TALEN- and CRISPR-enhanced DNA homologous recombination for gene editing in zebrafish
- Zhang, Y., Huang, H., Zhang, B., Lin, S.
- Methods in cell biology 135: 107-20 (Chapter)
- Registered Authors
- Huang, Haigen, Lin, Shuo, Zhang, Bo, Zhang, Ying
- CRISPR/Cas9, DNA double-strand breaks, Homologous recombination, In vitro fertilization, Twist2, Zebrafish
- MeSH Terms
- 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
- 27443922 Full text @ Meth. Cell. Biol.
Zhang, Y., Huang, H., Zhang, B., Lin, S. (2016) TALEN- and CRISPR-enhanced DNA homologous recombination for gene editing in zebrafish. Methods in cell biology. 135:107-20.
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.
Genes / Markers
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Engineered Foreign Genes