Adenine base editor in genome in plant organelle and application method of adenine base editor

Abstract

The invention provides two novel adenine base editors for rice mitochondria and chloroplast and an application field of the novel adenine base editors. On the first aspect, the invention provides a recombinant protein which is used for editing genes in plant organelles and comprises organelle positioning signal peptide, TALE protein, single-stranded DNA adenine deaminase and catalytic inactivation double-stranded DNA cytosine deaminase variants. The two editing systems provided by the invention can realize effective editing from A / T to G / C on rice mitochondrial sites, can quickly obtain rich mutants, and are beneficial to improvement of crop traits and research on organelle specific protein directed evolution and rice organelle gene functions.

Description

Technical Field

[0001] The present invention relates to an adenine base editor in a genome within a plant organelle and an application method thereof, and relates to the technical field of gene editing. Background Art

[0002] Mitochondria and chloroplasts are important semi-autonomous organelles in plants. They each have their own genomes, which encode many proteins required for photosynthesis and respiration, and work together with proteins produced by the nuclear genome to maintain the functions of organelles. However, the current progress in the functional genomics of mitochondria and chloroplasts is slow, which is not conducive to crop breeding and the study of gene function. With the development of genome editing technology, the third-generation gene editing technology represented by the CRISPR gene editing system has greatly promoted the research of functional genomics and crop breeding. CRISPR genome editing tools currently mainly target the editing of the nuclear genome, but due to the difficulty of its gRNA delivery, its application in organelle genome editing is limited.

[0003] The organelle editing vector mitoTALENs fuses TALE proteins that can target DNA sequences, mitochondrial signal peptides, and restriction endonucleases FokI, and produces fragment deletions at the target location by introducing double-strand DNA breaks (DSBs) in the plant mitochondrial genome. However, this editing tool may also cause the loss of up to thousands of bases around the cutting site and changes in chromosome structure while producing mutations. Therefore, a method for editing organelle genomes that can produce more precise changes is needed to provide new tools and research ideas for gene function research and crop breeding.

[0004] Base editing is a relatively mild genome editing method based on the CRISPR genome editing system that replaces bases at the target location. It uses deaminases to edit the target location without generating DSBs and does not cause changes in chromosome structure. It has been widely used in targeting nuclear genomes. However, most deaminases are single-strand specific and cannot target double-stranded DNA. Currently, a bacterial toxin protein DddA from Burkholderia cenocepacia has been discovered. tox , which is a cytidine deaminase that targets double-stranded DNA. toxIt is divided into two halves (DddA-G1397N and DddA-G1397C) to reduce toxic effects on cells, and is combined with a TALE sequence targeting the target DNA sequence and a uracil glycosylase inhibitor (UGI) to assemble a cytosine base editor DdCBE, which can achieve site-specific C / G to T / A mutations in mammalian cell mitochondria. sTALED, developed by fusing the cytosine deaminase DddA that can unwind some dsDNA and the single-stranded specific adenine deaminase TadA8e, can achieve base mutations from A / T to G / C at the target site in plant chloroplasts. However, at present, adenine base editors in plants can only be edited in the mitochondria and chloroplasts of the model plant Arabidopsis thaliana and rice chloroplasts, and there are no reports of adenine base editing in rice mitochondria. In addition, DddA tox Whether the split form of -E1347A can play a role in partially unwinding dsDNA and realize adenine base editing has not yet been reported. The dual-targeting vector can achieve efficient and precise editing of the target position, but the DddA in the dual-targeting vector tox -E1347A has not been reported to be able to achieve partial unwinding in plants. The present invention has achieved adenine base editing in rice chloroplasts and mitochondria and verified that DddA tox -E1347A and the effectiveness of the split form. Summary of the invention

[0005] The present invention provides an adenine base editor in a genome within a plant organelle and an application method thereof.

[0006] In a first aspect, the present invention provides a double-stranded DNA cytosine deaminase DddAtox-E1347A split form G1397N, the amino acid sequence of the double-stranded DNA cytosine deaminase DddAtox-E1347A split form G1397N is shown in SEQ ID NO:1.

[0007] The second aspect of the present invention provides a nucleic acid molecule encoding the above-mentioned double-stranded DNA cytosine deaminase DddAtox-E1347A split form G1397N.

[0008] The third aspect of the present invention provides a base editor for editing adenine bases in genes in plant organelles, comprising a double fusion protein or a single fusion protein of the above-mentioned double-stranded DNA cytosine deaminase DddAtox-E1347A split form G1397N.

[0009] Furthermore, the single fusion protein includes an organelle localization signal peptide, a TALE protein, a single-stranded DNA adenine deaminase, and a double-stranded DNA cytosine deaminase variant DddAtox-E1347A that loses deamination activity, wherein:

[0010] The TALE protein includes an N-terminal sequence, a repeating sequence that specifically recognizes the targeting sequence, and a C-terminal sequence, wherein the amino acid sequence of the N-terminal sequence is shown in SEQ ID NO: 2, and the amino acid sequence of the C-terminal sequence is shown in SEQ ID NO: 3;

[0011] The amino acid sequence of the single-stranded DNA adenine deaminase is shown in SEQ ID NO:4.

[0012] Furthermore, the double fusion protein comprises a first fusion protein and a second fusion protein, wherein:

[0013] The first fusion protein includes an organelle localization signal peptide, a TALE protein, and the double-stranded DNA cytosine deaminase DddAtox-E1347A split form G1397N; the second fusion protein includes an organelle localization signal peptide, a TALE protein, and a single-stranded DNA adenine deaminase;

[0014] The TALE protein includes an N-terminal sequence, a repeating sequence that specifically recognizes the targeting sequence, and a C-terminal sequence, wherein the amino acid sequence of the N-terminal sequence is shown in SEQ ID NO: 2, and the amino acid sequence of the C-terminal sequence is shown in SEQ ID NO: 3;

[0015] The amino acid sequence of the single-stranded DNA adenine deaminase is shown in SEQ ID NO:4.

[0016] Furthermore, the double fusion protein comprises a first fusion protein and a second fusion protein, wherein:

[0017] The first fusion protein includes an organelle localization signal peptide, a TALE protein, and the double-stranded DNA cytosine deaminase DddAtox-E1347A split form G1397N; the second fusion protein includes an organelle localization signal peptide, a TALE protein, the double-stranded DNA cytosine deaminase DddAtox-E1347A split form G1397C and a single-stranded DNA adenine deaminase;

[0018] The TALE protein includes an N-terminal sequence, a repeating sequence that specifically recognizes the targeting sequence, and a C-terminal sequence, wherein the amino acid sequence of the N-terminal sequence is shown in SEQ ID NO: 2, and the amino acid sequence of the C-terminal sequence is shown in SEQ ID NO: 3;

[0019] The amino acid sequence of the single-stranded DNA adenine deaminase is shown in SEQ ID NO:4.

[0020] Furthermore, the organelle localization signal peptide includes one of a mitochondrial localization signal peptide and a chloroplast localization signal peptide;

[0021] The mitochondrial localization signal peptide is a mitochondrial targeting sequence MTS derived from Arabidopsis thaliana, and the amino acid sequence of the mitochondrial targeting sequence MTS is shown in SEQ ID NO: 5;

[0022] The chloroplast localization signal peptide is a chloroplast transit peptide CTP derived from rice, and the amino acid sequence of the chloroplast transit peptide CTP is shown in SEQ ID NO:6.

[0023] Furthermore, the C-terminal sequence of the TALE protein is connected to the double-stranded DNA cytosine deaminase DddAtox-E1347A split form G1397N via a connecting peptide, and the amino acid sequence of the first connecting peptide is shown in SEQ ID NO: 7;

[0024] And / or, the single-stranded DNA adenine deaminase and the double-stranded DNA cytosine deaminase DddAtox-E1347A split form G1397N are connected via a second connecting peptide, and the amino acid sequence of the second connecting peptide is shown in SEQ ID NO:8.

[0025] The fourth aspect of the present invention provides a nucleic acid encoding the above-mentioned fusion protein.

[0026] The fifth aspect of the present invention provides a vector comprising the above-mentioned nucleic acid.

[0027] A sixth aspect of the present invention provides a plant expressing the above recombinant protein.

[0028] Furthermore, the plant is one or more of a dicotyledonous plant and a monocotyledonous plant.

[0029] Furthermore, the plant is rice.

[0030] A seventh aspect of the present invention provides the use of the above-mentioned recombinant protein in editing the genome of a plant organelle, wherein the editing comprises mutating adenine or thymine into guanine or cytosine.

[0031] The eighth aspect of the present invention provides a method for editing the genome of a plant organelle, so that the plant expresses the above-mentioned recombinant protein, which specifically comprises the following steps:

[0032] determining a target sequence to be edited, wherein the target sequence is located within a plant organelle genome;

[0033] According to the targeting sequence, designing a repeat sequence in the TALE protein that specifically recognizes the targeting sequence;

[0034] Connecting nucleic acid molecules encoding organelle localization signal peptide, N-terminal sequence of TALE protein, repeat sequence of specific recognition targeting sequence of TALE protein, C-terminal sequence of TALE protein, single-stranded DNA adenine deaminase, and double-stranded DNA cytosine deaminase variant DddAtox-E1347A to obtain a nucleic acid complex; inserting the nucleic acid complex into a plant expression vector to obtain a recombinant vector;

[0035] The recombinant vector is introduced into a plant for expression, thereby completing the editing of the plant organelle genome. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Edit the working diagram of the mt-dTALED system;

[0037] Figure 2 The working diagram of the cp-dTALED editing system;

[0038] Figure 3 This is a working diagram of the cp-sTALED editing system;

[0039] Figure 4 Schematic diagram of the working system of mt-sTALED editing system;

[0040] Figure 5 are the editing efficiencies of dTALED and sTALED at the rice mitochondrial ccmFn site and chloroplast psaA site, respectively;

[0041] Figure 6 The editing pattern of dTALED and sTALED at the ccmFn site is shown; the first base after the upstream target sequence is position 1, and the 16 bp interval sequence is represented as 1C to 16A;

[0042] Figure 7 The editing pattern of dTALED and sTALED at the psaA site; the first base after the upstream target sequence is position 1, and the 16bp interval sequence is represented as 1C to 16C. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] Figure 1 Edit the working diagram of the mt-dTALED system, such as Figure 1 As shown, mt-dTALED includes a double fusion protein, the first fusion protein includes, from N-terminus to C-terminus, a mitochondrial localization peptide MTS, an N-terminal sequence of a TALE protein, a repeating sequence that specifically recognizes a targeting sequence (Left TALErepeats), a C-terminal sequence of a TALE protein, and a double-stranded DNA cytosine deaminase DddA. tox (E1347A); the second fusion protein includes, from N-terminus to C-terminus, the mitochondrial localization peptide MTS, the N-terminal sequence of the TALE protein, the repeat sequence that specifically recognizes the targeting sequence (Right TALErepeats), the C-terminal sequence of the TALE protein, and the single-stranded DNA adenine deaminase TadA8e.

[0045] Figure 2 The working diagram of the cp-dTALED editing system is shown in Figure 2. Figure 2 As shown in the figure, the cp-dTALED editing system includes a double fusion protein. The first fusion protein includes, from the N-terminus to the C-terminus, a chloroplast localization peptide CTP, an N-terminal sequence of a TALE protein, a repeat sequence that specifically recognizes the targeting sequence (Left TALE repeats), a C-terminal sequence of a TALE protein, and a double-stranded DNA cytosine deaminase DddA. tox (E1347A); the second fusion protein includes, from N-terminus to C-terminus, the chloroplast localization peptide CTP, the N-terminal sequence of the TALE protein, the repeat sequence that specifically recognizes the targeting sequence (Right TALErepeats), the C-terminal sequence of the TALE protein, and the single-stranded DNA adenine deaminase TadA8e.

[0046] Figure 3 The working diagram of the cp-sTALED editing system is shown in Figure 2. Figure 3 As shown, cp-sTALED includes a double fusion protein, the first fusion protein includes a chloroplast localization peptide CTP, an N-terminal sequence of a TALE protein, a repeat sequence that specifically recognizes a targeting sequence (Left TALErepeats), a C-terminal sequence of a TALE protein, and a double-stranded DNA cytosine deaminase DddA from the N-terminus to the C-terminus. tox -E1347A split form G1397N; the second fusion protein includes chloroplast localization peptide CTP, N-terminal sequence of TALE protein, repeat sequence that specifically recognizes the targeting sequence (Right TALErepeats), C-terminal sequence of TALE protein, double-stranded DNA cytosine deaminase DddA from N-terminus to C-terminus tox-E1347A split form G1397C, single-stranded DNA adenine deaminase TadA8e.

[0047] Figure 4 The working diagram of the mt-sTALED editing system is as follows: Figure 4 As shown, mt-sTALED includes a double fusion protein, the first fusion protein includes, from N-terminus to C-terminus, a mitochondrial localization peptide MTS, an N-terminal sequence of a TALE protein, a repeat sequence that specifically recognizes the targeting sequence (Left TALErepeats), a C-terminal sequence of a TALE protein, and a double-stranded DNA cytosine deaminase DddA. tox -E1347A split form G1397N; the second fusion protein includes from N-terminus to C-terminus the mitochondrial localization peptide MTS, the N-terminal sequence of the TALE protein, the repeat sequence that specifically recognizes the targeting sequence (Right TALErepeats), the C-terminal sequence of the TALE protein, and the double-stranded DNA cytosine deaminase DddA tox -E1347A split form G1397C, single-stranded DNA adenine deaminase TadA8e.

[0048] The amino acid sequences of the various parts of the above fusion protein are shown in Table 1.

[0049] Table 1 Amino acid sequences of various parts of the above fusion protein

[0050]

[0051]

[0052] mt-sTALED backbone vector 1:

[0053]

[0054]

[0055]

[0056] mt-sTALED backbone vector 2:

[0057]

[0058]

[0059]

[0060]

[0061] mt-dTALED backbone vector 1:

[0062]

[0063]

[0064]

[0065]

[0066] mt-dTALED backbone vector 2:

[0067]

[0068]

[0069]

[0070]

[0071] The amino acid sequences for recognizing the TALE binding sequences of the ccmFn gene are "CGCCTTCTCTTCGCT" and "CCCTCCCCGTGTCCAT", respectively:

[0072]

[0073] The amino acid sequences that recognize the TALE binding sequences of the psaA gene are "TGTCCGGAAAGAGGAG" and "TCTGGTTCCGGCGAAC", respectively.

[0074]

[0075]

[0076] In the process of constructing the vector, the mitochondrial localization peptide MTS, the N-terminal sequence of the TELA protein, the C-terminal sequence of the TELA protein, the single-stranded DNA adenine deaminase TadA8e, and the double-stranded DNA cytosine deaminase DddA can be first constructed. tox -E1347A recombinant plasmid, and there are restriction sites between the N-terminal sequence of TELA protein and the C-terminal sequence of TELA protein. When the repeat sequence of the specific recognition target sequence in TELA protein is determined, double restriction enzyme cutting is used to connect to a specific position to improve the construction efficiency.

[0077] Example 1 The ccmFn gene in rice mitochondria was edited using the WE-mtABE editing system.

[0078] 1.1、Construction of mt-dTALED editing system:

[0079] The target gene on rice mitochondrial DNA (reference sequence version: NC_011033.1) was selected and the TALE sequence for recognition was designed. The TALE binding sequences of the ccmFn gene were "CGCCTTCTCTTCGCT" and "CCCTCCCCGTGTCCAT".

[0080] The RVD modules that recognize different DNA bases were assembled into the BsmBI restriction sites of the mitochondrial-localized mt-dTALED backbone vectors 1 and 2. The detailed assembly method and the various primers used can be found in the literature: Tomas C, Doyle E L, Michelle C, et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting [J]. Nucleic Acids Research, 2011, 39 (12): e82-e82. Addgene: Golden Gate TALEN and TAL Effector Kit 2.0.

[0081] The plasmid with correct sequencing was used for further experiments. The backbone vectors 1 and 2 of mt-dTALED connected with TALE recognition sequence were digested with SpeI-HF (NEB) + BstEⅡ-HF (NEB) and XmaⅠ (NEB) + KpnⅠ (NEB), respectively, and the final vector p1301-TALEN was digested with corresponding enzymes. The vector and fragments were recovered and connected with T4 DNA ligase (NEB). 5uL of the reaction solution was transformed into Escherichia coli competent DH5a (Shanghai Weidi Biotechnology Co., Ltd.) and coated with Kana resistance LB plate. After picking the resistant colony, PCR was used to detect the positive clones. The plasmids of the positive clones with correct sequencing were extracted for subsequent experiments.

[0082] 1.2、Construction of mt-sTALED editing system:

[0083] The construction steps were the same as those for mt-dTALED, and the backbone vectors used were mt-sTALED backbone vector 1 and backbone vector 2, with the same insertion sites.

[0084] 1.3. Editing the ccmFn gene in rice mitochondria using the above two editing systems:

[0085] The obtained mitochondrial gene editing vectors mt-dTALED and mt-sTALED were transformed into Agrobacterium (EHA105) by Agrobacterium-mediated transformation, and then infect rice callus. Detailed transformation steps and various culture medium formulas used are referenced in: Komari T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. [J]. Plant Journal for Cell & Molecular Biology, 2010, 6 (2): 271-282.

[0086] The genomic DNA of the transgenic rice regenerated plants was extracted by TPS method, and the vector sequence was amplified by Hieff PCR Master Mix (With Dye) (Yi Sheng) using primers SEQ ID NO: 11-12 to detect the positive transgenic plants. For transgenic rice plants that tested positive by PCR, the target fragment containing the editing site on the mitochondrial DNA was amplified, and then used for Sanger sequencing to analyze the editing situation.

[0087] The target gene of the transgenic seedlings with the mt-dTALED gene editing system at the ccmFn gene target site was amplified and sequenced. The editing efficiency and editing pattern were shown in Figure 5 , Figure 6 . Figure 5 In the experiment, mt-dTALED achieved base editing at the ccmFn site with an editing efficiency of 23.1% (6 / 26). Figure 6 In the figure, with the first base after the upstream target sequence as position 1, mt-dTALED edits multiple A / T in the TALE binding sequence and the intermediate spacer sequence, and the 5A and 7A positions of the intermediate spacer sequence (16bp) are the most efficient. It can be seen that gene editing in the target region can be achieved.

[0088] The target gene of the transgenic seedlings with the mt-sTALED gene editing system at the ccmFn gene target site was amplified and sequenced. The editing efficiency and editing pattern were shown in Figure 5 , Figure 6 . Figure 5 In the experiment, mt-sTALED achieved A-to-G base conversion at the ccmFn site with an editing efficiency of 38.7% (12 / 31). Figure 6In the figure, the first base after the upstream target sequence is position 1, and mt-sTALED achieves effective editing in the target sequence and the intermediate spacer sequence, and has a higher efficiency in the intermediate spacer sequence. It can be seen that gene editing can be achieved on the intermediate spacer sequence and the target sequence.

[0089] Example 2 The rps14 gene in rice chloroplasts was edited using the WE-cpABE editing system.

[0090] 2.1、Construction of cp-dTALED editing system:

[0091] The psaA gene was edited using the same method as in Example 1, the TALE binding sequences were both "TGTCCGGAAAGAGGAG" and "TCTGGTTCCGGCGAAC", and the spacer sequence length was 16 bp. The signal peptide of the backbone vector was the amino acid sequence of the chloroplast transit peptide CTP.

[0092] 2.2、Construction of cp-sTALED editing system:

[0093] The psaA gene was edited using the same method as in Example 1, and the TALE binding sequence was the same as in 2.1. The signal peptide of the backbone vector was the amino acid sequence of the chloroplast transit peptide CTP.

[0094] 2.3. Editing the psaA gene in rice chloroplasts using the above two editing systems:

[0095] The rice genetic transformation and positive strain identification were carried out according to the same operation procedures as in Example 1, and the target gene was amplified and sequenced by Sanger method for the positive transgenic seedlings, and the sequencing results were analyzed.

[0096] The target gene of the transgenic seedlings with the cp-dTALED gene editing system at the psaA gene target site was amplified and sequenced. The editing efficiency and editing pattern were shown in Figure 5 , Figure 7 . Figure 5 In the experiment, cp-dTALED achieved base editing at the psaA site with an editing efficiency of 25.0% (4 / 16). Figure 6 In the figure, the first base after the upstream target sequence is position 1, multiple A / Ts are edited in the TALE binding sequence and the intermediate spacer sequence, and the 8T position of the intermediate spacer sequence (16bp) has the highest efficiency. It can be seen that gene editing in the target region can be achieved.

[0097] The target gene of the transgenic seedlings with the cp-sTALED gene editing system at the psaA gene target site was amplified and sequenced. The editing efficiency and editing pattern were shown in Figure 5 , Figure 7 . Figure 5In the experiment, cp-sTALED achieved base editing at the psaA site with an editing efficiency of 26.3% (5 / 19). Figure 6 In the figure, the first base after the upstream target sequence is position 1, multiple A / Ts are edited in the TALE binding sequence and the intermediate spacer sequence, and the 8T position of the intermediate spacer sequence (16bp) has the highest efficiency. It can be seen that gene editing on the intermediate spacer sequence and the target sequence can be achieved.

[0098] In summary, the present invention provides two new adenine base editors for plant, especially rice mitochondrial and chloroplast DNA. Experiments have confirmed that this method can successfully achieve effective A / T to G / C gene editing on rice mitochondrial and chloroplast genes through two TALErepeats targeting.

[0099] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A base editor for editing adenine bases in genes in plant organelles, characterized in that: comprising a first fusion protein and a second fusion protein; The first fusion protein includes an organelle localization signal peptide and a TALE protein from the N-terminus to the C-terminus; the second fusion protein includes an organelle localization signal peptide, a TALE protein, and a single-stranded DNA adenine deaminase from the N-terminus to the C-terminus; The base editor also includes a double-stranded DNA cytosine deaminase DddAtox-E1347A, and the double-stranded DNA cytosine deaminase DddAtox-E1347A is located at the C-terminus of the TALE protein in the first fusion protein; or the split form of the double-stranded DNA cytosine deaminase DddAtox-E1347A is located at the C-terminus of the TALE protein in the first fusion protein and the second fusion protein.

2. The base editor according to claim 1, characterized in that The organelle localization signal peptide includes one of a mitochondrial localization signal peptide and a chloroplast localization signal peptide; The mitochondrial localization signal peptide is a mitochondrial targeting sequence MTS derived from Arabidopsis thaliana, and the amino acid sequence of the mitochondrial targeting sequence MTS is shown in SEQ ID NO: 5; The chloroplast localization signal peptide is a chloroplast transit peptide CTP derived from rice, and the amino acid sequence of the chloroplast transit peptide CTP is shown in SEQ ID NO:

6.

3. The base editor according to claim 1, characterized in that The TALE protein includes an N-terminal sequence, a repeating sequence that specifically recognizes a targeting sequence, and a C-terminal sequence. The amino acid sequence of the N-terminal sequence is shown in SEQ ID NO:2, and the amino acid sequence of the C-terminal sequence is shown in SEQ ID NO:

3.

4. The base editor according to claim 1, characterized in that The amino acid sequence of the single-stranded DNA adenine deaminase is shown in SEQ ID NO:

4.

5. The base editor according to claim 1, characterized in that The split forms of the double-stranded DNA cytosine deaminase DddAtox-E1347A are divided into a split form of DddAtox-E1347A G1397N with an amino acid sequence as shown in SEQ ID NO:1 and a split form of DddAtox-E1347A G1397C with an amino acid sequence as shown in SEQ ID NO:

9.

6. The base editor according to claim 5, characterized in that The C-terminal sequence of the TALE protein is connected to the double-stranded DNA cytosine deaminase DddAtox-E1347A split form G1397N via a first connecting peptide, and the amino acid sequence of the first connecting peptide is shown in SEQ ID NO: 7; And / or, the double-stranded DNA cytosine deaminase DddAtox-E1347A split form G1397C and the single-stranded DNA adenine deaminase are connected via a second connecting peptide, and the amino acid sequence of the second connecting peptide is shown in SEQ ID NO:

8.

7. A nucleic acid, characterized in that Encoding the base editor described in any one of claims 1-6.

8. A carrier, characterized in that Comprising the nucleic acid of claim 7.

9. Use of the base editor according to any one of claims 1 to 6 in editing a plant organelle genome, characterized in that: The editing involves mutating adenine or thymine to guanine or cytosine.

10. A method for editing a plant organelle genome, characterized in that: The step of causing the plant to express the base editor according to any one of claims 1 to 6 comprises the following steps: determining a target sequence to be edited, wherein the target sequence is located within a plant organelle genome; According to the targeting sequence, designing a repeat sequence in the TALE protein that specifically recognizes the targeting sequence; Constructing a nucleic acid molecule encoding the base editor according to any one of claims 1 to 6; inserting the nucleic acid molecule into a plant expression vector to obtain a recombinant vector; The recombinant vector is introduced into a plant for expression, thereby completing the editing of the plant organelle genome.

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