A high-fidelity LnCBE system for efficient C / G to T / A editing at the gC motif in DNA

Abstract

The present invention discloses a high-fidelity LnCBE system for realizing efficient C / G to T / A editing on the gC motif of DNA. The editing system constructs a backbone vector by engineering the segmentation protein of the DddA domain of the double-stranded DNA cytosine deaminase from Lachnospiraceae bacterium sunii NSJ‑8, which can realize more efficient on-target editing and extremely low off-target editing on DNA compared with the original version of the deaminase. At the same time, a DNA double-stranded deaminase immunosuppressive protein is disclosed, which can effectively inhibit the off-target editing of nuclear DNA in the LnCBE editing system. The LnCBE editing system can be used to prepare a gene editing kit, construct an animal model of mitochondrial DNA pathogenic mutation from C / G to T / A, and provide a new tool for gene editing technology.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene editing and is a high-fidelity LnCBE system for realizing efficient C / G to T / A editing on the gC motif of DNA. Background Art

[0002] Mitochondrial diseases are a type of maternal genetic disease that seriously endangers human health and can cause disability or death, such as mitochondrial encephalomyopathy, ataxia, etc. Mitochondrial diseases are often caused by nuclear gene mutations and mitochondrial DNA mutations. In particular, mitochondrial diseases caused by mitochondrial DNA (mtDNA) mutations are even more helpless. The most important reason is the lack of mitochondrial gene editing tools to construct mitochondrial disease animal models, so as to carry out systematic molecular mechanism research and treatment exploration.

[0003] Since RNA cannot enter mitochondria, existing CRISPR-based editing tools cannot be used to edit mtDNA. Restriction endonucleases, mito-ZFNs, and mito-TALENs fused to mitochondrial localization signal peptides can introduce DNA double-strand breaks (DSBs) into mutant mtDNA to reduce the proportion of mutant mtDNA. However, these methods cannot achieve precise editing of single bases in mtDNA, so they cannot be used to construct animal disease models of mtDNA mutations, nor can they be used to explore precise treatments for mitochondrial diseases.

[0004] DddA domain of bacterial toxin protein from Burkholderia cenocepacia tox It has double-stranded DNA deaminase activity and can achieve site-specific C / G to T / A mutations in mitochondria by connecting to TALE to form a cytosine base editor (DdCBE). However, the original version of DdCBE can only edit C in the "tC" motif on mitochondrial DNA. The DdCBE mutants V6 and V11 obtained through phage evolution have improved the editing efficiency and broadened the editing sequence preference, but the efficiency of editing C in the "gC" motif is still very low, and it will cause off-target editing on the entire mitochondrial DNA, making it impossible to achieve precise editing. It will also cause nuclear off-target on nuclear DNA, seriously affecting its safety in biological applications. Summary of the invention

[0005] The purpose of the present invention is to provide an accurate and efficient DNA cytosine editing system LnCBE in view of the above-mentioned deficiencies of the prior art, and to A Inhibiting its nuclear off-target editing can achieve efficient and precise editing of the C in the "gC" motif.

[0006] Another object of the present invention is to provide an application of the system.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] The invention discloses a double-stranded DNA cytosine deaminase DddA domain, wherein the DddA domain is a double-stranded DNA cytosine deaminase DddA domain from Lachnospiraceaebacterium sunii NSJ-8, the Uniprot ID is A0A7G9FZY2, and the gene name is abbreviated as FZY2.

[0009] The amino acid sequence of the cytosine deaminase DddA domain of the present invention is shown in SEQ ID NO: 1.

[0010] A gene encoding the DddA domain of cytosine deaminase described in the present invention.

[0011] As a preferred embodiment of the present invention, the coding gene sequence of the cytosine deaminase DddA domain is shown in SEQ ID NO:2.

[0012] The segmentation protein from the DddA domain of the present invention is split at the S100 site according to the amino acid structural characteristics of the DddA domain to obtain a pair of half DddA segmentation proteins: S100N and S100C, and the segmentation proteins are used in pairs; the amino acid sequence of S100N is shown in SEQ ID NO:3, and the amino acid sequence of S100C is shown in SEQ ID NO:5.

[0013] A gene encoding the segmentation protein of the present invention.

[0014] As a preferred embodiment of the present invention, the nucleotide sequence encoding S100N is shown in SEQ ID NO:4, and the nucleotide sequence encoding S100C is shown in SEQ ID NO:6.

[0015] The enhanced segmentation protein introduces mutations in the amino acid sequence of the above-mentioned segmentation protein to increase the deaminase activity of the original protein. The amino acid sequence of the obtained enhanced segmentation protein is as follows: the S100N-mut1 amino acid sequence is shown in SEQ ID NO:7, the S100N-mut2 amino acid sequence is shown in SEQ ID NO:9, and the S100C-mut3 amino acid sequence is shown in SEQ ID NO:11.

[0016] A gene encoding the enhanced segmentation protein of the present invention.

[0017] As a preferred embodiment of the present invention, the S100N-mut1 encoding gene sequence is shown in SEQ ID NO:8, the S100N-mut2 encoding gene sequence is shown in SEQ ID NO:10; and the S100C-mut3 encoding gene sequence is shown in SEQ ID NO:12.

[0018] The use of the double-stranded DNA cytosine deaminase DddA domain or its encoding gene, the segmentation protein or its encoding gene, and the enhanced segmentation protein or its encoding gene of the present invention in constructing a high-fidelity editing system LnCBE that achieves efficient C / G to T / A editing of C in the gC motif, and the use does not include the treatment of human diseases.

[0019] The cytosine editing system LnCBE is constructed using the gene encoding the enhanced segmentation protein described in the present invention.

[0020] As a preferred embodiment of the present invention, the cytosine editing system LnCBE includes an RVD library, a mitochondrial localization backbone vector and / or a nuclear localization backbone vector. The cytosine editing system LnCBE used to achieve C / G to T / A editing of mitochondrial DNA adopts an MTS-LnCBE backbone vector, and the cytosine editing system LnCBE used to achieve C / G to T / A editing of nuclear DNA adopts an NLS-LnCBE backbone vector.

[0021] As a preferred embodiment of the present invention, the five mitochondrial localization skeleton vectors (i.e., the MTS-LnCBE skeleton vector) are: MTS-ccdb-S100N-mut1-UGI, MTS-ccdb-S100N-mut2-UGI, MTS-ccdb-S100N-UGI, MTS-ccdb-S100C-mut3-UGI, and MTS-ccdb-S100C-UGI; the five nuclear localization skeleton vectors (i.e., the NLS-LnCBE skeleton vector) are: NLS-ccdb-S100N-mut1-UGI, NLS-ccdb-S100N-mut2-UGI, NLS-ccdb-S100N-UGI, NLS-ccdb-S100C-mut3-UGI, and NLS-ccdb-S100C-UGI.

[0022] Since the segmentation proteins are used in pairs, the corresponding mitochondrial localization skeleton vectors or nuclear localization skeleton vectors should also be used in pairs according to the principle of pairing S100N-mut1 with S100C, S100N-mut2 with S100C, S100N-mut1 with S100C-mut3, and S100N-mut2 with S100C-mut3. That is, MTS-ccdb-S100N-mut1-UGI ​​and MTS-ccdb-S100C-UGI are used in pairs, MTS-ccdb-S100N-mut2-UGI and MTS-ccdb-S100C-UGI are used in pairs, MTS-ccdb-S100N-mut1-UGI ​​and MTS-ccdb-S100C-mut3-UGI are used in pairs, and MTS-ccdb-S100N-mut2-UGI and MTS-ccdb-S100C-mut3-UGI are used in pairs. ; NLS-ccdb-S100N-mut1-UGI ​​and NLS-ccdb-S100C-UGI are used in pairs, NLS-ccdb-S100N-mut2-UGI and NLS-ccdb-S100C-UGI are used in pairs, NLS-ccdb-S100N-mut1-UGI ​​and NLS-ccdb-S100C-mut3-UGI are used in pairs, and NLS-ccdb-S100N-mut2-UGI and NLS-ccdb-S100C-mut3-UGI are used in pairs.

[0023] As a preferred embodiment of the present invention, the nucleotide sequence of the MTS-TALE backbone vector is shown in SEQ ID NO:13; the nucleotide sequence of the NLS-TALE backbone vector is shown in SEQ ID NO:14; and the nucleotide sequence of the UGI encoding gene is shown in SEQ ID NO:15.

[0024] A double-stranded DNA cytosine deaminase immunosuppressive protein DddI A The protein is a double-stranded DNA cytosine deaminase immunosuppressive protein derived from Lachnospiraceaebacterium sunii NSJ-8, with a Uniprot ID of A0A7G9FY10, and can inhibit off-target editing of the LnCBE editing system in nuclear DNA; the amino acid sequence of the immunosuppressive protein is shown in SEQ ID NO: 18.

[0025] Encoding the double-stranded DNA cytosine deaminase immunosuppressive protein DddI A genes.

[0026] As a preferred embodiment of the present invention, the double-stranded DNA cytosine deaminase immunosuppressive protein DddI is encoded A The nucleotide sequence of the gene is shown in SEQ ID NO:19.

[0027] Using the deaminase immunosuppressive protein DddI of the present invention A , or its gene construct NLS-FZY2-DddI A carrier.

[0028] As a preferred embodiment of the present invention, the NLS-FZY2-DddI A The vector contains a nuclear localization skeleton vector, a Flag tag protein, and a protein encoding FZY2-DddI A of the gene sequence.

[0029] As a preferred embodiment of the present invention, the nucleotide sequence of the cell nucleus localization skeleton vector is shown in SEQ ID NO:20; the nucleotide sequence of the Flag tag protein is shown in SEQ ID NO:21.

[0030] The cytosine editing system LnCBE and / or the NLS-FZY2-DddI of the present invention A Application of vectors in C / G to T / A editing of mitochondrial DNA or nuclear DNA.

[0031] The double-stranded DNA cytosine deaminase DddA domain or its encoding gene, the segmentation protein or its encoding gene, the enhanced segmentation protein or its encoding gene, the cytosine editing system LnCBE and / or the NLS-FZY2-DddI of the present invention A The use of the vector in preparing a kit for C / G to T / A editing of mitochondrial DNA or nuclear DNA.

[0032] A kit for C / G to T / A editing of mitochondrial DNA or nuclear DNA, the kit comprising the double-stranded DNA cytosine deaminase DddA domain or its encoding gene, the segmentation protein or its encoding gene, the enhanced segmentation protein or its encoding gene, the cytosine editing system LnCBE and / or the NLS-FZY2-DddI of the present invention A carrier.

[0033] As a preferred embodiment of the present invention, the kit contains any one of the following components:

[0034] (1)MTS-ccdb-S100N-mut1-UGI ​​and MTS-ccdb-S100C-UGI,

[0035] (2)MTS-ccdb-S100N-mut2-UGI and MTS-ccdb-S100C-UGI,

[0036] (3)MTS-ccdb-S100N-mut1-UGI ​​and MTS-ccdb-S100C-mut3-UGI,

[0037] (4) MTS-ccdb-S100N-mut2-UGI and MTS-ccdb-S100C-mut3-UGI;

[0038] (5) NLS-ccdb-S100N-mut1-UGI ​​and NLS-ccdb-S100C-UGI,

[0039] (6) NLS-ccdb-S100N-mut2-UGI and NLS-ccdb-S100C-UGI,

[0040] (7)NLS-ccdb-S100N-mut1-UGI ​​and NLS-ccdb-S100C-mut3-UGI,

[0041] (8)NLS-ccdb-S100N-mut2-UGI and NLS-ccdb-S100C-mut3-UGI,

[0042] (9)NLS-ccdb-S100N-mut1-UGI, NLS-ccdb-S100C-UGI and NLS-FZY2-DddI A ,

[0043] (10)NLS-ccdb-S100N-mut2-UGI, NLS-ccdb-S100C-UGI and NLS-FZY2-DddI A ,

[0044] (11)NLS-ccdb-S100N-mut1-UGI, NLS-ccdb-S100C-mut3-UGI and NLS-FZY2-DddI A , (12)NLS-ccdb-S100N-mut2-UGI, NLS-ccdb-S100C-mut3-UGI and NLS-FZY2-DddI A .

[0045] Further explanation:

[0046] The cytosine editing system LnCBE for realizing C / G to T / A editing of DNA provided by the present invention is to engineer the double-stranded DNA cytosine deamination domain DddA found in the Lachnospiraceae bacterium sunii NSJ-8 genus. tox (Uniprot ID: A0A7G9FZY2), the gene name is abbreviated as FZY2, and the DddA of the enhanced version of FZY2 is fused by TALE tox The LnCBE system composed of segmentation protein and glycosylase inhibitor (UGI) can enhance the deaminase activity of the original FZY2 protein and achieve efficient editing of the C in the gC motif of the target site.

[0047] The engineered domain has not been published as a DddA domain, but the sequence of the original protein exists in protein databases such as Uniprot and NCBI, with the annotated name A0A7G9FZY2.

[0048] The present invention splits the DddA domain (amino acids 1-125) in FZY2 from Lachnospiraceae bacterium sunii NSJ-8 at amino acid 100 to produce two half DddA, and engineered multiple single amino acid mutations of the two segmentation proteins. The modified versions of the segmentation proteins with enhanced effects were obtained through screening. The modified segmentation proteins and glycosylase inhibitors (UGI) were fused with linker amino acid sequences and inserted into the MTS-TALE backbone vector and the NLS-TALE backbone vector respectively. According to the principle of pairing S100N-mut1 with S100C, S100N-mut2 with S100C, S100N-mut1 with S100C-mut3, and S100N-mut2 with S100C-mut3, four editing systems, LnCBE-v1.6, LnCBE-v2.3, LnCBE-v2.3, and LnCBE-v2.4, were constructed, and finally 5 MTS backbone vectors and 5 NLS backbone vectors (such as Figure 1 and Figure 2 shown).

[0049] The cytosine editing system LnCBE for realizing C / G to T / A editing of DNA provided by the present invention comprises the following 5 MTS-LnCBE backbone vectors (see Figure 2), MTS-ccdb-S100N-mut1-UGI, MTS-ccdb-S100N-mut2-UGI, MTS-ccdb-S100N-UGI, MTS-ccdb-S100C-mut3-UGI, MTS-ccdb-S100C-UGI; and / or, 5 NLS-LnCBE backbone vectors (see Figure 2 )NLS-ccdb-S100N-mut1-UGI, NLS-ccdb-S100N-mut2-UGI, NLS-ccdb-S100N-UGI, NLS-ccdb-S100C-mut3-UGI, NLS-ccdb-S100C-UGI.

[0050] NLS-FZY2-DddI for inhibiting nuclear off-target of LnCBE editing system provided by the present invention A , a newly discovered double-stranded DNA deaminase immunosuppressive protein DddI from the Lachnospiraceae bacterium sunii NSJ-8 A , Uniprot ID is A0A7G9FY10. Nuclear localization signal protein and Flag tag protein were fused by homologous recombination to form NLS-FZY2-DddI A (See Figure 2 ), which can effectively inhibit the off-target editing of the LnCBE editing system on the nuclear genome.

[0051] This immunosuppressive protein does not act as DddI A It has been made public, but the sequence of the entire protein exists in protein databases such as Uniprot and NCBI, and its function has not been explored.

[0052] All backbone vectors of the present invention are matched to a TALE assembly system. The two sides of the backbone vector toxin gene ccdb contain Bsa I restriction sites respectively. After restriction cleavage, different sticky ends are produced for specific connection to TALE sequences that recognize specific DNA sequences. The assembly method is described in the authorized patent: A mitochondrial DNA editing system based on TALE assembly, patent number ZL202110688797.9.

[0053] The four LnCBE editing systems described in the present invention are used for C / G to T / A editing of double-stranded DNA, especially for editing mitochondrial DNA, and are used for efficient editing of C in the "gC" motif.

[0054] According to the principle of pairing S100N-mut1 with S100C, pairing S100N-mut2 with S100C, pairing S100N-mut1 with S100C-mut3, and pairing S100N-mut2 with S100C-mut3, the present invention selects two corresponding LnCBE vectors described in the system of the present invention for pairing, transfects them into cells to be edited, screens the successfully transfected cells by puromycin, and realizes the editing of C in the DNA "gC" motif.

[0055] Furthermore, the present invention pairs S100N with S100C and NLS-FZY2-DddI A The vector is co-transfected into cells to achieve editing of nuclear DNA, efficiently edit the C in the "gC" motif, and effectively inhibit off-target editing of the LnCBE editing system on the nuclear genome.

[0056] The four LnCBE editing systems described in the present invention can be used to explore the function of mitochondrial or nuclear DNA mutations. At the same time, the tools of the present invention can be used to repair mutated DNA in cells.

[0057] The working principle of the present invention is as follows: after the LnCBE system is transfected into cells, the editing site can be edited from C / G to T / A, mainly by deaminating the C to be edited to form dU through double-stranded DNA cytosine deaminase, and further editing from C / G to T / A is achieved through cell replication.

[0058] To achieve C / G to T / A editing of mitochondrial DNA, TALE-DddA reported by Mok BY et al. tox It can achieve C / G to T / A editing in the "tC" sequence on mitochondrial DNA (Nature. 2020 Jul; 583 (7817): 631-637.), and Simiaoa sunii (Ddd_Ss) reported by Li Mi et al. can achieve C / G to T / A editing in the "gC" sequence on mitochondrial DNA (Nat Commun. 2023 Feb; 14 (1): 874.). The DNA editing system LnCBE developed by the present invention can further enhance the efficient C / G to T / A editing in the "gC" sequence on mitochondrial DNA compared to Ddd_Ss.

[0059] Beneficial effects of the present invention:

[0060] The main innovative value of the present invention lies in the engineering modification of a double-stranded DNA deaminase, which enhances the functional activity of the original deaminating protein and can achieve efficient editing of C in the "gC" motif at any position on the double-stranded DNA. It also discloses a DNA double-stranded deaminase immunosuppressive protein that can effectively inhibit the nuclear off-target effects of the LnCBE editing system, so that LnCBE can be designed as a mtDNA cytosine editor with better gC compatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 Schematic diagram of engineering transformation of LnCBE editing system

[0062] A is the alignment result of DddA, DddA-V6, DddA-V11 and FZY2 amino acid sequences; B is the 3D structure diagram of the FZY2 protein after engineering modification; C is the first round of FZY2 amino acid single mutation version; D is the second round of FZY2 amino acid double mutation and triple mutation versions.

[0063] Figure 2 Schematic diagram of the MTS and NLS backbone vectors of the four LnCBE editing systems

[0064] A is the working diagram of LnCBE system; B is NLS-FZY2-DddI A Vector schematic diagram; C represents four LnCBE editing systems with different segmentation types of mitochondrial localization signals (MTS) and nuclear localization signals (NLS). The vector carries the ccdb gene element, which is located between two Bsa I restriction sites. The vector also carries an ampicillin resistance element.

[0065] Figure 3 T-Pool Characterization of LnCBE Editing System Characteristics

[0066] A is a comparison of the editing activities of different mutant versions of FZY2 in T-Pool; B is the editing efficiency of four different NC-motifs of four enhanced versions of LnCBE.

[0067] Figure 4 Editing of GC sites on mitochondria by four LnCBE editing systems

[0068] A, the upper figure shows the sequence information of the mitochondrial m.G3635A site editing window, and the lower figure shows the editing status of all sequences in the m.G3635A site window by the DddA-V11 and LnCBE editing systems; B is a comparison of the editing efficiency of the DddA-V11 and LnCBE editing systems at the m.G3635A site; C, the upper figure shows the sequence information of the mitochondrial m.G8313A site editing window, and the lower figure shows the editing status of all sequences in the m.G8313A site window by the LnCBE editing system; D is a comparison of the editing efficiency of the LnCBE editing system at the m.G8313A site.

[0069] Figure 5 Off-target situation of LnCBE editing system at mitochondrial sites m.G3460A and m.G3635A A is the number of off-targets and the average off-target rate of LnCBE editing system at mitochondrial m.G3635A site; B is the number of off-targets and the average off-target rate of LnCBE editing system at mitochondrial m.G8313A site; C is the editing and off-target situation of all mitochondrial target sites of LnCBE editing system at mitochondrial m.G3635A site

[0070] Figure 6 Co-expression of FZY2-DddI A Nuclear off-target inhibition of the LnCBE editing system

[0071] A is DddA-DddI A With FZY2-DddI A Protein structure alignment; B is co-expression of DddA-DddI A With FZY2-DddI A The nuclear off-target inhibition of DddA and FZY2 respectively; C is the co-expression of DddA-DddI A With FZY2-DddI A The nuclear off-target inhibition of DddA-G1397NC respectively; D is the co-expression of DddA-DddI A Nuclear off-target inhibition of DddA and FZY2 respectively; E is co-expression of FZY2-DddI A Nuclear off-target inhibition of FZY2; F is co-expression of FZY2-DddI at the nuclear genomic site JAK2 A Nuclear off-target inhibition of FZY2; G is co-expression of FZY2-DddI A Nuclear off-target inhibition of the mitochondrial G8313 site. DETAILED DESCRIPTION

[0072] The present invention is described in detail below in conjunction with the embodiments, so that how to apply the present invention to achieve C / G to T / A editing of DNA can be fully understood and implemented accordingly.

[0073] The present invention is further described below with reference to the accompanying drawings. The present invention provides a novel DNA editing tool: LnCBE can achieve C / G to T / A editing and co-express NLS-FZY2-DddI A To inhibit its nuclear off-target effect, the LnCBE system includes: 5 vectors for mitochondrial localization and 5 vectors for nuclear localization. The LnCBE for mitochondrial localization can be selected to edit mitochondrial DNA, and the LnCBE for nuclear localization can be selected to edit the nuclear genome. The RVD library can select any library that is compatible with the editing system. The library used in the embodiment of the present invention can be found in CellDiscov.2021Sep3;7(1):78, and the assembly method can be found in the authorized patent: A mitochondrial DNA editing system based on TALE assembly, patent number ZL202110688797.9. The steps, reagents, modules, etc. not described in detail in the embodiments are well known to those skilled in the art and will not be repeated in this application.

[0074] Example 1

[0075] The construction method of the MTS-LnCBE and NLS-LnCBE backbone vectors of the cytosine editing system LnCBE is as follows:

[0076] Splitting the DddA domain of double-stranded DNA cytosine deaminase from Lachnospiraceae bacterium sunii NSJ-8 into two halves DddA, engineered amino acid mutations were performed on the two segmentation proteins to obtain enhanced versions of the segmentation proteins, which were paired with each other and fused with the glycosylase inhibitor (UGI) respectively, and then inserted between the two restriction sites Nhe I and Pme I of the MTS-TALE backbone vector to obtain 5 MTS-LnCBE backbone vectors respectively; specific steps 1. According to the amino acid homology comparison of FZY2 and DddA and the secondary structure of the protein, the FZY2-S100 protein segmentation site corresponding to DddA-G1397 was selected for segmentation to obtain two protein segmentation pairs, and then gene fragments of different segmentation pairs were synthesized; 2. Primers with 16 bp were designed to perform PCR amplification on the synthesized gene fragments and the glycosylase inhibitor (UGI), so that one end of the different protein gene fragments had a 16 bp homology arm with the other end of the glycosylase inhibitor (UGI), and the other ends of the two had Nhe I and Pme I restriction site homology arms respectively; 3. Primers with mitochondrial localization signals (MTS), Nhe I and Pme I restriction site, ccdb toxin gene and ampicillin resistance gene vector were digested to obtain DNA backbone fragments with homology arms matching protein gene fragment and glycosylase inhibitor (UGI) gene fragment at both ends; 4. The backbone fragments, protein gene fragments and glycosylase inhibitor (UGI) gene fragments were homologously recombined; 5. The multi-fragment recombination products were transferred into competent cells trans DB3 with ccdb toxin resistance.1 After ampicillin screening, single clones were selected for Sanger sequencing verification, and 5 MTS-LnCBE backbone vectors with correct sequencing results were obtained: MTS-ccdb-S100N-mut1-UGI, MTS-ccdb-S100N-mut2-UGI, MTS-ccdb-S100N-UGI, MTS-ccdb-S100C-mut3-UGI, and MTS-ccdb-S100C-UGI; Based on the same method, the double-stranded DNA cytosine deaminase DddA domain from Lachnospiraceae bacterium sunii NSJ-8 was split into two halves DddA, engineered amino acid mutations were performed on the two segmentation proteins to obtain enhanced versions of the segmentation proteins, which were paired with each other and fused with glycosylase inhibitors (UGI) respectively, and then inserted between the two restriction sites NheⅠ and PmeⅠ of the NLS-TALE backbone vector to obtain five NLS-LnCBE backbone vectors: NLS-ccdb-S100N-mut1-UGI, NLS-ccdb-S100N-mut2-UGI, NLS-ccdb-S100N-UGI, NLS-ccdb-S100C-mut3-UGI, NLS-ccdb-S100C-UGI. .

[0077] The main elements contained in the MTS-TALE backbone vector described above from 5' to 3' are: mitochondrial localization signal (MTS) sequence, TALE-N-terminus (NTD) nucleotide sequence of 136 amino acids, ccdb toxin gene, TALE-C-terminus (CTD) nucleotide sequence of 41 amino acids, puromycin cell drug screening gene, and ampicillin resistance element; the main elements contained in the NLS-TALE backbone vector from 5' to 3' are: nuclear localization signal (NLS) sequence, TALE-N-terminus (NTD) nucleotide sequence of 136 amino acids, ccdb toxin gene, TALE-C-terminus (CTD) nucleotide sequence of 63 amino acids, puromycin cell drug screening gene, and ampicillin resistance element.

[0078] The nucleotide sequence of S100N is shown in SEQ ID NO:4, the nucleotide sequence of S100C is shown in SEQ ID NO:6, the nucleotide sequence of S100N-mut1 is shown in SEQ ID NO:8, and the nucleotide sequence of S100N-mut2 is shown in SEQ ID NO:10; the nucleotide sequence of S100C-mut3 is shown in SEQ ID NO:12, the nucleotide sequence of the MTS-TALE backbone vector is shown in SEQ ID NO:13, the nucleotide sequence of the NLS-TALE backbone vector is shown in SEQ ID NO:14, and the nucleotide sequence of the UGI encoding gene is shown in SEQ ID NO:15.

[0079] All backbone vectors are matched to a TALE assembly system. The two sides of the backbone vector toxin gene ccdb contain Bsa I restriction sites. After restriction digestion, different sticky ends are produced for specific connection to TALE sequences that recognize specific DNA sequences.

[0080] Example 2

[0081] Targeted T vector editing was performed in human 293FT cells using the nuclear-localized NLS version of LnCBE.

[0082] (I) Design of TALE sequences and assembly of LnCBE

[0083] T vectors containing different motifs (6xNNC, 6xNCN, 6xCNN, JAK2, SIRT6) were constructed in Spacer. The two ends of Spacer also had the same recognition sequence and 4bp base sequences of different lengths as the subsequent splitting index. T vectors with different motifs (38 in total) were mixed into T-Pool according to the same nanogram number, and the overall concentration was measured. The RVD modules (Cell Discov. 2021 Sep3; 7(1):78) targeting T vectors to recognize different DNA bases were assembled onto the LnCBE backbone vector by the Golden Gate method. The assembly method is described in the authorized patent: A mitochondrial DNA editing system based on TALE assembly, patent number ZL202110688797.9.

[0084] The assembled product was transformed into DH5α and screened using a solid LB culture plate containing ampicillin. After that, a single clone was picked and identified by PCR using the following primers, the primer sequences of which are shown in SEQ ID NO: 16 and 17. According to the number of RVDs, the positive clones were judged, such as 16 RVDs should have a size of 1759 bp, and 15 RVDs should have a size of 1657 bp. After that, the above primers were used for forward and reverse sequencing, and the positive clones with correct sequences were extracted with plasmids for subsequent experiments.

[0085] (II) Transfected cells to detect the editing efficiency of different combinations of LnCBE

[0086] Select 293FT cells as the editing object. When the cell density reaches 70-90%, change the medium 2 hours in advance. After counting the cells, take 1.5x10 5 Cells were used for cell electrotransfection (Instrument: Lonza4D-nucleofector). The vectors of 5 NLS versions of Half-DddA were combined according to the principle of pairing S100N-mut1 with S100C, S100N-mut2 with S100C, S100N-mut1 with S100C-mut3, and S100N-mut2 with S100C-mut3, and electrotransfected with T vectors containing different motifs into 293FT cells. 400ng of the vectors at the left and right ends were transfected each, and 50ng of T-Pool was electrotransfected into cells using SF-Cell Line4D-nucleofector X-kit.

[0087] After electrotransfection, the cells were plated in a 12-well plate and cultured for 24 hours, followed by cell screening using 1 μg / mL of puromycin. After 72 hours of culture, the cells were digested and centrifuged, and the cells were collected for testing DNA mutation efficiency.

[0088] The DNA extraction method of the collected cell samples is as follows: 30 μL of QuickExtract TM The cells were reselected with DNA Extraction Solution (Lucigen) and then heated at 65°C for 45 min and then at 98°C for 2 min.

[0089] Adoption 2 Green Taq Mix (Vazyme) was used to amplify the target fragment containing the editing site on the T vector or mitochondrial DNA, and then used for Sanger sequencing; or Phanta Super-Fidelity DNA polymerase (Vazyme) and high-throughput sequencing library primers were used for high-throughput sequencing to analyze the editing efficiency. Figure 2 and Figure 3As shown, L-S100N-mut1+R-S100C (LnCBE-v1.6); L-S100N-mut2+R-S100C (LnCBE-v2.1); L-S100N-mut1+R-S100C-mut3 (LnCBE-v2.3); L-S100N-mut2+R-S100C-mut3 (LnCBE-v2.4) (L is the left end, R is the right end). The results of high-throughput sequencing showed that the LnCBE enhanced version system (v1.6, v2.1, v2.3, v2.4) has a significantly improved ability to efficiently edit C in the "gC" motif compared to the original LnCBE editing system (FZY2).

[0090] Example 3

[0091] Mitochondrial DNA editing was performed in human 293FT cells using the mitochondrial-targeted MTS version of LnCBE.

[0092] Select the m.G8313A and m.G3635A sites on human mitochondrial DNA (reference sequence version: NC_012920.1) for editing (e.g. Figure 4 According to the DNA sequences near these sites, the recognized TALE sequences were designed, and the RVD modules of different recognized DNA bases were assembled onto the backbone vectors of 5 mitochondrial-localized LnCBEs by the Golden Gate method.

[0093] The assembled product was transformed into DH5α and screened using a solid LB culture plate containing ampicillin. After that, a single clone was picked and identified by PCR using the following primers. The primer sequences are shown in SEQ ID NO: 16 and SEQ ID NO: 17. According to the number of RVDs, the positive clones were judged, such as 16 RVDs should have a size of 1759 bp, and 15 RVDs should have a size of 1657 bp. After that, the above primers were used for forward and reverse sequencing, and the positive clones with correct sequences were extracted with plasmids for subsequent experiments.

[0094] According to the same operation process as in Example 2, the four combinations of each site were transfected into 293FT cells, and the cells were collected and DNA was extracted to construct a full mitochondrial library and analyze the mutation efficiency. Figure 4As shown, L-S100N-mut1+R-S100C (LnCBE-v1.6); L-S100N-mut2+R-S100C (LnCBE-v2.1); L-S100N-mut1+R-S100C-mut3 (LnCBE-v2.3); L-S100N-mut2+R-S100C-mut3 (LnCBE-v2.4) (L is the left end, R is the right end), the LnCBE enhanced version system (v1.6, v2.1, v2.3, v2.4) compared to the original LnCBE editing system (FZY2), its ability to efficiently edit the endogenous "gC" site of mitochondria is significantly improved, showing its efficient and specific editing effect on C in the "gC" motif. Figure 5 As shown, the average off-target rate and number of off-targets corresponding to the editing of the "gC" site were lower for the LnCBE editing system than for DddA-V11, indicating that the LnCBE editing system is efficient, specific and high-fidelity for "gC".

[0095] Example 4

[0096] The construction method of the NLS-FZY2-DddIA vector for nuclear off-target inhibition of the LnCBE editing system is as follows:

[0097] The double-stranded DNA deaminase immunosuppressive protein FZY2-DddI from Lachnospiraceae bacterium sunii NSJ-8 A After being fused with the Flag tag protein, it was inserted between the two restriction sites of NheI and PmlI of the NLS-backbone vector to obtain NLS-FZY2-DddI A The specific steps are as follows: design primers with 16 bp homology arms to synthesize the gene fragment DddI A PCR amplification with Flag tag protein to synthesize DddI A One end has a 16bp homology arm with the other end of Flag, and both have two restriction site homology arms, NheI and PmlI respectively; then the backbone fragment, Flag and DddI A The gene fragments were homologously recombined, and the recombinant products were transferred into competent cells trans 5 and screened with ampicillin. Single clones were selected for Sanger sequencing verification to obtain NLS-FZY2-DddI with the correct sequencing results. A .

[0098] The above FZY2-DddI AThe amino acid sequence is SEQ ID NO:18, the coding sequence is SEQ ID NO:19, the NLS-skeleton vector nucleotide sequence is SEQ ID NO:, 20, and the nucleotide sequence of the Flag tag protein is SEQ ID NO:21.

[0099] According to the same operation process as in Example 2, NLS-FZY2-DddI A The vector was co-transfected with the LnCBE system of Example 2 into 293FT cells, and the cells were collected and DNA was extracted to construct a long fragment amplicon library for mutation efficiency analysis. Figure 6 As shown, by co-expression of NLS-FZY2-DddI A The off-target editing of nuclear DNA by the LnCBE editing system was significantly inhibited, indicating that FZY2-DddI A Immunosuppressive proteins can effectively inhibit the nuclear off-target effect of the LnCBE editing system.

[0100] In the embodiment of the present invention, only one coding sequence is used to construct a vector to verify the feasibility and technical effect of the technical solution. Any coding sequence encoding the double-stranded DNA cytosine deaminase DddA domain, partition protein, enhanced segmentation protein, double-stranded DNA cytosine deaminase immunosuppressive protein DddI of the present invention is A The genes are all within the protection scope of the present invention, and can be used to implement the technical solution of the present invention, produce similar or identical technical effects to the above embodiments, and solve the technical problems of the present invention.

Claims

1. A pair of segmentation proteins from the DddA domain of double-stranded DNA cytosine deaminase, characterized in that According to the amino acid structural characteristics of the DddA domain, the S100 site was selected for segmentation to obtain a pair of half DddA segmentation proteins: S100N and S100C. The segmentation proteins are used in pairs; the amino acid sequence of S100N is shown in SEQ ID NO:3, and the amino acid sequence of S100C is shown in SEQ ID NO:

5.

2. A gene encoding a pair of segmentation proteins according to claim 1.

3. Enhanced segmentation protein, characterized in that: A mutation is introduced into the amino acid sequence of the partition protein described in claim 1 to improve the deaminase activity of the original protein, and the amino acid sequence of the enhanced partition protein obtained is as follows: the S100N-mut1 amino acid sequence is shown in SEQ ID NO: 7, the S100N-mut2 amino acid sequence is shown in SEQ ID NO: 9, and the S100C-mut3 amino acid sequence is shown in SEQ ID NO: 11; wherein, S100N-mut1 pairs with S100C, S100N-mut2 pairs with S100C, S100N-mut1 pairs with S100C-mut3, and S100N-mut2 pairs with S100C-mut3.

4. A gene encoding the enhanced segmentation protein according to claim 3.

5. Use of the pair of segmentation proteins described in claim 1, the gene described in claim 2, the enhanced segmentation protein described in claim 3, and the gene described in claim 4 in constructing a high-fidelity editing system LnCBE that realizes efficient C / G to T / A editing of C in the gC motif.

6. A cytosine editing system LnCBE constructed using the gene described in claim 4.

7. The cytosine editing system LnCBE according to claim 6, comprising an RVD library, a mitochondrial localization backbone vector and / or a nuclear localization backbone vector, characterized in that: The cytosine editing system LnCBE used to achieve C / G to T / A editing of mitochondrial DNA adopts the MTS-LnCBE backbone vector, and the cytosine editing system LnCBE used to achieve C / G to T / A editing of nuclear DNA adopts the NLS-LnCBE backbone vector.

8. The cytosine editing system LnCBE according to claim 7, characterized in that: The five MTS-LnCBE backbone vectors are: MTS-ccdb-S100N-mut1-UGI, MTS-ccdb-S100N-mut2-UGI, MTS-ccdb-S100N-UGI, MTS-ccdb-S100C-mut3-UGI, MTS-ccdb-S100C-UGI; the five NLS-LnCBE backbone vectors are: NLS-ccdb-S100N-mut1-UGI, NLS-ccdb-S100N-mut2-UGI, NLS-ccdb-S100N-UGI, NLS-ccdb-S100C-mut3-UGI, NLS-ccdb-S100C-UGI.

9. The cytosine editing system LnCBE and / or NLS-FZY2-DddI according to any one of claims 6 to 8 A The use of the vector in the C / G to T / A editing of mitochondrial DNA or nuclear DNA, the application does not include the treatment of diseases in humans and animals; the NLS-FZY2-DddI A The vector contains a nuclear localization skeleton vector, a Flag tag protein, and a protein encoding FZY2-DddI A Gene sequence of FZY2-DddI A The amino acid sequence is shown in SEQ ID NO:

18.

10. The pair of segmentation proteins according to claim 1, the gene according to claim 2, the enhanced segmentation protein according to claim 3, the gene according to claim 4, the cytosine editing system LnCBE according to any one of claims 6 to 8, and / or the NLS-FZY2-DddI according to claim 9 A The use of the vector in preparing a kit for C / G to T / A editing of mitochondrial DNA or nuclear DNA.

11. A kit for editing C / G to T / A of mitochondrial DNA or nuclear DNA, characterized in that: The kit contains a pair of segmentation proteins according to claim 1, the gene according to claim 2, the enhanced segmentation protein according to claim 3, the gene according to claim 4, and the cytosine editing system LnCBE according to any one of claims 6-8.

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