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.
CRISPR-based genome editors are fundamentally limited by their requirement for double-strand DNA breaks (DSBs), restricted transgene cargo capacity, and reliance on error-prone endogenous DNA repair mechanisms. Non–long terminal repeat (non-LTR) retrotransposons—especially the site-specific R2 element—offer a mechanistically distinct and potentially safer choice for programmable genomic integration. These elements employ target-primed reverse transcription (TPRT)—an RNA-templated integration mechanism that circumvents DSB formation and supports amplification of self-copy. This review delineates the molecular mechanism of R2 retrotransposons, emphasizing their highly specific integration into the 28 S ribosomal DNA locus—a recognized genomic safe harbor. We describe the functional domains of the R2 protein, including the reverse transcriptase, restriction-like endonuclease, and nucleic acid-binding motifs, and explain how they coordinate to achieve precise DNA cleavage and cDNA synthesis. Recent cryo–electron microscopy (cryo-EM) structures have revealed discrete RNA-protein complex that orchestrate the stepwise progression of TPRT. Informed by these mechanistic insights, researchers have engineered programmable platforms—including PRINT and STITCHR—that enable RNA-directed transgene integration in mammalian systems. These platforms establish R2 as a viable all-RNA programmable system for targeted genomic integration. Future directions include reprogramming the DNA-binding specificity of R2 through protein engineering to target loci, optimizing integration fidelity and efficiency, and mining diverse R2-like elements from metagenomic data. With continued optimization and rigorous safety validation, R2-derived platforms could supplant current nuclease-dependent editors in applications requiring high-fidelity, large-cargo integration.
Unknown authors· Frontiers in Genome Editing· 0 citations
Using high-throughput junction mapping together with large-scale comparative genomics, this work redefined the in vivo structural boundaries, growth, and mobilization of IS110 elements and uncovered a previously unrecognized size continuum extending to ∼100 kb, driven by progressive additions.
Kuang Hu, Bingliang Xie, HengYi Yang et al.· bioRxiv· 1 citation
The potential of terminally engineered PAM-less dsDNA as a structural handle for programming Cas12a activity is highlighted and useful insight is provided for the design of CRISPR-based biosensing strategies.
Genomic manipulation has advanced from stochastic nuclease‐mediated disruption toward programmable, deterministic precision. Early clustered regularly interspaced short palindromic repeats (CRISPR) strategies enabled targeted mutagenesis through double‐strand breaks; however, their therapeutic application is limited by genotoxicity, chromosomal instability, and dependence on endogenous repair pathways that are difficult to predict. In this review, we examined the transition from gene editing to genome writing, an approach that decouples genomic modification from host repair pathways to better balance efficiency, precision, and payload delivery. We also discussed the principles of precision technologies, including base and prime editors, and described emerging large‐scale writers, such as CRISPR‐associated transposases and recombinase‐based bridge RNAs, which enable the integration of multi‐kilobase synthetic modules. Beyond enzymatic mechanisms, we further considered the combined use of generative artificial intelligence, structural biology, and novel delivery architectures as potential strategies to overcome current biological limitations. Taken together, these developments point toward Generative Biology, in which computational design and high‐throughput screening transform the genome from a static substrate into a more dynamic model for complex, synthetic functional design.
Base editors (BEs) enable efficient A-to-G or C-to-T conversions without double-stranded DNA cleavage, but their editing windows remain difficult to tune, limiting genome engineering flexibility. Here, we engineered CRISPR/Cas12b sgRNA by introducing MS2 hairpins to recruit an MS2-N55K-cytidine deaminase-UGI complex, enabling programmable control of the editing window. Three modified sgRNAs were generated by replacing two loop regions, each producing distinct editing hotspots in E. coli. The AID*Δ-MSBE system (sgRNA1.1) generated a window near the PAM with peak activity at C7-C9, while the CDA-MSBE system (sgRNA1.2) produced a distal window with peak activity at C20-C23. Both systems exhibited identical editing patterns in Bacillus subtilis. A dual-orthogonal system (MS2 and PP7) was constructed to simultaneously recruit two deaminase complexes, restoring the classic dCas12b CBE editing pattern. Rifampicin resistance assays confirmed high targeting specificity with low off-target effects. As proof of concept, the MSBEs were successfully employed for the flexible reprogramming of sfGFP fluorescence and the targeted evolution of the endogenous gene rpsE, respectively. Collectively, we developed the MSBEs with tunable editing hotspots, providing innovative tools to enhance the flexibility and accessibility of BEs for genome engineering.
