CRISPR-iPSC Example: Targeted Heterozygous Point Mutation Generation Without Silent Mutation

Targeted Heterozygous Point Mutation Generation Without Silent Mutation in a Human iPSC Line Using CRISPR/Cas9

Goal: To engineer a heterozygous point mutation WITHOUT a silent mutation in a control iPSC line

ASC’s control iPSC line (ASE-9203) was used to engineer a precise heterozygous point mutation in a gene associated with neuronal development and dysfunction of which has been implicated in neurological disorders. The gRNA was designed without a silent mutation to generate heterozygous only mutations according to the specifications of the customer. Immunohistochemical characterization of the engineered iPSC line was done to ensure pluripotency of the line was preserved after genome editing.

Figure. (A) gRNA validation using next generation sequencing (NGS) identified gRNA #2 (g2); (B) Sequence chromatogram showing heterozygous point mutation, CCG > TCG (green box). The orange box highlights the heterozygous C/T base change. The ssODN was designed without silent mutation and precisely modified the gene to yield heterozygous clones; (C) Immunostaining staining of engineered iPSCs for pluripotency markers (red), OCT4, SOX2, and TRA-1-60 and corresponding nuclear staining with Hoechst dye (blue).