Canonical CRISPR–Cas systems provide adaptive immunity against mobile genetic elements¹. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion^2,3,4,5,6,7. Type V-K CRISPR-associated transposons rely on the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ⁷, but the molecular mechanism of RNA-directed DNA transposition has remained elusive. Here we report cryo-electron microscopic structures of a Cas12k-guide RNA–target DNA complex and a DNA-bound, polymeric TnsC filament from the CRISPR-associated transposon system of the photosynthetic cyanobacterium Scytonema hofmanni. The Cas12k complex structure reveals an intricate guide RNA architecture and critical interactions mediating RNA-guided target DNA recognition. TnsC helical filament assembly is ATP-dependent and accompanied by structural remodelling of the bound DNA duplex. In vivo transposition assays corroborate key features of the structures, and biochemical experiments show that TniQ restricts TnsC polymerization, while TnsB interacts directly with TnsC filaments to trigger their disassembly upon ATP hydrolysis. Together, these results suggest that RNA-directed target selection by Cas12k primes TnsC polymerization and DNA remodelling, generating a recruitment platform for TnsB to catalyse site-specific transposon insertion. Insights from this work will inform the development of CRISPR-associated transposons as programmable site-specific gene insertion tools.

规范的 CRISPR–Cas 系统提供针对移动遗传元件的适应性免疫¹。然而，Tn7 样转座子已采用 IF、IB 和 VK 型系统来指导 RNA 引导的转座子插入^2,3,4,5,6,7。VK 型 CRISPR 相关转座子依赖于假核酸酶 Cas12k、转座酶 TnsB、AAA+ ATP 酶 TnsC 和锌指蛋白 TniQ 7 ^，但 RNA 指导的 DNA 转座的分子机制仍然难以捉摸。在这里，我们报告了 Cas12k 引导 RNA-靶 DNA 复合物和来自光合蓝藻 Scytonema hofmanni 的 CRISPR 相关转座子系统的 DNA 结合聚合 TnsC 丝的低温电子显微结构. Cas12k 复杂结构揭示了复杂的向导 RNA 结构和介导 RNA 引导的目标 DNA 识别的关键相互作用。TnsC 螺旋丝组件依赖于 ATP，并伴随着结合的 DNA 双链体的结构重塑。体内转座分析证实了结构的关键特征，生化实验表明 TniQ 限制 TnsC 聚合，而 TnsB 直接与 TnsC 丝相互作用，在 ATP 水解时触发它们的分解。总之，这些结果表明，通过 Cas12k 进行的 RNA 定向目标选择启动了 TnsC 聚合和 DNA 重塑，为 TnsB 生成了一个募集平台，以催化位点特异性转座子插入。这项工作的见解将为 CRISPR 相关转座子的开发提供信息，作为可编程的位点特异性基因插入工具。