{Reference Type}: Journal Article
{Title}: Generation and application of endogenously floxed alleles for cell-specific knockout in zebrafish.
{Author}: Shin M;Yin HM;Shih YH;Nozaki T;Portman D;Toles B;Kolb A;Luk K;Isogai S;Ishida K;Hanasaka T;Parsons MJ;Wolfe SA;Burns CE;Burns CG;Lawson ND;
{Journal}: Dev Cell
{Volume}: 58
{Issue}: 22
{Year}: 2023 Nov 20
{Factor}: 13.417
{DOI}: 10.1016/j.devcel.2023.07.022
{Abstract}: 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.