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Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio
Published on: August 28, 2018
CRISPR-Cas13d Induces Efficient mRNA Knockdown in Animal Embryos
Gopal Kushawah1, Luis Hernandez-Huertas2, Joaquin Abugattas-Nuñez Del Prado3
1Stowers Institute for Medical Research, 1000 E 50th Street, Kansas City, MO 64110, USA.
Abstract:
Early embryonic development is driven exclusively by maternal gene products deposited into the oocyte. Although critical in establishing early developmental programs, maternal gene functions have remained elusive due to a paucity of techniques for their systematic disruption and assessment. CRISPR-Cas13 systems have recently been employed to degrade RNA in yeast, plants, and mammalian cell lines. However, no systematic study of the potential of Cas13 has been carried out in an animal system. Here, we show that CRISPR-RfxCas13d (CasRx) is an effective and precise system to deplete specific mRNA transcripts in zebrafish embryos. We demonstrate that zygotically expressed and maternally provided transcripts are efficiently targeted, resulting in a 76% average decrease in transcript levels and recapitulation of well-known embryonic phenotypes. Moreover, we show that this system can be used in medaka, killifish, and mouse embryos. Altogether, our results demonstrate that CRISPR-RfxCas13d is an efficient knockdown platform to interrogate gene function in animal embryos.
Insights
CRISPR-RfxCas13d (CasRx) effectively depletes RNA in zebrafish embryos, enabling the study of gene function during early development. This RNA knockdown system works across multiple animal models, including fish and mice.
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- Early embryonic development relies on maternal gene products.
- Understanding maternal gene function is crucial but challenging due to limited disruption techniques.
- CRISPR-Cas13 systems show promise for RNA degradation in various cell types.
Purpose of the Study:
- To evaluate CRISPR-RfxCas13d (CasRx) as a tool for systematic gene function analysis in animal embryos.
- To assess the efficacy of CasRx in depleting both zygotically expressed and maternally provided transcripts.
- To determine the applicability of CasRx in diverse animal models.
Main Methods:
- Utilized CRISPR-RfxCas13d (CasRx) to target specific mRNA transcripts in zebrafish embryos.
- Quantified transcript level reduction following CasRx treatment.
- Assessed the resulting embryonic phenotypes.
- Tested CasRx efficacy in medaka, killifish, and mouse embryos.
Main Results:
- Achieved efficient and precise depletion of target mRNA transcripts.
- Demonstrated an average decrease of 76% in transcript levels.
- Successfully recapitulated known embryonic phenotypes.
- Confirmed CasRx effectiveness in medaka, killifish, and mouse embryos.
Conclusions:
- CRISPR-RfxCas13d (CasRx) is a highly effective RNA knockdown platform for interrogating gene function in animal embryos.
- CasRx provides a powerful new tool for developmental biology research across multiple species.
- This system overcomes previous limitations in studying maternal gene function during embryogenesis.
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