CRISPR Guide Design
CRISPR genome editing uses a nuclease such as Cas9 that is steered to a place in the DNA by a short guide RNA. The guide is about 20 letters long and matches the protospacer in the DNA it should cut. Designing an experiment mostly means choosing a good guide.
NGG immediately after the 20 letters (N is any base). Guides can therefore only be designed where a PAM exists.Where it cuts
SpCas9 makes a blunt double-strand break about 3 bp before the PAM, between the 17th and 18th base of the guide. Cells repair the break, usually imprecisely, and small insertions or deletions that shift the reading frame can knock a gene out. Cas12a instead cuts further from its PAM and leaves staggered ends.
Nucleases and their PAMs
| Nuclease | PAM | Guide | Notes |
|---|---|---|---|
| SpCas9 | NGG (after the guide) | 20 nt | The most widely used |
| SpCas9-NG | NG (after the guide) | 20 nt | Engineered to accept a shorter PAM, so more sites are available |
| SaCas9 | NNGRRT (after the guide) | 21 nt | Small enough to fit in AAV vectors |
| Cas12a (Cpf1) | TTTV (before the guide) | 23 nt | Staggered cuts |
What makes a good guide
Guides with moderate GC content (roughly 40–70%) tend to work best. A run of four Ts (TTTT) ends transcription from the U6 promoter that is often used to express the guide, long runs of one base (especially GGGG) are best avoided, and a guide that folds back on itself works poorly. The most important design check is off-targets: places other than the intended one that differ by only a few letters and may also be cut.
How Aaron's Archive does it
| Item | What the tool does |
|---|---|
| Search | Scans both strands for every valid PAM and lists the guide next to it, with the position of the cut. |
| Score | Starts at 100 and loses points for GC content outside 40–70%, TTTT, GGGG, long single-base runs, guides that can fold on themselves, and exact or near copies elsewhere in your sequence. |
| Off-targets | Every other site in the pasted sequence that differs by 3 or fewer letters is found exactly, not approximately. |
| Limits | Plain A/C/G/T DNA, up to 500,000 bp. A region of interest can be selected. |
What it cannot tell you
The score is a design checklist, not a prediction of cutting efficiency; measured models such as Rule Set 2 or CRISPRscan are not used. And off-targets are only searched in the sequence you pasted, not in a whole genome. Before ordering, check candidate guides against the genome of your organism with a genome-wide tool such as CRISPOR or Cas-OFFinder.
Common questions about CRISPR guides
What is a PAM?
A short sequence next to the target that the nuclease must see before it cuts. SpCas9 needs NGG, SpCas9-NG accepts NG, SaCas9 needs NNGRRT, and Cas12a needs TTTV in front of the target.
How long is a guide?
20 bases for SpCas9 and SpCas9-NG, 21 for SaCas9 and 23 for Cas12a, as used in the tool here.
Where does Cas9 cut?
About three bases from the PAM, inside the target. Cas12a sits the other way round: its PAM is before the guide, and it makes a staggered cut.
Do I still need to check off-targets elsewhere in the genome?
Yes. The tool only counts near-identical sites inside the sequence you paste. Check shortlisted guides against your organism's genome before ordering.
Where can I find guides?
Use the CRISPR guide RNA finder.