All spCas9s were expressed and purified as described previously (2). A pET-based expression vector, which consisted of a sequence encoding Cas9 (Cas9 residues 1 to 1368 from S. pyogenes) and an N-terminal His6-tag followed by a peptide sequence containing a tobacco etch virus protease cleavage site, was used. The proteins were expressed in Escherichia coli strain BL21 Rosetta 2 (DE3) (TransGen Biotech) cells that were grown in LB at 37°C for a few hours. When the optical density at 600 nm reached 0.6, protein expression was performed at 18°C for 12 to 16 hours following induction with 0.2 mM isopropyl β-d-1-thiogalactopyranoside. The medium was then discarded, and the cells were harvested. The harvested cells were lysed in 20 mM tris-HCl (pH 8.0), 250 mM NaCl, 5 mM imidazole (pH 8.0), and 1 mM phenylmethylsulfonyl fluoride and passed through a homogenizer three times at ~1000 bar. The lysed dilution was then ultracentrifuged at 13,000g for 30 to 60 min, and the clarified supernatant from the cell lysate was separated from the cellular debris and bound in batch to Ni-NTA Agarose (Qiagen). The resin was washed extensively with 20 mM tris-HCl (pH 8.0), 250 mM NaCl, and 10 mM imidazole (pH 8.0), and the bound protein was eluted in a single step with 20 mM tris-HCl (pH 8.0), 250 mM NaCl, and 250 mM imidazole (pH 8.0). The dialysis of Cas9 in dialysis buffer [20 mM Hepes-KOH (pH 7.5), 150 mM KCl, 10% (v/v) glycerol, 1 mM dithiothreitol (DTT), and 1 mM EDTA] was performed overnight at 4°C. Cas9 was further purified with a HiTrap SP HP Sepharose column (GE Healthcare) and was subject to gel filtration chromatography with a Superdex 200 16/60 column (GE Healthcare) in Cas9 storage buffer [20 mM Hepes-KOH (pH 7.5), 500 mM KCl, and 1 mM DTT], and it was then stored at −80°C. The truncation mutant BLM642–1290 (core-BLM) encompassing the region homologous to the RecQ catalytic core was purified as previously described (36). Phi29 DNAP was purchased from NEB (M0269S).

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