LAUSR

Identifying and validating targets that underlie disease mechanisms and can be addressed to provide efficacious therapies remains a significant challenge in the drug discovery and development process. Use of siRNA and shRNA to knock-down RNA and suppress gene function, have provided insights into mechanism of action, but depending on the nature of the targets, cells, biology and end-point assays these approaches may suffer variously from their transient nature, design complexity, incomplete knock-down or off-target effects. The use of CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas9 nuclease and guide RNA (gRNA) provides a strong alternative that can produce long-lasting impact, straightforward design, knock-out of genes and increased specificity. A number of laboratories have already published reports demonstrating how pools of gRNA can be delivered to cells and “hits” can be established through enrichment or depletion of cells following a “survival” assay and identified by sequencing the introduced gRNAs in the remaining cell population. Here we demonstrate a knock-out screening approach that utilizes the Invitrogen™ LentiArray™ CRISPR library to interrogate the impact of individual gene knock-outs on the NFκB pathway as measured by a functional cell-based assay. We describe the library design concepts, the assay development, initial screening results and validation of specific identified hits. The gRNAs are designed to primarily 5’ coding exons of a target gene using our CRISPR design tool to maximize knock-out efficiency and minimize off-target effects. Each gRNA is delivered as a separate lentiviral particle including an antibiotic-resistant marker and each gene is targeted by 4 gRNAs per well, delivered in a 96-well plate. We tested the approach using a library that targets the human kinome and developed a loss-of-function assay using our CellSensor® NF-κB-bla ME180 cell line, which is based on the ratiometric blue/green reporter assay and easily enables 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 these optimized parameters, we screened the Invitrogen™ LentiArray™ CRISPR kinome library that targets >800 kinases and demonstrate how we followed-up on and validated a subset of the identified hits. We expect these approaches to be scalable to the entire human genome and portable to multiple cell types and end-point assays including both high-throughput plate-based assays and high-content imaging based assays. Citation Format: Chetana M. Revankar, Justin Wetter, Julia Braun, Natasha Roark, Veronica Magnon, LaiYee Wong, Yanfei Zou, Namritha Ravinder, Jian-Ping Yang, Jonathan Chesnut, David Piper. Functional genomics screening using LentiArray™ CRISPR libraries and CellSensor™ assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-116. doi:10.1158/1538-7445.AM2017-LB-116