Generating single cell-derived knockout clones in mammalian cells with CRISPR/Cas9

CRISPR/Cas9 technology enables the rapid and efficient generation of total loss-of- function mutations in a targeted gene in mammalian cells. A single cell that harbors those mutations can be used to establish a new cell line, thereby creating a CRISPR-induced knockout clone. These clonal cell lines serve as crucial tools for exploring protein function, analyzing the consequences of gene loss, and investigating the specificity of various biological reagents. However, the successful derivation of knockout clones may be technically challenging and can be complicated by multiple factors, including incomplete target ablation and inter-clonal heterogeneity. Here, we describe optimized protocols and plasmids for generating clonal knockouts in mammalian cell lines. We provide strategies for guide RNA design, CRISPR delivery, and knockout validation that facilitate the derivation and identification of true knockout clones and that are amenable to multiplexed gene targeting. These protocols will be broadly useful for researchers seeking to apply CRISPR to study gene function in mammalian cells.