Dynamic observations of CRISPR-Cas target recognition and cleavage heterogeneities
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
Affiliations
Zhang Zhijia
Department of Electrical & Computer Engineering, Biomedical Engineering, Applied Physics, Biointerfaces Institute, Macromolecular Science & Engineering, University of Michigan, Ann Arbor, USA
Jeong Haechan
Department of Electrical & Computer Engineering, Biomedical Engineering, Applied Physics, Biointerfaces Institute, Macromolecular Science & Engineering, University of Michigan, Ann Arbor, USA
Zu Di
Department of Electrical & Computer Engineering, Biomedical Engineering, Applied Physics, Biointerfaces Institute, Macromolecular Science & Engineering, University of Michigan, Ann Arbor, USA
Zhao Xintao
Department of Electrical & Computer Engineering, Biomedical Engineering, Applied Physics, Biointerfaces Institute, Macromolecular Science & Engineering, University of Michigan, Ann Arbor, USA
Senaratne Pramith
Department of Electrical & Computer Engineering, Biomedical Engineering, Applied Physics, Biointerfaces Institute, Macromolecular Science & Engineering, University of Michigan, Ann Arbor, USA
Filbin John
Department of Electrical & Computer Engineering, Biomedical Engineering, Applied Physics, Biointerfaces Institute, Macromolecular Science & Engineering, University of Michigan, Ann Arbor, USA
Silber Brett
Department of Electrical & Computer Engineering, Biomedical Engineering, Applied Physics, Biointerfaces Institute, Macromolecular Science & Engineering, University of Michigan, Ann Arbor, USA
Kang Sarah
Department of Electrical & Computer Engineering, Biomedical Engineering, Applied Physics, Biointerfaces Institute, Macromolecular Science & Engineering, University of Michigan, Ann Arbor, USA
Gladstone Ann
Department of Electrical & Computer Engineering, Biomedical Engineering, Applied Physics, Biointerfaces Institute, Macromolecular Science & Engineering, University of Michigan, Ann Arbor, USA
Lau Matthew
Department of Electrical & Computer Engineering, Biomedical Engineering, Applied Physics, Biointerfaces Institute, Macromolecular Science & Engineering, University of Michigan, Ann Arbor, USA
Cui Guangjie
Department of Electrical & Computer Engineering, Biomedical Engineering, Applied Physics, Biointerfaces Institute, Macromolecular Science & Engineering, University of Michigan, Ann Arbor, USA
Park Younggeun
Department of Mechanical Engineering, University of Michigan, Ann Arbor, USA
Lee Somin Eunice
Department of Electrical & Computer Engineering, Biomedical Engineering, Applied Physics, Biointerfaces Institute, Macromolecular Science & Engineering, University of Michigan, Ann Arbor, USA
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.
