ZFIN ID: ZDB-PUB-160725-31
TALEN- and CRISPR-enhanced DNA homologous recombination for gene editing in zebrafish
Zhang, Y., Huang, H., Zhang, B., Lin, S.
Date: 2016
Source: Methods in cell biology   135: 107-20 (Chapter)
Registered Authors: Huang, Haigen, Lin, Shuo, Zhang, Bo, Zhang, Ying
Keywords: CRISPR/Cas9, DNA double-strand breaks, Homologous recombination, In vitro fertilization, Twist2, Zebrafish
MeSH Terms:
  • Animals
  • CRISPR-Cas Systems/genetics*
  • DNA Repair/genetics
  • Gene Editing/methods*
  • Gene Targeting/methods*
  • Homologous Recombination/genetics*
  • Mutagenesis, Site-Directed/methods
  • Transcription Activator-Like Effector Nucleases/genetics
  • Zebrafish/genetics
PubMed: 27443922 Full text @ Meth. Cell. Biol.
The TALE nuclease and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas) have been developed as important tools for genome editing in zebrafish. Here we describe a CRISPR/Cas9-based approach for generating site-specific gene targeting in zebrafish using DNA double-strand breaks induced homologous recombination (HR)-dependent repair mechanism. Through comicroinjection of Cas9 mRNA, guide RNA targeting genomic DNA sequence corresponding to the twist2 gene and the corresponding double-strand long arm donor template with a point mutation identified in human, HR-mediated knock-in of the expected targeting sequence was obtained. To facilitate identification of germline transmission of targeted mutation, a method of screening sperms of male founder fish is designed.