CRISPR Efficiency Calculator
Calculate CRISPR on-target efficiency, indel frequency, and guide RNA scoring.
Editing Efficiency Versus Knockout Efficiency
These are different numbers and confusing them is the most common error in CRISPR analysis. Editing efficiency is the fraction of alleles carrying any indel. Knockout efficiency is the fraction carrying a frameshift.
Indels come in all sizes, and only those not divisible by three shift the reading frame. Roughly one third of random indels are in-frame, so they alter the protein without abolishing it.
| Indel size | Frame effect | Protein outcome |
|---|---|---|
| 1, 2, 4, 5 bp | Frameshift | Premature stop — likely knockout |
| 3, 6, 9 bp | In-frame | Residues removed; protein may still function |
So an 80% editing efficiency corresponds to roughly 53% frameshift alleles. A population reported as "80% edited" may contain a substantial fraction of functional protein.
Why Nonsense-Mediated Decay Matters
A premature stop codon usually triggers nonsense-mediated decay, degrading the transcript — which is what makes frameshifts effective knockouts. But if the stop falls in the last exon or within about 50 nucleotides of the final exon junction, NMD is not triggered and a truncated protein may be produced.
That truncated protein can retain function, or occasionally act as a dominant negative. This is why guides are normally targeted to early constitutive exons rather than wherever the best-scoring protospacer happens to be.
| Guide design factor | Target |
|---|---|
| GC content | 40–60% |
| Target exon | Early, constitutive, all isoforms |
| Off-target score | Minimise; check genome-wide |
| Distance to PAM | Cut site 3 bp upstream of PAM |
Validation matters as much as design. TIDE and ICE infer indel spectra from Sanger traces and are quick, but amplicon deep sequencing gives the actual distribution — and for any claim of knockout, protein-level confirmation by western blot is the standard.
Worked Examples
Common Mistakes
About a third of indels are in-frame and do not abolish the protein. An 80% edited population is roughly 53% frameshifted.
A premature stop in the final exon escapes nonsense-mediated decay, so a truncated and possibly functional protein is produced. Target early constitutive exons.
Indel frequency does not prove protein loss. Western blot or functional assay is needed to confirm knockout.
They can still disrupt function if they remove catalytic or binding residues, and can occasionally produce dominant negatives.