Targeted candidate gene screens using CRISPR/Cas9 technology

Abstract

In the postgenomic era, the ability to quickly, efficiently, and inexpensively assign function to the zebrafish proteome is critical. Clustered regularly interspaced short palindromic repeats (CRISPRs) have revolutionized the ability to perform reverse genetics because of its simplicity and broad applicability. The CRISPR system is composed of an engineered, gene-specific single guide RNA (sgRNA) and a Cas9 enzyme that causes double-stranded breaks in DNA at the targeted site. This simple, two-part system, when injected into one-cell stage zebrafish embryos, efficiently mutates target loci at a frequency such that injected embryos phenocopy known mutant phenotypes. This property allows for CRISPR-based F0 screening in zebrafish, which provides a means to screen through a large number of candidate genes for their role in a phenotype of interest. While there are important considerations for any successful genetic screen, CRISPR screening has significant benefits over conventional methods and can be accomplished in any lab with modest molecular biology experience.

Keywords: CRISPR; Cas9; Genome editing; Indel; Multiplex; Mutation; RNA-guided nuclease; Reverse genetic screen; Zebrafish; sgRNA.