Cytosine deamination editor and editing system

Abstract

The invention relates to the field of genome editing, in particular to a cytosine deamination editor and an editing system. The cytosine deamination editor is formed by sequentially connecting a deaminase evoCDA1, an XTEN sequence and an enUn1Cas12f1 protein from an N end to a C end, and the amino acid sequences of the deaminase evoCDA1 and the enUn1Cas12f1 protein are sequentially shown as SEQ ID NO. 4 and SEQ ID NO. 5. The cytosine deamination editor has the advantages that the cytosine deamination editor can be used for preparing the cytosine deamination editor, and the cytosine deamination editor can be used for preparing the cytosine deamination editor; compared with EnAsCas12f and enUn1Cas12f with the best performance at present, higher double-chain cutting activity is shown, C-to-T editing of a targeted chain can be achieved, an editing window far away from a PAM end is provided, and compared with an editing window of an existing editor, the application scene of an editing tool is expanded.

Description

Technical Field

[0001] The present invention relates to the field of genome editing, and in particular to a cytosine deamination editor and an editing system. Background Art

[0002] The emergence of base editing tools has significantly broadened the application of the CRISPR / Cas system, from the initial gene knockout and gene regulation to more precise base-level editing. Currently, base editing is mainly achieved through tools such as deaminases and glycosylases, which support base conversion or transversion. Because these enzymes usually prefer to act on single-stranded DNA and combine with the R-loop structure formed by Cas9 during the targeting process, existing base editors mainly edit bases on the non-targeted strand (NTS), including the classic deaminase base editing tools BE4max and ABE8e, which can achieve C-to-T and A-to-G conversions, respectively. It is worth noting that DddA deaminase, as a double-stranded deaminase, is different in editing mode.

[0003] In addition, glycosylase base editors such as gGBE, AYBE, CGBE, and gTBE cut the target base in the non-targeted strand (NTS) to generate an apurinic (AP) site, and use the DNA repair mechanism to achieve base conversion or transversion, including C-to-G, T-to-S, A-to-Y, and G-to-Y editing. However, the existing base editing tools still lack a tool that can efficiently achieve G-to-A editing on non-targeted strands.

[0004] Previous studies have found that base editors based on inactivated SpCas9 (dSpCas9) occasionally have rare off-target activity, causing C-to-T editing on the template strand (TS), thereby indirectly leading to G-to-A changes on the non-target strand. However, conventional base editing systems usually use Cas9Nickase mutants, under which conditions DNA repair is more likely to use the non-target strand as a template, making these off-target edits difficult to detect after repair. Summary of the invention

[0005] In view of the limitation that existing cytosine base editing tools cannot achieve non-targeted chain G-to-A editing, the present invention provides a cytosine deamination editor and editing system.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] In the first aspect, the present invention provides a cytosine deamination editor, which is composed of a deaminase evoCDA1, an XTEN sequence and an enUn1Cas12f1 protein connected in sequence from the N-terminus to the C-terminus, and the amino acid sequences of the deaminase evoCDA1, the XTEN sequence and the enUn1Cas12f1 protein are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively. Compared with the existing Un1Cas12f1 protein (NCBI accession number is A0A482D308.1), the enUn1Cas12f1 has 8 amino acid mutations, which are 143R, 244R, 393R, 437R, 447R, 331E, 147R, and 203R, respectively.

[0008] The present invention can effectively increase the stability of the protein-nucleic acid complex and thus enhance its editing efficiency by combining amino acid mutations. The optimized enUn1Cas12f1 has the ability to efficiently cut double-stranded DNA, and the optimized protein can be combined with the evoCDA1 deaminase with efficient cytosine deamination ability to achieve preferential C-to-T editing of the TS chain.

[0009] As a preferred cytosine deamination editor described in the present invention, the amino acid at position 473 of the enUn1Cas12f1 protein also has an alanine mutation. Further alanine mutation of the amino acid at position 473 of enUn1Cas12f1 can achieve more efficient C-to-T editing of the target site targeting chain and efficient G-to-A editing of the non-targeting chain, thereby reducing the amino acid mutation of the C-to-T editing efficiency of the non-targeting chain, so as to achieve an improvement in the purity of the editing product.

[0010] As a further preferred embodiment of the cytosine deamination editor of the present invention, the C-terminus of the cytosine deamination editor is further connected to a UGI, whose amino acid sequence is shown in SEQ ID NO. 3. The cytosine deamination editor has the ability to edit the C-to-T of the target site targeting chain and an editing window far away from the PAM end, which effectively expands the application scope of existing base editing tools.

[0011] As a further preference of the cytosine deamination editor of the present invention, the cytosine deamination editor further comprises a DNA binding protein, the DNA binding protein is HMG-D protein, its encoding gene sequence is shown in SEQ ID NO.7, and the DNA binding protein is located between the enUn1Cas12f1 protein and the deaminase evoCDA1.

[0012] By adding HMG-D protein to the cytosine deamination editor, higher targeted chain editing efficiency and editing product purity can be obtained, and the editing window range does not change. With the help of amino acid mutations near the PAM region of enUn1Cas12f1, the PAM recognition sequence can be extended from TTTR to NTTR.

[0013] In a second aspect, the present invention provides a cytosine deamination editing system comprising the cytosine deamination editor, wherein the cytosine deamination editor is guided to the target editing site by sgRNA.

[0014] In a third aspect, the present invention provides a polynucleotide encoding the cytosine deamination editor or the cytosine deamination editing system.

[0015] In a fourth aspect, the present invention provides a vector comprising the polynucleotide.

[0016] In a fifth aspect, the present invention provides a cell comprising the vector.

[0017] In a sixth aspect, the present invention provides a gene editing pharmaceutical composition comprising the cytosine deamination editor, the cytosine deamination editing system, the polynucleotide, the vector or the cell.

[0018] Based on the fact that the cytosine deamination editor performs C-to-T editing on the targeted chain during deamination catalysis and indirectly causes G-to-A changes in the non-targeted chain, the pharmaceutical composition provided by the present invention is expected to further expand the application scenarios in this field and promote the widespread application of precision gene editing in disease treatment, basic research and other fields.

[0019] The present invention has the following beneficial effects:

[0020] Compared with the existing cytosine base tools, the cytosine deamination editor proposed in the present invention has a change in its target object, from editing the non-targeted chain to editing the targeted chain. The original base editing tool cannot achieve the editing of the non-targeted chain G-to-A, and the cytosine deamination editor of the present invention can be indirectly achieved through the C-to-T editing of the targeted chain. The editing window of the cytosine deamination editor of the present invention is located at 11-25nt away from the PAM end, which complements the editing window of the existing base editing tool. At the same time, the improved enUn1Cas12f1 used in the present invention itself has a higher DNA double-stranded cutting activity than the existing Cas12f tool, which expands the application potential of Cas12f, and because some amino acid mutations are located near the PAM sequence, the PAM recognition range of enUn1Cas12f1 is extended from TTTR to NTTR, with a wider targeting range. In summary, the development of this tool has expanded the application range of existing cytosine base editing tools and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of achieving targeted strand C-to-T editing for TSminiCBE.

[0022] Figure 2 This is a graph showing the editing efficiency of different combinations of deaminases and Un1Cas12f1 protein at two gene loci.

[0023] Figure 3 The comparison results of the editing efficiency of the improved enUn1Cas12f and EnAsCas12f. A is the comparison of the editing efficiency of each site in HEK293T cells; B is the editing efficiency statistics in HEK 293T cells; C is the comparison of the editing efficiency of each site in HeLa cells; D is the editing efficiency statistics in HeLa cells.

[0024] Figure 4 Evaluation of the average editing efficiency of TSminiCBE for 12 targets in different cell lines, A is HeLa cells, B is AGS cells.

[0025] Figure 5 Comparison of editing efficiency between HMG-TSminiCBE and TSminiCBE after adding DNA binding proteins. A is a schematic diagram of the editing tool after adding different DNA binding proteins; B is a comparison of editing efficiency at each site in HEK293T cells; C is the editing efficiency statistics of different editors in HEK 293T cells; D is the purity of editing products of different editors in HEK 293T cells; E is the Indel frequency statistics of different editors in HEK 293T cells.

[0026] Figure 6 This is an exploration of the target adjacent motif (PAM) sequence of enUn1Cas12f1 and HMG-TSminiCBE. A is a comparison of the editing efficiency of enUn1Cas12f1 and Un1Cas12f1 in the reporter system under the PAM of NTTR in HEK293T cells; B is the editing efficiency statistics of enUn1Cas12f1 and Un1Cas12f1 under the PAM of NTTR in the reporter system; C is a comparison of the editing efficiency of enUn1Cas12f1, Un1Cas12f1 and enAsCas12f at the endogenous gene loci under the PAM of NTTR in HEK 293T cells; D is the editing efficiency statistics of enUn1Cas12f1, Un1Cas12f1 and enAsCas12f under the PAM of NTTR; E is the editing efficiency of HMG-TSminiCBE under the PAM of NTTR in HEK 293T cells. DETAILED DESCRIPTION

[0027] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0028] The carrier sources involved in the following examples are:

[0029] pCMV-Un1Cas12f1 vector: Addgene, catalog number: 174820.

[0030] pGL3-U6-gRNA4.1 vector: Addgene, catalog number: 107721.

[0031] pCMV-enAsCas12f vector: Addgene, catalog number: 204637, which contains EnAsCas12f (or enAsCas12f) protein, for comparison, the effect is compared with the cytosine deamination editor of the present invention.

[0032] U6-AsCas12f-v4.1 vector: Addgene, catalog number: 204636.

[0033] Example 1: Combining different deaminases with Un1Cas12f1 protein to achieve C-to-T editing of targeted strands

[0034] Two sites of the RIT1 gene in the HEK 293T cell genome were used as target editing sites, and different deaminases were combined with the Un1Cas12f1 protein for C-to-T editing. Based on the characteristics of Un1Cas12f1 single RuvC domain completing DNA double-strand cleavage, the possible working mode of Un1Cas12f1-deaminase for C-to-T editing of TS strands was speculated, such as Figure 1 shown.

[0035] 1. Preparation of different deaminase and Un1Cas12f1 protein combinations and sgRNA expression vectors

[0036] 1. Construction of expression vectors for different deaminases and Un1Cas12f1 protein combinations

[0037] Based on the nucleotide sequence of Un1Cas12f1 protein (amino acid sequence such as SEQ ID NO.1) and the nucleotide sequence of deaminase (evoCDA1, hA3A, rAPOBEC, Anc689), XTEN (amino acid sequence such as SEQ ID NO.2) was used to connect at the N-terminus of Un1Cas12f1 protein. Homologous recombination was used to construct a recombinant vector, and XTEN and deaminase were inserted into pCMV-Un1Cas12f1 to obtain vectors Un1Cas12f1-evoCDA1, Un1Cas12f1-hA3A, Un1Cas12f1-rAPOBEC1, Un1Cas12f1-Anc689, wherein Un1Cas12f1 introduced D326A and D510A mutations to become nuclease inactive forms. Except for Un1Cas12f1-Anc689, which was connected to 2 UGI proteins (amino acid sequence such as SEQ ID NO.3) at the C-terminus, the remaining vectors were only connected to 1 UGI protein at the C-terminus. After all vectors are constructed, plasmids must be tested to confirm that there are no mutants before they can be used in downstream experiments.

[0038] The sequence of the evoCDA1 is shown in SEQ ID NO. 4, the NCBI accession number of the Anc689 is WYV95014.1, the NCBI accession number of hA3A is NP_001180218.1, and the NCBI accession number of rAPOBEC1 is WYV95015.1.

[0039] SEQ ID NO.1:MAKNTITKTLKLRIVRPYNSAEVEKIVADEKNNREKIAL EKNKDKVKEACSKHLKVAAYCTTQVERNACLFCKARKLDDKFYQKLRGQFPDAVFWQEISEIFRQLQKQAAEIYNQSLIELYYEIFIKGKGIANASSVEHYLSDVCYTRAAELFKNAAIASGLRSKIKSNFRLKELKNMKSGLPTTKSDNFPIPLVKQKGGQYTGFEISNHNSDFIIKIPFGRWQVKKEIDKYRPWEKFDFEQVQKSPKPISLLLSTQRRKRNKGWSKDEGTEAEIKKVMNGDYQTSYIEVKRGSKIGEKSAWMLNLSIDVPKIDKGVDPSIIGGIDVGVKSPLVCAINNAFSRYSISDNDLFHFNKKMFARRRILLKKNRHKRAGHGAKNKLKPITILTEKSERFRKKLIERWACEIADFFIKNKVGTVQMENLESMKRKEDSYFNIRLRGFWPYAEMQNKIEFKLKQYGIEIRKVAPNNTSKTCSKCGHLNNYFNFEYRKKNKFPHFKCEKCNFKENADYNAALNISNPKLKSTKEEP。

[0040] SEQ ID NO.2:SGSETPGTSESATPES。

[0041] SEQ ID NO.3:SGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKP ESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGS。

[0042] SEQ ID NO.4:STDAEYVRIHEKLDIYTFKKQFSNNKKSVSHRCYVLFEL KRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWVCKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMFQVKILHTTKSPAV。

[0043] 2. Construction of sgRNA expression vector targeting the target site

[0044] (1) Design and synthesize spacer oligonucleotides targeting two target sites (Table 1). The lowercase letters in the sequence represent the linker part.

[0045] Table 1 Spacer oligonucleotide sequences at different sites of RIT1 gene

[0046]

[0047]

[0048] (2) Prepare the Buffer used for annealing. The formula is shown in Table 2:

[0049] Table 2 Buffer composition

[0050] Reagents Dosage NaCl 0.08766g 10 mM Tris-HCl buffer (pH = 8.5) 0.2mL <![CDATA[ddH2O]]> 30mL

[0051] (3) Annealing the spacer oligonucleotide chain to obtain a spacer annealing product. The annealing system and procedure are shown in Tables 3 and 4.

[0052] Table 3 Annealing system

[0053] Reagents Dosage Oligonucleotide sequence forward (10μM) 1μL Oligonucleotide sequence reverse (10μM) 1μL Annealing Buffer 2μL <![CDATA[ddH2O]]> 6μL Overall system 10μL

[0054] Table 4 Annealing schedule

[0055] temperature time Number of cycles 95℃ 5min 1 95℃ 30s 1 85℃ down to 25℃ -1℃ / cycle 60cycles 25℃ to 5℃ -2℃ / cycle 10cycles 4℃ Forever 1

[0056] (5) Using the pGL3-U6-gRNA4.1 plasmid as a template, an expression plasmid with the target spacer sequence was obtained by enzyme ligation. The specific operation is as follows:

[0057] 1) Use F primer: 5'-agctaggtctccttttatttttttaaagaattctcgacctcgagacaaatg-3', R primer: 5'-tctctcggtctcagttgcattcctttctttgtttcgagggttactttc-3' to amplify pGL3-U6-gRNA4.1, linearize it and add Bsa I restriction site at the end. The amplification system and amplification program are shown in Tables 5 and 6.

[0058] Table 5pGL3-U6-gRNA4.1 plasmid PCR amplification system

[0059] Reagents Dosage 2×Phanta Flash Master Mix(Dye Plus) 12.5μL Template plasmid 1μL F(10μM) 0.5μL R(10μM) 0.5μL <![CDATA[ddH2O]]> 10.5μL Overall system 25μL

[0060] 2) Based on the linearized vector, a single restriction enzyme digestion reaction was performed using Bsa I enzyme to produce sticky ends.

[0061] Table 6 pGL3-U6-gRNA4.1 plasmid PCR amplification program

[0062]

[0063] 3) The backbone of the sgRNA expression plasmid after enzyme digestion was purified and recovered, and the spacer annealing product targeting the target site was connected to the backbone of the sgRNA expression plasmid at 16° C. overnight using T4 DNA Ligase. The connection system is shown in Table 7 below.

[0064] Table 7 Connection system

[0065]

[0066]

[0067] (6) The ligation product was transformed into DH5α competent cells, and a single clone was picked for Sanger sequencing the next day. The bacterial solution with the correct sequencing results was expanded and the plasmid was extracted. The obtained plasmid was the sgRNA expression plasmid targeting the target site.

[0068] 2. HEK 293T cell transfection to verify editing efficiency

[0069] Plasmid components targeting the same locus were transiently transfected and the editing efficiency was determined.

[0070] 1. Seeding Cells

[0071] The frozen HEK 293T cells were revived and cultured in a 10 cm culture dish, and 7 mL of complete culture medium (90% high-glucose DMEM + 10% fetal bovine serum + working concentration of penicillin-streptomycin) was added and cultured at 37°C and 5% CO2. When the cell density reached 90%, the cells were inoculated into a 24-well plate and continued to be cultured.

[0072] 2. Cell Transfection and Sorting

[0073] (1) When the cell density of the 24-well plate grows to 75% to 85%, use EZ transfection reagent according to the instructions to co-transfect the expression plasmids of each component into the cells. The transfection mixture system is shown in Table 8 below:

[0074] Table 8 Transfection mixed system

[0075] composition Dosage pCMV-Un1Cas12f1-Deaminase 900ng sgRNA expression plasmid 300ng EZ transfection reagent 2.5μL DMEM culture medium 100μL

[0076] (2) Let the mixed system stand at room temperature for 10 minutes.

[0077] (3) Add the transfection solution that has been mixed and allowed to stand to each well of cells.

[0078] (4) 8 h after transfection, remove the culture medium containing the transfection reagent and add 700 μL of complete culture medium.

[0079] (5) 72 hours after transfection, the culture medium was removed, and the cells in each well were washed with 200 μL PBS solution. The cells were then digested and collected into a 1.5 mL centrifuge tube, and the cell pellet was resuspended with 260 μL PBS solution.

[0080] (6) The resuspended cells were filtered to form a single cell suspension, which was added to a flow cytometer for FACS sorting, and 10,000 to 20,000 cells with the top 20% of GFP fluorescence intensity were collected.

[0081] 3. C-to-T editing efficiency detection

[0082] The collected cells were centrifuged and then added to a cell lysis solution for lysis. Phanta Super-Fidelity DNA Polymerase was used to amplify the DNA sequence containing the target site. The PCR reaction system and conditions are shown in Tables 9 and 10 below.

[0083] Table 9 PCR amplification system of DNA sequences containing target sites

[0084] composition Dosage Buffer 12.5μL dNTP Mix 0.5μL Primer F 0.5μL Primer R 0.5μL Phanta Super-Fidelity DNA Polymerase 0.5μL Cell lysis buffer 1-2 μL <![CDATA[ddH2O]]> Add to 25 μL

[0085] Table 10 PCR amplification program of DNA sequences containing target sites

[0086]

[0087] The PCR products were purified and recovered after specific band detection by agarose gel electrophoresis, and then subjected to Sanger sequencing and targeted deep sequencing and editing efficiency analysis. Figure 2 As shown, TS is marked in red and NTS is marked in black. The results show that the combination of Un1Cas12f1-evoCDA1 can effectively achieve G-to-A editing of the NTS chain (C-to-T editing of the TS chain).

[0088] Example 2: Comparison of editing efficiency between enUn1Cas12f1 and enAsCas12f

[0089] Based on the combination of computational simulation and saturation mutation strategy, this study found that the combination of 8 point mutations can greatly improve the cutting efficiency of Un1Cas12f1 at the eukaryotic cell level. The present invention named the mutant protein enUn1Cas12f1 (amino acid sequence such as SEQ ID NO.5). enAsCas12f is the Cas12f protein with the best editing efficiency at present. In order to illustrate that enUn1Cas12f1 developed by the present invention has better editing performance, this example compares the editing efficiency of enUn1Cas12f1 and enAsCas12f at the cellular level.

[0090] SEQ ID NO.5:MAKNTITKTLKLRIVRPYNSAEVEKIVADEKNNREKIAL EKNKDKVKEACSKHLKVAAYCTTQVERNACLFCKARKLDDKFYQKLRGQFPDAVFWQEISEIFRQLQKQAAEIYNQSLIELYYEIFIKGKGIANASSVEHYLSRVCYRRAAELFKNAAIASG LRSKIKSNFRLKELKNMKSGLPTTKSDNFPIPLVKQKGGQYRGFEISNHNSDFIIKIPFGRWQVKKEIDKYRPWEKFDFEQVRKSPKPISLLLSTQRRKRNKGWSKDEGTEAEIKKVMNGDYQ TSYIEVKRGSKIGEKSAWMLNLSIDVPKIDKGVDPSIIGGIDVGVKEPLVCAINNAFSRYSISDNDLFHFNKKMFARRRILLKKNRHKRAGHGAKNKLKPITILTEKSRRFRKKLIERWACEI ADFFIKNKVGTVQMENLESMKRKEDSYFNRRLRGFWPYARMQNKIEFKLKQYGIEIRKVAPNNTSKTCSKCGHLNNYFNFEYRKKNKFPHFKCEKCNFKENADYNAALNISNPKLKSTKEEP.

[0091] 1. Development of enUn1Cas12f1

[0092] 1. Obtain candidate mutation sites using computational simulation

[0093] (1) Preparation steps before mutation simulation in Discovery Studio 2019 software: After entering the software interface, first load the protein structure file (7l49), select the Prepare Protein option under the Macromolecules column, and pull down to select the Clean Protein operation. Enter the Change Forcefield option under the Simulation column, and pull down to select the CHARMm force field in Forcefield to assign the protein structure.

[0094] (2) Discovery Studio 2019 software mutation process: select all atoms in the entire protein, enter the Design Protein option under the Macromolecules column, select Calculation Mutation Energy (Stability) in Mutation Energy from the drop-down menu, select all amino acid sites, and select all amino acid mutation types to start the calculation. The results are output as an Excel file. The smaller the value, the more stable the structure after mutation.

[0095] 2. Saturation mutagenesis screening for positive single-point mutations

[0096] (1) A fluorescent reporter system was constructed for rapid characterization of editing efficiency at the cellular level. The reporter system architecture was CMV-BFP-P2A-Target-GFP, where the Target length before GFP was 31 bp, causing a frameshift mutation in GFP, which would not emit light under normal circumstances. After using Un1Cas12f1 to target and cut the target, the Indel produced by DNA repair would make the downstream GFP likely to return to the normal coding frame, which would emit light normally. The higher the cutting activity, the higher the GFP fluorescence ratio, which could be quickly detected using a flow cytometer.

[0097] (2) Saturation mutation screening step, using two degenerate bases KNB and MNB to cover 20 kinds of amino acids, for candidate mutation sites, using two degenerate bases KNB and MNB to construct a small amino acid mutation library, extracting plasmids and transfecting cells with the above-mentioned reporter system, and the specific transfection steps are consistent with Example 1. After 48 hours of transfection, digest the cells into a flow tube, and use a flow analyzer to perform GFP fluorescence ratio detection to know which mutation library in which amino acid site can improve the editing efficiency, and then split all the amino acid types contained in the library to construct a single point mutation plasmid, and perform transfection and fluorescence ratio analysis here to obtain a positive single point mutation that can improve the editing efficiency.

[0098] (3) Single-point mutation combinations were used to construct enUn1Cas12f1. Based on the single-point mutations obtained by saturation mutation screening, the energy of the combined mutations was calculated using Discovery Studio 2019 software, following the same process as above. The top-ranked mutation combinations were selected for experimental analysis, and the three-mutation combination with the highest efficiency improvement was selected. Un1Cas12f1 variants containing more mutation sites were randomly designed for fluorescent reporter system testing, and the variant with the best efficiency was selected as enUn1Cas12f1.

[0099] 2. Construction of gRNA4.1 and As-sgRNA vectors

[0100] Design and synthesize spacer oligonucleotides targeting 8 genes (Table 11), the lowercase letters in the sequence are the linker part (As-sgRNA replaces the linker sequence Forward with GAAC, and the U6-AsCas12f-v4.1 after enzyme digestion is used as the backbone, and the rest of the information remains unchanged). The subsequent connection, transformation, monoclonal sequencing and plasmid acquisition steps are consistent with Example 1.

[0101] Table 11 Spacer oligonucleotide sequences of eight target genes

[0102]

[0103]

[0104] 3. Cell transfection and sorting

[0105] A 24-well plate was used for cell transfection. The specific steps of HEK 293T cell culture and transfection were consistent with those in Example 1. The culture conditions and transfection steps of HeLa cells were consistent with those of HEK 293T cells.

[0106] 4. Editing efficiency test

[0107] The genome of the positive cells after lysis and sorting was obtained, and the steps of PCR amplification of the target fragment and library construction and sequencing were consistent with those in Example 1. The statistical results of the editing efficiency were as follows: Figure 3 As shown, enUn1Cas12f1 exhibited higher editing efficiency than EnAsCas12f in both HEK 293T and HeLa cell lines.

[0108] Example 3: Development of TSminiCBE and determination of editing efficiency

[0109] Based on the enUn1Cas12f1 developed in the above example, the evoCDA1 deaminase was combined to develop a TS chain base editor with higher editing activity. Alanine scanning mutations were performed on the domain that cuts the TS chain to construct a TSminiCBE editor with higher editing activity, and the editing efficiency of TSminiCBE was tested in multiple cell lines.

[0110] 1. Development of TSminiCBE

[0111] 1. enUn1Cas12f1 further improves editing efficiency

[0112] The 8 point mutations contained in enUn1Cas12f1 were introduced into the Un1Cas12f1-evoCDA1 vector constructed in Example 1 based on the homologous recombination method (while restoring the nuclease activity of Un1Cas12f1, A510D and A326D), and the editing efficiency was tested at 3 gene sites of HEK293T cells. The gene site information is as shown in the following table (Table 12).

[0113] Table 12 Nucleotide sequences targeting CBE genes

[0114]

[0115]

[0116] 2. Construction of TSminiCBE by Alanine Scanning Mutagenesis

[0117] For the domain (RuvC+TNB) that interacts with the TS chain, alanine scanning mutagenesis was performed to construct mutant proteins, and editing efficiency was tested at two gene sites in HEK 293T cells (gene site information is shown in Table 13 below), and it was determined that the 473A mutation can effectively improve the editing efficiency of the TS chain. Based on this, evoCDA1-enUn1Cas12f1-473A was determined to be TSminiCBE, and the amino acid sequence is shown in SEQ ID NO.6, which includes a signal peptide sequence and a linker sequence.

[0118] SEQ ID NO.6: MPKKKRKVSTDAEYVRIHEKLDIYTFKKQFSNNKKSVS HRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWVCKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMFQVKILHTTKSPAVSGSETPGTSESATPESPKKKRKVGIHGVPAAMAKNTITKTLKLRIVRPYNSAEVEKIVADEKNNREKIALEKNKDKVKEACSKHLKVAAYCTTQVERNACLFCKARKLDDKFYQKLRGQFPDAVFWQEISEIFRQLQKQAAEIYNQSLIELYYEIFIKGKGIANASSVEHYLSRVCYRRAAELFKNAAIASGLRSKIKSNFRLKELKNMKSGLPTTKSDNFPIPLVKQKGGQYRGFEISNHNSDFIIKIPFGRWQVKKEIDKYRPWEKFDFEQVRKSPKPISLLLSTQRRKRNKGWSKDEGTEAEIKKVMNGDYQTSYIEVKRGSKIGEKSAWMLNLSIDVPKIDKGVDPSIIGGIDVGVKEPLVCAINNAFSRYSISDNDLFHFNKKMFARRRILLKKNRHKRAGHGAKNKLKPITILTEKSRRFRKKLIERWACEIADFFIKNKVGTVQMENLESMKRKEDSYFNRRLRGFWPYARMQNKIEFKLKQYGIEIRKVAPNNTSATCSKCGHLNNYFNFEYRKKNKFPHFKCEKCNFKENADYNAALNISNPKLKSTKEEPKRPAATKKAGQAKKKKSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSPKKKRKV*。

[0119] Table 13 Nucleotide sequences targeting the DNMT1 gene and the HNRNPK gene

[0120]

[0121] 2. Testing editing efficiency in multiple cell lines

[0122] The gRNA4.1 vector used in HeLa and AGS cell lines was constructed, and the design principle was consistent with Example 1. The specific information is shown in Table 14 below, and the lowercase letters in the sequence are the linker parts. The subsequent construction process and plasmid acquisition were consistent with Example 1.

[0123] Table 14 Spacer oligonucleotide sequences at different sites in HeLa and AGS cell lines

[0124]

[0125]

[0126] AGS cells were cultured using RPM 1640 culture medium, and the rest of the culture methods were consistent with HeLa and HEK 293T cells. The cell transfection and sorting procedures were consistent with Example 1.

[0127] The steps of library construction, sequencing and editing efficiency detection were consistent with those in Example 1. The results were as follows: Figure 4 As shown, TSminiCBE can achieve an average editing efficiency of more than 30% in both cell lines, and the editing window is located after 11nt.

[0128] Example 4: Comparison of editing efficiency between HMG-TSminiCBE and TSminiCBE

[0129] Based on the DNA binding protein HMG-D (gene sequence as SEQ ID NO.7) and Ssod7, they were connected into TSminiCBE to construct an efficiency-enhanced version. Using the IRF6 and PCSK9 genes in the HEK 293T cell genome as target sites, three sgRNAs were designed to measure the editing efficiency of HMG-TSminiCBE and TSminiCBE.

[0130] SEQ ID NO.7: ATGAGCGACAAGCCAAAGAGACCTCTGAGCGCCTAC ATGCTGTGGCTGAACAGCGCTAGAGAATCCATCAAGCGCGAAAACCCCGGCATCAAGGTGACCGAGGTGGCCAAGCGGGGAGGCGAGCTGTGGCGGGCCATGAAGGACAAGTCTGAATGGGAGGCCAAGGCCGCTAAAGCCAAAGACGAC TACGACAGAGCCGTGAAGGAATTCGAGGCAAATGGCGGCAGCAGCGCCGCCAACGGCGGAGGCGCCAAGAAAAGAGCCAAGCCTGCTAAGAAGGTCGCCAAGAAGTCCAAGAAAGAGGAATCTGATGAGGACGATGATGACGAGAGCGAG.

[0131] 1. Construction of TSminiCBE vector with DNA binding protein

[0132] 1. Preparation of vectors with added DNA binding protein

[0133] Schematic diagram of TSminiCBE enhanced protein constructed based on HMG-D and Ssod7 protein Figure 5 As shown in A, the above vector was constructed using the homologous recombination method.

[0134] 2. Preparation of sgRNA vectors targeting three sites and determination of editing efficiency

[0135] Design and synthesize spacer oligonucleotide sequences targeting three sites (Table 15), where the lowercase letters in the sequence are linker parts. The annealing connection of gRNA4.1 vector and the subsequent plasmid extraction related experimental procedures are consistent with Example 1.

[0136] Table 15 Spacer oligonucleotide sequences targeting three target sites

[0137]

[0138] 3. Editing efficiency detection in HEK 293T cells

[0139] The HEK 293T cell transfection and sorting steps were consistent with those in Example 1. The sorted cells were lysed to obtain the genome and amplify the DNA sequence near the editing site. The PCR steps and procedures were consistent with those in Example 1. The amplified product library was constructed for deep sequencing, and the relevant steps were consistent with those in Example 1. The results are shown in Figure 5As shown, the addition of HMG-D can further improve the G-to-A editing efficiency of TSminiCBE for the NTS chain (C-to-T editing of the TS chain), and the HMG-DM (amino acid sequence such as SEQ ID NO.8, gene sequence such as SEQ ID NO.9) with HMG-D located between evoCDA1 and enUn1Cas12f1-473A has the highest editing efficiency.

[0140] SEQ ID NO.8:MPKKKRKVSTDAEYVRIHEKLDIYTFKKQFSNNKKSVS。

[0141] SEQ ID NO.9: ATGCCCAAGAAGAAGAGGAAAGTC AGTACCGACGCCGAGTACGTGCGGATCCACGAGAAGCTGGATATCTATACATTCAAGAAGCAGTTTAGCAACAATAAGAAGTCCGTGTCTCACAGATGCTACGTGCTGTTCGAGCTGAAGCGGAGAGGAGAGAGGCGCGCCTGTTTTTGGGGCTATGCCGTGAACAAGCCACAGTCTGGAACCGAGAGGGGAATCCACGCAGAGATCTTCAGCATCAGGAAGGTGGAGGAGTACCTGCGCGACAACCCCGGCCAGTTTACAATCAATTGGTATAGCTCCTGGAGCCCTTGCGCCGATTGTGCCGAGAAGATCCTGGAGTGGTACAACCAGGAGCTGAGGGGCAATGGCCACACCCTGAAGATCTGGGTGTGCAAGCTGTACTATGAGAAGAACGCCAGGAATCAGATCGGCCTGTGGAACCTGCGCGACAATGGCGTGGGCCTGAACGTGATGGTGTCCGAGCACTATCAGTGCTGTCGCAAGATCTTTATCCAGTCTAGCCACAATCAGCTGAACGAGAATCGGTGGCTGGAGAAGACACTGAAGAGAGCCGAGAAGCGGAGAAGCGAGCTGTCCATCATGTTTCAGGTGAAGATCCTGCACACCACAAAGTCTCCCGCCGTG AGCGGCAGCGAGACTCCCGGGACC TCAGAGTCCGCCACACCCGAAAGTATGAGCGACAAGCCAAAGAGACCTCTGAGCGCCTACATGCTGTGGCTGAACAGCGCTAGAGAATCCATCAAGCGCGAAAACCCCGGCATCAAGGTGACCGAGGTGGCCAAGCGGGGAGGCGAGCTGTGGCGGGCCATGAAGGACAAGTCTGAATGGGAGGCCAAGGCCGCTAAAGCCAAAGACGACTACGACAGAGCCGTGAAGGAATTCGAGGCAAATGGCGGCAGCAGCGCCGCCAACGGCGGAGGCGCCAAGAAAAGAGCCAAGCCTGCTAAGAAGGTCGCCAAGAAGTCCAAGAAAGAGGAATCTGATGAGGACGATGATGACGAGAGCGAG TCCGGCAGCGAGACACCCGGCACCA GCGAAAGCGCCACCCCTGAGTCTCCTAAGAAGAAACGGAAGGTGGGCATACACGGCGTGCCAGCCGCCATGGCCAAGAACACAATCACAAAAACCCTGAAGCTGAGAATCGTGCGGCCCTACAATAGCGCCGAAGTGGAAAAAATCGTGGCCGACGAGAAGAATAACCGGGAAAAAATCGCTCTGGAAAAGAACAAGGACAAGGTGAAGGAAGCCTGTAGCAAGCACCTGAAGGTGGCCGCCTACTGCACCACCCAGGTGGAAAGAAACGCCTGCCTGTTCTGCAAGGCCAGAAAGTTGGATGATAAGTTCTACCAGAAGCTGAGAGGCCAGTTCCCCGACGCCGTGTTCTGGCAGGAGATCAGCGAGATTTTCAGACAGCTGCAGAAGCAGGCCGCTGAGATCTACAACCAGAGCCTGATCGAGCTGTACTACGAGATCTTTATCAAGGGCAAGGGCATCGCTAATGCTTCTTCAGTGGAACACTACCTGAGCAGAGTGTGCTACAGAAGAGCCGCCGAGCTGTTTAAAAATGCCGCTATCGCCAGCGGCCTGAGAAGCAAGATCAAGTCTAATTTTCGGCTTAAGGAACTGAAGAACATGAAGTCCGGTCTTCCAACAACCAAGTCCGACAATTTCCCTATCCCTCTGGTGAAGCAGAAAGGAGGCCAGTATAGAGGCTTTGAGATCAGCAATCACAACTCCGACTTCATCATCAAGATCCCGTTCGGCAGATGGCAAGTGAAGAAAGAGATCGACAAGTACAGACCTTGGGAGAAGTTCGACTTCGAACAGGTTAGAAAGTCTCCTAAGCCTATCAGCCTGCTGCTGTCTACCCAGCGGAGAAAGCGGAACAAGGGATGGTCCAAAGATGAGGGCACAGAGGCGGAGATCAAGAAGGTGATGAACGGCGACTACCAGACCAGCTATATC

[0142] GAGGTGAAGCGCGGCAGCAAGATCGGCGAGAAGTCTGCCTGGATGCTG

[0143] AATCTGAGCATTGATGTGCCTAAGATCGACAAGGGCGTGGACCCTAGCA

[0144] TCATCGGCGGAATCGATGTTGGCGTCAAGGAGCCCCTGGTGTGTGCCAT

[0145] CAACAACGCCTTCAGCAGATACAGCATCTCTGATAACGACCTGTTCCACT

[0146] TCAACAAAAAGATGTTCGCCAGAAGACGCATCCTGCTGAAAAAAAACAG

[0147] ACACAAGCGGGCCGGACACGGCGCCAAGAACAAACTCAAACCTATTAC

[0148] AATCCTGACCGAGAAGTCTAGAAGATTCCGGAAGAAGCTGATCGAGAGA

[0149] TGGGCCTGTGAAATCGCCGACTTCTTCATCAAGAACAAGGTGGGAACCG

[0150] TGCAGATGGAAAACCTGGAAAGCATGAAGAGAAAGGAGGACAGCTACT

[0151] TCAACAGAAGACTGCGGGGCTTCTGGCCCTATGCCAGAATGCAGAACAA

[0152] GATCGAGTTCAAACTGAAACAGTACGGCATCGAAATCAGAAAAGTCGCC

[0153] CCTAACAACACCTCTGCTACCTGCAGCAAATGCGGCCATCTGAACAACT

[0154] ACTTTAACTTCGAGTACCGGAAAAAGAACAAGTTCCCCCACTTTAAGTG

[0155] CGAGAAGTGCAACTTCAAGGAGAACGCCGACTACAACGCCGCCCTGAAC

[0156] ATTAGCAACCCCAAGCTGAAGAGCACCAAGGAGGAACCT AAGCGGCCT

[0157] GCTGCTACAAAGAAGGCAGGCCAAGCCAAGAAGAAGAAG TCTGGTGGT

[0158] TCTACTAATCTGTCAGATATTATTGAAAAGGAGACCGGTAAGCAACTGG

[0159] TTATCCAGGAATCCATCCTCATGCTCCCAGAGGAGGTGGAAGAAGTCAT

[0160] TGGGAACAAGCCGGAAAGCGATATACTCGTGCACACCGCCTACGACGAG

[0161] AGCACCGACGAGAATGTCATGCTTCTGACTAGCGACGCCCCTGAATACA

[0162] AGCCTTGGGCTCTGGTCATACAGGATAGCAACGGTGAGAACAAGATTAA

[0163] GATGCTCTCTGGTGGTTCT CCCAAGAAGAAGAGGAAAGTCTAA .

[0164] The underlined part is the signal peptide NLS sequence, and the double underlined part is the linker region.

[0165] Example 5: PAM sequence analysis of HMG-TSminiCBE and enUn1Cas12f1

[0166] 1. Construction of reporter system plasmid

[0167] 16 reporter system plasmids were constructed based on the PAM sequence of NTTN and co-transfected with the enUn1Cas12f1 plasmid into HEK293T cells. The transfection steps were the same as in Example 1.

[0168] 48 hours after transfection, cells were collected for flow cytometry analysis. The analysis process was as follows:

[0169] (1) Digest the cells with 50 μL of trypsin for 4 min, stop the digestion with 350 μL of culture medium, and transfer to a flow tube;

[0170] (2) During flow cytometry analysis, live cell gates were first obtained based on FSC-A and SSC-A, and then transfection-positive cells were selected based on PE-Texas Red-A;

[0171] (3) Analyze the fluorescence ratio of FITC-A for transfected positive cells. The higher the fluorescence ratio, the higher the editing efficiency.

[0172] 2. Verify editing efficiency at endogenous gene sites

[0173] The sequence information of the endogenous gene locus is shown in Table 16. The cell transfection, sorting and subsequent editing efficiency determination are consistent with the steps of Example 1. The analysis results are as follows Figure 6 As shown, both HMG-TSminiCBE and enUn1Cas12f1 can recognize the NTTR PAM sequence.

[0174] Table 16 Targeted gene site nucleotide sequence

[0175]

[0176]

[0177]

[0178]

[0179] The above examples demonstrate an innovative tool, TSminiCBE, which has the capability of efficient targeted strand C-to-T editing. This tool not only changes the editing object of the traditional BE system, but also achieves efficient and high-purity editing of the target. The development of TSminiCBE technology completes a puzzle of the targeting range of existing cytosine base editing technology, and has broad application prospects in the field of cytosine bases.

[0180] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0181] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A cytosine deamination editor, characterized in that The cytosine deamination editor is sequentially connected from the N-terminus to the C-terminus by the deaminase evoCDA1, the XTEN sequence and the enUn1Cas12f1 protein, and the amino acid sequences of the deaminase evoCDA1 and the enUn1Cas12f1 protein are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively.

2. The cytosine deamination editor according to claim 1, characterized in that The amino acid at position 473 of the enUn1Cas12f1 protein also has an alanine mutation.

3. The cytosine deamination editor according to claim 2, characterized in that The C-terminus of the cytosine deamination editor is also connected to a UGI, whose amino acid sequence is shown in SEQ ID NO.

3.

4. The cytosine deamination editor according to claim 3, characterized in that The cytosine deamination editor also comprises a DNA binding protein, which is located between the enUn1Cas12f1 protein and the deaminase evoCDA1.

5. The cytosine deamination editor according to claim 4, characterized in that The DNA binding protein is HMG-D protein, and its encoding gene sequence is shown in SEQ ID NO.

7.

6. A cytosine deamination editing system comprising the cytosine deamination editor according to any one of claims 1 to 5, characterized in that: The cytosine deamination editor is guided by sgRNA to the target editing site.

7. A polynucleotide encoding the cytosine deamination editor according to any one of claims 1 to 5 or the cytosine deamination editing system according to claim 6.

8. A vector comprising the polynucleotide of claim 7.

9. A cell comprising the vector of claim 8.

10. A gene editing pharmaceutical composition, characterized in that: It comprises the cytosine deamination editor according to any one of claims 1 to 5, the cytosine deamination editing system according to claim 6, the polynucleotide according to claim 7, the vector according to claim 8 or the cell according to claim 9.

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