Mutated ddd a-del1334 domain, split-protein pair, editing system and applications thereof

Abstract

This invention relates to the mutated DddA-del1334 domain, protein pair splitting, editing systems, and their applications. This invention is based on the DddA domain in the editor DdCBE. tox A novel series of mitochondrial gene editors, collectively referred to as △DdCBE, was constructed by deleting proline at position 45 (corresponding to position 1334 in the intact DddA protein) and its highly efficient derivative, DddA6-del1334. Experiments demonstrated that △DdCBE can comprehensively reduce side-editing effects within the targeted editing window and significantly reduce off-target editing effects on both the mitochondrial and nuclear genomes. This invention can be used to construct various animal models to simulate diseases caused by human mtDNA mutations, and is expected to provide new strategies and technical tools for gene therapy of mitochondrial-related diseases.

Description

Technical Field

[0001] This invention belongs to the field of gene editing technology, specifically involving the mutated DddA-del1334 domain, the split protein pair, the editing system, and its applications. Background Technology

[0002] Mitochondria are vital organelles responsible for energy production within cells, and their mitochondrial DNA (mtDNA) encodes 37 genes. Mutations in mtDNA are closely associated with a variety of human diseases, including mitochondrial diseases, neurodegenerative diseases, and metabolic diseases. Therefore, in situ, precise studies of mtDNA are crucial. However, due to the barrier of the mitochondrial double membrane, RNA cannot enter the mitochondrial matrix, thus the CRISPR-Cas system, which relies on RNA recognition, cannot be used for mitochondrial gene editing.

[0003] In recent years, researchers have utilized the functional domain of the bacterial toxin protein DddA from Burkholderia cenocepacia (DddA... tox A novel gene-editing tool, the cytosine base editor (DdCBE), has been developed for editing mitochondrial DNA. This editor utilizes DddA... tox Its double-stranded DNA deaminase activity, combined with the TALE protein, enables C / G to T / A mutations at specific target sites in mitochondria.

[0004] However, the original version of DdCBE exhibits significant off-target effects, including non-specific editing of nuclear and mitochondrial genes, particularly the side editing of C in non-target "tC" motifs within the editing window.

[0005] To overcome this challenge, the present invention addresses DddA tox A deletion of proline at position 45 (corresponding to proline 1334 in the intact DddA protein, named DddA-del1334) was performed, and a novel editor, △DdCBE, was developed based on this. Experiments show that △DdCBE can effectively reduce side-editing effects within the editing window and significantly reduce off-target editing effects on the whole mitochondrial genome and the nuclear genome, providing a new tool and method for high-precision editing of mitochondrial DNA and opening new avenues for gene therapy of mitochondrial-related diseases. Summary of the Invention

[0006] Given that traditional gene editing techniques cannot achieve precise editing of mitochondrial DNA, this invention successfully reduces the side-editing effect within the targeted editing window by limiting the motif preference of the editor. To this end, we propose and develop a novel mitochondrial DNA gene editing tool.

[0007] To achieve the above objectives, this invention discloses DddA tox The deletion variant and its highly efficient derivative variant. The deletion variant, named DddA-del1334, was obtained by deleting proline at position 45 (corresponding to proline at position 1334 of the complete DddA protein). Further, this deletion was introduced into the highly efficient editing variant DddA6, resulting in the derivative variant DddA6-del1334. DddA-del1334 enables efficient and precise editing of cytosine (C) in specific "gtC" motifs on double-stranded DNA; DddA6-del1334, while retaining efficient editing of the "gtC" motif, further gains the ability to edit the "ctC" motif, while also maintaining strict sequence selectivity.

[0008] Another object of the present invention is to provide an editing system that includes the above-described deletion variants and its application.

[0009] Another object of the present invention is to provide a method for achieving precise C / G to T / A editing on mitochondrial DNA using this system.

[0010] The objective of this invention can be achieved through the following technical solutions:

[0011] A mutated DddA-del1334 domain, wherein the domain is a cytosine deaminase DddA from double-stranded DNA derived from Burkholderia cenocepacia. tox The 45th proline residue of the domain is deleted, which corresponds to position 1334 on the intact DddA protein, and its amino acid sequence is shown in SEQ ID NO: 1.

[0012] Based on the DddA-del1334 domain segmentation protein pair, the mutated DddA-del1334 domain is segmented at the G1397 site of the intact DddA protein to obtain a pair of Half-DddA-del1334 segmentation proteins: G1397N-del1334 and G1397C-del1334; the segmentation proteins are used in pairs, the amino acid sequence of G1397N-del1334 is shown in SEQ ID NO: 2, and the encoding gene is shown in SEQ ID NO: 3, the amino acid sequence of G1397C-del1334 is shown in SEQ ID NO: 4, and the encoding gene is shown in SEQ ID NO: 5.

[0013] An optimized cytosine deaminase DddA6-del1334 domain is obtained by introducing a highly efficient editing variant, DddA6, carrying a multi-amino acid mutation into the DddA-del1334 domain (Mok, Beverly Y et al. CRISPR-free base editors with enhanced activity and expanded targeting scope in mitochondrial and nuclear DNA[J]. Nature biotechnology vol. 40,9 (2022):1378-1387), the amino acid sequence of which is shown in SEQ ID NO: 6.

[0014] Based on the optimized cytosine deaminase DddA6-del1334 domain segmentation protein pair, the optimized cytosine deaminase DddA6-del1334 domain is segmented based on the G1397 site of the intact DddA protein to obtain a pair of Half-DddA6-del1334 segmentation proteins: G1397N(V6)-del1334 and G1397C(V6)-del1334; the segmentation proteins are used in pairs; the amino acid sequence of G1397N(V6)-del1334 is shown in SEQ ID NO: 7, and the encoding gene is shown in SEQ ID NO: 8; the amino acid sequence of G1397C(V6)-del1334 is shown in SEQ ID NO: 9, and the encoding gene is shown in SEQ ID NO: 10.

[0015] The application of the gene encoding the DddA-del1334 domain, or the gene encoding the DddA6-del1334 domain, or the gene encoding the segmentation protein pair in constructing the ΔDdCBE of DNA C / G to T / A editing.

[0016] A cytosine editing system, △DdCBE, comprises the coding gene sequence pair of the segmented protein, the glycosylation enzyme inhibitor UGI, the transcription activator-like effector TALE, and a subcellular localization signal; the subcellular localization signal is either the mitochondrial targeting signal MTS or the nuclear localization signal NLS. △DdCBE enables efficient and precise editing of cytosine in the “gtC” characteristic sequence.

[0017] As a preferred embodiment of the present invention, the system comprises four mitochondrial localization backbone vectors and / or four nuclear localization backbone vectors, wherein the backbone vectors, from the 5' end to the 3' end, sequentially comprise: the subcellular localization signal, a TALE-N-terminal domain, a ccdB gene, a TALE-C-terminal domain, a puromycin resistance gene, and an ampicillin resistance element; and the ccdB gene has Bsa I restriction sites on both sides for assembling a TALE sequence that recognizes the target DNA sequence.

[0018] As a preferred embodiment of the present invention, the method for constructing the mitochondrial targeting backbone vector is as follows: the gene sequences encoding the paired segmented proteins are fused with the gene sequences encoding the glycosylation inhibitor UGI and then inserted into the MTS-TALE backbone vector carrying mitochondrial targeting signals.

[0019] The method for constructing the nuclear localization backbone vector is as follows: the coding gene sequences of the paired segmented proteins are fused with the coding gene sequence of UGI and then inserted into a vector carrying nuclear localization signals. of NLS-TALE skeleton carrier.

[0020] As a preferred embodiment of the present invention, the four mitochondrial localization backbone carriers are: MTS-ccdB-G1397N-del1334-UGI, MTS-ccdB-G1397C-del1334-UGI, MTS-ccdB-G1397N(V6)-del1334-UGI, and MTS-ccdB-G1397C(V6)-del1334-UGI; and the four nuclear localization backbone carriers are: NLS-ccdB-G1397N-del1334-UGI, NLS-ccdB-G1397C-del1334-UGI, NLS-ccdB-G1397N(V6)-del1334-UGI, and NLS-ccdB-G1397C(V6)-del1334-UGI.

[0021] As a further preferred embodiment of the present invention, the nucleotide sequence of the encoding gene of UGI is shown in SEQ ID NO: 13; the nucleotide sequence of the MTS-TALE backbone vector is shown in SEQ ID NO: 11; and the nucleotide sequence of the NLS-TALE backbone vector is shown in SEQ ID NO: 12.

[0022] The application of the DddA-del1334 domain, the segmentation protein pair, the DddA6-del1334 domain, the segmentation protein pair, or the ΔDdCBE in the preparation of a toolkit for C / G to T / A editing of mitochondrial DNA.

[0023] A toolkit for C / G to T / A editing of mitochondrial DNA, comprising the DddA-del1334 domain, or the DddA6-del1334 domain, or the segmentation protein, or the ΔDdCBE.

[0024] A method for C / G to T / A editing of mitochondrial DNA includes the following steps: designing a TALE recognition sequence based on the target editing site; assembling the TALE sequence into a mitochondrial localization backbone vector or a nuclear localization backbone vector using the Golden Gate cloning method to obtain a △DdCBE editor plasmid; transfecting the △DdCBE editor plasmid into target cells in a G1397N to G1397C pairing manner; and screening and verifying the editing efficiency.

[0025] The method of using the cytosine editing system △DdCBE is characterized by comprising the following steps: Step 1: Based on the DNA sequence of the target editing site, design TALE sequences that recognize the sequences flanking the site; select four backbone vectors from the cytosine editing system △DdCBE containing either the mitochondrial targeting signal MTS or the nuclear localization signal NLS, and ligate the designed TALE sequences to the backbones using the Golden Gate cloning method to assemble the △DdCBE editor vector; after transformation, single-clone PCR verification, and Sanger sequencing, obtain the correctly assembled △DdCBE plasmid; Step 2: Transfect the correctly assembled △DdCBE plasmid into target cells according to the Half-DddA pairing method, and screen successfully transfected cells using 1 μg / mL puromycin; verify the editing efficiency of the cytosine editing system △DdCBE by Sanger sequencing or high-throughput sequencing, wherein the Half-DddA pairing method is G1397N and G1397C.

[0026] All backbone vectors of this invention are adapted to a TALE assembly system. The toxin gene ccdB in the backbone vector contains Bsa I restriction sites on both sides, which, after digestion, produce different sticky ends for specifically linking TALE sequences that recognize specific DNA sequences. For specific assembly methods, please refer to the authorized patent: A TALE-based mitochondrial DNA editing system (Patent No. ZL202110688797.9).

[0027] The △DdCBE editing system described in this invention can be used for C / G to T / A editing of double-stranded DNA, and is especially suitable for editing mitochondrial DNA. In particular, it can achieve efficient and precise editing of C in the “gtC” characteristic sequence.

[0028] This invention selects two corresponding ΔDdCBE vectors for pairing according to the principle of pairing G1397N-del1334 with G1397C-del1334 and G1397N(V6)-del1334 with G1397C(V6)-del1334, transfects them into target cells, and achieves DNA editing from C / G to T / A by screening with puromycin.

[0029] The △DdCBE editing system described in this invention can be used to explore the function of mitochondrial DNA mutations and to repair mutated DNA at the cellular level.

[0030] The working principle of this invention is as follows: After transfecting cells with the △DdCBE system, double-stranded DNA cytosine deaminase deaminates the target cytosine (C) to form dU, which is then used to edit C / G to T / A during cell replication.

[0031] Compared to the TALE-DddA reported by Mok, Beverly Y et al. A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing[J]. Nature vol. 583,7817(2020): 631-637, this study is significantly different. tox The system can perform C / G to T / A editing of the "tC" sequence on mitochondrial DNA. The △DdCBE editing system developed in this invention can achieve efficient and precise C / G to T / A editing of the "gtC" and "ctC" sequences on mitochondrial DNA.

[0032] Beneficial effects of this invention:

[0033] The core innovation of this invention lies in the DddA tox A deletion of amino acid position 45 (corresponding to proline position 1334 of the intact DddA protein) yielded a new protein deletion variant, DddA-del1334. This variant was further combined with the highly efficient editing variant DddA6 to obtain the derived variant DddA6-del1334 (both collectively referred to as △DdCBE). DddA-del1334 enables high-precision editing of the C / G to T / A sequence within the specific "gtC" motif on double-stranded DNA; DddA6-del1334, while retaining efficient editing of the "gtC" motif, further gains the ability to edit the "ctC" motif, while maintaining strict sequence selectivity. Experiments show that the △DdCBE system can comprehensively reduce the side-editing effect within the editing window and significantly reduce off-target editing effects on the entire mitochondrial genome and the nuclear genome. Attached Figure Description

[0034] Figure 1 △A schematic diagram of the DdCBE editing system in operation;

[0035] a is a schematic diagram of the original DddA protein, with the 45th proline position marked, corresponding to the 1334th amino acid in the full length; b is a schematic diagram of the △DdCBE editing system in operation.

[0036] Figure 2 Different segmentation types of △DdCBE with nuclear localization signals (NLS);

[0037] The vector carries the ccdB gene element, which is located between two BsaI restriction sites. The vector also carries an ampicillin resistance element.

[0038] Figure 3 Different segmentation types of △DdCBE with mitochondrial localization signal (MTS);

[0039] The vector carries the ccdB gene element, which is located between two BsaI restriction sites. The vector also carries an ampicillin resistance element.

[0040] Figure 4 NLS-DdCBE edits the C motif in the gtC, ctC, atC, ttC, tcC, and aC motifs on the T vector.

[0041] Figure 5 △DdCBE editing of the m.4309 site in human mitochondria;

[0042] a represents the sequence information of the m.4309 site editing window, b represents the editing status within the m.4309 window and the on-target / off-target editing ratio, and c represents the off-target editing status of the m.4309 site by different combinations of ΔDdCBE.

[0043] Figure 6 △DdCBE editing of the m.9544 site in human mitochondria;

[0044] a represents the sequence information of the m.9544 site editing window, b represents the editing status within the m.9544 window and the on-target / off-target editing ratio, and c represents the off-target editing status of the m.9544 site by different combinations of ΔDdCBE.

[0045] Figure 7 △DdCBE editing of the m.15723 site in human mitochondria;

[0046] a represents the sequence information of the m.15723 site editing window, b represents the editing status within the m.15723 window and the on-target / off-target editing ratio, and c represents the off-target editing status of the m.15723 site by different combinations of ΔDdCBE.

[0047] Figure 8 △DdCBE editing of the m.15762 site in human mitochondria;

[0048] a represents the sequence information of the m.15762 site editing window, b represents the editing status within the m.15762 window and the on-target / off-target editing ratio, and c represents the off-target editing status of the m.15762 site by different combinations of ΔDdCBE.

[0049] Figure 9 △DdCBE editing of the m.14279 site in human mitochondria;

[0050] a represents the target editing sites in mitochondrial DNA (mtDNA) and the corresponding nuclear pseudogenes with similar TALE binding sites; b represents the high-throughput sequencing results of on-target editing within the target window; and cd represents the high-throughput sequencing results of off-target editing at nuclear pseudogene sites.

[0051] Figure 10 △DdCBE editing of the m.14076 site on zebrafish mitochondria;

[0052] a) Sequence alignment of human m.G13513A (located in the gtC motif) with zebrafish homologous site m.G14076A; b) Editing of ΔDdCBE within the window of zebrafish embryo at the zebrafish mimicry of human endogenous disease site m.G14076A; c) Target / Off-target editing ratio of ΔDdCBE.

[0053] Figure 11 △DdCBE editing of the m.16323 site on zebrafish mitochondria;

[0054] a) Sequence alignment of human m.G15762A (located in the ctC motif) with zebrafish homologous site m.G16323A; b) Editing of ΔDdCBE within the window of zebrafish embryo at the zebrafish mimicry of human endogenous disease site m.G16323A; c) Target / Off-target editing ratio of ΔDdCBE. Detailed Implementation

[0055] The present invention will be described in detail below with reference to embodiments, so that those skilled in the art can fully understand and implement the DNA C / G to T / A editing technology described herein.

[0056] This invention provides a novel DNA editing tool: △DdCBE. This system comprises four mitochondrial targeting vectors and four nuclear targeting vectors, enabling selective editing of the mitochondrial DNA genome. The RVD library used is compatible with the editing system (see Guo, Jiayin et al. Precision modeling of mitochondrial diseases in zebrafish via DdCBE-mediated mtDNA base editing[J]. Cell discovery, vol. 7, 1 78. 3 Sep. 2021). The specific assembly method is detailed in the authorized patent: A TALE-based mitochondrial DNA editing system (patent number ZL202110688797.9). Steps, reagents, and modules not described in detail in the embodiments are well-known to those skilled in the art and will not be elaborated upon here.

[0057] Example 1

[0058] (a) Design the assembly of the TALE sequence and △DdCBE

[0059] A T-vector containing different motifs (gtC, ctC, atC, ttC) was constructed, with identical recognition sequences at both ends of the spacer (Sun, Haifeng et al. Developing mitochondrial base editors with diverse context compatibility and high fidelity via saturated spacer library[J]. Nature communications vol. 14, 16625. 19 Oct. 2023). RVD modules targeting different DNA bases on the T-vector (Guo, Jiayin et al. Precision modeling of mitochondrial diseases in zebrafish via DdCBE-mediated mtDNA base editing[J]. Cell discovery vol. 7, 178. 3 Sep. 2021) were assembled onto the ΔDdCBE backbone vector using the Golden Gate method. The assembly method is described in Chinese Patent No. ZL202110688797.9.

[0060] The assembled product was transformed into DH5α, plated on LB agar plates containing ampicillin for screening, and single clones were picked and identified by PCR using primers (SEQ ID NO: 14, 15). Positive clones were determined based on the number of RVDs (e.g., a positive clone with 16 RVDs is approximately 1759 bp in size, and one with 15 RVDs is approximately 1657 bp). Forward and reverse sequencing were then performed using the aforementioned primers, and plasmids were extracted from positive clones with correct sequences for subsequent experiments.

[0061] (II) Transfecting cells and detecting the editing efficiency of different combinations of △DdCBE

[0062] Select 293FT cells and change the medium 2 hours in advance when the cell density reaches 70-90%. After cell counting, take 1.5 x 10⁻⁶ cells. 5 293FT cells were electroporated using the Lonza 4D-nucleofector. Four NLS versions of the Half-DddA vector were co-transfected with T vectors containing different motifs, following the pairing principle of G1397N-del1334 with G1397C-del1334 and G1397N(V6)-del1334 with G1397C(V6)-del1334. 400 ng of each of the left and right vectors and 50 ng of the T vector were transfected. Electroporation was performed using the SF-Cell Line 4D-nucleofector X-kit.

[0063] After electroporation, cells were inoculated into 12-well plates and cultured for 24 hours. Then, puromycin was added to a final concentration of 1 μg / mL for selection. After 72 hours of culture, cells were digested and collected for subsequent DNA mutation efficiency assays.

[0064] The collected cell pellet was resuspended in 30 μL of QuickExtract™ DNA Extraction Solution (Lucigen), heated at 65°C for 45 min, centrifuged by vortexing, and then heated at 98°C for 2 min to extract DNA.

[0065] The target fragment containing the editing site on the T vector was amplified using 2×Green Taq Mix (Vazyme) and then subjected to Sanger sequencing; or the editing efficiency was analyzed by high-throughput sequencing using Phanta Super-Fidelity DNA polymerase (Vazyme) and high-throughput sequencing library preparation primers. Results are as follows: Figure 4As shown, high-throughput sequencing results indicate that ΔDdCBE can significantly narrow the editing window and reduce the side-editing effect within the window. Specifically, DddA-del1334 edits only the gtC motif, while DddA6-del1334 can efficiently edit both gtC and ctC motifs, but is insensitive to other motifs.

[0066] Example 2

[0067] Mitochondrial DNA editing was performed in human 293FT cells using mitochondrial-localized MTS-ΔDdCBE, and its effect in reducing in-window side-editing and off-target effects across the entire mitochondrial genome was identified.

[0068] Select m.4309 on human mitochondrial DNA (NC_012920.1) Figure 5 a), m.9544 ( Figure 6 a), m.15723 ( Figure 7 a), m.15762 ( Figure 8 The a) sites were edited. TALE recognition sequences were designed based on the DNA sequences near these sites, and the corresponding RVD modules were assembled into four mitochondrial-localized △DdCBE backbone vectors using the Golden Gate method.

[0069] Subsequent procedures were the same as in Example 1. Four combinations at each site were transfected into 293FT cells, cells were collected, DNA was extracted, a whole mitochondrial library was constructed, and mutation efficiency was analyzed. Figures 5-8 As shown, △DdCBE can significantly narrow the editing window, reduce in-window side-editing effects and mitochondrial whole-genome off-target effects.

[0070] Example 3

[0071] Mitochondrial DNA editing was performed in human 293FT cells using mitochondrial-localized MTS-ΔDdCBE, and its effect in reducing off-target effects on the nuclear genome was identified.

[0072] Editing was performed on the m.14279 site on human mitochondrial DNA (e.g.) Figure 9 (As shown in a). Following the procedure in Example 2, TALE sequences were designed, assembled, identified, and plasmids were extracted. Four combinations were transfected into 293FT cells, cells were collected, DNA was extracted, amplicon libraries were constructed, and mutation efficiency was analyzed. Figure 9 As shown, △DdCBE can significantly narrow the editing window, reducing in-window side-editing effects and off-target editing effects of the cell nucleus genome.

[0073] Example 4

[0074] (a) In vitro transcription using the △DdCBE editor

[0075] Disease mimicry sites homologous to human mitochondrial DNA were selected on zebrafish mitochondrial DNA (NC_002333.2) for editing (e.g.) Figure 10 a in Figure 11 (as shown in a). The TALE sequence was designed according to the procedure in Example 1, and the plasmid was assembled, identified, and extracted. Equal molar amounts of the ΔDdCBE plasmids containing the T7 promoter were mixed and linearized using Pme I (New England Biolabs).

[0076] ΔDdCBE was transcribed in vitro using the mMESSAGE mMACHINE T7 Ultra Kit (Life Technologies). Linearized DNA was purified using DNA Clean & Concentrator™-5 (Zymo Research), and 500 ng was used for transcription. The transcribed mRNA was recovered using RNA Clean & Concentrator™-5 (Zymo Research) and stored at -80°C for later use.

[0077] (II) Constructing a mitochondrial DNA mutant zebrafish model using △DdCBE

[0078] The transcribed mRNA was mixed with phenol red and co-injected into single-cell stage zebrafish zygotes, with 1 nL of solution injected into each embryo. After incubation at 28°C for 3 days, individual zebrafish embryos were collected.

[0079] DNA was extracted according to the method in Example 1, an amplicon library was constructed, and mutation efficiency analysis was performed. Figure 10 , Figure 11 As shown, △DdCBE can significantly narrow the editing window and reduce the side editing effect within the window.

Claims

1. A mutated DddA-del1334 domain, characterized in that, The domain is a cytosine deaminase DddA derived from Burkholderiacenocepacia double-stranded DNA. tox The mutated DddA-del1334 domain is obtained by deleting proline at position 45. This position corresponds to position 1334 on the intact DddA protein. The amino acid sequence of the mutated DddA-del1334 domain is shown in SEQ ID NO:

1.

2. A protein pair based on the mutated DddA-del1334 domain segmentation as described in claim 1, characterized in that, The mutated DddA-del1334 domain is split based on the G1397 site of the intact DddA protein to obtain a pair of Half-DddA-del1334 split proteins: G1397N-del1334 and G1397C-del1334; the split proteins are used in pairs, and the amino acid sequence of G1397N-del1334 is shown in SEQ ID NO: 2, and the amino acid sequence of G1397C-del1334 is shown in SEQ ID NO:

4.

3. An optimized cytosine deaminase DddA6-del1334 domain, characterized in that, The amino acid sequence is shown in SEQ ID NO:

6.

4. The protein pair based on the optimized cytosine deaminase DddA6-del1334 domain segmentation as described in claim 3, characterized in that, The optimized cytosine deaminase DddA6-del1334 domain is split based on the G1397 site of the intact DddA protein to obtain a pair of Half-DddA6-del1334 split proteins: G1397N(V6)-del1334 and G1397C(V6)-del1334, which are used in pairs; the amino acid sequence of G1397N(V6)-del1334 is shown in SEQ ID NO: 7, and the amino acid sequence of G1397C(V6)-del1334 is shown in SEQ ID NO:

9.

5. The application of the gene encoding the mutated DddA-del1334 domain of claim 1, or the gene encoding the optimized cytosine deaminase DddA6-del1334 domain of claim 3, or the gene encoding the split protein pair of claim 2 or 4 in constructing the cytosine editing system △DdCBE for C / G to T / A editing of DNA.

6. A cytosine editing system △DdCBE, characterized in that, It includes the gene sequence encoding the segmented protein pair as described in claim 2 or 4, the glycosylation inhibitor UGI, the transcription activator-like effector TALE, and a subcellular localization signal; the subcellular localization signal is the mitochondrial targeting signal MTS or the nuclear localization signal NLS.

7. The cytosine editing system △DdCBE according to claim 6, characterized in that, The system comprises four mitochondrial localization backbone vectors and / or four nuclear localization backbone vectors. Each backbone vector, from its 5' end to its 3' end, sequentially comprises: the subcellular localization signal, a TALE-N-terminal domain, a ccdB gene, a TALE-C-terminal domain, a puromycin resistance gene, and an ampicillin resistance element. Furthermore, the ccdB gene has Bsa I restriction sites flanking it for assembling a TALE sequence that recognizes the target DNA sequence.

8. The cytosine editing system △DdCBE according to claim 7, characterized in that, The method for constructing the mitochondrial targeting backbone vector is as follows: the gene sequence encoding the segmented protein pair described in claim 2 or 4 is fused with the gene sequence encoding the glycosylation inhibitor UGI and then inserted into the MTS-TALE backbone vector carrying the mitochondrial targeting signal. The method for constructing the nuclear localization backbone vector is as follows: the coding gene sequence of the segmented protein pair described in claim 2 or 4 is fused with the coding gene sequence of UGI and then inserted into an NLS-TALE backbone vector carrying a nuclear localization signal.

9. The cytosine editing system △DdCBE according to claim 8, characterized in that, The nucleotide sequence of the encoding gene of UGI is shown in SEQ ID NO: 13; the nucleotide sequence of the MTS-TALE backbone vector is shown in SEQ ID NO: 11; and the nucleotide sequence of the NLS-TALE backbone vector is shown in SEQ ID NO:

12.

10. The use of the mutated DddA-del1334 domain of claim 1, the segmented protein pair of claim 2, the optimized cytosine deaminase DddA6-del1334 domain of claim 3, the segmented protein pair of claim 4, or the cytosine editing system △DdCBE of any one of claims 6-9 in the preparation of a toolkit for C / G to T / A editing of mitochondrial DNA.

11. A toolkit for C / G to T / A editing of mitochondrial DNA, characterized in that, The cytosine editing system includes the mutated DddA-del1334 domain of claim 1, or the optimized cytosine deaminase DddA6-del1334 domain of claim 3, or the split protein pair of claim 2 or 4, or the cytosine editing system △DdCBE of any one of claims 6-9.

12. A method for C / G to T / A editing of mitochondrial DNA, characterized in that, Includes the following steps: Design a TALE recognition sequence based on the target editing site; assemble the TALE sequence into the mitochondrial localization framework vector or nuclear localization framework vector as described in any one of claims 7-9 using the Golden Gate cloning method to obtain the △DdCBE editor plasmid; transfect the △DdCBE editor plasmid into target cells in a G1397N to G1397C pairing manner; screen and verify the editing efficiency.

13. A method of using the cytosine editing system △DdCBE according to any one of claims 6-9, characterized in that, Includes the following steps: Step 1: Based on the DNA sequence of the target editing site, design TALE sequences that recognize the sequences flanking the site; select four backbone vectors from the cytosine editing system △DdCBE containing mitochondrial targeting signal MTS or nuclear localization signal NLS, and assemble the designed TALE sequences into the backbones using the Golden Gate cloning method to form the △DdCBE editor vector. After transformation, single-clone PCR verification, and Sanger sequencing, the correctly assembled △DdCBE plasmid was obtained. Step 2: Transfect the correctly assembled △DdCBE plasmid into target cells according to the Half-DddA pairing method, and screen successfully transfected cells using 1 μg / mL puromycin; verify the editing efficiency of the cytosine editing system △DdCBE by Sanger sequencing or high-throughput sequencing, wherein the Half-DddA pairing method is G1397N and G1397C.

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