|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.
|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|
|PubMed:||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.
ABSTRACTThe 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.
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