Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

• DOI: 10.3791/67616
• Publication Date: 2025-01-25T00:00:16Z
• Authors: Xuepu Jin; Binghuang Zhang; Yu Sun; Yahan Duan; Junchen Lu; Jiannan Liu; Jiahuai Han; Yingying Zhang
• Keywords: Fluorescent labelling

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism vivo imaging due to its transparency during the early developmental stage. Here, we provide detailed protocol performing high-efficiency fluorescence gene KI, rapid screening KI founders, and low-abundance protein tracing larvae, which will lay critical foundation subsequent physio-pathological studies zebrafish. The current includes complete steps sgRNA design of interest, vitro transcription, Cas9 mRNA screen one with highest efficiency, donor plasmid construction, microinjection founder live imaging. Critical steps, troubleshooting tips, quality control methods, advantages applications this are included discussed. This assures quick accurate results at low cost has been validated by multiple trials.