Diversity, evolution, and classification of the RNA-guided nucleases TnpB and Cas12.

The TnpB proteins are transposon-associated RNA-guided nucleases that are among the most abundant proteins encoded in bacterial and archaeal genomes, but whose functions in the transposon life cycle remain unknown. TnpB appears to be the evolutionary ancestor of Cas12, the effector nuclease of type V CRISPR-Cas systems. We performed a comprehensive census of TnpBs in archaeal and bacterial genomes and constructed a phylogenetic tree on which we mapped various features of these proteins. In multiple branches of the tree, the catalytic site of the TnpB nuclease is rearranged, demonstrating structural and probably biochemical malleability of this enzyme. We identified numerous cases of apparent recruitment of TnpB for other functions of which the most common is the evolution of type V CRISPR-Cas effectors on about 50 independent occasions. In many other cases of more radical exaptation, the catalytic site of the TnpB nuclease is apparently inactivated, suggesting a regulatory function, whereas in others, the activity appears to be retained, indicating that the recruited TnpB functions as a nuclease, for example, as a toxin. These findings demonstrate remarkable evolutionary malleability of the TnpB scaffold and provide extensive opportunities for further exploration of RNA-guided biological systems as well as multiple applications.

Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28.

CRISPR-Cas adaptive immune systems defend microbes against foreign nucleic acids via RNA-guided endonucleases. Using a computational sequence database mining approach, we identify two class 2 CRISPR-Cas systems (subtype VI-B) that lack Cas1 and Cas2 and encompass a single large effector protein, Cas13b, along with one of two previously uncharacterized associated proteins, Csx27 and Csx28. We establish that these CRISPR-Cas systems can achieve RNA interference when heterologously expressed. Through a combination of biochemical and genetic experiments, we show that Cas13b processes its own CRISPR array with short and long direct repeats, cleaves target RNA, and exhibits collateral RNase activity. Using an E. coli essential gene screen, we demonstrate that Cas13b has a double-sided protospacer-flanking sequence and elucidate RNA secondary structure requirements for targeting. We also find that Csx27 represses, whereas Csx28 enhances, Cas13b-mediated RNA interference. Characterization of these CRISPR systems creates opportunities to develop tools to manipulate and monitor cellular transcripts.

Widespread CRISPR-derived RNA regulatory elements in CRISPR-Cas systems.

CRISPR-cas loci typically contain CRISPR arrays with unique spacers separating direct repeats. Spacers along with portions of adjacent repeats are transcribed and processed into CRISPR(cr) RNAs that target complementary sequences (protospacers) in mobile genetic elements, resulting in cleavage of the target DNA or RNA. Additional, standalone repeats in some CRISPR-cas loci produce distinct cr-like RNAs implicated in regulatory or other functions. We developed a computational pipeline to systematically predict crRNA-like elements by scanning for standalone repeat sequences that are conserved in closely related CRISPR-cas loci. Numerous crRNA-like elements were detected in diverse CRISPR-Cas systems, mostly, of type I, but also subtype V-A. Standalone repeats often form mini-arrays containing two repeat-like sequence separated by a spacer that is partially complementary to promoter regions of cas genes, in particular cas8, or cargo genes located within CRISPR-Cas loci, such as toxins-antitoxins. We show experimentally that a mini-array from a type I-F1 CRISPR-Cas system functions as a regulatory guide. We also identified mini-arrays in bacteriophages that could abrogate CRISPR immunity by inhibiting effector expression. Thus, recruitment of CRISPR effectors for regulatory functions via spacers with partial complementarity to the target is a common feature of diverse CRISPR-Cas systems.

Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems.

Microbial CRISPR-Cas systems are divided into Class 1, with multisubunit effector complexes, and Class 2, with single protein effectors. Currently, only two Class 2 effectors, Cas9 and Cpf1, are known. We describe here three distinct Class 2 CRISPR-Cas systems. The effectors of two of the identified systems, C2c1 and C2c3, contain RuvC-like endonuclease domains distantly related to Cpf1. The third system, C2c2, contains an effector with two predicted HEPN RNase domains. Whereas production of mature CRISPR RNA (crRNA) by C2c1 depends on tracrRNA, C2c2 crRNA maturation is tracrRNA independent. We found that C2c1 systems can mediate DNA interference in a 5'-PAM-dependent fashion analogous to Cpf1. However, unlike Cpf1, which is a single-RNA-guided nuclease, C2c1 depends on both crRNA and tracrRNA for DNA cleavage. Finally, comparative analysis indicates that Class 2 CRISPR-Cas systems evolved on multiple occasions through recombination of Class 1 adaptation modules with effector proteins acquired from distinct mobile elements.

CRISPRidentify: identification of CRISPR arrays using machine learning approach.

CRISPR-Cas are adaptive immune systems that degrade foreign genetic elements in archaea and bacteria. In carrying out their immune functions, CRISPR-Cas systems heavily rely on RNA components. These CRISPR (cr) RNAs are repeat-spacer units that are produced by processing of pre-crRNA, the transcript of CRISPR arrays, and guide Cas protein(s) to the cognate invading nucleic acids, enabling their destruction. Several bioinformatics tools have been developed to detect CRISPR arrays based solely on DNA sequences, but all these tools employ the same strategy of looking for repetitive patterns, which might correspond to CRISPR array repeats. The identified patterns are evaluated using a fixed, built-in scoring function, and arrays exceeding a cut-off value are reported. Here, we instead introduce a data-driven approach that uses machine learning to detect and differentiate true CRISPR arrays from false ones based on several features. Our CRISPR detection tool, CRISPRidentify, performs three steps: detection, feature extraction and classification based on manually curated sets of positive and negative examples of CRISPR arrays. The identified CRISPR arrays are then reported to the user accompanied by detailed annotation. We demonstrate that our approach identifies not only previously detected CRISPR arrays, but also CRISPR array candidates not detected by other tools. Compared to other methods, our tool has a drastically reduced false positive rate. In contrast to the existing tools, our approach not only provides the user with the basic statistics on the identified CRISPR arrays but also produces a certainty score as a practical measure of the likelihood that a given genomic region is a CRISPR array.

PpCas9 from Pasteurella pneumotropica - a compact Type II-C Cas9 ortholog active in human cells.

CRISPR-Cas defense systems opened up the field of genome editing due to the ease with which effector Cas nucleases can be programmed with guide RNAs to access desirable genomic sites. Type II-A SpCas9 from Streptococcus pyogenes was the first Cas9 nuclease used for genome editing and it remains the most popular enzyme of its class. Nevertheless, SpCas9 has some drawbacks including a relatively large size and restriction to targets flanked by an 'NGG' PAM sequence. The more compact Type II-C Cas9 orthologs can help to overcome the size limitation of SpCas9. Yet, only a few Type II-C nucleases were fully characterized to date. Here, we characterized two Cas9 II-C orthologs, DfCas9 from Defluviimonas sp.20V17 and PpCas9 from Pasteurella pneumotropica. Both DfCas9 and PpCas9 cleave DNA in vitro and have novel PAM requirements. Unlike DfCas9, the PpCas9 nuclease is active in human cells. This small nuclease requires an 'NNNNRTT' PAM orthogonal to that of SpCas9 and thus potentially can broaden the range of Cas9 applications in biomedicine and biotechnology.

DNA targeting by Clostridium cellulolyticum CRISPR-Cas9 Type II-C system.

Type II CRISPR-Cas9 RNA-guided nucleases are widely used for genome engineering. Type II-A SpCas9 protein from Streptococcus pyogenes is the most investigated and highly used enzyme of its class. Nevertheless, it has some drawbacks, including a relatively big size, imperfect specificity and restriction to DNA targets flanked by an NGG PAM sequence. Cas9 orthologs from other bacterial species may provide a rich and largely untapped source of biochemical diversity, which can help to overcome the limitations of SpCas9. Here, we characterize CcCas9, a Type II-C CRISPR nuclease from Clostridium cellulolyticum H10. We show that CcCas9 is an active endonuclease of comparatively small size that recognizes a novel two-nucleotide PAM sequence. The CcCas9 can potentially broaden the existing scope of biotechnological applications of Cas9 nucleases and may be particularly advantageous for genome editing of C. cellulolyticum H10, a bacterium considered to be a promising biofuel producer.

Systematic prediction of genes functionally linked to CRISPR-Cas systems by gene neighborhood analysis.

The CRISPR-Cas systems of bacterial and archaeal adaptive immunity consist of direct repeat arrays separated by unique spacers and multiple CRISPR-associated (cas) genes encoding proteins that mediate all stages of the CRISPR response. In addition to the relatively small set of core cas genes that are typically present in all CRISPR-Cas systems of a given (sub)type and are essential for the defense function, numerous genes occur in CRISPR-cas loci only sporadically. Some of these have been shown to perform various ancillary roles in CRISPR response, but the functional relevance of most remains unknown. We developed a computational strategy for systematically detecting genes that are likely to be functionally linked to CRISPR-Cas. The approach is based on a "CRISPRicity" metric that measures the strength of CRISPR association for all protein-coding genes from sequenced bacterial and archaeal genomes. Uncharacterized genes with CRISPRicity values comparable to those of cas genes are considered candidate CRISPR-linked genes. We describe additional criteria to predict functionally relevance for genes in the candidate set and identify 79 genes as strong candidates for functional association with CRISPR-Cas systems. A substantial majority of these CRISPR-linked genes reside in type III CRISPR-cas loci, which implies exceptional functional versatility of type III systems. Numerous candidate CRISPR-linked genes encode integral membrane proteins suggestive of tight membrane association of CRISPR-Cas systems, whereas many others encode proteins implicated in various signal transduction pathways. These predictions provide ample material for improving annotation of CRISPR-cas loci and experimental characterization of previously unsuspected aspects of CRISPR-Cas system functionality.

Building Functional Nanodevices with Vesicle-Templated Porous Polymer Nanocapsules.

Vesicle-templated nanocapsules offer a unique combination of properties enabled by robust shells with single-nanometer thickness containing programmed uniform pores capable of fast and selective mass transfer. These capsules emerged as a versatile platform for creating functional devices, such as nanoreactors, nanosensors, and containers for the delivery of drugs and imaging agents. Nanocapsules are synthesized by a directed assembly method using self-assembled bilayers of vesicles as temporary scaffolds. In this approach, hydrophobic building blocks are loaded into the hydrophobic interior of vesicles formed from lipids or surfactants. Pore-forming templates are codissolved with the monomers and cross-linkers in the interior of the bilayer. The polymerization forms a cross-linked shell with embedded pore-forming templates. Removal of the surfactant scaffold and pore-forming templates leads to free-standing nanocapsules with shells containing uniform imprinted nanopores. Development of reliable and scalable synthetic methods for the modular construction of capsules with tunable properties has opened the opportunity to pursue practical applications of nanocapsules. In this Account, we discuss how unique properties of vesicle-templated nanocapsules translate into the creation of functional nanodevices. Specifically, we focus the conversation on applications aiming at the delivery of drugs and imaging agents, creation of fast-acting and selective nanoreactors, and fabrication of nanoprobes for sensing and imaging. We present a brief overview of the synthesis of nanocapsules with an emphasis on recent developments leading to robust synthetic methods including the synthesis under physiological conditions and creation of biodegradable nanocapsules. We then highlight unique properties of nanocapsules essential for practical applications, such as precise control of pore size and chemical environment, selective permeability, and ultrafast transport through the pores. We discuss new motifs for catch and release of small molecules with porous nanocapsules based on controlling the microenvironment inside the nanocapsules, regulating the charge on the orifice of nanopores in the shells, and reversible synergistic action of host and guest forming a supramolecular complex in nanocapsules. We demonstrate successful creation of fast-acting and selective nanoreactors by encapsulation of diverse homogeneous and nanoparticle catalysts. Due to unhindered flow of substrates and products through the nanopores, encapsulation did not compromise catalytic efficiency and, in fact, improved the stability of entrapped catalysts. We present robust nanoprobes based on nanocapsules with entrapped sensing agents and show how the encapsulation resulted in selective measurements with fast response times in challenging conditions, such as small volumes and complex mixtures. Throughout this Account, we highlight the advantages of encapsulation and discuss the opportunities for future design of nanodevices.

Recruitment of CRISPR-Cas systems by Tn7-like transposons.

A survey of bacterial and archaeal genomes shows that many Tn7-like transposons contain minimal type I-F CRISPR-Cas systems that consist of fused cas8f and cas5f, cas7f, and cas6f genes and a short CRISPR array. Several small groups of Tn7-like transposons encompass similarly truncated type I-B CRISPR-Cas. This minimal gene complement of the transposon-associated CRISPR-Cas systems implies that they are competent for pre-CRISPR RNA (precrRNA) processing yielding mature crRNAs and target binding but not target cleavage that is required for interference. Phylogenetic analysis demonstrates that evolution of the CRISPR-Cas-containing transposons included a single, ancestral capture of a type I-F locus and two independent instances of type I-B loci capture. We show that the transposon-associated CRISPR arrays contain spacers homologous to plasmid and temperate phage sequences and, in some cases, chromosomal sequences adjacent to the transposon. We hypothesize that the transposon-encoded CRISPR-Cas systems generate displacement (R-loops) in the cognate DNA sites, targeting the transposon to these sites and thus facilitating their spread via plasmids and phages. These findings suggest the existence of RNA-guided transposition and fit the guns-for-hire concept whereby mobile genetic elements capture host defense systems and repurpose them for different stages in the life cycle of the element.

Comparative genomics and evolution of trans-activating RNAs in Class 2 CRISPR-Cas systems.

Trans-activating CRISPR (tracr) RNA is a distinct RNA species that interacts with the CRISPR (cr) RNA to form the dual guide (g) RNA in type II and subtype V-B CRISPR-Cas systems. The tracrRNA-crRNA interaction is essential for pre-crRNA processing as well as target recognition and cleavage. The tracrRNA consists of an antirepeat, which forms an imperfect hybrid with the repeat in the crRNA, and a distal region containing a Rho-independent terminator. Exhaustive comparative analysis of the sequences and predicted structures of the Class 2 CRISPR guide RNAs shows that all these guide RNAs share distinct structural features, in particular, the nexus stem-loop that separates the repeat-antirepeat hybrid from the distal portion of the tracrRNA and the conserved GU pair at that end of the hybrid. These structural constraints might ensure full exposure of the spacer for target recognition. Reconstruction of tracrRNA evolution for 4 tight bacterial groups demonstrates random drift of repeat-antirepeat complementarity within a window of hybrid stability that is, apparently, maintained by selection. An evolutionary scenario is proposed whereby tracrRNAs evolved on multiple occasions, via rearrangement of a CRISPR array to form the antirepeat in different locations with respect to the array. A functional tracrRNA would form if, in the new location, the antirepeat is flanked by sequences that meet the minimal requirements for a promoter and a Rho-independent terminator. Alternatively, or additionally, the antirepeat sequence could be occasionally 'reset' by recombination with a repeat, restoring the functionality of tracrRNAs that drift beyond the required minimal hybrid stability.

Altered stoichiometry Escherichia coli Cascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation.

The Escherichia coli type I-E CRISPR-Cas system Cascade effector is a multisubunit complex that binds CRISPR RNA (crRNA). Through its 32-nucleotide spacer sequence, Cascade-bound crRNA recognizes protospacers in foreign DNA, causing its destruction during CRISPR interference or acquisition of additional spacers in CRISPR array during primed CRISPR adaptation. Within Cascade, the crRNA spacer interacts with a hexamer of Cas7 subunits. We show that crRNAs with a spacer length reduced to 14 nucleotides cause primed adaptation, while crRNAs with spacer lengths of more than 20 nucleotides cause both primed adaptation and target interference in vivo Shortened crRNAs assemble into altered-stoichiometry Cascade effector complexes containing less than the normal amount of Cas7 subunits. The results show that Cascade assembly is driven by crRNA and suggest that multisubunit type I CRISPR effectors may have evolved from much simpler ancestral complexes.

Phylogenomics of Cas4 family nucleases.

BACKGROUND: The Cas4 family endonuclease is a component of the adaptation module in many variants of CRISPR-Cas adaptive immunity systems. Unlike most of the other Cas proteins, Cas4 is often encoded outside CRISPR-cas loci (solo-Cas4) and is also found in mobile genetic elements (MGE-Cas4). RESULTS: As part of our ongoing investigation of CRISPR-Cas evolution, we explored the phylogenomics of the Cas4 family. About 90% of the archaeal genomes encode Cas4 compared to only about 20% of the bacterial genomes. Many archaea encode both the CRISPR-associated form (CAS-Cas4) and solo-Cas4, whereas in bacteria, this combination is extremely rare. The solo-cas4 genes are over-represented in environmental bacteria and archaea with small genomes that typically lack CRISPR-Cas, suggesting that Cas4 could perform uncharacterized defense or repair functions in these microbes. Phylogenomic analysis indicates that both the CRISPR-associated cas4 genes are often transferred horizontally but almost exclusively, as part of the adaptation module. The evolutionary integrity of the adaptation module sharply contrasts the rampant shuffling of CRISPR-cas modules whereby a given variant of the adaptation module can combine with virtually any effector module. The solo-cas4 genes evolve primarily via vertical inheritance and are subject only to occasional horizontal transfer. The selection pressure on cas4 genes does not substantially differ between CAS-Cas4 and solo-cas4, and is close to the genomic median. Thus, cas4 genes, similarly to cas1 and cas2, evolve similarly to 'regular' microbial genes involved in various cellular functions, showing no evidence of direct involvement in virus-host arms races. A notable feature of the Cas4 family evolution is the frequent recruitment of cas4 genes by various mobile genetic elements (MGE), particularly, archaeal viruses. The functions of Cas4 in these elements are unknown and potentially might involve anti-defense roles. CONCLUSIONS: Unlike most of the other Cas proteins, Cas4 family members are as often encoded by stand-alone genes as they are incorporated in CRISPR-Cas systems. In addition, cas4 genes were repeatedly recruited by MGE, perhaps, for anti-defense functions. Experimental characterization of the solo and MGE-encoded Cas4 nucleases is expected to reveal currently uncharacterized defense and anti-defense systems and their interactions with CRISPR-Cas systems.

Dynamics of Escherichia coli type I-E CRISPR spacers over 42 000 years.

CRISPR-Cas are nucleic acid-based prokaryotic immune systems. CRISPR arrays accumulate spacers from foreign DNA and provide resistance to mobile genetic elements containing identical or similar sequences. Thus, the set of spacers present in a given bacterium can be regarded as a record of encounters of its ancestors with genetic invaders. Such records should be specific for different lineages and change with time, as earlier acquired spacers get obsolete and are lost. Here, we studied type I-E CRISPR spacers of Escherichia coli from extinct pachyderm. We find that many spacers recovered from intestines of a 42 000-year-old mammoth match spacers of present-day E. coli. Present-day CRISPR arrays can be reconstructed from palaeo sequences, indicating that the order of spacers has also been preserved. The results suggest that E. coli CRISPR arrays were not subject to intensive change through adaptive acquisition during this time.

Tuning Optical Properties of Encapsulated Clusters of Gold Nanoparticles through Stimuli-Triggered Controlled Aggregation.

Gold nanoparticles entrapped in the hollow polymer nanocapsules undergo pH-mediated controlled aggregation. Encapsulated clusters of nanoparticles show absorbance at higher wavelengths compared with individual nanoparticles. The size of the aggregates is controlled by the number of nanoparticles entrapped in individual nanocapsules. Such controlled aggregation may permit small biocompatible nanoparticles exhibit desirable properties for biomedical applications that are typically characteristic of large nanoparticles.

Pervasive generation of oppositely oriented spacers during CRISPR adaptation.

During the process of prokaryotic CRISPR adaptation, a copy of a segment of foreign deoxyribonucleic acid referred to as protospacer is added to the CRISPR cassette and becomes a spacer. When a protospacer contains a neighboring target interference motif, the specific small CRISPR ribonucleic acid (crRNA) transcribed from expanded CRISPR cassette can protect a prokaryotic cell from virus infection or plasmid transformation and conjugation. We show that in Escherichia coli, a vast majority of plasmid protospacers generate spacers integrated in CRISPR cassette in two opposing orientations, leading to frequent appearance of complementary spacer pairs in a population of cells that underwent CRISPR adaptation. When a protospacer contains a spacer acquisition motif AAG, spacer orientation that generates functional protective crRNA is strongly preferred. All other protospacers give rise to spacers oriented in both ways at comparable frequencies. This phenomenon increases the repertoire of available spacers and should make it more likely that a protective crRNA is formed as a result of CRISPR adaptation.

Size-selective yolk-shell nanoreactors with nanometer-thin porous polymer shells.

Yolk-shell nanoreactors with metal nanoparticle core and ultrathin porous polymer shells are effective catalysts for heterogeneous reactions. Polymer shells provide size-selectivity and improved reusability of catalyst. Nanocapsules with single-nanometer porous shells are prepared by vesicle-templated directed assembly. Metal nanoparticles are formed either by selective initiation in pre-fabricated nanocapsules or simultaneously with the creation of a crosslinked polymer shell. In this study, we investigated the oxidation of benzyl alcohol and benzaldehyde catalyzed by gold nanoparticles and hydrogenation of cyclohexene catalyzed by platinum nanoparticles. Comparison of newly created nanoreactors with commercially available nanoparticles revealed superior reusability and size selectivity in nanoreactors while showing no negative effect on reaction kinetics.

Facile directed assembly of hollow polymer nanocapsules within spontaneously formed catanionic surfactant vesicles.

Surfactant vesicles containing monomers in the interior of the bilayer were used to template hollow polymer nanocapsules. This study investigated the formation of surfactant/monomer assemblies by two loading methods, concurrent loading and diffusion loading. The assembly process and the resulting aggregates were investigated with dynamic light scattering, small angle neutron scattering, and small-angle X-ray scattering. Acrylic monomers formed vesicles with a mixture of cationic and anionic surfactants in a broad range of surfactant ratios. Regions with predominant formation of vesicles were broader for compositions containing acrylic monomers compared with blank surfactants. This observation supports the stabilization of the vesicular structure by acrylic monomers. Diffusion loading produced monomer-loaded vesicles unless vesicles were composed from surfactants at the ratios close to the boundary of a vesicular phase region on a phase diagram. Both concurrent-loaded and diffusion-loaded surfactant/monomer vesicles produced hollow polymer nanocapsules upon the polymerization of monomers in the bilayer followed by removal of surfactant scaffolds.

Widespread CRISPR repeat-like RNA regulatory elements in CRISPR-Cas systems.

CRISPR- cas loci typically contain CRISPR arrays with unique spacers separating direct repeats. Spacers along with portions of adjacent repeats are transcribed and processed into CRISPR(cr) RNAs that target complementary sequences (protospacers) in mobile genetic elements, resulting in cleavage of the target DNA or RNA. Additional, standalone repeats in some CRISPR- cas loci produce distinct cr-like RNAs implicated in regulatory or other functions. We developed a computational pipeline to systematically predict crRNA-like elements by scanning for standalone repeat sequences that are conserved in closely related CRISPR- cas loci. Numerous crRNA-like elements were detected in diverse CRISPR-Cas systems, mostly, of type I, but also subtype V-A. Standalone repeats often form mini-arrays containing two repeat-like sequence separated by a spacer that is partially complementary to promoter regions of cas genes, in particular cas8 , or cargo genes located within CRISPR-Cas loci, such as toxins-antitoxins. We show experimentally that a mini-array from a type I-F1 CRISPR-Cas system functions as a regulatory guide. We also identified mini-arrays in bacteriophages that could abrogate CRISPR immunity by inhibiting effector expression. Thus, recruitment of CRISPR effectors for regulatory functions via spacers with partial complementarity to the target is a common feature of diverse CRISPR-Cas systems.

Cargo Genes of Tn7-Like Transposons Comprise an Enormous Diversity of Defense Systems, Mobile Genetic Elements, and Antibiotic Resistance Genes.

Transposition is a major mechanism of horizontal gene mobility in prokaryotes. However, exploration of the genes mobilized by transposons (cargo) is hampered by the difficulty in delineating integrated transposons from their surrounding genetic context. Here, we present a computational approach that allowed us to identify the boundaries of 6,549 Tn7-like transposons. We found that 96% of these transposons carry at least one cargo gene. Delineation of distinct communities in a gene-sharing network demonstrates how transposons function as a conduit of genes between phylogenetically distant hosts. Comparative analysis of the cargo genes reveals significant enrichment of mobile genetic elements (MGEs) nested within Tn7-like transposons, such as insertion sequences and toxin-antitoxin modules, and of genes involved in recombination, anti-MGE defense, and antibiotic resistance. More unexpectedly, cargo also includes genes encoding central carbon metabolism enzymes. Twenty-two Tn7-like transposons carry both an anti-MGE defense system and antibiotic resistance genes, illustrating how bacteria can overcome these combined pressures upon acquisition of a single transposon. This work substantially expands the distribution of Tn7-like transposons, defines their evolutionary relationships, and provides a large-scale functional classification of prokaryotic genes mobilized by transposition. IMPORTANCE Transposons are major vehicles of horizontal gene transfer that, in addition to genes directly involved in transposition, carry cargo genes. However, characterization of these genes is hampered by the difficulty of identification of transposon boundaries. We developed a computational approach for detecting transposon ends and applied it to perform a comprehensive census of the cargo genes of Tn7-like transposons, a large class of bacterial mobile genetic elements (MGE), many of which employ a unique, CRISPR-mediated mechanism of site-specific transposition. The cargo genes encompass a striking diversity of MGE, defense, and antibiotic resistance systems. Unexpectedly, we also identified cargo genes encoding metabolic enzymes. Thus, Tn7-like transposons mobilize a vast repertoire of genes that can have multiple effects on the host bacteria.