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 that 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 ↗