University of Montreal

Purpose

The CRISPR/Cas system has revolutionized the analysis of gene function in mammalian cells. Cas enzymes cut DNA at a specific genomic site as targeted by a single guide RNA (sgRNA) complimentary to the desired modification site. This system was rapidly adopted by the functional genomics community to perform pooled screens because of its simplicity and efficiency. However, current pooled screen read-outs are limited to assays with strong selective pressure, but are ill-suited to measure phenotypic effects on the state and differentiation of targeted cells. As such, arrayed screens are superior for primary cells, such as iPSCs, neurons, and immune cells. The lack of “whole-genome” arrayed sgRNA libraries, especially in ibonucleoprotein (RNP) formats that avoid introduction of foreign DNA elements, has impeded the functional characterization of multiple disease models targeted by the Cell and Gene Therapy (CGT) program. This proof of concept (PoC) study seeks to develop a sgRNA arrayed library for use in cell types relevant to the CGT program