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.

本研究中，作者对比了自动化机器人注射与手动注射方法针对斑马鱼胚胎中CRISPR-RfxCas13d（CasRx）系统的mRNA敲低效果，以及Cas9介导的DNA靶向效率。他们以无尾（TBXTA）基因为原理验证靶点，评估了诱导产生的胚胎表型。Cas9与CasRx系统均可使TBXTA基因产生功能丧失表型。Cas9蛋白相较mRNA引发严重表型的比例更高，而CasRx蛋白与mRNA的敲低效率相近。无论是机器人注射还是手动注射，其表型比例与死亡率均无显著差异。

本研究结果凸显了靶向RNA的CRISPR效应蛋白用于精准基因敲低的潜力，并认可了以每胚胎1.0秒的速度开展的自动化显微注射，可作为手动注射方法的高通量替代方案。此前作者已针对斑马鱼胚胎中CRISPR-RfxCas13d系统的mRNA敲低与Cas9介导的DNA靶向，再次对比了自动化机器人与手动注射的效率，结果显示二者的表型比例与死亡率仍无显著差异。研究结果表明，自动化显微注射非常适合作为斑马鱼胚胎基因沉默的高通量替代方案。

一款集成触摸屏、自动液滴计算与选择性安乐死功能的先进机器人系统的开发，提升了斑马鱼注射的效率与精准度。本研究展示了该机器人的分步操作流程，其注射速度可达59枚胚胎/分钟，且在斑马鱼胚胎注射过程中具备高精度。机器人注射速度较手动注射提升近两倍，可达59枚胚胎/分钟，而手动注射仅能达到20枚胚胎/分钟。

研究还探索了注射位置（卵黄中心或细胞界面）对表型的影响，结果表明注射位置对表型外显率无显著影响。RfxCas13d系统在RNA层面展现出良好的功效，无论是机器人注射还是手动注射，其在TBXTA基因敲低中均表现出较高的表型外显率。

本研究有助于理解靶向RNA的CRISPR效应蛋白以高通量方式实现精准基因敲低的潜力。相较于手动注射方法，本研究证实了自动化机器人注射在斑马鱼胚胎基因沉默中的有效性与优势。该机器人系统具备操作简便、精度高及独特功能等特点，可简化大规模注射流程。

总体而言，本研究推动了该领域的研究进展，为加速早期胚胎发育研究与大规模注射实验提供了极具前景的方案，其中包括将CRISPR效应蛋白用于高通量遗传与转录组筛选。