Structural basis for mismatch surveillance by CRISPR-Cas9.
Structural basis for mismatch surveillance by CRISPR-Cas9.
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DOI：
10.1038/s41586-022-04470-1
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发表时间：
2022-03
期刊：
影响因子：
64.8
通讯作者：
Taylor DW
中科院分区：
文献类型：
--
作者：
Bravo JPK;Liu MS;Hibshman GN;Dangerfield TL;Jung K;McCool RS;Johnson KA;Taylor DW
CRISPR-Cas9作为可编程基因组编辑工具受到脱靶DNA切割的阻碍，并且Cas9识别错配的潜在机制知之甚少。尽管已经设计了对错配具有更大区分力的Cas9变体，但这些变体遭受了靶向DNA切割速率的显著降低。在这里，我们使用动力学引导的冷冻电子显微镜来确定Cas9在错配切割的不同阶段的结构。我们观察到在存在错配的情况下形成的引导RNA-DNA双链体的独特的线性构象，这阻止了Cas9活化。虽然典型的扭结引导RNA-DNA双链体构象有利于DNA切割，但我们观察到，通过RuvC结构域中的环的重组，在原型间隔区邻近基序的远端含有错配的底物被稳定。错配稳定残基的突变减少了脱靶DNA切割，但维持了快速的中靶DNA切割。通过靶向专门参与错配耐受的区域，我们为下一代高保真Cas9变体的设计提供了概念验证。在错配切割期间Cas9的冷冻电子显微镜结构提供了对控制Cas9的脱靶效应的机制的洞察，这将有助于未来设计具有减少的脱靶切割的高保真Cas9变体。
CRISPR–Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage, and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood. Although Cas9 variants with greater discrimination against mismatches have been designed, these suffer from substantially reduced rates of on-target DNA cleavage. Here we used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. We observed a distinct, linear conformation of the guide RNA–DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA–DNA duplex conformation facilitates DNA cleavage, we observe that substrates that contain mismatches distal to the protospacer adjacent motif are stabilized by reorganization of a loop in the RuvC domain. Mutagenesis of mismatch-stabilizing residues reduces off-target DNA cleavage but maintains rapid on-target DNA cleavage. By targeting regions that are exclusively involved in mismatch tolerance, we provide a proof of concept for the design of next-generation high-fidelity Cas9 variants. Cryo-electron microscopy structures of Cas9 during mismatch cleavage provide insight into the mechanisms that control off-target effects of Cas9, which will aid in the future design of high-fidelity Cas9 variants with reduced off-target cleavage.
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