Off-target scoring
Off-target specificity comes from scanning each guide against an off-target index. The engine is
auto-detected per -i index (crispr-ots or Guidescan2), so passing one of each gives a
specificity_<index> column from each (0-1, where 1.0 = no off-targets).
crispr-ots
Built by index_genome. Rust scanner, exact mismatches only (no
bulges), ~6x faster than Guidescan2. Emits a CFD-style aggregate specificity, with bundled matrices
for SpCas9 (CFD) and Cas12a (enCas12a and 2xNLS-Cas12a). The Cas12a CFD matrices are from
DeWeirdt et al. 2021
(code); SpCas9 CFD from
Doench et al. 2016.
Guidescan2
A Guidescan2 index (built with Guidescan2’s own tooling, or a prebuilt download), which score_guides
auto-detects and scores. Trie-based; supports RNA/DNA bulges
(score_guides --rna_bulges / --dna_bulges) in addition to mismatches, at higher runtime/memory.
Schmidt et al. 2023. Prebuilt indices
for some genomes are available at https://guidescan.com/downloads.
Controls (in score_guides)
--mismatches N– search radius.--threshold N– discard a guide if any off-target sits within N mismatches (-1keeps all).--rna_bulges/--dna_bulges– guidescan2 indices only (crispr-ots requires 0).--alt_pams NAG ...– additional PAMs to consider as off-targets.