A resposta resumida
The guide RNA is the part that decides where the editing happens. About twenty of its letters match the DNA you want to reach; the rest is structure that grips the Cas protein. Change those twenty letters and you have aimed the whole system somewhere else — that is the entire reason CRISPR is easy to use.
A single guide RNA fuses the CRISPR RNA and trans-activating CRISPR RNA into one molecule. Its 5' spacer, typically 20 nucleotides, base-pairs with the target protospacer; the 3' scaffold binds Cas9. Guide design must balance on-target activity against off-target potential, accounting for PAM availability, chromatin accessibility, GC content and the genome-wide distribution of similar sequences.
Why guide design is not trivial
Try it: find a target the way a scientist would
Editing is not "point at a gene". The enzyme can only cut where a short tag sits beside the target, so the first job is finding legal positions. Paste any DNA sequence — or use the example — and this will scan both strands for you.
This is a teaching model of the first step only. A real design run also searches the whole genome for near-matches that could be cut by mistake, scores predicted efficiency, and is then confirmed by sequencing in cells — none of which can be done from a short sequence alone. The example is an illustrative sequence, not a real genomic locus.
Sources
- National Human Genome Research Institute
Talking Glossary of Genomic Terms ↗