Cas12m2 Protein Purification Protocol

The Cas12m2 protein for structural analysis was expressed and purified using the protocol reported previously^{14 (link),20 (link)}. Briefly, the N-terminally His₆-tagged Cas12m2 protein was expressed in E. coli Rosetta 2 (DE3). E. coli cells were cultured at 37 °C until the OD₆₀₀ reached 0.8, and protein expression was then induced by the addition of 0.1 mM isopropyl β-d-thiogalactopyranoside (Nacalai Tesque). E. coli cells were further cultured at 20 °C overnight and collected by centrifugation. The cells were then resuspended in buffer A (20 mM HEPES–NaOH, pH 7.6, 20 mM imidazole and 1 M NaCl), lysed by sonication and centrifuged. The supernatant was mixed with 3 ml Ni-NTA Superflow resin (Qiagen), and the mixture was loaded into an Econo-Column (Bio-Rad). The protein was eluted with buffer B (20 mM HEPES–NaOH, pH 7.6, 0.3 M imidazole, 0.3 M NaCl) and then loaded onto a 5-ml HiTrap SP HP column (GE Healthcare) equilibrated with buffer C (20 mM HEPES–NaOH, pH 7.6, and 0.3 M NaCl). The protein was eluted with a linear gradient of 0.3–2 M NaCl and further purified by chromatography on a Superdex 200 column (GE Healthcare) equilibrated in buffer D (20 mM HEPES–NaOH, pH 7.6, 0.5 M NaCl). The purified proteins were stored at −80 °C until use. The crRNA was transcribed in vitro with T7 RNA polymerase and purified by 10% denaturing (7 M urea) polyacrylamide gel electrophoresis.