A comprehensive CRISPR/Cas9-based RAS knock-in cell line platform (50+ models) for assessing efficacy of KRAS-targeted inhibitors in RAS-mutant malignancies.

The rat sarcoma gene (RAS) was the first discovered human proto-oncogene. Its product, the RAS protein, is a small guanine nucleotide-binding protein with intrinsic guanosine triphosphatase (GTPase) activity. Currently, three subtypes are known in the RAS family: KRAS, NRAS, and HRAS.RAS is a key mediator of the MAPK pathway. Activated RAS proteins bind GTP, while inactivated RAS proteins bind GDP; thus, the function of RAS proteins primarily depends on their GTP/GDP ratio. Missense mutations in the RAS protein alter the GDP/GTP homeostasis and lead to sustained activation of signaling pathways by reducing GTP hydrolysis (G12, Q61), increasing GTP loading rates (G13, K117), or affecting nucleotide exchange (A146). This uncontrolled signaling promotes unregulated cell proliferation and ultimately leads to carcinogenesis. RAS mutations occur most frequently in the KRAS subtype (>80%), followed by NRAS and HRAS. Notably, KRAS mutations are found in 97.7% of pancreatic ductal adenocarcinomas. In colorectal adenocarcinoma, multiple myeloma, lung adenocarcinoma, and cutaneous melanoma, the overall prevalence of RAS mutations is 52.2%, 42.6%, 32.2%, and 29.4%, respectively.For decades, RAS was notoriously considered an "undruggable" target, posing significant challenges for drug development. The approval of AMG510 (sotorasib) broke this barrier, proving that direct targeting of RAS is achievable. Since then, the field of RAS-targeted therapy has advanced remarkably, moving the "undruggable" label into the past. With deepening understanding, targeting RAS is poised to drive the next wave of breakthroughs in cancer treatment.Using CRISPR/Cas9 technology, we have developed over 50 knock-in cell line models with KRAS and NRAS mutations for evaluating KRAS-targeted inhibitors. These include common mutations such as G12C, G12D, and G12V, as well as various double and triple mutation combinations. This Ras knock-in mutant cell line platform stands as a powerful tool for developing and evaluating the next generation of RAS inhibitors.