Reproduction Abstracts

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. We construct short arm targeting vectors and sgRNA expression plasmids in 96-well format. Following co-transfection of the targeting vector with Cas9 and sgRNA expression plasmids, we screen for clones 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. Our aim is to generate and distribute arrays of human iPS cell knockouts that will be coupled to focused phenotyping screens in cultured cells. 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.