CRISPR Off-Target Score Calculator
Score guide RNA specificity from mismatch count and position.
About CRISPR Off-Target Score Calculator
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.
Formula
CRISPR Off-Target Score
Activity falls exponentially with mismatch count, weighted heavily toward the PAM-proximal seed
- seed region
- The eight to twelve bases nearest the PAM, where mismatches are least tolerated
- PAM-distal
- The far end of the guide, where mismatches are often tolerated
Worked example
With the default values below, CRISPR Off-Target Score Calculator returns Specificity Score of 91.68. Change any input above and every figure here is recalculated in your browser.
Inputs used
- Mismatches to the off-target siteaccepts 0–10
- 3
- Mismatches within the PAM-proximal seedaccepts 0–10
- 1
- Guide lengthaccepts 15–30 bases
- 20 bases
- Guide GC contentaccepts 0–100 %
- 55 %
Results
- Specificity Score
- 91.68 /100
- Estimated Off-Target Activity
- 8.3242%
- Risk Band
- Moderate off-target risk
- Seed Region Penalty
- 0.850
- Guidance
- Validate this site experimentally before relying on the prediction
Step by step
- 1Values used
- 2CRISPR Off-Target Score
- 3Specificity Score= 91.68 /100
- 4Estimated Off-Target Activity= 8.3242
- 5Risk Band= Moderate off-target risk
- 6Seed Region Penalty= 0.850
- 7Guidance= Validate this site experimentally before relying on the prediction
Assumptions and limits
- The result is derived only from the 4 inputs listed above. Anything not among them is not part of the calculation.
- In particular, this calculator does not take inflation, tax, fees or charges or compounding frequency into account.
- Figures are rounded for display to 4 decimal places, so totals built from several rounded parts can differ slightly from the unrounded arithmetic.
How these calculators are built and tested is described on the Methodology page. If a figure looks wrong, please report it.
Frequently Asked Questions
How is CRISPR Off-Target Score calculated?
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.
Why does CRISPR Off-Target Score matter?
Computational scores narrow the candidate list but cannot replace empirical validation, since chromatin state also affects accessibility at off-target sites.
What values do I need to enter?
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.