Guide RNA (gRNA) Analyzer
Analyze a CRISPR guide spacer: length, GC content, TTTT terminators, G runs, a 5′ G and seed-region GC, with the guide RNA and its target-strand sequence.
Formula
- the 17 to 24 nt of the guide that pairs with the target, not including the PAM
- the PAM-proximal part of the spacer, where mismatches are least tolerated
How it works
A guide's spacer determines where the nuclease cuts. Some features of the sequence are known to affect how a guide performs in cells: extreme GC content, a run of four or more T (which ends transcription from U6 and other Pol III promoters), and runs of G, which can fold. A G at the start helps transcription from the U6 promoter.
The page checks each feature against the commonly quoted range and shows the guide as RNA and the target-strand sequence it pairs with. These are sequence features, not an activity prediction: how well a guide cuts depends on its locus and cellular context, and dedicated models trained on large datasets do better than rules of thumb.
Worked example
The spacer GACGTTAAGCTTCAGGATCC.
- Length 20 nt; 10 of 20 bases are G or C, so GC = 50%.
- No TTTT; the longest G run is 2; it starts with G.
- The 10 bases next to the PAM are 50% GC.
All features are within the usual guidelines. As RNA the guide is GACGUUAAGCUUCAGGAUCC.
These are the values the calculator opens with, so you can check its output against this example.
Assumptions
- SpCas9-style guides of about 20 nt.
- The guide is transcribed from a Pol III promoter, which is what the TTTT check assumes; a synthetic guide is not affected by it.
- Guidelines come from published guide-design studies and vary between them.
Common mistakes
- Choosing a guide on these features alone without checking off-target sites in the genome.
- Forgetting that a synthetic guide does not need the 5′ G, while one expressed from U6 does.
- Reading a pass on every feature as a prediction of high activity.