PUBLICATION
An Efficient Platform for Generating Somatic Point Mutations with Germline Transmission in the Zebrafish by CRISPR/Cas9-mediated Gene Editing
- Authors
- Zhang, Y., Zhang, Z., Ge, W.
- ID
- ZDB-PUB-180304-1
- Date
- 2018
- Source
- The Journal of biological chemistry 293(17): 6611-6622 (Journal)
- Registered Authors
- Ge, Wei, Zhang, Yibo, Zhang, Zhiwei
- Keywords
- CRISPR/Cas, gene knockout, homologous recombination, site-directed mutagenesis, zebrafish
- MeSH Terms
-
- Animals
- CRISPR-Cas Systems*
- Embryo, Nonmammalian/metabolism*
- Gene Editing/methods*
- Germ-Line Mutation*
- Point Mutation*
- Zebrafish*/genetics
- Zebrafish*/metabolism
- PubMed
- 29500194 Full text @ J. Biol. Chem.
Citation
Zhang, Y., Zhang, Z., Ge, W. (2018) An Efficient Platform for Generating Somatic Point Mutations with Germline Transmission in the Zebrafish by CRISPR/Cas9-mediated Gene Editing. The Journal of biological chemistry. 293(17):6611-6622.
Abstract
Homology-directed recombination (HDR)-mediated genome editing is a powerful approach for both basic functional study and disease modeling. Although some studies have reported HDR-mediated precise editing in nonrodent models, the efficiency of establishing pure mutant animal lines that carry specific amino acid substitutions remains low. Furthermore, because the efficiency of nonhomologous end joining (NHEJ)-induced insertion and deletion (indel) mutations is normally much higher than that of HDR-induced point mutations, it is often difficult to identify the latter in the background of indel mutations. Using zebrafish as the model organism and Y box-binding protein 1 (Ybx1/ybx1) as the model molecule, we have established an efficient platform for precise CRISPR/Cas9-mediated gene editing in somatic cells, yielding an efficiency of up to 74% embryos. Moreover, we established a procedure for screening germline transmission of point mutations out of indel mutations even when germline transmission efficiency was low (<2%). To further improve germline transmission of HDR-induced point mutations, we optimized several key factors that may affect HDR efficiency, including the type of DNA donor, suppression of NHEJ, stimulation of HDR pathways, and use of Cas9 protein instead of mRNA. The optimized combination of these factors significantly increased the efficiency of germline transmission of point mutation up to 25%. In summary, we have developed an efficient procedure for creating point mutations and differentiating mutant individuals from those carrying knockouts of entire genes.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping