Wang Jimin, Arantes Pablo R, Ahsan Mohd, Sinha Souvik, Kyro Gregory W, Maschietto Federica, Allen Brandon, Skeens Erin, Lisi George P, Batista Victor S, Palermo Giulia
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, United States.
Department of Bioengineering and Department of Chemistry, University of California, Riverside, Riverside, CA, United States.
Front Mol Biosci. 2023 Jan 9;9:1072733. doi: 10.3389/fmolb.2022.1072733. eCollection 2022.
The CRISPR-associated protein 9 (Cas9) has been engineered as a precise gene editing tool to make double-strand breaks. CRISPR-associated protein 9 binds the folded guide RNA (gRNA) that serves as a binding scaffold to guide it to the target DNA duplex a RecA-like strand-displacement mechanism but without ATP binding or hydrolysis. The target search begins with the protospacer adjacent motif or PAM-interacting domain, recognizing it at the major groove of the duplex and melting its downstream duplex where an RNA-DNA heteroduplex is formed at nanomolar affinity. The rate-limiting step is the formation of an R-loop structure where the HNH domain inserts between the target heteroduplex and the displaced non-target DNA strand. Once the R-loop structure is formed, the non-target strand is rapidly cleaved by RuvC and ejected from the active site. This event is immediately followed by cleavage of the target DNA strand by the HNH domain and product release. Within CRISPR-associated protein 9, the HNH domain is inserted into the RuvC domain near the RuvC active site two linker loops that provide allosteric communication between the two active sites. Due to the high flexibility of these loops and active sites, biophysical techniques have been instrumental in characterizing the dynamics and mechanism of the CRISPR-associated protein 9 nucleases, aiding structural studies in the visualization of the complete active sites and relevant linker structures. Here, we review biochemical, structural, and biophysical studies on the underlying mechanism with emphasis on how CRISPR-associated protein 9 selects the target DNA duplex and rejects non-target sequences.
CRISPR相关蛋白9（Cas9）已被改造成为一种精确的基因编辑工具，用于产生双链断裂。CRISPR相关蛋白9与折叠后的向导RNA（gRNA）结合，gRNA作为一种结合支架，引导其靶向DNA双链体——这是一种类似RecA的链置换机制，但无需ATP结合或水解。靶向搜索始于原间隔序列临近基序或PAM相互作用结构域，在双链体的大沟处识别它，并使其下游双链体解链，在此处形成具有纳摩尔亲和力的RNA-DNA异源双链体。限速步骤是形成R环结构，其中HNH结构域插入到靶标异源双链体和被置换的非靶标DNA链之间。一旦R环结构形成，非靶标链就会被RuvC迅速切割，并从活性位点弹出。紧接着，靶标DNA链会被HNH结构域切割，产物释放。在CRISPR相关蛋白9中，HNH结构域插入到靠近RuvC活性位点的RuvC结构域中——两个连接环提供了两个活性位点之间的变构通讯。由于这些环和活性位点具有高度的灵活性，生物物理技术有助于表征CRISPR相关蛋白9核酸酶的动力学和机制，辅助结构研究以可视化完整的活性位点和相关的连接结构。在此，我们综述关于潜在机制的生化、结构和生物物理研究，重点关注CRISPR相关蛋白9如何选择靶标DNA双链体并排除非靶标序列。
