ZFIN ID: ZDB-PUB-210220-17
Cre-Controlled CRISPR mutagenesis provides fast and easy conditional gene inactivation in zebrafish
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.
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.
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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.
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