TALEN- and CRISPR-enhanced DNA homologous recombination for gene editing in zebrafish
- PMID: 27443922
- DOI: 10.1016/bs.mcb.2016.03.005
TALEN- and CRISPR-enhanced DNA homologous recombination for gene editing in zebrafish
Abstract
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.
Keywords: CRISPR/Cas9; DNA double-strand breaks; Homologous recombination; In vitro fertilization; Twist2; Zebrafish.
Copyright © 2016 Elsevier Inc. All rights reserved.
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