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.

与 CRISPR 相关的转座系统允许在距可编程靶序列固定距离的一个方向上引导 RNA 定向整合单个 DNA 货物。我们使用低温电子显微镜 (cryo-EM) 通过表征来自 VK 型 CRISPR 转座酶系统的转座调节因子 TnsC 来定义该过程的基础机制。在这种情况下，三磷酸腺苷结合的 TnsC 螺旋丝的聚合可以解释极性信息如何传递给转座酶。TniQ 覆盖 TnsC 灯丝，代表 Tn7/Tn7 类元素中目标信息传输的通用机制。转座酶驱动的拆卸仅将元素递送至未使用的原型间隔区。最后，TnsC 转换定义插入的固定点，₃ . 这些机制研究结果为用于研究和治疗应用的工程 CRISPR 相关转座系统提供了基础。