Recombinant LbCas12a Protein Production

The LbCas12a recombinant proteins were produced as previously described [5 (link)]. Briefly, DNA sequences encoding the C′-terminal 6 × His-tagged LbCas12a proteins were cloned into pET28a vector. Transformed E. coli BL21 (DE3) cells (EMD Millipore) were grown in TB medium with 50 μg/mL Kanamycin until OD₆₀₀ reaches 0.6–0.8. Cells were chilled at 4 °C for 30 min, and the protein expression was induced using 1 mM IPTG for 12–16 h at 4 °C. Cells were harvested by centrifugation (4000 × g, 20 min, 4 °C) and resuspended in the lysis buffer (20 mM NaPO₄, pH 6.8, 0.5 M NaCl, 15 mM imidazole, 10 mM CaCl₂, and 10% glycerol) supplemented with protease inhibitor cocktail (Sigma: 11,873,580,001), DNaseI and lysozyme. The resuspended cells were lysed by passing through Avestin Emulsiflex C3 three times at 15,000 psi, 4 °C. The cell lysate was centrifuged at 14,000 × g for 40 min, and the soluble fraction was sequentially purified by Nickle affinity (HisTrap HP, 5 mL, Cytiva) and cation exchange chromatography (HiTrap Heparin, 5 mL, Cytiva). Purified protein was concentrated (Amicon centrifugal filter, 10 kDa) and dialyzed against storage buffer overnight (20 mM TrisHCl, pH 7.4, 0.3 M NaCl, 0.1 mM EDTA, 50% Glycerol, and 1 mM DTT). The protein concentration was determined by Nanodrop using an extinction coefficient at 167,780 M⁻¹ cm⁻¹.