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.
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
Figures
Show all Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping