Since type V-K CAST does not comprise any other protein to replace TnsA endonuclease activity, its transposition produces co-integrates that need to be resolved 15, 16. However, type V-K CAST systems are different from Tn7 due to the lack of TnsA in their loci. The interaction of TniQ with the target DNA bound by the CRISPR-Cas complex is thought to create a distortion in the DNA structure, allowing TnsC to recognize both TniQ and the DNA 14, and leading to the insertion of the transposon at the attachment site. TnsC is part of the AAA+ ATPase family and directs TnsA/TnsB to the insertion site 11, 13. In the canonical Tn7 system, the interaction of TnsA and TnsB is necessary to activate catalysis 12. TnsB is a recombinase and catalyses the cleavage of the 3′-ends of the transposon. TnsA is an endonuclease that cleaves the 5′-ends of the transposon 9 and interacts with TnsB, TnsC and DNA 9, 10, 11. In an analogy with the Tn7 transposon systems, the CAST Tn 7 proteins are thought to assemble into a pre-integration nucleoprotein complex that involves TnsA (in Types I and IV), TnsB, TnsC and TniQ, to regulate transposition into the insertion site. ![]() coli TnsD), and, in certain cases, TnsA genes. This includes TnsB, TnsC, TniQ (a homologue of E. To date, all known CASTs are derived from Tn 7-like transposons and include the corresponding crRNAs and Cas genes necessary for target selection 6, 7, and the core transposition machinery within a Tn 7-like transposon locus. Therefore, CASTs are thought to provide a very promising system for the development of next-generation gene-editing tools.ĬAST I-F, I-B and V-K subtypes, from Vibrio cholerae (vc), Anabaena variabilis (av) and Scytonema hofmannii (sh), respectively, were the first ones to be discovered 3, 4, but recent bioinformatic searches of metagenomic databases have vastly expanded the known CAST repertoire to over 1000 non-redundant subsystems representing Types I, IV and V 6. They insert large DNA cargos (10–30 kb) at specific genome regions without the need for homology-directed repair 4, 5, 6, 7, combining the site-selection precision of CRISPR-Cas with the integration properties of transposons 8. These complexes do not degrade their target DNA and operate exclusively in prokaryotes. These CAST systems 3, 4 are a product of an evolutionary process by which Tn 7-like transposons recruited the CRISPR-Cas system for transposon mobilization. Recently, several CRISPR-Cas machineries have been found associated with Tn7-like transposon systems in types I, IV and V. Especially, Class 2 members have garnered much attention as they have been developed into versatile RNA-guided nucleases for RNA-guided genome editing, which has radically altered life sciences, enabling genome manipulation in living organisms 2. The two classes are further divided into six types (I–VI) depending on the identity of the nuclease module, and many subtypes depending on which other Cas proteins are present in other functional modules. They are divided into two classes, Class 1 and Class 2, the former including a multi-subunit effector complex and the later a single protein effector 1. CRISPR-Cas systems are highly diverse ribonucleoprotein (RNP) complexes with different evolutionary origins. The discovery of an adaptive prokaryotic immune system called Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), in which the repeats associate with Cas (CRISPR-associated) proteins, has constituted a revolution in life sciences. Our structure sheds light on the strand transfer reaction of DDE transposases and offers new insights into CAST transposition. ![]() DNA binding residue mutants reveal that lack of specificity decreases activity, but it could increase transposition in some cases. Collectively, the structural features suggest that catalysis is coupled to protein-DNA assembly to secure proper DNA integration. A singular in trans association of NTD1 domains of the catalytically competent subunits with the inactive DDE domains reinforces the assembly. Transposon end recognition is accomplished by the NTD1/2 helical domains. Two protomers involved in strand transfer display a catalytically competent active site composed by DDE residues, while other two, which play a key structural role, show active sites where the catalytic residues are not properly positioned for phosphodiester hydrolysis. The strand transfer complex displays an intertwined pseudo-symmetrical architecture. ![]() Here we present the 2.4 Å cryo-EM structure of the Scytonema hofmannii (sh) TnsB transposase from Type V-K CAST, bound to the strand transfer DNA. CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opted CRISPR-Cas systems for RNA-guided transposition.
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