DNA Damage (Comet) Assays.

Alkaline (pH 13, detects primarily single-stranded breaks) single-cell gel electrophoresis (Comet) assays were performed according to the manufacturer’s protocol (CometAssay Kit, cat. no. 4250-050-K; Trevigen, Gaithersburg, MD) and as previously described by our laboratory (Vlasova et al., 2011; Kanagasabai et al., 2017, 2018). Briefly, K562, K/VP.5, and K/VP.5/edit-3 cells were washed and resuspended in buffer (25 mM HEPES, 10 mM glucose, 1 mM MgCl₂, 5 mM KCl, 130 mM NaCl, 5 mM NaH₂PO₄, pH 7.4). Cells were subsequently incubated with 2 or 10 μM etoposide or DMSO (solvent control) for 30 minutes at 37°C. The treated cells were washed with ice-cold buffer and resuspended to 0.28 × 10⁶ cells per milliliter and then further diluted in low melt agarose. After alkaline electrophoresis (of ∼2000 cells) and subsequent staining with a fluorescent DNA intercalating dye, SYBR Gold, the migrating fragments (comet tail) from the nucleoid (comet head) were visualized and the images captured by fluorescence microscopy. The Olive tail moment (Olive, 2002) was quantified by the ImageJ processing program with the open-source software tool OpenComet (Gyori et al., 2014; www.cometbio.org). Olive tail moments from more than 100 cells per sample condition were determined.