Department of Biophysics and Chemical Biology, Seoul National University, Seoul 151-747, Republic of Korea.
Department of Chemistry, Seoul National University, Seoul 151-747, Republic of Korea.
Nat Commun. 2016 Nov 2;7:13350. doi: 10.1038/ncomms13350.
The type II CRISPR-associated protein Cas9 recognizes and cleaves target DNA with the help of two guide RNAs (gRNAs; tracrRNA and crRNA). However, the detailed mechanisms and kinetics of these gRNAs in the Cas9 nuclease activity are unclear. Here, we investigate the structural roles of gRNAs in the CRISPR-Cas9 system by single-molecule spectroscopy and reveal a new conformation of inactive Cas9 that is thermodynamically more preferable than active apo-Cas9. We find that tracrRNA prevents Cas9 from changing into the inactive form and leads to the Cas9:gRNA complex. For the Cas9:gRNA complex, we identify sub-conformations of the RNA-DNA heteroduplex during R-loop expansion. Our single-molecule study indicates that the kinetics of the sub-conformations is controlled by the complementarity between crRNA and target DNA. We conclude that both tracrRNA and crRNA regulate the conformations and kinetics of the Cas9 complex, which are crucial in the DNA cleavage activity of the CRISPR-Cas9 system.
II 型 CRISPR 相关蛋白 Cas9 在两条向导 RNA（tracrRNA 和 crRNA）的帮助下识别并切割靶 DNA。然而，这些 gRNA 在 Cas9 核酸酶活性中的详细机制和动力学尚不清楚。在这里，我们通过单分子光谱法研究了 gRNA 在 CRISPR-Cas9 系统中的结构作用，并揭示了一种新的无活性 Cas9 构象，其热力学上比活性 apo-Cas9 更有利。我们发现 tracrRNA 阻止 Cas9 转变为无活性形式，并导致 Cas9:gRNA 复合物。对于 Cas9:gRNA 复合物，我们在 R 环扩展过程中鉴定了 RNA-DNA 杂合双链的亚构象。我们的单分子研究表明，亚构象的动力学受 crRNA 和靶 DNA 之间互补性的控制。我们得出结论，tracrRNA 和 crRNA 都调节 Cas9 复合物的构象和动力学，这对于 CRISPR-Cas9 系统的 DNA 切割活性至关重要。
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