Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9.
Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9.
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DOI：
10.1038/ncomms12778
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发表时间：
2016-09-14
影响因子：
16.6
通讯作者：
Ha T
中科院分区：
文献类型：
--
作者：
Singh D;Sternberg SH;Fei J;Doudna JA;Ha T
Cas9 - 引导RNA复合物与DNA的结合特异性对基因组工程应用至关重要；然而，错配如何影响靶标识别/排斥动力学尚未得到很好的理解。在此我们使用单分子荧光共振能量转移（FRET）来探测Cas9 - RNA与DNA靶标之间的实时相互作用。双分子结合速率仅微弱地依赖于序列；然而，在靠近前间隔序列邻近基序（PAM）处引入错配时，解离速率从<0.006 s⁻¹大幅增加到>2 s⁻¹，这表明在异源双链形成早期遇到的错配会诱导对脱靶DNA的快速排斥。相反，长度达11个碱基对的PAM远端错配虽然阻止DNA切割，但仍允许形成稳定的复合物（解离速率<0.006 s⁻¹），这表明极其缓慢的排斥可能会隔离Cas9 - RNA，增加基因组编辑所需的Cas9表达水平，从而加剧脱靶效应。我们还观察到至少两种不同的结合FRET状态，它们可能代表了靶标搜索和校对过程中的不同步骤。
CRISPR - Cas9使人们拥有了前所未有的操纵基因组的能力，但在分子水平上靶标识别如何发挥作用仍未得到很好的理解。在此作者使用单分子FRET来探测Cas9 - 靶标相互作用，并识别出不同的搜索和校对状态。
Binding specificity of Cas9–guide RNA complexes to DNA is important for genome-engineering applications; however, how mismatches influence target recognition/rejection kinetics is not well understood. Here we used single-molecule FRET to probe real-time interactions between Cas9–RNA and DNA targets. The bimolecular association rate is only weakly dependent on sequence; however, the dissociation rate greatly increases from <0.006 s−1 to >2 s−1 upon introduction of mismatches proximal to protospacer-adjacent motif (PAM), demonstrating that mismatches encountered early during heteroduplex formation induce rapid rejection of off-target DNA. In contrast, PAM-distal mismatches up to 11 base pairs in length, which prevent DNA cleavage, still allow formation of a stable complex (dissociation rate <0.006 s−1), suggesting that extremely slow rejection could sequester Cas9–RNA, increasing the Cas9 expression level necessary for genome-editing, thereby aggravating off-target effects. We also observed at least two different bound FRET states that may represent distinct steps in target search and proofreading. CRISPR-Cas9 has enabled an unprecedented ability to manipulate the genome yet it is still poorly understood how target recognition functions at a molecular level. Here the authors use single-molecule FRET to probe Cas9-target interaction and identify distinct search and proofreading states.