guide RNA

The guide RNA is the address label that tells Cas9 where to go. Cas9 is the same for every job; what changes is the short RNA sequence it carries, which spells out the target. Swap the guide and you redirect the scissors to a completely different gene — that interchangeability is what makes CRISPR so flexible.

A guide RNA contains a roughly 20-nucleotide stretch (the spacer) that base-pairs with the DNA target by the same A–T, G–C complementarity that holds the double helix together. It also includes a structural scaffold portion that Cas9 grips. The spacer's sequence is chosen by the experimenter, so it can be programmed for almost any site that sits next to a suitable PAM.

In bacteria, two separate RNAs (crRNA and tracrRNA) form the guide. For convenience, lab tools usually fuse them into a single molecule called a single guide RNA. Designing a good guide matters: poorly chosen guides can bind similar but wrong sequences elsewhere, causing off-target edits.