Score guide RNA specificity from mismatch count and position.
Mismatch position matters more than count, because the PAM-proximal seed region tolerates almost no mismatches while the distal end tolerates several. That asymmetry is why simple mismatch counting predicts off-target activity poorly. Computational scores narrow the candidate list but cannot replace empirical validation, since chromatin state also affects accessibility at off-target sites.
CRISPR Off-Target Score
Activity falls exponentially with mismatch count, weighted heavily toward the PAM-proximal seed
Activity falls exponentially with mismatch count, weighted heavily toward the PAM-proximal seed Mismatch position matters more than count, because the PAM-proximal seed region tolerates almost no mismatches while the distal end tolerates several. That asymmetry is why simple mismatch counting predicts off-target activity poorly.
Computational scores narrow the candidate list but cannot replace empirical validation, since chromatin state also affects accessibility at off-target sites.
This calculator takes 4 inputs: Mismatches to the off-target site, Mismatches within the PAM-proximal seed, Guide length, Guide GC content. The pre-filled defaults are a realistic starting point — replace them with figures from your own environment for a result you can act on.