Dynamic observations of CRISPR-Cas target recognition and cleavage heterogeneities

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.

Automated summary

In this study, the conjugation, target recognition, and DNA cleavage processes of CRISPR-Cas system were dynamically observed using single particle spectroscopy. The findings show that the CRISPR-Cas system is stable and functional on single particle surfaces and there is heterogeneity in target recognition and DNA cleavage processes.