I've read about on-target efficiency and off-target effects in use of CRISPR/Cas9, and about tools that suggest good guide sequences. I am wondering: how many guide sequences do typical CRISPR users try -- i.e. is there any iteration or parallelism involved?
I think the answer is yes for some use cases, but I am not sure:
- Knocking out one particular locus: There are only a few possible guide sequences, so you can just make a library that includes all of those and run one experiment, without iteration. You would not know which guide sequence worked best, though, but users may not need this information.
- Knocking out one gene, no matter at which loci: Since there are many possible loci for indels, the key is to choose a locus for which the guide sequence is effective and produces the last off-target effects elsewhere in the genome. This would mean iterating/trying several of the top suggested guide sequences individually. These could be tried in parallel across several experiments.
- Knocking out multiple genes, aka multiplexing: The user would want to find good guide sequences for each gene separately to avoid any interactions. This would reduce to the previous example above.
Is my understanding above correct?