一种高效的β地中海贫血CD17(A>T)点突变293T细胞系的建立

Establishment of a highly efficient method for β-thalassemia CD17(A>T) point mutation in HEK293T cell line

目的 建立一种高效构建β-地中海贫血CD17(A>T)点突变基因型HEK293T细胞系的方法。方法 利用改良的CRISPR/Cas9基因编辑技术,即无痕基因组编辑(consecutive re-Guide or re-Cas steps to erase CRISPR/Cas-blocked targets, CORRECT),通过电转染CRISPR/Cas9质粒诱导HEK293T细胞HBB基因切割,同时以引入有CD17(A>T)点突变和同义突变碱基(G>T)的单链寡核苷酸(singlestranded oligo DNA nucleotides, ssODNs)作为同源模板进行重组,经单克隆筛选、测序验证获得β-珠蛋白基因(HBB)点突变CD17(A>T)基因型HEK293T细胞系。结果 利用“CORRECT”技术成功获得一株β-地贫CD17(A>T)基因型点突变的HEK293T细胞系,同义突变的引入减少Cas9蛋白对靶点不准确的再编辑,提高单碱基突变效率。结论 通过“CORRECT”技术可以高效获得点突变的293T细胞系,对单碱基突变疾病模型的细胞系及动物模型的建立具有重要意义。

Objective To establish an efficient method for constructing HEK293T cell line of β-thalassemia CD17(A>T) point mutation.Methods Using a modified CRISPR/Cas9 gene editing technology,termed‘CORRECT’(consecutive re-Guide or re-Cas steps to erase CRISPR/Cas-blocked targets)for scarless genome editing.Firstly,the cleavage of HBB gene in HEK293T cells was induced by electrotransfection of CRISPR/Cas9 plasmid.Then,single-stranded oligo DNA nucleotides(ssODNs) with CD17(A>T) point mutation and synonymous mutation(G>T) were used as homologous templates for repair.The HEK293 T cell line with β-globin CD17(A>T) point mutation was obtained by monoclonal screening and sequencing analysis.Results A HEK293T cell line with point mutation of β-thalassemia CD17(A>T)genotype was successfully obtained by ’CORRECT’ technique.The introduction of synonymous mutation might reduced re-editing of Cas9 protein to the target,which greatly improved the efficiency of single base mutation.Conclusions The point mutation HEK293T cell line can be efficiently obtained by ’CORRECT’technique,which is of great significance for the establishment of single base mutation cell lines and animal models.