Try It Yourself (7 steps)

We will design and analyze guide RNAs for the Brca2 gene using Benchling’s CRISPR tool.

1. Click the + on the left side panel, and then CRISPR and then CRISPR Guides

2. Search for the Brca2 gene. Ensure all settings (including the hg38 genome) are the same as the image below and save in the Training project and click Next.

3. Select Single guide, and ensure that the guide length is set to 20, the genome is hg38, and that the PAM is NGG, and click Finish. You can also input your own custom PAM site by typing into the bar.

4. Use the Annotations tab on the right side panel, and click on Exon 3 to select the third exon’s amino acids.

5. On the Design CRISPR tab, click on the + to set the target region as the selected Exon 3

6. Sort all sequences using the off-target score by clicking on the off-target-score column. Select the guides shown in the following image.When choosing guide RNAs, look at both the on-target and off-target scores. Off-target scores above 50 are considered to be good guides (see On-target scores above 60 are considered to be good guides. Use these scores to rank guides relative to each other.

7. Go to Save above your list of guides and select oligos on Benchling. Save the guides in the Training project. Go to the top of the Design CRISPR tab and save the analysis as “BRCA Exon 3 CRISPR Analysis”.

Pro Tip: CRISPR guide design offers flexibility in the Advanced Settings, letting you adjust whether you mask repeated regions, which cuts you show, and other options. 

The advanced settings panel:

Did this answer your question?