Rational engineering of a modular bacterial CRISPR-Cas activation platform with expanded target range

Area of Science:

• Synthetic Biology
• Molecular Biology
• Microbial Genetics

Background:

• CRISPR-Cas activator (CRISPRa) systems enable targeted gene transcription in eukaryotes but face limitations in bacteria.
• Bacterial CRISPRa systems are restricted by distance-dependent binding site requirements and need extensive customization.
• Existing systems lack the flexibility for diverse genetic and cellular applications in bacteria.

Purpose of the Study:

• To engineer a highly modular and customizable CRISPRa platform for bacterial applications.
• To overcome the targeting limitations of current bacterial CRISPRa systems.
• To expand the range and improve the performance of CRISPRa in diverse bacterial contexts.

Main Methods:

• Rational protein engineering of Cas proteins and transcription activation domains.
• Utilizing noncovalent protein-protein interactions for modular component assembly.
• Employing a library of circularly permuted Cas proteins and diverse activation domains for screening.

Main Results:

• Developed a modular CRISPRa platform with easily interchangeable plasmid elements.
• Demonstrated rapid screening of activation domains to create systems with distinct regulatory properties.
• Engineered CRISPRa systems with altered target binding site requirements, expanding targeting flexibility and range.

Conclusions:

• The new modular CRISPRa platform offers enhanced customization and targeting flexibility for bacterial gene activation.
• This platform addresses key limitations of existing bacterial CRISPRa systems, paving the way for broader applications.
• The engineered Cas proteins and modular design significantly expand the utility of CRISPRa in microbial synthetic biology.