Abstract LB397: Simultaneous CRISPR-Cas9 induced double strand breaks causes chromosomal catastrophe in pancreatic cancers: A parallel comparison with ionizing radiation

Abstract CRISPR-Cas9, a highly effective gene editing tool, is a molecular scissor that induces double strand breaks (DSBs) at its targeted genomic locations. Ionizing radiation (IR), in contrast, generates DSBs and other DNA lesions, making it potentially more toxic than CRISPR-Cas9 induced DSBs. Alternatively, CRISPR-Cas9 might be more toxic as the Cas9 activity would continue if the DSB were repaired to wild type, resulting in more scissions per target. We designed multitarget sgRNAs targeting 0-16 sites in the human genome and performed similar dose-response experiments with IR (0-10 Gy) over 21 days on two pancreatic cancer (PC) cell lines. Growth inhibition was measured through clonogenicity and alamarBlue assays, and γ-H2AX staining performed to calculate DSBs. Surviving colonies from sgRNA transduction underwent whole genome sequencing (WGS) and targeted deep sequencing to quantify mutations. Cell growth was assessed through cell counting and sgRNA tag survival over time. Chromosomal changes were elucidated through chromosome breakage assays and XY FISH. We found that cytotoxicity increased as a function of both IR dose and number of sgRNA target site. At a comparable number of DSBs, CRISPR-Cas9 demonstrated higher cytotoxicity (~44% growth inhibition from 5-target sgRNA, ~15 γ-H2AX foci in the PC cells) compared to IR (9% inhibition from 0. 5 Gy, ~25 foci). WGS of 5-cutter treated cells also revealed ~13 CRISPR-Cas9 induced DSBs in the PC cells. 99% cytotoxicity was achieved with 12-target sgRNA and 8 Gy. Moreover, sequencing of surviving colonies from sgRNA-treated cells confirmed lack of widespread off-target events. Notably, no surviving colonies could be obtained from the 12- and 16-cutter treated Panc10. 05 cells and 8-14 cutters treated TS0111 cells. Both CRISPR-Cas9 and IR assays demonstrated delayed cell death, and cell death began 7 days after transduction of multitarget sgRNAs. Cytogenetic analyses on cells transduced with a 14-cutter sgRNA over 21 days revealed accumulation of karyotypic abnormalities peaking at day 14, and polyploidization peaking at day 10 and decreasing by day 21. Structural variant (SV) analyses from WGS data showed that the number of SVs per colony increased and peaked in 6-cutter colonies (average = 42 SVs), then decreased in 8- and 14-cutter colonies. Finally, surviving colonies that were resistant to 5-, 8-, and 16-cutter sgRNAs were re-challenged with their original multitarget sgRNA, 12-, and 14-cutter sgRNAs. Re-treating with original sgRNA showed no growth inhibition, while 95% cytotoxicity was observed by re-challenging them with 12- or 14-cutter sgRNAs. In conclusion, CRISPR-Cas9 kills pancreatic cancers by inducing chromosomal catastrophe, and resistance to original set of CRISPR-Cas9 induced DSBs can be easily overcome by targeting different sites. For the same number of DSBs, CRISPR-Cas9 was surprisingly more toxic than IR. Citation Format: Selina Shiqing K. Teh, Akhil Kotwal, Eitan Halper-Stromberg, Laura Morsberger, Kirsten Bowland, Alexis Bennett, Michael Goldstein, Ying S. Zou, James R. Eshleman. Simultaneous CRISPR-Cas9 induced double strand breaks causes chromosomal catastrophe in pancreatic cancers: A parallel comparison with ionizing radiation abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (7Suppl): Abstract nr LB397.