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.
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
Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping