De Paula Viviane S, Dubey Abhinav, Arthanari Haribabu, Sgourakis Nikolaos G
Center for Computational and Genomic Medicine, The Children's Hospital of Philadelphia, 3501 Civic Center Boulevard, Philadelphia, PA 19104, USA; Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, 3620 Hamilton Walk, Philadelphia, PA 19104-6059, USA.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.
Cell Chem Biol. 2025 Feb 20;32(2):267-279.e5. doi: 10.1016/j.chembiol.2024.10.001. Epub 2024 Oct 28.
CRISPR-Cas9 has revolutionized genome engineering applications by programming its single-guide RNA, where high specificity is required. However, the precise molecular mechanism underscoring discrimination between on/off-target DNA sequences, relative to the guide RNA template, remains elusive. Here, using methyl-based NMR to study multiple holoenzymes assembled in vitro, we elucidate a discrete protein conformational state which enables recognition of DNA mismatches at the protospacer adjacent motif (PAM)-distal end. Our results delineate an allosteric pathway connecting a dynamic conformational switch at the REC3 domain, with the sampling of a catalytically competent state by the HNH domain. Our NMR data show that HiFi Cas9 (R691A) increases the fidelity of DNA recognition by stabilizing this "surveillance state" for mismatched substrates, shifting the Cas9 conformational equilibrium away from the active state. These results establish a paradigm of substrate recognition through an allosteric protein-based switch, providing unique insights into the molecular mechanism which governs Cas9 selectivity.
CRISPR-Cas9通过对其单导向RNA进行编程，彻底改变了基因组工程应用，而在这些应用中需要高特异性。然而，相对于导向RNA模板，区分靶标/脱靶DNA序列的精确分子机制仍不清楚。在这里，我们使用基于甲基的核磁共振来研究体外组装的多种全酶，阐明了一种离散的蛋白质构象状态，该状态能够识别原间隔相邻基序（PAM）远端的DNA错配。我们的结果描绘了一条变构途径，该途径将REC3结构域的动态构象转换与HNH结构域对催化活性状态的采样联系起来。我们的核磁共振数据表明，HiFi Cas9（R691A）通过稳定错配底物的这种“监测状态”，将Cas9构象平衡从活性状态转移，从而提高了DNA识别的保真度。这些结果建立了一种基于变构蛋白开关的底物识别模式，为控制Cas9选择性的分子机制提供了独特的见解。
