LSC-EMBL Partnership Institute for Genome Editing Technologies, Life Sciences Center, Vilnius University, Vilnius, Lithuania.
Department of Biology, Philipps-Universität Marburg, Marburg, Germany.
Nat Commun. 2024 Oct 29;15(1):9306. doi: 10.1038/s41467-024-53778-1.
CRISPR-Cas mediated DNA-interference typically relies on sequence-specific binding and nucleolytic degradation of foreign genetic material. Type IV-A CRISPR-Cas systems diverge from this general mechanism, using a nuclease-independent interference pathway to suppress gene expression for gene regulation and plasmid competition. To understand how the type IV-A system associated effector complex achieves this interference, we determine cryo-EM structures of two evolutionarily distinct type IV-A complexes (types IV-A1 and IV-A3) bound to cognate DNA-targets in the presence and absence of the type IV-A signature DinG effector helicase. The structures reveal how the effector complexes recognize the protospacer adjacent motif and target-strand DNA to form an R-loop structure. Additionally, we reveal differences between types IV-A1 and IV-A3 in DNA interactions and structural motifs that allow for in trans recruitment of DinG. Our study provides a detailed view of type IV-A mediated DNA-interference and presents a structural foundation for engineering type IV-A-based genome editing tools.
CRISPR-Cas 介导的 DNA 干扰通常依赖于对外源遗传物质的序列特异性结合和核酸酶降解。IV-A 型 CRISPR-Cas 系统与这种一般机制不同，它使用一种不依赖于核酸酶的干扰途径来抑制基因表达，以实现基因调控和质粒竞争。为了了解 IV-A 型系统相关效应复合物如何实现这种干扰，我们确定了两种进化上不同的 IV-A 型复合物（IV-A1 和 IV-A3）与同源 DNA 靶标结合的冷冻电镜结构，同时存在和不存在 IV-A 型特征性 DinG 效应物解旋酶。这些结构揭示了效应复合物如何识别前导序列相邻基序和靶链 DNA 以形成 R 环结构。此外，我们还揭示了 IV-A1 和 IV-A3 之间在 DNA 相互作用和结构模体方面的差异，这些差异允许 DinG 在转录水平募集。我们的研究提供了对 IV-A 型介导的 DNA 干扰的详细观察，并为基于 IV-A 型的基因组编辑工具的工程提供了结构基础。
