CRISPR/Cas9-mediated gene correction in iPSCs derived from Dominant megacolon mouse skin fibroblasts
Refereed conference paper presented and published in conference proceedings


摘要Hirschsprung's disease (HSCR) is a common congenital gastrointestinal motility disorder presented with the regional absence or reduction of enteric neurons in the colon. The Dominant megacolon (Sox10Dom) mouse 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, provided that the gene mutation in patient-derived iPSCs can be corrected before transplantation. In this study, as a proof of principle, we made use of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) gene editing technology to correct the Sox10 mutation in iPSCs generated from Sox10Dom/Dom embryos. We first designed 4 sgRNAs targeting the mutation site, and constructed a common donor plasmid carrying the correct sequence for the homology directed repair (HDR). We obtained 8 homozygous and 4 heterozygous isogenic lines with corrected Sox10 sequence, among which 3 homozygous corrected lines did not show meaningful off-targets. Then we used a three-step protocol to induce the mouse iPSCs into neural crest cells (NCCs), which give rise to enteric neurons and glia. Real-time RT-PCR showed rescued expression of nestin, p75 and Ednrb in iPSCs-induced NCCs carrying the corrected sequence. Our results demonstrated that we have successfully used CRISPR/Cas9-mediated gene editing to correct the Sox10 mutation in Sox10Dom/Dom miPSCs.
著者Yuejiao LI, Bo FENG, Hui ZHAO, Wood Yee CHAN
會議名稱Keystone Symposia on Molecular and Cellular Biology 2017
會議地點Hong Kong

上次更新時間 2018-27-11 於 10:54