Dual-mode application of transcriptional regulation and DNA editing using the ultra-compact TnpB system
TL;DR
A dual-mode genome regulation platform using ISDge10 TnpB effectors and engineered ωRNAs is established, in which modulation of the ωRNA guide length enables switching between programmable transcriptional activation and genome editing.
Abstract
Abstract RNA-guided obligate mobile element guided activity systems derived from transposable elements have emerged as compact genome-editing tools that may replace clustered regularly interspaced short palindromic repeats platforms. We established a dual-mode genome regulation platform using ISDge10 TnpB effectors and engineered ωRNAs, in which modulation of the ωRNA guide length enables switching between programmable transcriptional activation and genome editing. TnpB programmed with a 10-nt guide region of the ωRNA engages target DNA without inducing double-strand breaks. Fusion of transcriptional activators with Sso7d (DNA-binding protein from Sulfolobus solfataricus) enables specific transcriptional upregulation across endogenous loci. Restoring the ωRNA guide length to 20 nt triggers DNA cleavage, thereby supporting homology-directed repair-mediated sequence correction. A catalytically inactivated TnpB-based adenine base editor enabled A-to-G base conversion at genomic targets. TnpB shows strict ωRNA-dependent mismatch sensitivity with low off-target effects, suggesting its potential as a high-fidelity genome regulation platform. Compact ISDge10 TnpB facilitates co-packaging of effector and ωRNA in a single adeno-associated virus vector and co-expression of large functional domains. Thus, this study expands RNA-guided genome-editing capabilities.