Abstract

High-throughput Biallelic Modification of Human iPS Cells

William Skarnes, Senior Group Leader, Wellcome Trust Sanger Institute

The advent of site-specific nucleases and improved conditions for human iPSC culture now permits efficient engineering of human stem cells. CRISPR-Cas9 technology, in particular, provides a facile tool for the generation of a range of alleles in human stem cells with little risk of off-target damage. We established a high-throughput pipeline for the generation of homozygous knockout human iPSCs where one allele is targeted by homologous recombination and the second allele is damaged by non-homologous end joining. Our method lends itself to high-throughput genotyping: biallelic events are identified by Sanger sequencing of the non-targeted allele. Bi-allelic knockout of genes is observed in 10-30% of the colonies screened. Currently, we are developing a vector-free method using Cas9 ribonucleoprotein and single strand oligonucleotides for fluent generation of biallelic point mutations and revertants for disease modelling.