CN117106758B - RiCBE system for realizing C/G to T/A editing on gC motif of DNA - Google Patents

RiCBE system for realizing C/G to T/A editing on gC motif of DNA Download PDF

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CN117106758B
CN117106758B CN202311075175.4A CN202311075175A CN117106758B CN 117106758 B CN117106758 B CN 117106758B CN 202311075175 A CN202311075175 A CN 202311075175A CN 117106758 B CN117106758 B CN 117106758B
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沈彬
沈李宓妮
韩露
孙海峰
刘单鹏
王兆君
程凯
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Nanjing Medical University
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Abstract

The invention belongs to the technical field of gene editing, and discloses a RiCBE editing system for realizing C/G to T/A editing on C in a gC motif. The system adopts the split protein of the double-chain DNA cytosine deaminase DddA structural domain from Roseburia_intestinalis to construct a skeleton carrier, can realize high-efficiency editing from C/G to T/A on C in gC motif, and has low off-target rate. The invention can be used for preparing a gene editing kit, constructing a mitochondrial DNA pathogenic mutation animal model from C/G to T/A, and providing a new tool for researching and treating gene diseases.

Description

RiCBE system for realizing C/G to T/A editing on gC motif of DNA
Technical Field
The invention belongs to the technical field of gene editing, and relates to a RiCBE editing system which is used for efficiently and accurately editing C/G to T/A aiming at C in a gC motif and has low off-target.
Background
Mitochondria are a double-membrane coated organelle that provides energy support for various cellular metabolism. Mitochondrial diseases caused by mitochondrial DNA mutations are a class of maternal genetic diseases that severely compromise human health and can cause disability and lethality. Mitochondrial diseases caused by mutations in mitochondrial DNA (mtDNA) are even more forensic, the most important reason of which is the lack of mitochondrial gene editing tools for constructing animal models of mitochondrial diseases to develop systematic molecular mechanism studies and therapeutic approaches exploration.
Because guide RNAs of Cas9 systems are difficult to enter mitochondria, existing CRISPR-based editing tools cannot be used to edit mtDNA. A DNA Double Strand Break (DSB) can be introduced into the mutated mtDNA by fusing restriction enzymes of mitochondrial localization signal peptide, mito-ZFN and mito-TALEN to reduce the proportion of mutated mtDNA. However, these methods cannot achieve precise editing of single bases of mtDNA, and thus cannot be used to construct an animal disease model of mtDNA mutations, nor to explore precise treatment of mitochondrial disease.
The DddA domain of bacterial toxin protein from Burkholderia cenocepacia, dddA tox, has double-stranded DNA deaminase activity and can be linked to TALE to form a cytosine base editor (DdCBE) to effect site-directed C/G to T/a mutation in mitochondria. However, the DdCBE of the original version prefers to edit C in the "tC" motif on the mtDNA, and the versions of DdCBE mutants V6 and V11 obtained through phage evolution improve the editing efficiency and widen the preference of the editing sequence, but also cause serious bystander mutation in an editing window, so that the accurate editing cannot be realized.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides a DNA cytosine editing system RiCBE which is accurate and efficient and only aims at a gC motif, so that efficient and accurate editing of C in the gC motif can be realized.
It is a further object of the invention to provide an application of the system.
It is a further object of the present invention to provide a method for effecting C/G to T/A editing on mitochondrial DNA using the system.
The aim of the invention can be achieved by the following technical scheme:
A double-stranded DNA cytosine deaminase DddA domain, the DddA domain is a double-stranded DNA cytosine deaminase DddA domain derived from Roseburia_intestinalis, the gene sequence number is SAMEA3134499_274314, and the gene name is SA071.
The amino acid sequence of the DddA domain of the cytosine deaminase DddA domain is shown as SEQ ID NO. 1.
The split proteins from the DddA domain were split at the G105 site selected according to the amino acid structural features of the DddA domain to give a pair of half DddA split proteins: G105N and G105C, and a pair of half DddA split proteins N169N and N169C are obtained by splitting at the N169 site according to the amino acid structural characteristics of the DddA domain; split proteins are used in pairs.
The amino acid sequence of the split protein is as follows: the amino acid sequence of G105N is shown as SEQ ID NO. 2; the amino acid sequence of G105C is shown as SEQ ID NO. 3; the N169N amino acid sequence is shown as SEQ ID NO. 4; the amino acid sequence of N169C is shown in SEQ ID NO. 5.
The split protein, in which a single amino acid is mutated to increase the activity of the original protein, has the amino acid sequence shown below: the amino acid sequence of the N169N-mut1 is shown in SEQ ID NO. 6; the amino acid sequence of N169N-mut2 is shown in SEQ ID NO. 7.
Use of said DddA domain or said split protein in the construction of C/G to T/a edited cytosine editing system RiCBE.
Cytosine editing system RiCBE constructed using the DddA domain or the split protein.
The cytosine editing system RiCBE comprises an RVD library, a mitochondrial localization framework vector or a cell nucleus localization framework vector, wherein the cytosine editing system RiCBE for realizing efficient and accurate C/G-to-T/A editing of the specificity of mitochondrial DNA only aiming at C in a gC motif adopts an MTS-RiCBE framework vector; the cytosine editing system RiCBE for realizing efficient and accurate C/G to T/A editing of the specificity of the nuclear DNA only for C in the "gC" motif adopts NLS-RiCBE backbone vector.
The cytosine editing system RiCBE, wherein the 4 MTS-RiCBE backbone vectors are: MTS-ccdb-G105N-UGI, MTS-ccdb-G105C-UGI, MTS-ccdb-N169N-UGI, MTS-ccdb-N169C-UGI; the 2 NLS-RiCBE framework vectors are respectively: NLS-ccdb-N169N-UGI and NLS-ccdb-N169C-UGI.
The specificity of the cytosine editing system RiCBE in mitochondrial DNA or cell nuclear DNA is only applied to the efficient and accurate editing from C/G to T/A of C in a gC motif.
The use of the DddA domain, or the split protein, or the cytosine editing system RiCBE in a kit for preparing mitochondrial DNA or nuclear DNA with specificity for efficient and precise C/G to T/a editing only for C in the "gC" motif.
A kit for efficient, accurate C/G to T/a editing of C in a mitochondrial DNA or nuclear DNA specific for only C in the "gC" motif, the kit comprising said DddA domain, or said split protein, or said cytosine editing system RiCBE.
Further description:
The cytosine editing system RiCBE for realizing C/G to T/A editing of DNA provided by the invention is characterized in that a newly discovered double-stranded DNA cytosine deaminase DddA structural domain from Roseburia_intestinalis genus has a gene sequence number of SAMEA3134499_274314, the gene name is SA071, and a RiCBE system is formed by a DddA tox segmentation protein of TALE fusion SA071 and a glycosylase inhibitor (Uracil glycosylase inhibitor, UGI), so that efficient and accurate editing of C in a gC motif of a target site can be realized.
This domain was not disclosed as DddA domain, but its entire protein sequence was found in the GMGC, GEMs and TG2G protein databases, and its function was not explored.
SA071 double-stranded DNA cytosine deamination domain DddAtox is derived from amino acids 95-295 of Roseburia_intestinalis genus, and has only 42.857% amino acid sequence similarity compared with DddA tox of Burkholderia cenocepacia.
The invention cuts DddA structural domain (95-295 amino acids) in SA071 from Roseburia_intestinalis at 105 th amino acid and 169 th amino acid to generate 4 kinds of Half DddA, respectively fuses Half DddA and glycosylase inhibitor (UGI) through linker amino acid sequences and inserts the fused products into MTS-TALE framework vectors and NLS-TALE framework vectors to finally obtain 4 MTS framework vectors and 2 NLS framework vectors (see figure 2).
Description: the 95 th to 295 th positions in the full-length sequence of the double-stranded DNA cytosine deaminase (gene sequence No. SAMEA 3134499-274314, GMGC database) protein from Roseburia_intetinalis are DddA homologous domain complete sequence amino acids of the strain to which SA071 belongs, total 201aa, the splitting points of 105 and 169, namely G105 and N169 are calculated by taking the domain sequence as the start, and the splitting points of 153-295 in the full-length sequence of the protein are calculated by taking the SEQ ID NO:1 sequence as the start. The results of these two positioning modes are consistent, as follows.
The cytosine editing system RiCBE for implementing C/G to T/A editing of DNA provided by the invention comprises the following 4 MTS-RiCBE skeleton carriers (see figure 2), namely MTS-ccdb-G105N-UGI, MTS-ccdb-G105C-UGI, MTS-ccdb-N169N-UGI and MTS-ccdb-N169C-UGI; and/or 2 NLS-RiCBE backbone vectors (see FIG. 2) NLS-ccdb-N169N-UGI, NLS-ccdb-N169C-UGI.
All framework vectors of the invention are matched with a TALE assembly system, and the two sides of a toxin gene ccdb of the framework vector respectively contain Bsa I enzyme cutting sites, and different sticky ends are generated after enzyme cutting and are used for specifically connecting and identifying TALE sequences of specific DNA sequences, and the assembly method is as follows in the issued patent: mitochondrial DNA editing system based on TALE assembly, patent number ZL202110688797.9.
The RiCBE editing system is used for the application from C/G to T/A editing of double-stranded DNA, particularly the application for editing mitochondrial DNA, and is specific and efficient for editing only C in a gC motif.
The invention selects the corresponding 2 RiCBE vectors in the system of the invention to pair according to the pairing principle of G105N and G105C and the pairing principle of N169N and N169C, and transfects the vectors to cells to be edited, and the cells successfully transfected are screened by puromycin, so that the editing of C in the DNA gC motif is realized.
The enhanced version RiCBE editing system provided by the invention is matched with N169N-mut1 and N169N-mut2 according to the matching principle of N169C, so that the specific and efficient editing capability of gC is further improved.
The RiCBE editing system described in this invention can be used to explore the function of mitochondrial or nuclear DNA mutations. Meanwhile, the tool of the invention can be used for repairing mutant DNA on cells.
The working principle of the invention is as follows: after the RiCBE system is transfected into cells, the target site can be edited from C/G to T/A, the target site C is deaminated to form dU mainly through double-stranded DNA cytosine deaminase, and the transformation from C/G to T/A is further realized through the replication of the cells.
To achieve C/G to T/A editing of mitochondrial DNA, TALE-DddA tox reported BY Mok BY et al can achieve C/G to T/A editing in the mitochondrial DNA "tC" motif (Nature.2020)
Jul;583 (7817): 631-637.) the DNA editing system RiCBE developed by the present invention enables efficient C/G to T/A editing specifically for C in mitochondrial DNA "gC" sequences.
The invention has the beneficial effects that:
The main innovation value of the invention is to provide a novel double-stranded DNA deaminase DddA structural domain and a related editing system, which can realize high-efficiency and accurate editing specifically aiming at C in a double-stranded DNA gC motif. Some editing systems disclosed in the prior art are directed to NC (AC, TC, CC, GC), including gC, which can cause a large number of side edits within the editing window; riCBE of the present invention is directed to gC only and does not result in a large number of side edits.
Drawings
FIG. 1RiCBE is a schematic diagram of the operation of the editing system.
FIG. 1A, DNA double stranded cytosine deaminase SA071 metagenome mining flow chart; FIG. 1B, alignment of amino acid sequence homology of Roseburia_intestinalis-derived DddA domain SA071 with DddAtox and mutants DddAtox-V6, dddAtox-V11 thereof in Burkholderia cenocepacia genus; FIG. 1C, a schematic diagram of the operation of the RiCBE system; FIG. 1D, a schematic representation of the operation of the DNA double stranded cytosine deamination protein DddA domain in the absence of TALE localization.
FIG. 2DddA-V11, riCBE edits MTS and NLS backbone vectors of the system.
FIG. 2A shows various split types DddA-V11 with mitochondrial localization signals (MTS) and Nuclear Localization Signals (NLS), the vector carrying the ccdb gene element, ccdb located between two Bsa I cleavage sites, the vector carrying an ampicillin resistance element; FIG. 2B shows a RiCBE editing system with different partition types of mitochondrial localization signals (MTS) and Nuclear Localization Signals (NLS), the vector carrying ccdb gene elements, ccdb located between two Bsa I cleavage sites, and the vector carrying ampicillin resistance elements.
FIG. 3T-pool screening strategy.
All motif characteristics of 6xNNC, 6xNCN, 6xCNN are covered in T-pool, as well as JAK2, SIRT6 two positive control T vectors.
FIG. 4T-pool of SA071 and DdA, ddA-V6, dddA-V11 editing cases
FIG. 4A screens out the editing efficiency of SA071 and four different NC-motifs of DddA, dddA-V6, dddA-V11 by T-Pool; FIG. 4B shows the ratio of four different NC-motifs from SA071 and DddA, dddA-V6, dddA-V11 by T-Pool screening.
FIG. 5DddA-V11 and RiCBE editing System edits GC sites on mitochondria.
Sequence information of mitochondrial m.C1634T locus editing window is shown in FIG. 5A, and the editing conditions of DddA-V11 and RiCBE of different combinations on all sequences in the m.C1634T locus window are shown in FIG. 5A; sequence information of mitochondrial m.G7486A site editing window is shown in FIG. 5B, and the editing conditions of DddA-V11 and RiCBE of different combinations on all sequences in m.G 74886A site window are shown in FIG. 5B; FIG. 5C shows sequence information of mitochondrial m.G3255A site editing window, and FIG. 5C shows the editing of all sequences in m.G3255A site window by DddA-V11 and RiCBE in different combinations; sequence information of mitochondrial m.G14831A site editing window is shown in FIG. 5D, and the editing conditions of DddA-V11 and RiCBE of different combinations on all sequences in m.G14831A site window are shown in FIG. 5D; sequence information of mitochondrial m.C8393T site editing window is shown in FIG. 5E, and the editing conditions of DddA-V11 and RiCBE of different combinations on all sequences in the m.C8393T site window are shown in FIG. 5E; the sequence information of the mitochondrial m.G9139A site editing window is shown in FIG. 5F, and the editing conditions of DddA-V11 and RiCBE of different combinations on all sequences in the m.G9139A site window are shown in FIG. 5F.
FIG. 6DddA-V11 vs RiCBE comparison of the editing system at mitochondrial site m.C1634T, m.G7486A, m.G3255A, m.G14831A, m.C8393T and m.G9139A edits.
FIG. 7DddA-V11 compares the average off-target rate of whole mitochondria edited by the RiCBE editing system at mitochondrial locus m.C1634T, m.G7486A, m.G3255A, m.G14831A, m.C8393T and m.G 9139A.
FIG. 8DddA-V11 compares the number of total mitochondrial destarget sites edited by the RiCBE editing system at mitochondrial locus m.C1634T, m.G7486A, m.G3255A, m.G14831A, m.C8393T and m.G 9139A.
Fig. 9DddA-V11, dddA, riCBE edit system each split combination was compared against mitochondrial edits without TALE localization.
FIG. 10DddA-V11 and RiCBE edit system nuclear off-target contrast cases.
FIG. 10A is a diagram of a combination of DddA-V11 and RiCBE editing systems. FIG. 10B is a sequence of a mitochondrial site m.C8393T TALE recognition and editing window and a highly similar sequence thereof on the nuclear genome, and FIG. 10B is a comparison of DddA-V11 with the nuclear off-target condition of the RiCBE editing system when the mitochondrial site m.C8393T is edited; FIG. 10C is a sequence of a mitochondrial site m.G7486A TALE recognition and editing window and a highly similar sequence thereof on the nuclear genome, and FIG. 10C is a comparison of DddA-V11 with the nuclear off-target condition of the RiCBE editing system when editing at mitochondrial site m.G 7486A; FIG. 10D shows the mitochondrial site m.G9139A TALE recognition and editing window sequence and its highly similar sequence on the nuclear genome, and FIG. 10D shows the comparison of DddA-V11 with the nuclear off-target condition of the RiCBE editing system when editing the mitochondrial site m.G 9139A; FIG. 10E shows the mitochondrial site m.C1624T TALE recognition and editing window sequence and its highly similar sequence on nuclear genome chromosome 3, and FIG. 10E shows the comparison of DddA-V11 with the nuclear chromosome 3 off-target condition of the RiCBE editing system when the mitochondrial site m.C1624T is edited; FIG. 10F shows the mitochondrial site m.C1624T TALE recognition and editing window sequence and its highly similar sequence on chromosome 5 of the nuclear genome, and FIG. 10F shows the comparison of DddA-V11 with the nuclear chromosome 5 off-target condition of the RiCBE editing system when the mitochondrial site m.C1624T is edited; on fig. 10G is a mitochondrial site m.c1624t TALE recognition and editing window sequence and its highly similar sequence on chromosome 11 of the nuclear genome, and on fig. 10G is a DddA-V11 comparison with the nuclear chromosome 11 off-target condition of the RiCBE editing system when mitochondrial site m.c1624t was edited.
FIG. 11 enhanced version RiCBE editing system
FIG. 11A, dddA, dddA, dddA11 and SA071 amino acid sequence alignment; FIG. 11B illustrates the editing of the SA071 original version and enhanced version on four NC motifs by the T-Pool strategy.
Detailed Description
The present invention will be described in detail with reference to examples, whereby the C/G to T/A editing of DNA is achieved and practiced with the present invention.
The application is further described below with reference to the accompanying drawings. The application provides a novel DNA editing tool: riCBE can implement C/G to T/a editing, the system comprising: there are 4 vectors located in mitochondria and 2 vectors located in nuclei. Editing mitochondrial localized RiCBE can be accomplished by selecting mitochondrial localized RiCBE can be accomplished by selecting nuclear localized mitochondrial DNA. RVD library any library compatible with the editing system can be selected, and the RVD library adopted in the embodiment of the application is shown in Cell discovery.2021Sep3; 78, the assembly method is as follows: mitochondrial DNA editing system based on TALE assembly, patent number ZL202110688797.9. Examples steps, reagents, modules, etc. not described in detail are well known to those skilled in the art, and the present application will not be repeated.
[ Example 1]
The construction method of the MTS-RiCBE and NLS-RiCBE backbone vectors of the cytosine editing system RiCBE is described as follows:
4 split protein pairs which are obtained by splitting a double-stranded DNA cytosine deaminase DddA domain derived from Roseburia_intetinalis into half DddA are respectively fused with a glycosylase inhibitor (UGI) and then respectively inserted between two cleavage sites of NheI and Pme I of the MTS-TALE backbone vector to respectively obtain 4 MTS-RiCBE backbone vectors;
The method comprises the following specific steps:
1. According to the amino acid homology comparison of SA071 and DddA and the secondary structure of protein, the SA071-G105 and SA071-N169 protein segmentation sites corresponding to DddA-G1333 and DddA-G1397 are selected for segmentation to obtain two protein segmentation pairs, and then gene fragments of different segmentation pairs are synthesized;
2. Designing a primer with 16bp, and carrying out PCR amplification on the synthesized gene fragment and a glycosylase inhibitor (UGI), so that one end of different protein gene fragments and one end of the glycosylase inhibitor (UGI) have 16bp homology arms, and the other ends of the two have Nhe I and Pme I enzyme cutting site homology arms respectively;
3. Selecting a vector with mitochondrial localization signals (MTS), nhe I and Pme I cleavage sites, ccdb toxin gene and ampicillin resistance gene for enzyme digestion to obtain a DNA skeleton fragment of which two ends are matched with a protein gene fragment and a glycosylase inhibitor (UGI) gene fragment respectively;
4. homologous recombination is carried out on the framework fragment, the protein gene fragment and the glycosylase inhibitor (UGI) gene fragment;
5. Transferring the product of the multi-fragment recombination into a competent cell trans DB3.1 with ccdb toxin resistance, screening by ampicillin, selecting a monoclonal to perform Sanger sequencing verification, and obtaining 4 MTS-RiCBE framework vectors with correct sequencing results: MTS-ccdb-G105N-UGI, MTS-ccdb-G105C-UGI, MTS-ccdb-N169N-UGI, MTS-ccdb-N169C-UGI;
According to the same method, 4 split protein pairs obtained by splitting the double-stranded DNA cytosine deaminase DddA domain derived from Roseburia_intestinalis into half DddA are fused with glycosylase inhibitor (UGI) respectively, and then inserted into the two cleavage sites of Nhe I and Pme I of NLS-TALE backbone vector respectively to obtain 2 NLS-RiCBE backbone vectors respectively: NLS-ccdb-N169N-UGI and NLS-ccdb-N169C-UGI.
The MTS-TALE backbone vector comprises the main elements from the 5 'end to the 3' end in sequence: mitochondrial localization signal (MTS) sequence, TALE-N-terminal (NTD) 136 amino acid nucleotide sequence, ccdb toxin gene, TALE-C-terminal (CTD) 41 amino acid nucleotide sequence, puromycin cytosolic screen gene, ampicillin resistance element; the NLS-TALE skeleton carrier comprises the main elements from the 5 'end to the 3' end in sequence: a Nuclear Localization Signal (NLS) sequence, a nucleotide sequence of 136 amino acids at the TALE-N end (NTD), a ccdb toxin gene, a nucleotide sequence of 63 amino acids at the TALE-C end (CTD), a puromycin cytospin gene, and an ampicillin resistance element.
The nucleotide sequence of the MTS-TALE framework vector is shown as SEQ ID NO. 8; the nucleotide sequence of the NLS-TALE skeleton vector is shown as SEQ ID NO. 9; the nucleotide sequence of the UGI encoding gene is shown as SEQ ID NO. 10.
All framework vectors are matched with a TALE assembly system, and Bsa I enzyme cleavage sites are respectively contained at two sides of a toxin gene ccdb of the framework vectors, and different sticky ends are generated after enzyme cleavage and are used for specifically connecting and identifying TALE sequences of specific DNA sequences.
[ Example 2]
Targeted T vectors were edited in human 293FT cells using RiCBE of the nuclear localized NLS version.
Design of TALE sequence and RiCBE Assembly
T vectors (6 xNNC, 6xNCN, 6xCNN, JAK2 and SIRT 6) containing different motifs in a Spacer are constructed, the same recognition sequences and base sequences with different lengths of 4bp are arranged at two ends of the Spacer to be used as follow-up splitting index, the T vectors (total 38) of the different motifs are mixed into T-Pool according to the number of nanograms, and the total concentration is measured. RVD modules (Cell discover.2021 Sep3;7 (1): 78) for identifying different DNA bases of the targeting T vector are respectively assembled on RsCBE framework vectors by a Golden Gate method, and the assembling method is disclosed in the issued patent: mitochondrial DNA editing system based on TALE assembly, patent number ZL202110688797.9.
The assembled product was transformed into DH 5. Alpha. And screened using solid LB plates containing ampicillin, after which the monoclonal was picked and PCR identified using the following primers, the sequences of which were as follows, seq-For: TGACCGCAGTGGAGGCAGTG (SEQ ID NO: 11); seq-Rev: TTCACTGCATCCAGCGCAGG (SEQ ID NO: 12). Based on the number of RVDs, positive clones were judged, for example 16 RVDs would be 1759bp in size and 15 RVDs would be 1657bp in size. Then, forward and reverse sequencing is carried out by using the primer, and plasmids are extracted from positive clones with correct sequences for subsequent experiments.
(II) detection of the edit efficiency of different combinations RiCBE by transfected cells
293FT cells were selected as the object of editing, and when the cell density reached 70-90%, the liquid was changed 2 hours in advance, and after cell counting, 1.5x10 5 cells were taken for cell electrotransfection (instrument: lonza
4D-nucleofector). The vectors of 2 NLS versions of Half-DddA were combined according to the principle of N169N and N169C pairing with T vectors containing different motif for electrotransfer of 293FT cells. 400ng of each vector was transfected at the left and right ends, and 50ng of T-Pool was used to electrotransfect cells using SF-Cell Line 4D-nucleofector X-kit.
After completion of electrotransfection, cells were inoculated into 12-well plates and cultured for 24 hours, followed by cell screening using puromycin containing 1. Mu.g/mL, and after culturing for 72 hours, the cells were digested and centrifuged, and the cells were harvested for detection of DNA mutation efficiency.
The DNA extraction of the harvested cell samples was as follows: cells were reselected with 30. Mu.L QuickExtract TM DNA Extraction Solution (Lucigen), followed by heating at 65℃for 45min and then 98℃for 2min.
Amplifying the target fragment containing the editing site on the T vector or mitochondrial DNA by using 2×Green Taq Mix (Vazyme), and then using for Sanger sequencing; or carrying out high-throughput sequencing analysis and editing efficiency by adopting Phanta Super-FIDELITY DNA polymerase (Vazyme) and high-throughput sequencing library-building primers. As shown in fig. 4, the results of high throughput sequencing show RiCBE that the system can efficiently edit for C in the "gC" motif.
[ Example 3]
Mitochondrial DNA was edited in human 293FT cells using the RiCBE version of MTS with mitochondrial localization.
The m.C1624T, m.G7486A, m.G3255A, m.G14831A, m.C8393T, m.G9139A locus on human mitochondrial DNA (reference sequence version: NC_ 012920.1) was selected for editing (as shown in the upper part of FIG. 5), the identified TALE sequences were designed based on the DNA sequences in the vicinity of these loci, and the RVD modules of the identified different DNA bases were assembled onto the backbone vector of 4 mitochondrially located RiCBE by the Golden Gate method, respectively.
The assembled product was transformed into DH 5. Alpha. And screened using solid LB plates containing ampicillin, after which the monoclonal was picked and PCR identified using the following primers, the sequences of which were set forth as Seq ID NO:11 and Seq ID NO: shown at 12. Based on the number of RVDs, positive clones were judged, for example 16 RVDs would be 1759bp in size and 15 RVDs would be 1657bp in size. Then, forward and reverse sequencing is carried out by using the primer, and plasmids are extracted from positive clones with correct sequences for subsequent experiments.
The 293FT cells were transfected with each of the four combinations of sites according to the same procedure as the following example 2, and cells were collected, and DNA was extracted to construct an amplicon library, and mutation efficiency analysis was performed. L-G105C+R-G105N as shown in FIGS. 5 and 6; L-G105N+R-G105C; L-N169N+R-N169C; L-N169C+R-N169N) (L is the left end, R is the right end), riCBE can efficiently edit against the endogenous "gC" locus of mitochondria, and the efficient and specific editing effect on C in the "gC" motif is shown. As shown in fig. 7 and 8, the average off-target rate and the number of off-target sites corresponding to editing of the "gC" sites are lower than DddA-V11, which indicates that the RiCBE editing system has high efficiency, specificity and high fidelity to the "gC".
[ Example 4]
Targeted T vectors were edited in human 293FT cells using enhanced version RiCBE of the nuclear localized NLS version.
Single amino acids were selected based on the alignment of dda, dda6, dddA and SA071 amino acid sequences (as shown in fig. 11A), mutated one by PCR amplification, and assembled as in example 1 (one).
The assembled product was transformed into DH 5. Alpha. And screened using ampicillin-containing solid LB plates, after which the monoclonal was picked up and sequenced using the following primer sequence CAGGGTCAAGGAAGGCAC (SEQ ID NO: 13), the positive clone with the correct sequence was used to extract the plasmid for the subsequent experiments.
The principle of pairing SA071-N169C with SA071-N169N-mut1 and SA071-N169N-mut2 was used for combined electrotransfection of 293FT cells according to the same procedure as in example 2 (II), and the cells were collected, DNA was extracted to construct amplicon libraries for mutation efficiency analysis.
As shown in FIG. 11B, enhanced versions RiCBE (SA 071-N169N-mut1 and SA071-N169N-mut 2) have significantly improved editing capabilities for "gC" compared to the original RiCBE version, revealing accurate and efficient "gC" editing of enhanced version RiCBE.
The foregoing description shows and describes embodiments of the invention, and in summary, the present invention provides a novel DNA editor: riCBE. Experiments prove that the novel DNA editor provided by the invention comprises: riCBE can successfully realize efficient and accurate editing aiming at C in the gC motif.

Claims (11)

1. A domain of double-stranded DNA cytosine deaminase DddA, characterized in that the DddA domain is a domain of double-stranded DNA cytosine deaminase DddA derived from Roseburia intestinalis, the amino acid sequence of which corresponds to positions 95-295 in the full-length sequence of the double-stranded DNA cytosine deaminase protein with the sequence number SAMEA3134499_ 274314.
2. The DddA domain according to claim 1, wherein the amino acid sequence of the DddA domain is as shown in SEQ ID NO. 1.
3. The split protein from the DddA domain of claim 1 or 2, wherein the split protein from the G47 site of the sequence shown in SEQ ID No. 1 is selected for split according to the amino acid structural characteristics of the DddA domain to provide a pair of half DddA split proteins, which are: G47N and G47C, and selecting N111 site of the sequence shown in SEQ ID NO. 1 according to the amino acid structural characteristics of the DddA domain to split to obtain a pair of half DddA split proteins, namely N111N and N111C; split proteins are used in pairs; the amino acid sequence of the split protein is as follows: the amino acid sequence of G47N is shown as SEQ ID NO. 2; the amino acid sequence of G47C is shown as SEQ ID NO. 3; the N111N amino acid sequence is shown as SEQ ID NO. 4; the N111C amino acid sequence is shown in SEQ ID NO. 5.
4. A split protein according to claim 3, wherein a single amino acid is mutated to increase the activity of the original protein, the amino acid sequence of the enhanced split protein being as follows:
the amino acid sequence of the N111N-mut1 is shown in SEQ ID NO. 6; the N111N-mut2 amino acid sequence is shown in SEQ ID NO. 7.
5. Use of the DddA domain of claim 1 or 2 or the split protein of claim 3 or4 in constructing a C/G to T/a edited cytosine editing system RiCBE.
6. Cytosine editing system RiCBE constructed using the DddA domain of claim 1 or 2 or the split protein of claim 3 or 4.
7. The cytosine editing system RiCBE as claimed in claim 6 comprising a RVD library, a mitochondrial localization backbone vector or a nuclear localization backbone vector, wherein the cytosine editing system RiCBE for effecting efficient and accurate C/G to T/a editing of mitochondrial DNA only for C in the "gC" motif employs a MTS-RiCBE backbone vector; the cytosine editing system RiCBE for realizing efficient and accurate C/G to T/A editing of the specificity of the nuclear DNA only for C in the "gC" motif adopts NLS-RiCBE backbone vector.
8. The cytosine editing system RiCBE as claimed in claim 7 wherein the 4 MTS-RiCBE backbone vectors are each: MTS-ccdb-G47N-UGI, MTS-ccdb-G47C-UGI, MTS-ccdb-N111N-UGI, MTS-ccdb-N111C-UGI; the 2 NLS-RiCBE framework vectors are respectively: NLS-ccdb-N111N-UGI and NLS-ccdb-N111C-UGI.
9. Use of the cytosine editing system RiCBE as defined in any one of claims 6-8 for efficient and accurate editing of C/G to T/a with respect to C in a "gC" motif only, with specificity of mitochondrial DNA or nuclear DNA.
10. Use of the DddA domain of claim 1 or 2, or the split protein of claim 3 or 4, or the cytosine editing system RiCBE of any one of claims 6-8 in a kit for preparing a highly efficient, accurate C/G to T/a editing of mitochondrial DNA or nuclear DNA specific for C in the "gC" motif only.
11. A kit for efficient and accurate C/G to T/a editing of mitochondrial DNA or nuclear DNA specific for C in the "gC" motif only, characterized in that the kit comprises the DddA domain of claim 1 or 2, or the split protein of claim 3 or 4, or the cytosine editing system RiCBE of any one of claims 6-8.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114736893A (en) * 2022-03-04 2022-07-12 南京医科大学 Method for realizing A/T to G/C editing on mitochondrial DNA
WO2022155265A2 (en) * 2021-01-12 2022-07-21 Mitolab Inc. Context-dependent, double-stranded dna-specific deaminases and uses thereof
WO2022178124A1 (en) * 2021-02-17 2022-08-25 The Curators Of The University Of Missouri Chloroplast cytosine base editors and mitochondria cytosine base editors in plants
CN116004592A (en) * 2022-11-18 2023-04-25 南京医科大学 RsCBE system for realizing C/G to T/A editing on DNA

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022155265A2 (en) * 2021-01-12 2022-07-21 Mitolab Inc. Context-dependent, double-stranded dna-specific deaminases and uses thereof
WO2022178124A1 (en) * 2021-02-17 2022-08-25 The Curators Of The University Of Missouri Chloroplast cytosine base editors and mitochondria cytosine base editors in plants
CN114736893A (en) * 2022-03-04 2022-07-12 南京医科大学 Method for realizing A/T to G/C editing on mitochondrial DNA
CN116004592A (en) * 2022-11-18 2023-04-25 南京医科大学 RsCBE system for realizing C/G to T/A editing on DNA

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