Analysis of protein expression levels by western blot

Proteins were isolated 48 h after transfection. Cells were resuspended in lysis buffer (Tris-HCl 50 mM, pH 7, NaCl 150 mM or KCl 300 mM, Triton-X-100 1%, SDS O 1%, EDTA 1 mM, DTT 1 mM, aprotinin 1 μg/μl, pepstatin 10 μg/μl, and leupeptin 1 μg/μl), centrifuged at 15,000 × g for 15 min at 4 °C, and supernatants were used. We observed that lysis with buffer containing 300 mM KCl, which is known to favor the release of chromatin-bound proteins, allowed better extraction of Cas9–HE and derivatives compared to lysis buffer with 150 mM NaCl. Western blots were performed by standard Tris-glycine SDS-PAGE followed by transfer to nitrocellulose membranes. Following blocking with 5% BSA in TBS-T (Tris 24 mM, NaCl 137 mM, KCL 2.68 mM, and Tween-20 0.1%), membranes were probed with anti-Cas9 antibody (Novus Biologicals, NBP2-36440SS) at 1 μg/ml and anti-tubulin antibody (Sigma, T6074200UL) at 0.1 μg/ml, and visualized by chemiluminescence with a GBox (Syngene) or exposure to the X-ray film. Uncropped scans are provided as Supplementary Fig. ⁸.