Fig. 4: Cas9^D10A-mediated DNA damage promotes replication stress, hyperactivation of the ATR- mediated DNA damage repair pathway and G₂/M cell cycle arrest.

A–D Flow cytometric cell cycle analysis of SK-N-BE(2)C cells expressing LINE-1, MYCN or AAVS1 targeting sgRNA at 1-, 2-, and 3 days post-treatment with Cas9^D10A mRNA (30 nM). LINE-1 and MYCN targeted cells display an extension of S-phase at 1 day post-treatment and eventual arrest in G₂/M at days 2 and 3 (n = 3 biological replicates). AAVS1 targeted cells demonstrate no apparent alteration in cell cycle progression. Data in panels (B–D) are presented as mean ± s.d. E Western blot of DNA damage markers from SK-N-BE(2)C cells expressing LINE-1, MYCN or AAVS1 sgRNA at 3 days post-treatment with Cas9^D10A. LINE-1 and MYCN targeted cells demonstrate a substantial elevation in DNA damage markers. F, G Cell viability assessment for SK-N-BE(2)C, or SK-N-BE(2)C-RPA(123) cells expressing (F) LINE-1 or (G) MYCN targeting sgRNA treated with Cas9^D10A, Cas9^D10A + siRNA, or siRNA only. Cells treated with both Cas9^D10A and siRNA were pre-incubated with siRNA for 24 h prior to the delivery of Cas9^D10A – mRNA (30 nM). For each condition, cell viability was assessed at 3 days post-treatment (n = 3 biological replicates). Data are presented as mean ± s.d. normalized relative to viability of cells expressing AAVS1 targeting sgRNA treated with Cas9^D10A. Data were analyzed multiple unpaired t-tests (two-tailed); ns, P > 0.05; *, P ≤ 0.05; ** P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Source data are provided as a Source Data file.