CRISPR/Cas9 methods for identification and validation of genes regulating BCR-mediated antigen uptake

Abstract

Genome-wide CRISPR screens are a powerful tool to interrogate and identify gene function in a wide variety of applications and cell types. CRISPR-Cas9 technology using pooled CRISPR single guide RNA (sgRNA) libraries enables genetic editing in bulk in a large population of cells of interest. After selection of gene-edited cells, phenotyping effects can be evaluated by quantifying abundance (over- or under-representation) of individual sgRNAs using DNA sequencing. In addition to cell survival, these assays can be applied to investigations of drug sensitivity, as well as almost any cellular process with a clear phenotypic read out, for example, receptor internalization, migration, autophagy, and differentiation. Here, taking as an example the identification of molecular components governing B-cell antigen uptake through the B-cell receptor, we describe whole-genome, small-scale, and in vivo validation methods to identify and validate genes regulating BCR-mediated antigen uptake.</p