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 [Display omitted] •Construction of tissue-specific mutagenesis vector based on CRISPR-Cas9 strategy•Protocol for CRISPR-Cas9-based tissue-specific mutagenesis in zebrafish•Identification of tissue-specific gene mutagenesis in heterozygous zebrafish•Pipeline for the establishment of the stable homozygous zebrafish line Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.