No abstract

Functional genomic screening with CRISPR 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.Until now, these exquisitely powerful screening technologies have been deployed predominantly in simple in vitro analyses. Whilst these in vitro approaches allow for high quality hit discovery and at whole-genome level scale, they are not able to adequately capture the impact of tumour microenvironment and heterogeneity on the genetic perturbations under study. To address this, we present here the development and validation of a platform for pooled CRISPR knock-out screening in xenograft models of tumour growth. A critical step in appropriate study design is the evaluation of the cell uptake following implantation. Since this directly contributes to the tumour growth and cell number, understanding cell survival dynamics post-implantation is necessary to determine the appropriate experimental scope. We used a barcoding approach coupled to next generation sequencing to determine the response and contribution of individual A375 melanoma cells to final tumour composition and used this to calibrate the experimental design for robust pooled-based CRISPR screening analysis. Following the determination of experimental conditions, a proof-of-concept drug mechanism of action analysis was conducted using a custom designed cancer genome CRISPR knock-out library. A375 cells were first infected with the CRISPR library and selected ex vivo ahead of implantation and treatment of established tumour-bearing nude mice with a defined therapeutic regime of the BRAFV600E selective inhibitor, Vemurafenib. Tumours were harvested and the identification of genes which selectively modulate the tumour response to drug were identified by next generation sequencing using our optimised screening analysis platform. Our results provide a robust experimental validation and define a novel platform which will allow the rapid and definitive in vivo evaluation of both drug and gene interactions using powerful CRISPR-based screening tools. Citation Format: Michele Melton, Athena Bast, Alastair Leeson-Payne, Maximilian Blanck, Steffen Lawo, Deeds Stacy, Benedict C.S. Cross. CRISPR screening in a xenograft model for in vivo drug moa analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3704.