- Which vector system should I use for my experiment?
- Which marker should I put in my vector?
- How is vector name assigned?
- How is the species reflected in the vector name?
- Should I use IRES or 2A in my polycistronic expression cassette?
- Why is the yield of my plasmid prep so low?
- How to use PB transposon system in vitro and in vivo?
How is gRNA specificity score calculated?
VectorBuilder follows the algorithm utilized in CRISPR library design (CLD) to calculate specificity scores for gRNAs. Briefly, for a given gRNA intended to target a N(20)NGG sequence in a species, we search for all potential off-target sites in the genome of that species that have ≤3 mismatches with the target sequence. For each potential off-target site identified this way, a single off-target score is calculated. Scores for all the off-target sites are then used in aggregate to calculate the final specificity score of the gRNA, which is between 0 and 100, with higher values indicating greater targeting specificity.
Please note that specificity scores are only a rough guide. Actual targeting efficiency and specificity could depart from what the scores predict. gRNAs with low scores may still work well.
