Structural Molecular Biology Group, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark.
Structural Molecular Biology Group, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark; Core Facility for Integrated Microscopy (CFIM), Faculty of Health and Medical Sciences University of Copenhagen; Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.
Mol Cell. 2024 Jun 20;84(12):2353-2367.e5. doi: 10.1016/j.molcel.2024.05.005. Epub 2024 Jun 3.
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 (att) 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 att 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.
CRISPR 相关转座子（CASTs）是一类可移动的遗传元件，它们利用 CRISPR-Cas 系统进行 RNA 引导的 DNA 转位。CASTs 将大型 DNA cargos 独立于同源定向修复整合到附着（att）位点，因此有望用于真核生物基因组工程。然而，CASTs 的功能多样性和复杂性阻碍了对其机制的理解。在这里，我们呈现了重构的 ∼1 MDa 类型 V-K CAST 转位后复合物的高分辨率冷冻电镜（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合物的形成。突变实验探测特定残基和 TnsB 结合位点的作用的结果表明，转位活性可以增强，并表明 PAM 和 att 位点之间的距离由 TnsB C 末端和 TnsC 丝的长度决定。这种独特的 RNA 引导转位模型为重新利用该系统进行基因组编辑应用提供了基础。
