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). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA on PAM binding, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryogenic-electron microscopy (cryo-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 nontarget 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.

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，以定位邻接原型间隔子相邻基序 (PAM) 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。在这里，我们展示了 Cas9 在 PAM 结合时急剧弯曲和扭转 DNA，从而将 DNA 核苷酸翻转出双链体并朝向引导 RNA 进行序列询问。捕获在询问途径不同状态的 Cas9-RNA-DNA 复合物的低温电子显微镜 (cryo-EM) 结构，结合溶液构象探测，揭示了整体蛋白质重排伴随着未堆叠的 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，这一过程对于细菌抗病毒免疫和基因组编辑都至关重要。该机制为互补引导 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 几何结构如何影响基因组编辑效率。