Generating Custom Pooled CRISPR Libraries for Genetic Dissection of Biological Pathways

Abstract

Genetic screens are a classic approach to dissecting biological pathways including membrane trafficking. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 have enabled the utility of this approach in diploid models, including cultured mammalian cells. Here, we present detailed protocols for generating custom CRISPR libraries. These methods are useful for generating genome-wide libraries for new model organisms that lack an existing genome-wide library, and for generating smaller focused libraries.