Abstract
Genomic DNA replacement for achieving sophisticated genetic manipulation is implemented currently through homogenous recombination/homology-dependent repair (HR/HDR). Here we report a novel 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 of zebrafish. Functional assays 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. We further extended the application by making two EGFP reporter lines, in which the EGFP expression patterns were consistent with those of endogenous genes. Furthermore, combining double-fluorescence expression donor vectors, we showed that the DNA replacement efficiency of this NHEJ-mediated approach was around 3 times larger than that of HR/HDR-mediated approach. Together, we provides a feasible strategy for genomic DNA replacement in zebrafish, which can be applicable for other organisms as well.
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