Engineered targeted enhancement of translation in mammalian cell

BACKGROUND: While the central dogma outlines DNA-to-protein information flow, existing gene regulators mainly target transcription. Here, we developed the λN-Guided RNA Targeting System (λGRTS), a CRISPR-independent platform enhancing mammalian mRNA translation via specific translation-guiding RNAs (tgRNAs). RESULTS: λGRTS integrates λN (high-affinity BoxB binder), mutated eIF4E1 (ablated non-specific 5' cap binding, retains TIC recruitment), and auxiliary factors (HuR for dsRNA stabilization, PABP and RRM2-RRM3 for mRNA closed loops). Optimized 22-nt tgRNAs (targeting 58 bp upstream of mRNA ATG in 5' UTR) and NES-tagged proteins maximized efficacy. λGRTS outperformed dCasRX (smaller ~ 50 kDa vs. ~ 150 kDa, single tgRNA vs. crRNA-tracrRNA), boosting functional proteins (e.g., GFP). It activated P53/PTEN, suppressing GBM cell proliferation, inducing G1 arrest, and reducing invasion in vitro. In vivo, lentiviral λGRTS inhibited orthotopic GBM in nude mice, extended survival by approximately 35-40 days, with IHC confirming P53/PTEN upregulation. Mass spectrometry showed no off-target effects. Cross-species tests (human, mouse, bovine cells) validated broad applicability via conserved eIF4E1. CONCLUSIONS: λGRTS offers a specific, safe translation-centric tool for gene regulation and oncology research.