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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
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Comparing robotic and manual injection methods in zebrafish embryos for high-throughput RNA silencing using
Joaquin Abugattas-Nuñez Del Prado1,2, Yi Ding3, Jan de Sonneville3
1Institute of Biology Leiden, Animal Science & Health, Leiden University, Einsteinweg 55, Leiden, 2333CC, The Netherlands.
Biotechniques
|February 29, 2024
Summary
Automated robotic and manual injections showed similar efficiency for CRISPR gene editing in zebrafish embryos. RNA-targeting CRISPR (CasRx) and DNA-targeting CRISPR (Cas9) systems effectively knocked down the no tail gene.
Area of Science:
- Molecular Biology
- Genetics
- Developmental Biology
Background:
- CRISPR gene editing systems offer powerful tools for genetic manipulation.
- CRISPR-RfxCas13d (CasRx) enables RNA targeting, complementing DNA-targeting Cas9.
- Efficient delivery methods are crucial for CRISPR applications in model organisms like zebrafish.
Purpose of the Study:
- To compare the efficiency of automated robotic versus manual injection methods for CRISPR systems.
- To evaluate the efficacy of CRISPR-RfxCas13d (CasRx) for mRNA knockdown and Cas9 for DNA targeting.
- To assess the impact of different delivery methods and CRISPR systems on zebrafish embryonic development.
Main Methods:
- Zebrafish embryos were injected with either CRISPR-RfxCas13d (CasRx) or Cas9 systems targeting the no tail (TBXTA) gene.
- Both automated robotic and manual microinjection techniques were employed for delivery.
- Embryonic phenotypes and mortality rates were evaluated to assess system efficiency and delivery method impact.
Main Results:
- Both Cas9 and CasRx systems successfully induced loss-of-function phenotypes for the TBXTA gene.
- Cas9 protein resulted in a higher percentage of severe phenotypes compared to Cas9 mRNA.
- CasRx demonstrated comparable efficiency whether delivered as protein or mRNA, and both injection methods yielded similar results in terms of phenotypes and mortality.
Conclusions:
- RNA-targeting CRISPR effectors like CasRx show promise for precise gene knockdown.
- Automated microinjection is a viable, high-throughput alternative to manual injection for CRISPR applications.
- The study validates the use of both Cas9 and CasRx systems and automated injection for zebrafish gene editing.
