Section outline

  • Dual Cas13a is a free, web-based tool (part of the crisprtools.org suite) designed to help researchers build highly mismatch-sensitive assays for high-precision RNA detection. By pinpointing specific positions within an RNA sequence, this tool streamlines the generation of optimized guide RNAs, linkers, and primers necessary for your diagnostic or detection experiments.

    Key features of the tool include:

    • Streamlined Setup: Simply upload your target RNA via FASTA file and define the exact mismatch position you want to detect—no login required.
    • Customizable Configurations: Easily adjust guide lengths, wobble offsets (overlap positions), and T7 promoter or linker sequences to suit your specific assay.
    • Advanced Thermodynamic Modeling: Utilizes RNAfold and RNAup algorithms to evaluate internal structure stability and target binding energy, ensuring your guides bind the target strongly without forming unwanted internal hairpins.
    • Intelligent Ranking & Output: Automatically filters candidates based on Minimum Free Energy (MFE) and binding strength, while providing lab-ready forward and reverse primers that can be easily exported.

    DUAL-CAS13a



    How to Design Dual Cas13a Assays for High-Precision RNA Detection

    Welcome to this comprehensive tutorial on using the Dual Cas13a design tool available at crisprtools.org. Dual Cas13a is a powerful, highly mismatch-sensitive technology used to detect RNA with exceptional precision.

    In this guide, we will walk you through how to use our free, web-based tool, no login required, to design and optimize your Dual Cas13a experiments.

    Step 1: Setting Up Your Job and Configuring Parameters

    To get started, you can explore the tool manually or use the built-in example. For this tutorial, we will click the Load Example button to automatically populate the required fields.

    Here is what you need to set up a job: * Job Name: Every analysis requires a unique name (e.g., "Example Job"). * FASTA File: Upload the file containing your transcript RNA of interest. * Target Position: Define the specific position where you want to target and detect a mismatch in the RNA.

    Configuration Options

    Before running the analysis, it is important to review the default configurations. We have pre-selected the recommended settings for optimal results: * Guide Length: Set to 28 base pairs by default for Cas13a. * Primers per Guide: By default, the tool designs 2 primers per guide. * Offset (Wobble): This allows for a slight shift around the guide. We select positions 3 to 5. This means that for a 28-bp guide, positions 3 to 5 of the spacer will overlap over your specified target position. * T7 Promoter & Linker Sequence: The default sequences provided are highly recommended for standard assays.

    Step 2: Running the Analysis

    Once your settings are confirmed, click Analyze the sequence.

    • Cached Results: If another user has previously run a job with the exact same settings, your results will load immediately.
    • New Calculations: If your sequence and settings are unique, the calculation will take some time. You will be placed in a queue, and your results will appear as soon as the computation is complete.

    Step 3: Navigating the Results Page

    The results page is packed with data to help you choose the best guides. Here is how to navigate it:

    1. The "Info" Tab: We highly recommend reading this section. It explains how to interpret the complex data and different selection choices available.
    2. Exporting Data: You can easily download the full results table by clicking the Download button.
    3. Citations: If you use this tool for your research, please use the citation tool on this page to cite the original Dual Cas13a strategy paper. The paper also contains vital details on how to implement this system in the lab. https://doi.org/10.1093/nar/gkag161

    💡 Important Pro-Tip: Lab Validation is Essential This tool does not predict a single, perfect optimal design. It is a highly accurate theoretical model. We strongly recommend screening multiple candidate guides and primers in the laboratory to validate which ones yield the best efficiency for your specific cell type and cellular context.

    Step 4: Interpreting the Metrics and Selecting the Best Guides

    When you look at the results table, you will see a list of candidate designs. For each, you will see which position of the spacer overlaps with your RNA target (e.g., position 4). You are also provided with Forward and Reverse primers, which you can copy simply by clicking on them.

    To help you select the best options, the tool provides structural and thermodynamic predictions using RNAfold and RNAup software:

    1. Internal Structure (RNAfold)

    You want to ensure that your spacer and linker do not bind too strongly to each other. If they create a strong internal hairpin, it will be difficult for them to unwind and bind to your target RNA. * Minimum Free Energy (MFE): Look for MFE values closer to zero (e.g., greater than -8). A value closer to zero indicates a less stable internal structure, meaning the guide won't easily form unwanted hairpins. * Structure Visualization: The table shows visual predictions of paired vs. unpaired bases.

    2. Target Binding Energy (RNAup)

    While you want the internal structure to be weak, you want the binding to your actual RNA target to be strong. * Binding Energy & Duplex Energy: You want these values to be lower (more negative). * Open Spacer & Open Target: You also want these values to be lower.

    How Results are Sorted

    By default, the tool filters and sorts your candidates intelligently: 1. MFE Filter: Prioritizes guides with an MFE > -8 to eliminate those that create strong hairpins. 2. Binding Energy: Sorts by the lowest binding energy possible. 3. Tiebreaker: Uses the lowest "Open Target" value as a tiebreaker.

    Custom Sorting: You don't have to stick to our defaults! You can click any column header to sort by that metric. To sort by multiple columns, simply hold down CTRL (or Command on Mac) while clicking additional columns. Play around and prioritize the metrics that fit your specific experimental needs.

    Conclusion

    Thank you for reading this quick tutorial on designing Dual Cas13a systems. This tool is designed to save you time and provide highly efficient theoretical candidates for your lab work.

    If you found this helpful, please support my work by following the channel and be sure to check out crisprtools.org for your next experiment!

    This tutorial was created with the grant from COST Action: CA21113, Reference: E-COST-GRANT-CA21113-55536e26.