CRISPR-Based Lentiviral Knockout Libraries for Functional Genomic Screening and Identification of Phenotype-Related Genes
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.
Key wordsCRISPR/Cas9 Lentiviral sgRNA Genome-wide screening Forward genetics
The authors are grateful to the laboratories of Feng Zhang and John G. Doench for making constructs and libraries available through Addgene. Genome-wide CRISPR screens performed in the laboratory of Jacob Giehm Mikkelsen are made possible through funding by the Danish Council for Independent Research │ Medical Sciences (grant DFF-4004-00220), The Lundbeck Foundation (grant R126-2012-12456), the Novo Nordisk Foundation, Else og Mogens Wedell-Wedellsborgs Fond, Einar Willumsens Mindelegat, Emil C. Hertz og hustru Inger Hertz’s Fond, Holger Hjortenberg og Hustru Dagmar Hjortenbergs Fond, Krista og Viggo Petersens Fond, and Andersen-Isted Fonden.
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