PUBLICATION
Fast, precise and cloning-free knock-in of reporter sequences in vivo with high efficiency
- Authors
- Zhang, Y., Marshall-Phelps, K., Almeida, R.G.
- ID
- ZDB-PUB-230614-32
- Date
- 2023
- Source
- Development (Cambridge, England) 150(12): (Journal)
- Registered Authors
- Almeida, Rafael
- Keywords
- HDR, Knock-in, Neuron, Synaptobrevin, Vamp, Zebrafish
- MeSH Terms
-
- Animals
- CRISPR-Cas Systems*/genetics
- Gene Editing
- Gene Knock-In Techniques
- Genome
- Zebrafish*/genetics
- PubMed
- 37309812 Full text @ Development
Citation
Zhang, Y., Marshall-Phelps, K., Almeida, R.G. (2023) Fast, precise and cloning-free knock-in of reporter sequences in vivo with high efficiency. Development (Cambridge, England). 150(12):.
Abstract
Targeted knock-in of fluorescent reporters enables powerful gene and protein analyses in a physiological context. However, precise integration of long sequences remains challenging in vivo. Here, we demonstrate cloning-free and precise reporter knock-in into zebrafish genes, using PCR-generated templates for homology-directed repair with short homology arms (PCR tagging). Our novel knock-in reporter lines of vesicle-associated membrane protein (vamp) zebrafish homologues reveal subcellular complexity in this protein family. Our approach enables fast and efficient reporter integration in the zebrafish genome (in 10-40% of injected embryos) and rapid generation of stable germline-transmitting lines.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping