In the previous exercise you scored all of the "GN(20)GG" sites on the LYP1 gene for off-target binding against the Saccharomyces cerevisiae genome. This search was relatively fast as the Saccharomyces cerevisiae genome is reasonably small and there are only a small number of "GN(20)GG" sites on the LYP1 gene. However, if you are testing for off-target binding against a larger genome (for example, the human genome) and/or are scoring a larger number of CRISPR sites, this search may take substantially longer. In this exercise we will use one of the alternative search strategies in the Find CRISPR sites tool to discard some sites and speed up the search.
Select the LYP1 CDS document and go to Cloning→Find CRISPR sites.
As in the previous exercises, 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".
In this exercise we want to use one of the alternative search strategies to discard CRISPR sites with strong offsite matches, so under Speed and Filter strategy select Fast - discard more sites. The off-target database should still be set to the Yeast genome folder.
The dialog box should now look like the image below. Click OK to run the search.
Using this search strategy, Geneious has only annotated five of the CRISPR sites on this sequence.
Any sites which were found to have an off-target match where there are no mismatches within the PAM site or within the first 8bp next to the PAM site were dropped from the search. Geneious did not search for further off-target matches for these sites and the CRISPR sites were not annotated on the sequence.
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.