Generation of floxed alleles for cell-specific knockout in zebrafish

Abstract

A benefit of the zebrafish as a model is its amenability to genetic approaches. However, a lack of conditional deletion alleles makes it a challenge to perform stage- or cell-specific knockout. Here, we initially applied an existing protocol to establish a floxed allele for gata2a and encountered several issues, including off-target integration and incomplete knock-in. To address these problems, we developed a protocol incorporating simultaneous co-targeting with Cas12a to insert loxP sites in cis, together with transgenic counter-screening to identify off-target insertions. We applied this protocol to establish a floxed allele of foxc1a in a single generation. We demonstrate the utility of our floxed 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 straightforward generation and application of floxed alleles in zebrafish.