Fig. 2: Cas9^D10A eliminates neuroblastoma cells in a dose dependent manner.

A Positive control for Cas9^D10A nickase – mediated cell-killing. Long interspersed nuclear element 1 (LINE-1) is a widespread endogenous transposable element where there are >5000 canonical LINE-1 repeats in the reference human genome. Dose dependent nickase toxicity is observed in all cell lines expressing LINE-1 targeting sgRNA (sgLINE-1) at 3 days post-treatment (n = 3 biological replicates). BMYCN-amplified neuroblastoma cells expressing MYCN targeting sgRNA (sgMYCN-1) are rapidly depleted in a dose-dependent manner whereas MYCN non-amplified (SH-SY5Y) and non-neuroblastoma (HEK293T) cells expressing sgMYCN-1 are not appreciably affected at 3 days post-treatment (n = 3 biological replicates). Focal MYCN amplifications present as a homogeneously staining region (HSR) or ecDNA produce toxicity when targeted by Cas9 nickase. Data in panels (A and B are presented as mean ± s.d. and normalized relative to viability of cells expressing AAVS1 targeting sgRNA treated with Cas9^D10A. C MYCN copy number variance determined by real-time qPCR demonstrates a reduction in MYCN copy number 3 days following treatment with Cas9^D10A-mRNA (30 nM) targeting MYCN in MYCN-amplified cell lines but no change in MYCN non-amplified SH-SY5Y cells (n = 3 biological replicates with technical duplicates). D Western blot of N-MYC protein expression levels from SK-N-BE(2)C or NGP cells expressing MYCN or AAVS1 sgRNA at 24 h post-treatment with Cas9^D10A. E, F MYCN expression analysis by qRT-PCR demonstrates a modest reduction in MYCN expression in SK-N-BE(2)C or NGP MYCN-amplified neuroblastoma cells up to 72-h post-treatment with Cas9^D10A-mRNA (30 nM) when targeting LINE-1 or MYCN (n = 4 biological replicates with technical duplicates). G, H MCM7 expression analysis by qRT-PCR demonstrates an increase in MCM7 expression in SK-N-BE(2)C or NGP MYCN-amplified neuroblastoma cells up to 72 h post-treatment with Cas9^D10A-mRNA (30 nM) when targeting LINE-1 or MYCN (n = 4 biological replicates with technical duplicates). Data are presented as individual data points around the mean ± s.d. and normalized to untreated cells as a baseline control. Data analyzed by multiple unpaired t-tests (two-tailed); ns, P > 0.05; *, P ≤ 0.05; ** P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001 relative to untreated cells. Source data are provided as a Source Data file.