Peek Behind the Paper: Comparing injection methods for high-throughput RNA silencing using CRISPR-rfxCas13d

In this Peek Behind the Paper, we learn more about a recently published paper by Joaquin Abugattas-Nuñez Del Prado (Leiden University, The Netherlands; Universidad Peruana Cayetano Heredia, Lima, Perú) and Herman Spaink (Leiden University). The pair worked together to compare robotic and manual injection methods in zebrafish embryos using CRISPR-RfxCas13d (CasRx) for RNA silencing.

Joaquin Abugattas-Nuñez Del Prado (left) is a PhD candidate whose work focuses on investigating angiogenesis and its cellular, molecular and mechanical interactions with the extracellular matrix. He employs the intersegmental vessel model in the zebrafish, coupled with high-resolution microscopy and cutting-edge CRISPR-CasRx systems for transcriptomic modifications, to elucidate the cellular underpinnings of this complex biological process.

Herman Spaink’s (right) research is focused on understanding how biological signal transduction functions at a molecular level. A central theme in his work is the investigation of intercellular communication, particularly the communication between cells of one organism and another, which requires a highly multidisciplinary approach using methods from biology, chemistry, physics and bioinformatics.

Please provide a short summary of your paper.

We compared the efficiency of automated robotic and manual injection methods for the CRISPR-CasRx system for mRNA knockdown and Cas9-mediated DNA targeting in zebrafish embryos. We targeted the no tail (TBXTA) gene in a proof-of-principle experiment, 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 to mRNA, while CasRx protein and mRNA showed similar efficiency. Both robotic and manual injections demonstrated comparable phenotype percentages and mortality rates. Our findings highlight the potential of RNA-targeting CRISPR effectors for precise gene knockdown and endorse automated microinjection at a speed of 1 second per embryo as a high-throughput alternative to manual methods.

What inspired you to compare the methods?

The inspiration for developing this comparative study, which demonstrates various methodologies, stemmed from the opportunity to conduct automated robotic injections and mRNA knockdown via the CRISPR-CasRx system within our team. However, these methods had not yet been combined. We believe that offering other researchers in the field the ability to perform mRNA knockdown in a high-throughput manner would open new horizons for them.

What impact do you hope it will have on laboratory researchers?

We hope our developed technology will have a significant impact. Through demonstrating the efficacy and advantages of robotic automation in zebrafish experimentation, especially in high-throughput screening research, we aim to streamline various laboratory processes. These include drug screening, gene and transcript function analysis, toxicity assessments and pathogen studies. By doing so, we aim to enable researchers to conduct experiments more efficiently and on a larger scale, ultimately accelerating the pace of scientific discovery in these critical areas.

Do you have any tips for best practice for researchers looking to use robotic injection methods for gene silencing experiments?

When employing gene silencing techniques with the CRISPR-CasRx toolbox, we highly recommend testing 3–4 guide RNAs (gRNAs) for each gene target. Following this initial screening, it is essential to validate the efficacy of the selected gRNAs using RT-qPCR. Once validated, a single gRNA demonstrating the highest efficiency can be utilized for subsequent experiments, similar to our approach with the previously validated TBXTA gRNA. This systematic approach ensures the effectiveness of the gene knockdown process, enhancing the reliability of experimental results. You can follow this protocol here.

Regarding robotic injections, we advise preparing the injection solutions while the robot is performing the injections; this strategy will save time. By synchronizing the injection preparation process with the robotic injection procedure, researchers can effectively streamline their experimental workflow, ultimately maximizing the productivity and efficiency of the robotic system.

What are you hoping to do next in this area?

Our approach lays the groundwork for future technical advancements in zebrafish automation, including improvements in throughput levels, injection accuracy and needle-loading ease, which could benefit researchers working with other model organisms, such as organoid cultures. Furthermore, the integration of CRISPR-Cas technology, particularly the exploration of the CasRx system, holds promise for precise and potent gene knockdown, offering researchers new tools for modifying DNA, RNA and protein functions with higher accuracies and throughput. Overall, we anticipate that our research will catalyze innovation and facilitate breakthroughs in laboratory research across various disciplines.