Design guide RNAs (gRNAs)

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Learn how to use the guide RNA tool on Benchling.

We will walk you through an example, designing and analyzing example guide RNAs for the Brca2 gene.
Click the + on the left side panel, and then CRISPR and then CRISPR Guides,

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.

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. 

Benchling’s CRISPR tool supports various Cas enzymes targeting different PAM sites. To specify your custom PAM, select the “Custom PAM” option in the “PAM” dropdown selection. 

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

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

Sort all sequences using the on-target score by clicking on the on-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.



Go to Save above your list of guides to save your guides in Benchling. Save the guides in the Training project. 

Note[Enterprise]: If you have access to Worklists in Benchling, then you will also see an option to select/create a Worklist to save your guides into. Worklists are a great way to collect and keep track of entities created in Benchling, and can be used to populate tables and in tools like Bulk Assembly.


Now 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. 

Here are the options in the Advanced Settings:


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