A thermostable Cas9 with increased lifetime in human plasma

Area of Science:

• Molecular Biology
• Biotechnology
• Genomics

Background:

• CRISPR-Cas9 genome editing is widely used but limited by enzyme thermostability.
• Existing Cas9 enzymes derived from mesophilic bacteria degrade at elevated temperatures.
• This restricts CRISPR applications in environments requiring heat stability.

Purpose of the Study:

• To identify and characterize a thermostable Cas9 enzyme for expanded CRISPR applications.
• To evaluate the performance of the thermophilic GeoCas9 in genome editing.
• To assess the potential of GeoCas9 for in vivo applications.

Main Methods:

• Isolation and characterization of Cas9 from the thermophilic bacterium Geobacillus stearothermophilus (GeoCas9).
• Assays for RNA-guided DNA cleavage activity at various temperatures.
• Testing GeoCas9 efficacy in mammalian genome editing using ribonucleoprotein (RNP) complexes.
• Evaluation of GeoCas9 stability in human plasma.

Main Results:

• GeoCas9 exhibits RNA-guided DNA cleavage activity at temperatures up to 70°C, significantly higher than SpyCas9 (45°C).
• GeoCas9 effectively edits mammalian genomes when delivered as an RNP complex.
• The enzyme demonstrates increased stability in human plasma compared to mesophilic Cas9 variants.
• Thermostable GeoCas9 expands the operational temperature range for CRISPR-Cas9 applications.

Conclusions:

• GeoCas9 is a thermostable CRISPR-associated protein with robust genome editing capabilities at high temperatures.
• Its enhanced stability and efficacy in RNP complexes offer advantages for in vivo genome editing.
• GeoCas9 broadens the utility of CRISPR technology for diverse experimental and therapeutic applications.