CRISPR gRNA Designer
Free CRISPR guide RNA design tool. Finds every PAM site for SpCas9, SaCas9 and Cas12a, ranks candidates and outputs cloning oligos.
How it works
The target sequence is scanned on both strands for protospacer adjacent motifs — NGG for SpCas9, NNGRRT for SaCas9, TTTV for Cas12a. Candidate guides are scored by a heuristic combining GC content, homopolymer runs, poly-T termination signals that would truncate transcription, and seed-region composition. This is a screening aid informed by the published literature, not an implementation of Doench 2016 Rule Set 2, and it performs no genome-wide off-target search — validate shortlisted guides in a dedicated off-target tool before ordering. Sequences are retrieved live from NCBI RefSeq; when NCBI cannot be reached the tool reports that plainly rather than substituting a placeholder sequence.
Frequently asked questions
What makes a good CRISPR guide RNA?
An effective guide typically has 40–65% GC content, no run of four or more thymines (which acts as a Pol III termination signal), and no long homopolymer stretches. Position matters as much as composition: guides targeting early constitutive exons are more likely to produce a functional knockout, and the seed region — the roughly ten bases adjacent to the PAM — dominates both on-target activity and off-target risk.
What PAM sequence does Cas9 require?
Streptococcus pyogenes Cas9 (SpCas9) requires an NGG protospacer adjacent motif immediately 3′ of the 20-nucleotide protospacer, where N is any base. Staphylococcus aureus Cas9 requires NNGRRT and is smaller, which makes it easier to package in AAV. Cas12a (formerly Cpf1) instead recognises a 5′ TTTV motif and produces staggered rather than blunt cuts.