Краткий ответ
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
Where the analogy breaks downGuides are often described as 'GPS coordinates'. GPS is exact; a guide is a similarity search. It will sometimes stop at an address that merely resembles the one you asked for, which is precisely what off-target editing is.
Sources
- National Human Genome Research Institute
Talking Glossary of Genomic Terms ↗