Adenine-to-guanine base editing method of plant organelle DNA

Abstract

The present invention relates to a composition for base editing of plant organelle DNA and, in particular, to a composition and editing method for editing adenine of plant organelle DNA into guanine.

Description

Technical Field

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

[0002] Fusion proteins linked to DNA-binding proteins and deaminases enable single-nucleotide conversion in a targeted manner to perform targeted nucleotide substitutions or base editing in the genome without generating DNA double-strand breaks (DSBs), thereby editing point mutations that cause genetic diseases or introducing desired single-nucleotide variations in prokaryotes, humans, and other eukaryotes.

[0003] Program (set) genome editing tools such as ZFN (zinc finger nuclease), TALEN (transcription activator-like effector nuclease), CRISPR (clustered regularly interspaced short palindromic repeats) system and CRISPR-associated protein 9 (Cas9) variants without nucleolytic efficiency, as well as base editing technology of nucleotide deaminase protein, have been developed for genetic research of plants and for improving crop traits by changes in base sequences. However, these tools are not suitable for editing DNA sequences in plant organelles (including mitochondria and chloroplasts), mainly because it is difficult to deliver guide RNA to organelles or co-express two compounds in organelles. Plant organelles encode many essential genes required for photosynthesis and respiration. Methods or tools for editing organelle genes are very necessary for studying the function of genes or improving crop yields and traits. For example, targeted mutations in the mitochondrial atp6 gene may lead to male sterility, which is a useful trait in reproduction, and specific point mutations in the 16SrRNA gene of the chloroplast genome may lead to antibiotic resistance.

[0004] Bacterial toxin DddA tox It is the enzymatic part of a bacterial toxin derived from Burkholderia cenocepacia and is responsible for the deamination of cytosine within double-stranded DNA. As an example of a deaminase, DddA tox is toxic in cells, and therefore, to circumvent toxicity in host cells, DddA tox Split into two inactive parts, each part is linked to a DNA-binding protein designed to bind to DNA to form a functional DdCBE pair.

[0005] In this technical context, it has been demonstrated that adenine in plant organelle DNA can be edited into guanine, thereby completing the present invention. Summary of the invention

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

[0007] Another object of the present invention is to provide a method for editing adenine into guanine in plant organelle DNA.

[0008] Another object of the present invention is to provide a herbicide-resistant plant, wherein the adenine base of the chloroplast psbA gene is edited into guanine by the above method.

[0009] Another object of the present invention is to provide a spectinomycin-resistant plant, wherein the adenine base of 16S rRNA is edited into guanine by the above method.

[0010] Still another object of the present invention is to provide a plant having an albino phenotype, wherein the adenine base of the psaA gene is edited into guanine by the above method.

[0011] In order to achieve the above-mentioned objectives, the present invention provides a composition for editing adenine bases in plant organelle DNA, comprising a DNA binding protein or a nucleic acid encoding the same, 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, wherein each of the first cleavage body and the second cleavage body is bound to the DNA binding protein.

[0012] The present invention also provides a method for editing adenine in plant organelle DNA into guanine, comprising treating plant cells with the above composition.

[0013] The present invention also provides a plant resistant to atrazine herbicide, wherein the adenine base of the chloroplast psbA gene including the GTTGAAAGC sequence is edited into guanine by the above method.

[0014] The present invention also provides a spectinomycin-resistant plant, wherein the adenine base of the 16s rRNA including the CGTCATCCTCA sequence is edited into guanine by the above method.

[0015] The present invention also provides a plant with an albino phenotype, wherein the adenine base of the psaA gene including ATG is edited into guanine by the above method. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic representation of an editor in which DddAtox and adenine deaminase are attached to a TALE;

[0017] Figure 2The location of psbA of lettuce to be base edited is shown;

[0018] Figure 3 Results demonstrating the efficiency of adenine base editing after obtaining callus tissue by culture after mRNA delivery are shown;

[0019] Figure 4 Results demonstrating adenine base editing efficiency 7 days after delivery of in vitro synthesized mRNA to lettuce protoplasts are shown;

[0020] Figure 5 shows the results of confirming the efficiency of adenine base editing after obtaining callus tissue by culture after mRNA delivery; and

[0021] Figure 6 The results of alignment of psbA DNA sequences of various plants are shown. DETAILED DESCRIPTION

[0022] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the terms used herein are well known and typical in the art.

[0023] The only way to edit the bases of plant organelle DNA is to edit cytosine into thymine by attaching DddAtox to TALE. The technical task according to the present invention aims to edit the adenine base in organelle DNA into guanine by attaching DddAtox and adenine deaminase to TALE. By base editing the adenine of organelle DNA (which is traditionally impossible), functional plants can be cultivated, such as plants with herbicide resistance.

[0024] The inventors of the present application prepared a base editor capable of editing base A by connecting DddAtox cytosine deaminase and adenine deaminase that can cause A to G conversion to TALE or ZFP proteins that can bind to DNA.

[0025] Conventional deaminase using DddAtox (DdCBE) is a cytosine deaminase that uses TALE repeats as DNA binding modules. Unlike DdCBE, which only causes C to T conversion, DdABE can cause A to G conversion, thus generating different mutation patterns.

[0026] DdABE recognizes double-stranded DNA by itself and causes deamination, and therefore does not have additional components such as RNA. For mitochondria or chloroplasts, the RNA delivery mechanism is unknown, so the CRISPR system cannot be applied. However, DdABE without such a component can target not only genomic DNA in cells, but also DNA in organelles such as mitochondria or chloroplasts, thereby causing A to G conversion in specific DNA.

[0027] DdABE is highly compatible as it can be used with either the DddAtox split or full-length DddAtox variants as desired.

[0028] As used herein, the term "editing" is used interchangeably with "proofreading" and refers to a method of altering a nucleic acid sequence by selectively deleting a specific genomic target. Specific genomic targets include, but are not limited to, chromosomal regions, genes, promoters, open reading frames, or any nucleic acid sequence.

[0029] As used herein, "single base" refers to one and only one nucleotide in a nucleic acid sequence. In the context of single base editing, this means that the base at a specific position in a nucleic acid sequence is replaced by a different base. This replacement can occur through many mechanisms, including but not limited to substitution or modification.

[0030] As used herein, the term "target" or "target site" refers to any nucleic acid sequence of predetermined composition and / or length. Target sites include, but are not limited to, chromosomal regions, genes, promoters, open reading frames, or any nucleic acid sequence.

[0031] As used herein, the term "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.

[0032] As used herein, the term "off-target" refers to a subsequence of a specific genomic target that may be partially complementary to a programmable DNA binding domain and / or a single guide RNA sequence.

[0033] The present invention includes a first split body and a second split body derived from cytosine deaminase or variants thereof, wherein each of the first split body and the second split body is bound to a DNA binding protein.

[0034] Cytosine deaminase is an enzyme that removes the amino group and converts cytosine (C) to uridine (U).

[0035] Cytosine deaminase can be used. For example, there are many types of cytosine deaminase, such as APOBEC1 (apolipoprotein B editing complex 1) and AID (activation-induced deaminase), but most DNA deaminases only act on single-stranded DNA and may not be suitable for base editing by connecting to DNA binding proteins. Specifically, cytosine deaminase can be derived from a deaminase (DddA) or its ortholog acting on double-stranded DNA. More specifically, cytosine deaminase can be a double-stranded DNA-specific bacterial cytosine deaminase.

[0036] The cytosine deaminase is provided in a split body form, the cytosine deaminase includes a first split body and a second split body, and each of the first split body and the second split body does not have a deaminase activity.

[0037] The cytosine deaminase may include a sequence of SEQ ID NO: 1 corresponding to the tox split body in the full-length cytosine deaminase. The cytosine deaminase includes a first split body and a second split body, and each of the first split body and the second split body does not have deaminase activity.

[0038] [SEQ ID NO:1]

[0039] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGC

[0040] In one embodiment, the first split body or the second split body of cytosine deaminase may include a sequence from the N-terminus to at least one of the group consisting of G33, G44, A54, N68, G82, N98, and G108 in the sequence of SEQ ID NO: 1. The first split body or the second split body of cytosine deaminase may include a sequence from at least one of the group consisting of G34, P45, G55, N69, T83, A99, and A109 to the C-terminus in the sequence of SEQ ID NO: 1.

[0041] Specifically, the cytosine deaminase can include a first split body (G1333-N) of SEQ ID NO:2 and a second split body (G1333-C) of SEQ ID NO:3, a first split body (G1397-N) of SEQ ID NO:4 and a second split body (G1397-C) of SEQ ID NO:5, a first split body (G1333-N) of SEQ ID NO:2 and a second split body (G1397-C) of SEQ ID NO:5, or a first split body (G1397-N) of SEQ ID NO:4 and a second split body (G1333-C) of SEQ ID NO:2.

[0042] (SEQ ID NO: 2) Wild-type DddAtox G1333-N

[0043] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGG

[0044] GGCTCTGGTTCCTACGCCCTGGGTCCATATCAGATTAGTGCTCCCCAACTCCCCGCCTACAACGGTCAGACAGTGGGGACCTTTTACTATGTCAACGACGCCGGGGGATTGGAATCCAAGGTTTTTCTCTAGCGGTGGG

[0045] (SEQ ID NO: 3) Wild type G1333-C

[0046] PTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGC

[0047] CCAACACCTTATCCTAACTACGCTAACGCCGGGCACGTCGAGGGGCAGTCAGCTCTTTTTATGAGAGATAACGGCATTAGCGAAGGGCTTGTGTTCCATAATAATCCTGAGGGCACCTGTGGCTTCTGTGTAAATATGACC GAAACACTTCTGCCTGAGAACGCTAAAATGACTGTCGTACCACCCGAAGGCGCAATCCCAGTTAAACGGGGCGCAACCGGCGAAACCAAAGTATTCACCGGAAACAGCAATAGTCCAAAGTCCCCACCAAGGGAGGTTGC

[0048] (SEQ ID NO: 4) Wild-type DddAtox G1397-N

[0049] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETL

[0050] LPENAKMTVVPPEG

[0051] GGTAGCTACGCACTTGGTCCTTACCAGATTAGCGCACCCCAACTCCCCGCCTATAATGGTCAAACCGTCGGGACCTTTTACTACGTAAACGATGCTGGTGGGCTGGAATCCAAAGTATTCTCCTCAGGGGGCCCTACACCCTACCCCAACTACGCCAATGCT GGTCATGTAGAAGGGCAGTCAGCACTGTTTATGCGCGATAATGGTATAAGCGAGGGGTTGGTCTTCCATAACAACCCAGAGGGTACTTGTGGCTTCTGTGTGAATATGACTGAAACCCTTCTGCCCGAAAATGCCAAGATGACTGTCGTCCCACCTGAAGGC

[0052] (SEQ ID NO:5) Wild-type DddAtox G1397-C

[0053] AIPVKRGATGETKVFTGNSNSPKSPTKGGC

[0054] GCCATACCTGTGAAGCGGGGAGCAACAGGGGAGACAAAGGTGTTCACAGGCAACTCTAACAGTCCAAAGAGCCCCACCAAAGGCGGGTGT

[0055] The combination of G1333N, G1333C, G1397N and G1397C can be used as a split body deaminase. Specifically, it can be provided 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.

[0056] The DNA binding protein can be, for example, a zinc finger protein, a TALE (transcription activator-like effector) array, or a combination thereof.

[0057] 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. A DNA binding protein comprising 3-6 zinc finger motifs recognizes a DNA sequence.

[0058] In one embodiment, each of the first cleavage form of cytosine deaminase and the second cleavage form of cytosine deaminase may be bound to the N-terminus or the C-terminus of the zinc finger protein.

[0059] The C-terminus of the zinc finger protein (ZF-left) binds to the N-terminus of the first cleavage complex of cytosine deaminase, and the C-terminus of the zinc finger protein (ZF-right) binds to the N-terminus of the second cleavage complex of cytosine deaminase (CC configuration);

[0060] The N-terminus of the zinc finger protein (ZF-left) binds to the C-terminus of the first cleavage complex of cytosine deaminase, and the C-terminus of the zinc finger protein (ZF-right) binds to the N-terminus of the second cleavage complex of cytosine deaminase (NC configuration);

[0061] The C-terminus of the zinc finger protein (ZF-left) is bound to the N-terminus of the first cleavage complex of cytosine deaminase, and the N-terminus of the zinc finger protein (ZF-right) is bound to the C-terminus of the second cleavage complex of cytosine deaminase (CN configuration); or

[0062] The N-terminus of the zinc finger protein (ZF-left) binds to the C-terminus of the first cleavage complex of CDase, and the N-terminus of the zinc finger protein (ZF-right) binds to the C-terminus of the second cleavage complex of CDase (NN configuration).

[0063] ZF-left can include the following sequence:

[0064] GIHGVPAAMAERPFQCRICMRNFSDRSNLSRHIRTHTGEKPFACDIC GRKFAISSNLNSHTKIHTGSQKPFQCRICMRNFSRSDNLARHIRTHTGEKP FACDICGRKFATSGNLTRHTKIHLR.

[0065] ZF-right can include the following sequence:

[0066] GIHGVPAAMAERPFQCRICMRNFSRSDNLSVHIRTHTGEKPFACDIC GRKFAQKINLQVHTKIHTGEKPFQCRICMRNFSRSDVLSEHIRTHTGEKPF ACDICGRKFAQRNHRTTHTKIHLR.

[0067] The sequence of ZF can be changed according to the DNA target. ZF can be customized according to the DNA target sequence. Since ZF recognizes 3bp of DNA, by connecting 3-6 ZFs, a ZF combination that recognizes 9-18bp of DNA can be constructed. For example, a library including modules such as GNN, TNN, CNN or ANN can be used for construction.

[0068] In some cases, 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. The linker can be, for example, a linker having a length of 2aa, 5aa, 10aa, 16aa, 24aa or 32aa, but is not limited thereto.

[0069] In one embodiment, the linker may include:

[0070] 2a.a Connector: GS

[0071] 5a.a Connector: TGEKQ

[0072] 10a.a Connector: SGAQGSTLDF

[0073] 16a.a Connector: SGSETPGTSESATPES

[0074] 24a.a Connector: SGTPHEVGVYTLSGTPHEVGVYTL or

[0075] 32a.a Connector: GGGSSGGSSSGSETPGTSESATPESSGGSSGGS.

[0076] In a specific embodiment according to the present invention, the split deaminase and the zinc finger protein can be connected via a linker, the zinc finger protein is bound to the N-terminus of the semi-deaminase comprising the first split body, and the zinc finger protein is bound to the N-terminus of the semi-deaminase comprising the second split body. Therefore, the conversion of C to T bases can occur in the spacer between the sites to which the left and right ZFPs are attached. It was confirmed that when the left and right ZFPs were connected to the semi-deaminase comprising the first split body and the semi-deaminase comprising the second split body via a 24a.a linker, they all showed high editing efficiency.

[0077] TAL effectors (TALEs) consist of repeat sequences, each of which is 33-34 amino acids long and has a domain of about 9 repeats (repeat variant domains (RVDs)). Each domain of the TAL effector can recognize one nucleotide and can bind to a specific DNA sequence based on the 12th-13th amino acid sequence (HD->cytosine, NI->adenine, NG->thymine, NN->guanine). TAL effectors (TALEs) recognize a single DNA chain within the target site. The distance between target sites can be 12-14 nucleotides.

[0078] The TALE domain refers to a protein domain that binds to nucleotides in a sequence-specific manner through a combination of at least one TALE repeat sequence unit. The TALE domain includes at least one TALE repeat sequence unit, particularly 1 to 30 TALE repeat sequence units, but is not limited thereto. The TALE repeat sequence is a site that recognizes a specific nucleotide sequence in the TALE domain.

[0079] The TALE domain includes a region containing the N-terminus of TALE and a region containing the C-terminus of TALE as a backbone structure.

[0080]

[0081] Depending on the binding position of the TALE domain based on the cleavage site, a single TALE array or a first TALE array and a second TALE array can be combined.

[0082] The first TALE (left TALE) can bind to the first split body of cytosine deaminase, and the second TALE (right TALE) can bind to the second split body of cytosine deaminase, thereby forming the structures of N'-TALE-first split body-C' and N'-TALE-second split body-C', respectively.

[0083] TALE arrays can be customized according to the target DNA sequence.TALE arrays are configured to repeat modules consisting of 33-35 amino acid residues.TALE is derived from the plant pathogen Xanthomonas, and each module recognizes each of A, C, G and T bases and binds to DNA.The base specificity of each module is determined by the 12th and 13th amino acid residues, which are referred to as repeated variable di-residues (RVD).For example, the module with RVD NN recognizes G, the module with RVD NI recognizes A, the module with RVD HD recognizes C, and the module with RVD NG recognizes T.TALE arrays consist of at least 14 to 18 modules and can be designed to recognize a target DNA sequence of 15-20bp.

[0084] The Cas protein may be included in a mutant form, which may mean that it is mutated to eliminate the endonuclease activity of cutting double-stranded DNA, and exemplifies at least one selected from a mutated mutant target-specific nuclease and a mutated form, wherein the mutated mutant target-specific nuclease is mutated to eliminate the endonuclease activity and has nickase activity, and the mutated form is mutated to eliminate both the endonuclease activity and the nickase activity.

[0085] When having nickase activity, regardless of the order, a nick can be introduced into the strand where the base conversion occurs or the opposite strand (e.g., the strand opposite to the strand where the base conversion occurs) simultaneously or sequentially with base conversion (e.g., conversion of cytosine to uridine) by cytosine deaminase (e.g., introducing a nick between the third nucleotide and the fourth nucleotide in the 5' end direction of the PAM sequence on the strand opposite to the strand where the PAM is located). Such mutations (e.g., amino acid substitutions, etc.) can occur in the catalytically active domain (e.g., the RuvC catalytic domain in Cas9). In addition, the Cas9 derived from Streptococcus pyogenes may include a mutation in which at least one of the catalytic aspartic acid residues (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), aspartic acid at position 986 (D986) etc. is replaced by any different amino acid. Here, the different amino acid replaced may be alanine, but is not limited thereto.

[0086] In some cases, the Streptococcus pyogenes-derived Cas9 protein can be mutated to recognize NGA (wherein N is any base selected from A, T, G, and C) different from the PAM sequence (NGG) of wild-type Cas9 by replacing one or more (e.g., all three amino acids) selected from aspartic acid at position 1135 (D1135), arginine at position 1335 (R1335), and threonine at position 1337 (T1337) with a different amino acid.

[0087] For example, in the amino acid sequence of the Cas9 protein derived from Streptococcus pyogenes, amino acid substitutions may occur in:

[0088] (1) D10, H840, or D10+H840;

[0089] (2) D1135, R1335, T1337, or D1135+R1335+T1337; or

[0090] (3) Both residues (1) and (2).

[0091] Here, "different amino acids" can be alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, asparagine, cysteine, glutamine, glycine, serine, threonine, tyrosine, aspartic acid, glutamic acid, arginine, histidine or lysine, and can refer to amino acids selected from amino acids other than the amino acids at the original mutation positions in the wild-type protein from all the known amino acid variants mentioned above. For example, "different amino acids" can be alanine, valine, glutamine or arginine.

[0092] In some cases, a guide RNA may be further included. Here, the guide RNA may be at least one selected from the group consisting of, for example, CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA) and single guide RNA (sgRNA). Specifically, the guide RNA may be a double-stranded crRNA:tracrRNA complex, in which crRNA and tracrRNA are connected to each other, or a single-stranded guide RNA (sgRNA), in which crRNA or a portion thereof and tracrRNA or a portion thereof are connected via an oligonucleotide linker.

[0093] The present invention includes adenine deaminase. The adenine deaminase can be selected from the group consisting of, for example, APOBEC1 (apolipoprotein B editing complex 1), AID (activation-induced deaminase) and tadA (tRNA-specific adenosine deaminase), and can specifically be tadA (tRNA-specific adenosine deaminase). The adenine deaminase can be, for example, a deoxyadenine deaminase that is a variant of Escherichia coli (E. coli) TadA.

[0094] In the structure in which cytosine deaminase is included in the form of split bodies; the DNA binding protein is a zinc finger protein; 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.

[0095] Even in which the C-terminus of the zinc finger protein (ZF-left) binds to the N-terminus of the first cleavage body of cytosine deaminase and the C-terminus of the zinc finger protein (ZF-right) binds to the N-terminus of the second cleavage body of cytosine deaminase (CC configuration); the C-terminus of the zinc finger protein (ZF-left) binds to the N-terminus of the first cleavage body of cytosine deaminase and the N-terminus of the zinc finger protein (ZF-right) binds to the C-terminus of the second cleavage body of cytosine deaminase (CN configuration); or the zinc finger protein In the 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 cytosine deaminase and the N-terminus of the zinc finger protein (ZF-right) is bound to the C-terminus of the second split body 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 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.

[0096] When cytosine deaminase is included in the form of a split body and the DNA binding protein is a TALE, the first TALE can bind to the first split body of the cytosine deaminase, and the second TALE can bind to the second split body of the cytosine deaminase, thereby forming the corresponding N'-TALE-first split body DDDA-C' and N'-TALE-second split body DDDA-C' structures. Adenine deaminase can bind to the N-terminus or C-terminus of the first split body of the cytosine deaminase, or to the N-terminus or C-terminus of the second split body of the cytosine deaminase.

[0097] The present invention relates to a composition for performing A to G base editing in plant organelle DNA (without UGI), comprising 1) a DNA binding protein, 2) a bacterial cytosine deaminase specific for splitting double-stranded DNA, and 3) a deoxyadenine deaminase derived from Escherichia coli TadA, wherein the DNA binding protein is a zinc finger protein (ZFP) or a transcription activator-like effector (TALE) array, and the bacterial cytosine deaminase specific for splitting double-stranded DNA is DddAtox derived from Burkholderia cepacia.

[0098] In the structure in which cytosine deaminase is included in the form of split bodies; the DNA binding protein is a zinc finger protein; 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.

[0099] Even in which the C-terminus of the zinc finger protein (ZF-left) binds to the N-terminus of the first cleavage body of cytosine deaminase and the C-terminus of the zinc finger protein (ZF-right) binds to the N-terminus of the second cleavage body of cytosine deaminase (CC configuration); the C-terminus of the zinc finger protein (ZF-left) binds to the N-terminus of the first cleavage body of cytosine deaminase and the N-terminus of the zinc finger protein (ZF-right) binds to the C-terminus of the second cleavage body of cytosine deaminase (CN configuration); or the zinc finger protein In the 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 cytosine deaminase, and the N-terminus of the zinc finger protein (ZF-right) is bound to the C-terminus of the second split body 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 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.

[0100] When cytosine deaminase is included in the form of a split body and the DNA binding protein is a TALE, the first TALE can bind to the first split body of the cytosine deaminase, and the second TALE can bind to the second split body of the cytosine deaminase, thereby forming the corresponding N'-TALE-first split body DDDA-C' and N'-TALE-second split body DDDA-C' structures. Adenine deaminase can bind to the N-terminus or C-terminus of the first split body of the cytosine deaminase, or to the N-terminus or C-terminus of the second split body of the cytosine deaminase.

[0101] The present invention relates to a composition for A to G base editing in plant organelles, wherein the DNA binding protein is a zinc finger protein or a TALE array, and the first and second split bodies derived from cytosine deaminase are of bacterial origin and are specific for double-stranded DNA.

[0102] The present invention relates to a composition for performing A to G base editing in a plant organelle, wherein the DNA binding protein is a zinc finger protein or a TALE array, the first and second cleavage bodies derived from cytosine deaminase are of bacterial origin and are specific for double-stranded DNA, the DNA binding protein is attached to the N-terminus of the first cleavage body, and the DNA binding protein is attached to the C-terminus of the adenine deaminase.

[0103] The present invention relates to a composition for A to G base editing in plant organelles, comprising 1) a DNA binding protein, 2) a bacterial cytosine deaminase specific for splitting double-stranded DNA, and 3) a deoxyadenine deaminase derived from Escherichia coli TadA, wherein the composition does not contain UGI, the DNA binding protein is a zinc finger protein (ZFP) or a TALE array, and the bacterial cytosine deaminase specific for splitting double-stranded DNA is DddAtox derived from Burkholderia cepacia.

[0104] The present invention relates to a method for performing A to G base editing in a plant organelle, comprising treating a plant cell with a DNA binding protein or a nucleic acid encoding the same, a first cleavage body and a second cleavage body derived from 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 cleavage body and the second cleavage body derived from cytosine deaminase are of bacterial origin and are specific to double-stranded DNA.

[0105] The present invention relates to a method for performing A to G base editing in a plant organelle, comprising treating a plant cell with a DNA binding protein or a nucleic acid encoding the same, a first cleavage body and a second cleavage body derived from 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 cleavage body and the second cleavage body derived from cytosine deaminase are of bacterial origin and are specific to double-stranded DNA, the DNA binding protein is attached to the N-terminus of the first cleavage body, and the DNA binding protein is attached to the C-terminus of the adenine deaminase.

[0106] The present invention relates to a method for performing A to G base editing in a plant organelle, comprising treating plant cells with a DNA binding protein or a nucleic acid encoding the same, 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, in particular with a composition for performing A to G base editing in a plant organelle, the composition comprising 1) a DNA binding protein, 2) a bacterial cytosine deaminase specific for splitting double-stranded DNA, and 3) a deoxyadenine deaminase derived from Escherichia coli TadA, wherein the composition does not contain UGI, the DNA binding protein is a zinc finger protein (ZFP) or a TALE array, and the bacterial cytosine deaminase specific for splitting double-stranded DNA is DddAtox derived from Burkholderia cepacia.

[0107] The present invention may further include a chloroplast transit peptide or a mitochondrial targeting signal (MTS).

[0108] For example, chloroplast transit peptide (CTP) or mitochondrial targeting signal (MTS) is bound and delivered to chloroplasts and mitochondria in plant cells. When delivered to chloroplasts and mitochondria, the rest of the protein except the N-terminal CTP or MTS protein is delivered to the chloroplasts and mitochondria in the form of preprotein. During the process of entering chloroplasts and mitochondria, the transporter part is detached, and the specific part can be base edited by targeting chloroplasts and mitochondria.

[0109] With respect to nucleic acids, "polynucleotide," "nucleotide," "nucleotide sequence," and "oligonucleotide" are used interchangeably. Polymeric forms of nucleotides of any length, deoxyribonucleotides or ribonucleotides or their analogs may be included. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. Polynucleotides may include one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modification of the nucleotide structure may occur before or after polymer assembly.

[0110] The polynucleotide can be an RNA sequence, a DNA sequence, or a combination thereof (RNA-DNA hybrid sequence).

[0111] As means for expressing the fusion protein, known expression vectors such as plasmid vectors, cosmid vectors, phage vectors, etc. can be used, and the vector can be easily prepared by those skilled in the art using recombinant DNA technology according to any known method.

[0112] The vector may be a plasmid vector or a viral vector, and specific examples of the viral vector may include, but are not limited to, adenovirus, adeno-associated virus, lentivirus, and retrovirus vectors.

[0113] The recombinant expression vector may contain a nucleic acid in a form suitable for expression of the nucleic acid in a host cell, which means that it contains at least one regulatory element that can be selected based on the host cell so that the recombinant expression vector is used for expression, i.e., is operably linked to the nucleic acid sequence to be expressed.

[0114] In the context of a recombinant expression vector, "operably linked" means that (for example, in an in vitro transcription / translation system, or in a host cell when the vector is introduced into a host cell) the nucleotide sequence of interest is linked to regulatory elements in a manner that permits expression of the nucleotide sequence.

[0115] The recombinant expression vector may comprise a format suitable for messenger RNA synthesis, including a T7 promoter, which means that it comprises at least one regulatory element enabling in situ mRNA synthesis, ie, messenger RNA synthesis by T7 polymerase.

[0116] "Regulatory elements" can include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences). Regulatory elements include elements that direct the induction or constitutive expression of nucleotide sequences in many types of host cells, and elements that direct the expression of nucleotide sequences only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can direct expression primarily in the desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). Regulatory elements can also direct expression in a time-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.

[0117] In some cases, the vector includes at least one pol III promoter, at least one pol II promoter, at least one pol I promoter, 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, retroviral Rous sarcoma virus (RSV) LTR promoters (optionally with RSV enhancers), cytomegalovirus (CMV) promoters (optionally with CMV enhancers) (e.g., Boshart et al. (1985) Cell 41: 521-530), SV40 promoters, dihydrofolate reductase promoters, β-actin promoters, phosphoglycerol kinase (PGK) promoters, and EF1α promoters.

[0118] "Regulatory elements" include enhancers such as WPRE; CMV enhancer; R-U5' fragment in the LTR of HTLV-I; SV40 enhancer; and intronic sequences between exons 2 and 3 of rabbit β-globin. Those skilled in the art will appreciate that the design of the expression vector can depend on factors such as the choice of the host cell to be transformed, the expression level of the desired protein, etc. The vector can be introduced into the host cell to produce a transcript, protein or peptide, including a fusion protein or peptide encoded by a nucleic acid as described herein (e.g., a clustered regularly interspaced short palindromic repeat (CRISPR) transcript, protein, enzyme, mutant thereof, fusion protein thereof, etc.). Advantageous vectors include lentiviruses and adeno-associated viruses, and these types of vectors can also be selected to target a certain type of cell.

[0119] The vector can be delivered in vivo or into cells by local injection (e.g., directly into a lesion or target site), electroporation, liposome transfection, viral vectors, nanoparticles, PTD (protein transduction domain) fusion protein methods, etc.

[0120] The nucleic acid can be injected in the form of RNA (such as messenger RNA mRNA) to enable unrestricted gene base editing in cells (e.g., in animal cells or plant cells).

[0121] The nucleic acid according to the present invention may be in the form of mRNA, and when delivered in the form of mRNA, the transcription into mRNA is not required compared to delivery in the form of a vector using DNA, so gene editing can be initiated quickly. The possibility of transient protein expression is high.

[0122] The inventors of the present application have found that when a cytosine base editor is introduced into a plant cell in the form of ribonucleic acid, such as messenger RNA, for editing plant organelle genes, off-target effects are reduced compared to delivery via a plasmid. For the first time, it has been demonstrated that when a cytosine base editor is transformed into a plant cell in the form of mRNA, it has an advantage over a plasmid in terms of off-target effects in editing plant organelle genes.

[0123] The mRNA can be delivered directly or via a carrier. In some cases, the mRNA of the nuclease and / or cleavage factor can be chemically modified or delivered directly in the form of a synthetic self-replicating RNA.

[0124] 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 of organisms such as humans or animals in vivo. For example, mRNA molecules can be delivered to cells, including lipids (e.g., liposomes, micelles, etc.), nanoparticles or nanotubes, cationic compounds (e.g., polyethyleneimine or PEI), or cationic compounds (e.g., polyethyleneimine or PEI). In some cases, gene gun method (a method using a gene gun or biolistic particle delivery system) can be performed to deliver mRNA to cells.

[0125] Examples of carriers may include, but are not limited to, cell penetrating peptides (CPPs), nanoparticles, and polymers.

[0126] CPPs are short peptides that facilitate the cellular uptake of a variety of molecular cargoes, from nanoscale particles to small chemical molecules and large DNA fragments.

[0127] About nanoparticles, the composition according to the present invention can be delivered via polymer nanoparticles, metal nanoparticles, metal / inorganic nanoparticles or lipid nanoparticles.Polymer nanoparticles can be, for example, DNA nanoparticles, yarn-like DNA nanoparticles synthesized by rolling circle amplification.DNA nanoparticles, yarn-like DNA nanoparticles, can be loaded with mRNA and coated with PEI to improve endosomal escape ability.These complexes bind to the cell membrane, are internalized, and then are transported to the nucleus by endosomal escape, where they can be delivered.

[0128] Regarding metal nanoparticles, gold particles can be linked to and complexed with cationic endosomal disrupting polymers and thus delivered to cells. Examples of cationic endosomal disrupting polymers can include polyethyleneimine, poly(arginine), poly(lysine), poly(histidine), poly-[2-{(2-aminoethyl)amino}-ethyl-asparagine] (pAsp(DET)), block copolymers of poly(ethylene glycol) (PEG) and poly(arginine), block copolymers of PEG and poly(lysine), and block copolymers of PEG and poly{N-[N-(2-aminoethyl)-2-aminoethyl]asparagine} (PEG-pAsp(DET)).

[0129] Regarding metal / inorganic nanoparticles, mRNA can be encapsulated by, for example, ZIF-8 (zeolitic imidazolate framework-8).

[0130] In some cases, mRNA can be negatively charged and coupled to cationic materials to form nanoparticles that can penetrate cells via receptor-mediated endocytosis or phagocytosis.

[0131] Examples of cationic polymers may include polyallylamine (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 alicyclic 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-alkylammonium halide); homopolymers or copolymers of quaternized di-C1-C4-alkyl-aminoethyl acrylate or methacrylate; POLYQUAD TM ; Polyaminoamide, etc.

[0132] Cationic lipids can include cationic liposome preparations. The lipid bilayer of the liposome can protect the encapsulated nucleic acid from degradation and can prevent specific neutralization by antibodies that can bind to the nucleic acid. During endosomal maturation, the endosomal membrane and the liposome fuse, allowing the cationic lipid nuclease to effectively escape from the endosome. Examples of cationic lipids can include polyethyleneimine, starburst polyamidoamine (PAMAM) dendrimers, lipofection (a combination of DOTMA and DOPE), lipofectase, (For example, 2000, 3000, RNAiMAX, 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-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP), 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2,3-dioleoyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propylammonium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide or dioctadecyldimethylammonium bromide (DDAB).

[0133] About lipid nanoparticles, liposomes can be used as carriers for delivery.Liposomes are spherical vesicle structures, consisting of a monolayer or multilayer lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable external lipophilic phospholipid bilayer.Liposome preparations can mainly contain natural phospholipids and lipids such as 1,2-distearoyl-sn-glycerol-3-phosphatidylcholine (DSPC), sphingomyelin, phosphatidylcholine or monosialic acid ganglioside.In some cases, cholesterol or 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE) can be added to the lipid membrane to address the instability in blood plasma.Adding cholesterol reduces the rapid release of encapsulated bioactive compounds into blood plasma, or 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE) is used to increase stability.

[0134] The composition can be delivered to plant cells by:

[0135] Gene gun bombardment;

[0136] PEG-mediated protoplast transfection;

[0137] Protoplast transfection by electroporation; or

[0138] Protoplast injection by microinjection.

[0139] The polynucleotide sequence encoding the fusion protein according to the present invention may be an RNA sequence, a DNA sequence or a combination thereof (RNA-DNA hybrid sequence).

[0140] Nucleic acids can be delivered to plant cells by:

[0141] Transformation with Agrobacterium , such as Agrobacterium tumefaciens, Agrobacterium rhizogenes, and the like, is used.

[0142] - binary vector,

[0143] -Viral vectors: Geminivirus, tobacco rattle virus (TRV), tomato mosaic virus (ToMV), foxtail mosaic virus (FoMV), barley yellow streak mosaic virus (BYSMV), syringa yellow web rhabdovirus (SYNV), etc.;

[0144] Virus transfection;

[0145] Gene gun bombardment;

[0146] PEG-mediated protoplast transfection;

[0147] Protoplast transfection by electroporation; or

[0148] Protoplast injection by microinjection.

[0149] Examples of viruses may include viral vectors: geminivirus, tobacco rattle virus (TRV), tomato mosaic virus (ToMV), foxtail mosaic virus (FoMV), barley yellow streak mosaic virus (BYSMV), sowthistle yellow web rhabdovirus (SYNV), and the like.

[0150] The vector can be delivered into cells by local injection (eg, directly into a lesion or target site), electroporation, lipofection, viral vectors, nanoparticles, PTD (protein transduction domain) fusion protein methods, and the like.

[0151] The present invention relates to a method for editing adenine in plant organelle DNA into guanine, comprising treating plant cells with the above composition.

[0152] The present invention relates to a plant resistant to atrazine herbicide, wherein the adenine base of the chloroplast psbA gene including the GTTGAAAGC sequence is edited into guanine by the above method.

[0153] The present invention relates to a plant resistant to spectinomycin, wherein the adenine base of 16s rRNA including the CGTCATCCTCA sequence is edited into guanine by the above method.

[0154] The present invention relates to a plant with an albino phenotype, wherein the adenine base of the psaA gene including ATG is edited into guanine by the above method.

[0155] By editing the 295th amino acid phenylalanine of psbA in chloroplast DNA to serine, plants resistant to the herbicide atrazine can be bred. By editing the 16s rRNA and psaA and adenine bases of psbA in chloroplasts, plants resistant to spectinomycin and albino plants can be bred.

[0156] The present invention relates to a plant in which the adenine base of the chloroplast RuBisCO (ribulose diphosphate carboxylase) encoding gene is edited into guanine by the above method. Since base editing of the RuBisCO large unit involved in photosynthesis is feasible, it is expected that plant yield will be increased by controlling photosynthesis efficiency.

[0157] The base sequence of chloroplasts has a high degree of homology among plants, so not only the lettuce (Lactuca sativa cv. Cheongchima) used in the present invention but also plants such as rice, wheat, potatoes, tomatoes, etc. can be bred to have resistance to atrazine.

[0158] Example

[0159] A better understanding of the present invention can be obtained by the following examples. These examples are only provided to illustrate the present invention and should not be construed as limiting the scope of the present invention, which is obvious to those skilled in the art.

[0160] Example 1.

[0161] According to a paper published in Scientific Reports 8:5179 (2018), it is reported that Escherichia coli becomes resistant to spectinomycin by replacing uracil at positions 1183 and 1189 of 16s rRNA of Escherichia coli with cytosine alone or simultaneously. When adenine is replaced with guanine at positions 1183 and 1189 of 16s rRNA present in plant chloroplasts, plants resistant to spectinomycin can be cultivated.

[0162] The sequence of each component of the adenine base editor used in the example is as follows. Figure 1 A configuration in which DddAtox and adenine deaminase are attached to a TALE is schematically shown.

[0163] CTS

[0164] MDSQLVLSLKLNPSFTPLSPLFPFTPCSSFSPSLRFSSCYSRRLYSPVTVYAAK

[0165] NTD

[0166] DLRTLGYSQQQQEKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLN

[0167] CTD

[0168] LTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG

[0169] AD

[0170] SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN

[0171] Linker 1 (TALE array-Linker 1-DddA tox )

[0172] GS

[0173] Connector 2 (DddA tox -Connector 2-AD)

[0174] SGSETPGTSESATPES

[0175] DdD tox G1347N

[0176] GSGSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG

[0177] DddA tox G1397C

[0178] GSAIPVKRGATGETKVFTGNSNSPKSPTKGGC

[0179] Left TALE repeat sequence of psbA

[0180] LTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIAS

[0181] NGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHG

[0182] Right TALE repeat sequence of psbA

[0183] LTPDQVVAIASNNGGQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLPVLCQDHGLTQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHLTPDQV IASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIAASNGGGKQ ALETVQRLPVLCQAHGLTQVVAIASNIGGKQALETVQRLLPVQDHGLTPAQVVAASNGGGQALETVQRLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRL LPVLCQDHGLTPDQVVAIASNNGGQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHG

[0184] 16s rRNA sequence

[0185] LTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLPVLCQDHGLTQVVAIASNNGGKQALETVQRLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLPVLCQAHGLTPDGQVVAIASN ALETVQRLPVLCQDHGLTPEQVVAIASNNGGKALETVQRLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVIAASNGGKQALEQTVAHQRLLPV LTPAQVVAIASNNGGQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLPVLCQDHGLTPAQKQVAIASNNGGKQALETVQRLPVLCQDHGLTPAQKQVAIA ALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLPVLCQAHGLTPAQVVAIASNGGKQALETVQRLLPVLCQAHGLTQVVAIASNNGGQVALEQGRLQ

[0186] 16s rRNA TALE

[0187] LTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPD

[0188] QVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHG

[0189] Left TALE repeat sequence of psaA

[0190] LTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHG

[0191] The right TALE repeat sequence of psaA

[0192] LTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQ ALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHG LTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQ ALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHG

[0193] The efficiency of adenine base editing was confirmed 7 days after delivery of 30 μg of in vitro synthesized mRNA to lettuce protoplasts, and the results are shown in Figure 2 a to 2c. Figure 2 a shows the binding of the left and right TALE arrays to the DNA sequence in blue. A5 and A11 marked in red are the target adenines of 16sRNA. Figure 2 b, the base editing rates for A5 were 17.27% and 6.91%, and the base editing rates for A11 were 18.17% and 7.7%. Figure 2 In c, the adenine targeted to WT is marked in blue, and the alleles in which the target was altered are allele-3, allele-4, and allele-9.

[0194] The efficiency of adenine base editing was confirmed after obtaining callus tissue by culture after mRNA delivery. Figure 3a, the base editing rates for A5 were 9.7% and 2.9%, and the base editing rates for A11 were 9.15% and 3.06%. Figure 3 b, Adenine targeted to WT is labeled in blue, and the alleles in which the target was altered are allele-1, allele-4, and allele-6.

[0195] Example 2.

[0196] Mutation of psaA in chloroplasts produces plants with an albino phenotype. In the present invention, plants with an albino phenotype can be bred by disrupting the ATG in the start codon of psaA.

[0197] The efficiency of adenine base editing was confirmed 7 days after 30 μg of in vitro synthesized mRNA was delivered to lettuce protoplasts. Figure 4 shown. Figure 4 a shows the binding of the left and right TALE arrays to the DNA sequence in blue. A5 and A6 are the target adenines of the start codon. Figure 4 b, the base editing rates for A5 were 10.67% and 7.68%, and the base editing rates for A6 were 14.66% and 12.27%. Figure 4 c, Adenine targeted to WT is labeled in blue, and the alleles in which the target was altered are allele-3, allele-5, allele-6, and allele-9.

[0198] The efficiency of adenine base editing was confirmed after obtaining callus tissue by culture after mRNA delivery. Figure 5 a, the base editing rates for A5 were 8.4% and 7.5%, and the base editing rates for A6 were 12.2% and 10.0%. Figure 5 b, Adenine targeted to WT is labeled in blue, and the alleles in which the target was altered are allele-4 and allele-8.

[0199] Example 3.

[0200] Plant chloroplast DNA has a high degree of homology. Figure 6The psbA DNA sequences of various plants are shown, where the red a indicates the target position. Herbicide-resistant plants can be cultivated by base editing the chloroplast DNA of commercially valuable corn, tomatoes, potatoes, rice, wheat, etc. using mRNA delivery, RNP and Agrobacterium methods. Sequences of maize-NC_00166.2, japonica rice-NC_001320.1, cucumber-NC_007144, common wheat-NC_002762.1, potato-NC_008096, tomato-NC_007898, Arabidopsis-AP000423, lettuce-AP007232.1 were used.

[0201] Industrial Applicability

[0202] The only base editing of plant organelle DNA is the deamination of cytosine to thymine using DddAtox. The present invention expands the scope of organelle DNA base editing by editing adenine to guanine.

[0203] Although herbicide-resistant plants have so far been bred by base editing of DNA in the nucleus, base editing of psbA in chloroplasts makes it possible to breed new herbicide-resistant plants.

[0204] By editing not only psbA in chloroplasts, but also other chloroplast genes, it may be possible to breed new plant traits.

[0205] Since base editing of large units of RuBisCO involved in photosynthesis is feasible, plant yield is expected to increase by controlling photosynthesis efficiency.

[0206] Adenine in plant mitochondrial DNA can be edited using MTS (mitochondrial targeting signal) instead of CTS.

[0207] The specific parts of the present invention have been described in detail above, and 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 essential scope of the present invention is defined by the appended claims and their equivalents.

[0208] Sequence Listing Free Text

[0209] Attach electronic file.

Claims

1. A composition for editing adenine bases in plant organelle DNA, comprising: A DNA binding protein or a nucleic acid encoding the same; A first cleavage complex and a second cleavage complex derived from cytosine deaminase or a nucleic acid encoding the same; and Adenine deaminase or a nucleic acid encoding the same, Wherein, each of the first split body and the second split body is connected to the DNA binding protein binding.

2. The composition according to claim 1, wherein Each of the first split body and the second split body lacks cytosine deaminase activity.

3. The composition according to claim 1, wherein The cytosine deaminase is derived from a deaminase acting on double-stranded DNA (DddA) or a direct homolog thereof.

4. The composition according to claim 1, wherein The first split body includes a sequence from the N-terminus to at least one selected from the group consisting of G33, G44, A54, N68, G82, N98 and G108 in the sequence of SEQ ID NO:

1.

5. The composition according to claim 1, wherein The second split body includes a sequence from at least one selected from the group consisting of G34, P45, G55, N69, T83, A99 and A109 to the C-terminus in the sequence of SED ID NO:

1.

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

7. The composition according to claim 1, wherein The DNA binding proteins are linked via a peptide linker comprising 2 to 40 amino acid residues.

8. The composition according to claim 7, wherein 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 according to claim 1, wherein The DNA binding protein is a zinc finger protein, and each of the first split body and the second split body binds to the N-terminus or the C-terminus of the zinc finger protein.

10. The composition according to claim 1, wherein The DNA binding protein is a TALE array, and a single TALE array is bound to one end of the split body or a first TALE array and a second TALE array are bound to the first split body and the second split body.

11. The composition according to claim 1, wherein The adenine deaminase binds to the N- or C-terminus of the DNA binding protein or the cytosine deaminase.

12. The composition according to claim 1, wherein The adenine deaminase is a deoxyadenine deaminase derived from TadA.

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

14. The composition of claim 1, comprising a mitochondrial targeting signal (MTS) or a nucleic acid encoding the same.

15. The composition of claim 1, which is delivered to plant cells by: Gene gun bombardment; PEG-mediated protoplast transfection; Protoplast transfection by electroporation; or Protoplast injection by microinjection.

16. The composition according to claim 1, wherein The nucleic acid is delivered to the plant cell by: transformation using Agrobacterium including Agrobacterium tumefacien or Agrobacterium rhizogenes; Virus transfection; Gene gun bombardment; PEG-mediated protoplast transfection; Protoplast transfection by electroporation; or Protoplast injection by microinjection.

17. A method for editing adenine into guanine in plant organelle DNA, comprising treating the plant cell with the composition according to any one of claims 1 to 16.

18. A plant resistant to atrazine herbicide, wherein: The adenine base of the chloroplast psbA gene including the GTTGAAAGC sequence is edited into guanine by the method according to claim 17.

19. A plant resistant to spectinomycin, wherein: The adenine base of 16s rRNA including the CGTCATCCTCA sequence is edited into guanine by the method according to claim 17.

20. A plant having an albino phenotype, wherein: The adenine base of the psaA gene including ATG is edited into guanine by the method according to claim 17.

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