Combining Multiplexed CRISPR/Cas9-Nickase and PARP Inhibitors Efficiently and Precisely Targets Cancer Cells

Abstract

Triggering cancer cell death by inducing DNA damage is the primary aim of radiotherapy; however, normal cells are also damaged. In this study, we showed that delivery of only four synthetic guide RNAs with Cas9 endonuclease efficiently induced simultaneous DNA double-strand breaks, resulting in efficient cell death in a cell type-specific manner. Off-target effects of Cas9 endonuclease were prevented by using Cas9-nickase to induce DNA single-strand breaks and blocking their repair with PARP inhibitors (PARPi). When recombinant Cas9-nickase protein and multiple synthetic guide RNAs were delivered with PARPis into cultured cells, in vivo xenografts, and patient-derived cancer organoids via lipid nanoparticles, cancer cells were unable to tolerate the induced DNA damage even in the presence of a functional BRCA2 gene. This approach has the potential to expand the use of PARPis with verified safety and thus is a potentially powerful tool for personalized genome-based anticancer therapy.

Significance: Targeting cancer-specific variants with CRISPR/Cas9-nickase induces cancer-specific cell death in combination with DNA repair pathway inhibitors, demonstrating the potential of CRISPR cancer therapy for treating a broad range of cancers.

Figure 1.

RNP-based delivery of multiplexed CRISPR/Cas induces cell death. A, Schematic of cancer-specific cell death induced by multiplexed CRISPR/Cas targeting cancer-specific indel mutations. B, Relative viability of HCT116, HeLa, SW-480, and MDA-MB-231 cells transfected with sgRNAs and Cas9^WT protein (top) or mRNA (bottom). C, Left, schematic of qPCR to investigate relative cleavage of genomic DNA following CRISPR/Cas9-mediated induction of DSBs. Relative cleavage of genomic DNA upon transfection of individual sgRNAs in Hmix-4 (middle) or cotransfection of four sgRNAs (right). The quantitative value was normalized to the nontarget locus corresponding to each target locus. D, Bar plots representing the mutation efficiency at on-target sites and five predicted candidate off-target sites for each sgRNA in Hmix-4. On-target sequences of Hmix-4 are not present in the genome of HeLa cells; therefore, 20-nt sequences adjacent to the PAM sequences according to the genomic coordinates were considered as on-target sequences and analyzed. Cas9^WT protein along with individual (left) or multiplexed (right) sgRNAs were transfected into HCT116 or HeLa cells. The sgRNA targeting the HPRT1 gene was used as a positive control for efficient RNP transfection. Mutation efficiencies were measured by targeted sequencing of each target site of Hmix-4. E, Chromosomal map of the four sgRNAs included in Umix-4. In the sky-blue box, the positions of the sgRNA targeting C4BPB and its neighboring sgRNAs are indicated (left). Relative viability of cells transfected with CRISPR/Cas9^WT targeting C4BPB and neighboring sgRNAs with Umix-3 (right). NT, nontarget control sgRNA. Umix, multiplexed sgRNAs targeting universally conserved human genome sequences. Hmix, multiplexed sgRNAs targeting HCT116 cell–specific sequences. MT-50, a single sgRNA targeting 50 loci in the human genome. Cell viability was measured using the CellTiter-Glo assay at 72 hours after transfection and normalized to that observed following treatment with a nontarget sgRNA control. Error bars, the mean ± SD of three independent biological replicates. In B, D, and E, statistical significance was calculated using an unpaired two-tailed Student t test: n.s., not significant (P ≥ 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 2.

Assessment of in vivo antitumor effects in a mouse xenograft model after delivery of CRISPR-loaded LNPs. A, Schematic illustrating preparation of the mouse xenograft model for treatments and analyses. B, Timelines of treatments and tumor growth curve of HCT116 cell–derived xenografts in mice following administration of CRISPR/Cas9^WT RNP-loaded LNPs, which were delivered every 2 days (eight injections in total). Tumor volume was 80 mm³ at day 0. C, Weights and images of tumors obtained from mice at day 25 after the first injection. Scale bar, 20 mm. D, Body weight changes during the treatment period. In B–D, error bars indicate the mean ± SE of independent biological replicates. Number of mice: n = 7 in the PBS group and n = 5 in the other groups. E, Mutation frequencies at on-target sites and five predicted candidate off-target sites for each sgRNA of Hmix-4 in remaining cancer cells in each mouse at 24 days after injection. Mutation frequencies were measured by targeted sequencing of each target site of Hmix-4. NT, nontarget control sgRNA. Error bars, mean ± SD of three independent biological replicates. All statistical significance was calculated using an unpaired two-tailed Student t test; n.s., not significant (P ≥ 0.05). A, Created in BioRender. Joo, J. (2025) https://BioRender.com/td206tx.

Figure 3.

Target-specific cell death induced by a combination of multiplexed CRISPR/nCas9^D10A and PARPis. A, Relative viability of cells treated with multiplexed CRISPR/nCas9^D10A RNPs and 1 µmol/L olaparib. B, Colony formation assay using HeLa and HCT116 cells transfected with multiplexed CRISPR/Cas9^WT or CRISPR/nCas9^D10A RNPs, with or without 1 µmol/L olaparib. Cells were plated on 6-well plates at an initial density of 10,000 cells/well (HeLa cells) or 3,000 cells/per well (HCT116 cells), followed by serial dilution (10-fold). Each colony formation assay was performed in duplicate. The brightness of the images was adjusted for visibility. C, Relative cell viability in the presence of different concentrations of olaparib in combination with multiplexed CRISPR/nCas9^D10A RNPs. A siRNA or shRNA targeting the BRCA2 gene was used as a positive control for the synthetic lethality of olaparib. D, Cytotoxicity of PARPis combined with multiplexed CRISPR/nCas9^D10A RNPs. NT, nontarget control sgRNA. E, A model for induction of cytotoxicity following CRISPR/Cas9^WT or CRISPR/nCas9^D10A treatment. Bold text indicates the dominant repair pathways of each damage type. F, Mutation efficiency at each target sequence following transfection of RNPs containing each sgRNA and Cas9^WT or nCas9^D10A, with or without olaparib treatment, into HeLa and HCT116 cells. Cell viability was measured in a CellTiter-Glo assay performed at 72 hours after transfection, and data were normalized to those observed following treatment with a nontarget sgRNA control. Error bars, mean ±SD of three independent biological replicates. All statistical significances were calculated using an unpaired two-tailed Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 4.

Evaluation of targeted cell death with multiplexed CRISPR/Cas9^D10A and PARPis in a mouse xenograft model and human colorectal cancer organoids. A, Timeline of treatment and tumor growth curves for HCT116 cell–derived xenografts following administration of olaparib (1.5 mg/kg) and/or LNPs containing RNPs consisting of CRISPR/Cas9^D10A and sgRNAs (delivered every 2 days; eight injections in total). Left, tumor volume was 80 mm³ at day 0. Right, images of tumors at day 25 after the first injection. Scale bar, 2 cm. B, Tumor growth inhibition following each treatment. C, IHC staining for caspase-3 and Ki-67 and a TUNEL assay in ex vivo tumor tissues. Bottom left, hematoxylin was used as a nuclear stain. The areas shown in the images are indicated by white boxes in the whole-slide images. Positive cells are stained brown. Scale bars, 50 µm. Quantitative analysis of the area positive for caspase-3, Ki-67, or TUNEL staining was performed using ImageJ software (bottom). Number of mice used for Cas9^WT: n = 7 in the PBS group and n = 5 in the other groups. Number of mice used for nCas9^D10A: n = 6 in the PBS group, n = 7 in the olaparib-only group, n = 8 in the Hmix-4 with olaparib group, and n = 4 in the other groups. D, Cell viability in human colorectal cancer organoids following transfection of multiplexed CRISPR RNPs along with Cas9^WT or nCas9^D10A, with or without 1 µmol/L olaparib. Scale bars, 500 µm. The CellTiter-Glo assay was performed at 6 days after transfection, and the results were normalized to a nontreatment control. Number of independent transfections: n = 4 for Cas9^WT and n = 3 for nCas9^D10A. CRC-mix4, colorectal cancer–mix4; NT, nontarget control sgRNA. All statistical significances were calculated using an unpaired two-tailed Student t test. n.s., not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars, mean ± SE of biological replicates.

Figure 5.

Synthetic lethality of combined CRISPR/Cas9 and DNA damage–inducing treatments. A, Bar plots showing the viability of cells treated with multiplexed CRISPR/Cas9^WT RNPs plus irradiation. Cell viability was measured using the CellTiter-Glo assay at 3 days after transfection, and data were normalized to those observed following treatment with a NT sgRNA control without irradiation. Cell viability was also measured at 72 hours after transfection, and data were normalized to those observed following treatment with a nontarget sgRNA control. Error bars, mean ± SD of three independent biological replicates. B, Schematic illustrating synthetic lethality of CRISPR/Cas9 targeting, followed by DNA damage–inducing treatment or DNA repair inhibitors such as additional CRISPR/Cas, irradiation, or a PARPi. NT, nontarget control sgRNA. Statistical significance was calculated using an unpaired two-tailed Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.