Exercise 1: Finding CRISPR sites and checking for off-target matches

In this exercise you will run the Find CRISPR Sites function to find "GN(20)GG" CRISPR sites on the LYP1 CDS sequence. For each site identified within LYP1, you will also search for non-target binding sites within the Saccharomyces cerevisiae genome. If a gRNA sequence binds to other regions of the genome, Cas9 will also affect the sequence in these regions, and this may have serious unintended consequences. For each CRISPR site, an Activity score, which predicts the activity at the target site, and a Specificity score, which is based on the presence of off-target binding sites elsewhere in the genome, is calculated.

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 Find CRISPR Targets select Anywhere in sequence.

The Geneious Prime CRISPR tool can be used to find 3' Cas9 sites, or 5' Cpf1 sites. In the PAM Site Location selector, make sure 3' (Cas9) is selected.

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.

To score the sites based on their off-target binding, check Score against an off-target database under Specificity Scoring.

The off-target database is typically the whole genome of your organsim of interest, but can include other sequences, for example, the targeting vector. You can make an off-target database by creating a new, empty folder in your Geneious database and importing the sequences you wish to use. As there are a wide variety of genomes researchers may wish to test against, genome sequences can be very large, and new versions of genome assemblies may be released, Geneious does not contain inbuilt copies of any full genome sequences. Genomes can be downloaded directly from NCBI using the NCBI folders at the bottom of the Sources panel. Commonly studied genomes (for example, the human, zebrafish and the rat genomes) can be downloaded from NCBI using the links found in the Genomes folder in the Geneious Sample Documents. Genomes can be also be downloaded from other sources and imported into Geneious using common file formats.

In this tutorial the off-target database will be the Saccharomyces cerevisiae S288c genome, and this has been provided for you in the Yeast genome subfolder within this tutorial. Click the folder icon in the Specificity Scoring panel and choose the Yeast genome folder from the folder selector. Also ensure Color CRISPR sites by (at the bottom of the set up dialog) is set to Specificity Score.

Your settings should now look as in the screenshot below, and you can click OK to run the analysis.



Note: if we had chosen to look for sites within a selected subregion of a larger sequence, but had not selected an off target database for Specificity Scoring, Geneious would automatically test against the unselected region of the sequence for off-target binding.


Exercise 1 continued: the results

Exercise 2: Finding paired CRISPR sites