How is gRNA specificity score calculated?

VectorBuilder follows the algorithm developed in Feng Zhang’s lab to calculate specificity scores for gRNAs. Briefly, for a given gRNA intended to target a N(20)NGG sequence in a species, we search for all potential off-target sites in the genome of that species that have ≤3 mismatches with the target sequence. For each potential off-target site identified this way, a single off-target score is calculated. Scores for all the off-target sites are then used in aggregate to calculate the final specificity score of the gRNA, which is between 0 and 100, with higher values indicating greater targeting specificity.

Please note that specificity scores are only a rough guide. Actual targeting efficiency and specificity could depart from what the scores predict. gRNAs with low scores may still work well.