GE-ON CRISPR Tools

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.

CHOPCHOP is a comprehensive, user-friendly web tool (part of the crisprtools.org suite) used to design and evaluate guide RNAs (gRNAs) for CRISPR experiments. Whether you are using standard Cas9, Cas12, Cas13 (RNA targeting), or Nickases, CHOPCHOP streamlines the design process by generating an interactive map of potential guides ranked by efficiency and safety.

Key features of the tool include:

• Flexible Inputs: Search by gene name, FASTA sequence, or genomic coordinates across a vast library of species.
• Highly Customizable: Adjust target regions, PAM sequences, guide lengths, and scoring algorithms to fit your specific experimental needs.
• Comprehensive Outputs: Evaluate off-target mismatch scores, predict editing efficiency, design flanking primers, and easily download your results.

(Note: For advanced off-target screening that includes DNA/RNA bulges, users should pair CHOPCHOP with its sister tool, CHOPOFF).

CHOPCHOP

CHOPCHOP Web Tool Tutorial: Designing guide RNAs for CRISPR

Welcome to this short tutorial on using the CHOPCHOP web tool for designing guide RNAs (gRNAs) for your CRISPR experiments. CHOPCHOP is part of the crisprtools.org suite of tools, though it opens as a separate webpage.

Let's walk through how to navigate the main page, configure your options, and interpret your results.

1. Basic Inputs & Settings

On the main page, you'll start by defining your basic experimental parameters: * Target: For this example, we will use the gene MT2A. * Species: Select the genome you want to target (e.g., Homo sapiens). * CRISPR Type: Choose your experiment style. Options include standard CRISPR (Cas9), Nickases, Cas12, Cas13 (for RNA targeting), or TALENs (our legacy design tool). * Purpose: Choose from our presets. Selecting a preset will automatically fill in the appropriate fields in the "Options" tab, but you can always adjust these manually.

Inputting Your Target

Often, just typing your favorite gene name is enough. However, you have other options: * FASTA Sequence: If you have a FASTA sequence, clear the gene name box, click "Paste Target," and paste your FASTA sequence into the text field. * Transcripts/Gene Names: You can specify a transcript from GENCODE or Ensembl. Note: CHOPCHOP does not recognize version numbers (the extra numbers at the end of an identifier, like .1). If you include the version number, the tool will return an error, so be sure to remove it. * Chromosme:start-stop You can also search using "chromsome_name:start-stop" style of inputs, for many species it requires you to know which chromsome naming style was submitted to the webpage. Note: You can figure it out by for example typing anything e.g. "dada" for your gene name, the website will then return an erro message with example gene names for that species, then you can copy any of these example gene names and use it for a query. Next, you can then see what kind of genomic locations naming we use for chromosomes and then you know whether its Ensembl or RefSeq, you can then use that naming convention for your inputs.

2. Configuring Options

Before running your design, always click the Options button to review your settings. You can hit "Reset Options" at any time to return to the defaults.

General Options

• Target Region: By default, this is the coding region. You can change this to include all exons, splice sites, promoters, or a specific target exon.
• Restrict Targeting: By default, the tool searches exons and short flanking regions (allowing the guide to overlap slightly on the outside). You can restrict this to search only within the exon.
• Consensus (Intersection vs. Union): This setting often confuses users.
  • Intersection (Default): The tool searches only for regions present in all transcripts/isoforms of a gene. This works beautifully for well-annotated genes. However, if the gene annotation is highly complex or flawed, the tool may throw an error.
  • Union: If you get an error using Intersection, switch to Union. This searches all exons across all isoforms. Use this when targeting complex genes with highly variable isoforms.
• Pre-filtering: You can set minimum/maximum thresholds for GC content and self-complementarity. (Note: Changing your primary purpose, like selecting "Nanopore Enrichment," will automatically adjust these thresholds).
• Other Settings: You can also select your preferred restriction enzymes (New England BioLabs is the default), FASTA coloring, and sequence display lengths.

Cas9 Specific Options

When designing for standard Cas9 (NGG PAM on the 3' end, default length of 20bp), you can customize: * Guide Length: e.g., 16bp for truncated guides. * Custom PAMs: You can type in an exotic PAM (e.g., NYNY) or select from popular presets. * Scoring & Efficiency: Choose your preferred algorithms for determining targets, off-target mismatches (default is max 3 mismatches), and efficiency scoring. * Repair Profile Prediction: You can choose to calculate this or turn it off to save processing time.

Other CRISPR System Options

• Cas13 (RNA Targeting): Because this targets RNA, there is no PAM. Instead, you select a Protospacer Flanking Sequence (PFS), choose whether it's on the 5' or 3' end, and set the length.
• Cas12 / Cpf1 / CasX: For this family, the PAM is located on the 5' end. You can select specific enzymes, guide lengths, and efficiency models.
• Nickases: Options are similar to Cas9 but optimized for paired designs.
• Primers: You can ask the tool to design primers for your guides, specifying amplicon size, melting temperatures, and distance to the target site.

3. Running the Job and Viewing Results

Once your settings are configured, click to run the targets.

Job Sharing & Storage: The URL for your results page is shareable. Jobs are saved in our database for 2 to 3 days. If you (or someone you shared the link with) actively accesses the page, it remains saved. If left inactive, the job will be deleted, and you will need to rerun it.

The Results Page (Standard Cas9)

On the results page for our gene (MT2A), you will see an interactive map of the transcripts and a list of predicted guides. * Coloring & Sorting: Guides are color-coded (which visually represents their score), numbered, and sorted. You can click on column headers to sort by efficiency, off-targets, GC content, etc. * Evaluating Guides: The table displays off-target scores (showing how many potential off-targets exist with 0, 1, 2, or 3 mismatches) and an efficiency score. If optimizing for safety, sort by off-targets; if optimizing for editing, sort by efficiency. * Downloading: You can download all results in various formats using the download button.

Detailed Guide View

Clicking on a specific guide opens a detailed view showing: * The target sequence and the Protospacer Adjacent Motif (PAM). * Lab Tip: When ordering guides for your lab, do not order the PAM sequence. The Cas9 protein recognizes the PAM in the genome natively; you only order the guide RNA sequence itself. * The predicted cut site, restriction enzyme sites, and surrounding primers. * A table of potential off-targets.

4. Checking Off-Targets with CHOPOFF

Important: CHOPCHOP only predicts off-targets based on mismatches. It ignores the possibility of bulges (DNA/RNA insertions or deletions).

To ensure maximum safety, take your top guide and verify it using CHOPOFF: 1. Copy your top guide sequence from CHOPCHOP. 2. Go to crisprtools.org and select the CHOPOFF tool. 3. Paste your guide, name your job (e.g., "Rank 1 MT2A"), select your species, and run the tool. 4. CHOPOFF will search for off-targets including bulges.

If CHOPOFF reveals that your guide targets another unintended gene, you can use the tool to design specific primers to test those off-target loci in the lab. (For more details, see our dedicated CHOPOFF tutorial).

5. Other CRISPR Systems in CHOPCHOP

• Cas13 Results: When targeting RNA, the results will highlight which specific isoforms are targeted by the guide. Instead of an editing efficiency model, guides are scored on the local structure of the RNA (lower local structure is better).
• Nickase Results: Results will display pairs of guides (e.g., a blue guide and a red guide) that create a single-strand cut on opposite strands, creating an overhang. The tool will show off-targets for individual guides, but more importantly, it tracks dangerous off-targets for the pair itself.

6. Additional Features & Resources

• UCSC Genome Browser: Click the browser button to view your guides in their wider genomic context (currently supported for human and mouse).
• Instructions & Protocols: At the bottom/side of the page, you will find detailed instructions, scoring methodologies, and lab protocols.
• Genome Submissions: If you need a genome that isn't currently listed, read the "Genome Submissions" section, fill out the form, and email us to have it included.
• Citation Info: Information on how to cite CHOPCHOP in your publications is also available.

Please follow and support the crisprtools.org channel for more updates.

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

SNIPSNP is an advanced computational web tool (part of the crisprtools.org suite) designed to automate the creation of Homology-Directed Repair (HDR) templates. Whether you are correcting existing genetic mutations or introducing new ones, SNIPSNP streamlines the design process by intelligently introducing synonymous variants into your template—effectively preventing Cas nucleases from re-cutting the DNA after a successful edit.

Key features of the tool include:

• Intelligent Optimization: Choose between Safety First, Disruption First, or Balanced strategies to dictate how the algorithm introduces protective synonymous variants.
• Complex Edits Made Simple: Input multiple variants simultaneously within a 50-bp window and easily toggle whether they should be repaired, retained, or newly introduced.
• Advanced Predictive Modeling: Evaluate guide efficiency and safety using the CRISPR-MFH algorithm, and predict cellular variant risks using alphaGenome Splicing Predictions.
• Comprehensive Analytics: Visualize your edits on an interactive genomic map, assess the toxicity of added variants using CADD and dbSNP data, and automatically generate lab-ready primers and probes to verify your edits.

(Note: SNIPSNP seamlessly integrates with the CHOPOFF tool, allowing you to instantly perform deep-dive off-target analyses on your chosen guides).

SNIPSNP

A Complete Guide to Using SNIPSNP for HDR Template Design

SNIPSNP is a powerful computational tool available on crisprtools.org designed to help researchers build Homology-Directed Repair (HDR) templates. Whether you want to repair existing genetic variants or create entirely new ones, SNIPSNP automates the process of designing templates and adding synonymous variants to prevent Cas nucleases from re-cutting the edited DNA.

This guide covers all the features, inputs, and analytical tools available within the SNIPSNP platform.

1. Getting Started: Access and Setup

To access all features, start by clicking Login with ORCID. Logging in allows you to interact with the website (such as voting on features) and, most importantly, saves your search history. Once approved, you will see notifications and your past jobs.

When starting a new design, always assign a clear Job Name (e.g., "Example Job One"). Because complex jobs can take up to 30 minutes to run, giving them an identifiable name ensures you can easily find them in your history and view the results later without having to run them again. If you ever need a quick refresher on the UI, you can click the Instructions button or the Tutorial button.

2. Core Configuration

Before inputting variants, you need to establish the basic parameters of your design.

Optimization Schemes

This is the most important selection. It dictates how the algorithm chooses extra synonymous variants to add to your template. These extra variants disrupt the guide RNA's binding site to prevent re-cutting. You have three options: * Balanced (Default): The recommended choice. It provides an optimal balance between patient safety, PAM disruption, and variant quality. * Safety First: Ideal if you are editing real human cells. This strategy strictly avoids non-coding overlaps, prefers known-benign variants, and prioritizes genome safety over maximal guide disruption. * Disruption First: Focuses heavily on disrupting the guide first, with safety as an afterthought. This is useful for model organisms where absolute patient safety is not the primary concern.

Species and Chromosome Selection

Select your target Species. While SNIPSNP supports multiple species, the Human and Mouse pipelines are fully featured. These organisms have enhanced design capabilities because the tool can access comprehensive, specific annotation databases like dbSNP.

Next, specify the Chromosome (you can type to search or select from a dropdown, which also includes extra loci). All variants you input must reside on this selected chromosome.

3. Inputting Variants (The Core Feature)

One of the most powerful features of SNIPSNP is the ability to input multiple variants at once, provided they fall within a short 50-base-pair window. For example, if you sequenced a patient and found a cluster of variants in a specific locus, you can input every single one of them. Variants must be inputted using the official VCF/dbSNP formatting perspective (from the sense / plus strand).

For each variant, you input the Reference allele (automatically fetched) and the Alternate allele (what is replacing it, e.g., "T" replacing "C").

Crucially, you must use the toggle switches to tell the algorithm where this variant exists: * Genome ON / Template ON: The variant currently exists in the patient's genome (so guides must be designed assuming the mutation is there). You also want this variant to remain present on the template after the HDR edit. * Genome ON / Template OFF: The variant exists in the patient's genome, but you want to fix it. The template will carry the clean reference sequence to replace the variant. * Genome OFF / Template ON: The genome is currently clean/reference, but you want to introduce this variant via the HDR template (e.g., creating a designer organism).

Handling Indels (Insertions and Deletions): If you are inputting an insertion (e.g., Reference "G" becomes Alternate "GAGA") or a large chunk deletion, the variant must be anchored to a specific reference base. In the insertion example, "G" at position 120 is the anchor base, and "AGA" is the actual insert.

4. Advanced Design Options

SNIPSNP offers deep customization for your template architecture and predictive models:

• Distance to Cut & Max Variants: By default, the tool designs a maximum of 3 extra synonymous variants. It is best to use the least amount of extra variants possible to do the job; adding too many mutates the genome in unexpected ways.
• Template & Homology Arm Size: The default HDR template size is 120 base pairs. By default, 30 base pairs on each end are protected as Homology Recombination arms. These protected outer regions will not be edited with extra variants.
• Guide Filtering: You can restrict the design engine to build templates for one specific, concrete guide.
• Intron Exclusion: By default, the tool excludes 6 base pairs at intron-exon boundaries to prevent the accidental disruption of splicing mechanics.
• Primers and Probes: Disabled by default because they are computationally time-consuming to design. (However, they are pre-enabled when loading the example dataset).
• CRISPR-MFH Algorithm: This is our proprietary predictive model for guide off-target interactions and guide mismatch disruption. You can disable it if you prefer older, handcrafted rule based system.
• alphaGenome Splicing Predictions: Predicts variant risks based on cellular context. Warning: alphaGenome is not licensed for commercial applications. Non-commercial users can optimize predictions by selecting their specific cellular context (e.g., T-cell, HEK cell, bone, etc.). To disable it entirely, leave the field empty.

5. Interpreting the Results

Normal jobs take time, but if a job has been run previously (or if you click "Load Example"), the pre-calculated results load instantly.

The Genomic Context View

At the top of the results is the Genomic Context View. This visual map is highly interactive and connected to the data tables below. If you hover over or click a template in the table (e.g., Template "CCN_3"), it will highlight the corresponding guide ("CCN3") and visually isolate the specific variants designed for that template. You can zoom in and out to inspect the loci.

Evaluating Guides (Safety First)

When reviewing the data tables, it is highly recommended to look at the Guides table first. Safety is paramount, and you want a guide with high on-target efficiency and minimal off-targets. * The top 5 safest guides are highlighted by default. * You can sort tables by clicking the columns (hold Control/Command to sort by multiple columns simultaneously). * Guides are scored by efficiency predictors (Doench 2014, Moreno Mateos, Labuhn 2018) and ranked. * Off-targets are summarized by exact distance (Distance 0, Distance 1, Distance 2) and whether they overlap known genes.

Deep Dive into Guides (CHOPOFF Tool): By clicking "View Details" on any guide, you enter the CHOPOFF tool interface. Here, you can see if a guide has known lab activity. It provides exact CRISPR-MFH alignment scores, showing the precise alignments that create the off-target distance. It also lists the specific genes overlapped by off-targets, providing direct links to the NCBI Gene Card, Uniprot, Ensemble, and the UCSC Genome Browser. You can even click "Design Primers" for specific off-target sites to generate 5 lab-ready primers to test that specific off-target risk experimentally.

The Editing Strategy: The "Zero-Variant" Template

Sometimes, the variant you are intentionally editing is enough to disable the guide on its own.

For example, look at Guide "CCN_1". It is a highly safe guide with very few off-targets. It happens to physically overlap the variant we want to fix. Because our HDR template will remove the patient's mutation and restore the reference sequence, the guide loses its binding match. Therefore, SNIPSNP will recommend a template for CCN_1 with zero extra synonymous variants designed for it, because your primary edit inherently disables the guide.

Evaluating Extra Variants

If your chosen guide does require extra synonymous variants, you must evaluate what you are introducing into the cell using the Variant Scores tables: * CADD Score: Predicts toxicity (a score of 0 means low toxicity). * dbSNP Priority Tier: Tier 1 means the exact variant and allele already exists naturally and safely in the human population. Tier 2 means a variant exists at that specific spot in the population, but with a different allele (e.g., a "Y" instead of an "A"). * Frameshifts: If your primary intended edit creates a frameshift, the entire gene is essentially disabled. In this scenario, downstream metrics like codon number or synonymous status will simply read as "NA" (Not Available), because the gene is already broken.

Exporting and Verification (Primers/Probes)

At the bottom of the results, you will find the Primers (marked in blue) designed for the whole loci to help you confirm that your overall editing was successful. You will also find Probes designed specifically for each Guide + Template combination to detect if the specific edit worked in the lab.

Every data table includes an Info (i) button explaining each column's metrics, as well as a Download button so you can export the data as a CSV for your records.

With this information, you can copy your optimized template sequences and move confidently into the lab to start your experiments!

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

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.

OVERHANG is a dedicated, tag-based community forum (integrated directly into the crisprtools.org suite) designed specifically for genome engineers. It serves as a central hub for researchers to collaborate, network, ask questions about CRISPR experiments, and request new tool features. By requiring a secure ORCID login, the forum guarantees a professional, bot-free environment without ever spamming your email.

Key features of the platform include:

• Tag-Based Navigation: Easily curate your custom feed by following or blocking specific tags, allowing you to focus on the topics, tools, or job postings most relevant to you.
• Integrated Workspace: Seamlessly connected to the rest of crisprtools.org, the forum's top menu allows you to receive notifications when your complex tool queries (like SNIPSNP or Dual Cas13a) finish running and easily access your past job history.
• Professional Networking: Set up a dedicated "Profile Post" that acts as your professional bio, easily mention colleagues, and filter the feed to discover and connect with other researchers.
• Rich Interactions: Create informative posts featuring custom polls, embedded images, and user mentions. You can upvote content, engage in threaded discussions, and "stash" important posts to read later.

OVERHANG Forum

Tutorial: How to Use the OVERHANG Forum

Welcome everyone to this tutorial about the OVERHANG Forum, which you can find on the crisprtools.org website. This forum was created with the idea that genome engineers can collaborate, find jobs, ask questions, and build a community together.

Here is a complete guide on how to navigate and use the platform.

1. Logging In and Authentication

First, we need to log into the forum. The forum is not accessible, if you are not logged in.

I highly recommend logging in using ORCID. When you click to log in, it just takes a moment. You simply approve the ORCID website pop-up, and crisprtools.org will access your credentials. This confirms that you are a real person, which helps protect the forum from bots.

Privacy Note: When you use your ORCID ID to log in, we only get public information from your profile. We never email you anything, there is no email capability on the website at all. We only know your full name and that you are an authenticated user.

2. Navigating the Top Menu

Once you log in, you get access to a special top menu with several helpful buttons:
Home: Use this to easily return to the main webpage. * Toggle Theme: Switch the website between Dark Mode and Light Mode. * Notifications: Here you can see when someone has messaged you, tagged you in a post, or when posts containing tags you follow are created. You will also receive notifications here when your tool queries finish running (for example, CHOPOFF, SNIPSNP, or Dual Cas13a jobs). * History: This shows the history of your queries and jobs you’ve run. Thanks to this feature, you can access previously run jobs without needing to rerun them. * Stash: Access posts that you have saved to read later. * Profile: Access your personal profile and settings. * Log Out:* Securely exit your account.

3. The Main Feed, Searching, and Sorting

When you enter the forum, you are greeted by the main feed. At the top is the logo, followed by a search menu, and then the feed itself.

By default, posts are sorted by the newest first. However, administrators can "pin" important threads (like polls or announcements) so they stay at the top.

You can use the search menu to filter the main feed. For example, if you search for "amplican", the feed will only show posts related to that topic. You can also change how posts are sorted, such as sorting by the "most upvoted" posts rather than the newest.

4. The Tagging System (How to Follow and Block)

OVERHANG is primarily a tag-based forum. While it shares some similarities with Reddit, tags play the main role here in managing different threads.

When you type a tag into the search bar, autocomplete will show you available tags. You can also interact directly with tags attached to posts: * Follow a tag: Click on a tag once. You are now following it. * Block a tag: Click the tag a second time. You will no longer see posts with this tag. * Neutral: Click it a third time to return your relationship with the tag to normal.

We also have quick-tag buttons. For example, the "People" button automatically filters for the #person tag, allowing you to browse user profiles and personal posts.

5. Managing Your Profile and Settings

If you click on your Profile, you will see the information we pulled from ORCID, such as your name and ORCID ID.

Your Profile Post: Here, you can edit your special "Profile Post." This post is always tagged with #person (you cannot add other tags to it), and comments on it are disabled. This acts as your bio. When someone clicks your name, they see this post alongside your recent activity. Whenever you update it, it briefly shows up in the main feed so other people can find you.

Settings Menu: * Tag Preferences: Manage the tags you recently followed or blocked. * Notification Settings: Disable certain types of notifications if they get too noisy. * Secret Management (Passwords): Because you log in with ORCID, you don't have a standard password. However, in case you ever lose access to your ORCID, you can generate a "Secret" here. This is a string of letters that acts as a backup password. Save it to your browser, and you can use it on the login screen to access your account without ORCID.

6. How to Create a New Post

To make a new post, simply click the Plus (+) button. This opens the post creation menu. Please note the following formatting rules, as this forum is designed to be informative rather than meme-based: * Title: The first sentence you type automatically becomes the title of the post in the feed. * Preview: The second and third sentences become the text preview. * Images: Photos are supported in the post body, but they are hidden from the main feed view to keep the interface clean. * Tags: You must add at least one tag to create a post. You can search for existing tags, or create a new one by typing it out and pressing Enter. * Mentions: Type the @ symbol followed by two letters to pull up a list of users. If you mention someone, they will receive a notification. * Emojis & Polls: You can type emojis (like a smiley face) and easily attach polls with custom options to your post. * Comments: You have the option to disable comments on your post if you wish.

Before posting, please read the rules. They are short, but they clearly explain how to behave and properly tag your posts.

7. Interacting with Posts

When browsing the feed, you can interact with existing posts in several ways: * Upvote: Click on a post to vote for it. (Clicking again removes your vote; you cannot vote on your own posts). * Reply: Click the reply button. This automatically quotes the previous post (which you can delete if you want) and opens a text box. When users reply back and forth, it creates a comment thread. * Hide Threads: You can collapse or hide comment threads using the anchor buttons next to them. * Stash: Save a post to your personal stash to read later. * Report: If you find a post inappropriate, you can report it to the administrators, providing a reason why. * Delete: You can delete your own posts at any time.

Conclusion

Currently, the forum is in its early stages, but it has the potential to be a highly useful space. You can use it to ask questions about CRISPR tools, request new features for crisprtools.org, and interact with me and other genome engineers.

Together, we can build a great community here. It is completely free and available to you right now.

That’s it for this tutorial! If you found this helpful and enjoy my work, please subscribe to the channel and support the project. Thank you!

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