"A Genome-wide CRISPRi Screen for Discovering Novel Factors of iPSC Growth & Survival"
Host Mentor: Bruce Conklin, M.D.
Gladstone Institute of Cardiovascular Disease
In order to study disease in physiologically relevant cell models, it is important to develop technologies that enable efficient control of gene expression. Using the newly developed CRISPR interference (CRISPRi) technology, we aim to interrogate genes involved in iPSC growth and maintenance at the whole-genome level. CRISPRi utilizes a deactivated Cas9 enzyme with a fused KRABs domain (dCas9-KRAB) for gene silencing. We have generated a genome-wide kinase and phosphatase library of 13,000 single guide RNAs designed to target 2600 genes. Using inducible CRISPRi iPSC, we have used our library to dissect genes paramount to the maintenance of pluripotency. Several highly confident gene hits were identified in our screen. Our highest ranking gene hits include genes validated in pluripotent cells, such as ATR, CHEK1. Several genes of unknown relevance have also been identified, with some of them being novel hits with little literature on their role in growth and survival. Overall this projects will improve our understanding of cellular pluripotency and differentiation.