Calculation of ON:OFF target cleavage ratios and specificity scores

For each comparable pair of sequence-similar on- and off-target sites in DNA, an ON:OFF ratio of the on-target cleavage yield versus the off-target cleavage yield is calculated by simple division. By contrast, a specificity score is calculated as the odds ratio of both yields. That is, if and are the average cleavage yields for the comparable on-target and off-target sites, respectively, then the specificity score is defined as

This formula is also suitable for evaluating specificity when measuring indel frequencies in CRISPR-treated cells, in which case and are the average indel frequencies at the on-target and off-target sites, respectively. The specificity score is symmetrical with respect to on-target and off-target cleavage and therefore can be a more informative metric when both are important. When the on-target cleavage rate is roughly constant, and the off-target rate is close to zero, the specificity score tracks the simple ON:OFF ratio, but it is more sensitive to changes in the on-target rate than the simple ratio assessment, as the latter is less sensitive to losses in cleavage activity.