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.
TnpB proteins are among the most abundant genes encoded in bacterial and archaeal genomes, yet their function in the transposon life cycle remained unclear for decades. Recognition that TnpB is the likely evolutionary ancestor of type V CRISPR-Cas12 effector nucleases, followed by experimental demonstration that TnpB is itself a compact RNA-guided DNA endonuclease, has transformed these previously regarded accessory transposon proteins into a promising frontier in genome engineering. At roughly 350-410 amino acids, TnpB is less than half the size of Cas12a and approximately one-third the size of Cas9, making it well suited to delivery vehicles with constrained cargo capacity, such as adeno-associated virus (AAV). This review discusses recent progress in TnpB biology and technology. It first describes the biochemical and structural basis of RNA-guided DNA cleavage by TnpB and its role in transposon homing. It then summarises comparative genomic analyses that reveal the diversity of TnpB, repeated independent evolutionary transitions from TnpB to Cas12, and recurrent exaptation of TnpB for cellular functions unrelated to transposition. The review also considers how mining natural TnpB diversity and high-throughput protein engineering have produced compact editors with activity and specificity approaching established CRISPR-Cas tools in selected contexts, including early demonstrations of TnpB-mediated editing in animals and crop plants. It concludes by outlining the principal challenges, including transposon-associated motif (TAM) restriction, off-target activity, delivery and mechanistic understanding, that must be addressed before the therapeutic and agricultural potential of TnpB-derived technologies can be fully realised.
Sanchit Pal Singh, Shruti Gupta, Rohit Solanki et al.· Journal of Advances in Biolo...· 0 citations
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
A protein design strategy is used that couples a structure-guided inverse-folding model with evolution-informed residue constraints to generate active, divergent variants of TnpB, a minimal CRISPR-Cas12-like nuclease, termed SynTnpBs, establishing a strategy for creating non-natural RNA-guided nucleases and conformationally active nucleic acid binders, enlarging the designable protein space.
Petr Skopintsev, Isabel Esain-Garcia, Evan C. DeTurk et al.· Science· 2 citations
An RNA-guided bridge recombinase system is engineered through rational mutagenesis and AI-assisted directed evolution, enabling programmable chromosomal rearrangements in both plant and mammalian cells and achieving up to a 29.8-fold increase in activity.
Rui Gao, Jingjing Wei, Chao Sun et al.· Trends in Biotechnology· 0 citations
Viruses encode diverse regulatory elements, but their breadth and mechanisms remain poorly defined. To address this gap, we performed massively parallel reporter assays spanning ∼200,000 genomic segments from 297 vertebrate-infecting viral genera. We identified numerous viral elements that enhance RNA stability and translation through TENT4-mediated mixed tailing, distributed across 19 genera and grouped into six distinct subclasses, indicating extensive convergent evolution. We also found diverse TENT4-independent elements acting through alternative pathways. One such element, Pt1 from Potamipivirus, stabilizes linear mRNA to levels comparable to circular RNA, suggesting its potential for RNA therapeutics. Pt1 directly recruits canonical poly(A) polymerases (PAPγ/α)-previously thought to function exclusively in transcription-coupled nuclear pre-mRNA processing-to drive cytoplasmic polyadenylation. Together, these findings chart the rich landscape of viral regulation, extend the scope of poly(A)-tail biology, and establish the virome as a valuable source for uncovering host RNA regulatory mechanisms.
Jenny J Seo, Chemin Lee, Dongbin Lim et al.· Cell· 0 citations
Sequence-programmable directed evolution systems have great potential to accelerate bioengineering. Diversity-generating retroelements (DGRs) are natural hypermutation systems widely distributed in prokaryotes and bacteriophages with the capacity to introduce diverse mutations at template-specified sites of target genes. Here, we show that DGRs can be installed in E. coli and reprogrammed for the continuous, iterative mutagenesis of user-defined target genes. We show that the DGR template RNA can be reprogrammed for gene- and residue-specific mutagenesis, leaving untargeted, adjacent residues unchanged. Furthermore, we establish continuous DGR-enabled mutagenesis with conjugation-mediated horizontal gene transfer of target genes (HGT-DGR) into a new host for the progressive accumulation of target-specific mutations. Iterative HGT-DGR mutagenesis over seven cycles yielded an average mutation load of approximately 6% across adenine positions in the target segment, generating a diverse library of variants comprising 40% mutant sequences, with a median pairwise Hamming distance of 4 among mutant variants. HGT-DGR enables iterative diversification of either the same or different user-specified segments of the target gene, as demonstrated with the directed evolution of the M. mazei pyrrolysyl-tRNA synthetase for non-canonical amino acid incorporation. HGT-DGR provides a simple, low-cost, sequence-programmable system that enables iterative, position-specific and tunable in vivo mutagenesis of any target sequence for applications in biotechnology and medicine.
Yang Liu, Yang-Qi Gu, Ganesh Agam et al.· bioRxiv· 0 citations