CRISPR Induced ‘Second Hit’ Mutations in APC Cause Differentiation of FAP Human Embryonic Stem Cells

Introduction
Patients carrying a germline mutation in the APC gene will all develop colon cancer, and PGD is thus their best fertility option. APC is a tumor suppressor gene implicated in FAP as well as in sporadic colorectal cancer, is a major controller of Wnt signaling, one of the main pathways regulating development.

Aim
To study the role of APC in hESCs differentiation potential by CRISPR genome editing technology.

Materials & Methods
hESCs, carrying a germline mutation in the APC gene, were derived in our lab following PGD. We used CRISPR with specific gRNAs to target the second allele of APC in order to mimic cancer and induce the somatic mutation. Single cell clones were isolated and successful targeting was evaluated by Sanger, NGS and western blot analysis. To further study mutation effect on Wnt signaling and hESCs self-renewal, β-catenin transcriptional activity was analyzed by FACS analysis and sorting.

Results
CRISPR was targeted to loci along the APC gene in FAP-hESCs, and 59 clones were isolated, and sequencing showed that 19% carried CRISPR induced mutations. Though, all clones expressed the full-length APC protein. Follow up analysis of FAP-hESCs carrying a β-catenin reporter after CRISPR revealed that the proportion of APC mutated cells significantly declined with time, a result that was also confirmed by NGS. Isolation of double mutated cells directly after targeting demonstrated that the cells undergo rapid differentiation.

Conclusions
APC induced mutation in FAP-hESCs results in Wnt signaling activation and differentiation. The use of CRISPR in FAP-hESCs provides a novel research model to study cancer development.