Generation of isogenic iPSC with RET mutation using CRISPR-Cas9

Abstract

The Hirschsprung (HSCR) disease is a congenital disorder that causes serve intestinal obstruction in affected patients due to absence of neuronal cells in the distal part of the gut. Although mutations in the RET-proto tyrosine kinase receptor have frequently been associated with this disease, it is still not entirely clear what is the precise underlying molecular mechanism. Evidences from genetic and animal studies suggest mutations in the RET gene can result in reduced expression level or comprised protein function that might be the cause of HSCR. To date, advancements in stem cell technology allow us to directly address individual gene function using an in vitro neuronal differentiation system that can mimics this process during embryonic development. In this study, we aim to define the role of RET signalling in human neural crest cell (NCC) development by generating an isogenic human iPS cell harbouring a RET deletion mutation using CRISPR-Cas9. By comparing the in vitro neuronal differentiation capabilities of RET mutant cells to that of isogenic wild type control cell, we found that iPS cells carrying coding sequence mutation in RET was able to generate NCCs in a comparable manner to the control. However, when these RET mutant NCCs were subjected to further neuronal differentiation, we observed reduced neuronal differentiation as compared to the control. We also found reduced cell migration and increased cell death in the RET mutant NCCs. Together, our data suggests proper RET function is essential for neuronal differentiation and migration of human NCCs.