Journal: Nature Communications

Article Title: Selective targeting of genome amplifications and repeat elements by CRISPR-Cas9 nickases to promote cancer cell death

doi: 10.1038/s41467-025-60160-2

Figure Lengend Snippet: A , B SK-N-BE(2)C, NGP, and SH-SY5Y cells expressing ( A ) LINE-1 or ( B ) MYCN targeting sgRNA were treated with Cas9 D10A -mRNA at increasing doses in the absence or presence of a CHK1 inhibitor, MK8776 (500 nM). Co-treatment with CHK1i potentiated Cas9 D10A -mediated cell-killing across cell lines at 3 days post-treatment ( n = 3 biological replicates). Data were analyzed by multiple unpaired t -tests (two-tailed); ns, P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. C Average MYCN and ALK genome copy number in IMR-32 cells determined by qPCR ( n = 4 biological replicates). Data are presented normalized to HFF cells as a baseline control where for each box plot the horizontal line represents the median, the top and bottom of the box represent the upper and lower quartiles, respectively, and the top and bottom whiskers represent the maximum and minimum values, respectively. D Targeting of LINE-1 , MYCN , ALK , or MYCN and ALK in IMR-32 cells (2 × 105) with Cas9 D10A demonstrates a dose-dependent cytotoxic effect. Multiplex targeting of MYCN and ALK increases Cas9 D10A -mediated cell-killing relative to targeting MYCN or ALK individually ( n = 3 biological replicates). Data were analyzed using a two-way ANOVA with a Tukey’s multiple comparison test; ns, P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. E No additional cellular toxicity is observed for multiplex targeting of an amplified ( MYCN or ALK ) locus with a non-amplified ( AAVS1 ) locus in IMR-32 cells with Cas9 D10A -mRNA (15 nM; n = 3 biological replicates). Data were analyzed by multiple unpaired t -tests (two-tailed); ns, P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. F Multiplex targeting of non-amplified loci in SH-SY5Y cells with Cas9 D10A -mRNA (30 nM) resulted in similar rates of cell viability to targeting individual loci. Cells were assessed for changes in cell viability at 3 days post-treatment with Cas9 D10A -mRNA ( n = 3). Data in panels ( A , B , and E , F ) is presented as mean ± s.d. and normalized relative to viability of cells expressing AAVS1 targeting sgRNA treated with Cas9 D10A . Source data are provided as a Source Data file.

Article Snippet: Cell proliferation was monitored at 1-, 2-, and 3 days post-treatment with Cas9 nickase mRNA or by QIBC using the Celigo Image Cytometer (Nexcelcom Bioscience, USA) to measure the cell population following staining cell with Hoecsht 33342 (1 μg/mL; Invitrogen, #H1399) at 37 °C for 30 min. Where applicable, cells were supplemented with various reagents: in the presence of MK8776 (MedChemExpress, #HY-15532), rucaparib (MedChemExpress, #HY-10617A), PDD00017273 (MedChemExpress, # HY-108360), berzosertib (Selleck, #S7102), calpastatin (Millipore Sigma, #208902), alisertib (MedChemExpress, #HY-10971), etoposide (MedChemExpress, #HY-13629R), or topotecan (MedChemExpress, #HY-13768A) at concentrations ≤IC50 to prevent confounding toxicity.

Techniques: Expressing, Two Tailed Test, Control, Multiplex Assay, Comparison, Amplification