Human cell culture and transfection. U2OS cells (obtained from Toni Cathomen, Freiburg) and HEK293 cells were cultured at 37 °C with 5% CO2 in Advanced DMEM supplemented with 10% heat-inactivated FBS, 2 mM GlutaMax, and penicillin/streptomycin (all cell culture products were obtained from Life Technologies). Cell line identity was validated by STR profiling (ATCC), and mycoplasma testing was performed twice per month. 2 × 105 cells were transfected using the SE Cell Line Nucleofector Kit with 500 ng Cpf1- or SpCas9-encoding plasmid and 250 ng gRNA expression plasmid and the DN-100 program for U2OS cells, or with 300 ng Cpf1- or SpCas9-encoding plasmid and 150 ng gRNA expression plasmid using the CM-137 program for HEK293 cells, on a 4D-Nucleofector according to the manufacturer’s instructions (Lonza). For negative control transfections, Cpf1- or SpCas9-encoding plasmids were co-transfected with a U6-null plasmid that did not encode a crRNA. Genomic DNA extraction was performed approximately 72 h following nucleofection using an Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter).