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.
Yeounsun Oh, Se‑Been Jeon, Lee Wha Gwon et al.· Nucleic Acids Research· 0 citations
Efficient delivery of CRISPR components remains a major determinant of genome editing outcomes. In this study, we compared conventional lipofection with magnetic nanoparticle-assisted gene delivery (magnetofection) for CRISPR-mediated genome editing efficiency using SpCas9 and AsCas12a systems. Based on the average values obtained from multiple independent targets, lipofection resulted in relatively low indel efficiencies, with mean values of average 8.1%–12.47%. In contrast, magnetofection markedly enhanced genome editing outcomes, yielding average indel efficiencies of average 42.29%–45.04%, representing a substantial increase (3.39- and 5.56-fold, respectively) compared with lipofection. This enhancement was consistently observed across both SpCas9-and AsCas12a-mediated editing, indicating that the improved efficiency conferred by magnetic nanoparticle delivery is independent of the nuclease platform. Furthermore, the increased performance of magnetofection was reproducible across multiple genomic loci and cell lines and was also effective under RNP-based delivery conditions, demonstrating its robustness and reliability. In addition to indel-based genome disruption, magnetofection also significantly improved prime editing efficiency (13.95% on average) compared to lipofection (3.81% on average). Overall, our results demonstrate that magnetic nanoparticle-mediated delivery enables highly efficient and reproducible CRISPR genome editing, substantially outperforming conventional lipofection for both indel formation and prime editing. Magnetofection therefore represents a powerful and broadly applicable delivery strategy for next-generation genome editing applications.