WO2023142594A1 - 一种精确无pam限制的腺嘌呤碱基编辑器及其应用 - Google Patents

一种精确无pam限制的腺嘌呤碱基编辑器及其应用 Download PDF

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WO2023142594A1
WO2023142594A1 PCT/CN2022/131039 CN2022131039W WO2023142594A1 WO 2023142594 A1 WO2023142594 A1 WO 2023142594A1 CN 2022131039 W CN2022131039 W CN 2022131039W WO 2023142594 A1 WO2023142594 A1 WO 2023142594A1
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tada8e
spry
editing
pam
adenine
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姚远
李果
李叶秋
程亚仙
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浙江大学杭州国际科创中心
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Definitions

  • the invention relates to the technical field of gene editing, in particular to a precise adenine base editor without PAM restriction and its application.
  • point mutations also known as single nucleotide polymorphisms (SNPs)
  • SNPs single nucleotide polymorphisms
  • Gene editing is a molecular biology technique developed since the late 1980s. It is a technology to artificially modify a specific gene through a certain way. Early gene editing mainly used the principle of DNA homologous recombination to design homologous fragments to replace target gene fragments, so as to achieve the purpose of gene editing. At present, the relatively successful gene knockout technologies mainly include: Zinc-finger, TALEN and CRISPR/Cas9. CRISPR/Cas is a technique for specific DNA modification of targeted genes by RNA-guided Cas nucleases. It is an adaptive immune defense mechanism evolved by bacteria and archaea in response to the constant attack of phages and foreign plasmids.
  • the CRISPR/Cas system can realize efficient gene knockout, knockin, replacement and transcriptional regulation.
  • the performance of the traditional CRISPR/Cas system is not satisfactory for the correction of genes with single base mutations.
  • HENJ non-homologous end repair
  • HDR homologous recombination
  • BE single base editor
  • BE4 does not generate double-strand breaks (DSBs) during the editing process, and only needs a DNA single-stranded nick to achieve single-base precise editing, which can effectively avoid genome damage during the editing process.
  • the BE4 single base editor can achieve safe, efficient, high specificity and high security by fusing cytosine deaminase APOBEC1 and uracil glycosylase inhibitor UGI to the Cas9n (D10A) protein in the traditional CRISPR-Cas9 system.
  • Authentic C->T base substitution editing, and ABE7.10 can convert adenine into inosine to form A->G mutation, but both of the above two edits have editing windows and can only do purine-purine Or the problem of pyrimidine-pyrimidine mutual conversion, the precise editing of a single base and the conversion between multiple bases cannot be realized.
  • the PE system can mediate targeted insertions, deletions, and mutual substitutions between arbitrary bases. Also, it can combine different types of editing. All of these can be performed without DSB or donor DNA templates (Anzalone AV, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019, 576(7785): 149- 157), but the current PE system still has defects in editing efficiency and targeting range. At present, there is still no single base editing tool with broad-spectrum targeting, high efficiency and specificity.
  • the purpose of the present invention is to provide an accurate adenine base editor without PAM restriction and its construction method.
  • This gene editing tool can be applied to the repair of G>A single base mutation and the treatment of diseases.
  • the applicant's variant SpRY(D10A) combined with SpCas9 has broad-spectrum targeting without PAM restriction and the specificity of TadA8e F148A deaminase to precisely target the A>G mutation at a specific site, providing an accurate adenocarcinoma without PAM restriction.
  • Purine base editor construction method through the constructed TadA8e-SpRY and TadA8e F148A -SpRY base editors, it was verified that the sgRNA targeting the human gene VISTA enhancer hs267 (NCBI ID: NG_053265.1) sequence had different PAMs A>G DNA editing efficiency within the editing window.
  • the present invention provides an accurate adenine base editor without PAM restriction
  • the adenine base editor comprises the coding gene of the adenine deaminase mutant TadA8e F148A and the coding gene of the SpRY nuclease mutant, wherein , the adenine deaminase mutant TadA8e F148A is obtained by mutating the amino acid Phe at position 148 of the amino acid sequence of the adenine deaminase TadA8e to Ala, and the amino acid sequence of the adenine deaminase TadA8e is shown in SEQ ID NO.21.
  • the coding gene of the adenine deaminase mutant TadA8e F148A is located at the upstream end of the coding gene of the SpRY nuclease mutant.
  • the upstream end refers to the end where transcription starts, that is, the 5' end
  • the downstream end refers to the 3' end.
  • nucleotide sequence of the coding gene of the adenine deaminase mutant TadA8e F148A is shown in SEQ ID NO.2
  • nucleotide sequence of the coding gene of the SpRY nuclease mutant is shown in SEQ ID NO.3 .
  • the precise adenine base editor without PAM restriction also includes a nuclear localization signal fragment NLS, and the nucleotide sequence of the gene encoding the nuclear localization signal fragment NLS is shown in SEQ ID NO.4.
  • the coding gene of the adenine deaminase mutant TadA8e F148A is located at the upstream end of the coding gene of the SpRY nuclease mutant, and the coding gene of the nuclear localization signal fragment NLS is located at the downstream end of the coding gene of the SpRY nuclease mutant.
  • the present invention further provides the application of the precise adenine base editor without PAM restriction in the preparation of gene editing reagents.
  • the present invention further provides the application of the precise adenine base editor without PAM restriction in gene editing for the purpose of non-disease treatment.
  • the present invention also provides a gene editing method, using the precise adenine base editor without PAM restriction for gene editing.
  • the present invention has at least the following advantages and beneficial effects:
  • the present invention provides a method for constructing an adenine base editor without PAM restriction.
  • the SpRY protein without PAM restriction is fused with TadA8e deaminase that introduces the mutation of the 148th amino acid Phe to Ala.
  • the adenine base editor TadA8e F148A -SpRY has the characteristics of no PAM-restricted target editing range, high editing accuracy and high editing efficiency, and provides a new way for the repair of G>A single base mutation and disease treatment tool.
  • the TadA8e F148A -SpRY base editor of the present invention can mediate efficient and precise repair of any G>A mutation.
  • Statistics of the editing efficiency of 16 different PAM sgRNAs by the TadA8e F148A -SpRY base editor the editing window of TadA8e F148A -SpRY is reduced from 3-11 of TadA8e-SpRY to 3-10, while reducing the non-editing window
  • Parallel editing outside the system improves the accuracy of single base editing.
  • A>G editing at the fifth position in the editing window was favored among all sgRNAs. Since TadA8e F148A -SpRY has no restriction of PAM, any G>A mutation base can be set at the fifth position of sgRNA, which can realize efficient and precise repair of all G>A mutations.
  • TadA8e F148A -SpRY of the present invention has lower RNA off-target editing efficiency, further improving the accuracy of editing.
  • Figure 1 is a schematic diagram of TadA8e-SpRY and TadA8e F148A -SpRY base editors.
  • Figure 2 is a graph showing the average editing efficiency results of TadA8e-SpRY and TadA8e F148A -SpRY targeting 16 PAM sgRNAs in Example 2.
  • Fig. 3 is a graph of the editing efficiency results of TadA8e-SpRY and TadA8e F148A -SpRY targeting the fifth position A>G of 16 PAM sgRNAs in Example 2.
  • Fig. 4 is a graph showing the average editing efficiency results of 16 PAM sgRNAs targeting endogenous genes of HEK293T cells by TadA8e-SpRY and TadA8e F148A -SpRY in Example 3, where AD is NAN, NCN, NGN, NTN, respectively.
  • Figure 5 is a summary statistical chart of the results in Figure 4, where A is the overall statistical results, and B is the statistical results of the four types of sequences.
  • Fig. 6 is a graph showing the average editing efficiency results of 16 PAM sgRNAs targeting Hela cell endogenous genes by TadA8e-SpRY and TadA8e F148A -SpRY in Example 3, where AD is NAN, NCN, NGN, NTN, respectively.
  • Figure 7 is a summary statistical chart of the results in Figure 6, where A is the overall statistical results, and B is the statistical results of the four types of sequences.
  • Fig. 8 is a graph showing the statistical results of the editing window of 16 kinds of PAM sgRNA targeting endogenous genes of HEK293T cells by TadA8e-SpRY and TadA8e F148A -SpRY in Example 3.
  • Fig. 9 is a graph showing the statistical results of editing windows of 16 PAM sgRNAs targeting Hela cell endogenous genes in Example 3 by TadA8e-SpRY and TadA8e F148A -SpRY.
  • Fig. 10 is a graph showing the statistical results of the RNA off-target rate of TadA8e-SpRY and TadA8e F148A -SpRY targeting HEK293T cells in Example 3.
  • Fig. 11 is a graph showing the results of TadA8e F148A -SpRY base editor in the application of disease-causing site repair in Example 3.
  • a method for constructing an adenine base editor that is accurate without PAM restriction comprising:
  • TadA8e-SpRY and TadA8e F148A -SpRY editor expression vectors were constructed, which also contained a nuclear localization signal fragment NLS, and the nucleotide sequence was shown in SEQ ID NO.4.
  • the PAM sequences are TAA, AAC, CAG, CAT, AGA, TGC, GGG, AGT, TCA, CCC, ACG, GCT, TTA, ATC, CTG and ATT , using the sgRNA sequence to locate TadA8e-SpRY and TadA8e F148A -SpRY to the target sequence for A>G DNA single base editing within the editing window.
  • the TadA8e target sequence can be selected from the backbone vector: ABE8e.
  • the SpRY target sequence can be selected from the backbone vector: pCMV-T7-ABEmax(7.10)-SpRY-P2A-EGFP.
  • the DNA sequence that can be used for A>G single base editing comes from the following genes: human gene VISTA enhancer hs267 (NCBI ID: NG_053265.1), and other 48 human genes.
  • the above method is provided to perform A>G editing of sgRNAs with 16 different PAMs by TadA8e_SpRY and TadA8e F148A_SpRY editors in cell lines HEK293T and Hela, and verify the efficiency and efficiency of the two DNA editing tools. precision.
  • TadA8e target fragment As shown in Figure 1, after performing PCR amplification on the TadA8e target fragment (as shown in SEQ ID NO.1), use the point mutation kit (Vazyme, C215-01) to design PCR amplification primers to deaminate adenine
  • the 148th amino acid Phe in the sequence of the enzyme TadA8e (amino acid sequence shown in SEQ ID NO.21) is mutated to Ala, and the nucleotide sequence is TTC mutated to GCC to obtain TadA8e F148A (nucleotide sequence shown in SEQ ID NO.2 shown), form the following two deaminases: (a) TadA8e; (b) TadA8e F148A ;
  • TadA8e and TadA8e F148A is fused to the carbon terminus (C terminus) of the SpRY (D10A) gene (nucleotide sequence shown in SEQ ID NO.3) to form the following two adenine base editors: (1) TadA8e_SpRY; (2) TadA8e F148A _SpRY.
  • sgRNA sequences were designed for the sequence of the human gene VISTA enhancer hs267 (NCBI ID: NG_053265.1), which can represent any sequence of the target genome.
  • the present invention selects and designs sgRNA as follows:
  • NAN NAN
  • NAC NAC
  • NAG NAG
  • NAT NGN
  • NGC NGG
  • NGT NCN
  • NCC NCC
  • NCG NCG
  • NCT NTN
  • NTC NTC, NTG, NTT
  • NAA-sgRNA TTGAAAGACTAAACAAACCT (TAA);
  • NAC-sgRNA ACCAACAATAGAGGCCCATT (AAC);
  • NAG-sgRNA GTTTACATAAAAGATTCTTCA (CAG);
  • NAT-sgRNA ACTAAACAAACCTTAACTGT(CAT);
  • NGA-sgRNA ATAAAATAAATGCATTAAAA(AGA);
  • NGC-sgRNA CTGGAACACAAAGCATAGAC (TGC);
  • NGG-sgRNA GAACACAAAGCATAGACTGC (GGG);
  • NGT-sgRNA GAAAAATGATATCCATTATT(AGT);
  • NCA-sgRNA AATGAAGTATTGTTATTGCC (TCA);
  • NCC-sgRNA AAAGATCTTCACAGGCTACC (CCC);
  • NCG-sgRNA TGGTAGAATGGCAGTGCAAT(ACG);
  • NCT-sgRNA TCCTAAAACCAGTGTCAGGGA (GCT);
  • NTA-sgRNA ACAAAAAAAAAGCCTTCTTT(TTA);
  • NTC-sgRNA GGGAAAAATTGTCCAGCCCC (ATC);
  • NTG-sgRNA GGAAACAATGATAACAAGAC (CTG);
  • NTT-sgRNA CTTTAAACGTGTTCTTAACT (ATT).
  • sgRNA expression vector For the above selected target gene sequences, a total of 16, the corresponding sgRNA expression vectors were constructed, and different sgRNAs were respectively introduced into pGL3-U6-sgRNA-PGK-puromycin (Addgene, Plasmid #51133) mammalian gene editing plasmid (sgRNA expression vector ).
  • the DNA single base editing of the cell line (by electroporation or lipofection) is carried out, and the lipofection is taken as an example.
  • HEK293T cells are inoculated and cultured in DMEM high-glucose culture medium ( HyClone, SH30022.01B), which contains penicillin (100U/mL) and streptomycin (100 ⁇ g/mL).
  • DMEM high-glucose culture medium HyClone, SH30022.01B
  • penicillin 100U/mL
  • streptomycin 100 ⁇ g/mL
  • b Resuspend the cells and collect them in a PCR tube, centrifuge at 800rpm for 5 minutes, remove the liquid with a suction pump, add 20 ⁇ L of cell lysate, and place in a PCR instrument to fully lyse the cells.
  • Lysis program 68°C, 30min; 16°C, 2min; 98°C, 2min; 16°C hold.
  • PCR amplification of the target fragment design PCR amplification primers for the mutation site, and perform PCR amplification of the target fragment.
  • PCR system 50 ⁇ L: Super-Fidelity DNA Polymerase, 1 ⁇ L; dNTP, 1 ⁇ L; 2x Buffer, 25 ⁇ L; cell lysate, 4 ⁇ L; PCR amplification primer F/R (10nM), 2 ⁇ L; ddH 2 O, 15 ⁇ L.
  • PCR reaction program 95°C, 3min; first cycle (10 cycles): 95°C, 15s; 68°C, 15s; 72°C, 30s; second cycle (30 cycles): 95°C, 15s; 58 °C, 15s; 72°C, 30s; 72°C, 5min; 16°C maintained.
  • the PCR primers are as follows:
  • the PCR product was purified and recovered, and Sanger sequencing of the amplified fragment was performed using Forward: AAAGCCTCAACAATGTTGCC. Results Use the online statistical tool EditR (https://moriaritylab.shinyapps.io/editr_v10/) to conduct specific editing efficiency statistics.
  • TadA8e-SpRY and TadA8e F148A -SpRY editors edited the sgRNAs of 16 kinds of PAMs with A>G DNA single base editing in the 4-8 editing window.
  • the average editing efficiencies of TadA8e-SpRY to A>G in NAN, NGN, NCN and NTN PAM windows were 45.7%, 41.0%, 58.3% and 25.7% respectively; TadA8e F148A -SpRY to NAN, NGN, NCN and NTN PAM windows
  • the average editing efficiencies of inner A>G were 40.3%, 42.0%, 52.7% and 20.0%, respectively (Fig. 2).
  • the average editing efficiencies of A base A>G at each position in the TadA8e-SpRY editor sgRNA sequence are: A1, 0.0%; A2, 1.3%; A3, 1.0%; A4, 41.3%; A5, 43.0%; A6, 45.0%; A7, 42.7%; A8, 39.3%; A9, 19.3%; A10, 1.7%; A11, 0.7%; A12, 0.3%; A13, 0.0%; A14, 0.0%; A15, 0.0%; A16 , 0.0%; A17, 0.0%; A18, 0.0%; A19, 0.0%; A20, 0.0%; A1, 0.0%; A2, 0.7%; A3, 0.7%; A4, 17.3%; A5, 49.0%; A6, 18.7%; A7, 16.0%; A8, 9.7%; A9, 3.3%; A10, 2.3% A11, 0.0%; A12, 0.0%; A13, 1.0%; A14, 0.5%; A15, 0.0%; A16, 0.0%; A17, 0.0%; A18, 0.0%; A
  • TadA8e F148A -SpRY is more inclined to A>G editing of A5 (the fifth base A) in the editing window, which reduces collateral editing and improves the editor's ability to edit a single base accuracy ( Figure 3).
  • sgRNA sequences were designed for 48 human gene sequences, which can represent any sequence of the target genome.
  • the present invention selects and designs sgRNA as follows:
  • NAN NAN
  • NAC NAC
  • NAG NAG
  • NAT NGN
  • NGC NGG
  • NGT NCN
  • NCC NCC
  • NCG NCG
  • NCT NTN
  • NTC NTC
  • NTT a total of 16 PAM 20bp targeting sequences, forming a total of 48 sgRNA sequences.
  • N means any one of the four bases A/T/G/C.
  • the corresponding coding sequences of the 48 sgRNAs selected in the present invention are shown in Table 1, and the sequence order is 5'-3'.
  • NC_000015 GATTGAGCAGGGAAATACTG GAA SHANK3 NC_000022.11 GTTTATGTAACTCTTCCACT CAA MECP2 NC_000023.11 GCAGATACTGAGTTTTTAAC AAA RUNX1 NC_000021.9 GGCCTCATAAACAACCACAG AAC CUL3 NC_000002.12 GGTCCAGTAGATATTGAAGT TAC HEKsite4 NC_000003.12 GGGTCAGACGTCCAAAACCA GAC FANCF NC_000011.10 GCAGGAGGTGGGGAAGGCCG AAG SHANK3 NC_000022.11 GGTCCCCAGGAACCTCTCCG AAG DYRK1A NC_000021.9 GGTTTGCAGCCTAAGAGCAG TAG FANCF NC_000011.10 GAGACGTTCATGACTGGCAT CAT CUL3 NC_000002.12 GTATTCAGCATATTGACATG TAT ZSCAN2 NC_000015.
  • sgRNA expression vector For the above-mentioned selected target gene sequences, a total of 48, the corresponding sgRNA expression vectors were constructed, and different sgRNAs were respectively introduced into pGL3-U6-sgRNA-PGK-GFP (Addgene, Plasmid#107721) mammalian gene editing plasmid (sgRNA expression vector ).
  • the DNA single base editing of the cell line (by electroporation or lipofection) is carried out, and the lipofection is taken as an example.
  • HEK293T cells are inoculated and cultured in DMEM high-glucose culture medium ( HyClone, SH30022.01B), which contains penicillin (100U/mL) and streptomycin (100 ⁇ g/mL).
  • DMEM high-glucose culture medium HyClone, SH30022.01B
  • penicillin 100U/mL
  • streptomycin 100 ⁇ g/mL
  • Lysis program 68°C, 30min; 16°C, 2min; 98°C, 2min; 16°C hold.
  • PCR amplification of the target fragment design PCR amplification primers for the mutation site, and perform PCR amplification of the target fragment.
  • PCR system 50 ⁇ L: Super-Fidelity DNA Polymerase, 1 ⁇ L; dNTP, 1 ⁇ L; 2x Buffer, 25 ⁇ L; cell lysate, 4 ⁇ L; PCR amplification primer F/R (10nM), 2 ⁇ L; ddH 2 O, 15 ⁇ L.
  • PCR reaction program 95°C, 3min; first cycle (10 cycles): 95°C, 15s; 68°C, 15s; 72°C, 30s; second cycle (30 cycles): 95°C, 15s; 58 °C, 15s; 72°C, 30s; 72°C, 5min; 16°C maintained.
  • PCR primers are shown in Table 2 below:
  • Upstream primer (5'-3') Downstream primer (5'-3') UBE3A ACTGCTTTCTGTCTTCTGGC CAGCAGCTATTCCAAAAATC SHANK3 GGCAGGCACCGTCTTTGTCG CATGTACGTTCGTCAAGGTT MECP2 GCCTCTTGGTTGTAATATGC CATCAGAGAGCATTGATCAC RUNX1 CTGACCACTATGCTGGGTTC TTTCTTGCACAGCCTGGGGG CUL3 GCCACCTGGTTTATGGGATT CCAAGTTTTGGGCTCCAGTA HEKsite4 GAGGTGGGGGTTAAAGCGGA CAGTGAAATCACCCCTGGGGG FANCF CTCTCCAGGTGATTTGTGTGGA GGAGGACTCTCTGATGAAGA SHANK3 CCCATCTTCCCGAGCATTCT CGCCAGCTTCTCGTCCTCCC DYRK1A CAAATAATGAGGGTTACAGT AACATCACTGAGTATACACTGCA FANCF GGAGGGAGCAGATGTAGG AG
  • the average editing efficiency of TadA8e F148A -SpRY on A>G in NAN, NGN, NCN and NTN PAM windows was 42.57%, 34.74%, 36.85% and 36.68%, respectively.
  • the TadA8e F148A -SpRY editor has no PAM restriction, and has high A>G DNA editing efficiency in most sites, which greatly broadens the targeted editing range of the genome.
  • the main active editing window of TadA8e-SpRY in HEK293T and Hela cells is A3-A11 (calculated from the far end of PAM, the first base A is marked as A1), and at some sites such as NCN PAM, there are also Collateral editing of sites outside the window such as A15 and A18.
  • the main active editing window of the novel base editor TadA8e F148A -SpRY in HEK293T and Hela cells is A3-A10, and there is almost no paralogous editing outside the window.
  • TadA8e F148A -SpRY narrows the editing window, reduces paralogous editing, and improves the editor's precision for single-base editing (Fig. 8 and Fig. 9).
  • RNA-seq data analysis process RNA-seq data analysis process.
  • the RNA mutation identification adopts the GATK standardized process, and the gvcf file is merged to obtain the VCF collection of the joint call to ensure the comparability of SNPs within the batch. After obtaining the SNP collection, carry out SNP annotation and clarify the position.
  • FANCF-sgRNA+TadA8e-SpRY and FANCF-sgRNA+SpRY-AB8e F148A were the experimental groups, and eGFP was used as the control group, with 3 samples in each group.
  • Basic logic determine the positive and negative chain ⁇ determine the genotype of the eGFP group ⁇ determine the genotype of each sample in the experimental group ⁇ perform in-depth filtering on the mutation site ⁇ calculate the mutation frequency. Firstly, it is judged whether the mRNA template strand of each SNP mutation is the positive strand or the negative strand of the reference genome.
  • RNA-seq sequencing analysis of endogenous gene-edited cells showed that compared with TadA8e-SpRY, TadA8e F148A -SpRY editor did not significantly reduce the total number of A>G RNA off-targets, but significantly reduced the average editing efficiency of A>G RNA off-targets , generally reducing the RNA off-target rate and further improving the accuracy (Figure 10).
  • APOC3 p.D65N, c.G2871A
  • SCN9A p.R896Q, c.G98851A
  • SLC30A8 p.M50I, c.G196816A
  • pCAG-CBE4max-SpRY-P2A-EGFP (RTW5133) (Addgene #139999) mediated C>T DNA single base editing to construct the G>A mutation model of the above three sites.
  • APOC3 p.D65N, c.G2871A
  • SCN9A p.R896Q, c.G98851A
  • SLC30A8 p.M50I, c.G196816A
  • the plasmid combination of sgRNA+TadA8e F148A -SpRY was transfected into mutated HEK293T cells. After 48 hours of transfection, GFP-positive cells were sorted by flow cytometry, 10,000 cells/sample. Cells were lysed, target fragments were amplified by PCR using identification primers, Sanger sequencing, and A>G repair efficiency statistics were performed using EditR.
  • the repaired sgRNA sequence is shown in Table 3.
  • the present invention fuses the PAM-free SpCas9 variant SpRY (D10A) with the deaminase TadA8e (F148A) that introduces key amino acid mutations, and constructs a novel adenine editor TadA8e F148A -SpRY that can efficiently target the genome for A>G's DNA single base editing, almost without PAM restrictions. Narrowed the editing window of TadA8e-SpRY from 3-11 to 3-10, reduced the paralogous editing outside the editing window, and reduced the tie editing efficiency of A>G RNA off-target, and improved the adenine base editor accuracy, and successfully applied it to the repair editing of G>A pathogenic mutant cells.
  • the novel adenine editor TadA8e F148A -SpRY provided by the present invention has the advantages of no PAM restriction and both editing efficiency and specificity. It provides new ideas and methods for the optimization of gene editing tools for the subsequent precise repair of endogenous gene G>A point mutations, the simulation or repair of pathogenic sites in genetic diseases, and clinical research.

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Abstract

提高了一种精确无PAM限制的腺嘌呤碱基编辑器及其应用，该腺嘌呤碱基编辑器包含核苷酸序列如SEQ ID NO.2所示的腺嘌呤脱氨酶突变体TadA8eF148A的编码基因和核苷酸序列如SEQ ID NO.3所示的SpRY核酸酶突变体的编码基因。该腺嘌呤碱基编辑器可用于制备基因编辑试剂或非疾病治疗目的的基因编辑，编辑范围无PAM限制、编辑精确性和效率高，可用于G>A单碱基修复。

Description

一种精确无PAM限制的腺嘌呤碱基编辑器及其应用 技术领域
本发明涉及基因编辑技术领域，具体涉及一种精确无PAM限制的腺嘌呤碱基编辑器及其应用。
背景技术
迄今为止，已知人类致病突变的最大类别是点突变(也称为单核苷酸多态性(SNP))，尽管由于广泛使用短阅读测序来分析基因组多样性而导致的采样偏差可能会扭曲这种分布。因此，高效、干净地安装或逆转致病SNPs对于遗传疾病的研究和治疗非常有意义，并且需要一种方法来特异性地改变基因组中个体碱基对的序列。
基因编辑是自80年代末发展起来的一种分子生物学技术。它是一种通过一定的途径实现人为修改特定基因的技术。早期的基因编辑主要是利用DNA同源重组原理，通过设计同源片段替代靶基因片段，从而达到基因编辑的目的。目前应用比较成功的基因敲除技术主要有：Zinc-finger、TALEN和CRISPR/Cas9。CRISPR/Cas是一种由RNA指导的Cas核酸酶对靶向基因进行特定DNA修饰的技术。它是细菌和古生菌为了应对噬菌体和外源质粒的不断攻击而演化来的获得性免疫防御机制。CRISPR/Cas系统可实现高效的基因敲除、敲入、替换及转录水平的调控。然而，针对单个碱基突变的基因的修正，传统CRISPR/Cas系统的表现却不尽如人意。由于DNA双链断裂时，细胞更倾向于利用非同源末端修复(HENJ)原理对DNA进行修复,因而利用同源重组(HDR)进行的单个碱基的替换过程往往十分低效。而单碱基编辑器(BE)系统的出现成功攻克了这一技术壁垒，实现了高效且安全的单个碱基的替换编辑。BE4，ABE7.10，PE系统是目前最先进的可实现单个碱基精准替换的基因编辑工具。BE4编辑过程中不产生双链断裂(DSB)，仅需一个DNA单链切口就能实现单碱基精准编辑，可有效避免编辑过程中产生基因组损伤。BE4单碱基编辑器通过在传统CRISPR-Cas9系统中的Cas9n(D10A)蛋白上融合胞嘧啶脱氨基酶APOBEC1及尿嘧啶糖基化酶抑制剂UGI，可实现安全、高效、高特异性、高保真性的C->T的碱基替换编辑，而ABE7.10可将腺嘌呤转化为肌苷，从而形成A->G突变，但上述两种编辑都存在编辑窗口及只能做到嘌呤-嘌呤或者嘧啶-嘧啶的互相转变的问题，无法实现单个碱基的精确编辑及多碱基间的相互转换。PE系统可以介导靶向的插入、缺失以及任意碱基间的相互替换。而且，它可以将不同类型的编辑相结合。所有这些都可以在没有DSB或供体DNA模板的情况下进行(Anzalone AV,et al.Search-and-replace genome editing without double-strand breaks or donor DNA.Nature.2019,576(7785):149-157)，但目前的PE系统在编辑效率及靶向范围等方面仍存在缺陷。目前仍然没有一种兼具广谱靶向性、高效性与特异性的单碱基编辑工具。
发明内容
本发明的目的是提供一种精确无PAM限制的腺嘌呤碱基编辑器及其构建方法，这种基因编辑工具可以应用于G>A单碱基突变的修复与疾病相关的治疗。
申请人结合SpCas9的变体SpRY(D10A)具有无PAM限制的广谱靶向性与TadA8e F148A脱氨酶精确靶向特定位点A>G突变的特异性，提供一种精确无PAM限制的腺嘌呤碱基编辑器的构建方法：通过构建的TadA8e-SpRY与TadA8e F148A-SpRY碱基编辑器，验证其靶向人源基因VISTA增强子hs267(NCBI ID：NG_053265.1)序列具有不同PAM的sgRNA编辑窗口内的A>G DNA编辑效率。
本发明提供了一种精确无PAM限制的腺嘌呤碱基编辑器，所述腺嘌呤碱基编辑器包含 腺嘌呤脱氨酶突变体TadA8e F148A的编码基因和SpRY核酸酶突变体的编码基因，其中，腺嘌呤脱氨酶突变体TadA8e F148A为腺嘌呤脱氨酶TadA8e氨基酸序列148位的氨基酸Phe突变为Ala获得，腺嘌呤脱氨酶TadA8e的氨基酸序列如SEQ ID NO.21所示。优选的，腺嘌呤脱氨酶突变体TadA8e F148A的编码基因位于SpRY核酸酶突变体的编码基因的上游端。对于基因序列来说，上游端是指转录起始的一端，即5’端，下游端是3’端。
优选的，腺嘌呤脱氨酶突变体TadA8e F148A的编码基因的核苷酸序列如SEQ ID NO.2所示，SpRY核酸酶突变体的编码基因的核苷酸序列如SEQ ID NO.3所示。
所述精确无PAM限制的腺嘌呤碱基编辑器还包含核定位信号片段NLS，所述核定位信号片段NLS的编码基因的核苷酸序列如SEQ ID NO.4所示。
更优选的，腺嘌呤脱氨酶突变体TadA8e F148A的编码基因位于SpRY核酸酶突变体的编码基因的上游端，核定位信号片段NLS的编码基因位于SpRY核酸酶突变体的编码基因的下游端。
本发明又提供了所述精确无PAM限制的腺嘌呤碱基编辑器在制备基因编辑试剂中的应用。
本发明又提供了所述精确无PAM限制的腺嘌呤碱基编辑器在非疾病治疗为目的的基因编辑中的应用。
本发明还提供了一种基因编辑方法，通过所述精确无PAM限制的腺嘌呤碱基编辑器进行基因编辑。
与现有技术相比，本发明至少具有下列优点及有益效果：
(1)本发明提供一种无PAM限制的腺嘌呤碱基编辑器的构建方法，本方法通过将无PAM限制的SpRY蛋白与引入第148位氨基酸Phe突变为Ala的TadA8e脱氨酶融合，构建的腺嘌呤碱基编辑器TadA8e F148A-SpRY，具有无PAM限制的靶向编辑范围、高编辑精确性与编辑效率高的特点，为G>A单碱基突变的修复与疾病治疗提供了新的工具。
(2)本发明的TadA8e F148A-SpRY碱基编辑器可介导任意G>A突变的高效、精确修复。通过TadA8e F148A-SpRY碱基编辑器对16种不同PAM的sgRNA的编辑效率统计，TadA8e F148A-SpRY的编辑窗口由TadA8e-SpRY的3-11位缩小为3-10位，同时减少了非编辑窗口外的旁系编辑，提高了单碱基编辑的精确性。在所有sgRNA中偏向于编辑窗口内第五位的A>G编辑。由于TadA8e F148A-SpRY无PAM的限制，因此可以将任意G>A突变碱基设置于sgRNA的第五位，可实现所有G>A突变的高效、精确修复。
(3)通过分析RNA-seq的实验结果，相较于TadA8e-SpRY，本发明的TadA8e F148A-SpRY具有更低的RNA脱靶编辑效率，进一步提高了编辑的精确性。
附图说明
图1为TadA8e-SpRY与TadA8e F148A-SpRY碱基编辑器的示意图。
图2为实施例2中TadA8e-SpRY与TadA8e F148A-SpRY靶向16种PAM sgRNA的平均编辑效率结果图。
图3为实施例2中TadA8e-SpRY与TadA8e F148A-SpRY靶向16种PAM sgRNA第五位A>G的编辑效率结果图。
图4为实施例3中TadA8e-SpRY与TadA8e F148A-SpRY靶向HEK293T细胞内源基因16种PAM sgRNA的平均编辑效率结果图，其中，A-D分别为NAN、NCN、NGN、NTN。
图5为图4中结果的总结统计图，其中A为总体统计结果，B为四种序列分类别统计结果。
图6为实施例3中TadA8e-SpRY与TadA8e F148A-SpRY靶向Hela细胞内源基因16种PAM sgRNA的平均编辑效率结果图，其中，A-D分别为NAN、NCN、NGN、NTN。
图7为图6中结果的总结统计图，其中A为总体统计结果，B为四种序列分类别统计结果。
图8为实施例3中TadA8e-SpRY与TadA8e F148A-SpRY靶向HEK293T细胞内源基因16 种PAM sgRNA的编辑窗口统计结果图。
图9为实施例3中TadA8e-SpRY与TadA8e F148A-SpRY靶向Hela细胞内源基因16种PAM sgRNA的编辑窗口统计结果图。
图10为实施例3中TadA8e-SpRY与TadA8e F148A-SpRY靶向HEK293T细胞的RNA脱靶率统计结果图。
图11为实施例3中TadA8e F148A-SpRY碱基编辑器在致病位点修复应用中的结果图。
具体实施方式
根据本发明的第一方面，提供一种精确无PAM限制的腺嘌呤碱基编辑器的构建方法，其包括：
构建TadA8e-SpRY与TadA8e F148A-SpRY编辑器表达载体，还包含核定位信号片段NLS，核苷酸序列如SEQ ID NO.4所示。
设计、构建具有不同PAM识别序列的sgRNA，共16种，PAM序列分别为TAA、AAC、CAG、CAT、AGA、TGC、GGG、AGT、TCA、CCC、ACG、GCT、TTA、ATC、CTG和ATT，利用sgRNA序列将TadA8e-SpRY与TadA8e F148A-SpRY定位到目标序列进行编辑窗口内的A>G DNA单碱基编辑。
TadA8e目的序列可选自骨架载体：ABE8e。SpRY目的序列可选自骨架载体：pCMV-T7-ABEmax(7.10)-SpRY-P2A-EGFP。
根据本发明的方法，可用于A>G单碱基编辑的DNA序列来自如下基因：人源基因VISTA增强子hs267(NCBI ID：NG_053265.1)，以及其他48个人源基因。
根据本发明的第二方面，提供上述方法在细胞系HEK293T与Hela中进行TadA8e_SpRY与TadA8e F148A_SpRY编辑器对具有16种不同PAM的sgRNA的A>G编辑，验证两个DNA编辑工具的高效性与精确性。
实施例1
一种无PAM限制的腺嘌呤碱基编辑器的构建方法。
(1)构建TadA8e_SpRY与TadA8e F148A_SpRY碱基编辑器
如图1所示，将TadA8e目的片段(如SEQ ID NO.1所示)进行PCR扩增后，使用点突变试剂盒(Vazyme，C215-01)说明书设计PCR扩增引物，将腺嘌呤脱氨酶TadA8e(氨基酸序列如SEQ ID NO.21所示)序列中的第148位氨基酸Phe突变为Ala，核苷酸序列为TTC突变为GCC，得到TadA8e F148A(核苷酸序列如SEQ ID NO.2所示)，形成如下两种脱氨酶：(a)TadA8e；(b)TadA8e F148A；
从pCMV-T7-ABEmax(7.10)-SpRY-P2A-EGFP(Addgene，Plasmid#140003)基因编辑质粒中扩增去除ABEmax(7.10)序列的pCMV-T7-SpRY-P2A-EGFP，分别将TadA8e与TadA8e F148A融合至SpRY(D10A)基因(核苷酸序列如SEQ ID NO.3所示)的碳端(C端)，形成如下两种腺嘌呤碱基编辑器：(1)TadA8e_SpRY；(2)TadA8e F148A_SpRY。
实施例2
一、进行sgRNA质粒的设计。
为了验证两种编辑器的靶向编辑范围，针对人源基因VISTA增强子hs267(NCBI ID：NG_053265.1)序列共设计了16种不同PAM的sgRNA序列，可代表靶向基因组的任意序列。本发明如下选择设计sgRNA：
根据hs267基因序列，分别设计了具有NAN(NAA，NAC，NAG，NAT)，NGN(NGA，NGC，NGG，NGT)，NCN(NCA，NCC，NCG，NCT)，NTN(NTA，NTC，NTG，NTT)共16种PAM的20bp靶向序列，共形成16条sgRNA序列。其中的“N”表示A/T/G/C四种碱基中的任意一种。
本发明选定的16条sgRNA对应编码序列(括号内为标注的PAM序列，序列顺序为5’-3’)：
NAA-sgRNA：TTGAAAGACTAAACAAACCT(TAA)；
NAC-sgRNA：ACCAACAATAGAGGCCCATT(AAC)；
NAG-sgRNA：GTTTACATAAAAGATCTTCA(CAG)；
NAT-sgRNA：ACTAAACAAACCTTAACTGT(CAT)；
NGA-sgRNA：ATAAAATAAATGCATTAAAA(AGA)；
NGC-sgRNA：CTGGAACACAAAGCATAGAC(TGC)；
NGG-sgRNA：GAACACAAAGCATAGACTGC(GGG)；
NGT-sgRNA：GAAAAATGATATCCATTATT(AGT)；
NCA-sgRNA：AATGAAGTATTGTTATTGCC(TCA)；
NCC-sgRNA：AAAGATCTTCACAGGCTACC(CCC)；
NCG-sgRNA：TGGTAGAATGGCAGTGCAAT(ACG)；
NCT-sgRNA：TCCTAAACCAGTGTCAGGGA(GCT)；
NTA-sgRNA：ACAAAAAAAAAGCCTTCTTT(TTA)；
NTC-sgRNA：GGGAAAAATTGTCCAGCCCC(ATC)；
NTG-sgRNA：GGAAACAATGATAACAAGAC(CTG)；
NTT-sgRNA：CTTTAAACGTGTTCTTAACT(ATT)。
针对上述选定的目标基因序列，共16条，构建相应的sgRNA表达载体，将不同的sgRNA分别导入pGL3-U6-sgRNA-PGK-puromycin(Addgene，Plasmid#51133)哺乳基因编辑质粒(sgRNA表达载体)。
二、在细胞株上进行TadA8e-SpRY与TadA8e F148A-SpRY碱基编辑器针对不同PAM sgRNA的A>G编辑，验证两种编辑器在不同PAM sgRNA序列中的靶向编辑效率。
按常规操作，进行细胞株的DNA单碱基编辑(通过电转或脂质体转染)，以脂质体转染为例。
(1)以HEK293T细胞为例，本发明进行真核生物细胞的培养与转染：HEK293T细胞接种培养于添加10％(以体积百分比计)胎牛血清(FBS)的DMEM高糖培养液中(HyClone，SH30022.01B)，其中含青霉素(100U/mL)和链霉素(100μg/mL)。
(2)在转染前传至24孔板中，待密度达到80％-90％时进行转染。
(3)转染以脂质体转染为例。按照Lipofectamine TM2000 Transfection Reagent(Invitrogen，11668-019)的操作手册，将660ng TadA8e-SpRY或TadA8e F148A-SpRY质粒与330ng sgRNA表达质粒混匀，共转染至每孔细胞中，6h后换液，转染12h后，添加2ng/μL puromycin抗生素维持培养，72h后进行编辑效率的检测。
(4)编辑效率的检测与分析
a.转染细胞72h后，使用PBS清洗细胞三次，去除死细胞。滴加胰酶(覆盖细胞表面即可)消化细胞2-3min，以体积百分比计，添加200μL 10％FBS的DMEM完全培养基终止消化，重悬细胞；
b.重悬细胞收集至PCR管中，800rpm离心5min，使用吸液泵去掉液体，添加20μL细胞裂解液，置于PCR仪中充分裂解细胞。
裂解程序：68℃，30min；16℃，2min；98℃，2min；16℃保持。
c.PCR扩增目的片段：针对突变位点，设计PCR扩增引物，进行目的片段的PCR扩增。
PCR体系(50μL)：
Figure PCTCN2022131039-appb-000001
Super-Fidelity DNA Polymerase，1μL；dNTP，1μL；2x Buffer，25μL；细胞裂解产物，4μL；PCR扩增引物F/R(10nM)，分别2μL；ddH 2O，15μL。
PCR反应程序：95℃，3min；第一轮循环(10个循环)：95℃，15s；68℃，15s；72℃，30s；第二轮循环(30个循环)：95℃，15s；58℃，15s；72℃，30s；72℃，5min；16℃维持。
PCR引物如下：
Forward：AAAGCCTCAACAATGTTGCC；
Reverse：TTCCCAAGTGAGAAGCCAGT。
d.编辑效率的统计
将PCR产物进行纯化回收，使用Forward：AAAGCCTCAACAATGTTGCC，进行扩增片段的Sanger测序。结果使用在线统计工具EditR(https://moriaritylab.shinyapps.io/editr_v10/)进行具体编辑效率的统计。
结果表明，TadA8e-SpRY与TadA8e F148A-SpRY编辑器对16种PAM的sgRNA，在4-8位编辑窗口内均有A>G的DNA单碱基编辑。其中TadA8e-SpRY对NAN，NGN，NCN与NTN PAM窗口内A>G的平均编辑效率分别为45.7％，41.0％，58.3％与25.7％；TadA8e F148A-SpRY对NAN，NGN，NCN与NTN PAM窗口内A>G的平均编辑效率分别为40.3％，42.0％，52.7％与20.0％(图2)。
TadA8e-SpRY编辑器sgRNA序列中内各个位置的A碱基A>G的平均编辑效率分别为：A1，0.0％；A2，1.3％；A3，1.0％；A4，41.3％；A5，43.0％；A6，45.0％；A7，42.7％；A8，39.3％；A9，19.3％；A10，1.7％；A11，0.7％；A12，0.3％；A13，0.0％；A14，0.0％；A15，0.0％；A16，0.0％；A17，0.0％；A18，0.0％；A19，0.0％；A20，0.0％；TadA8e F148A-SpRY编辑器sgRNA序列中内各个位置的A碱基A>G的平均编辑效率分别为，A1，0.0％；A2，0.7％；A3，0.7％；A4，17.3％；A5，49.0％；A6，18.7％；A7，16.0％；A8，9.7％；A9，3.3％；A10，2.3％；A11，0.0％；A12，0.0％；A13，1.0％；A14，0.5％；A15，0.0％；A16，0.0％；A17，0.0％；A18，0.0％；A19，0.0％；A20，0.0％。相较于TadA8e-SpRY编辑器，TadA8e F148A-SpRY更倾向于编辑窗口内A5(第五位的碱基A)的A>G编辑，减少了旁系编辑，提高了编辑器针对单一碱基编辑的精确性(图3)。
实施例3
一、进行sgRNA质粒的设计。
为了验证两种编辑器的靶向编辑范围，针对48个人源基因序列共设计了16种不同PAM的sgRNA序列，可代表靶向基因组的任意序列。本发明如下选择设计sgRNA：
根据选择的基因序列，分别设计了具有NAN(NAA，NAC，NAG，NAT)，NGN(NGA，NGC，NGG，NGT)，NCN(NCA，NCC，NCG，NCT)，NTN(NTA，NTC，NTG，NTT)共16种PAM的20bp靶向序列，共形成48条sgRNA序列。其中的“N”表示A/T/G/C四种碱基中的任意一种。
本发明选定的48条sgRNA对应编码序列详见表1，序列顺序为5’-3’。
表1内源基因与对应的sgRNA信息表
靶标基因 NCBI ID sgRNA序列 PAM
UBE3A NC_000015.10 GATTGAGCAGGGAAATACTG GAA
SHANK3 NC_000022.11 GTTTATGTAACTCTTCCACT CAA
MECP2 NC_000023.11 GCAGATACTGAGTTTTTAAC AAA
RUNX1 NC_000021.9 GGCCTCATAAACAACCACAG AAC
CUL3 NC_000002.12 GGTCCAGTAGATATTGAAGT TAC
HEKsite4 NC_000003.12 GGGTCAGACGTCCAAAACCA GAC
FANCF NC_000011.10 GCAGGAGGTGGGGAAGGCCG AAG
SHANK3 NC_000022.11 GGTCCCCAGGAACCTCTCCG AAG
DYRK1A NC_000021.9 GGTTTGCAGCCTAAGAGCAG TAG
FANCF NC_000011.10 GAGACGTTCATGACTGGCAT CAT
CUL3 NC_000002.12 GTATTCAGCATATTGACATG TAT
ZSCAN2 NC_000015.10 GACAGTGTCCTGGAAATGAG GAT
UBE3A NC_000015.10 GTAAGCATAGAGGTGCTATG GGA
VEGFA NC_000006.12 GGTCAGAAATAGGGGGTCCA GGA
GRIN2B NC_000012.12 GCTGTAACAGGAGGGCCAGG AGA
RUNX1 NC_000021.9 GACTCAAATATGCTGTCTGA AGC
FANCF NC_000011.10 GAGACACTCCAAGAGAGCCT GGC
FANCF NC_000011.10 GCTCGGAAAAGCGATCCAGG TGC
EMX1 NC_000002.12 GTTCCAGAACCGGAGGACAA AGT
GRIN2B NC_000012.12 GCAAATACCAGAGATAAGAG AGT
DYRK1A NC_000021.9 GTACCTATCTGAGCATACCG TGT
EMX1 NC_000002.12 GCAACCACAAACCCACGAGG GCA
HEKsite3 NC_000003.12 GCTGGAGAAGCAGAAAAAAA GCA
MECP2 NC_000023.11 ACTCAGATGACTTTTATATG GCA
FANCF NC_000011.10 GGAGGACTCTCTGATGAAGA CCC
SHANK3 NC_000022.11 GGACTGACAGAACTGTAAAG GCC
EMX1 NC_000002.12 GTAGAGCAAACGCGTTCAGG GCC
FANCF NC_000011.10 GGGCCATGCCGACCAAAGCG CCG
HEKsite4 NC_000003.12 GCTTTAACCCCCACCTCCAG CCG
MECP2 NC_000023.11 TGTGATACTGAGTGGCCTAG ACG
MECP2 NC_000023.11 GCACACACATCCCTCGTGCA GCT
EMX1 NC_000002.12 GTCCGAGCAGAAGAAGAAGG GCT
DYRK1A NC_000021.9 GTAAACGCCCACACAAGTGA TCT
RUNX1 NC_000021.9 GTAAGTAATCCAATAGACTT GTA
RUNX1 NC_000021.9 GAAGAAAGAGAGATGTAGGG CTA
RUNX1 NC_000021.9 GCAAAGCTGAGCAAAAGTAG ATA
针对上述选定的目标基因序列，共48条，构建相应的sgRNA表达载体，将不同的sgRNA分别导入pGL3-U6-sgRNA-PGK-GFP(Addgene，Plasmid#107721)哺乳基因编辑质粒(sgRNA表达载体)。
二、在细胞株上进行TadA8e-SpRY与TadA8e F148A-SpRY碱基编辑器针对不同PAM sgRNA的A>G编辑，验证两种编辑器在不同PAM sgRNA序列中的靶向编辑效率。
按常规操作，进行细胞株的DNA单碱基编辑(通过电转或脂质体转染)，以脂质体转染为例。
(1)以HEK293T细胞为例，本发明进行真核生物细胞的培养与转染：HEK293T细胞接种培养于添加10％(以体积百分比计)胎牛血清(FBS)的DMEM高糖培养液中(HyClone，SH30022.01B)，其中含青霉素(100U/mL)和链霉素(100μg/mL)。
(2)在转染前传至24孔板中，待密度达到70％-80％时进行转染。
(3)转染以脂质体转染为例。按照Lipofectamine TM2000 Transfection Reagent(Invitrogen，11668-019)的操作手册，将660ng TadA8e-SpRY或TadA8e F148A-SpRY质粒与330ng sgRNA表达质粒混匀，共转染至每孔细胞中，6h后换液，转染12h后，添加2ng/μL puromycin抗生素维持培养，72h后进行编辑效率的检测。
(4)编辑效率的检测与分析
a.转染细胞72h后，使用PBS清洗细胞三次，去除死细胞。滴加胰酶(覆盖细胞表面即可)消化细胞2-3min，以体积百分比计，添加200μL 10％FBS的DMEM完全培养基终止消化，重悬细胞，细胞悬液通过70μm细胞筛，去掉培养基中的杂质，将细胞滤液置于流式管中；
b.使用流式细胞分选仪BD Aria III进行细胞分选，每个样品分选10,000个GFP阳性的细胞，分选的细胞进行1000rpm,5min的离心沉淀处理后，移除上清，添加20μL细胞裂解液重悬细胞，置于PCR仪中充分裂解细胞。
裂解程序：68℃，30min；16℃，2min；98℃，2min；16℃保持。
c.PCR扩增目的片段：针对突变位点，设计PCR扩增引物，进行目的片段的PCR扩增。
PCR体系(50μL)：
Figure PCTCN2022131039-appb-000002
Super-Fidelity DNA Polymerase，1μL；dNTP，1μL；2x Buffer，25μL；细胞裂解产物，4μL；PCR扩增引物F/R(10nM)，分别2μL；ddH 2O，15μL。
PCR反应程序：95℃，3min；第一轮循环(10个循环)：95℃，15s；68℃，15s；72℃，30s；第二轮循环(30个循环)：95℃，15s；58℃，15s；72℃，30s；72℃，5min；16℃维持。
PCR引物如下表2所示：
表2内源基因编辑效率检测PCR扩增引物序列表
靶标基因 上游引物(5’-3’) 下游引物(5’-3’)
UBE3A ACTGCTTTCTGTCTTCTGGC CAGCAGCTATTCCAAAAATC
SHANK3 GGCAGGCACCGTCTTTGTCG CATGTACGTTCGTCAAGGTT
MECP2 GCCTCTTGGTTGTAATATGC CATCAGAGAGCATTGATCAC
RUNX1 CTGACCACTATGCTGGGTTC TTTCTTGCACAGCCTGGGGG
CUL3 GCCACCTGGTTTATGGGATT CCAAGTTTTGGGCTCCAGTA
HEKsite4 GAGGTGGGGGTTAAAGCGGA CAGTGAAATCACCCTGGGGG
FANCF CTCTCCAGGTGATTTGTGGA GGAGGACTCTCTGATGAAGA
SHANK3 CCCATCTTCCCGAGCATTCT CGCCAGCTTCTCGTCCTCCC
DYRK1A CAAATAATGAGGGTTACAGT AACATCACTGAGTATACACTGCA
FANCF GGAGGGAGAGCAGATGTAGG AGAGCGTTTCCTCACGTCAC
CUL3 GGAATAGCACCAGAATGTTC GCCTACACTTAAAAACTTGACGT
ZSCAN2 GGACTGGCCTGGAGTGGGAG CCTTCCACGCCTATGCCCTG
UBE3A GGCCTCTCTCCAAGTTTCTG GGACAGTGAGATTAGGCAGA
VEGFA GGGCTCTCTGTACATGAAGC GAAGACGCTGCTCGCTCCAT
GRIN2B GGAAAAGAGGTTGTGAGTGG AGAATGCAGGGCTTGTGTAC
RUNX1 CAAACAAGACAGGGAACTGG CCCCGCCTTCAGAAGAGGGT
FANCF CCACCTCCTGCAGACGCTCC GGTGCAGCAACTCTTTCCCG
FANCF GGTCCCAGGTGCTGACGTAG CACGGATAAAGACGCTGGGA
EMX1 GGGGCCTCCTGAGTTTCTCA GGTTGCCCACCCTAGTCATT
GRIN2B GGACCTTATCTCCTTTCATTGAG CATACTCGCATGGCTACCTG
DYRK1A CCAACCCCTGCCTGTGGAAT GCAATGTGAAGGTCTACGAACA
EMX1 GAAGCAGGCCAATGGGGAGG CTTGTCCCTCTGTCAATGGC
HEKsite3 CCTAGAAAGGCATGGATGAG CCTTTCCTCTGCCATCACGT
MECP2 ACCAGATGGGGCAAGTTCAT CTCGGGGTCCATACTTAGCA
FANCF CCGCTATCACCTTCAGGAAG GGCGGTCTGCGGTGCACATG
SHANK3 GGCAGGCACCGTCTTTGTCG CATGTACGTTCGTCAAGGTTAAAG
EMX1 CAGAGCCTGGGGTGGTAGAT GGCCCTTCCCTATGTCTAGC
FANCF CTTGCCTCCACTGGTTGTGC GCGCACCTCATGGAATCCCT
HEKsite4 GGCGAGGCAGAGGGTCCAAA CTCCTTCTGGGGCCTTTTTC
MECP2 CTCTGCCGAGCCTTTCACAC GCCATGGAACCCAAAATTCT
MECP2 CCAGCTCTGTGGGAAGCAAC GCTGTTTTCCCCTCTGAGCT
EMX1 CAGAACCGGAGGACAAAGTA GCAGCAAGCAGCACTCTGCC
DYRK1A CCCATTGCAACTTCCAGTCC CGCTAGACGGTAGAGCCTAC
RUNX1 CCAGCACAACTTACTCGCAC GAGATGCCTCGGTGCCTGCC
RUNX1 CCTCCTGAAAATGCACCCTC GGTGCATTTTTTAATAGGGC
RUNX1 CAAACAAGACAGGGAACTGG CCCCGCCTTCAGAAGAGGGT
CUL3 GGAATAGCACCAGAATGTTC GCCTACACTTAAAAACTTGACG
DYRK1A TCGCCAGCCAAACATAAGTG CCCAATCCATAATCCCACGT
MECP2 AAAAAGCTCATTCTGGAATT ACACAACGTGTGAAAGGCTC
MECP2 GCCTCTTGGTTGTAATATGCAG CATCAGAGAGCATTGATCACAG
EMX1 CCTGGGACCACTTGGCCTTC AGCTGGATGCCCGTGTCATT
FANCF GGGACTCAGTTCCAACCCAA GGCATCCACAAATCACCTGG
GRIN2B ACGAGGATGACAGCAATGCC CTAGCCTCTTCTAAGACAGGTTAC
UBE3A CTTACCCGGACAAGTGCATC CATGTCCCTTTATATTGAATGCTGT
CUL3 TTGGGAGCACTTCCAGGTTC CTGCACTCCAGCCTTGGTGA
AAVS1 GGCCCAGACTAGCCCAGTTG CCACCTGCCTTGGCCTCTCA
GRIN2B CCAATCATGACCAATTGCCA CACAGCTTCATCCCTGAGCC
EMX1 GAAGCAGGCCAATGGGGAGG CTTGTCCCTCTGTCAATGGC
d.编辑效率的统计
将PCR产物进行纯化回收，使用Primer-F进行扩增片段的Sanger测序。结果使用在线统计工具EditR(https://moriaritylab.shinyapps.io/editr_v10/)进行具体编辑效率的统计。
结果表明，TadA8e-SpRY与TadA8e F148A-SpRY编辑器对在16种PAM的所有48个位点的sgRNA均有A>G的DNA单碱基编辑效率，几乎无PAM的限制，拓宽了编辑基因组范围。如图4和图5所示，针对HEK293T细胞，TadA8e F148A-SpRY对NAN，NGN，NCN与NTN PAM窗口内A>G的平均编辑效率分别为50.73％，46.43％，40.59％与22.50％。如图6和图7所示，针对Hela细胞，TadA8e F148A-SpRY对NAN，NGN，NCN与NTN PAM窗口内A>G的平均编辑效率分别为42.57％，34.74％，36.85％与36.68％。TadA8e F148A-SpRY编辑器无PAM限制，在大多数位点中具有较高的A>G DNA编辑效率，极大的拓宽了基因组的靶向编辑范围。
TadA8e-SpRY在HEK293T与Hela细胞中的主要活性编辑窗口均为A3-A11(从PAM远端开始计算，第一位的碱基A标记为A1)，同时在NCN PAM等部分位点，还存在A15与A18等窗口外位点的旁系编辑。而新型碱基编辑器TadA8e F148A-SpRY在HEK293T与Hela细胞中的主要活性编辑窗口均为A3-A10，且几乎不存在窗口外的旁系编辑。TadA8e F148A-SpRY缩小了编辑窗口，减少了旁系编辑，提高了编辑器针对单一碱基编辑的精确性(图8和图9)。
(4)RNA脱靶检测与分析
a.选择高效编辑位点FANCF(sgRNA:GAGACACTCCAAGAGAGCCT,PAM:GGC)进行TadA8e-SpRY与SpRY-AB8e F148A的RNA脱靶检测、分析。
b.将2μg FANCF-sgRNA+4μg TadA8e-SpRY，2μg FANCF-sgRNA+4μg SpRY-AB8e F148A和6μg pCMV-GFP的质粒组合分别转染至70-80％密度的6cm皿的HEK293T细胞中，3重复/样品，共9个细胞样品。转染6h后换液，转染48h后，通过流式细胞分选，每个细胞样品分选10 6GFP阳性细胞，分选细胞提取总RNA，建库进行RNA-seq测序。
c.RNA-seq数据分析流程。RNA突变鉴定采用GATK标准化流程，合并gvcf文件得到joint call的VCF合集，保证批次内SNP的可比性。得到SNP集合后进行SNP注释，明确位置。在9个样本中，FANCF-sgRNA+TadA8e-SpRY和FANCF-sgRNA+SpRY-AB8e F148A是实验组，以eGFP为对照组，每组内3个样本。基本逻辑：判定正负链→判定eGFP组基因型→判定实验组每个样本基因型→突变的位点进行深度过滤→计算突变频率。首先判断每个SNP突变的mRNA模板链是参考基因组的正链还是负链。如果是正链上发生的SNP突变，则在eGFP组中寻找UU纯合子位点(需保证所有control组的基因型均为UU纯合)，判定这些位点在实验组的每个样本中的基因型，当三个样本任意一个中存在测序深度大于20x的UC突变，则记录该位点位置，利用两种碱基的reads数目之比计算突变频率。如果是负链上发生的 SNP突变，则在eGFP组中寻找AA纯合子位点，判定该位点在实验组中是否存在AG突变，其余分析条件同上。
内源基因编辑细胞的RNA-seq测序分析结果显示，相较于TadA8e-SpRY，TadA8e F148A-SpRY编辑器没有显著减少A>G RNA脱靶总数，但显著降低了A>G RNA脱靶的平均编辑效率，总体上降低了RNA脱靶率，进一步提高了精确性(图10)。
(5)G>A突变位点的修复
a.从ClinVar(https://www.ncbi.nlm.nih.gov/clinvar/,accessed Feb,2022)数据库中获得APOC3(p.D65N,c.G2871A),SCN9A(p.R896Q,c.G98851A)和SLC30A8(p.M50I,c.G196816A)三个致病位点的基因序列信息。根据其序列，分别设计构建突变位点的mut-sgRNA，构建表达载体。使用pCAG-CBE4max-SpRY-P2A-EGFP(RTW5133)(Addgene#139999)介导的C>T DNA单碱基编辑进行上述三个位点的G>A突变模型构建。将mut-sgRNA+pCAG-CBE4max-SpRY-P2A-EGFP的质粒组合转染至70-80％密度的HEK293T细胞中，转染48h后，使用流式细胞分选GFP阳性的单克隆细胞至96孔板中。培养细胞约两周后，将生长的单克隆细胞传代至24孔板中。待细胞长满24孔板后，取约1/4进行裂解，使用鉴定引物进行PCR扩增，Sanger测序，获得C>T编辑(G>A致病突变)的单克隆细胞。mut-sgRNA与鉴定引物序列信息见表3。
b.针对APOC3(p.D65N,c.G2871A),SCN9A(p.R896Q,c.G98851A)和SLC30A8(p.M50I,c.G196816A)的序列信息，分别设计5条靶向致病位点修复的sgRNA1-5，构建表达载体。将sgRNA+TadA8e F148A-SpRY的质粒组合分别转染至突变的HEK293T细胞中，转染48h后，使用流式细胞分选GFP阳性细胞，10,000细胞/样品。细胞裂解，使用鉴定引物PCR扩增目的片段，Sanger测序，使用EditR进行A>G修复效率统计。修复sgRNA序列见表3。
表3突变位点与sgRNA序列信息表
Figure PCTCN2022131039-appb-000003
结果表明，在对APOC3(p.D65N,c.G2871A),SCN9A(p.R896Q,c.G98851A),and SLC30A8(p.M50I,c.G196816A)等G>A突变细胞的单碱基致病位点修复中，TadA8e F148A-SpRY分别展现出了较高的A>G DNA修复编辑效率，分别可达29.33％，22.33％与27.67％(图11)。
总结：本发明将无PAM限制的SpCas9变体SpRY(D10A)与引入关键氨基酸突变的脱氨酶TadA8e(F148A)融合，构建的新型腺嘌呤编辑器TadA8e F148A-SpRY可高效靶向基因组进行A>G的DNA单碱基编辑，几乎无PAM限制。将TadA8e-SpRY的编辑窗口由3-11位缩小至3-10位，减少了编辑窗口外的旁系编辑，同时降低了A>G RNA脱靶的平局编辑效率，提高了腺嘌呤碱基编辑器的精确性，并成功将其应用于G>A致病突变型细胞的修复编辑。本发明提供的新型腺嘌呤编辑器TadA8e F148A-SpRY具有无PAM限制，兼具编辑高效性与特异性的优势。为后续内源性基因G>A点突变精确修复基因编辑工具的优化，基因疾病致病性位点的模拟或修复及临床研究及提供新的思路与方法。

Claims (8)

  1. 一种精确无PAM限制的腺嘌呤碱基编辑器，其特征在于，所述腺嘌呤碱基编辑器包含腺嘌呤脱氨酶突变体TadA8e F148A的编码基因和SpRY核酸酶突变体的编码基因，其中，腺嘌呤脱氨酶突变体TadA8e F148A为腺嘌呤脱氨酶TadA8e氨基酸序列148位的氨基酸Phe突变为Ala获得，腺嘌呤脱氨酶TadA8e的氨基酸序列如SEQ ID NO.21所示。
  2. 如权利要求1所述精确无PAM限制的腺嘌呤碱基编辑器，其特征在于，腺嘌呤脱氨酶突变体TadA8e F148A的编码基因位于SpRY核酸酶突变体的编码基因的上游端。
  3. 如权利要求1所述精确无PAM限制的腺嘌呤碱基编辑器，其特征在于，腺嘌呤脱氨酶突变体TadA8e F148A的编码基因的核苷酸序列如SEQ ID NO.2所示，SpRY核酸酶突变体的编码基因的核苷酸序列如SEQ ID NO.3所示。
  4. 如权利要求1所述精确无PAM限制的腺嘌呤碱基编辑器，其特征在于，还包含核定位信号片段NLS，所述核定位信号片段NLS的编码基因的核苷酸序列如SEQ ID NO.4所示。
  5. 如权利要求4所述精确无PAM限制的腺嘌呤碱基编辑器，其特征在于，腺嘌呤脱氨酶突变体TadA8e F148A的编码基因位于SpRY核酸酶突变体的编码基因的上游端，核定位信号片段NLS的编码基因位于SpRY核酸酶突变体的编码基因的下游端。
  6. 如权利要求1-5任一项所述精确无PAM限制的腺嘌呤碱基编辑器在制备基因编辑试剂中的应用。
  7. 如权利要求1-5任一项所述精确无PAM限制的腺嘌呤碱基编辑器在非疾病治疗为目的的基因编辑中的应用。
  8. 一种基因编辑方法，其特征在于，通过权利要求1-5任一项所述精确无PAM限制的腺嘌呤碱基编辑器进行基因编辑。
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