PO-327 Whole-genome CRISPR-CAS9 screening: the wellcome sanger institute cellular generation and phenotyping pipeline

Abstract

IntroductionThe aim of this project was to perform genome-wide loss-of-function CRISPR-Cas9 screens across hundreds of genetically-annotated human cancer cell lines to identify synthetic-lethal interaction between cancer genomes and targeted gene deletion. This poster details the pipeline and processes used to successfully screen a large collection of diverse human cancer cell lines.Material and methodsA whole genome CRISPR-Cas9 screening pipeline was set up in the Cellular Generation and Phenotyping cell culture core facility at the Wellcome Sanger Institute. The pipeline consists of the following three stages:Antibiotic titration of parental cancer cell lines to determine the correct kill concentration for selection markers.Production and banking of stable Cas9-expressing lines via lentiviral transduction. Cas9 activity was determined using a BFP-GFP-gGFP reporter vector. Lines which had Cas9 activity of greater than 75% were used for downstream screening.33 million Cas9-expressing cells were transduced in triplicate with a lentivirally delivered genome wide guide RNA library at an efficiency of 15%–50% as determined by BFP reporter.Successfully transduced cells were selected with puromycin on day 3 and expanded, maintaining library representation at x500 coverage. Following two weeks screening, cells were harvested and pelleted for DNA extraction and sequencing to identify essential genes.Multiple process improvements increased pipeline efficiency, increased Cas9 activity and improved accuracy of scale up of guide RNA library transductions.Results and discussionsThe screening pipeline is highly robust and consistent with an overall success rate of >80% of lines successfully Cas9 transduced and screened. The screen has identified hundreds of essential genes across our cancer types, including many core essential genes.ConclusionCancer cell lines can be successfully screened at scale to generate robust essentiality data.