Jul 2026· Journal of Advances in Biology & Biotechnology· Vol 29, pp. 872-882· 0 citations
TL;DR
The principal challenges, including transposon-associated motif (TAM) restriction, off-target activity, delivery and mechanistic understanding, that must be addressed are outlined before the therapeutic and agricultural potential of TnpB-derived technologies can be fully realised.
Abstract
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.
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
Simple Summary The CRISPR-Cas9 and CRISPR-Cas12 systems recognize target DNA through base pairing with their guide RNAs, revolutionarily simplifying the design of DNA endonucleases with novel target-sequence specificity for genome editing and gene therapy, compared with endonucleases that rely on protein-based target recognition. The OMEGA (Obligate Mobile Element-Guided Activity) system, considered a precursor to Cas12, and likely to Cas9, in the CRISPR-Cas system, is widely present in the three domains of life as an auxiliary component of transposons. Recent studies have shown that the OMEGA system cleaves the site from which a transposon was excised, thereby inducing transposon restoration via gene conversion. It is now evident that the OMEGA system acts selfishly as a homing endonuclease. This article will focus on the selfish behavior of classical homing endonucleases and the OMEGA system, and discuss how it has evolved, been maintained, and adapted to new genomic environments.
Genome size variation in eukaryotes is driven largely by transposable elements (TEs), yet the biological mechanisms that initiate their proliferation remain understudied. Here, we identify a recurrent association between bacterial horizontal gene transfer (HGT) and bursts of TE activity that contribute to genome expansion. By leveraging comparative genomics and genus-level pangenome analyses across three species of the nut weevil, Curculio, we detect extensive bacterially derived DNA sequences embedded within structurally dynamic genomic regions. These HGT-associated regions are dominated by a small number of young, proliferating TE families, particularly DNA type II Mavericks, which encapsulate transferred bacterial sequences and comprise a substantial fraction of recent genomic DNA in derived lineages. Analyses of codon usage bias, intron length, and functional enrichment suggest that most transferred genes undergo progressive pseudogenization over evolutionary time, whereas a subset of selectively advantageous HGTs persist. Together, our findings support a model linking foreign DNA invasion with TE proliferation, genome size variation, and molecular innovation.
Z. Cohen, L. Perkin, P. Frandsen et al.· bioRxiv· 0 citations
This work establishes TIGR-Tas as a valuable addition to the yeast genome engineering toolbox, particularly for applications requiring PAM-independent targeting or compact delivery.
Natural transformation is a key mechanism of bacterial adaptation in which exogenous DNA (eDNA) is taken up, processed into single-stranded DNA (ssDNA), and integrated into the genome. While earlier studies primarily focused on uptake mechanisms, transport proteins, and recombination processes, exonucleases were long regarded as merely nonspecific degradation enzymes in DNA uptake. However, recent studies show that nucleases, partly related to the SOS response, play a key role in processing uptake ssDNA. They affect the imported DNA, thereby promoting efficient recombination. This review highlights the interactions between nucleases and taken-up ssDNA, discusses the functional link between natural competence and the bacterial SOS damage response, and demonstrates that key components of these processes have been conserved in bacteria. This suggests a possible universal principle in which ssDNA-specific nucleases serve as switches between the DNA damage response, competence, and horizontal gene transfer.
Rebecca Hinrichs· Biochimica et biophysica act...· 0 citations
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
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