Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish

TLDR

CRISPR/Cas9 enables genome editing in zebrafish, yet precise in‑frame integration of exogenous DNA at targeted loci has been limited by mutation‑prone homology‑independent repair. The study aims to develop a precise CRISPR/Cas9-mediated integration strategy using short (10–40 bp) homology arms flanking the target locus. The authors employed a donor vector containing 10–40 bp homology arms flanking the genomic target, with Cas9 cuts introduced on both sides of the left arm to drive precise integration. They achieved high‑efficiency, precise in‑frame integration of mCherry or eGFP into tyrosinase and krtt1c19e genes without vector backbone, confirmed heritability, and demonstrated a simple method for precise targeted gene knock‑in in zebrafish.

Abstract

Abstract The CRISPR/Cas9 system provides a powerful tool for genome editing in various model organisms, including zebrafish. The establishment of targeted gene-disrupted zebrafish (knockouts) is readily achieved by CRISPR/Cas9-mediated genome modification. Recently, exogenous DNA integration into the zebrafish genome via homology-independent DNA repair was reported, but this integration contained various mutations at the junctions of genomic and integrated DNA. Thus, precise genome modification into targeted genomic loci remains to be achieved. Here, we describe efficient, precise CRISPR/Cas9-mediated integration using a donor vector harbouring short homologous sequences (10–40 bp) flanking the genomic target locus. We succeeded in integrating with high efficiency an exogenous mCherry or eGFP gene into targeted genes ( tyrosinase and krtt1c19e ) in frame. We found the precise in-frame integration of exogenous DNA without backbone vector sequences when Cas9 cleavage sites were introduced at both sides of the left homology arm, the eGFP sequence and the right homology arm. Furthermore, we confirmed that this precise genome modification was heritable. This simple method enables precise targeted gene knock-in in zebrafish.