Secrets of a fast base editor CRISPR-Cas9 base editors comprise RNA-guided Cas proteins fused to an enzyme that can deaminate a DNA nucleoside. No natural enzyme deaminates adenine in DNA, and so a breakthrough came when a natural transfer RNA deaminase was fused to Cas9 and evolved to give an adenine base editor (ABE) that works on DNA. Further evolution provided the enzyme ABE8e, which catalyzes deamination more than 1000 times faster than early ABEs. Lapinaite et al. now present a 3.2-angstrom resolution structure of ABE8e bound to DNA in which the target adenine is replaced with an analog designed to trap the catalytic conformation. The structure, together with kinetic data comparing ABE8e to earlier ABEs, explains how ABE8e edits DNA bases and could inform future base-editor design. Science, this issue p. 566 A cryo–electron microscopy structure of the base editor ABE8e in a substrate-bound state elucidates base-editing mechanisms. CRISPR-Cas–guided base editors convert A•T to G•C, or C•G to T•A, in cellular DNA for precision genome editing. To understand the molecular basis for DNA adenosine deamination by adenine base editors (ABEs), we determined a 3.2-angstrom resolution cryo–electron microscopy structure of ABE8e in a substrate-bound state in which the deaminase domain engages DNA exposed within the CRISPR-Cas9 R-loop complex. Kinetic and structural data suggest that ABE8e catalyzes DNA deamination up to ~1100-fold faster than earlier ABEs because of mutations that stabilize DNA substrates in a constrained, transfer RNA–like conformation. Furthermore, ABE8e’s accelerated DNA deamination suggests a previously unobserved transient DNA melting that may occur during double-stranded DNA surveillance by CRISPR-Cas9. These results explain ABE8e-mediated base-editing outcomes and inform the future design of base editors.

中文翻译：

---

由 CRISPR-Cas9 引导的腺嘌呤碱基编辑器捕获 DNA

快速碱基编辑器的秘密 CRISPR-Cas9 碱基编辑器由 RNA 引导的 Cas 蛋白与一种酶融合而成，该酶可以使 DNA 核苷脱氨基。没有天然酶可以使 DNA 中的腺嘌呤脱氨，因此当一种天然转移 RNA 脱氨酶与 Cas9 融合并进化为对 DNA 起作用的腺嘌呤碱基编辑器 (ABE) 时，取得了突破。进一步的进化提供了酶 ABE8e，它催化脱氨基的速度比早期的 ABE 快 1000 倍以上。拉皮奈特等人。现在提出了与 DNA 结合的 ABE8e 的 3.2 埃分辨率结构，其中目标腺嘌呤被设计用于捕获催化构象的类似物取代。该结构以及将 ABE8e 与早期 ABE 进行比较的动力学数据，解释了 ABE8e 如何编辑 DNA 碱基，并可以为未来的碱基编辑器设计提供信息。科学，本期第 3 页。566 碱基编辑器 ABE8e 在底物结合状态下的低温电子显微镜结构阐明了碱基编辑机制。CRISPR-Cas 引导的碱基编辑器将细胞 DNA 中的 A•T 转换为 G•C，或 C•G 转换为 T•A，以进行精确的基因组编辑。为了了解腺嘌呤碱基编辑器 (ABE) 进行 DNA 腺苷脱氨的分子基础，我们确定了 ABE8e 在底物结合状态下的 3.2 埃分辨率低温电子显微镜结构，其中脱氨酶结构域与暴露在 CRISPR-Cas9 内的 DNA 结合。 R 环复合体。动力学和结构数据表明，ABE8e 催化 DNA 脱氨的速度比早期的 ABE 快约 1100 倍，因为突变可以将 DNA 底物稳定在受限制的转移 RNA 构象中。此外，ABE8e 加速的 DNA 脱氨作用表明，在 CRISPR-Cas9 监测双链 DNA 期间可能发生以前未观察到的瞬时 DNA 熔解。这些结果解释了 ABE8e 介导的碱基编辑结果，并为碱基编辑器的未来设计提供了信息。