Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering

TLDR

CRISPR/Cas9 is rapidly adopted for genome engineering because of its simplicity, flexibility, and high efficiency, and has recently enabled homology‑independent knock‑in of long DNA fragments in zebrafish. The study aims to assess whether CRISPR/Cas9 can efficiently generate knock‑in transgenic zebrafish and to evaluate its broad applicability, reporting successful cell‑type specific Gal4 or reporter expression. The method uses a heat‑shock promoter donor plasmid co‑injected with sgRNAs targeting the genome and the donor plasmid, together with Cas9 mRNA. Stable knock‑in transgenic fish were established at four loci with frequencies exceeding 25%, supporting the proposal that CRISPR/Cas9‑mediated knock‑in will become a standard zebrafish transgenesis method.

Abstract

Abstract The type II bacterial CRISPR/Cas9 system is rapidly becoming popular for genome-engineering due to its simplicity, flexibility and high efficiency. Recently, targeted knock-in of a long DNA fragment via homology-independent DNA repair has been achieved in zebrafish using CRISPR/Cas9 system. This raised the possibility that knock-in transgenic zebrafish could be efficiently generated using CRISPR/Cas9. However, how widely this method can be applied for the targeting integration of foreign genes into endogenous genomic loci is unclear. Here, we report efficient generation of knock-in transgenic zebrafish that have cell-type specific Gal4 or reporter gene expression. A donor plasmid containing a heat-shock promoter was co-injected with a short guide RNA (sgRNA) targeted for genome digestion, a sgRNA targeted for donor plasmid digestion and Cas9 mRNA. We have succeeded in establishing stable knock-in transgenic fish with several different constructs for 4 genetic loci at a frequency being exceeding 25%. Due to its simplicity, design flexibility and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic zebrafish.