SeqBench

CRISPR Guide RNA Designer & PAM Finder

Scan a sequence for SpCas9, SaCas9 or Cas12a guide candidates with PAMs and scoring.

🌐 Nothing you paste is logged or stored — every tool is also callable via REST & MCP, and in bulk from the batch tools

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 the tool 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.

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Paste a target sequence to find guide candidates.

On-target efficiency models. Doench ’14(Rule Set 1) and CRISPRscan are published regression models, shown side-by-side with the heuristic Score. Both apply only to SpCas9 with an NGG PAM and a 20 nt spacer, and both need the guide’s flanking genomic context (Doench: a 30-mer = 4 nt 5′ + spacer + PAM + 3 nt 3′; CRISPRscan: a 35-mer = 6 nt 5′ + spacer + PAM + 6 nt 3′). Guides too close to the ends of the pasted sequence, or any non-SpCas9 nuclease, show “—” rather than a padded (and silently wrong) value. Doench is a logistic score in 0–1 (shown as %); CRISPRscan is CRISPOR’s integer ~0–100. Expand a Doench cell to see its GC term and top position contributions. These are legacy/complementary heuristics (Spearman ~0.3–0.5 vs. measured activity) — Doench RS1 is superseded by Rule Set 2/Azimuth, and CRISPRscan is tuned for T7-transcribed sgRNA in zebrafish. Refs: Doench et al. 2014,Nat Biotechnol (PMID 25184501); Moreno-Mateos et al. 2015,Nat Methods (PMID 26322839). Weights via the open-source CRISPOR.
Scope: the score is a transparent rule-of-thumb (GC content, poly-T terminators, homopolymer runs) to help you triage candidates — it is not a validated on-target efficiency prediction like Doench 2016. Off-target risk is not assessed: that requires aligning each guide against your target genome. Always verify shortlisted guides with a genome-aware specificity tool before ordering.

Working on a whole construct rather than one guide search? Open SeqStudio — the full editor, with live feature annotation, plasmid maps, restriction and primer panels, undo/redo, multi-document tabs and GenBank / SnapGene import and export.

How to use the CRISPR gRNA Designer tool

  1. 1Paste the target DNA region (raw or FASTA).
  2. 2Pick the nuclease / PAM and whether to search both strands.
  3. 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 penalises 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 on-target efficiency model like Doench 2016.

Does it check off-target sites?

No. Off-target assessment requires aligning each guide against your target genome, which an in-browser tool can't do. Use this to shortlist candidates, then verify them in a genome-aware specificity tool before ordering.

More

Guides

References

CRISPR PAM table

PAM sequences for common Cas nucleases and guide design.

CRISPR gRNA design rules

Spacer length, GC, poly-T and seed-region rules for guide RNA design.

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