Abstract B163: Genetic screening with CRISPR-Cas9: Proof and principles

The RNA-guided CRISPR-Cas9 gene editing technology can be adapted to provide a loss-of-function genetic screening platform. CRISPR-Cas9 Knockout (KO) screening uses sequence-specific gene disruption and circumvents many of the problems encountered by RNAi technologies. In particular, complete ablation of gene expression allows the opportunity to examine the cellular physiological effects resulting from loss of targets rather than partial knockdown of target, which might not have a phenotypic effect. Our screens are conducted with large-scale complex libraries using a pooled lentivirus transduction strategy. We have developed several of our own custom libraries in addition to recapitulating a previously published whole-genome library for use in-house at Horizon. Next generation sequencing is used to quantitatively identify changes in the abundance of individual sgRNA sequences in each treatment population. As our libraries target each gene with multiple sgRNAs, we can score targets based on several biostatistical metrics to identify targets, including the adaptation of recently published ranking formulae designed specifically for CRISPR-Cas9 KO datasets. To critically evaluate the power of CRISPR-Cas9 KO screening in functional genomics, we have completed several proof-of-principle and novel screening programmes. We find that CRISPR-Cas9 KO screening is exceptionally powerful for identifying genetic factors for drug resistance. This is evident when using small ultra-complex libraries in addition to whole-genome screening libraries in response to a variety of drugs, including vemurafenib, paclitaxel and 6-TG treatment. Although sensitivity (drop-out) screening is more technically challenging, we have nevertheless been able to adapt the CRISPR-Cas9 KO technology to successfully identify sensitising factors in response to compound treatment. Using an ultra-complex library in a high coverage screen we identified BRCA1, BRCA2 and PTEN as sensitising with olaparib and uncovered several compelling novel hits. We have further explored library design and drop-out screening optimisation using a custom, ultra-complex library targeting an essential gene collection. We conclude that CRISPR-Cas9 KO screening offers a substantive increment in performance over existing functional genomic screening platforms, and if deployed astutely can offer researchers a rapid insight into complex biological problems. Citation Format: Benedict CS Cross, Steffen Lawo, Tim ME Scales, Caroline Archer, Jessica Hunt, Alessandro Riccombeni, Leigh Brody, Neil Humphryes, Riley Doyle, Victor B. Dillard, Nicola J. McCarthy, Jonathan D. Moore. Genetic screening with CRISPR-Cas9: Proof and principles. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B163.