Abstract 6846: Using CRISPR/Cas9 to edit KRAS in a small cell lung cancer cell line

Abstract Introduction: Small cell lung cancer (SCLC) accounts for 10-15% of lung cancers and is the most aggressive subtype. Approximately 16% of SCLCs contain mutations in the KRAS oncogene. The KRASG12C activating mutation is now clinically targetable. There are limited SCLC models containing the KRASG12C mutation available to generate pre-clinical rationale for clinical studies. This study aimed to develop a protocol for the insertion of the KRASG12C mutation in the KRAS wild-type SCLC cell line NCI-H1048 using CRISPR/Cas9 technology. Methods: A protospacer adjacent motif (PAM) was identified near the KRASG12C mutation site and a single guide RNA (sgRNA) was designed using SnapGene software. The efficiency and specificity of the sgRNA were determined using CRISPRon and CRISPRoff webservers (https://rth.dk/resources/crispr/). A repair template containing the nucleotide substitution was created. The CRISPR/Cas9 plasmid pSpCas9(BB)-2A-GFP (PX458, Addgene) was assembled with a single-stranded DNA oligo matching the sgRNA sequence. The NCI-H1048 cell line was transfected with the DNA assembly product (pKRASm) or the PX548 empty plasmid (control) using Lipofectamine 3000. Cells were sorted by GFP fluorescence 3 days after transfection using a BD FACS Aria III cell sorter. pKRASm- and PX458-transfected cells were seeded for colony formation in 10 cm tissue culture plates. Several pKRASm and PX458 clones were isolated and Sanger sequenced to determine KRASG12C mutation status. Codons affected by genetic alterations were determined by DNA local alignment using SnapGene software. Cell line growth rates were assessed over 7 days using the Incucyte® live-imaging system. Results: The transfection efficiency in NCI-H1048 was 0.6% for pKRASm and 1.2% for PX458. No clones with a KRASG12C mutation were isolated. However, two clones had alterations in the KRAS exon 2 sequence. Clone NCI-H1048-29 contained a 15bp in-frame deletion (impacting codons 9-13), and clone NCI-H1048-32 had heterozygous alleles from position 27 (impacting codons 9-37). The regions affected are the P-loop (codons 10-14, phosphate-binding loop) and switch I (codons 30-40, binding interface for KRAS effectors and regulators). The growth of clones NCI-H1048-29 and NCI-H1048-32 was compared to a control clone (PX458-transfected NCI-H1048-9). The KRAS alteration significantly reduced the growth of NCI-H1048-29 (p=0.023) and NCI-H1048-32 (p=0.004) compared to the NCI-H1048-9 control confirming phenotypic impact of the KRAS alterations. Conclusion: The CRISPR/Cas9 method employed resulted in the creation of novel SCLC cell lines with KRAS alterations, but further optimization of the protocol is needed to introduce KRAS point mutations. The CRISPR/Cas9-edited cell lines created will be used for further in vitro study of KRAS biology in SCLC. Citation Format: Myra Elise Castel, Neil T. Conlon, Georg F. Bischof, Ke Wei Gong, Neil A. O'Brien, John P. Crown, Denis M. Collins. Using CRISPR/Cas9 to edit KRAS in a small cell lung cancer cell line [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 6846.