Protocol for tissue-specific mutagenesis with fluorescent labeling in zebrafish

Here, we present a protocol for tissue-specific mutagenesis in zebrafish. We describe the preparation of the Tol2 transposase donor vector containing a U6 promoter that drives the transcription of single-guide RNAs (sgRNAs) and Cas9 under the control of a tissue-specific promoter. We then detail the establishment, identification, and phenotypic analysis of the stable tissue-specific mutagenesis zebrafish line. This protocol is useful for generating stable tissue-specific knockout lines to analyze mosaic loss-of-function phenotypes. For complete details on the use and execution of this protocol, please refer to Luo et al.1.