New Functional Genomics toolsets: Arrayed loss of function screening with LentiArray CRISPR libraries
Recorded On: 02/07/2018
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. Mechanisms of RNAi have provided the use of siRNA and shRNA to knock-down RNA and suppress gene function. However, 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 transient or 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. 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 and provide data from initial screens using the Invitrogen™ LentiArray™ CRISPR kinome library. 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.
Thermo Fisher Scientific
Cell and Molecular Biology, Genome Editing.