George D. Lampe

Defense-associated reverse transcriptases (DRTs) employ diverse and distinctive mechanisms of cDNA synthesis to protect bacteria against viral infection¹. However, the functions and mechanisms of many DRTs remain unknown². Here we identify a new antiviral defense system, DRT10, that associates with a noncoding RNA (ncRNA) to catalyze processive, protein-primed synthesis of tandem-repeat DNA. Reverse transcription is templated by a defined ncRNA region with a characteristic sequence pattern and template boundary elements, both of which are conserved and essential for DNA repeat addition. Cryo-EM reconstructions reveal an unanticipated 2:1 architecture, wherein two RT monomers bind opposite sides of a pseudo-symmetric ncRNA. Biochemical experiments further demonstrate that each RT monomer reverse transcribes the template encoded on its respective side of the ncRNA, but only one generates the long repetitive product, whose template is defined by the distance between two flanking stem-loop anchors. This mode of iterative, boundary-delimited cDNA synthesis exhibits direct parallels to the mechanism of telomeric repeat addition by eukaryotic telomerase, and phylogenetic analyses of RTs across domains of life place telomerase in a monophyletic clade with the DRT family, suggesting a common origin for their DNA polymerization activities. Together with earlier studies of DRT2, DRT3, and DRT9^3–5, our findings identify a conserved mechanistic logic underlying RNA-templated DNA repeat addition across diverse DRTs, despite vastly different architectural solutions, and expand the repertoire of reverse transcription-based strategies for antiviral immunity in bacteria.

Genome editing technologies have advanced from nuclease-based reagents that generate programmed DNA double-strand breaks, which can cause deleterious effects, to next-generation reagents that perform controlled DNA modification through double-strand break-independent mechanisms, such as base editing and prime editing. Although these approaches enable precise small-scale sequence changes, methods for programmable insertion of large DNA cargos have been limited. The ability to write entire genes or large regions into the genome could transform the treatment of genetically heterogeneous disorders, for which numerous pathogenic variants underlie a common disease and mutation-specific editing strategies are impractical. Recent advances in computational genome mining have accelerated the discovery of naturally occurring enzymes with novel biochemical and functional properties, including recombinases and transposases capable of large-scale modifications. Moreover, directed evolution, rational engineering and expanded homologue discovery are enabling the repurposing and optimization of these systems for genome engineering. Here we review recent technology development efforts that harness diverse enzymes for kilobase-scale genome engineering, with a particular focus on CRISPR-associated transposase systems.

CRISPR-associated transposons (CASTs) are Tn7-like elements that have co-opted RNA-guided CRISPR effectors for targeted DNA insertion. CASTs have been adapted as genome editing tools for programmable, site-specific integration. Among them, the type I-F system from Ps e udoalteromonas ( Pse CAST) shows exceptionally robust activity in human cells, yet its mechanistic basis remains poorly understood. Here, we present structural and biochemical analysis of the Pse CAST transposase TnsAB. Biochemical reconstitution of transposon DNA excision defines key characteristics of the transposition mechanism. Cryogenic electron microscopy (cryo-EM) structures of Pse TnsAB paired-end complexes reveal molecular determinants of transpososome assembly, transposon end recognition and cleavage. We validate these findings using biochemical and in vivo assays of structure-based transposase mutants, and provide mechanistic insights into the enhanced activity of a laboratory-evolved TnsAB variant. Together, our studies highlight molecular features underlying the efficiency of natural and engineered type I-F transposases and establish a mechanistic framework for their continued rational optimization.

Defense-associated reverse transcriptases (DRTs) employ DNA synthesis to protect bacteria against phage infection. We previously showed that DRT10, a tripartite system comprising an RT, a noncoding RNA (ncRNA), and a SLATT effector protein, catalyzes protein-primed, tandem-repeat DNA synthesis in a mechanism strikingly analogous to eukaryotic telomerase. However, the structural basis by which the RT-ncRNA complex directs repeat addition processivity and controls repeat length remains unknown. Here we present cryo-EM reconstructions of two evolutionarily diverse DRT10 RT-ncRNA systems that reveal an unanticipated 2:1 architecture, wherein two RT monomers bind opposite sides of a single, pseudo-symmetric ncRNA. Biochemical experiments demonstrate that each RT monomer reverse transcribes the template encoded on its respective side of the ncRNA, but only one generates the long repetitive product, with the template sequence defined by the distance between two flanking stem-loop anchors. Together with earlier studies of DRT2, DRT3, and DRT9, our findings identify a conserved mechanistic logic underlying ncRNA-templated tandem-repeat synthesis across Class 2 UG antiviral systems, despite vastly different architectural solutions.

CRISPR-associated transposases (CASTs) achieve site-specific DNA integration by coupling the RNA-guided targeting action of a nuclease-deficient CRISPR-Cas system with the assembly of a Tn7-like transpososome complex ^1,2 . Understanding the detailed mechanisms of this elaborate process is paramount to engineering CAST systems into programmable genetic tools ^3–6 . The type I-F Pseudoalteromonas CAST ( Pse CAST) displays the highest activity in mammalian cells to date ⁷ and has been the subject of extensive directed evolution ⁸ , but efforts to rationally engineer further improvements have been hampered by critical gaps in our understanding of transpososome assembly and activation ⁹ . Here we use cryo-EM structural analysis, validated by DNA transposition assays, to visualize the Pse CAST system in a series of functional states that define the stepwise mechanism of RNA-guided DNA integration. The structure of a target DNA-bound Cascade-TniQ-TnsC complex reveals that conformational changes induced by R-loop formation are coupled to target DNA stabilization and TnsC heptamerization, which in turn recruits the TnsAB transposase via conserved interactions with its C-terminal tail. Finally, the structure of the 1.2 MDa Pse CAST transpososome holocomplex reveals specific TnsC-TnsB and TnsB-target DNA interactions that drive allosteric remodelling of the TnsB catalytic site to activate donor DNA integration. Together, these findings establish a unified structural and mechanistic blueprint for RNA-guided DNA integration and lay the foundation for engineering next-generation DNA insertion systems for genome editing applications.

CRISPR-associated transposons (CASTs) are Tn7-like elements that have co-opted RNA-guided CRISPR effectors for targeted DNA insertion. CASTs have been adapted as genome editing tools for programmable, site-specific integration. Among them, the type I-F system from Ps e udoalteromonas ( Pse CAST) shows uniquely robust activity in human cells, yet its mechanistic basis remains poorly understood. Here, we present structural and biochemical analysis of the Pse CAST transposase TnsAB. Biochemical reconstitution of transposon DNA excision defines key characteristics of the transposition mechanism. Cryogenic electron microscopy (cryo-EM) structures of Pse TnsAB paired-end complexes reveal molecular determinants of transpososome assembly, transposon end recognition and cleavage. We validate these findings using biochemical and in vivo assays of structure-based transposase mutants, and provide mechanistic insights into the enhanced activity of a laboratory-evolved TnsAB variant. Together, our studies highlight molecular features underlying the efficiency of natural and engineered type I-F transposases and establish a mechanistic framework for their continued rational optimization.

Bacterial transcription initiation is a tightly regulated process that canonically relies on sequence-specific promoter recognition by dedicated sigma (σ) factors, leading to functional DNA engagement by RNA polymerase (RNAP)¹. Although the seven σ factors in Escherichia coli have been extensively characterized², Bacteroidetes species encode dozens of specialized, extracytoplasmic function σ factors (σ^E) whose precise roles are unknown, pointing to additional layers of regulatory potential³. Here we uncover a mechanism of RNA-guided gene activation involving the coordinated action of σ^E factor in complex with nuclease-dead Cas12f (dCas12f). We screened a large set of genetically linked dCas12f and σ^E homologues in E. coli using RNA and chromatin immunoprecipitation experiments, revealing systems that exhibit robust guide RNA enrichment and DNA target binding with a minimal 5′-G target-adjacent motif. Recruitment of σ^E was dependent on dCas12f and guide RNA, suggesting direct protein–protein interactions, and co-expression experiments demonstrated that the dCas12f–gRNA–σ^E ternary complex was competent for programmable recruitment of the RNAP holoenzyme. Remarkably, dCas12f–RNA–σ^E complexes drove potent gene expression in the absence of any requisite promoter motifs, with de novo transcription start sites defined exclusively by the relative distance from the dCas12f-mediated R-loop. Our findings highlight a new paradigm of RNA-guided transcription that embodies natural features reminiscent of CRISPR activation (CRISPRa) technology^4,5.

Different forms of epithelial-to-mesenchymal transition (EMT) manifest during tumor progression. Little is known about the mechanistic basis and functional role of these distinct EMTs. We explored this question in lung adenocarcinoma (LUAD) primitive progenitors, which are competent to enter dormancy in response to transforming growth factor-β (TGFβ) upon metastatic dissemination. The TGFβ response in these cells includes growth arrest and a full EMT that subsequently transitions into an atypical mesenchymal state of round morphology and lacking actin stress fibers. TGFβ drives this transition by inducing expression of the actin depolymerizing protein gelsolin, which converts a migratory, stress-fiber-rich phenotype into a cortical actin-rich, spheroidal state. This transition lowers the biomechanical stiffness of metastatic progenitors and protects them from killing by cytotoxic lymphocytes. Gelsolin-deficient LUAD progenitors can enter dormancy but succumb to immune surveillance. Thus, quiescent LUAD metastatic progenitors undergo an atypical EMT to avert immune surveillance during TGFβ-driven metastatic dormancy.

Conventional genome editing tools rely on DNA double-strand breaks (DSBs) and host recombination proteins to achieve large insertions, resulting in heterogeneous mixtures of undesirable outcomes. We recently leveraged a type I-F CRISPR-associated transposase, PseCAST, for DSB-free DNA integration in human cells, albeit at low efficiencies; multiple lines of evidence suggest DNA binding may be a bottleneck for higher efficiencies. Here we report structural determinants of DNA recognition by the PseCAST QCascade complex using single-particle cryogenic electron microscopy (cryoEM), revealing subtype-specific interactions and RNA-DNA heteroduplex features. By combining structural data, library screens, and rationally engineered mutants, we uncover variants with increased integration efficiencies and modified PAM stringencies. We further leverage transpososome structural predictions to build hybrid CASTs that combine orthogonal DNA binding and integration modules. Our work provides unique structural insights into type I-F CASTs and showcases diverse strategies to investigate and engineer RNA-guided transposase architectures for human genome editing applications.

Bacterial transcription initiation is a tightly regulated process that canonically relies on sequence-specific promoter recognition by dedicated sigma (σ) factors, leading to functional DNA engagement by RNA polymerase (RNAP) ¹ . Although the seven σ factors in E. coli have been extensively characterized ² , Bacteroidetes species encode dozens of specialized, extracytoplasmic function σ factors (σ ^E ) whose precise roles are unknown, pointing to additional layers of regulatory potential ³ . Here we uncover an unprecedented mechanism of RNA-guided gene activation involving the coordinated action of σ ^E factor in complex with nuclease-dead Cas12f (dCas12f). We screened a large set of genetically-linked dCas12f and σ ^E homologs in E. coli using RIP-seq and ChIP-seq experiments, revealing systems that exhibited robust guide RNA enrichment and DNA target binding with a minimal 5ʹ-G target-adjacent motif (TAM). Recruitment of σ ^E was dependent on dCas12f and guide RNA (gRNA), suggesting direct protein-protein interactions, and co-expression experiments demonstrated that the dCas12f-gRNA-σ ^E ternary complex was competent for programmable recruitment of the RNAP holoenzyme. Remarkably, dCas12f-RNA-σ ^E complexes drove potent gene expression in the absence of any requisite promoter motifs, with de novo transcription start sites defined exclusively by the relative distance from the dCas12f-mediated R-loop. Our findings highlight a new paradigm of RNA-guided transcription (RGT) that embodies natural features reminiscent of CRISPRa technology developed by humans ^4,5 .