CN117106758A - RiCBE system for realizing C/G to T/A editing specifically on gC motif of DNA - Google Patents

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

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CN117106758A
CN117106758A CN202311075175.4A CN202311075175A CN117106758A CN 117106758 A CN117106758 A CN 117106758A CN 202311075175 A CN202311075175 A CN 202311075175A CN 117106758 A CN117106758 A CN 117106758A
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ricbe
editing
domain
amino acid
ddda
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CN117106758B (en
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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 application 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 division protein of the DddA domain of the double-stranded DNA cytosine deaminase from Roseburia_intestinalis to construct a skeleton carrier, can realize the efficient editing from C/G to T/A of C in gC motif, and has low off-target rate. The application 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 specifically on gC motif of DNA
Technical Field
The application belongs to the technical field of gene editing, and relates to a RiCBE editing system which can efficiently and accurately edit 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.
DddA domain of bacterial toxin protein from Burkholderia cenocepacia, i.e., dddA tox With double-stranded DNA deaminase activity, site-directed C/G to T/A mutations in mitochondria can be achieved by ligation to TALE to form a cytosine base editor (DdBE). However, the original version of DdBE prefers to edit C in the "tC" motif on mtDNA, and versions of DdBE mutants V6 and V11 obtained by phage evolution improve editing efficiency, widen editing sequence preference, but also cause serious bystander mutation in an editing window, and cannot realize accurate editing.
Disclosure of Invention
The application aims at overcoming the defects of the prior art, and provides a precise and efficient DNA cytosine editing system RiCBE only aiming at a gC motif, which can realize the precise and efficient editing of C in the gC motif.
It is a further object of the application to provide an application of the system.
It is a further object of the present application to provide a method for effecting C/G to T/A editing on mitochondrial DNA using the system.
The aim of the application can be achieved by the following technical scheme:
a double-stranded DNA cytosine deaminase DddA domain is derived from Roseburia_intestinalis, and has a gene sequence number of SAMEA3134499_274314 and a gene name of SA071.
The amino acid sequence of the DddA structural domain of the cytosine deaminase is shown as SEQ ID NO. 1.
The split proteins from the dda domain are split at the G105 site according to the amino acid structural features of the dda domain to obtain a pair of half dda split proteins, which are: G105N and G105C, and a pair of split proteins of half DddA, N169N and N169C, are obtained by splitting at the N169 site according to the amino acid structural characteristics of the domain of DddA; 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 dda domain or said split protein in the construction of a C/G to T/a edited cytosine editing system RiCBE.
Cytosine editing system RiCBE constructed using the dda 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 high-efficiency and accurate editing of C/G to T/A 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 high-efficiency and accurate editing of C/G to T/A only aiming at C in a gC motif adopts NLS-RiCBE skeleton carrier.
The cytosine editing system RiCBE, wherein the 4 MTS-RiCBE framework vectors are respectively: 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 nucleus 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 said dda domain, or said split protein, or said cytosine editing system RiCBE in a kit for preparing mitochondrial DNA or nuclear DNA with specificity for efficient and accurate C/G to T/a editing only for C in the "gC" motif.
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, the kit comprising the dda domain, or the split protein, or the cytosine editing system RiCBE.
Further description:
the application provides a cytosine editing system RiCBE for realizing C/G to T/A editing of DNA, which is characterized in that a newly discovered double-stranded DNA cytosine deaminase DddA structural domain from Roseburia_intestinalis genus, the gene sequence number is SAMEA3134499_274314, the gene name is SA071, and DddA of SA071 is fused through TALE tox The split protein and glycosylase inhibitor (Uracil glycosylase inhibitor, UGI) form a RiCBE system, which can achieve high C content in the gC motif of the target siteAnd (5) effective and accurate editing.
This domain was not disclosed as a dda domain, but its entire protein sequence was found in 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 DddA of Burkholderia cenocepacia tox The similarity of the amino acid sequences is only 42.857%.
The application cuts DddA domain (95-295 amino acids) in SA071 from Roseburia_intestinalis at 105 th amino acid and 169 amino acids to generate 4 Half DddA, respectively fuses Half DddA and glycosylase inhibitor (UGI) through linker amino acid sequences and inserts the fused Half DddA and glycosylase inhibitor (UGI) 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).
The application provides a cytosine editing system RiCBE for realizing C/G to T/A editing of DNA, which 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 application 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 No. ZL202110688797.9.
The RiCBE editing system is used for the application from C/G to T/A editing of double-stranded DNA, especially the application for editing mitochondrial DNA, and is specific and efficient for editing only C in a gC motif.
The application selects the corresponding 2 RiCBE vectors in the system of the application to pair according to the pairing principle of G105N and G105C and the pairing principle of N169N and N169C, and transfects the corresponding 2 RiCBE 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 application 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 of the application can be used for exploring the functions of mitochondrial or nuclear DNA mutations. Meanwhile, the tool of the application can be used for repairing mutant DNA on cells.
The working principle of the application 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 reported BY Mok BY et al tox C/G to T/A editing in the mitochondrial DNA "tC" motif can be achieved (Nature.2020
Jul;583 (7817): 631-637.) the DNA editing system RiCBE developed by the present application enables efficient C/G to T/A editing specifically for C in mitochondrial DNA "gC" sequences.
The application has the beneficial effects that:
the application has main innovation value of providing a novel double-stranded DNA deaminase DddA structural domain and a related editing system, and 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 an editing window; the RiCBE of the present application is directed to gC only and does not result in a significant amount of side editing.
Drawings
FIG. 1 is a schematic diagram of the operation of the RiCBE editing system.
FIG. 1A, DNA double stranded cytosine deaminase SA071 metagenome mining flow chart; FIG. 1B, alignment of DddA domain SA071 from Roseburia_intestinalis with DddAtox and its mutants DddAtox-V6, dddAtox-V11 amino acid sequence homology 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 DddA domain in the absence of TALE localization.
FIG. 2 shows the MTS and NLS backbone vectors of DddA-V11, riCBE editing system.
FIG. 2A shows different split types of DddA-V11 with mitochondrial localization signals (MTS) and Nuclear Localization Signals (NLS), with the vector carrying the ccdb gene element, ccdb located between two Bsa I cleavage sites, with the ampicillin resistance element on the vector; FIG. 2B shows different split types of RiCBE editing systems with mitochondrial localization signals (MTS) and Nuclear Localization Signals (NLS), with a vector carrying ccdb gene elements, ccdb located between two Bsa I cleavage sites, with ampicillin resistant elements on the vector.
FIG. 3 is a T-pool screening strategy.
All motif characteristics of 6xNNC, 6xNCN, 6xCNN, and two positive control T vectors of JAK2 and SIRT6 are covered in the T-pool.
FIG. 4 shows the editing of SA071, ddA, dddA-V6 and DddA-V11 in T-pool
FIG. 4A screens SA071 and four different NC-motif editing efficiency conditions of DddA, dddA-V6, and DddA-V11 by T-Pool; FIG. 4B shows the ratio of four different NC-motifs from SA071, dddA-V6 and DddA-V11 by T-Pool screening.
FIG. 5 shows the editing of GC sites on mitochondria by DddA-V11 and RiCBE editing system.
FIG. 5A shows sequence information of mitochondrial m.C1634T locus editing window, and FIG. 5A shows editing conditions of DddA-V11 and RiCBE of different combinations on all sequences in m.C1634T locus window; FIG. 5B shows sequence information of mitochondrial m.G7486A site editing window, and FIG. 5B shows the editing of all sequences in m.G74886A site window by DddA-V11 and RiCBE in different combinations; 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; FIG. 5D shows sequence information of mitochondrial m.G14831A site editing window, and FIG. 5D shows the editing of all sequences in m.G14831A site window by DddA-V11 and RiCBE in different combinations; FIG. 5E shows sequence information of mitochondrial m.C8393T site editing window, and FIG. 5E shows the editing of all sequences in m.C8393T site window by DddA-V11 and RiCBE in different combinations; the sequence information of the mitochondrial m.G9139A site editing window is shown in FIG. 5F, and the editing of all sequences in the m.G9139A site window by different combinations of DddA-V11 and RiCBE is shown in FIG. 5F.
FIG. 6 is a comparison of DddA-V11 with RiCBE editing system editing at mitochondrial site m.C1634T, m.G7486A, m.G3255A, m.G14831A, m.C8393T and m.G9139A.
FIG. 7 is a graph showing the comparison of DddA-V11 and the average total mitochondrial miss rate of the RiCBE editing system at mitochondrial site m.C1634T, m.G7486A, m.G3255A, m.G14831A, m.C8393T and m.G 9139A.
FIG. 8 is a comparison of DddA-V11 and the number of total mitochondrial decoys edited by the RiCBE editing system at mitochondrial site m.C1634T, m.G7486A, m.G3255A, m.G14831A, m.C8393T and m.G 9139A.
FIG. 9 is a comparison of the editing of various segmentation combinations on mitochondria without TALE localization by DddA-V11, dddA and RiCBE editing systems.
FIG. 10 is a comparison of nuclear off-target of DddA-V11 with RiCBE editing systems.
FIG. 10A shows the 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 nuclear off-target conditions of the RiCBE editing system when editing at mitochondrial site m.C 8393T; FIG. 10C is a sequence of a mitochondrial site m.G748ia TALE recognition and editing window and its highly similar sequence on the nuclear genome, and FIG. 10C is a comparison of DddA-V11 with nuclear off-target conditions of the RiCBE editing system when editing at mitochondrial site m.G748ia; FIG. 10D is a sequence of a mitochondrial site m.G9139A TALE recognition and editing window and a highly similar sequence thereof on the nuclear genome, and FIG. 10D is a comparison of DddA-V11 with nuclear off-target conditions of the RiCBE editing system when editing at 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 nuclear chromosome 3 off-target condition of the RiCBE editing system when editing at mitochondrial site m.C1624T; 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 editing at mitochondrial site m.C1624T; on FIG. 10G is a mitochondrial site m.C1624T TALE recognition and editing window sequence and its highly similar sequence on nuclear genome chromosome 11, and on FIG. 10G is a comparison of DddA-V11 with nuclear chromosome 11 off-target for the RiCBE editing system at mitochondrial site m.C1624T.
FIG. 11 is an enhanced version RiCBE editing system
FIG. 11A, amino acid sequence alignment of DddA, dddA6, dddA11 and SA 071; 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 application 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 application.
The application is further described below with reference to the accompanying drawings. The application provides a novel DNA editing tool: riCBE may implement C/G to T/A editing, the system comprising: there are 4 vectors located in mitochondria and 2 vectors located in nuclei. The selection of mitochondrial localized ricba can effect editing of mitochondrial DNA and the selection of nuclear localized ricba can effect editing on the nuclear genome.
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 No. 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 MTS-RiCBE and NLS-RiCBE skeleton vector of 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_intestinalis into half DddA are respectively fused with glycosylase inhibitors (UGI) and then respectively inserted into two cleavage sites of NheI and PmeI of the MTS-TALE skeleton vector to respectively obtain 4 MTS-RiCBE skeleton vectors;
the method comprises the following specific steps:
1. according to the amino acid homology comparison of SA071 and DdA and the secondary structure of the 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 multi-fragment recombined product 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 a double-stranded DNA cytosine deaminase DddA domain derived from Roseburia_intestinalis into half DddA are fused with glycosylase inhibitors (UGI) respectively, and then inserted between 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 nuclear localized NLS versions of RiCBE.
Design of the assembly of TALE sequence and RiCBE
T vectors (6 xNNC, 6xNCN, 6xCNN, JAK2 and SIRT 6) containing different motifs in the 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 Sep 3;7 (1): 78) for identifying different DNA bases of the targeting T vector are respectively assembled on the RsCBE skeleton vector by a Golden Gate method, and the assembling method is disclosed in the issued patent: mitochondrial DNA editing system based on TALE assembly, patent No. 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) transfection of cells to detect the editing efficiency of different combinations of RiCBE
Selecting 293FT cells as editing target, changing liquid 2 hr before cell density reaches 70-90%, counting, and collecting 1.5x10 5 Individual cells were used for cell electrotransfection (instrument: lonza 4D-nucleic acid). The vectors of 2 NLS versions of Half-DddA were combined with T vectors containing different motif according to the principle of N169N and N169C pairing to electrotransfer 293FT cells. Each of the left and right vectors was transfected with 400ng, and 50ng of T-Pool was used to electrotransfect cells using SF-Cell Line 4D-nucleic acid 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: 30. Mu.L of QuickExract was used TM DNA Extraction Solution (Lucigen) reselects cells, followed by heating at 65℃for 45min and then 98℃for 2min.
The target fragment containing the editing site on the T vector or mitochondrial DNA was amplified using 2 XPreen Taq Mix (Vazyme) and then used for Sanger sequencing; or high throughput sequencing analysis editing efficiency is carried out by using 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 that the RiCBE system allows efficient editing of C in the "gC" motif.
[ example 3 ]
Mitochondrial DNA was edited in human 293FT cells using mitochondrial localized MTS versions of RiCBE.
The m.C1624T, m.G7486A, m.G3255A, m.G14831A, m.C8393T, m.G9139A site 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 sequence was designed based on the DNA sequence in the vicinity of these sites, and 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 be efficiently edited 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 figures 7 and 8, the RiCBE editing system is lower than DddA-V11 aiming at the average off-target rate and the number of off-target sites corresponding to the editing of the 'gC' sites, which shows the characteristics of high efficiency, specificity and high fidelity of the RiCBE editing system to the 'gC'.
[ example 4 ]
Targeted T vectors were edited in human 293FT cells using the enhanced version of RiCBE of the nuclear localized NLS version.
Single amino acids were selected based on the amino acid sequence alignment of dda, dda6, dda11 and SA071 (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 primers with the sequence CAGGGTCAAGGAAGGCAC (SEQ ID NO: 13), and the plasmid was extracted from the positive clone with the correct sequence 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, the enhanced versions RiCBE (SA 071-N169N-mut1 and SA071-N169N-mut 2) have significantly improved editing capabilities specific to "gC" compared to the original RiCBE version, revealing accurate and efficient editing of "gC" by the enhanced version RiCBE.
The foregoing description shows and describes embodiments of the application, and in summary, the present application provides a novel DNA editor: riCBE. Experiments prove that the novel DNA editor provided by the application comprises: riCBE can successfully realize high-efficiency and accurate editing aiming at C in a gC motif.

Claims (12)

1. A double-stranded DNA cytosine deaminase DddA domain is characterized in that the DddA domain is a double-stranded DNA cytosine deaminase DddA domain derived from Roseburia_intestinalis, and the gene sequence number is SAMEA3134499_274314, and the gene name is SA071.
2. The cytosine deaminase dda domain as defined in claim 1 wherein the amino acid sequence of the dda domain is as set forth in SEQ ID No. 1.
3. A split protein from the dda domain of claim 1, wherein the split protein is selected to split at the G105 site according to the amino acid structural characteristics of the dda domain to provide a pair of half dda, which is: G105N and G105C, and a pair of split proteins of half DddA, N169N and N169C, are obtained by splitting at the N169 site according to the amino acid structural characteristics of the domain of DddA; split proteins are used in pairs.
4. A split protein according to claim 3, wherein 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.
5. The split protein of claim 4, wherein the single amino acid is mutated to increase the activity of the original protein, wherein the amino acid sequence of the enhanced split protein is as follows:
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.
6. Use of the dda domain of claim 1, 2 or the split protein of claim 3, 4, 5 for constructing a C/G to T/a edited cytosine editing system RiCBE.
7. Cytosine editing system RiCBE constructed using the dda domain of claim 1, 2 or the split protein of claim 3, 4, 5.
8. The cytosine editing system RiCBE according to claim 7, 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 specific for only C in the "gC" motif employs a MTS-RiCBE backbone vector; the cytosine editing system RiCBE for realizing high-efficiency and accurate editing of C/G to T/A only aiming at C in a gC motif adopts NLS-RiCBE skeleton carrier.
9. The cytosine editing system RiCBE according to claim 8, wherein the 4 MTS-RiCBE backbone vectors are each: 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.
10. Use of the cytosine editing system RiCBE as defined in claims 7-9 for efficient and accurate C/G to T/a editing of only C in the "gC" motif with specificity of mitochondrial DNA or nuclear DNA.
11. Use of the dda domain of claim 1, 2, or the split protein of claim 3, 4, 5, or the cytosine editing system RiCBE of claim 7,8, 9 in a kit for preparing mitochondrial DNA or nuclear DNA with specificity for efficient and accurate C/G to T/a editing of C in the "gC" motif only.
12. 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 it contains the DddA domain according to claim 1, 2, or the split protein according to claim 3, 4, 5, or the cytosine editing system RiCBE according to claim 7,8, 9.
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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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