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

Area of Science:

• Biotechnology
• Molecular Biology
• Gene Therapy

Background:

• CRISPR/Cas systems offer efficient gene editing but can have off-target effects.
• Real-time monitoring of Cas9 delivery and activity is crucial for safer gene therapy but remains challenging.

Purpose of the Study:

• To engineer a Cas9 system, GlowCas9, for real-time chemical probing and theratracking.
• To enhance Cas9 thermostability and gene editing efficiency for improved safety and efficacy.

Main Methods:

• Rational engineering of the Cas9 system to create GlowCas9 with enhanced thermostability.
• Testing GlowCas9's real-time probing capabilities across various assay systems (cell lysate, gels, ex vivo, in vivo).
• Assessing GlowCas9's gene editing activity, precision (HDR knock-in), and performance compared to wild-type Cas9 (WTCas9).

Main Results:

• GlowCas9 enables real-time chemical probing in diverse biological samples and conditions.
• The engineered system exhibits remarkable thermostability and enhanced gene editing activities.
• GlowCas9 demonstrates precise homology-directed repair (HDR)-based gene knock-in and outperforms WTCas9.

Conclusions:

• GlowCas9 is a novel, thermostable engineered SpCas9 system suitable for real-time theratracking.
• This system offers non-invasive, real-time monitoring capabilities for gene therapy development.
• GlowCas9 enhances safety and efficacy by providing precise tracking and improved gene editing performance.