In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide-RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM)¹. Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism^2,3. Here we show that Cas9 sharply bends and undertwists DNA at each PAM, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryo-electron-microscopy (EM) structures of Cas9:RNA:DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 “reads” snippets of DNA to locate target sites within a vast excess of non-target DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.

中文翻译：

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CRISPR-Cas9 弯曲和扭曲 DNA 以读取其序列

在细菌防御和基因组编辑应用中，CRISPR 相关蛋白 Cas9 搜索数百万个 DNA 碱基对，以定位与原始间隔区相邻基序 (PAM) ¹相邻的 20 个核苷酸的引导 RNA 互补靶序列。目标捕获需要 Cas9 使用未知的 ATP 独立机制在候选序列处解开 DNA ^2,3. 在这里，我们展示了 Cas9 在每个 PAM 处急剧弯曲和扭转 DNA，从而将 DNA 核苷酸从双链体中翻转出来并朝向引导 RNA 进行序列查询。Cas9:RNA:DNA 复合物的冷冻电子显微镜 (EM) 结构被困在询问途径的不同状态，连同溶液构象探测，揭示了全局蛋白质重排伴随着未堆积的 DNA 铰链的形成。弯曲诱导的碱基翻转解释了 Cas9 如何“读取”DNA 片段以在大量非目标 DNA 中定位目标位点，这一过程对细菌抗病毒免疫和基因组编辑都至关重要。这种机制为互补引导的 DNA 搜索问题建立了物理解决方案，并展示了询问速度和局部 DNA 几何形状如何影响基因组编辑效率。