Arrayed CRISPR/Cas9 Screening for the Functional Validation of Cancer Genetic Dependencies

CRISPR/Cas9 screening has revolutionized functional genomics in biomedical research and is a widely used approach for the identification of genetic dependencies in cancer cells. Here, we present an efficient and versatile protocol for the cloning of guide RNAs (gRNA) into lentiviral vectors, the production of lentiviral supernatants, and the transduction of target cells in a 96-well format. To assess the effect of gene knockouts on cellular fitness, we describe a competition-based cell proliferation assay using flow cytometry, enabling the screening of many genes at the same time in a fast and reproducible manner. This readout can be extended to any parameter that is accessible to flow-based measurements, such as protein expression and stability, differentiation, cell death, and others. In summary, this protocol allows to functionally assess the effect of a set of 50-300 gene knockouts on various cellular parameters within eight weeks.

Automated summary

CRISPR/Cas9 screening is a revolutionary method in functional genomics that has been widely used to identify genetic dependencies in cancer cells. This article presents an efficient protocol for cloning guide RNAs into lentiviral vectors, producing lentiviral supernatants, and transducing target cells in a 96-well format. The authors also describe a competitive cell proliferation assay using flow cytometry, which allows for the simultaneous screening of multiple genes in a fast and reproducible manner. This protocol can be extended to measure other parameters accessible to flow-based measurements.