Dynamics of Target DNA Binding and Cleavage by Staphylococcus aureus Cas9 as Revealed by High-Speed Atomic Force Microscopy
Dynamics of Target DNA Binding and Cleavage by Staphylococcus aureus Cas9 as Revealed by High-Speed Atomic Force Microscopy
复制标题
高速原子力显微镜揭示金黄色葡萄球菌 Cas9 靶标 DNA 结合和切割的动力学
DOI：
10.1021/acsnano.2c10709
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发表时间：
2023
期刊：
影响因子：
17.1
通讯作者：
Shibata Mikihiro
中科院分区：
文献类型：
--
作者：
Puppulin Leonardo;Ishikawa Junichiro;Sumino Ayumi;Marchesi Arin;Flechsig Holger;Umeda Kenichi;Kodera Noriyuki;Nishimasu Hiroshi;Shibata Mikihiro
CRISPR-Cas9的可编程DNA结合和切割已经给生命科学带来了革命性的变化。然而，在与目标有一定同源性的DNA序列中观察到的脱靶切割仍然是Cas9在生物学和医学上更广泛使用的主要限制因素。因此，完全了解Cas9的DNA结合、询问和切割的动力学对于提高基因组编辑的效率至关重要。在这里，我们使用高速原子力显微镜(HS-AFM)来研究金黄色葡萄球菌Cas9(SaCas9)及其DNA结合和切割的动力学。当与单导RNA(SgRNA)结合时，SaCas9形成一个封闭的双叶结构，瞬时和灵活地采用开放的构型。SaCas9介导的DNA切割的特征是裂解的DNA被释放并立即解离，证实了SaCas9是一种多周转的核酸内切酶。根据目前的知识，寻找靶DNA的过程主要是由三维扩散控制的。独立的HS-AFM实验表明，SaCas9-sgRNA与其靶DNA之间存在潜在的远程吸引相互作用。这种相互作用在稳定的三元络合物形成之前就已经发生，并且只在原空间基邻近基序(PAM)附近观察到，最远可达几纳米。通过连续的地形图像直观地显示这一过程，表明SaCas9-sgRNA首先与目标序列结合，而随后的PAM结合伴随着局部DNA弯曲和稳定络合物的形成。总而言之，我们的HS-AFM数据揭示了SaCas9在寻找DNA目标过程中的潜在和意想不到的行为。
Programmable DNA binding and cleavage by CRISPR-Cas9 has revolutionized the life sciences. However, the off-target cleavage observed in DNA sequences with some homology to the target still represents a major limitation for a more widespread use of Cas9 in biology and medicine. For this reason, complete understanding of the dynamics of DNA binding, interrogation and cleavage by Cas9 is crucial to improve the efficiency of genome editing. Here, we use high-speed atomic force microscopy (HS-AFM) to investigateStaphylococcus aureusCas9 (SaCas9) and its dynamics of DNA binding and cleavage. Upon binding to single-guide RNA (sgRNA), SaCas9 forms a close bilobed structure that transiently and flexibly adopts also an open configuration. The SaCas9-mediated DNA cleavage is characterized by release of cleaved DNA and immediate dissociation, confirming that SaCas9 operates as a multiple turnover endonuclease. According to present knowledge, the process of searching for target DNA is mainly governed by three-dimensional diffusion. Independent HS-AFM experiments show a potential long-range attractive interaction between SaCas9-sgRNA and its target DNA. The interaction precedes the formation of the stable ternary complex and is observed exclusively in the vicinity of the protospacer-adjacent motif (PAM), up to distances of several nanometers. The direct visualization of the process by sequential topographic images suggests that SaCas9-sgRNA binds to the target sequence first, while the following binding of the PAM is accompanied by local DNA bending and formation of the stable complex. Collectively, our HS-AFM data reveal a potential and unexpected behavior of SaCas9 during the search for DNA targets.