U2OS-Cas9 and U2OS-Cas9-NLS cell line generation

Lentivector expressing Cas9 without nuclear localization sequence was generated by subcloning Cas9 without NLS from lenti-Cas9-blast (26), using primers described in Supplementary Table 1 , into pLenti CMV/TO Puro (27). Restriction cloning was performed using BamHI/XbaI restriction enzymes. Lentivirus was generated by transfecting use Lenti-X 293T cell line (Takarabio) by transfecting cells with pCMV-TO Puro-Cas9 or pLenti-Cas9-Blast (contains NLS), pSPAX2, and pMD.2 (3:2:1 ratio) using Lipofectamine 2000 (2 µL/1ug DNA). Supernatants were collected approximately 48 and 72h hours after transfection and passed through a 0.45-μm filter and added to cells. Appropriate antibiotic cell selection was performed to isolate stably transduced cell lines. Clonal selection of U2OS-Cas9 cells was performed by limiting dilution and immunoblot analyses was performed to identify clones with the highest level of Cas9 expression (refer to Supplementary Figure 1 ). U2OS-Cas9 cells were maintained under puromycin selection (1 μg/mL).