Zhang Zhijia, Jeong Haechan, Zu Di, Zhao Xintao, Senaratne Pramith, Filbin John, Silber Brett, Kang Sarah, Gladstone Ann, Lau Matthew, Cui Guangjie, Park Younggeun, Lee Somin Eunice
Department of Electrical & Computer Engineering, Biomedical Engineering, Applied Physics, Biointerfaces Institute, Macromolecular Science & Engineering, University of Michigan, Ann Arbor, USA.
Department of Mechanical Engineering, University of Michigan, Ann Arbor, USA.
Nanophotonics. 2022 Aug 30;11(19):4419-4425. doi: 10.1515/nanoph-2022-0286. eCollection 2022 Sep.
CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats) have shown great potential as efficient gene editing tools in disease therapeutics. Although numerous CRISPR-Cas systems have been developed, detailed mechanisms of target recognition and DNA cleavage are still unclear. In this work, we dynamically observe the entire process of conjugation, target recognition and DNA cleavage by single particle spectroscopy of CRISPR-Cas systems on single particle surfaces (gold) with the unique advantage of extended time periods. We show the CRISPR-Cas system, comprised of Cas endonuclease and single guide RNA, is stable and functional on single particle surfaces. Owing to the photostability of single particle surfaces, we directly observe in real time the entire dynamic process of conjugation, target recognition and DNA cleavage without photobleaching. We find heterogeneity in target recognition and DNA cleavage processes in which individual spectra vary significantly from one another as well as from the ensemble. We believe an in depth understanding of heterogeneities in CRISPR-Cas systems can overcome potential barriers in precision medicine and personalized disease therapeutics.
CRISPR-Cas系统（成簇规律间隔短回文重复序列）作为疾病治疗中高效的基因编辑工具已展现出巨大潜力。尽管已开发出众多CRISPR-Cas系统，但靶标识别和DNA切割的详细机制仍不清楚。在这项工作中，我们利用单颗粒表面（金）上CRISPR-Cas系统的单颗粒光谱学，通过延长时间周期的独特优势，动态观察了结合、靶标识别和DNA切割的全过程。我们表明，由Cas核酸内切酶和单向导RNA组成的CRISPR-Cas系统在单颗粒表面是稳定且有功能的。由于单颗粒表面的光稳定性，我们能够实时直接观察结合、靶标识别和DNA切割的整个动态过程而不会发生光漂白。我们发现在靶标识别和DNA切割过程中存在异质性，其中各个光谱彼此之间以及与总体光谱都有显著差异。我们相信，深入了解CRISPR-Cas系统中的异质性能够克服精准医学和个性化疾病治疗中的潜在障碍。
