Gene correction of a Sox10 mutation in mouse iPSCs using CRISPR/Cas9 gene editing system
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摘要Hirschsprung’s disease (HSCR) is a congenital gastrointestinal motility disorder characterized by the regional absence or reduction of enteric neurons in the colon. The Dominant megacolon (Dom) mouse mutant is an animal model of HSCR, where a frameshift mutation in the Sox10 gene has been located. Recently, the generation of induced pluripotent stem cells (iPSCs) from patients has raised the possibility of using autologous cell transplantation to treat HSCR patients, with the pre-requisite that the gene mutation in patient’s iPSCs has to be corrected before transplantation. In this study, as a proof of principle, we made use of the CRISPR/Cas9 gene editing technology to correct the Sox10 mutation in iPSCs generated from the Dom mouse. We first designed 3 sgRNAs to target the mutation site, and constructed a common donor plasmid to carry a correct sequence for the homology directed repair. We obtained 8 homozygous and 4 heterozygous isogenic lines with correct Sox10 transcripts, among which 5 homozygous corrected lines did not show any meaningful off-targets. Real-time PCR showed rescued expression of Sox10 and Nestin in corrected miPSC-induced neural precursors. Our results demonstrated that we have successfully used CRISPR/Cas9-mediated gene editing to correct the Sox10 mutation in Dom miPSCs.
著者LI Yuejiao, FENG Bo, CHAN Wood Yee
會議名稱The 25th International Symposia on Morphological Sciences

上次更新時間 2018-20-01 於 19:01