High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9

TLDR

CRISPR/Cas9 has revolutionized targeted mutagenesis across model organisms. The study introduces a high‑throughput CRISPR/Cas9 pipeline for zebrafish enabling genome‑wide saturation mutagenesis and large‑scale phenotyping. The pipeline uses a cloning‑free sgRNA synthesis and rapid mutant detection via fluorescent PCR and multiplexed sequencing. The method achieved a 99 % mutation success rate, 28 % average germline transmission, produced 678 alleles from 58 genes, enabled efficient multiplexed targeting and F1 phenotyping, and proved CRISPR/Cas9 to be sixfold more efficient than TALENs/ZFNs with low off‑target risk.

Abstract

The use of CRISPR/Cas9 as a genome-editing tool in various model organisms has radically changed targeted mutagenesis. Here, we present a high-throughput targeted mutagenesis pipeline using CRISPR/Cas9 technology in zebrafish that will make possible both saturation mutagenesis of the genome and large-scale phenotyping efforts. We describe a cloning-free single-guide RNA (sgRNA) synthesis, coupled with streamlined mutant identification methods utilizing fluorescent PCR and multiplexed, high-throughput sequencing. We report germline transmission data from 162 loci targeting 83 genes in the zebrafish genome, in which we obtained a 99% success rate for generating mutations and an average germline transmission rate of 28%. We verified 678 unique alleles from 58 genes by high-throughput sequencing. We demonstrate that our method can be used for efficient multiplexed gene targeting. We also demonstrate that phenotyping can be done in the F1 generation by inbreeding two injected founder fish, significantly reducing animal husbandry and time. This study compares germline transmission data from CRISPR/Cas9 with those of TALENs and ZFNs and shows that efficiency of CRISPR/Cas9 is sixfold more efficient than other techniques. We show that the majority of published "rules" for efficient sgRNA design do not effectively predict germline transmission rates in zebrafish, with the exception of a GG or GA dinucleotide genomic match at the 5' end of the sgRNA. Finally, we show that predicted off-target mutagenesis is of low concern for in vivo genetic studies.