Figure 3.

Auxotrophic screening and target efficiency in B. thailandensis. (A) Auxotrophic CAST mutants with the integrated resistance marker are spotted on LB-agar plate plus antibiotic. Subsequently, mutants are passed to M9 agar with and without the cognate amino acid. Selection plates for B. thailandensistrpA-1 target that results in tryptophan auxotrophy. M9 minimal media plates with 0.2% glucose (Glc) with or without 0.8 mM tryptophan (l-Trp). (B) Map of sgRNA targets (arrows), PAM site (diamonds), and insertion direction (arrow point). (C) Relative frequency table of on-target gene disruption results with auxotrophy complementation (n = mutants collected) and genotype (n = 24).