (E. coli). The composition does not contain UGI, and the DNA binding protein is a zinc finger protein (ZFP) or a transcription activator-like effector (TALE) array; the split double-stranded DNA-specific bacterial cytosine deaminase is DddA derived from Burkholderia cenocepacia. tox .

[0121] The present invention relates to a method, which is a plant organelle A to G base editing method, characterized in that it includes: a step of treating plant cells with a DNA binding protein or a nucleic acid encoding the same; a first segment and a second segment derived from a cytosine deaminase or a nucleic acid encoding the same; and an 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 segment and the second segment derived from the cytosine deaminase are derived from bacteria and are double-stranded DNA specific.

[0122] The present invention relates to a method, which is a plant organelle A to G base editing method, comprising: a step of treating plant cells with a DNA binding protein or a nucleic acid encoding the same; a first segment and a second segment derived from a cytosine deaminase or a nucleic acid encoding the same; and an 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 segment and the second segment derived from the cytosine deaminase are derived from bacteria and are double-stranded DNA specific, and the N-terminus of the first segment is bound to the DNA binding protein, and the C-terminus of the adenine deaminase is bound to the DNA binding protein.

[0123] The present invention relates to a method, which is a method for editing plant organelle A to G bases, comprising: a step of treating plant cells with a DNA binding protein or a nucleic acid encoding the same; a first segment and a second segment derived from a cytosine deaminase or a nucleic acid encoding the same; and an adenine deaminase or a nucleic acid encoding the same, comprising 1) a DNA binding protein, 2) a segmented double-stranded DNA-specific bacterial cytosine deaminase, and 3) a deoxyadenine deaminase derived from Escherichia coli (E. coli) TadA, a composition for editing plant organelle A to G bases, which does not contain UGI, and the DNA binding protein is a zinc finger protein (ZFP) or a transcription activator-like effector (TALE) array, and the segmented double-stranded DNA-specific bacterial cytosine deaminase is DddA derived from Burkholderia cenocepacia. tox .

[0124] Previously, TALEDs that did not contain UGI only caused A-to-G editing in animal cell mitochondria, but not C-to-T editing.

[0125] The present invention may also include a chloroplast transit peptide or a mitochondrial transport protein (Mitochondrial Targeting Signal (MTS)).

[0126] For example, it is delivered to chloroplasts and mitochondria in plant cells by binding to chloroplast transport proteins (Chloroplast Transit Peptide, CTP) or mitochondrial transport signal proteins (Mitochondrial Targeting Signal, MTS). When delivered to chloroplasts and mitochondria, the remaining part except the N-terminal CTP or MTS protein is delivered to the inside of the chloroplasts and mitochondria in the form of preprotein. In the process of entering the chloroplasts and mitochondria, the transport protein part is detached and the specific part is targeted to the chloroplasts and mitochondria for base editing.

[0127] With respect to the nucleic acid, "polynucleotide", "nucleotide", "nucleotide sequence" and "oligonucleotide" are used interchangeably. Polynucleotides of any length may be included, deoxyribonucleotides or ribonucleotides or their analogs. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. A polynucleotide may contain one or more modified nucleotides, for example, methylated nucleotides and nucleotide analogs. Modification of the nucleotide structure can be performed before or after polymer assembly.

[0128] The polynucleotide may be an RNA sequence, a DNA sequence or a combination thereof (RNA-DNA combined sequence).

[0129] As a means for expressing the fusion protein, known expression vectors such as plasmid vectors, cosmid vectors, and phage vectors can be used, and the vector can be easily prepared by those skilled in the art according to any known method using DNA recombination technology.

[0130] The vector may be a plasmid vector or a viral vector, and the viral vector may specifically be an adenovirus vector, an adeno-associated virus vector, a lentivirus vector, and a retrovirus vector, but is not limited thereto.

[0131] The recombinant expression vector may include the nucleic acid in a form suitable for expressing the nucleic acid in a host cell, and this means comprising one or more regulatory elements selected based on the host cell in a manner operably linked to the nucleic acid sequence to be expressed so that the recombinant expression vector is used for expression.

[0132] In a recombinant expression vector, "operably linked" means that the target nucleotide sequence is connected to the regulatory elements in a manner that allows expression of the nucleotide sequence (for example, in an in vitro transcription / translation system or in a host cell when the vector is introduced into a host cell).

[0133] The recombinant expression vector may be in a form suitable for synthesizing messenger RNA by containing a T7 promoter, which means that it contains one or more regulatory elements that can synthesize messenger RNA by T7 polymerase, so that mRNA can be synthesized in vitro.

[0134] "Regulatory elements" may include promoters, enhancers, internal ribosome entry sites (IRES), and other expression regulatory elements (e.g., transcription termination signals, e.g., polyadenylation signals and poly-U sequences). Regulatory elements include elements that direct the induction or constitutive expression of nucleotide sequences in various types of host cells and elements that direct the expression of nucleotide sequences in specific host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters may primarily direct expression in desired target tissues, e.g., muscle, neurons, bone, skin, blood, specific organs (e.g., liver and pancreas), or specific cell types (e.g., lymphocytes). Regulatory elements may also direct expression in a transient-dependent manner (e.g., cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue or cell type specific.

[0135] Depending on the circumstances, 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, 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 RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with 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.

[0136] "Regulatory elements" may include enhancers, such as WPRE; CMV enhancer; R-U5' fragment in the LTR of HTLV-I; SV40 enhancer; and the intron sequence between rabbit β-globin exons 2 and 3, etc. It will be clear to those skilled in the art that the design of the expression vector may depend on factors such as the choice of host cells and the desired expression level. The vector is introduced into the host cell to generate fusion proteins or peptide transcripts, proteins or peptides (e.g., clustered regularly interspaced short palindromic repeats (CRISPR) transcripts, proteins, enzymes, their mutants, their fusion proteins, etc.) encoded by the nucleic acid as described in the present invention. Useful vectors include lentiviruses and adeno-associated viruses, and the types of these vectors can be selected to target specific types of cells.

[0137] The vector can be delivered into the body or into cells not only by local injection (e.g., direct injection into the lesion or target site), electroporation, lipofection, viral vector, nanoparticles, but also by protein translocation domain (PTD) fusion protein method.

[0138] The nucleic acid can be injected in the form of ribonucleic acid (eg, messenger ribonucleic acid mRNA) to perform gene base editing in cells, for example, gene base editing can be performed in animal cells or plant cells without limitation.

[0139] The nucleic acid of the present invention may be in the form of mRNA. When delivered in the form of mRNA, compared with delivery in the form of a DNA vector, there is no need for transcription into mRNA, so gene editing can be started more quickly. The possibility of achieving transient protein expression is high.

[0140] The inventors of the present invention have demonstrated that, for plant organelle gene editing, when a cytosine base editor is injected into a plant cell in the form of RNA (e.g., messenger RNA), the off-target effect is reduced compared to when it is delivered as a plasmid. That is, it has been demonstrated for the first time that, in the process of plant organelle gene editing, when a cytosine base editor is transformed into a plant cell in the form of mRNA, it has an advantage in terms of off-target effects compared to when a plasmid is used.

[0141] The mRNA can be delivered directly or via a carrier. Depending on the situation, the mRNA of the nuclease and / or cleavage factor can be chemically modified, or it can be delivered directly in the form of a synthetic self-replicative RNA.

[0142] Methods for delivering mRNA molecules to cells in vitro or in vivo, including methods for delivering mRNA to cells in vivo or in vivo, can be considered. For example, mRNA molecules can be delivered to cells by including lipids (e.g., liposomes, micelles, etc.), or nanoparticles or nanotubes, or cationic compounds (e.g., polyethyleneimine or PEI), or cationic compounds (e.g., polyethyleneimine or PEI). Depending on the circumstances, in order to deliver mRNA to cells, a bioballistic method (bolistics method) (such as a gene gun or a bioballistic particle delivery system) can be used.

[0143] For example, the carrier may include, but is not limited to, a cell penetrating peptide (CPP), a nanoparticle or a polymer.

[0144] The CPP is a short peptide that facilitates the cellular uptake of a variety of molecular cargoes, from nanoscale particles to small chemical molecules and large fragments of DNA.

[0145] As for the nanoparticles, the composition of the present invention can be delivered via polymer nanoparticles, metal nanoparticles, metal / inorganic nanoparticles or lipid nanoparticles. For example, the polymer nanoparticles can be DNA nanoclews and linear DNA nanoparticles synthesized by rolling circle amplification. mRNA is loaded on DNA nanoclews and linear DNA nanoparticles, and PEI is coated to improve the ability to escape from the endosomal structure. This complex can bind to the cell membrane and be internalized, and then escape from the endosomal structure and move to the nucleus, thereby achieving delivery.

[0146] The metal nanoparticles can be linked to gold particles and form a complex with a cationic endosomal disruptive polymer before being delivered to cells. For example, the cationic endosomal disruptive polymer can be polyethylene imine, poly(arginine), poly(lysine), poly(histidine), poly-[2-{(2-aminoethyl)amino}-ethyl-aspartamide] (pAsp(DET)), a block co-polymer of poly(ethylene glycol, PEG) and poly(arginine), a block co-polymer of PEG and poly(lysine), or a block co-polymer of PEG and poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PEG-pAsp(DET)).

[0147] With respect to the metal / inorganic nanoparticles, for example, mRNA can be encapsulated by zeoliticimidazolate framework-8 (ZIF-8).

[0148] Depending on the situation, the mRNA is negatively charged and can form nanoparticles by combining with a cationic substance, and can penetrate cells through receptor-mediated endocytosis or phagocytosis.

[0149] Cationic polymers may include: polyacrylamine (PAH); polyethyleneimine (PEI); poly(L-lysine) (PLL); poly(L-arginine) (PLA); homopolymers or copolymers of polyvinylamine; poly(vinylbenzyl-tri-C1-C4-alkylammonium salt); polymers of aliphatic or aromatic-aliphatic dihalides and aliphatic N,N,N',N'-tetra-C1-C4-alkyl-alkylenediamine; poly(vinylpyridine) or poly(vinylpyridinium salt); poly(N,N-diallyl-N,N-di-C1-C4-alkyl-ammonium halide); homopolymers or copolymers of quaternized di-C1-C4-alkyl-aminoethylacrylate or methacrylate; polyquaternary amine salt (POLYQUAD) TM ; Polyaminoamide, etc.

[0150] The cationic lipids may comprise cationic liposome preparations. The lipid bilayer of the liposomes protects the encapsulated nucleic acid from being decomposed and prevents specific neutralization based on antibodies that can bind to the nucleic acid. During endosomal maturation, the endosomal membrane and the liposomes may fuse, thereby enabling effective endosomal escape of the cationic lipid-nuclease. The cationic lipids may comprise: polyethyleneimine, polyamidoamine (PAMAM) modified dendrimers, liposomes (LIPOFECTIN) (a combination of DOTMA and DOPE), LIPOFECTASE, (For example 2000, 3000, RNAiMAX, LTX), SAINT-RED (Synvolux Therapeutics, Groningen, The Netherlands), DOPE, CYTOFECTIN (Gilead Sciences, Foster City, CA), and EuPectin (JBL, San Luis Obispo, CA). Representative cationic liposomes can be prepared from N-[1-(2,3-dioleoyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoyloxy)-propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), 3β-[N-(N',N'-dimethylaminoethyl))carbamoyl]cholesterol (DC-Chol), dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide (DMRIE); or dimethyldioctadecyl ammonium bromide (DDAB).

[0151] For lipid nanoparticles, liposomes can be used as carriers for delivery. Liposomes are spherical vesicle structures composed of a single or multi-lamellar lipid bilayer that encapsulates the internal aqueous space and a relatively impermeable outer lipophilic phospholipid bilayer. Liposome dosage forms can mainly include natural phospholipids and lipids, such as 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, phosphatidylcholine or monosialoganglioside. Depending on the situation, in order to solve the instability in plasma, cholesterol or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) can be added to the lipid membrane. The addition of cholesterol will reduce the rapid release of encapsulated bioactive compounds into plasma, or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) can increase its stability.

[0152] The composition can be delivered into plant cells by:

[0153] Bombardment using a gene gun;

[0154] Protoplast transfection, PEG-mediated;

[0155] Protoplast transfection (electroporation) based on electroporation; or

[0156] Protoplast injection, microinjection.

[0157] The polynucleotide sequence encoding the fusion protein of the present invention can be an RNA sequence, a DNA sequence or a combination thereof (RNA-DNA combined sequence).

[0158] The nucleic acid can be delivered into the plant cell by:

[0159] Transformation using Agrobacterium (e.g., Agrobacterium tumefaciens and Agrobacterium rhizogenes);

[0160] - binary vector;

[0161] -Viral vectors: 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), etc.;

[0162] Transfection using viruses;

[0163] Bombardment (Gene gun);

[0164] Protoplast transfection, PEG-mediated;

[0165] Protoplast transfection (electroporation) based on electroporation; or

[0166] Protoplast injection, microinjection.

[0167] For example, the virus may 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 netrhabdovirus (SYNV), etc.

[0168] The vector can be delivered into the body or into cells not only by local injection (e.g., direct injection into the lesion or target site), electroporation, lipofection, viral vector, nanoparticles, but also by protein translocation domain (PTD) fusion protein method.

[0169] The present invention relates to a method for base editing of plant organelle DNA, which comprises the step of treating plant cells with the composition.

[0170] The present invention relates to a plant having atrazine herbicide resistance, wherein the bases of a chloroplast psbA gene including a GTTGAAAGC sequence are edited by the method.

[0171] The present invention relates to a plant having spectinomycin resistance, wherein the base of 16s rRNA including a CGTCATCCTCA sequence is edited by the method.

[0172] The present invention relates to a plant, wherein the bases of atp6 gene are edited by the method.

[0173] The present invention relates to a plant having an albino phenotype, wherein the bases of the psaA gene including ATG are edited by the method.

[0174] By base editing phenylalanine (amino acid at position 295 of psbA) in chloroplast DNA to serine, plants with resistance to atrazine (a type of herbicide) can be developed. Not only by base editing psbA in chloroplasts, but also by base editing 16s rRNA and psaA, plants with spectinomycin resistance and albino forms can be developed.

[0175] Since base editing can be performed on the Rubisco large subunit (rbcL) gene involved in photosynthesis, it is expected that plant yields can be increased or plants with high carbon dioxide absorption efficiency can be produced by regulating photosynthesis efficiency.

[0176] The present invention relates to a plant in which the bases of a gene encoding chloroplast ribulose bisphosphate carboxylase (rubisco) are edited by the method. By performing base editing on the Rubisco Large Unit involved in photosynthesis, it is expected that the yield of the plant can be increased by regulating the photosynthesis efficiency.

[0177] The chloroplast base sequence has a high degree of homology among plants, so that not only the head lettuce (Lactuca sativa cv. Cheongchima) used in the present invention but also rice, wheat, potato, tomato and other plants can be used to develop plants having atrazine resistance.

[0178] The plasmid was delivered into lettuce protoplasts, and 7 days later, adenine base editing was confirmed to have occurred in the target atp6 gene in mitochondrial DNA.

[0179] Example

[0180] The present invention is described in more detail below by way of examples. These examples are only used to illustrate the present invention, and it is obvious to those skilled in the art that the scope of the present invention should not be construed as being limited to these examples.

[0181] Example 1.

[0182] exist Figure 1A schematic diagram of DNA cloning for producing transformed plants using Agrobacterium is shown in the figure. In order to clone the TALE pair with repeated base sequences into one vector, the RPS5A promoter-PTP-3xFlag-Left TALE-1397N and the RPS5A promoter-PTP-3xFlag-Right TALE-1397C-ABE8.0 are cloned into vectors with different positions of the Aat II and Pme I sites, respectively. After digesting the two plasmids with Aat II and Pme I, respectively, the RPS5A promoter-PTP-3xFlag-Right TALE-1397C-ABE8.0-35S terminator is ligated into the plasmid with the Left TALE, thereby cloning them all into one plasmid. The above Figure 1 Just for illustration, 1397C-ABE8.0 can be cloned on the Left TALE and 1397N on the Right TALE.

[0183] By using Agrobacterium transformation, the first-generation transformants targeting the psaA, rbcL, and rrn16S genes in the chloroplasts of Arabidopsis thaliana were obtained, and 20, 6, and 37 plants were obtained, respectively. Figure 2 The results of transformant plants targeting the psaA gene are shown in FIG, where a is the base editing efficiency and position of each plant, b is the phenotype of the plant, and c is the change in the amino acid base sequence by DNA base editing. Figure 2 From a, it can be confirmed that C-12, C-11, and C2 (marked as G2) in the base sequence markers have been edited.

[0184] Figure 3 ] The results of transformant plants targeting the rbcL gene are shown in FIG, wherein a represents the base editing efficiency and position of each plant, and b represents the change in the amino acid base sequence caused by DNA base editing.

[0185] Figure 4 The figure shows the base editing efficiency and location of the transformed plants with the rrn16S gene as the target.

[0186] Figure 5The figure shows the base editing efficiency and phenotype of the first-generation transformants targeting psaA, rbcL and rrn16S genes in the chloroplasts of Arabidopsis thaliana. a, the base editing efficiency and position of psaA#1, 2, and 3, not only base editing of adenine, but also base editing of cytosine. b shows that the phenotype of psaA#3 can show green, chimeric and pale green traits, c is the base editing efficiency, position and phenotype of rbcL#1 and 2, and d is the base editing efficiency of the first-generation transformants targeting the rrn16S gene.

[0187] Confirmable Figure 4 C-2 Figure 5 C2 and C-2 in a and d are also edited respectively.

[0188] according to Figure 6 , showing the base editing efficiency and phenotype of the second-generation transformed plants. Among them, a is the phenotype of the second-generation transformed psaA#3, b is the base editing efficiency and position, c is the phenotype of the second-generation transformed rrn16S resistant to spectinomycin, d is the base editing efficiency and position, and e is the result of confirming whether the external gene was introduced into the transformed plant by PCR, confirming that rrn16S#9-1, 3 has no external gene.

[0189] pass Figure 6 It was confirmed that chloroplast base editing would be passed to the next generation, and part b confirmed that A to G and C to T base editing would be passed to the next generation together.

[0190] This confirmed that, unlike animals, A to G base editing and C to T base editing occur simultaneously in plants.

[0191] Figure 7 Shown are the base editing positions and efficiencies of rrn16S transformed second generation plants that are resistant and sensitive to spectinomycin.

[0192] according to Figure 8 The results of off-target base mutations were confirmed by full-length chloroplast genome analysis of psaA#1 (a), psaA#3 chimeric (b), psaA#3 palegreen (c), rrn16s#1 (d), rrn16s#6 (e), and wild-type Col-0 (f). No significant off-target base mutations were observed in transformed plants compared to the wild-type.

[0193] according to Fig. 9, showing the base editing efficiency of lettuce mitochondrial gene atp6. 15ug 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, PcUBi promoter-MTS-3xFlag-right TALE-1397N-Pea3A terminator (30ug in total) were delivered to lettuce protoplasts. The adenine base editing efficiency was measured 7 days later, and the results showed that the base editing efficiency of A6 was 0.99% and that of A9 was 1.12%.

[0194] The sequences of the various components used in the examples are as follows:

[0195] CTS (or PTP: SEQ ID NO.16)

[0196] MDSQLVLSLKLNPSFTPLSPLFPFTPCSSSFSPSLRFSSCYSRRLYSPVT VYAAK.

[0197] MTS (SEQ ID NO. 17)

[0198] MFKQASRLLSRSVAAASSKSVTTRAFSTELPSTLDS.

[0199] NTD(TALE N-terminal SEQ ID NO.18)

[0200] DLRTLGYSQQQQEKPKVRSTVAQHHEALVGHGFTHAHIVALSQH PAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGEL RGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLN.

[0201] CTD (TALE C-terminus SEQ ID NO. 19)

[0202] LTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACL GGRPALDAVKKGLG。

[0203] AD (ABE8.0 or TadA8e: SEQ ID NO.20)

[0204] SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN。

[0205] Linker 1 (TALE array - Linker 1 - DddA tox )

[0206] GS。

[0207] Linker 2 (DddA tox - Linker 2 - AD)

[0208] SGSETPGTSESATPES。

[0209] DddA tox 1397N (SEQ ID NO.21)

[0210] GSGSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGG PTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTET LLPENAKMTVVPPEG。

[0211] DddA tox 1397C (SEQ ID NO.22)

[0212] GSAIPVKRGATGETKVFTGNSNSPKSPTKGGC。

[0213] psbA Left TALE repeat (SEQ ID NO.23)

[0214] LTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHG。

[0215] Right TALE repeat of psbA (SEQ ID NO.24)

[0216] LTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTP AQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHG。

[0217] rrn16S Left TALE repeat (SEQ ID NO.25)

[0218] LTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHG。

[0219] rrn16S Right TALE repeat (SEQ ID NO.26)

[0220] LTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQA LETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHG。

[0221] Left TALE repeat of psaA (SEQ ID NO.27)

[0222] LTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHG。

[0223] Right TALE repeat of psaA (SEQ ID NO.28)

[0224] LTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQ ALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHG。

[0225] rbc L Left TALE repeat (SEQ ID NO.29)

[0226] LTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHG。

[0227] rbc L Right TALE repeat (SEQ ID NO.30)

[0228] LTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQAL ETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHG。

[0229] atp6 Left TALE repeat (SEQ ID NO.31)

[0230] LTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHG。

[0231] atp6 Right TALE repeat (SEQ ID NO.32)

[0232] LTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALE TVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHG。

[0233] RPS5A promoter (SEQ ID NO.41)

[0234] CTCAACTTTTGATTCGCTATTTGCAGTGCACCTGTGGCGTTCATCACATCTTTTGTGACACTGTTTGCACTGGTCATTGCTATTACAAAGGACCTTCCTGATGTTGAAGGAGATCGAAAGTAAGTAACTGCACGCATAACCATTTTCTTTCCGCTCTTTGGCTCAATCCATTTGACAGTCAAAGACAATGTTTAACCAGCTCCGTTTGATATATTGTCTTTATGTGTTTGTTCAAGCATGTTTAGTTAATCATGCCTTTGATTGATCTTGAATAGGTTCCAAATATCAACCCTGGCAACAAAACTTGGAGTGAGAAACATTGCATTCCTCGGTTCTGGACTTCTGCTAGTAAATTATGTTTCAGCCATATCACTAGCTTTCTACATGCCTCAGGTGAATTCATCTATTTCCGTCTTAACTATTTCGGTTAATTAAAGCACGAACACCATTACTGCATGTAGAAGCTTGATAAACTATCGCCACCAATT TATTTTTGTTGCGATATTGTTACTTTCCTCAGTATGCAGCTTTGAAAAGACCAACCCTCTTATCCTTTAACAATGAACAGGTTTTTAGAGGTAGCTTGATGATTCCTGCACATGTGATCTTGGCTTCAGGCTTAATTTTCCAGGTAAAGCATTATGAGATACTCTTATATCTCTTACATACTTTTGAGATAATGCACAAGAACTTCATAACTATATGCTTTAGTTTCTGCATTTGACACTGCCAAATTCATTAATCTCTAATATCTTTGTTGTTGATCTTTGGTAGACATGGGTACTAGAAAAAGCAAACTACACCAAGGTAAAATACTTTTGTACAAACATAAACTCGTTATCACGGAACATCAATGGAGTGTATATCTAACGGAGTGTAGAAACATTTGATTATTGCAGGAAGCTATCTCAGGATATTATCGGTTTATATGGAATCTCTTCTACGCAGAGTATCTGTTATTCCCCTTCCTCTAGCTTTCAATTTCATGGTGAGGATATGCAGTTTTCTTTGTATATCATTCTTCTTCTTCTTTGTAGCTTGGAGTCAAAATCGGTTCCTTCATGTACATACATCAAGGATATGTCCTTCTGAATTTTTATATCTTGCAATAAAAATGCTTGTACCAATTGAAACACCAGCTTTTTGAGTTCTATGATCACTGACTTGGTTCTAACCAAAAAAAAAAAAATGTTTAATTTACATATCTAAAAGTAGGTTTAGGGAAACCTAAACAGTAAAATATTTGTATATTATTCGAATTTCACTCATCATAAAAACTTAAATTGCACCATAAAATTTTGTTTTACTATTAATGATGTAATTTGTGTAACTTAAGATAAAAATAATATTCCGTAAGTTAACCGGCTAAAACCACGTATAAACCAGGGAACCTGTTAAACCGGTTCTTTACTGGATAAAGAAATGAAAGCCCATGTAGACAGCTCCATTAGAGCCCAAACCCTAAATTTCTCATCTATATAAAAGGAGTGACATTAGGGTTTTTGTTCGTCCTCTTAAAGCTTCTCGTTTTCTCTGCCGTCTCTCTCATTCGCGCGACGCAAACGATCTTCAGGTGATCTTCTTTCTCCAAATCCTCTCTCATAACTCTGATTTCGTACTTGTGTATTTGAGCTCACGCTCTGTTTCTCTCACCACAGCC。

[0235] 35S terminator (SEQ ID NO.42)

[0236] CGGCCATGCTAGAGTCCGCAAAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCTATTTTTCTCCAGAATAATGTGTGAGTAGTTCCCAGATAAGGGAATTAGGGTTCTTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTGACCT。

[0237] PcUbi promoter (SEQ ID NO.43)

[0238] CTAGCAACGATTGTACAATTGCTTCTTTAAAAAAGGAAGAAAGAAAGAAAGAAAAGAATCAACATCAGCGTTAACAAACGGCCCCGTTACGGCCCAAACGGTCATATAGAGTAACGGCGTTAAGCGTTGAAAGACTCCTATCGAAATACGTAACCGCAAACGTGTCATAGTCAGATCCCCTCTTCCTTCACCGCCTCAAACACAAAAATAATCTTCTACAGCCTATATATACAACCCCCCCTTCTATCTCTCCTTTCTCACAATTCATCATCTTTCTTTCTCTACCCCCAATTTTAAGAAATCCTCTCTTCTCCTCTTCATTTTCAAGGTAAATC TCTCTCTCTCTCTCTCTCTCTGTTATTCCTTGTTTTAATTAGGTATGTATTATTGCTAGTTTGTTAATCTGCTTATCTTATGTATGCCTTATGTGAATATCTTTATCTTGTTCATCTCATCCGTTTAGAAGCTATAAATTTGTTGATTTGACTGTGTATCTACACGTGGTTATGTTTATATCTAATCAGATATGAATTTCTTCATATTGTTGCGTTTGTGTGTACCAATCCGAAATCGTTGATTTTTTTCATTTAATCGTGTAGCTAATTGTACGTATACATATGGATCTACGTATCAATTGTTCATCTGTTTGTGTTTGTATGTATACAGATCTGAAAACATCACTTCTCTCATCTGATTGTGTTGTTACATACATAGATATAGATCTGTTATATCATTTTTTTTATTAATTGTGTATATATATATGTGCATAGATCTGGATTACATGATTGTGATTATTTACATGATTTTGTTATTTACGTATGTATATATGTAGATCTGGACTTTTTGGAGTTGTTGACTTGATTGTATTTGTGTGTGTATATGTGTGTTCTGATCTTGATATGTTATGTATGTGCAGC。

[0239] Pea3A terminator (SEQ ID NO.44)

[0240] CAGGCCTCCCAGCTTTCGTCCGTATCATCGGTTTCGACAACGTTCGTCAAGTTCAATGCATCAGTTTCATTGCCCACACACCAGAATCCTACTAAGTTTGAGTATTATGGCATTGGAAAAGCTGTTTTCTTCTATCATTTGTTCTGCTTGTAATTTACTGTGTTCTTTCAGTTTTTGTTTTCGGACATCAAAATGCAAATGGATGGATAAGAGTTAATAAATGATATGGTCCTTT TGTTCATCTCAAATTATTATTATCTGTTGTTTTTACTTTAATGGGTTGAATTTAAGTAAGAAAGGAACTAACAGTGTGATATTAAGGTGCAATGTTAGACATATAAAACAGTCTTTCACCTCTCTTTGGTTATGTCTTGAATTGGTTTGTTCTTCACTTATCTGTGTAATCAAGTTTACTATGAGTCTATGATCAAGTAATTATGCAATCAAGTTAAGTACAGTATAGGCTT.

[0241] Industrial Applicability

[0242] As a base editor for plant organelle DNA, DddA was originally used tox Base editing of cytosine deamination to thymine. The present invention can edit adenine base to guanine, thereby expanding the scope of organelle DNA base editing. A to G base editing and C to T base editing can occur simultaneously.

[0243] Above, specific parts of the present invention are described in detail. It is obvious to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereto. Therefore, the actual scope of the present invention is defined by the appended claims and their equivalents.

[0244] Sequence Catalog (Free text)

[0245] See electronic attachment.

Claims

1. A composition for base editing of plant organelle DNA, characterized in that: Include: A DNA binding protein, or a nucleic acid encoding the same; Cytosine deaminase or a first segment and a second segment derived from cytosine deaminase, or nucleic acids encoding the same; and Adenine deaminase, or a nucleic acid encoding the same, The first segment and the second segment are respectively bound to the DNA binding protein, The composition edits adenine into guanine and cytosine into thymine in the plant organelle DNA.

2. The composition for base editing of plant organellar DNA according to claim 1, characterized in that Each of the first segment and the second segment has no cytosine deaminase activity.

3. The composition for base editing of plant organelle DNA according to claim 1, characterized in that The cytosine deaminase is derived from a deaminase acting on double-stranded DNA or an ortholog thereof.

4. The composition for base editing of plant organelle DNA according to claim 1, characterized in that The first segment includes the sequence of SEQ ID NO. 1 from the N-terminus to one or more sequences selected from the group consisting of G33, G44, A54, N68, G82, N98 and G108.

5. The composition for base editing of plant organelle DNA according to claim 1, characterized in that The second segment includes a sequence from one or more selected from the group consisting of G34, P45, G55, N69, T83, A99 and A109 to the C-terminus of the sequence of SEQ ID NO.

1.

6. The composition for base editing of plant organellar DNA according to claim 1, characterized in that The DNA binding protein is a ZF protein or a TALE array.

7. The composition for base editing of plant organelle DNA according to claim 1, characterized in that The DNA binding proteins are linked via a peptide linker comprising 2 to 40 amino acid residues.

8. The composition for base editing of plant organelle DNA according to claim 7, characterized in that The connector comprises: 2a.a Connector: GS; 5a.a Connector: TGEKQ; 10a.a Connector: SGAQGSTLDF; 16a.a Linker: SGSETPGTSESATPES; 24a.a Connector: SGTPHEVGVYTLSGTPHEVGVYTL; or 32a.a Connector: GGGSSGGSSSGSETPGTSESATPESSGGSSGGS.

9. The composition for base editing of plant organelle DNA according to claim 1, characterized in that The DNA binding protein is a zinc finger protein, and the first segment and the second segment are in a form of being bound to the N-terminus or the C-terminus of the zinc finger protein, respectively.

10. The composition for base editing of plant organellar DNA according to claim 1, characterized in that The DNA binding protein is a TALE array and is in the form of a single TALE array bound to one end of the segment or a first TALE array and a second TALE array bound to a first segment and a second segment.

11. The composition for base editing of plant organelle DNA according to claim 1, characterized in that The adenine deaminase binds to the N- or C-terminus of a DNA binding protein or a cytosine deaminase.

12. The composition for base editing of plant organelle DNA according to claim 1, characterized in that The adenine deaminase is a TadA-derived deoxyadenine deaminase.

13. The composition for base editing of plant organelle DNA according to claim 1, characterized in that The composition comprises a chloroplast transit peptide, or a nucleic acid encoding the same.

14. The composition for base editing of plant organelle DNA according to claim 1, characterized in that The composition comprises a mitochondrial transporter (mitochondrial targeting signal (MTS)), or a nucleic acid encoding the same.

15. The composition for base editing of plant organelle DNA according to claim 1, characterized in that The composition does not include a uracil glycosylase inhibitor.

16. The composition for base editing of plant organellar DNA according to claim 1, characterized in that The composition is delivered into plant cells by: Bombardment using a gene gun; PEG-mediated protoplast transfection; Protoplast transfection based on electroporation; or Protoplast injection based on microinjection.

17. The composition for base editing of plant organellar DNA according to claim 1, characterized in that The nucleic acid is delivered into the plant cell by: Transformation of Agrobacterium using Agrobacterium tumefaciens or Agrobacterium rhizogenes; Virus transfection; Bombardment using a gene gun; PEG-mediated protoplast transfection; Protoplast transfection based on electroporation; or Protoplast injection based on microinjection.

18. A method for base editing of plant organelle DNA, characterized in that: Comprising the step of treating plant cells with a composition according to any one of claims 1 to 17.

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