nCas9-RT protein purification

The nCas9-RT fragment from pCMV-PE2 was retrieved by BglII digestion and then cloned into the pET30a(+) expression vector (Novagen 69909) in the frame between the NotI and NdeI sites using NEBuilder HiFi DNA Assembly Master Mix (NEB) with bridging gblocks (Supplementary file 1) to encode a version of the protein bearing a C-terminal His6-tag. For protein expression, the plasmid was introduced into Rosetta 2 (pLysS). The cells were grown at 37°C and shaken at 175 rpm. Isopropyl β- d-1-thiogalactopyranoside (IPTG) (0.5 mM) was added at an OD600 of 0.6 and the cells were grown for 16 hr at 18°C. The cells were harvested by centrifugation (5000 × g) for 10 min at 4°C. Harvested cell pellets were washed with phosphate-buffered saline (PBS) and snap-frozen in liquid nitrogen for later purification. Cell pellets were thawed on ice, disrupted in 35 mL lysis buffer (25 mM HEPES-KOH pH 7.6, 1 M KCl, 20 mM imidazole, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 × protease inhibitor cocktail), briefly sonicated, then clarified by centrifugation at 25,000 × g for 30 min. Supernatants were filtered through a 0.22 μm syringe filter before application to 5 mL of nickel resin (Ni-NTA Superflow, QIAGEN) equilibrated in loading buffer (25 mM HEPES-KOH pH 7.6, 150 mM KCl, 20 mM imidazole, 1 mM DTT, and 1 mM PMSF). The resin was washed with 100 mL loading buffer followed by 50 mL wash buffer (25 mM HEPES-KOH, pH 7.6, 150 mM KCl, 40 mM imidazole, 1 mM DTT, and 1 mM PMSF). Protein was eluted in batch six times with 10 mL elution buffer (wash buffer + 500 mM imidazole). The eluted protein was diluted into a low-salt buffer (25 mM HEPES-KOH pH 7.6, 100 mM KCl, 1 mM DTT, and 1 mM PMSF), then loaded onto a 1 mL HiTrap heparin HP column (GE Healthcare) pre-equilibrated in low-salt buffer and eluted with a linear gradient of 100 mM to 1 M KCl over 40 column volumes. Peak fractions were concentrated to 8 mg/mL using a Spin-X UF 20 50 kDa molecular weight cutoff (MWCO) (Corning). Protein concentration was determined by UV absorbance at a wavelength of 280 nm. The final protein purity was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Coomassie staining to be around 90%. The C-terminal His6-tag was not removed prior to the experiments. Detailed protocols can be found on protocols.io (https://doi.org/10.17504/protocols.io.b4yxqxxn).