CRISPR/Cas9-Directed Gene Editing for the Generation of Loss-of-Function Mutants in High-Throughput Zebrafish F 0 Screens

The ability to perform reverse genetics in the zebrafish model organism has been greatly advanced with the advent of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated) system. The high level of efficiency in generating mutations when using the CRISPR/Cas9 system combined with the rapid generation time of the zebrafish model organism has made the possibility of performing F screens in this organism a reality. This unit describes a detailed protocol for performing an F screen using the CRISPR/Cas9 system in zebrafish starting with the design and production of custom CRISPR/Cas9 reagents for injection. Next, two approaches for determining the efficiency of mutation induction by the custom CRISPR/Cas9 reagents that are easily performed using standard molecular biology protocols are detailed. Finally, screening for F induced phenotypes using the zebrafish flh gene as an example is discussed. © 2017 by John Wiley & Sons, Inc.