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To spread, transposons must integrate into target sites without disruption of essential genes while avoiding host defense systems. Tn7-like transposons employ multiple mechanisms for target-site selection, including protein-guided targeting and, in CRISPR-associated transposons (CASTs), RNA-guided targeting. Combining phylogenomic and structural analyses, we conducted a broad survey of target selectors, revealing diverse mechanisms used by Tn7 to recognize target sites, including previously uncharacterized target-selector proteins found in newly discovered transposable elements (TEs). We experimentally characterized a CAST I-D system and a Tn6022-like transposon that uses TnsF, which contains an inactivated tyrosine recombinase domain, to target the comM gene. Additionally, we identified a non-Tn7 transposon, Tsy, encoding a homolog of TnsF with an active tyrosine recombinase domain, which we show also inserts into comM. Our findings show that Tn7 transposons employ modular architecture and co-opt target selectors from various sources to optimize target selection and drive transposon spread. [Display omitted] • A broad survey of Tn7-like transposons clarifies their target selection strategies • Type I-D CASTs transpose via both RNA-guided and protein-mediated mechanisms • Transposon-encoded TnsF is a novel target selector that targets the comM gene • Tsy is a tyrosine recombinase transposon that shares TnsF with Tn7-like Tn6022 Faure et al. use phylogenomic and structural approaches to systematically survey transposon target selectors, revealing a modular architecture that promotes transposon spread. Through this work, they identified a new target selector, TnsF, and a previously uncharacterized transposable element, Tsy. [ABSTRACT FROM AUTHOR]

CRISPR-associated transposons (CASTs) are natural RNA-directed transposition systems. We demonstrate that transposon protein TniQ plays a central role in promoting R-loop formation by RNA-guided DNA-targeting modules. TniQ residues, proximal to CRISPR RNA (crRNA), are required for recognizing different crRNA categories, revealing an unappreciated role of TniQ to direct transposition into different classes of crRNA targets. To investigate adaptations allowing CAST elements to utilize attachment sites inaccessible to CRISPR-Cas surveillance complexes, we compared and contrasted PAM sequence requirements in both I-F3b CAST and I-F1 CRISPR-Cas systems. We identify specific amino acids that enable a wider range of PAM sequences to be accommodated in I-F3b CAST elements compared with I-F1 CRISPR-Cas, enabling CAST elements to access attachment sites as sequences drift and evade host surveillance. Together, this evidence points to the central role of TniQ in facilitating the acquisition of CRISPR effector complexes for RNA-guided DNA transposition. [Display omitted] • Structures of I-F3 CAST effector module reveal roles of transposon protein TniQ • TniQ participates in complete R-loop formation by engaging with target DNA • TniQ regulates target-site choice through interactions with crRNA • Structural basis of PAM ambiguity in I-F3 CAST compared with canonical CRISPR-Cas Park et al. investigate the mechanisms of the DNA-recognition module from I-F3 CRISPR-Cas-associated transposons, Cascade-TniQ. They demonstrate that TniQ functions in target engagement and regulation of target-site choice with Cascade components. Comparing PAM recognition of I-F3 Cascade with canonical CRISPR-Cas reveals the structural basis of PAM ambiguity. [ABSTRACT FROM AUTHOR]

Tn7 is a bacterial transposon with relatives containing element-encoded CRISPR-Cas systems mediating RNA-guided transposon insertion. Here, we present the 2.7 Å cryoelectron microscopy structure of prototypic Tn7 transposase TnsB interacting with the transposon end DNA. When TnsB interacts across repeating binding sites, it adopts a beads-on-a-string architecture, where the DNA-binding and catalytic domains are arranged in a tiled and intertwined fashion. The DNA-binding domains form few base-specific contacts leading to a binding preference that requires multiple weakly conserved sites at the appropriate spacing to achieve DNA sequence specificity. TnsB binding imparts differences in the global structure of the protein-bound DNA ends dictated by the spacing or overlap of binding sites explaining functional differences in the left and right ends of the element. We propose a model of the strand-transfer complex in which the terminal TnsB molecule is rearranged so that its catalytic domain is in a position conducive to transposition. [Display omitted] • Cryo-EM structure of Tn7 transposase TnsB in complex with transposon end DNA • TnsB chains interact with its binding sites in DNA in a tiled and intertwined fashion • TnsB exhibits a DNA sequence preference rather than strict specificity • Overlap of TnsB-binding sites in the right Tn7 end is key for its functionality Kaczmarska et al. present a structure of Tn7 transposon transposase TnsB bound to transposon end DNA. Tn7 is a bacterial transposon. Its relatives containing CRISPR-Cas machinery are promising tools for gene editing. TnsB molecules interact with repeating binding sites in the transposon end in an intertwined fashion for specific end recognition. [ABSTRACT FROM AUTHOR]

CRISPR-Cas biology and technologies have been largely shaped to date by the characterization and use of single-effector nucleases. By contrast, multi-subunit effectors dominate natural systems, represent emerging technologies, and were recently associated with RNA-guided DNA transposition. This disconnect stems from the challenge of working with multiple protein subunits in vitro and in vivo. Here, we apply cell-free transcription-translation (TXTL) systems to radically accelerate the characterization of multi-subunit CRISPR effectors and transposons. Numerous DNA constructs can be combined in one TXTL reaction, yielding defined biomolecular readouts in hours. Using TXTL, we mined phylogenetically diverse I-E effectors, interrogated extensively self-targeting I-C and I-F systems, and elucidated targeting rules for I-B and I-F CRISPR transposons using only DNA-binding components. We further recapitulated DNA transposition in TXTL, which helped reveal a distinct branch of I-B CRISPR transposons. These capabilities will facilitate the study and exploitation of the broad yet underexplored diversity of CRISPR-Cas systems and transposons. [Display omitted] • PAM-DETECT for rapid determination of PAMs for Type I CRISPR-Cas systems in TXTL • Mining of Type I orthologs and characterization of extensively self-targeting systems • TXTL-based assessment of DNA target recognition and transposition by CASTs • Identification of a distinct branch of Type I-B CRISPR transposons Wimmer et al. report how cell-free transcription-translation (TXTL) systems can be utilized to rapidly characterize multi-subunit CRISPR effectors. The provided methodologies allow comprehensive PAM determination and the interrogation of DNA transposition by CRISPR-associated transposons, accelerating the characterization of difficult-to-study systems that are highly prevalent and hold technological promise. [ABSTRACT FROM AUTHOR]

Canonical CRISPR-Cas systems utilize RNA-guided nucleases for targeted cleavage of foreign nucleic acids, whereas some nuclease-deficient CRISPR-Cas complexes have been repurposed to direct the insertion of Tn 7 -like transposons. Here, we established a bioinformatic and experimental pipeline to comprehensively explore the diversity of Type I-F CRISPR-associated transposons. We report DNA integration for 20 systems and identify a highly active subset that exhibits complete orthogonality in transposon DNA mobilization. We reveal the modular nature of CRISPR-associated transposons by exploring the horizontal acquisition of targeting modules and by characterizing a system that encodes both a programmable, RNA-dependent pathway, and a fixed, RNA-independent pathway. Finally, we analyzed transposon-encoded cargo genes and found the striking presence of anti-phage defense systems, suggesting a role in transmitting innate immunity between bacteria. Collectively, this study substantially advances our biological understanding of CRISPR-associated transposon function and expands the suite of RNA-guided transposases for programmable, large-scale genome engineering. [Display omitted] • A diverse suite of 20 CRISPR-associated transposons function robustly in E. coli • Some systems exhibit unique modularity and possess multiple DNA targeting pathways • RNA-guided transposons encode diverse anti-phage defense systems • A selection of efficient, orthogonal transposases provides new genome editing tools Klompe et al. explore the natural diversity and function of CRISPR-associated transposons, which direct RNA-guided DNA integration. Further investigation of eighteen new systems reveals the modular nature of molecular machineries that perform DNA targeting and DNA insertion. A selection of high-efficiency, high-fidelity, and fully orthogonal systems is reported. [ABSTRACT FROM AUTHOR]

The type V-K CRISPR-Cas system, featured by Cas12k effector with a naturally inactivated RuvC domain and associated with Tn7-like transposon for RNA-guided DNA transposition, is a promising tool for precise DNA insertion. To reveal the mechanism underlying target DNA recognition, we determined a cryoelectron microscopy (cryo-EM) structure of Cas12k from cyanobacteria Scytonema hofmanni in complex with a single guide RNA (sgRNA) and a double-stranded target DNA. Coupled with mutagenesis and in vitro DNA transposition assay, our results revealed mechanisms for the recognition of the GGTT protospacer adjacent motif (PAM) sequence and the structural elements of Cas12k critical for RNA-guided DNA transposition. These structural and mechanistic insights should aid in the development of type V-K CRISPR-transposon systems as tools for genome editing. [Display omitted] • Cryo-EM structure of the Cas12k-sgRNA-target DNA complex is presented at 3.6 Å • Cas12k uses an unusually large sgRNA for RNA-guided DNA transposition • Identification of key residues for recognition of the PAM sequence • Cryo-EM structure of the Cas12k-sgRNA complex is solved at 3.8 Å CRISPR-associated transposase (CAST) systems allow for RNA-guided DNA insertion at precise locations in genomes and are promising tools for genome editing. Xiao et al. report the structure of Cas12k, the CRISPR effector protein of the type V-K CAST system, and present mechanistic insights into target DNA recognition and RNA-guided transposition. [ABSTRACT FROM AUTHOR]