PUBLICATION
One-step efficient generation of dual-function conditional knockout and geno-tagging alleles in zebrafish
- Authors
- Li, W., Zhang, Y., Han, B., Li, L., Li, M., Lu, X., Chen, C., Lu, M., Zhang, Y., Jia, X., Zhu, Z., Tong, X., Zhang, B.
- ID
- ZDB-PUB-191031-9
- Date
- 2019
- Source
- eLIFE 8: (Journal)
- Registered Authors
- Zhang, Bo
- Keywords
- genetics, genomics, zebrafish
- MeSH Terms
-
- Alleles*
- Animals
- Gene Editing/methods*
- Gene Knockout Techniques/methods*
- Genotype
- Staining and Labeling/methods*
- Transcription Factors/genetics
- Zebrafish/genetics*
- Zebrafish Proteins/genetics
- PubMed
- 31663848 Full text @ Elife
Citation
Li, W., Zhang, Y., Han, B., Li, L., Li, M., Lu, X., Chen, C., Lu, M., Zhang, Y., Jia, X., Zhu, Z., Tong, X., Zhang, B. (2019) One-step efficient generation of dual-function conditional knockout and geno-tagging alleles in zebrafish. eLIFE. 8:.
Abstract
CRISPR/Cas systems are widely used to knockout genes by inducing indel mutations, which are prone to genetic compensation. Complex genome modifications such as knockin (KI) might bypass compensation, though difficult to practice due to low efficiency. Moreover, no 'two-in-one' KI strategy combining conditional knockout (CKO) with fluorescent gene-labeling or further allele-labeling has been reported. Here, we developed a dual-cassette-donor strategy and achieved one-step and efficient generation of dual-function KI alleles at tbx5a and kctd10 loci in zebrafish via targeted insertion. These alleles display fluorescent gene-tagging and CKO effects before and after Cre induction, respectively. By introducing a second fluorescent reporter, geno-tagging effects were achieved at tbx5a and sox10 loci, exhibiting CKO coupled with fluorescent reporter switch upon Cre induction, enabling tracing of three distinct genotypes. We found that LiCl purification of gRNA is critical for highly efficient KI, and preselection of founders allows the efficient germline recovery of KI events.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping