CRISPR-Cas9 mediated correction of Hirschsprung-associated mutations in human iPSC restores neural crest cell functions

Abstract

Hirschsprung (HSCR) disease is caused by the failure of enteric neural crest cells (ENCCs) to fully colonize the bowel, thus leading to bowel obstruction. Heterozygous mutations in the coding region of the RET gene account for the severe form of HSCR (total colonic aganglionosis, TCA). However, 80% of HSCR patients have short segment HSCR (S-HSCR), whose genetic etiology remains unclear. Here, we aimed to establish a new strategy for the functional identification of novel mutation(s) in HSCR disease. Three HSCR-iPSC lines (1 TCA-HSCR patient with a RET G731del mutation and 2 S-HSCR patients without RET mutation) and two RET isogenic mutant iPSC lines (RET+/- and RET-/-) were established to generate disease-relevant ENCCs. ENCCs derived from all diseased iPSC lines exhibited defects in migration and neuronal lineage differentiation. RET mutations were functionally associated with cellular phenotypes of ENCCs in vitro. Integration of genetic and transcriptomic data further identified a novel mutation in Vinculin (VCL M209L) associated with S-HSCR. Correcting the RET and VCL mutations in HSCR-iPSCs restored the ENCC functions. Taken together, we identified VCL as a novel HSCR gene. More importantly, our results also illustrate the value of human iPSCs for defining mutations that functionally contribute to a complex disease.