Chen Janice S, Dagdas Yavuz S, Kleinstiver Benjamin P, Welch Moira M, Sousa Alexander A, Harrington Lucas B, Sternberg Samuel H, Joung J Keith, Yildiz Ahmet, Doudna Jennifer A
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
Biophysics Graduate Group, University of California, Berkeley, California 94720, USA.
Nature. 2017 Oct 19;550(7676):407-410. doi: 10.1038/nature24268. Epub 2017 Sep 20.
The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity are unknown. Here, using single-molecule Förster resonance energy transfer experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we design a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.
来自化脓性链球菌的RNA引导的CRISPR-Cas9核酸酶（SpCas9）已被广泛用于基因组编辑。高保真（SpCas9-HF1）和增强特异性（eSpCas9(1.1)）变体在人类细胞中表现出显著降低的脱靶切割，但靶点识别机制以及进一步提高保真度的潜力尚不清楚。在这里，我们使用单分子Förster共振能量转移实验表明，当与错配靶点结合时，SpCas9-HF1和eSpCas9(1.1)都被困在无活性状态。我们发现，Cas9中的一个非催化结构域REC3识别靶点互补性并控制HNH核酸酶来调节整体催化能力。利用这一观察结果，我们设计了一种新的超精确Cas9变体（HypaCas9），它在人类细胞中表现出高全基因组特异性，且不影响靶向活性。这些结果提供了一个更全面的模型，以合理化和修改靶点识别与核酸酶激活之间的平衡，用于精确基因组编辑。
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