Abstract

Genomic DNA replacement for achieving sophisticated genetic manipulation is implemented currently through homogenous recombination/homology-dependent repair (HR/HDR). Here we report an efficient DNA fragment replacement method that is mediated by non-homologous end joining (NHEJ)-dependent DNA repair at two sites of CRISPR/Cas9-induced double-strand breaks at non-coding genomic regions flanking the exons of targeted genes. We demonstrated this method by generating three conditional alleles and two reporter lines of zebrafish. Functional assays of the conditional alleles proved that the genomic sequence between two inserted loxP sites was deleted by the Cre recombinase, and the phenotype after Cre-induced excision was comparable to previously reported mutants or morphants. Furthermore, combining double-fluorescence expression donor vectors, we showed that the efficiency of this NHEJ-mediated DNA replacement was around 3 times larger than that of HR/HDR-mediated approach. Our method provides a feasible strategy for genomic DNA replacement in zebrafish, which can be applicable for other organisms as well.