One method for improving the specificity of CRISPR/Cas9 targeting is by using the mutant Cas9-D10A nickase with a pair of guide RNAs complementary to opposite strands of the target site. The two individual nicks on opposite strands simulate a double stranded break, which then leads to non-homologous end joining. Off-target interactions are minimised because any single off-target nick will be repaired with much higher fidelity by the base excision repair pathway.
In this exercise you will use Find CRISPR Sites to find paired sites on the LYP1 CDS. Select the LYP1 CDS document and go to Cloning→Find CRISPR sites.
Keep the same settings as for the previous exercise, and also tick the box next to Pair CRISPR sites. You can specify the maximum allowable overlap of the sites returned, and the maximum space allowed between the paired sites. The maximum overlap and maximum space between sites are measured from the 5', PAM-distal end of the CRISPR sites. We will use the default options of 6 and 16 respectively.
The dialog box should now look like the image below. Click OK to run the search.
After the analysis has run you should see a second CRISPR sites track containing three pairs of CRISPR sites.
When using the Pair CRISPR sites option, sites will only be annotated if they have at least one pair conforming to the maximum overlap and maximum space between sites settings. Sites which do not have a pair within these settings are not annotated.
Each pair of CRISPR sites has a combined "paired CRISPR score". If you hold your mouse over one of the CRISPR annotations, you can see the paired CRISPR score for this site. This combined score is a mean of the specificity scores of each individual CRISPR site. The pairs of CRISPR sites with the highest combined scores are linked. Each CRISPR site will be linked to its highest scoring paired site, unless that second site has an even higher scored pairing with another site. The pair of sites are colored according to their paired score, instead of their individual CRISPR scores.
Congratulations, you have now completed the CRISPR tutorial.