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ZFIN ID: ZDB-PUB-131202-3
Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair
Auer, T.O., Duroure, K., De Cian, A., Concordet, J.P., and Del Bene, F.
Date: 2014
Source: Genome research 24(1): 142-53 (Journal)
Registered Authors: Auer, Thomas, Del Bene, Filippo, Duroure, Karine
Keywords: none
MeSH Terms:
  • Animals
  • CRISPR-Associated Proteins/genetics
  • CRISPR-Associated Proteins/metabolism*
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • DNA Breaks, Double-Stranded
  • DNA Repair*
  • Gene Knock-In Techniques*
  • Genetic Engineering/methods*
  • Genome
  • Mutagenesis
  • RNA, Guide/genetics
  • Recombinational DNA Repair
  • Zebrafish/embryology
  • Zebrafish/genetics*
PubMed: 24179142 Full text @ Genome Res.
ABSTRACT

Sequence-specific nucleases like TALENs and the CRISPR/Cas9 system have greatly expanded the genome editing possibilities in model organisms such as zebrafish. Both systems have recently been used to create knock-out alleles with great efficiency, and TALENs have also been successfully employed in knock-in of DNA cassettes at defined loci via homologous recombination (HR). Here we report CRISPR/Cas9-mediated knock-in of DNA cassettes into the zebrafish genome at a very high rate by homology-independent double-strand break (DSB) repair pathways. After co-injection of a donor plasmid with a short guide RNA (sgRNA) and Cas9 nuclease mRNA, concurrent cleavage of donor plasmid DNA and the selected chromosomal integration site resulted in efficient targeted integration of donor DNA. We successfully employed this approach to convert eGFP into Gal4 transgenic lines, and the same plasmids and sgRNAs can be applied in any species where eGFP lines were generated as part of enhancer and gene trap screens. In addition, we show the possibility of easily targeting DNA integration at endogenous loci, thus greatly facilitating the creation of reporter and loss-of-function alleles. Due to its simplicity, flexibility, and very high efficiency, our method greatly expands the repertoire for genome editing in zebrafish and can be readily adapted to many other organisms.

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