关键词: CRISPR/Cas9, Vel抗原, SMIM1基因, K562细胞系

Abstract: 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.

Key words: CRISPR/Cas9, Vel blood group, SMIM1 gene, K562 cell line