Target degradation of Type I CRISPR The CRISPR adaptive immune systems defend bacteria against invaders. Type I CRISPR-Cas systems, the most prevalent type, use a Cascade complex to search the target DNA that is then degraded by Cas3 protein. Xiao et al. report cryo–electron microscopy structures of the Type I-E Cascade/Cas3 complex in the pre– and post–DNA-nicking states. These structures reveal how Cas3 captures Cascade only in its correct conformation to reduce off-targeting and how Cas3 switches from the initial DNA-nicking mode to the processive DNA degradation mode. Science, this issue p. eaat0839 Structures of pre– and post–DNA-nicking states of the Type I-E CRISPR Cascade/Cas3 complex reveal mechanisms of target degradation. INTRODUCTION Type I CRISPR-Cas, the most prevalent CRISPR system, features a sequential target-searching and -degradation process. First, the multisubunit surveillance complex Cascade (CRISPR associated complex for antiviral defense) recognizes the matching double-stranded DNA target flanked by an optimal protospacer-adjacent motif (PAM), promotes the heteroduplex formation between CRISPR RNA (crRNA) and the target strand (TS) DNA, and displaces the nontarget strand (NTS) DNA, resulting in R-loop formation at the target site. The helicase-nuclease fusion enzyme Cas3 is then specifically recruited to Cascade/R-loop and nicks and processively degrades the DNA target. High-resolution structures of Type I-E Cascade/R-loop and Cas3/single-stranded DNA (ssDNA) complexes from Thermobifida fusca elucidate the PAM recognition and R-loop formation mechanism. However, the Cas3 recruitment and the DNA-nicking and -degradation mechanisms remain elusive. RATIONALE We reconstituted the TfuCascade/R-loop/Cas3 ternary complex and captured structures of the pre– and post–R-loop–nicking states using single-particle cryo–electron microscopy (cryo-EM). Together, these results provide the structural basis to understand crRNA-guided DNA degradation in Type I CRISPR-Cas systems. RESULTS We determined the TfuCascade/R-loop/Cas3 cryo-EM structure in the pre–NTS-nicking state at 3.7-Å resolution. Binding of Cas3 does not introduce further conformational changes to the R-loop–forming Cascade, suggesting that Cascade-Cas3 interaction for the most part features a conformation-capture rather than an induced-fit mechanism. The Cas3-Cascade interaction is exclusively mediated by the Cse1 subunit in Cascade. The recognitions are complementary in charge and surface contour to Cascade/R-loop but not to the apolipoprotein and seed-bubble states of Cascade. This is because before full R-loop formation, the C-terminal domain of Cse1 is in an alternative orientation. By making extensive contacts to both domains of Cse1, Cas3 is able to sense the altered surface landscape of Cse1 and reject the Cascade in such functional states. The conditional recruitment of Cas3 to Cascade serves as a mechanism to avoid mis-targeting a DNA with only partial complementarity [see the figure, (A)]. Moreover, we provided direct evidence that a substrate hand-over mechanism is essential for Type I-E CRISPR interference. The HD nuclease of Cas3 directly captures the NTS for strand-nicking, and this action bypasses the helicase moiety completely. Substrate capture relies on the presence of a flexible bulge in the NTS, and the nicking site preference is predetermined by the path of the recruitment pathway. We further determined the post–NTS-nicking structure at 4.7-Å resolution, which allowed us to identify structural changes accompanying the strand-nicking reaction. The structure reveals that the entire NTS strand in the R-loop region disappears from its original path because of increased flexibility. Upon adenosine 5′-triphosphate (ATP) hydrolysis, the PAM-proximal half of NTS spontaneously relocates to the opening of the Cas3 helicase. It follows that upon ATP hydrolysis, the Cas3 helicase would feed the ssDNA through itself and further into its HD nuclease, entering into a processive DNA degradation mode [see the figure, (B)]. CONCLUSION We completed the structure-function characterization of the molecular events that lead to Type I-E CRISPR interference. The onset of CRISPR interference is tightly controlled at the Cas3 recruitment step as a mechanism to reduce off-targeting. Upon NTS-nicking, however, Type I systems excel at target destruction because Cas3 degrades DNA processively rather than stopping at generating a double-strand break. Such characteristics may explain why Type I evolved to be the most prevalent CRISPR-Cas system found in nature. It would be interesting to see whether the Type I system may be repurposed into a genome-editing tool with distinct utilities from that of Cas9. Type I-E CRISPR interference by Cascade and Cas3 zooming into focus. (A) Mechanism for R-loop–dependent Cas3 recruitment explained. This prevents mistargeting partial-matching DNA sequences. (B) Structural rearrangements after R-loop–nicking by Cas3. The NTS DNA spontaneously relocates to the opening of Cas3 helicase, ready to be threaded for processive degradation. Type I CRISPR-Cas system features a sequential target-searching and degradation process on double-stranded DNA by the RNA-guided Cascade (CRISPR associated complex for antiviral defense) complex and the nuclease-helicase fusion enzyme Cas3, respectively. Here, we present a 3.7-angstrom-resolution cryo–electron microscopy (cryo-EM) structure of the Type I-E Cascade/R-loop/Cas3 complex, poised to initiate DNA degradation. Cas3 distinguishes Cascade conformations and only captures the R-loop–forming Cascade, to avoid cleaving partially complementary targets. Its nuclease domain recruits the nontarget strand (NTS) DNA at a bulged region for the nicking of single-stranded DNA. An additional 4.7-angstrom-resolution cryo-EM structure captures the postnicking state, in which the severed NTS retracts to the helicase entrance, to be threaded for adenosine 5′-triphosphate–dependent processive degradation. These snapshots form the basis for understanding RNA-guided DNA degradation in Type I-E CRISPR-Cas systems.

中文翻译：

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通过 Cascade 和 Cas3 进行 RNA 引导的 DNA 降解的结构基础

I 型 CRISPR 的目标降解 CRISPR 适应性免疫系统保护细菌免受入侵者的侵害。I 型 CRISPR-Cas 系统是最流行的类型，它使用 Cascade 复合物来搜索目标 DNA，然后被 Cas3 蛋白降解。肖等人。报告 IE 级联/Cas3 复合物在 DNA 切口前和后状态下的冷冻电子显微镜结构。这些结构揭示了 Cas3 如何仅以其正确构象捕获 Cascade 以减少脱靶，以及 Cas3 如何从初始 DNA 切口模式切换到进行性 DNA 降解模式。科学，这个问题 p。eaat0839 IE 型 CRISPR 级联/Cas3 复合物的 DNA 切口前和 DNA 后切口状态的结构揭示了目标降解的机制。介绍 I 型 CRISPR-Cas，最流行的 CRISPR 系统，具有顺序目标搜索和降级过程。首先，多亚基监视复合物级联（用于抗病毒防御的 CRISPR 相关复合物）识别匹配的双链 DNA 靶标，两侧是最佳的原型间隔区相邻基序 (PAM)，促进 CRISPR RNA (crRNA) 和靶标链之间的异源双链形成。 TS) DNA，并取代非目标链 (NTS) DNA，导致在目标位点形成 R 环。然后解旋酶-核酸酶融合酶 Cas3 被特异性募集到级联/R 环和切口，并逐步降解 DNA 目标。来自 Thermobifida fusca 的 IE 型级联/R 环和 Cas3/单链 DNA (ssDNA) 复合物的高分辨率结构阐明了 PAM 识别和 R 环形成机制。然而，Cas3 募集以及 DNA 切口和降解机制仍然难以捉摸。基本原理我们使用单粒子低温电子显微镜 (cryo-EM) 重建了 TfuCascade/R-loop/Cas3 三元复合物并捕获了 R-loop 前和后-切口状态的结构。总之，这些结果为理解 I 型 CRISPR-Cas 系统中 crRNA 引导的 DNA 降解提供了结构基础。结果我们以 3.7 Å 的分辨率确定了处于前 NTS 切口状态的 TfuCascade/R-loop/Cas3 冷冻电镜结构。Cas3 的结合不会对形成 R 环的 Cascade 引入进一步的构象变化，这表明 Cascade-Cas3 相互作用在很大程度上具有构象捕获而不是诱导拟合机制。Cas3-Cascade 相互作用完全由 Cascade 中的 Cse1 亚基介导。这些识别在电荷和表面轮廓上与 Cascade/R-loop 互补，但与 Cascade 的载脂蛋白和种子泡状态不互补。这是因为在完整的 R 环形成之前，Cse1 的 C 端域处于另一个方向。通过与 Cse1 的两个域进行广泛接触，Cas3 能够感知 Cse1 改变的表面景观，并在这种功能状态下拒绝级联。Cas3 有条件地募集到 Cascade 作为一种机制，可以避免错误靶向仅具有部分互补性的 DNA [见图，(A)]。此外，我们提供了直接证据，证明基板移交机制对于 IE 型 CRISPR 干扰至关重要。Cas3 的 HD 核酸酶直接捕获 NTS 以进行链切口，并且该动作完全绕过解旋酶部分。底物捕获依赖于 NTS 中灵活凸起的存在，而切口位点偏好由募集途径的路径预先确定。我们进一步确定了 4.7 Å 分辨率的后 NTS 切口结构，这使我们能够识别伴随链切口反应的结构变化。该结构表明，由于灵活性增加，R 环区域中的整个 NTS 链从其原始路径中消失。在腺苷 5'-三磷酸 (ATP) 水解后，NTS 的 PAM 近端一半自发地重新定位到 Cas3 解旋酶的开口处。因此，在 ATP 水解时，Cas3 解旋酶将通过自身供给 ssDNA 并进一步进入其 HD 核酸酶，进入进行性 DNA 降解模式 [见图，(B)]。结论 我们完成了导致 IE 型 CRISPR 干扰的分子事件的结构-功能表征。CRISPR 干扰的开始在 Cas3 募集步骤受到严格控制，作为减少脱靶的机制。然而，在 NTS 切割时，I 型系统在目标破坏方面表现出色，因为 Cas3 会持续降解 DNA，而不是在产生双链断裂时停止。这些特征可以解释为什么 I 型进化为自然界中发现的最普遍的 CRISPR-Cas 系统。看看 I 型系统是否可以重新用于基因组编辑工具，具有与 Cas9 不同的实用程序，这将会很有趣。通过级联和 Cas3 放大聚焦来键入 IE CRISPR 干扰。(A) 解释了 R 环依赖性 Cas3 募集的机制。这可以防止错误定位部分匹配的 DNA 序列。(B) Cas3 切割 R 环后的结构重排。NTS DNA 会自发地重新定位到 Cas3 解旋酶的开口处，准备好进行连续降解。I 型 CRISPR-Cas 系统的特点是分别通过 RNA 引导的级联（用于抗病毒防御的 CRISPR 相关复合物）复合物和核酸酶-解旋酶融合酶 Cas3 在双链 DNA 上进行顺序目标搜索和降解过程。在这里，我们展示了 IE 级联/R-loop/Cas3 复合物的 3.7 埃分辨率冷冻电子显微镜 (cryo-EM) 结构，准备启动 DNA 降解。Cas3 区分级联构象并且只捕获形成 R 环的级联，以避免切割部分互补的目标。它的核酸酶结构域在凸出区域募集非目标链 (NTS) DNA，用于切割单链 DNA。额外的 4.7 埃分辨率的冷冻电镜结构捕获了后切口状态，其中切断的 NTS 缩回到解旋酶入口，被穿入以进行依赖于腺苷 5'-三磷酸的持续降解。这些快照构成了理解 IE 型 CRISPR-Cas 系统中 RNA 引导的 DNA 降解的基础。