Abstract A186: Dual CRISPRi and CRISPRa screening reveals phenotypic switches in response to BRAF inhibition

Abstract

Abstract 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 has allowed us and others to exploit this technology in target ID, target validation, drug MOA analysis, and patient stratification. To date, the majority of screens have been conducted using loss-of-function perturbation driven by CRISPR-Cas9 enacted gene knockout. While powerful, this approach does not allow for the examination of activating gene function, leaving a salient hole in the functional genomic analysis. To address this, we have developed two new paired pooled screening platforms using catalytically inactivated Cas9 (dCas9) to control gene expression in both loss- and gain-of-function cell reprogramming. 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 synergistic activation mediator (SAM) system and both platforms have been adapted to use next-generation, highly optimized whole-genome libraries in order 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. We used these paired tools to explore the development of resistance mechanisms to the BRAF V600E inhibitor, vemurafenib. Unambiguous discovery of both expected hits and multiple novel genes involved in drug resistance was determined by both platforms. Those novel hits arising from the CRISPRi screen represented an enriched cohort of essential genes that have been missed by previous screens using RNAi and CRISPR-KO technologies. 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 sensitization or resistance. Thus, in contrast to loss-of-function-only analysis, this paired approach allows the discovery of key genes that control the drug response, but through an inhibitory mechanism. These genes were found to sit in the center of the hit nexus and revealed as key components only by the assimilation of both datasets, demonstrating the unique power of bidirectional functional genomic screening approaches. Citation Format: Carlos le Sage, Prince Panckier, Bendict CS Cross. Dual CRISPRi and CRISPRa screening reveals phenotypic switches in response to BRAF inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A186.