PUBLICATION
One-step generation of zebrafish carrying a conditional knockout-knockin visible switch via CRISPR/Cas9-mediated intron targeting
- Authors
- Li, J., Li, H.Y., Gu, S.Y., Zi, H.X., Jiang, L., Du, J.L.
- ID
- ZDB-PUB-191226-27
- Date
- 2019
- Source
- Science China. Life sciences 63(1): 59-67 (Journal)
- Registered Authors
- Du, Jiu Lin, Li, Hong-Yu
- Keywords
- NHEJ, conditional knockout, knockin, non-HR, visible switch, zCKOIS, zebrafish
- MeSH Terms
-
- Alleles
- Animals
- Animals, Genetically Modified
- Base Sequence
- Basic Helix-Loop-Helix Transcription Factors/genetics*
- CRISPR-Cas Systems/genetics*
- Gene Expression
- Gene Knock-In Techniques/methods
- Gene Knockout Techniques/methods
- Gene Targeting/methods*
- Genetic Engineering
- Genotype
- Green Fluorescent Proteins/genetics
- Homologous Recombination/genetics*
- Introns/genetics*
- Zebrafish
- Zebrafish Proteins/genetics*
- PubMed
- 31872378 Full text @ Sci. China Ser. C-Life Sci.
Citation
Li, J., Li, H.Y., Gu, S.Y., Zi, H.X., Jiang, L., Du, J.L. (2019) One-step generation of zebrafish carrying a conditional knockout-knockin visible switch via CRISPR/Cas9-mediated intron targeting. Science China. Life sciences. 63(1):59-67.
Abstract
The zebrafish has become a popular vertebrate animal model in biomedical research. However, it is still challenging to make conditional gene knockout (CKO) models in zebrafish due to the low efficiency of homologous recombination (HR). Here we report an efficient non-HR-based method for generating zebrafish carrying a CKO and knockin (KI) switch (zCKOIS) coupled with dual-color fluorescent reporters. Using this strategy, we generated hey2zKOIS which served as a hey2 KI reporter with EGFP expression. Upon Cre induction in targeted cells, the hey2zCKOIS was switched to a non-functional CKO allele hey2zCKOIS-invassociated with TagRFP expression, enabling visualization of the CKO alleles. Thus, simplification of the design, and the visibility and combination of both CKO and KI alleles make our zCKOIS strategy an applicable CKO approach for zebrafish.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping