CRISPR-associated transposition systems allow guide RNA–directed integration of a single DNA cargo in one orientation at a fixed distance from a programmable target sequence. We used cryo–electron microscopy (cryo-EM) to define the mechanism that underlies this process by characterizing the transposition regulator, TnsC, from a type V-K CRISPR-transposase system. In this scenario, polymerization of adenosine triphosphate–bound TnsC helical filaments could explain how polarity information is passed to the transposase. TniQ caps the TnsC filament, representing a universal mechanism for target information transfer in Tn7/Tn7-like elements. Transposase-driven disassembly establishes delivery of the element only to unused protospacers. Finally, TnsC transitions to define the fixed point of insertion, as revealed by structures with the transition state mimic ADP•AlF3. These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications.

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SummaryHorizontally acquired genetic information in bacterial chromosomes accumulates in blocks termed genomic islands. Tn7-like transposons form genomic islands at a programmed insertion site in bacterial chromosomes, attTn7. Transposition involves five transposon-encoded genes (tnsABCDE) including an atypical heteromeric transposase. One transposase subunit, TnsB, is from the large family of bacterial transposases, the second, TnsA, is related to endonucleases. A regulator protein, TnsC, functions with different target site selecting proteins to recognize different targets. TnsD directs transposition into attTn7, while TnsE encourages horizontal transmission by targeting mobile plasmids. Recent work suggests that distantly related elements with heteromeric transposases exist with alternate targeting pathways that also facilitate the formation of genomic islands. Tn6230 and related elements can be found at a single position in a gene of unknown function (yhiN) in various bacteria as well as in mobile plasmids. Another group we term Tn6022-like elements form pathogenicity islands in the Acinetobacter baumannii comM gene. We find that Tn6022-like elements also appear to have an uncharacterized mechanism for provoking internal transposition and deletion events that serve as a conduit for evolving new elements. As a group, heteromeric transposase elements utilize diverse target site selection mechanisms adapted to the spread and rearrangement of genomic islands.

Tn7 transposable elements are unique for their highly specific, and sometimes programmable, target-site selection mechanisms and precise insertions. All the elements in the Tn7-family utilize a AAA+ adaptor (TnsC) to coordinates target-site selection with transposase activation and prevent insertions at sites already containing a Tn7 element. Due to its multiple functions, TnsC is considered the linchpin in the Tn7 element. Here we present the high-resolution cryo-EM structure of TnsC bound to DNA using a gain-of-function variant of the protein and a DNA substrate that together recapitulate the recruitment to a specific DNA target site. We find that TnsC forms an asymmetric ring on target DNA that segregates target-site selection and transposase recruitment to opposite faces of the ring. Unlike most AAA+ ATPases, TnsC uses a DNA distortion to find the target site but does not remodel DNA to activate transposition. By recognizing pre-distorted substrates, TnsC creates a built-in regulatory mechanism where ATPhydrolysis abolishes ring formation proximal to an existing element. This work unveils how Tn7 and Tn7-like elements determine the strict spacing between the target and integration sites.

SummaryCRISPR-Cas defense systems have been coopted multiple times in nature for guide RNA-directed transposition by Tn7-like elements. Prototypic Tn7 uses dedicated proteins for two targeting pathways, one targeting a neutral and conserved attachment site in the chromosome and a second directing transposition into mobile plasmids facilitating cell-to-cell transfer. We show that Tn7-CRISPR-Cas elements evolved a system of guide RNA categorization to accomplish the same two-pathway lifestyle. Selective regulation of specialized guide RNAs allows long-term memory for access to chromosomal sites upon entry into a new host, while conventional CRISPR features maintain the ability to continually acquire guide RNAs to new plasmid and phage targets. Transposon-encoded guide RNAs are also privatized to be recognized only by the transposon-adapted system working with selective regulation to guard against toxic self-targeting by endogenous CRISPR-Cas defense systems. This information reveals new avenues to engineer guide RNAs for enhanced CRISPR-Cas functionality for genome modification.