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

Abstract

Focal gene amplification serves as an oncogenic driver during tumorigenesis and is a hallmark of many forms of cancer. Oncogene amplifications promote genomic instability, which is integral to cancer cell survival and evolution. However, focal gene amplification potentially affords an opportunity for therapeutic exploitation. As a proof-of-concept, we leverage CRISPR-Cas9 nickase to selectively promote cancer cell death in MYCN-amplified neuroblastoma in a gene amplification-dependent manner. Our analysis demonstrates that CRISPR-Cas9 nickase can generate a lethal number of highly toxic, replication-dependent double-strand breaks in cells harboring amplified loci. Furthermore, we demonstrate that Cas9 nickase-mediated toxicity can be modulated in combination with small molecule inhibitors targeting key regulators of the DNA-damage response or cell death pathways. Importantly, our findings in MYCN-amplified neuroblastoma translate to other cancer types with distinct oncogene amplifications.

Fig. 1. Generating lethal DNA damage in a genome amplification dependent manner.

A Top pathway: Transient single-strand breaks (SSBs) are largely innocuous and quickly repaired in non-dividing cells. Bottom pathway: Persistent SSBs at high copy number loci within the genome in proliferating cells can induce cellular toxicity through their conversion into single-ended double-strand breaks (seDSBs) during DNA replication. Created in BioRender. Wolfe, S. (2025) https://BioRender.com/ttl9vxt. B Average MYCN genome copy number across various neuroblastoma cell lines obtained by qPCR (n = ≥ 3 biological replicates). 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. C A schematic of piggyBac-integrated sgRNA expression cassettes used to generate stable sgRNA-expressing cell lines. D A schematic of in vitro transcribed Cas9 nickase – mRNA construct variants. Created in BioRender. Wolfe, S. (2025) https://BioRender.com/tgt62po. Source data are provided as a Source Data file.

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.

Fig. 3. Cas9^D10A generates double-strand breaks at highly amplified loci in MYCN-amplified neuroblastoma cell lines.

Cumulative DNA damage (alkaline) and DSBs (neutral) quantified by single cell gel electrophoresis (comet assay). Alkaline conditions (pH > 13) relax the supercoiled structure DNA allowing both SSBs and DSBs to be detected. Neutral conditions restrict detection to fragmented DNA from DSBs. A–C SK-N-BE(2)C, NGP, or SH-SY5Y cells expressing LINE-1, MYCN or AAVS1 targeting sgRNA were electroporated with Cas9^D10A-mRNA (30 nM). Individual cells were assessed for DNA damage at 3 days post-treatment by alkaline and neutral comet assay (n = 150). Representative images of Cas9^D10A-treated cell comets are shown below each graph, respectively. Data are presented as individual data points around the median (black line) and were analyzed using a one-way ANOVA; ns, P > 0.05; *, P ≤ 0.05; ** P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Significance was determined using untreated cells as a baseline control. Source data are provided as a Source Data file.

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.

Fig. 5. Cellular toxicity of Cas9^D10A in MYCN-amplified neuroblastoma cells is augmented by CHK1 inhibitors or multiplex targeting.

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.

Fig. 6. Cas9^D10A demonstrates efficacy in ERBB2 (HER2)-amplified breast cancer, MYC-amplified non-small cell lung cancer, and MYC-amplified colorectal cancer cells.

A, B qPCR analysis of gene copy number variance in (A) ERBB2 (HER2)-amplified breast cancer cell line, BT-474 and (B) MYC-amplified non-small-cell lung cancer and MYC-amplified colorectal cancer cell lines, NCI-H2170 and NCI-H716, respectively (n = 3 biological replicates with technical duplicates). Data are presented normalized to non-amplified human foreskin fibroblast (HFF) 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. C, D Targeting of (C) LINE-1 or (D) ERBB2 (HER2) locus in BT-474 or MYC locus in NCI-H716 or NCI-H2170 cells demonstrates a cytotoxic effect similar to that observed when targeting the MYCN locus in MYCN-amplified neuroblastoma cells. All cells were assessed for changes in cell viability at 3 days post-treatment with Cas9^D10A-mRNA (n = 3 biological replicates). Data are 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.