In addition to the gene of interest, a gRNA sequence may bind to other regions in the genome. Cas9 will also affect the sequence in these regions of the genome. Depending on the location of these other binding sites, this can have serious unintended consequences. When selecting CRISPR sites we are often interested in how many other locations in the genome that gRNA sequence may bind to and what effects this may have. The Find CRISPR sites tool in Geneious can search for non-target binding and uses this information to calculate a score for each CRISPR site.
In the first exercise, you used the Find CRISPR sites function to find all of the "GN(20)GG" sites in the LYP1 CDS. In this exercise, you will also check for offsite binding against the Saccharomyces cerevisiae genome
To do this, you will first need to create a database of the sequences you wish to test against for off-target binding sites - this is typically the whole genome of your organsim of interest, but can include other sequences, for example, the targeting vector. To make a database, create a new empty folder in Geneious and import the sequences you want to test against into this folder. 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 in Geneious (this is the panel on the left hand side of Geneious). 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 Sample Documents in Geneious. Genomes can be also be downloaded from other sources and imported into Geneious using common file formats.
In this tutorial the database of off-target sequences has been created for you. As this tutorial is identifying CRISPR sites in the LYP1 gene from Saccharomyces cerevisiae, we are interested in off-target binding in the Saccharomyces cerevisiae genome. The complete genome of Saccharomyces cerevisiae has been downloaded from NCBI and placed in the folder Yeast genome. To view these sequences, open this folder.
To run the Find CRISPR sites tool to identify a CRISPR sites and check for off-target binding, select the LYP1 CDS document and go to Cloning→Find CRISPR sites.
Next to Binding site select Anywhere in sequence. In the field next to Target type "GN(19)" and in the PAM Site field type "NGG". Leave Score sites by on-target activity checked.
Now check Score sites through off-target analysis and Score against an off-target database. Then select the folder containing the sequences we want to test against for non-target binding: click Select a folder and select the "Yeast genome" folder. For the Speed and Filter strategy select Slow - score all sites. Keep the maximum mismatch settings at their defaults of 3 for mismatches against the off-targets, and 0 allowed to be indels.
Note: if we did not select Score against an off-target database, but had chosen to look for sites within a selected subregion of a larger sequence, Geneious would automatically test against the unselected region of the sequence for off-target binding.
This time we want to color the results track by the off-target score, so ensure this option is set in Color CRISPR sites by. The dialog box should now look like the image below. Click OK to run the search.