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 [1]. Typically, LINE-1 is repressed in healthy tissues but may become activated in various human diseases. LINE-1 expression has been associated with aging [2–4], neurodegenerative disorders [5–7], cancer [8–10], and autoimmune diseases [11,12]. 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. Elucidating these mechanisms will deepen our understanding of 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 reporter system, we identified genes that control LINE-1 expression through multiple potential mechanisms, including their regulation at both RNA and protein levels. Besides known regulators like the HUSH complex, our screening uncovered previously unknown regulators of ORF1p and ORF2p, and revealed distinct mechanisms regulating expression of these proteins. We also identified genes whose disruption contributes to LINE-1 activity-dependent lethality. Conclusion This study offers a valuable resource for the retrotransposon field, providing new insights into the distinct molecular mechanisms regulating LINE-1-encoded ORF1p and ORF2p, and highlighting potential therapeutic targets for diseases driven by LINE-1 dysregulation.