Dynamic observations of CRISPR-Cas target recognition and cleavage heterogeneities
Author:
Zhang Zhijia1, Jeong Haechan1, Zu Di1, Zhao Xintao1, Senaratne Pramith1, Filbin John1, Silber Brett1, Kang Sarah1, Gladstone Ann1, Lau Matthew1, Cui Guangjie1, Park Younggeun2, Lee Somin Eunice1ORCID
Affiliation:
1. Department of Electrical & Computer Engineering, Biomedical Engineering, Applied Physics , Biointerfaces Institute, Macromolecular Science & Engineering, University of Michigan , Ann Arbor , USA 2. Department of Mechanical Engineering , University of Michigan , Ann Arbor , USA
Abstract
Abstract
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.
Publisher
Walter de Gruyter GmbH
Subject
Electrical and Electronic Engineering,Atomic and Molecular Physics, and Optics,Electronic, Optical and Magnetic Materials,Biotechnology
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