CHOPCHOP

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.