%0 Journal Article
%T Generation and application of endogenously floxed alleles for cell-specific knockout in zebrafish.
%A Shin M
%A Yin HM
%A Shih YH
%A Nozaki T
%A Portman D
%A Toles B
%A Kolb A
%A Luk K
%A Isogai S
%A Ishida K
%A Hanasaka T
%A Parsons MJ
%A Wolfe SA
%A Burns CE
%A Burns CG
%A Lawson ND
%J Dev Cell
%V 58
%N 22
%D 2023 Nov 20
%M 37633272
%F 13.417
%R 10.1016/j.devcel.2023.07.022
%X 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.