CRISPR guide finder
Finds every 20 nt SpCas9 protospacer with an NGG PAM, on both strands.
Finds every 20 nt SpCas9 protospacer with an NGG PAM, on both strands.
SpCas9 cuts wherever the 20 nucleotides of its guide RNA match the DNA and the three bases immediately after the match read NGG, the protospacer adjacent motif. Finding guides is therefore a search for NGG: every NGG with at least 20 bases in front of it gives one candidate.
The guide RNA itself is the 20 nt protospacer with U in place of T, joined to the constant scaffold; the PAM is in the DNA only and is never ordered as part of the oligo. Both strands carry PAMs, so the reverse complement is searched as well and its guides are reported at their position on the plus strand of what you pasted.
"Try an example" loads the 720 nt coding sequence of EGFP and finds 119 guides,47 on the plus strand and 72 on the minus strand. The first isAAGGGCGAGGAGCTGTTCAC at 10 to 29 with the PAM CGG, GC 60 %, cutting after base 26. GC across the set runs from 45 % to 80 % and no guide carries a warning, which is what a GC-rich coding sequence looks like. Turn "Search both strands" off and 47 are left.
| Flag | What it means | What to do |
|---|---|---|
| TTTT | A run of four or more T inside the protospacer | Avoid for a U6 or H1 expressed guide; fine as synthetic RNA |
| GC low | Under 30 % GC | Expect weak binding and low activity |
| GC high | Over 80 % GC | Expect more off-target cutting |
| Repeated | The same 20 nt occurs elsewhere in the input | It will cut in more than one place, so pick another |
To check a guide or a repair oligo for self-structure and dimers, use theprimer dimer and hairpin checker. To search the target for any other motif, including a different PAM written in IUPAC codes, usepattern find. To design the primers that amplify the region for sequencing, use the Tm calculator andPCR product size. To see where a cut falls in the reading frame, use DNA to protein translation.
The protospacer adjacent motif is a short sequence in the DNA, immediately 3-prime of the target, that the Cas9 protein itself recognises. Streptococcus pyogenes Cas9 needs NGG. Without it the protein does not unwind the duplex, so no guide can be designed for a site with no NGG next to it. The PAM is in the DNA only, never in the guide RNA.
Three base pairs from the PAM, between the 17th and 18th base of the 20 nt protospacer, and on both strands at the same place, leaving a blunt double-strand break. The tool gives that position on the plus strand for every guide, which is where an insertion or a deletion will be centred.
Between about 40 and 70 % works well for most guides. Below 30 % the guide binds weakly and tends to be inactive; above 80 % it binds too well and off-target cutting rises. Both extremes are flagged here. GC alone is a weak predictor, so treat it as a filter that removes bad guides rather than a score that finds good ones.
A guide expressed from a U6 or H1 promoter is made by RNA polymerase III, which stops at a run of four or more T. A TTTT inside the protospacer truncates the transcript, so the guide is never made at full length. Guides delivered as synthetic RNA or as ribonucleoprotein are not affected, but the flag is worth heeding for any plasmid or lentiviral construct.
Only inside the sequence you paste: a guide whose 20 nt protospacer occurs more than once in the input is flagged as repeated. Real off-target prediction needs the whole genome and a mismatch model, so run the shortlist from here through a genome-aware tool such as CRISPOR or Cas-OFFinder before ordering anything.