IV-A 型 CRISPR-Csf 复合物：组装、dsDNA 靶向和 CasDinG 募集。

Type IV-A CRISPR-Csf complex: Assembly, dsDNA targeting, and CasDinG recruitment.

机构信息

Department of Biochemistry, School of Medicine, Southern University of Science and Technology, Shenzhen 518055, China.

Key Laboratory of Molecular Design for Plant Cell Factory of Guangdong Higher Education Institutes, Department of Chemical Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518055, China.

出版信息

Mol Cell. 2023 Jul 20;83(14):2493-2508.e5. doi: 10.1016/j.molcel.2023.05.036. Epub 2023 Jun 20.

DOI:10.1016/j.molcel.2023.05.036

PMID:37343553

Abstract

Type IV CRISPR-Cas systems, which are primarily found on plasmids and exhibit a strong plasmid-targeting preference, are the only one of the six known CRISPR-Cas types for which the mechanistic details of their function remain unknown. Here, we provide high-resolution functional snapshots of type IV-A Csf complexes before and after target dsDNA binding, either in the absence or presence of CasDinG, revealing the mechanisms underlying Csf complex assembly, "DWN" PAM-dependent dsDNA targeting, R-loop formation, and CasDinG recruitment. Furthermore, we establish that CasDinG, a signature DinG family helicase, harbors ssDNA-stimulated ATPase activity and ATP-dependent 5'-3' DNA helicase activity. In addition, we show that CasDinG unwinds the non-target strand (NTS) and target strand (TS) of target dsDNA from the Csf complex. These molecular details advance our mechanistic understanding of type IV-A CRISPR-Csf function and should enable Csf complexes to be harnessed as genome-engineering tools for biotechnological applications.

摘要

IV 型 CRISPR-Cas 系统主要存在于质粒上，表现出强烈的质粒靶向偏好，是已知的六种 CRISPR-Cas 类型中唯一一种其功能的机制细节尚不清楚的类型。在这里，我们提供了 IV-A 型 Csf 复合物在结合或不结合 CasDinG 的情况下结合目标 dsDNA 前后的高分辨率功能快照，揭示了 Csf 复合物组装、“DWN”PAM 依赖性 dsDNA 靶向、R 环形成和 CasDinG 募集的机制。此外，我们确定了 CasDinG，一种特征性的 DinG 家族解旋酶，具有 ssDNA 刺激的 ATPase 活性和 ATP 依赖性 5'-3' DNA 解旋酶活性。此外，我们还表明 CasDinG 从 Csf 复合物中解开目标 dsDNA 的非靶链 (NTS) 和靶链 (TS)。这些分子细节提高了我们对 IV-A 型 CRISPR-Csf 功能的机制理解，并应该使 Csf 复合物能够被用作生物技术应用的基因组工程工具。