Exercise 1: Finding CRISPR sites

In this exercise you will run the Find CRISPR sites function to find "GN(20)GG" CRISPR sites on the LYP1 CDS sequence.

Select the LYP1 CDS document and then go to Cloning→Find CRISPR sites.

First, reset the options to the default settings. To do this, click on the cog in the bottom left hand corner of the window and select Reset to defaults (this will be grayed out if the current settings are the default settings).

As we want to find CRISPR sites anywhere in this sequence, next to Binding site select Anywhere in sequence.

In the Motif panel, use the Target and PAM fields to specify the gRNA sequence to search for. If you wish to evaluate all potential CRISPR sites enter N(20) in the Target box. Or, if you have a particular sequence you want to check for offsite binding, you can enter the exact sequence in the Target box. In this tutorial, we are interested in the "GN(20)GG" guide sequence. The target sequence for this is GN(19) and the PAM sequence is NGG so in the Target field type "GN(19)" and in the PAM Site field type "NGG". The preview underneath these fields shows what the guide sequence for this target and PAM sequence - ie GNNNNNNNNNNNNNNNNNNNNGG

In this exercise we are only interested in where the "GN(20)GG" sites are in the sequence, so uncheck the option to Score sites through off-target analysis. We want to assess the activity of each site, so keep Score sites through on-target activity checked. The dialog box should now look like the image below. Click OK to run the search.



Geneious will identify any CRISPR sites on this sequence with the "GN(20)GG" motif and annotate them. The annotations from each run of the "Find CRISPR sites" function are grouped in a new track labelled CRISPR sites. There are 41 "GN(20)GG" CRISPR sites in this CDS.

Mouse over one of these sites, and you will see a pop-up window with information about that site. This information includes the On-target activity score, which is a measure of the efficiency of gRNA binding and Cas9 cleavage at that site, as calculated as in Doench et al 2014. This score is a number between 0 and 1, with high numbers denoting higher expected activity. The CRISPR site annotations are automatically colored according to this score, with a gradient from green (high scores) through to yellow then red (poor scores).



To delete these annotations from the sequence, click the arrow to the left of the track name in the sequence view and select Delete Track.


Exercise 2: Finding CRISPR sites and checking for off-target matches
Exercise 3: Finding and filtering CRISPR sites and checking for off-target matches
Exercise 4: Finding paired CRISPR sites