Targeted transposition with Tn7 elements: safe sites, mobile plasmids, CRISPR/Cas and beyond

Abstract

Transposon Tn7 is notable for the control it exercises over where transposition events are directed. One Tn7 integration pathways recognizes a highly conserved attachment (att) site in the chromosome, while a second pathway specifically recognizes mobile plasmids that facilitate transfer of the element to new hosts. In this review, I discuss newly discovered families of Tn7-like elements with different targeting pathways. Perhaps the most exciting examples are multiple instances where Tn7-like elements have repurposed CRISPR/Cas systems. In these cases, the CRISPR/Cas systems have lost their canonical defensive function to destroy incoming mobile elements; instead, the systems have been naturally adapted to use guide RNAs to specifically direct transposition into these mobile elements. The new families of Tn7-like elements also include a variety of novel att sites in bacterial chromosomes where genome islands can form. Interesting families have also been revealed where proteins described in the prototypic Tn7 element are fused or otherwise repurposed for the new dual activities. This expanded understanding of Tn7-like elements broadens our view of how genetic systems are repurposed and provides potentially exciting new tools for genome modification and genomics. Future opportunities and challenges to understanding the impact of the new families of Tn7-like elements are discussed.

Figure 1 –

Representation of the two pathways found in Tn7 and Tn7-like elements. Tn7-like transposons newly entering the host on a plasmid, virus, or other DNA can transpose at a high frequency with the TnsABC + TnsD(TniQ) pathway into a host attachment site (attTn7/glmS in the case of Tn7)(green arrow). Different attachment sites are recognized by other Tn7-like elements as described in the text. Tn7 preferentially directs transposition events into mobile plasmids or filamentous phages with a second pathway using TnsABC + TnsE (Red arrow), a pathway that would facilitate rapid cell-to-cell transfer across bacterial populations. Tn7-CRISPR/Cas elements can utilize a TnsABC + TniQ + guide RNA complex to target protospacers recognized by spacers in the element-encoded CRISPR array. As described in the text, the majority of spacer matches identified from CRISPR arrays in CRISPR/Cas associated transposons are from mobile plasmids indicating that these systems likely facilitate distribution of the transposon via mobile plasmids. Insertion into the att site always occur with the right (R) and left (L) ends oriented as indicated for the Tn7-like element in the diagram.

Figure 2 –

Models for how target complexes are assembled at various preferred target sites with Tn7 and Tn7-like elements. The Left (L) and Right (R) ends of the Tn7/Tn7-like elements are indicated with grey rectangles and the body of the element in green. The ends of the element are synapsed with TnsB (purple ovals, labeled B) and shown interfaced at the blue target DNA with TnsC (blue oval) and TnsA (yellow ovals, labeled A) to the site of insertion as recruited by three different targeting mechanisms. A. Prototypic Tn7 transposition into the attTn7 site uses the sequence specific DNA binding protein TnsD(TniQ) (green oval) that recognizes the 3’ coding region of the glmS gene (yellow block arrow) to recruit and activate transposition. Insertion occurs at a single position with the right end of the element integrated closest to glmS. Tn7-like elements may use a similar process for recognizing other att sites using TniQ as described in the text. B. Prototypic Tn7 transposition that recognizes active DNA replication found during plasmid conjugation uses the TnsE proteins which recognizes two features of discontinuous DNA replication in recipient cell bacteria; interaction with the sliding clamp (DnaN – red ring) and 3’ recessed-end substrates to direct transposon integration. Tn7 insertion events occur at multiple positions, but in a single origination with the direction of discontinuous DNA replication in recipient bacteria. C. Tn7-like elements with the I-F variant CRISPR/Cas system use a guide RNA(gRNA) complex to recognize a protospacer with the correct PAM sequence as predominantly found in mobile plasmids to direct transposition events ~49 bp from the protospacer with the right end of the element proximal to the end of the protospacer through an association with TniQ.

Figure 3 –

Representation of the major Tn7-like element families identified in sequenced bacterial genomes. Schematic representation of Tn7 and Tn7-like elements as situated with the attachment site(s) used within each family (bright yellow block arrow)(named where known or indicated with a ?). Transposons are shown with their transposon end sequences (grey polygons) with the right (R) and left (L) ends indicated where relevant. Signature Tn7 proteins families are indicated; the endonuclease TnsA (orange block arrow – endo), the transposase TnsB (purple block arrow – rve), the regulator protein TnsC (blue block arrow - AAA+) and the TnsD/TniQ family protein (green block arrow). Note: one unnamed family of Tn7-like elements has naturally fused TnsA and TnsB proteins, TnsA-B(fusion). Where known, accessory proteins for targeting mobile DNA elements capable of cell-cell transport are indicated (hypothetical activities are indicated with a ?). The Tn5053 site-specific recombination system is indicated (with the tniR-encoded resolvase and the resolution site it recognizes for resolving cointegrates found following replicative transposition indicated (res - red)). Black rectangles indicate spacers found in the CRISPR arrays. Two slashes indicate where cargo genes have been omitted to simplify the figure. An artistic representation of the TnsA phylogenetic tree is shown (Blue), but is available in Peters et. al. 2017 along with information for downloading via ftp. See text for details.

Figure 4 –

Representation of typical examples of the type I-F and I-B CRISPR/Cas systems and the variants found in Tn7-like elements. CRISPR associated (cas) genes are indicated with block arrows. Black rectangles indicate spacers found in the CRISPR arrays. Genes maintained in the typical system and the transposon encoded systems are indicated in red, the spacer acquisition system is indicated in blue, and the nuclease component shown in orange. See text for details.