PUBLICATION
Advancing knock-in approaches for robust genome editing in zebrafish
- Authors
- Rodriguez-Parks, A., Beezley, E.G., Manna, S., Silaban, I., Almutawa, S.I., Cao, S., Ahmed, H., Guyer, M., Baker, S., Richards, M.P., Kang, J.
- ID
- ZDB-PUB-260203-27
- Date
- 2026
- Source
- Biology Open : (Journal)
- Registered Authors
- Kang, Junsu
- Keywords
- Amino acid substitution, Genome Editing, Knock-in, Zebrafish, foxd3, hbaa
- MeSH Terms
-
- Animals
- CRISPR-Cas Systems
- Exons
- Gene Editing*/methods
- Gene Knock-In Techniques*/methods
- Genetic Vectors
- Genome
- Zebrafish*/genetics
- PubMed
- 41630603 Full text @ Biol. Open
Citation
Rodriguez-Parks, A., Beezley, E.G., Manna, S., Silaban, I., Almutawa, S.I., Cao, S., Ahmed, H., Guyer, M., Baker, S., Richards, M.P., Kang, J. (2026) Advancing knock-in approaches for robust genome editing in zebrafish. Biology Open. :.
Abstract
Precise genome editing remains a major challenge in functional genomics, particularly for generating knock-in (KI) alleles in model organisms. Here, we introduce the mini-golden system, a versatile Golden Gate-based subcloning platform that enables rapid assembly of donor constructs containing homology arms and a gene of interest. This system offers a library of middle entry vectors including diverse genes, enhancing the preparation of donor minicircles for KI applications. Using the mini-golden system, we efficiently generated a foxd3CreER KI zebrafish line, allowing conditional recombination in neural crest cells. To further improve genome editing precision, we developed a synthetic exon-based donor template strategy combined with fluorescence screening. Using this approach, we successfully engineered a targeted isoleucine-to-valine substitution (Ile-to-Val) in hbaa1.2, one of the two adult hemoglobin alpha genes in zebrafish. Importantly, despite the high sequence similarity between hbaa1.2 and its paralog hbaa1.1, our strategy specifically edited hbaa1.2, demonstrating the effectiveness of the synthetic exon approach. This method minimized undesired recombination and significantly improved the identification of lines carrying the edited genome. Together, we provide a robust toolkit for efficient and precise genome engineering in zebrafish, with broad applicability to other model systems.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping