Navigating to the CRISPR Tab and Sequence View
In order to access the CRISPR-Cas features for designing and creating gRNAs, open a file and navigate to the “CRISPR” tab in the top banner. Note that the CRISPR-Cas features are only available in sequence view, this means you will need to use the bottom navigation bar and select “Sequence” as the default view is “Map” view. Once you have successfully navigated here the buttons in the top banner will no longer be grayed out.
Select the CRISPR System
To start designing your gRNAs first select your CRISPR-Cas or base editing system. Do this by navigating to the left of the banner and clicking the dropdown, note that by default SpCas9 is selected. The current options are broken into three subgroups CRISPR-Cas systems (this includes AsCas12a, LbCas12a, SaCas9 and SpCas9), ABE base editors (this includes ABE7.10, ABE8e and ABEmax) and CBE base editors (this includes A3A-BE4, AncBE4-max, BE3, BE3-NG, BE4, BE4-NG, BE4max, evoAPOBEC1-BE4max, HF-BE3, Target-AID, YE1-BE3 and YE1-BE4).
Creating a Custom CRISPR-Cas System
Alternatively, to selecting a pre-existing system, you have the option to add your own systems. Use the “Manage systems” button at the bottom of the dropdown to open the “Manage CRISPR systems” modal box. You will see all of the preset systems and can explore their configurations by selecting them from the left panel. Note that the preset files are may not be edited. To start creating your own system click the “+ Add new” button at the top left of the modal box. You can then input the systems’ data. This includes Name, PAM sequence, PAM location (after or before protospacer), Spacer length, sgRNA scaffold sequence, crRNA scaffold sequence, Scaffold position (at 3’ or 5’ end of spacer), tracrRNA sequence and Cut distance from PAM. Additionally, if you are creating a base editing system you can choose the type (cytosine or adenine base editor), if it edits the target or non-target strand and the editing window. Once finished make sure to save your new system using the “Save” button at the top of the modal box. If you would like to go back and edit a system you have previously created, select it from the left side bar and select “Edit”.
Visualizing PAM Sites
Once you have made your selection for the CRISPR system you would like to use, find the “Show PAM sites” button in the top banner. Toggling this button on will display all the PAM sites for the given system present in your current file. PAM sites are displayed as blue bars below the sequence. In order to visualize the cut site and the guide click on one of the blue pam site indicators. This will highlight the PAM site and show the associated guide (note that for base editors the editing window will be displayed). If you would like to create the guide and add it to your current file, first select it by clicking the blue indicator, then use the button “+ Add guide RNA”, you will be prompted to give the guide a name and save it to the file. Once saved the guide will be permanently displayed next to the sequence.
Filtering by Codon Conversion for Base Editors
If you are working with base editors, you can also use the “Codon conversion” filter. This allows you to filter the displayed PAM sites based on a desired codon conversion. Click the dropdown and select your filters, you can select which codon should be present before the editing and which codon should be present after editing. Additionally it is possible to select what should be affected by the edits, anywhere in sequence, a selected sequence, a coding sequence features or open reading frames. Once you have set your filters, you will see the number of PAM sites found that match your criteria, you can then quickly navigate through each site by using the “Previous site” and “Next site” buttons.
Editing, Deleting and Exporting Guide RNAs
If you would like to edit a gRNA’s name you can select it by clicking on the sequence, then select the “Edit guide RNA” button from the top ribbon, this will open the editing modal box. For deleting a guide, again first select it and then navigate to the top banner and select the “Remove guide RNA” button, you will be asked to confirm your decision and can then delete the guide. To export your guide sequence, select it by clicking on it, then navigate to the top banner and select the “Export” dropdown, here you will see the option to export “All guide RNAs” or “Selected guide RNAs”, you can also choose between the export format CSV, IDT ordering format (paste entry or plate file upload) and Synthego ordering (tubes or plates).
Guide RNA Overview
If you would like to see an overview of all guide RNAs associated with your file, you can use the bottom navigation bar and select “Guide RNAs” this will take you to the guides overview tab. Here you will see a table of all your file’s guides, their names, the spacer sequence and the CRISPR-Cas system used. If you would like more information on the sequence you can select the info icon next to the spacer sequence, this will give you an overview of the crRNA, tracrRNA and the full sgRNA sequence.
