Dual-Direction Crispr Transcriptional Regulation Screening Uncovers Gene Networks Driving Drug Resistance

Functional genomic screening with CRISPR-Cas9 has provided a powerful and precise new way to interrogate the phenotypic consequences of gene manipulation in high-throughput, unbiased analyses. Rapid development of pooled lentivirus and deep-sequencing-led approaches have allowed us and others to exploit this technology in target ID, target validation, drug MOA analysis and patient stratification. Many screens have been conducted using loss-of-function driven by CRISPR-Cas9 knock-out. Whilst powerful, this approach is not suitable for the examination of hypomorphic phenotypes, which include essential genes. In addition, CRISPR-Cas9 knock-out does not allow the analysis of gene activation, leaving a significant hole in the functional genomic analysis. To address this, we have developed two new paired pooled screening platforms using inactivated Cas9 (dCas9) to control gene expression in both loss- and gain-of-function screens. Our CRISPRi platform uses a streamlined and high-performance approach with an improved tracrRNA sequence incorporated into a single-shot transduction protocol. Our CRISPRa platform is based on the SAM system and both platforms have been adapted to use next generation, highly optimised whole-genome libraries to enact maximum gene expression modulation. Validation analysis of both tools revealed outstanding performance and sensitivity, with greater than ten-fold improvement in detection rates compared to existing platforms, including RNAi. We used these paired tools to explore the development of resistance mechanisms to the BRAF V600E inhibitor, vemurafenib. Both platforms allowed for the unambiguous discovery of expected hits, additional complex members not identified previously, as well as multiple novel genes involved in drug resistance. Most interestingly, simultaneous evaluation of both activating and inhibiting perturbations revealed direct and opposing phenotypic effects within complex gene networks, switching the response of affected cells to either sensitisation or resistance. In addition to providing a compelling strategy for the validation of drug resistance targets, the parallel analysis of CRISPRi and CRISPRa data sets also allows the identification of pathway modulators that affect both drug sensitivity and resistance, but in which sgRNAs are not reciprocally lost or increased in each screen. Taken together, our data indicate that CRISPRa and CRISPRi screens in parallel provide an exceptionally powerful dataset for target identification. Dual directional approaches such as ours provide the new opportunity to achieve increased depth of hit finding in the analysis and offer novel discovery pathways by exploring opposing function and more complete gene network perturbation. Citation Format: Carlos le Sage, Steffen Lawo, Prince Panckier, Tim M. Scales, Syed Asad Rahman, Annette S. Little, Nicola J. McCarthy, Jonathan D. Moore, Benedict C. Cross. Dual-direction CRISPR transcriptional regulation screening uncovers gene networks driving drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 409.