Comparing robotic and manual injection methods in zebrafish embryos for high-throughput RNA silencing using CRISPR-RfxCas13d.

Journal: BioTechniques
PMID: 38420933

Abstract

In this study, the authors compared the efficiency of automated robotic and manual injection methods for the CRISPR-RfxCas13d (CasRx) system for mRNA knockdown and Cas9-mediated DNA targeting in zebrafish embryos. They targeted the no tail () gene as a proof-of-principle, evaluating the induced embryonic phenotypes. Both Cas9 and CasRx systems caused loss of function phenotypes for . Cas9 protein exhibited a higher percentage of severe phenotypes compared with mRNA, while CasRx protein and mRNA showed similar efficiency. Both robotic and manual injections demonstrated comparable phenotype percentages and mortality rates. The findings highlight the potential of RNA-targeting CRISPR effectors for precise gene knockdown and endorse automated microinjection at a speed of 1.0 s per embryo as a high-throughput alternative to manual methods.

Authors

  • Joaquin Abugattas-Nuñez Del Prado
    Institute of Biology Leiden, Animal Science & Health, Leiden University, Einsteinweg 55, Leiden, 2333CC, The Netherlands.
  • Yi Ding
    Clinical and Research Center of AIDS, Beijing Ditan Hospital, Capital Medical University, Beijing, China.
  • Jan de Sonneville
    Life Science Methods BV, JH Oortweg 19, Leiden, 2333CH, The Netherlands.
  • Kees-Jan van der Kolk
    Life Science Methods BV, JH Oortweg 19, Leiden, 2333CH, The Netherlands.
  • Miguel A Moreno-Mateos
    Andalusian Center for Developmental Biology (CABD), Pablo de Olavide University/CSIC/Junta de Andalucía, Ctra. Utrera Km.1, Seville, 41013, Spain.
  • Edward Málaga-Trillo
    Department of Biology, Universidad Peruana Cayetano Heredia, Av. Honorio Delgado 430, Lima, 15102, Perú.
  • Herman P Spaink
    Institute of Biology, Leiden University, Leiden, the Netherlands.

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