PUBLICATION

Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish

Authors
Hisano, Y., Sakuma, T., Nakade, S., Ohga, R., Ota, S., Okamoto, H., Yamamoto, T., Kawahara, A.
ID
ZDB-PUB-150306-11
Date
2015
Source
Scientific Reports   5: 8841 (Journal)
Registered Authors
Kawahara, Atsuo, Okamoto, Hitoshi, Ota, Satoshi
Keywords
none
MeSH Terms
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • CRISPR-Cas Systems*
  • Gene Knock-In Techniques*
  • Gene Targeting/methods
  • Genes, Reporter
  • Genetic Loci
  • Genetic Vectors/genetics
  • Genome
  • Homologous Recombination
  • Molecular Sequence Data
  • Zebrafish/genetics*
PubMed
25740433 Full text @ Sci. Rep.
Abstract
The CRISPR/Cas9 system provides a powerful tool for genome editing in various model organisms, including zebrafish. The establishment of targeted gene-disrupted zebrafish (knockouts) is readily achieved by CRISPR/Cas9-mediated genome modification. Recently, exogenous DNA integration into the zebrafish genome via homology-independent DNA repair was reported, but this integration contained various mutations at the junctions of genomic and integrated DNA. Thus, precise genome modification into targeted genomic loci remains to be achieved. Here, we describe efficient, precise CRISPR/Cas9-mediated integration using a donor vector harbouring short homologous sequences (10-40 bp) flanking the genomic target locus. We succeeded in integrating with high efficiency an exogenous mCherry or eGFP gene into targeted genes (tyrosinase and krtt1c19e) in frame. We found the precise in-frame integration of exogenous DNA without backbone vector sequences when Cas9 cleavage sites were introduced at both sides of the left homology arm, the eGFP sequence and the right homology arm. Furthermore, we confirmed that this precise genome modification was heritable. This simple method enables precise targeted gene knock-in in zebrafish.
Genes / Markers
Figures
Show all Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping