PUBLICATION
CRISPR/Cas9-based genome engineering of zebrafish using a seamless integration strategy
- Authors
- Luo, J.J., Bian, W.P., Liu, Y., Huang, H.Y., Yin, Q., Yang, X.J., Pei, D.S.
- ID
- ZDB-PUB-180531-1
- Date
- 2018
- Source
- FASEB journal : official publication of the Federation of American Societies for Experimental Biology 32(9): 5132-5142 (Journal)
- Registered Authors
- Bian, Wanping, Liu, Yi, Luo, Juanjuan, Pei, Desheng
- Keywords
- MMEJ, gene editing, knock-in, transgenesis
- MeSH Terms
-
- Animals
- Animals, Genetically Modified/genetics
- CRISPR-Cas Systems/genetics*
- Clustered Regularly Interspaced Short Palindromic Repeats/genetics*
- Gene Editing/methods
- Gene Transfer Techniques
- Genetic Engineering/methods
- Genetic Therapy/methods
- Genome/genetics*
- Promoter Regions, Genetic/genetics
- Zebrafish/genetics*
- PubMed
- 29812974 Full text @ FASEB J.
Citation
Luo, J.J., Bian, W.P., Liu, Y., Huang, H.Y., Yin, Q., Yang, X.J., Pei, D.S. (2018) CRISPR/Cas9-based genome engineering of zebrafish using a seamless integration strategy. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 32(9):5132-5142.
Abstract
Numerous feasible methods for inserting large fragments of exogenous DNA sequences into the zebrafish genome have been developed, as has genome editing technology using programmable nucleases. However, the coding sequences of targeted endogenous genes are disrupted, and the expression patterns of inserted exogenous genes cannot completely recapitulate those of endogenous genes. Here we describe the establishment of a novel strategy for endogenous promoter-driven and microhomology-mediated end-joining-dependent integration of a donor vector using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9. We successfully integrated mCherry into the final coding sequence of targeted genes to generate seamless transgenic zebrafish lines with high efficiency. This novel seamless transgenesis technique not only maintained the integrity of the endogenous gene but also did not disrupt the function of targeted gene. Therefore, our microhomology-mediated end-joining-mediated transgenesis strategy may have broader applications in gene therapy. Moreover, this novel seamless gene-editing strategy in zebrafish provides a valuable new transgenesis technique, which was driven by endogenous promoters and in vivo animal reporter modes for translational medicine. It is expected to be a standard gene-editing technique in the field of zebrafish, leading to some important breakthroughs for studies in early embryogenesis.-Luo, J.-J., Bian, W.-P., Liu, Y., Huang, H.-Y., Yin, Q., Yang, X.-J., Pei, D.-S. CRISPR/Cas9-based genome engineering of zebrafish using a seamless integration strategy.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping