Engineered Thermostable Chemically Responsive GlowCas9 System for Real-Time Therapeutic Monitoring Applications

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) has revolutionized gene therapy applications due to its ease of design and efficiency, albeit accompanied by off-target effects. Spatiotemporally regulated Cas9 offers safer gene editing methods due to its lower off-target mediated genotoxicities; however, probing the Cas9 in real time for assessing the delivery efficiency, gene editing timeframe, and spatial target information remains elusive. Here, we report an engineered Cas9 system, GlowCas9, that can be probed chemically in real-time across all assay systems, including cell lysate, native gel, ex vivo, and in vivo. Importantly, we rationally engineered the Cas9 system to attain remarkable thermostability with enhanced gene editing activities. Besides its sensitive reporter and enhanced gene editing activity, the GlowCas9 system exhibits precision HDR-based gene knock-in ability in cells and significantly outperformed the WTCas9. Overall, we report a new thermostable engineered SpCas9 system for real-time theratracking applications compatible with diverse assay formats, including live ex vivo and in vivo. This GlowCas9 system will add a new dimension of non-invasive real-time tracking in gene therapy development, both ex vivo and in vivo.