Section outline

  • CHOPOFF is a powerful, free web-based tool (part of the crisprtools.org suite) dedicated to rigorously identifying and evaluating off-target sites for CRISPR guide RNAs. Designed to ensure maximum safety for your genome editing experiments, CHOPOFF goes beyond basic mismatch counting by uniquely detecting DNA bulges, predicting real-world cutting activity, and generating lab-ready primers for experimental validation.

    Key features of the tool include:

    • Advanced Off-Target Detection: Uniquely identifies and visualizes both standard mismatches and complex DNA bulges (insertions/deletions) across the genome for Cas9 and Cas12a systems.
    • Predictive Cutting Algorithm: Utilizes the proprietary CRISPR-MFH model to score the likelihood of a guide cutting at a specific off-target site, allowing you to prioritize the highest-risk loci for lab testing.
    • Seamless Primer Design: Select any high-risk off-target site and the tool will automatically design forward and reverse primers (customizable to your desired product size) for easy lab validation.
    • Deep Analytics & Community Integration: Sort results by mismatch distance or gene overlaps, link directly to databases like the UCSC Genome Browser, and log in securely via ORCID to save your job history and share real-world lab activity with the community.

    (Note: CHOPOFF is the perfect companion tool to CHOPCHOP and SNIPSNP, allowing you to take your top candidate guides and subject them to the most rigorous safety checks available).

    CHOPOFF



    How to Use CHOPOFF: A Complete Tutorial for Finding CRISPR Off-Targets

    Welcome to this tutorial on CHOPOFF, a powerful web-based tool for finding off-targets for CRISPR guide RNAs. You can access the tool for free at crisprtools.org.

    In this guide, we will walk you through how to configure your inputs, understand your results, predict cutting activity, and design primers for lab testing.

    Step 1: Configuring Your Input

    When you open the CHOPOFF tool, you will start at the main configuration page. Here is how to set up your search:

    • Job Name: Give your project a recognizable name.
    • Guide Sequences: Enter your sequences here.
      • Separate multiple sequences with spaces, commas, or new lines.
      • Do not include the PAM (Protospacer Adjacent Motif) sequence.
      • Input sequences must be in the 5' to 3' orientation.
      • Note: You can input non-standard bases (beyond just A, C, T, G) if needed.
    • Example Data: If you want to test the tool, click "Load Examples" to auto-fill the fields with sample guide sequences.
    • Species & Cas Type: Select your target species from the supported list. Then, select your Cas enzyme. The default is Cas9, but Cas12a is also supported.

    Once configured, click the Find off-targets button. Because the tool performs computationally intensive work, your job may be placed in a queue. Just wait a short period for it to complete!

    💡 Pro-Tip: Log in with ORCID We highly recommend logging in using the Login with ORCID button. This allows you to: * Receive notifications when your job is finished. * Save the history of all your previous jobs. * Upvote or downvote guide activity based on your real-world lab results, contributing to our community-driven database!

    Step 2: Navigating the Results Summary Page

    Once your job finishes, you will be taken to the results page. Here, you will see a summary table showing how many off-targets were found for each guide.

    • Understanding the Columns: If you forget what a column means, hover over the "Info" buttons. For example, OT0 means the number of off-targets at a mismatch distance of zero (this is normally your on-target site). GO0 This is Gene Overlapping off-targets at distance of zero.
    • Advanced Sorting: You can sort multiple columns at once. Just hold Control (or Command on Mac) and click the columns you want to sort by. For example, you can sort by the lowest number of off-targets, then by genomic overlaps.
    • Community Activity: You will see an "Activity" column where the community reports whether a guide works in the lab.
    • Exporting: You can easily download the summary table or download all off-targets for all guides using the download buttons.

    Step 3: Exploring Detailed Results for a Specific Guide

    Click on a specific guide to see a detailed breakdown of all its potential off-targets.

    • Identifying the On-Target Site: Usually, the site with a distance of zero and no mismatches is your actual on-target site.
    • External Links: The tool provides quick links to the NCBI Gene Card, Ensembl, UniProt, and the UCSC Genome Browser so you can easily investigate where the guide lands (e.g., examining gene overlaps).
    • Visual Alignments & Bulges: You will see a visual alignment between your guide and the reference genome, with red letters marking the differences. Importantly, CHOPOFF is able to find and display DNA bulges. This is the main feature that differentiates CHOPOFF from other web tools!

    Step 4: Predicting Activity with the CRISPR-MFH Algorithm

    Some guides can return thousands of potential off-targets. To help you figure out which loci are actually dangerous and worth testing in the lab, CHOPOFF features the CRISPR-MFH algorithm.

    • This model predicts the likelihood of the guide cutting at that specific site.
    • The closer the number is to 1, the more likely the guide is to cut. The closer it is to 0, the less likely. Note On-target sites are usually predicted around 0.3 and you should know the model is not optimized for predicting the activity on on-target sites.
    • Best Practice: Use this score to prioritize checking off-targets that have a high MFH score and overlap with known genomic entities (like transcripts or genes).

    Step 5: Designing Primers for Lab Testing

    Once you have identified off-targets you want to test in the lab, CHOPOFF can help you design primers to validate them.

    1. Expand the view on an off-target to see if primers have already been pre-calculated. If they have, you will see the left and right primer sequences immediately.
    2. If they haven't been designed yet, simply select the off-targets you are interested in via the checkboxes.
    3. Click the Design Primers button. You can specify the exact product size you want.
    4. If you change your product size requirements, just click Refresh to update the calculations. Sometimes you have to wait a minute and Refresh for the results to show up.

    Conclusion

    CHOPOFF gives you all the data you need, from bulge detection to predictive scoring and primer design, to ensure you are designing the safest CRISPR guides possible.

    If you use this tool in your research, please use the citation button on the website to cite our BioRxiv paper.

    If you found this tutorial helpful, please subscribe to the channel and support my work. Thank you for watching!

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