Mammalian cell culture

All cells were cultured and maintained at 37 °C with 5% CO₂. Antibiotics were not used for cell culture. HEK293T cells (ATCC CRL-3216) and HeLa cells (ATCC CCL-2) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) plus GlutaMax (ThermoFisher) supplemented with 10% (v/v) fetal bovine serum (FBS). K562 cells (ATCC CCL-243) were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium plus GlutaMax (ThermoFisher) supplemented with 10% (v/v) FBS. U2OS cells were cultured in MyCoy’s 5A Medium plus GlutaMax (ThermoFisher) supplemented with 10 % (v/v) FBS.

hiPS cells (human episomal iPS cell line; A18945; ThermoFisher) were cultured in Essential 8 Flex Medium (ThermoFisher) supplemented with RevitaCell after passaging (ThermoFisher) according to the manufacturer’s directions. Versene (Thermo Fisher) was used for cell passaging and dissociation. Prior to nucleofection, cells were harvested with Accutase (ThermoFisher).

For data shown in Fig. 5i, j, nuclease expression plasmids were constructed whereby the Cas-enzyme construct (Cas9 or RDN(A89E)) was proceeded by P2A-GFP to enable isolation of transfected cells. iPS cells were flow sorted at the MIT FACS core 3–5 days after nucleofection and genomic DNA was isolated directly after sorting.