Abstract 5527: Optimization of CRISPR-Cas9 library screens in pancreatic cancer organoid models targeting KRASG12D

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) has a critical need in systemic therapeutic development, with hallmark activation of the mitogen-activated protein kinase pathway. Novel mutant-specific KRAS inhibitors have been developed, however no method to date can predict patient-specific resistance mechanisms. Here, we optimize a CRISPR-Cas9 lentivirus screen in 3D patient-derived cancer organoids (PCOs) to study resistance mechanisms and guide combination therapy using the KRASG12D inhibitor MRTX1133. Methods: PDAC PCOs were derived from surgical specimens. Puromycin (puro) dose-response curves were established using cell viability assays. PCOs were transfected with a lentiviral-based ‘druggable genome’ CRISPR-Cas9 library of 2, 292 unique targets (Millipore-Sigma) overnight (18h) in a liquid phase with the addition of polybrene (5ug/mL). Puro selection was compared for efficiency at 6 days and 12 days. Western blotting was used to confirm Cas9 expression. Multiplicity of infection (MOI) optimization was performed by digital droplet PCR (ddPCR) on selected cells to measure lentiviral genome copy number efficiency. MOI was compared across traditional cell viability assays using CellTiterGlo (CTG, 50% v/v) and cell counting methods. Results: Molecular profiling of the PDAC PCO line confirmed KRASG12D mutant by DNA sequencing (SureSelect Cancer, Agilent). Puro dose was selected at 2 μM to exceed the IC90 of 1.95 ± 0.1 μM. Western blots confirmed Cas9 presence in selected cells. The KRASG12D inhibitor MRTX1133 revealed an IC50 of 11.16 ± 0.36 nM. The 1:5 cell-to-lentivirus ratio after 6 days of puro selection was selected based on copy number analysis by ddPCR (0.93 ± 0.01). The control, 1:2, and 1:10 ratios after 6 days of selection exhibited copy numbers of 0 ± 0, 0.53 ± 0.13, and 1.52 ± 0.38, respectively. Selection for 12 days yielded higher copy numbers (1.13 ± 0.38 at 1:2, 1.19 ± 0.11 at 1:5, and 1.72 ± 0.43 at 1:5) compared to the 6 day puro selection. Increased selection time also revealed reduced viability compared to the 6 day selection. The 1:5 ratio showing 16.8% (p<0.0001) and 22.65% (p<0.0001) viability, respectively. Post-selection, the viability of puro-selected, non-transfected cells was 0%, confirming selection efficiency. Conclusions: Here, we demonstrate the optimization of CRISPR-Cas9 screening with 3D PDAC PCOs using ddPCR to validate the efficiency of transfection. Importantly, the degree of puro selection and lentivirus transfection did confer multiplicity in viral transfections. The ongoing work is aimed at understanding the time course dynamics of gene editing between early (14 day) and late (28 day) resistance to KRASG12D inhibition using the Model-based Analysis of the Genome-wide CRISPR/Cas9 Knockout (MAGeCK) method. This work extends the efficiency of novel gene editing technologies for screening applications in organoid models of pancreatic cancer. Citation Format: Md Shahadat Hossan, Lauryn Flannagan, Molly A. Nellen, Austin Stram, Ethan S. Lin, Michela Cadarso, C. Dustin Rubinstein, Sean M. Ronnekleiv-Kelly, Jeremy D. Kratz. Optimization of CRISPR-Cas9 library screens in pancreatic cancer organoid models targeting KRAS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5527.