The Governing Council of the University of Toronto

Purpose

The CRISPR/Cas system has revolutionized the analysis of gene function in mammalian cells. Cas cuts DNA at specific genomic region by associating with a single guide RNA (sgRNA) molecule whose sequence is complimentary to the genomic DNA to be targeted. This system was rapidly adopted by the functional genomic community to perform pooled screens because of its simplicity and efficiency. However, current pooled screen read-outs are limited to assays with strong selective ressure, but are ill fitted to measure phenotypic effect on state and differentiation of targeted cells. As such, arrayed screens are better suited for primary cells, such as iPSCs, neurons, and immune cells. Thus, the lack of a “whole-genome” arrayed sgRNA libraries, especially ribonucleoprotein (RNP) compatible, has impeded the functional characterization of multiple disease models targeted by the Cell and Gene Therapy (CGT) program. This proof of concept proof of concept (PoC) study seeks to develop a sgRNA arrayed library for use in cell types relevant to the CGT program.