Protocol for generating mutant zebrafish using CRISPR-Cas9 followed by quantitative evaluation of vascular formation

The use of vascular-specific transgenic zebrafish provides advantages for identifying new mutations affecting angiogenesis and vascular development. Here, we present a protocol for establishing, screening, and phenotyping CRISPR-Cas9-based mutagenesis in fluorescently labeled transgenic zebrafish. We describe steps for designing single-guide RNA (sgRNA) oligos, synthesizing sgRNA and Cas9 mRNA, and microinjection and generation of mutant lines. We then detail procedures for visualizing dynamic vasculature and quantitatively evaluating vascular formation in transgenic zebrafish. For complete details on the use and execution of this protocol, please refer to Luo et al.¹.