Once the analysis has run, you'll see the following message:
This message appears because the LYP1 CDS sequence we are searching for CRISPR sites in is also present in the off-target database, as it is part of the yeast genome. Matches within this region are ignored for off-target scoring as they are likely to be the guide site itself.
Click OK to clear this message, and you should now see a new annotation track containing CRISPR sites with the "GN(20)GG motif. There should be 41 CRISPR site annotations on this track.
Mouse over one of these sites, and you will see a pop-up window with information about that site. This information includes the 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 Specificity Score is a measure of how many off-target sites the gRNA potentially will bind to, and how similar the off-target sites are to the original sequence. This is calculated according to the method developed by the Zhang lab at MIT (for more information about this, click here). Each off-target site is given a score based on how similar it is to the original CRISPR site and where any mismatches occur (mismatches near the PAM site will affect binding more than mismatches further away from the PAM site). A higher score for an off-target site indicates a higher similarity to the original CRISPR site (and thus a higher likelihood of the CRISPR/Cas complex binding to the off target). The overall specificity score for a CRISPR site is 100% minus a weighted sum of off-target scores in the target genome. Thus, a higher specificity score indicates a better CRISPR site with few or weak potential offsite targets.
The CRISPR annotations on the sequence are colored according to their Specificity score. This color scheme uses a gradient of red to green, where the annotation is pure red when the CRISPR score is 20%, moving to pure yellow at 85%, then up to pure green at 100%.
Click the Save button to save the annotation track on the sequence.
Open the Annotations tab. To show only the CRISPR annotations in the annotations table, click Type and select CRISPR. Click Columns and tick Specificity score and #Off-target sites (these may already be selected). These columns should now be visible in the annotations table.
Clicking the name of a column in the annotations table will sort the rows of the table by the values in that column. A small triangle will appear next to the column name indicating whether the rows have been sorted from smallest to largest value or vice versa. Clicking the column name again will reverse the direction the rows are sorted. Sort the rows of the annotations table by lowest to highest Specificity Score.
From this table you'll see that many CRISPR sites have specificity scores of 100%, meaning they have no off-target matches that fit the criteria of no indels and 3 or fewer mismatches with the guide RNA (however they may have additional off-target sites with more than 3 mismatches). CRISPR guide 1 has the lowest off-target score. Select this row in the table and return to the Sequence View. The "CRISPR guide 1" annotation should now be selected in the sequence view. Hold the mouse over the annotation so a yellow tool tip appears. This tool tip contains more information about this CRISPR site.
The Specificity score for this site is 83.33%. There is only one off-target binding site for this sequence in the Saccharomyces cerevisiae genome, but it is an exact match for the CRISPR guide so has an off-target score of 100%. This site is in the ALP1 CDS at position 136,903→136,925 of Chromosome 14.
For CRISPR guides with multiple off-target sites, only the top five will be listed in the tool tip. This information can also be viewed, sorted and exported from the Annotations tab.