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 insertion in one orientation at a fixed distance from a programmable target sequence. We define the mechanism explaining this process by characterizing the transposition regulator, TnsC, from a Type V-K CRISPR-transposase system using cryo-EM. Polymerization of ATP-bound TnsC helical filaments explains how polarity information is passed to the transposase. Our Cryo-EM structure of TniQ-TnsC reveals that TniQ caps the TnsC filament, establishing 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, structures with the transition state mimic, ADPᐧAlF3, reveals how TnsC transitions to define the fixed point of insertion. These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications.

Investigating the DNA Damage Response Elicited by Transposable Element Integration

“…CRISPR/Cas12k is the newest candidate for high precision and fidelity gene editing. The CRISPR/Cas12k system acts similarly to the E. coli indigenous TE Tn7, due to its use of Tn7-like proteins TnsA, TnsB, TnsC, and TniQ (Park et al, 2021). However, like Tn7, research on Cas12k is only known in prokaryotic organisms thus far.…”

“…Instead of the typical gRNA to direct the Cas machinery as seen in many other Cas effector proteins, Cas12k utilizes Tn7-like TnsC and TniQ (similar to TnsD in Tn7) to work together as the target site selector machinery. TnsC/TniQ define the fixed point of insertion of the transposon by recognizing the specific attachment site in the host genome to create a controlled gene modification (Park et al, 2021). After TnsC/TniQ localization, CRISPR/Cas12k utilizes a cassette of transposases (TnsA/TnsB) similar to Tn7 to execute DNA integration at a precise distance and orientation (Park et al, 2021).…”

“…TnsC/TniQ define the fixed point of insertion of the transposon by recognizing the specific attachment site in the host genome to create a controlled gene modification (Park et al, 2021). After TnsC/TniQ localization, CRISPR/Cas12k utilizes a cassette of transposases (TnsA/TnsB) similar to Tn7 to execute DNA integration at a precise distance and orientation (Park et al, 2021). Furthermore, TnsC/TniQ function is multifaceted.…”

“…Furthermore, TnsC/TniQ function is multifaceted. Alongside target site selection, TniQ recruits TnsA and TnsB transposases to bind to the ends of the substrate DNA to integrate into the host genome target-site (Park et al, 2021). Lastly, TnsC induces a distortion (unwinding) in the DNA in order for the transposon to insert; however, the implications are unknown at this time (Park et al, 2021).…”