About This Tool

This page provides computationally predicted CRISPR guide RNA and primer designs for endogenous protein tagging in C. elegans. Each design includes guide RNAs, CRISPR primers for homology arms, and genotyping primers.

How to Use This Tool

1. Search for a gene: Use the search box to find genes by name (e.g., unc-6) or WormBase ID (e.g., WBGene00006746).

2. Select a guide design: Click on a row in the table to view the primers and guide RNA in the genome browser. Click the ⊕ symbol to expand and see full primer sequences.

3. Review the genome browser: The interactive IGV browser shows guide RNA locations (green), CRISPR primers (orange/red), and genotyping primers (purple).

Understanding the Primers

Primer 1: Outer primer for left homology arm amplification.

Primer 2/2B: Inner primer for left homology arm. Lowercase bases indicate silent mutations to prevent Cas9 from cutting the knock-in vector.

Primer 3/3B: Inner primer for right homology arm. Lowercase bases indicate silent mutations.

Primer 4: Outer primer for right homology arm amplification.

Primer 2A/3A: Additional primers for cases where the guide sequence is too far from the insertion site for the standard inner primers to reach.

Genotyping Primers: Forward and reverse primers to verify successful insertion via PCR. They can also be used for amplifying the genomic region for subsequent homology arm amplification, to avoid tricky PCR reactions with long/mutated/overhang-containing primers on genomic DNA directly.

Important Notes

• Overhang addition: When ordering primers, append the appropriate overhangs to your knock-in vector sequence to Primers 1-4. If Primer 2A or 3A exists, only append overhangs to Primers 2B and 3B (not to 2A and 3A).
• Lowercase bases: These indicate silent mutations introduced to disrupt the PAM site and prevent Cas9 from cutting your knock-in vector.
• Off-targets: Check the off-target column to see the number of off-target sequences with up to 4 bases mismatched. Designs with fewer off-targets are preferred.
• Distance to insert: Shows how far the guide cuts from the desired insertion site.
• Amplicon size: Expected PCR product size.
• Check before ordering: While every effort has been made to ensure the predicted sequences are correct and specific, errors are possible, so always verify the sequences before ordering and beginning cloning experiments.

Workflow Summary

1. Design vector overhang sequences onto the 5' end of Primers 1-4 (try to keep total length below ~60bp to reduce cost)

2. Order guide DNA and all required primers

3a. (Optional) Amplify the genomic region using the Genotyping Primers

3b. Amplify left and right homology arms using Primers 1+2 and Primers 3+4

3c. If a Primer 2A/3A exists, amplify with Primers 1+2A or Primers 3A+4 first, then add the rest of the HA sequence using Primers 1+2B or Primers 3B+4

4. Clone homology arms into your knock-in vector with appropriate overhangs (Gibson assembly)

5. Inject the completed repair template into young adult worms along with the guide and Cas9 (on DNA vector or as in vitro translated guide RNA + Cas9 protein)

6. Use genotyping primers to validate successful insertions