Genome Editing and Cell Viability Assay

Approximately 400,000 HEK293T cells were nucleofected with 400 ng SpCas9 plasmid, 40 ng GAPDH-targeting gRNA plasmid and 40 pmol single-stranded oligodeoxynucleotide using a SF cell line 4D-Nucleofector X kit (Lonza) following the pulse program DS-150. For RNP-based genome editing, 10 pmol Cas9 and 12 pmol gRNA were mixed and incubated for 5 min. Next, 20 pmol single-stranded oligodeoxynucleotide was added. The cells were nucleofected with the resulting mixture using the same pulse program. The cells were then transferred to a 96-well plate at a density of 35,000 cells per well and incubated with the indicated amount of compounds for 24 h. Cell viability was measured using PrestoBlue reagent (Thermo) with a SpectraMax M5 reader operated by SoftMax Pro 7.0 (Molecular Devices) at excitation and emission wavelengths of 544 and 590 nm, respectively. Luminescence measurements were then performed using a Nano-Glo HiBiT lytic detection system (Promega) according to the manufacturer’s protocol with an EnVision multilabel plate reader (PerkinElmer) at an integration time of 0.5 s per well. The resulting luminescence signals were normalized based on the cell viability^{34 (link)}.