Purpose

This SOP provides step-by-step instructions for designing CRISPR guide RNAs (gRNAs) using the CHOPCHOP web tool. The goal is to automate gRNA selection for knockout experiments while reinforcing understanding of how CRISPR-Cas9 functions at the molecular level.

Background Video: How CRISPR-Cas9 Works

Watch the short video above to understand Cas9’s role as a DNA-cutting enzyme guided by RNA sequence complementarity. The animation highlights:

• The importance of the PAM sequence (NGG) for Cas9 binding.
• How double-stranded DNA breaks can cause frameshift mutations and gene disruption.

Principle

CRISPR-Cas9 gene editing relies on a 20-nucleotide gRNA adjacent to a PAM site recognized by Cas9. When targeted to early coding exons, the repair of the Cas9-induced DNA break by non-homologous end joining (NHEJ) introduces indels that inactivate the gene. CHOPCHOP automates discovery, scoring, and ranking of these gRNA targets, minimizing manual design steps.

Materials

• Computer with internet access
• Web browser (Chrome, Firefox, Safari)
• Target gene name or genomic sequence
• CHOPCHOP website: https://chopchop.cbu.uib.no/
• Benchling account for visualization

Procedure

1. Access CHOPCHOP

Open https://chopchop.cbu.uib.no/. The homepage displays four main input boxes labeled Target, In, Using, and For.

2. Enter Gene and Select Options

Click Find Target Sites! to begin the automated design.

3. Review Output Page

CHOPCHOP displays:

• Exon Map – graphical view of your target gene and gRNA positions.
• Guide Table – ranked list of all predicted gRNAs with scores, GC content, strand, and coordinates.

4. Selecting the Final gRNA

Review the ranked guide list and choose only one gRNA for your knockout experiment. This selection should meet all of the following criteria:

• Efficiency ≥ 60%
• MM0–MM2 = 0 in exonic regions (no perfect or near-perfect off-targets)
• GC content 40–70%
• Located in an early coding exon shared across all major transcript isoforms

Click on your chosen gRNA in the results table to open its Detailed Guide View.

The detailed view shows:

• A visual map of the exon structure and target cut site (black bar).
• A table of PCR primer pairs with coordinates, melting temperature (Tm), and product size.
• Predicted repair profile statistics such as frameshift frequency.

5. Downloading Files

While viewing your selected gRNA’s detailed page, scroll to the “Download:” menu above the primer table. Download both of the following files:

1. GenBank annotated file → BRCA1_gRNA1_CHOPCHOP.gb
2. Results table as .tsv → BRCA1_gRNA1_CHOPCHOP.tsv

These files contain:

• The complete genomic context, cut site, and primer annotations (.gb)
• Primer sequences, coordinates, and product sizes (.tsv)

6. Importing the GenBank File into Benchling

1. Log in to Benchling and open your project folder.
2. Click + Create → Import DNA Sequence → Upload File.
3. Select BRCA1_gRNA1_CHOPCHOP.gb and click Import.
4. Benchling will display exons (blue), PAM (NGG), and cut site (scissors icon).
5. Rename the file to BRCA1_gRNA1_Final_Target and confirm annotations.

7. Exporting from Benchling as PDF

1. Open the annotated sequence in Benchling.
2. Click File → Export → PDF.
3. Select Include annotations and Include map.
4. Save as BRCA1_gRNA1_Benchling_Map.pdf.
5. Check that the exon map, gRNA, and PAM labels are visible.

8. Final Submission to Google Form

After completing your CHOPCHOP and Benchling work, submit all required files via the Google Form:

Submission Checklist

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