Gene Editing with CRISPR/Cas9 Technology before Cell Transplantation Using a Mouse Model of Hirschsprung's Disease

Abstract

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, if the gene mutation in patient‐derived iPSCs can be corrected before transplantation. In this study, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated 9 (Cas9) gene editing technology was used to correct the Sox10 mutation in iPSCs derived from Sox10Dom/Dom embryos. 4 sgRNAs targeting the mutation site were designed, and a common donor plasmid carrying the correct sequence for the homology directed repair (HDR) was constructed. A SmaI digestion site was introduced to the correct sequence of the donor plasmid for detecting the HDR efficiency. The T7E1 assay demonstrated that the sgRNAs 1, 2 and 3 had high indel efficiency, and SmaI digestion showed 23.9% HDR efficiency. Using SmaI digestion, 8 homozygous and 4 heterozygous isogenic lines with the corrected Sox10 sequence were obtained, among which 3 homozygous corrected lines did not show meaningful off‐targets. Immunofluorescence staining showed that stem cell markers Oct4, Sox2 and SSEA‐1 were highly expressed in corrected iPSCs (Sox10Cor/Cor iPSCs). Neural crest cells (NCCs) are the progenitors of enteric neurons and glia cells. To determine the ability of iPSCs to differentiate into NCCs before and after the gene correction, a three‐step protocol was used to induce mouse iPSCs into NCCs, which were found to exhibit properties similar to enteric neural crest cells within the developing gut. Immunofluorescence staining showed that NCC markers Sox10, Nestin, AP2α and Ednrb were expressed in NCCs derived from Sox10+/+ iPSCs. Real time RT‐PCR also demonstrated that Sox10, p75 and Nestin were highly expressed in Sox10+/+ iNCCs. Flow cytometric cell analyses showed 25% p75‐positive cells in Sox10+/+ iPSCs‐induced NCCs. Immunofluorescence staining also showed that NCC markers Sox10, Nestin and Ednrb were expressed in NCCs derived from Sox10Dom/Dom and Sox10Cor/Cor iPSCs. Sox10+/+, Sox10Dom/Dom and Sox10Cor/Cor iPSCs‐derived NCCs were successfully directed to differentiate into neurons, glia, myocytes, osteoblasts and chondrocytes in vitro. Real‐time RT‐PCR showed rescued expression of nestin, p75 and Ednrb in Sox10Cor/Cor iPSCs‐derived NCCs when compared those derived from Sox10Dom/Dom. Our results demonstrated the successful use of CRISPR/Cas9‐mediated gene editing to correct the Sox10 mutation in Sox10Dom/Dom mouse iPSCs. After gene editing, the iPSCs could be induced to form neural crest cells for the potential use in cell transplantation to generate enteric neurons in the gut.Support or Funding InformationThe work was supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, China (Project No.: CUHK 14102214)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.