ZFIN ID: ZDB-PUB-160725-35
Multiplex conditional mutagenesis in zebrafish using the CRISPR/Cas system
Yin, L., Maddison, L.A., Chen, W.
Date: 2016
Source: Methods in cell biology   135: 3-17 (Chapter)
Registered Authors: Chen, Wenbiao
Keywords: CRISPR, Cas9, Conditional mutagenesis, Mutagenesis, Transgenesis, sgRNA
MeSH Terms:
  • Animals
  • Animals, Genetically Modified
  • CRISPR-Cas Systems/genetics*
  • Gene Editing/methods*
  • Gene Transfer Techniques
  • Genetic Engineering/methods*
  • Mutagenesis/genetics*
  • Mutation/genetics
  • Zebrafish/genetics
  • Zebrafish/growth & development
PubMed: 27443918 Full text @ Meth. Cell. Biol.
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is a powerful tool for genome editing in numerous organisms. However, the system is typically used for gene editing throughout the entire organism. Tissue and temporal specific mutagenesis is often desirable to determine gene function in a specific stage or tissue and to bypass undesired consequences of global mutations. We have developed the CRISPR/Cas system for conditional mutagenesis in transgenic zebrafish using tissue-specific and/or inducible expression of Cas9 and U6-driven expression of sgRNA. To allow mutagenesis of multiple targets, we have isolated four distinct U6 promoters and designed Golden Gate vectors to easily assemble transgenes with multiple sgRNAs. We provide experimental details on the reagents and applications for multiplex conditional mutagenesis in zebrafish.