利用CRISPR/Cas9技术敲除K562细胞中的SMIM1基因

To Knock Out the SMIM1 Gene in K562 Cells by CRISPR/Cas9

目的利用慢病毒介导的CRISPR/Cas9基因编辑技术,在K562细胞系中敲除编码Vel血型抗原的SMIM1基因。方法设计5条sgRNA序列,构建Cas9-sgRNA共表达质粒,在293T细胞中初步筛选切割效率较高的sgRNA序列。将筛选后的sgRNA通过第二代慢病毒包装系统包装慢病毒并感染K562细胞,提取细胞基因组DNA,进行Sanger测序和TA克隆检测。结果使用易转染的293T细胞建立sgRNA的初步筛选平台,筛选出切割效率较高的sgRNA4序列。CRISPR/Cas9系统成功在K562细胞中SMIM1基因的sgRNA4识别位点发挥基因编辑活性。结论证实了利用CRISPR/Cas9技术在K562细胞中敲除SMIM1基因的可行性,为构建Vel抗原表型阴性的细胞模型奠定基础,也为后续编辑其他血型抗原的基因提供实验依据。

Objective To knock out the SMIM1 gene encoding the Vel blood group antigen in the K562 cell line by lentivirus-mediated CRISPR/Cas9 gene editing.Methods We designed five sgRNA sequences and constructed Cas9-sgRNA co-expression plasmids to initially screen sgRNA sequences with higher cutting efficiency in 293T cells.The sgRNA which had been screened out was packaged with the second-generation lentivirus packaging system and then infected K562 cells.We extracted the genomic DNA for Sanger sequencing and TA cloning detection.Results We established a preliminary screening platform for sgRNA using easy-to-transfect 293T cells,and screened out sgRNA4 sequences with higher cutting efficiency.The CRISPR/Cas9 system successfully exerted gene editing activity at the sgRNA4 recognition site of SMIM1 gene in K562 cells.Conclusion We confirmed the feasibility of using CRISPR/Cas9 technology to knock out the SMIM1 gene in K562 cells,which was a foundation for constructing a cell model with negative Vel antigen phenotype and providing experimental basis for subsequent editing genes of other blood group antigens.