Identification of novel regulators of LINE-1 expression via CRISPR/Cas9 screening.

BACKGROUND: Long Interspersed Nuclear Elements-1 (LINE-1, L1) are transposable elements that make up roughly 17% of the human genome. These elements can copy and insert themselves into new genomic locations (Kazazian and Moran, N Engl J Med 377:361-370, 2017). Typically, LINE-1 is repressed in healthy tissues but may become activated in various human diseases. LINE-1 expression has been associated with aging (Simon, et al., Cell Metab 29:871-885.e5, 2019; De Cecco et al. Nature 566:73-78, 2019; Della Valle et al. Nat Rev Genet 26:1-12, 2025), neurodegenerative disorders (Roy et al., Acta Neuropathol 148:75, 2024;Frost and Dubnau, Annu Rev Neurosci 47:123-143, 2024; Ravel-Godreuil et al. FEBS Lett 595:2733-2755, 2021), cancer (Rodriguez-Martin et al., Nat Genet 52:306-319, 2020; Taylor et al. Cancer Discov 13:2532-2547, 2023; Solovyov et al. Nat Commun 16:2049, 2025), and autoimmune diseases (Rice et al., N Engl J Med 379:2275-2277, 2018), (Carter et al., Arthritis Rheumatol 72:89-99, 2020). Despite the strong association between LINE-1 expression and disease, the regulatory mechanisms controlling the expression of LINE-1-encoded ORF1p and ORF2p and the link between LINE-1 activity and cancer cell survival remain poorly understood. Gaining insights into these regulatory pathways may help elucidate how LINE-1 contributes to disease pathogenesis. RESULTS: To identify upstream regulators of LINE-1 and genes associated with LINE-1 activity-dependent lethality, we developed a dual-reporter system that simultaneously monitors the protein levels of LINE-1-encoded ORF1p and ORF2p (wild-type or catalytically inactive EN/RT mutant). Using genome-wide CRISPR/Cas9-based screens with this system, we identified candidate genes that may influence LINE-1 regulation at multiple levels, including RNA and protein expression. Alongside known factors such as the HUSH complex, the screens revealed additional genes not previously linked to LINE-1 regulation, suggesting possible new regulatory mechanisms for ORF1p and ORF2p expression. We also identified genes whose loss correlated with reduced viability in a manner dependent on LINE-1 activity. These findings collectively provide a broad resource for exploring cellular factors that may modulate LINE-1 expression and activity. CONCLUSION: This study provides a resource for investigating the cellular regulation of LINE-1, highlighting distinct candidate factors that may modulate ORF1p and ORF2p expression and influence LINE-1 activity-associated cytotoxicity. While functional validation of these candidate regulators remains necessary, the findings offer a foundation for future studies aimed at experimentally confirming their roles and elucidating the molecular mechanisms underlying LINE-1 regulation and its potential contributions to disease contexts.