Characterizing a thermostable Cas9 for bacterial genome editing and silencing

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• CRISPR-Cas9 systems are powerful genome engineering tools but are limited by their mesophilic nature.
• Applications requiring enhanced stability, such as high-temperature engineering, are hindered by existing Cas9 systems.

Purpose of the Study:

• To identify and characterize a thermophilic CRISPR-Cas9 system for high-temperature genome engineering.
• To develop and validate ThermoCas9-based tools for bacterial gene editing and transcriptional silencing.

Main Methods:

• Isolation and characterization of ThermoCas9 from Geobacillus thermodenitrificans T12.
• In vitro activity assays across a temperature range (20-70°C).
• Development and application of ThermoCas9 for gene deletion and silencing in Bacillus smithii and Pseudomonas putida.

Main Results:

• ThermoCas9 exhibits activity from 20 to 70°C with stringent PAM preference at lower temperatures.
• Its efficiency at elevated temperatures is dependent on sgRNA structure.
• Successful gene deletion and transcriptional silencing were achieved at 55°C in B. smithii and 37°C in P. putida.

Conclusions:

• ThermoCas9 is a functional thermophilic CRISPR-Cas family member.
• This provides a robust Cas9-based tool for bacterial genome editing and silencing across a broad temperature range.
• Expands the utility of CRISPR technology in biotechnological applications requiring thermal stability.