Adaptation of the CRISPR system has enabled scientists to probe the genome and interfere with gene function at an unprecedented scale. Adding to the use of CRISPR for generation of individual gene knockout, which is by now conventional, the CRISPR system enables high-throughput functional screening of the genome. By combining the integrative properties of lentiviral vector delivery with the disruptive nature of the CRISPR system, genome-wide CRISPR libraries provide the power to screen among thousands of genes despite the high complexity of the entire genome and identify a list of genes potentially affecting a certain phenotype. Genome-wide CRISPR screening is an advanced technology compiling numerous practical aspects and a series of molecular biology techniques. In this protocol, we describe all steps toward implementing CRISPR knockout screens in your research; we describe the core procedures and key information as well as some tricks and tips needed to successfully perform a CRISPR screen.
Keywords: CRISPR/Cas9; Forward genetics; Genome-wide screening; Lentiviral; sgRNA.