CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargo using an RNA-guided mechanism^1-3. CAST elements contain multiple conserved genes, including: a CRISPR effector (Cas12k or Cascade), a AAA+ regulator (TnsC), a transposase (TnsA/TnsB), and a target site associated factor (TniQ). These components are thought to cooperatively integrate DNA via formation of a multisubunit transposition integration complex (transpososome). Here we reconstitute the approximately 1 MDa type V-K CAST transpososome from Scytonema hofmannii (ShCAST) and determined the structure to near-atomic resolution (3.5 Å) using single particle cryo-EM. Transpososome architecture reveals modular association between components. Cas12k forms a complex with ribosomal subunit S15 and TniQ, stabilizing formation of a full R-loop. TnsC has dedicated interaction interfaces with TniQ and TnsB. Interestingly, we observe novel TnsC-TnsB interactions at the C-terminal face of TnsC, which contribute to stimulation of ATPase activity. Although the TnsC oligomeric assembly slightly deviates from the helical configuration found in isolation, the TnsC-bound target DNA conformation differs dramatically in the transpososome. As a consequence, TnsC makes new protein-DNA interactions throughout the transpososome that are important for transposition activity. Finally, we discover two distinct transpososome populations which differ in their DNA contacts near TniQ. This suggests that associations with the CRISPR effector can be flexible. This ShCAST transpososome structure significantly enhances our understanding of CAST transposition systems and suggests avenues for improving CAST transposition for precision genome-editing applications.

CRISPR 相关转座子 (CAST) 是可编程的移动遗传元件，可使用 RNA 引导机制插入大型 DNA 货物^1-3 。 CAST 元件包含多个保守基因，包括：CRISPR 效应子（Cas12k 或 Cascade）、AAA+ 调节子 (TnsC)、转座酶 (TnsA/TnsB) 和靶位点相关因子 (TniQ)。这些成分被认为通过形成多亚基转座整合复合物（转座体）来协同整合 DNA。在这里，我们重建了来自Scytonema hofmannii (ShCAST) 的大约 1 MDa 型 VK CAST 转座体，并使用单粒子冷冻电镜确定了近原子分辨率 (3.5 Å) 的结构。转座体架构揭示了组件之间的模块化关联。 Cas12k 与核糖体亚基 S15 和 TniQ 形成复合物，稳定完整 R 环的形成。 TnsC 具有与 TniQ 和 TnsB 的专用交互接口。有趣的是，我们在 TnsC C 端面观察到新的 TnsC-TnsB 相互作用，这有助于刺激 ATP 酶活性。尽管 TnsC 寡聚体组装与分离中发现的螺旋构型略有不同，但转座体中 TnsC 结合的靶 DNA 构象却存在显着差异。因此，TnsC 在整个转座体中产生新的蛋白质-DNA 相互作用，这对于转座活性很重要。最后，我们发现了两个不同的转座体群体，它们在 TniQ 附近的 DNA 接触不同。这表明与 CRISPR 效应子的关联可以是灵活的。这种 ShCAST 转座体结构显着增强了我们对 CAST 转座系统的理解，并为精确基因组编辑应用改进 CAST 转座提供了途径。