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

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 (<i>TBXTA</i>) gene as a proof-of-principle, evaluating the induced embryonic phenotypes. Both Cas9 and CasRx systems caused loss of function phenotypes for <i>TBXTA</i>. 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. The authors compared the efficiency of automated robotic and manual injection methods for the CRISPR-RfxCas13d system for mRNA knockdown and Cas9-mediated DNA targeting in zebrafish embryos. Both robotic and manual injections demonstrated comparable phenotype percentages and mortality rates. The findings show that automated microinjection is highly suitable as a high-throughput alternative to manual methods of gene silencing in zebrafish embryos. The development of an advanced robotic system with an integrated touch screen, automated droplet calculation and selective euthanasia has enhanced the efficiency and precision of zebrafish injection. Here we present a stepwise procedure showcasing the robot's efficiency of speed of 59 embryos/min and high precision in zebrafish embryo injection procedures. The robotic injections demonstrated a three-times faster injection speed, achieving a rate of 59 embryos per minute, compared to manual injections, which managed only 20 embryos per minute. Exploration of injection positions, wether at the yolk center or cell interface, was conducted to assess their impact on phenotypes; demonstating that the position of injection has not efect in the phenotype penetrance. RfxCas13d system demonstrated efficacy at the RNA level, exhibiting high phenotypic penetrance in <i>TBXTA</i> gene knockdown in both robotic and manual injections. This study contributes to understanding the potential of RNA-targeting CRISPR effectors for precise gene knockdown in a highthroughput manner. The study establishes the efficacy and advantages of robotic automated injection methods for gene silencing in zebrafish embryos, compared to manual injection methods. The robotic system offers user-friendly features, precision and unique capabilities, streamlining large-scale injections. Overall, the study advances understanding in the field, providing a promising approach for accelerating research in early embryonic development and large-scale injections; including, the use of CRISPR effectors for highthroughput genetic and transcriptomic screenings.