PUBLICATION
Cre-Controlled CRISPR mutagenesis provides fast and easy conditional gene inactivation in zebrafish
- Authors
- Hans, S., Zöller, D., Hammer, J., Stucke, J., Spieß, S., Kesavan, G., Kroehne, V., Eguiguren, J.S., Ezhkova, D., Petzold, A., Dahl, A., Brand, M.
- ID
- ZDB-PUB-210220-17
- Date
- 2021
- Source
- Nature communications 12: 1125 (Journal)
- Registered Authors
- Brand, Michael, Hans, Stefan, Kesavan, Gokul, Kroehne, Volker
- Keywords
- none
- MeSH Terms
-
- Animals
- Base Sequence
- Clustered Regularly Interspaced Short Palindromic Repeats/genetics*
- Eye/embryology
- Eye/metabolism
- Gene Silencing*
- Green Fluorescent Proteins/metabolism
- Integrases/metabolism*
- Monophenol Monooxygenase/genetics
- Mutagenesis/genetics*
- Pigmentation/genetics
- RNA, Messenger/genetics
- RNA, Messenger/metabolism
- Time Factors
- Transgenes
- Zebrafish/genetics*
- PubMed
- 33602923 Full text @ Nat. Commun.
Citation
Hans, S., Zöller, D., Hammer, J., Stucke, J., Spieß, S., Kesavan, G., Kroehne, V., Eguiguren, J.S., Ezhkova, D., Petzold, A., Dahl, A., Brand, M. (2021) Cre-Controlled CRISPR mutagenesis provides fast and easy conditional gene inactivation in zebrafish. Nature communications. 12:1125.
Abstract
Conditional gene inactivation is a powerful tool to determine gene function when constitutive mutations result in detrimental effects. The most commonly used technique to achieve conditional gene inactivation employs the Cre/loxP system and its ability to delete DNA sequences flanked by two loxP sites. However, targeting a gene with two loxP sites is time and labor consuming. Here, we show Cre-Controlled CRISPR (3C) mutagenesis to circumvent these issues. 3C relies on gRNA and Cre-dependent Cas9-GFP expression from the same transgene. Exogenous or transgenic supply of Cre results in Cas9-GFP expression and subsequent mutagenesis of the gene of interest. The recombined cells become fluorescently visible enabling their isolation and subjection to various omics techniques. Hence, 3C mutagenesis provides a valuable alternative to the production of loxP-flanked alleles. It might even enable the conditional inactivation of multiple genes simultaneously and should be applicable to other model organisms amenable to single integration transgenesis.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping