Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage.
Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage.
复制标题
DOI：
10.1093/nar/gkv892
复制
发表时间：
2015-10-15
影响因子：
14.9
通讯作者：
Marszalek PE
中科院分区：
文献类型：
--
作者：
Josephs EA;Kocak DD;Fitzgibbon CJ;McMenemy J;Gersbach CA;Marszalek PE
CRISPR 相关核酸内切酶 Cas9 在由 Cas9 结合的 RNA 分子指定的可变靶位点切割 DNA。 Cas9 能够被单个“引导 RNA”分子引导以靶向几乎任何序列，最近已被用于许多新兴的生物和医学应用。因此，了解Cas9脱靶活性的本质对其实际应用至关重要。使用原子力显微镜 (AFM)，我们可以直接解析单个 Cas9 和无核酸酶活性的 dCas9 蛋白，因为它们与工程 DNA 底物结合。高分辨率成像使我们能够确定它们与不同引导 RNA 变体结合到目标或脱靶序列的相对倾向。将 Cas9 和 dCas9 的结构特性映射到各自的结合位点，揭示了 DNA 位点的渐进构象转变，与其靶标的序列相似性不断增加。通过动力学蒙特卡罗 (KMC) 模拟，这些结果提供了由引导 RNA 与目标 DNA 的第 14-17 个核苷酸区域之间的相互作用驱动的“构象门控”机制的证据，我们发现其稳定性与报告的脱靶切割率显着相关。 KMC 模拟还揭示了通过考虑引导 RNA 侵入目标 DNA 双链体来设计具有更高特异性的引导 RNA 序列的潜在方法。
CRISPR-associated endonuclease Cas9 cuts DNA at variable target sites designated by a Cas9-bound RNA molecule. Cas9's ability to be directed by single ‘guide RNA’ molecules to target nearly any sequence has been recently exploited for a number of emerging biological and medical applications. Therefore, understanding the nature of Cas9's off-target activity is of paramount importance for its practical use. Using atomic force microscopy (AFM), we directly resolve individual Cas9 and nuclease-inactive dCas9 proteins as they bind along engineered DNA substrates. High-resolution imaging allows us to determine their relative propensities to bind with different guide RNA variants to targeted or off-target sequences. Mapping the structural properties of Cas9 and dCas9 to their respective binding sites reveals a progressive conformational transformation at DNA sites with increasing sequence similarity to its target. With kinetic Monte Carlo (KMC) simulations, these results provide evidence of a ‘conformational gating’ mechanism driven by the interactions between the guide RNA and the 14th–17th nucleotide region of the targeted DNA, the stabilities of which we find correlate significantly with reported off-target cleavage rates. KMC simulations also reveal potential methodologies to engineer guide RNA sequences with improved specificity by considering the invasion of guide RNAs into targeted DNA duplex.