Induction method of gene point mutation
A technology of mutants and variants, applied in the field of induction of fusion proteins and gene point mutations, can solve the problems of limited application of single-base editing tools editing ability, and achieve the effect of fine C-T single-base replacement
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Embodiment 1
[0083] Embodiment 1 constructs fusion protein
[0084]The open reading frames (ORFs) of adenosine deaminase (TadA) and deoxyadenosine deaminase (TadA*) were synthesized with codon-optimized human origin, and their nucleotide sequences are shown in SEQ ID NO:1-2 respectively , and the encoded amino acid sequences are respectively shown in SEQ ID NO: 3-4. Synthesize cytidine deaminase LpCDA1L1-1(8) and cytidine deaminase APOBEC3A3-Y130F (A3A(Y130F)), the nucleotide sequences of which are shown in SEQ ID NO:5-6 respectively, and the encoded amino acid sequences are respectively As shown in SEQ ID NO:7-8.
[0085] In this experiment, spCas9 and SaCas9 were used, and specific mutations were introduced to obtain nspCas9 (Addgene #48137; NCBI ACCESSION QAB34447.1), nSaCas9 (Addgene #61593; NCBI ACCESSION J7RUA5). Restriction sites (Table 1) were introduced into nspCas9 through point mutations to fuse cytidine deaminase or deoxyadenosine deaminase to obtain fusion proteins for subse...
Embodiment 2
[0093] Example 2 Construction of sgRNA expression vector
[0094] A sgRNA expression vector was constructed to express UGI-2A-mCherry while expressing a specific sgRNA. UGI can inhibit the activity of uracil glycosylase and improve the efficiency of C-T mutation. The red fluorescent protein mCherry is used to indicate the expression of the vector and can be used for subsequent flow cytometry (see figure 1 ).
[0095] A variety of sgRNAs (see Table 3 for details) were selected to construct sgRNA expression vectors (see Table 4 for details) to analyze the frequency of mutation of cytosine to thymine in the prespacer sequence.
[0096] Table 3 sgRNA information
[0097] S16 GGGAATAAATCATAGAATCC SEQ ID NO:12 sgA tGCCCCTCCCCTCCCTGGCCC SEQ ID NO: 13 sgB AGAGCCCCCCCTCAAAGAGA SEQ ID NO: 14 HEK4 GGCACTGCGGCTGGAGGTGG SEQ ID NO: 15 FANCFN GGAATCCCTTCTGCAGCACC SEQ ID NO: 16 EMXNGG GAGTCCGAGCAGAAGAAGAA SEQ ID NO: 17 site1 G...
Embodiment 3
[0102] Example 3 Different ABE internal fusion single base editing vectors and sgRNA expression vectors were transfected into HEK293T cells to detect the frequency and characteristics of A-G mutations
[0103] The HEK293T cell line was planted on a 24-well plate and placed in a 37°C 5% carbon dioxide incubator for culture, and the transfection experiment was carried out 8-16 hours later. The expression vector (SEQ ID NO: 9) of the ABE internal fusion protein system and the expression vector of specific sgRNA were co-transfected with Lipofectamine 3000, cultured at 37°C for 72 hours in a 5% carbon dioxide incubator, and sorted by flow cytometry (flow cytometry) to enrich cells expressing both GFP and mCherry fluorescent proteins. After the genome is extracted, PCR primers for specific sgRNAs are used for fragment amplification, and sent to Sanger sequencing to analyze the A-G mutations in the pre-spacer sequence targeted by the sgRNA. For the editing efficiency of each fusion ...
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