Optimized CRISPR–Cas9 System for Genome Editing in Zebrafish

TLDR

CRISPRscan, built on an integrated analysis of over 1,000 zebrafish sgRNAs, predicts factors that influence Cas9 activity and guides the design of highly efficient guide RNAs. The protocol aims to generate and genotype mutant zebrafish lines using an optimized CRISPR–Cas9 system. It describes generating sgRNAs in vitro, injecting them in vivo, targeting Cas9 to the germline to reduce somatic mutations, and using Mut‑Seq and PCR‑based fragment analysis to identify F1 heterozygotes. Highly efficient sgRNAs can produce biallelic mutations in embryos, leading to lethality.

Abstract

This protocol describes how to generate and genotype mutants using an optimized CRISPR–Cas9 genome-editing system in zebrafish (CRISPRscan). Because single guide RNAs (sgRNAs) have variable efficiency when targeting specific loci, our protocol starts by explaining how to use the web tool CRISPRscan to design highly efficient sgRNAs. The CRISPRscan algorithm is based on the results of an integrated analysis of more than 1000 sgRNAs in zebrafish, which uncovered highly predictive factors that influence Cas9 activity. Next, we describe how to easily generate sgRNAs in vitro, which can then be injected in vivo to target specific loci. The use of highly efficient sgRNAs can lead to biallelic mutations in the injected embryos, causing lethality. We explain how targeting Cas9 to the germline increases viability by reducing somatic mutations. Finally, we combine two methods to identify F 1 heterozygous fish carrying the desired mutations: (i) Mut-Seq, a method based on high-throughput sequencing to detect F 0 founder fish; and (ii) a polymerase chain reaction–based fragment analysis method that identifies F 1 heterozygous fish characterized by Mut-Seq. In summary, this protocol includes the steps to generate and characterize mutant zebrafish lines using the CRISPR–Cas9 genome engineering system.