Journal
SCIENTIFIC REPORTS
Volume 5, Issue -, Pages -Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/srep08841
Keywords
-
Categories
Funding
- Special Postdoctoral Researcher Program
- Research Fellow of Japan Society for the Promotion of Science
- Japan Society for the Promotion of Science
- Program for Next Generation World-Leading Researchers (NEXT Program)
- Grants-in-Aid for Scientific Research [25890014, 26870845, 13J04914] Funding Source: KAKEN
Ask authors/readers for more resources
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available