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

BioTechniques, vol. 76, pp. 185–193

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 (TBXTA) gene as a proof-of-principle, evaluating the induced embryonic phenotypes. Both Cas9 and CasRx systems caused loss of function phenotypes for TBXTA. 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 7

1. Joaquín Abugattas-Núñez del Prado Aachen Institute of Biology Leiden

   Leiden University · Universidad Peruana Cayetano Heredia

   Affiliation as printed

   Department of Biology, Universidad Peruana Cayetano Heredia, Av. Honorio Delgado 430, Lima, 15102, Perú

   Institute of Biology Leiden, Animal Science & Health, Leiden University, Einsteinweg 55, Leiden, 2333CC, The Netherlands

2. Yi Ding
   Affiliation as printed

   Life Science Methods BV, JH Oortweg 19, Leiden, 2333CH, The Netherlands

3. Jan de Sonneville
   Affiliation as printed

   Life Science Methods BV, JH Oortweg 19, Leiden, 2333CH, The Netherlands

4. Kees-Jan van der Kolk
   Affiliation as printed

   Life Science Methods BV, JH Oortweg 19, Leiden, 2333CH, The Netherlands

5. Miguel A. Moreno-Mateos

   Consejo Superior de Investigaciones Científicas · Centro Andaluz de Biología del Desarrollo · Universidad Pablo de Olavide

   Affiliation as printed

   Andalusian Center for Developmental Biology (CABD), Pablo de Olavide University/CSIC/Junta de Andalucía, Ctra. Utrera Km.1, Seville, 41013, Spain

   Department of Molecular Biology & Biochemical Engineering, Pablo de Olavide University, Ctra. Utrera Km.1, Seville, 41013, Spain

6. Edward Málaga‐Trillo

   Universidad Peruana Cayetano Heredia

   Affiliation as printed

   Department of Biology, Universidad Peruana Cayetano Heredia, Av. Honorio Delgado 430, Lima, 15102, Perú

7. Herman P. Spaink Aachen Institute of Biology Leiden

   Leiden University

   Affiliation as printed

   Institute of Biology Leiden, Animal Science & Health, Leiden University, Einsteinweg 55, Leiden, 2333CC, The Netherlands