CRISPR Guide RNA Designer & PAM Finder
Scan a sequence for SpCas9, SaCas9 or Cas12a guide candidates with PAMs and scoring.
Predicted, not measured
- How good is it?
- A ranking aid, not an efficiency prediction: no accuracy is claimed for either of these two scores on guides they were not fitted to, and both predate the current models. Use them to shortlist, and read the Rule Set 3 section below for SpCas9 guides with an NGG PAM, which is the one score here with a held-out calibration.
- Only valid for:
- SpCas9 with an NGG PAM and a 20 nt spacer, and only when enough genomic flanking context is present to build the model's 30-mer / 35-mer window — both scores are null rather than padded otherwise. Nothing is predicted for SaCas9, Cas12a or SpCas9-NG.
How this was measured, and what it was fitted on
No held-out skill statistic is claimed. Both are pre-2016 models, superseded by Rule Set 3 (DeWeirdt et al., Nat Commun 2022) and by DeepHF. Rule Set 3 IS now shipped here, as crispr_ontarget, and unlike these two it carries a held-out calibration: on an independent tiling library, 87.7% / 74.9% / 82.0% of its lowest-scoring guides landed in the bottom two activity quintiles. Prefer it for SpCas9 with an NGG PAM; DeepHF is still not shipped. Treat these two as a ranking aid, not an efficiency prediction.
Two published regressions over measured cutting: Doench et al. 2014 Rule Set 1 (human and mouse cell assays) and CRISPRscan (Moreno-Mateos et al. 2015, zebrafish embryo assays). SeqBench's own composite score on top of them is a transparent rule-of-thumb, not a fitted model.
Find protospacer and PAM candidates in a target DNA sequence. Paste a sequence, choose a nuclease (SpCas9 NGG, SpCas9-NG, SaCas9 NNGRRT or Cas12a TTTV), and this gRNA designer lists candidate guides on both strands with position, spacer sequence, PAM and GC content. Warnings for poly-T terminators and homopolymer runs, plus a transparent heuristic score, help you shortlist candidates for downstream off-target checking.
0 bp
Paste a target sequence above to list every guide with a valid PAM, on both strands.
Paste a target sequence to find guide candidates.
Score the shortlist with Rule Set 3
The composite above is a triage heuristic. Rule Set 3 is the only score here with a held-out calibration, and is the one to rank SpCas9 guides on. It is reported beside the other scores rather than blended with them: a composite of three models is a fourth, unvalidated model, and the three are expected to disagree.
A guide needs 4 bases before its spacer and 3 after its PAM to be scored; guides without that flank are skipped.
The tracrRNA is not cosmetic — the score depends on which scaffold you use, and it gets less accurate when the wrong one is given.
Paste a target region above, and this ranks the guides in it.
Now check the guide you picked for off-targets
Every score above describes the intended site. None assesses off-target risk, which requires a genome rather than a spacer. This section screens a protospacer against curated lab reference genomes for matches carrying a valid PAM on either strand.
A small curated set of common lab reference genomes: bacterial chromosomes plus the human mitochondrion, roughly 9.7 Mbp in total. Not the human, mouse or rat nuclear genome. For mammalian guide design this is a check on the host and vector side only; the genome-wide search must be run elsewhere. Every run lists the genomes it covered, and anything absent from that list was not searched.
Paste a target region above and every guide it finds is listed here. Screened against SpCas9, the nuclease chosen above.
Design, edit, and verify complete constructsSeqStudio combines sequence editing and annotation, plasmid maps, primer/cloning/CRISPR design, Sanger verification, and GenBank/SnapGene files in one workspace.
How to use the CRISPR gRNA Designer tool
- 1Paste the target DNA region (raw or FASTA).
- 2Pick the nuclease / PAM and whether to search both strands.
- 3Review the ranked guide candidates, their PAMs, GC and warning flags, then shortlist for off-target checking.
Frequently asked questions
Which nucleases and PAMs are supported?
SpCas9 (NGG, 20 nt spacer), SpCas9-NG (NG), SaCas9 (NNGRRT, 21 nt) and Cas12a/Cpf1 (TTTV, 23 nt, 5′ PAM). Both strands are scanned and coordinates are reported on the forward strand.
What does the score mean?
It is a transparent rule-of-thumb (0–100) that penalizes GC content outside the usable range, poly-T tracts that terminate Pol III transcription, and long homopolymer runs. It is a triage aid, not a validated efficiency prediction.
Does it check off-target sites?
Yes, on this page, but against a small curated genome set rather than a mammalian one. The off-target section screens a protospacer against four bundled reference genomes (E. coli K-12 MG1655, B. subtilis 168, the human mitochondrion (rCRS) and Mycoplasma hyorhinis, ~9.7 Mbp in total) for protospacer matches carrying a valid PAM on either strand, up to 4 mismatches, and scores each site of a 20 nt SpCas9 guide so the list ranks by how likely that site is to be cut rather than by mismatch count. It is not a human, mouse or other large nuclear-genome search, and it finds substitutions only, not DNA/RNA bulges, so a guide bound for a mammalian experiment still needs a genome-aware specificity tool before you order it.