CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opt CRISPR-Cas systems for RNA-guided DNA transposition. CASTs integrate large DNA cargos into the attachment () site independently of homology-directed repair and thus hold promise for eukaryotic genome engineering. However, the functional diversity and complexity of CASTs hinder an understanding of their mechanisms. Here, we present the high-resolution cryoelectron microscopy (cryo-EM) structure of the reconstituted ∼1 MDa post-transposition complex of the type V-K CAST, together with different assembly intermediates and diverse TnsC filament lengths, thus enabling the recapitulation of the integration complex formation. The results of mutagenesis experiments probing the roles of specific residues and TnsB-binding sites show that transposition activity can be enhanced and suggest that the distance between the PAM and sites is determined by the lengths of the TnsB C terminus and the TnsC filament. This singular model of RNA-guided transposition provides a foundation for repurposing the system for genome-editing applications.

中文翻译：

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VK 型 CRISPR 相关转座子整合组装的构象景观


CRISPR 相关转座子 (CAST) 是利用 CRISPR-Cas 系统进行 RNA 引导的 DNA 转座的移动遗传元件。 CAST 将大 DNA 货物整合到附着 () 位点，独立于同源定向修复，因此为真核基因组工程带来了希望。然而，CAST 的功能多样性和复杂性阻碍了对其机制的理解。在这里，我们展示了 VK CAST 型重建的~1 MDa 转座后复合物的高分辨率冷冻电子显微镜 (cryo-EM) 结构，以及不同的组装中间体和不同的 TnsC 丝长度，从而能够重现整合复杂的形成。探究特定残基和 TnsB 结合位点作用的诱变实验结果表明，转座活性可以增强，并表明 PAM 和位点之间的距离由 TnsB C 末端和 TnsC 丝的长度决定。这种 RNA 引导转座的独特模型为重新利用该系统进行基因组编辑应用奠定了基础。