CRISPR/Cas9介导的同源重组插入敲除人SH2B3基因

CRISPR/Cas9 Mediated Homology Recombination Insertion Knockout of Human SH2B3 Gene

SH2B3基因的突变可以显著提高人干细胞向红细胞诱导分化的效率。该研究利用CRISPR/Cas9技术介导的同源重组插入敲除策略,建立了一种高效获得特定基因编辑类型的敲除SH2B3基因的方法。通过设计两个含有不同荧光标记和不同抗性基因的同源重组筛选载体和一个靶向SH2B3基因的CRISPR/Cas9敲除载体,共转染HeLa细胞,然后用嘌呤霉素和新霉素进行筛选,两周后一部分细胞用于分子生物学检测,另一部分细胞通过有限稀释法分离单细胞克隆。结果显示,在药物抗性筛选两周的HeLa细胞中,野生型SH2B3基因转录产物几乎检测不到,可以检测到重组型转录产物的表达。敲除效率的统计结果显示,在获得的19株SH2B3基因敲除细胞中,有11株细胞为双等位基因插入敲除。另外8株细胞为单等位基因插入敲除,其中有2株细胞的等位基因没有检测到突变,而剩余的6株细胞的等位基因都检测到有突变。因此,该研究的双插入敲除率为57.9%,双敲除率达到89.5%。该研究为构建SH2B3基因敲除的人多能干细胞系奠定了基础,也为建立一种高效、低成本的诱导红细胞的技术体系提供了有效的工具。

Mutation of SH2B adapter protein 3（SH2B3） gene resulted in significantly increasing the induced efficiency of red blood cells derived from human pluripotent stem cells. We established a high-efficiency method through certain type of gene editing to knockout SH2B3 gene in human cells via CRISPR/Cas9-mediated homology-dependent DNA repair. Two homology-dependent repair screening plasmids, which retain different fluorescent genes and different drug-resistant genes, and one plasmid targeting SH2B3 gene were designed and were cotransfected into He La cells that were cultured in medium with antibiotics puromycin and neomycin. After 2-week selection, one part of drug-resistant He La cells was used for the molecular biology assays, and rest of cells were sorted for colonies via limiting dilution. Results showed that expression of wild type SH2B3 gene could not be detected in the drug-resistant He La cells, however, the recombinant insertion was able to be detected in drugresistant He La cells. The statistical analysis of knockout efficiency showed that among the 19 SH2B3-KO clones,11 clones were double knockout via double knock-in of reporter genes, and 8 clones were knockout via single knock-in of reporter genes. Moreover, within these 8 clones, there were 2 clones that had the wild type gene in the allele, and 6 clones had site mutations in the allele. Therefore, the efficiency of double knockout via knock-in of reporter genes was 57.9%, and double knockout was 89.5%. This study provides a powerful tool to establish an efficient and low-cost means to improve the induction-efficiency of red blood cells derived in human pluripotent stem cells.