Generation and application of endogenously floxed alleles for cell-specific knockout in zebrafish

The zebrafish is amenable to a variety of genetic approaches. However, lack of conditional deletion alleles limits stage- or cell-specific gene knockout. Here, we applied an existing protocol to establish a floxed allele for gata2a but failed to do so due to off-target integration and incomplete knockin. To address these problems, we applied simultaneous co-targeting with Cas12a to insert loxP sites in cis, together with transgenic counterscreening and comprehensive molecular analysis, to identify off-target insertions and confirm targeted knockins. We subsequently used our approach to establish endogenously floxed alleles of foxc1a, rasa1a, and ruvbl1, each in a single generation. We demonstrate the utility of these alleles by verifying Cre-dependent deletion, which yielded expected phenotypes in each case. Finally, we used the floxed gata2a allele to demonstrate an endothelial autonomous requirement in lymphatic valve development. Together, our results provide a framework for routine generation and application of endogenously floxed alleles in zebrafish. •Improved methods for generating conditional deletion alleles in zebrafish•Generation of four zebrafish lines bearing endogenously floxed alleles•Blueprint for characterization and validation of floxed alleles in zebrafish•Use of a floxed allele to define cell-specific gene function in zebrafish The zebrafish is amenable to genetic approaches, but conditional gene knockout has been limited due to the lack of floxed deletion alleles. Shin et al. describe reliable methods for generating, validating, and applying zebrafish bearing endogenously floxed alleles, providing a foundation for widespread application of conditional knockout approaches in zebrafish.