Exercise 2 continued

After the CRISPR site finder runs, you'll see the following message:


This message appears because the sequence we are searching for CRISPR sites in, the LYP1 CDS, 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.

As in the previous exercise, Geneious has annotated the 41 "GN(20)GG" CRISPR sites on the LYP1 sequence and grouped them in a CRISPR Sites track.



Each CRISPR site in the sequence has been scored according to how many off-target sites it potentially will bind to, and how similar the off-target sites are to the original sequence. The "Off target score" 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 score for a CRISPR site is 100% minus a weighted sum of off-target scores in the target genome. Thus, a higher score indicates a better CRISPR site with few or weak potential offsite targets.

The CRISPR annotations on the sequence are now colored according to their CRISPR 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 Off-target 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 off-target score.



From this table you'll see that many CRISPR sites have off-target 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 41 has the lowest off-target score. Select this row in the table and return to the Sequence View. The "CRISPR guide 41" 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 CRISPR 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.

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 and click the Save button.


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