Structural basis for target site selection in RNA-guided DNA transposition systems

Abstract

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.

Cargo Genes of Tn 7 -Like Transposons Comprise an Enormous Diversity of Defense Systems, Mobile Genetic Elements, and Antibiotic Resistance Genes

“…CASTs and other Tn 7 -like transposons integrate at a fixed distance downstream from their target sites ( 27 , 54 ) ( Fig. 1B ), which is typically ∼50 bp in CASTs and likely corresponds to the footprint of the transposition complex ( 30 , 33 , 55 , 56 ). In total, the LEs/REs were predicted for 352/523 (67%) of the previously reported CASTs.…”

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

“…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).…”

CvkR, a novel MerR-type transcriptional regulator, is a repressor of class 2 type V-K CRISPR-associated transposase systems

“…The growth in the 5’ to 3’ direction is stopped by TniQ binding at the filament end concomitantly connecting the TnsC filament with Cas12k. On the other filament end, the Mu-like transposase TnsB then starts to integrate the transposon 15 . In addition to the genes encoding transposase and effector proteins, all of these systems contain various numbers of cargo genes.…”

“…Targeting transposition by these CAST systems depends on the DNA-crRNA interaction facilitated by the effector protein Cas12k 11 . The TnsC transposon then forms helical polymers around the DNA supported by ATP binding 15 . The growth in the 5' to 3' direction is stopped by TniQ binding at the filament end concomitantly connecting the TnsC filament with Cas12k.…”

“…On the other filament end, the Mu-like transposase TnsB then starts to integrate the transposon 15 . In addition to the genes encoding transposase and effector proteins, all of these systems contain various numbers of cargo genes.…”