Target site selection and remodelling by type V CRISPR-transposon systems

Structural basis for RNA-mediated assembly of type V CRISPR-associated transposons

“…The resulting atomic model of the complex comprises Cas12k and the sgRNA guide bound to the target site in the DNA, together with a single TniQ molecule and an emerging right-handed TnsC filament assembled on the PAM-distal region of the target DNA. Cas12k binds the DNA in a guide RNA- and PAM-dependent manner, as observed previously in the structure of the Cas12k-sgRNA-target DNA complex (Querques et al, 2021; Xiao et al, 2021). TniQ makes direct contacts with the tracrRNA part of the sgRNA and two TnsC protomers, thereby bridging Cas12k and the TnsC filament without directly interacting with Cas12k.…”

“…For type V-K systems, RNA-guided transposition relies on the CRISPR effector complex comprising Cas12k, a crRNA and a trans-activating RNA (tracrRNA), and three transposon proteins: the AAA+ ATPase TnsC, the transposase TnsB and the zinc-finger protein TniQ (Strecker et al, 2019). Biochemical and structural studies of a type V CAST system from Scytonema hofmanni (ShCAST) revealed that Cas12k-mediated DNA targeting depends on the intricate architecture of the tracrRNA that serves as a scaffold to correctly position Cas12k and the crRNA guide for the recognition of complementary targets (Park et al, 2021; Querques et al, 2021; Xiao et al, 2021). Upon binding to a 5’-GTN protospacer adjacent motif (PAM) in the target DNA, Cas12k initiates guide RNA hybridization that generates an incomplete R-loop structure, suggesting that further rearrangements occurring upon recruitment of the downstream transposon machinery are required to elicit further guide RNA-target DNA hybridization (Strecker et al, 2019).…”

“…Upon binding to a 5’-GTN protospacer adjacent motif (PAM) in the target DNA, Cas12k initiates guide RNA hybridization that generates an incomplete R-loop structure, suggesting that further rearrangements occurring upon recruitment of the downstream transposon machinery are required to elicit further guide RNA-target DNA hybridization (Strecker et al, 2019). In turn, structural and biochemical studies revealed that TnsC assembles ATP-dependent helical filaments on double-stranded DNA, remodeling its duplex geometry (Park et al, 2021; Querques et al, 2021). Based on homology to the prototypical E. coli Tn7 transposon (Peters and Craig, 2001) and analysis of transposase-mediated integration events (Vo et al, 2021a), the DDE-type TnsB transposase has been postulated to catalyze the 3′-DNA strand breakage and transfer reactions required for a replicative transposition mechanism, resulting in transposon end nicking and ligation to a target DNA at sites located 60-66 bp downstream of the PAM (Strecker et al, 2019).…”

“…TnsC is thought to recruit and activate TnsB at the target site (Peters and Craig, 2001). Concurrently, TnsB interacts with the TnsC filament to trigger its disassembly by stimulating the ATPase activity of TnsC (Park et al, 2021; Querques et al, 2021). This prevents insertion of additional transposon copies into the same site and ensures target immunity, which is a conserved feature of Tn transposons harboring coupled transposase and AAA+ ATPase components (Adzuma and Mizuuchi, 1988; Greene and Mizuuchi, 2002) (Skelding et al, 2003) (Klompe et al, 2019; Strecker et al, 2019).…”

“…The function of TniQ is also presently unclear. In type V-K systems, TniQ directly interacts with one end of the TnsC filament and has been implicated in the regulation of its polymerization (Park et al, 2021; Querques et al, 2021). Conversely, in type I-F3 systems a TniQ homodimer is an integral component of the DNA targeting complex together with the Cascade (Halpin-Healy et al, 2019; Jia et al, 2020; Li et al, 2020).…”