Studies in zebrafish have made substantial contributions to our understanding of gene function in vertebrates. Large-scale genetic screens have led to discoveries of components and pathways underlying essential biological processes. More recently the imprecise repair of targeted DSBs induced by programmable nucleases has expanded use of the zebrafish further by providing a means for creating loss of function mutations affecting selected genes of interest. Importantly, the DSB lesions induced by the programmable nucleases stimulate both repair and recombination pathways, dramatically increasing the chance of integrating donor DNA sequences at the targeted locus. Indeed, a handful of reports have described successful integration of donor sequences at the targeted locus in zebrafish using different molecular configurations, i.e., single-stranded oligodeoxynucleotides or double-stranded DNA (dsDNA), with or without homologous arms.
We have focused on sequence replacement or integration via homologous recombination between the host genome and exogenously supplied dsDNA as donor molecules. We found that digestion of donor ends with I-SceI enzyme in defined orientation enhances homologous recombination efficiency in zebrafish embryos. Under these conditions, several kilobase long stretches of sequence can be replaced efficiently. We used this approach to accomplish a variety of precise editing events, including: a single base substitution, in-frame integration of sequences encoding an epitope tag, and in-frame integration of reporter sequences accompanied by disruption of the targeted host gene. Taking advantage of the ability to integrate long donor dsDNA molecules, we also developed a method for efficiently isolating silent genome modifications, by incorporating a linked reporter within the donor sequences. Presence of the linked reporter, which can be excised subsequently by FLP/FRT-mediated recombination, makes it easy to recover and identify edited alleles. With these improvements, we created a conditional mutant in zebrafish in which a pair of loxP sequences flanked a target exon. The floxed allele maintains wild-type function but can be converted to a null mutation in the presence of Cre protein. Thus, genome editing mediated by programmable nucleases continues to advance our ability to perform genetic analysis in zebrafish.