PUBLICATION
Identifying optimal conditions for precise knock-in of exogenous DNA into the zebrafish genome
- Authors
- Oikemus, S., Hu, K., Shin, M., Idrizi, F., Goodman-Khan, A., Kolb, A., Ghanta, K.S., Lee, J., Wagh, A., Wolfe, S.A., Zhu, L.J., Watts, J.K., Lawson, N.D.
- ID
- ZDB-PUB-250531-6
- Date
- 2025
- Source
- Development (Cambridge, England) : (Journal)
- Registered Authors
- Kolb, Amy, Lawson, Nathan, Wolfe, Scot A.
- Keywords
- CRISPR, Genome editing, Homology-dependent repair, Zebrafish
- MeSH Terms
-
- CRISPR-Cas Systems/genetics
- DNA Breaks, Double-Stranded
- Gene Editing*/methods
- Gene Knock-In Techniques*/methods
- Genome*/genetics
- Zebrafish*/genetics
- DNA*/genetics
- Animals
- Recombinational DNA Repair/genetics
- PubMed
- 40446205 Full text @ Development
Citation
Oikemus, S., Hu, K., Shin, M., Idrizi, F., Goodman-Khan, A., Kolb, A., Ghanta, K.S., Lee, J., Wagh, A., Wolfe, S.A., Zhu, L.J., Watts, J.K., Lawson, N.D. (2025) Identifying optimal conditions for precise knock-in of exogenous DNA into the zebrafish genome. Development (Cambridge, England). :.
Abstract
CRISPR nucleases can be used to insert exogenous DNA into the zebrafish genome via homology-dependent repair (HDR), although germline transmission rates for precise edits remain quite low. Comparative studies to optimize HDR parameters for introducing base pair changes using short-read deep sequencing have been successful, but similar analysis for insertions is challenging due to read length constraints. Here, we quantified editing outcomes using long-read sequencing to identify optimal template and CRISPR parameters for precise targeted insertion in zebrafish. Through side-by-side comparisons, we found that chemically modified templates out-perform those released in vivo from a plasmid, while Cas9 and Cas12a nucleases performed similarly for targeted insertion. Consistent with previous studies, precise editing rates were dependent on the distance between a double-strand break and the inserted sequence. We further found that non-homologous base pairs in homology templates significantly reduced precise editing rates. Using optimized parameters, we consistently achieved germline founder rates of greater than twenty percent for precise insertions across four loci. Together, our quantitative analyses identified optimal conditions for precise insertion of exogenous DNA into the zebrafish genome.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping