Pooled cell-based screens of mammalian genetic perturbations enable systematic large-scale, even genome-scale, evaluation of gene function. Pooled screens introduce genetic perturbations into a cell population through viral transduction such that each cell integrates into its DNA a single or small number of library perturbations with barcodes identifying the perturbations. One then selects and physically isolates the subset of cells that exhibit the phenotype of interest. Sequencing the barcodes in the hit cells reveals which genes favored or inhibited the hit phenotype. Various genetic perturbations are possible, including CRISPR gene knockout, ectopic gene expression, and RNA interference. Regardless of the type of library being screened or the type of cell model being tested, such screens involve many common steps and procedures. This unit describes detailed experimental protocols for the key steps, and also highlights some of the key factors to achieving a well-powered, reproducible screen result. © 2018 by John Wiley & Sons, Inc.
Keywords: CRISPR screens; functional genomics; large-scale genetic screens; pooled screens.
Copyright © 2018 John Wiley & Sons, Inc.