PUBLICATION
Rapid generation of maternal mutants via oocyte transgenic expression of CRISPR-Cas9 and sgRNAs in zebrafish
- Authors
- Zhang, C., Lu, T., Zhang, Y., Li, J., Tarique, I., Wen, F., Chen, A., Wang, J., Zhang, Z., Zhang, Y., Shi, D.L., Shao, M.
- ID
- ZDB-PUB-210808-10
- Date
- 2021
- Source
- Science advances 7(32): (Journal)
- Registered Authors
- Shao, Ming
- Keywords
- none
- MeSH Terms
-
- Animals
- Animals, Genetically Modified
- CRISPR-Cas Systems*
- Gene Editing
- Oocytes
- RNA, Guide, Kinetoplastida/genetics
- Zebrafish*/genetics
- PubMed
- 34362733 Full text @ Sci Adv
Citation
Zhang, C., Lu, T., Zhang, Y., Li, J., Tarique, I., Wen, F., Chen, A., Wang, J., Zhang, Z., Zhang, Y., Shi, D.L., Shao, M. (2021) Rapid generation of maternal mutants via oocyte transgenic expression of CRISPR-Cas9 and sgRNAs in zebrafish. Science advances. 7(32).
Abstract
Maternal products are exclusive factors to drive oogenesis and early embryonic development. As disrupting maternal gene functions is either time-consuming or technically challenging, early developmental programs regulated by maternal factors remain mostly elusive. We provide a transgenic approach to inactivate maternal genes in zebrafish primary oocytes. By introducing three tandem single guide RNA (sgRNA) expression cassettes and a green fluorescent protein (GFP) reporter into Tg(zpc:zcas9) embryos, we efficiently obtained maternal nanog and ctnnb2 mutants among GFP-positive F1 offspring. Notably, most of these maternal mutants displayed either sgRNA site-spanning genomic deletions or unintended large deletions extending distantly from the sgRNA targets, suggesting a prominent deletion-prone tendency of genome editing in the oocyte. Thus, our method allows maternal gene knockout in the absence of viable and fertile homozygous mutant adults. This approach is particularly time-saving and can be applied for functional screening of maternal factors and generating genomic deletions in zebrafish.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping