Terrific broth medium

Cultivation of E. coli was done in LB medium (BD) or Terrific Broth medium (Fisher Scientific) at 37 °C. Whenever applicable, media were supplemented with 100 μg/ml ampicilin or 34 μg/ml chloramphenicol to ensure plasmid maintenance.
All in vivo experiments were performed in S. aureus RN4220 (Kreiswirth et al., 1983 (link)). Cultivation of S. aureus RN4220 was done in TSB medium (BD) at 37 °C. Whenever applicable, media were supplemented with chloramphenicol or erythromycin at 10 μg/ml to ensure plasmid maintenance. When appropriate, anhydrotetracycline (aTc) was used at a concentration of 0.25 μg/ml (unless otherwise indicated) to initiate transcription from the P_tet promoter.

pAS1 was transformed into E. coli BL21 (DE3) Rosetta 2 cells (Merck Millipore), grown in Terrific Broth medium (Fisher Scientific) containing 100 μg/mL ampicillin and 34 μg/mL chloramphenicol at 37 °C until A₆₀₀ reached 0.6. Cells were harvested and resuspended in a lysis buffer (50 mM Tris-HCl pH 7.5, 350 mM NaCl, 10 mM imidazole, 1 mM β-mercaptoethanol, 0.1% Triton X-100) after induction with 0.5 mM IPTG overnight at 16 °C. The lysate was sonicated and the supernatant was bound to Ni-NTA agarose (Qiagen), followed by wash flow using the lysis buffer containing 50 mM, 75 mM, and 100 mM imidazole in a stepwise manner. Cas10-Csm complexes loaded with mature crRNA were finally eluted from the Ni-NTA column with lysis buffer containing 250 mM imidazole and subsequently purified on a 1-mL Resource Q column (GE Healthcare). The peak fraction from the column was further purified by size exclusion chromatography using Superdex 200 10/300 GL (GE Healthcare) in a storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol). The mutant (Csm3^D32A), Sfp-tagged and SNAP-tagged protein complexes were purified using the same procedure.