Using the sequence of the guide RNAs, the CRISPR Design Tool identifies all the potential off-target sites across the same genome and determines the number of mismatches between each target guide RNA and all its potential off-target sites. Based on this information, the algorithm is able to score and rank the guides that have the least number of off-target sites.
The off-target sites are presented as a string of values separated by commas in the “All Guides” tab of the CRISPR Design Tool. Here is an example of how to interpret the string:
Off Targets: 0,0,1,0,35
This sequence has 0 identical matches, 0 sequences with single-mismatch, 1 sequence with two-mismatches, 0 sequences with three-mismatches, and 35 sequences with four-mismatches.