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

Abstract: Mutation in 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 HeLa cells that were cultured in medium with antibiotics puromycin and neomycin. After 2-week selection， one part of drug-resistant HeLa 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 HeLa cells， however， the recombinant insertion was able to be detected in drugresistant HeLa 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.

CSTR: 32200.14.cjcb.2018.02.0013