PUBLICATION
Optimal tagging strategies for illuminating expression profiles of genes with different abundance in zebrafish
- Authors
- Liu, J., Li, W., Jin, X., Lin, F., Han, J., Zhang, Y.
- ID
- ZDB-PUB-231222-10
- Date
- 2023
- Source
- Communications biology 6: 13001300 (Journal)
- Registered Authors
- Keywords
- none
- MeSH Terms
-
- Animals
- CRISPR-Cas Systems*
- Fluorescence
- Genome
- RNA, Guide, CRISPR-Cas Systems
- Zebrafish*/genetics
- PubMed
- 38129658 Full text @ Commun Biol
Citation
Liu, J., Li, W., Jin, X., Lin, F., Han, J., Zhang, Y. (2023) Optimal tagging strategies for illuminating expression profiles of genes with different abundance in zebrafish. Communications biology. 6:13001300.
Abstract
CRISPR-mediated knock-in (KI) technology opens a new era of fluorescent-protein labeling in zebrafish, a preferred model organism for in vivo imaging. We described here an optimized zebrafish gene-tagging strategy, which enables easy and high-efficiency KI, ensures high odds of obtaining seamless KI germlines and is suitable for wide applications. Plasmid donors for 3'-labeling were optimized by shortening the microhomologous arms and by reducing the number and reversing the sequence of the consensus Cas9/sgRNA binding sites. To allow for scar-less KI across the genome, linearized dsDNA donors with 5'-chemical modifications were generated and successfully incorporated into our method. To refine the germline screen workflow and expedite the screen process, we combined fluorescence enrichment and caudal-fin junction-PCR. Furthermore, to trace proteins expressed at a low abundance, we developed a fluorescent signal amplifier using the transcriptional activation strategy. Together, our strategies enable efficient gene-tagging and sensitive expression detection for almost every gene in zebrafish.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping