Optimized CRISPR-RfxCas13d system for RNA targeting in zebrafish embryos

CRISPR-Cas systems have been used to induce DNA mutagenesis for gene function discovery. However, the development of tools to eliminate RNAs provides complementary and unique approaches to disrupt gene expression. Here, we present a workflow to perform specific, efficient, and cost-effective mRNA kn...

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Bibliographic Details
Published in:STAR protocols 2022-03, Vol.3 (1), p.101058, Article 101058
Main Authors: Hernandez-Huertas, Luis, Kushawah, Gopal, Diaz-Moscoso, Alejandro, Tomas-Gallardo, Laura, Moreno-Sanchez, Ismael, da Silva Pescador, Gabriel, Bazzini, Ariel A., Moreno-Mateos, Miguel A.
Format: Article
Language:English
Subjects:
Animals
Biotechnology and bioengineering
CRISPR
CRISPR-Cas Systems - genetics
Developmental biology
Gene Expression
Genetics
Model Organisms
Molecular Biology
Protocol
RNA - genetics
RNA, Guide, CRISPR-Cas Systems - genetics
RNA, Messenger - genetics
Zebrafish - genetics
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Summary:CRISPR-Cas systems have been used to induce DNA mutagenesis for gene function discovery. However, the development of tools to eliminate RNAs provides complementary and unique approaches to disrupt gene expression. Here, we present a workflow to perform specific, efficient, and cost-effective mRNA knockdown in zebrafish embryos using our in vivo optimized CRISPR-RfxCas13d (CasRx) system. Although the described protocol focuses on mRNA knockdown in zebrafish embryos, it can also be applied to other vertebrates. For complete details on the use and execution of this protocol, please refer to Kushawah et al. (2020). [Display omitted] •CRISPR-RfxCas13d optimized protocol for RNA targeting in zebrafish embryos•Step-by-step protocol from gRNA design to mRNA targeting and validation•Two alternative approaches including using RfxCas13d mRNA or purified protein•Different strategies for mRNA knockdown validation CRISPR-Cas systems have been used to induce DNA mutagenesis for gene function discovery. However, the development of tools to eliminate RNAs provides complementary and unique approaches to disrupt gene expression. Here, we present a workflow to perform specific, efficient, and cost-effective mRNA knockdown in zebrafish embryos using our in vivo optimized CRISPR-RfxCas13d (CasRx) system. Although the described protocol focuses on mRNA knockdown in zebrafish embryos, it can also be applied to other vertebrates.
ISSN:2666-1667
2666-1667
DOI:10.1016/j.xpro.2021.101058