Abstract LB-112: Unraveling biology and identifying targets with functional genomics approaches using lentiarray crispr libraries

Abstract

Abstract The ability of CRISPR/Cas9, to efficiently and precisely edit a cell’s DNA and introduce a complete genetic knockout, while minimizing off-target effects, offers an improved approach to target identification. Identifying and validating targets that underlie disease mechanisms still remains a significant challenge in the drug discovery and development process. The transcription factor NF-κB is a crucial player in many aspects of cancer initiation and progression. Here we demonstrate a knock-out screening approach that utilizes an arrayed CRISPR library to interrogate the impact of individual gene knock-outs on the NF-κB pathway in a cervical carcinoma cell line as measured by a functional cell-based assay. We describe the library design concepts, the assay development, screening results and validation of specific identified hits. We tested the approach using a library that targets the human kinome and developed a loss-of-function assay using CellSensor™ NF-κB-bla ME-180 cell line, which stably expresses Cas9. The CellSensor™ reporter assay enables easy identification of genomic targets associated with the NF-κB pathway. We elucidate the key factors in developing a robust assay including both transduction and assay optimization to achieve the highest levels of transduction efficiency and assay window. Using the optimized parameters, we screened the LentiArray CRISPR human kinome library that targets >800 kinases and demonstrate how we followed-up on and validated a subset of the identified hits. The hits were further validated by confirming nucleotide(s) deletion/ insertion in target DNA and additional screening using target specific siRNA and chemical compounds. The ability to scale this approach through the generation of genome wide CRISPR libraries, coupled with the use of lentiviral delivery methods enables high-throughput (HTP) loss-of-function screens to be performed rapidly and identify genes whose activity is important for the specific endpoint being measured. These screenings can provide a wealth of data on the normal functioning of a target and in turn, should yield better validated targets for progression into full drug discovery. Ultimately, the hits from the HTP screenings can be followed up by using CRISPR technology to generate animal knockout models that would support translating the screen to the pre-clinical trials. This in turn could provide better correlation to the clinical setting and thereby reduce candidate drug attrition. Citation Format: Chetana M. Revankar, Julia Braun, Jian-Ping Yang, Justin Wetter, Tufan Gokirmak, Namritha Ravinder, Jon Chesnut, David Piper. Unraveling biology and identifying targets with functional genomics approaches using lentiarray crispr libraries [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 LB-112.