Highly efficient and specific regulation of gene expression using enhanced CRISPR-Cas12f system

Area of Science:

• Molecular Biology
• Gene Regulation
• Biotechnology

Background:

• CRISPR activator (CRISPRa) systems regulate gene expression without DNA alteration.
• Existing CRISPRa systems (Cas9/Cas12a) face limitations in efficacy and accuracy.
• Need for improved CRISPRa tools for precise transcriptional control.

Purpose of the Study:

• To develop a novel CRISPRa system with enhanced efficacy and accuracy.
• To engineer a CRISPR-Cas effector for improved gene regulation.
• To establish a precise tool for controlling endogenous gene transcription.

Main Methods:

• Engineered a small CRISPR-Cas effector, Candidatus Woesearchaeota Cas12f (CWCas12f).
• Developed the optimized eCWCas12f-VPR system by modifying the CRISPR-Cas module.
• Utilized various combinations of activation domains, linkers, and nuclear localization signals.

Main Results:

• The eCWCas12f-VPR system demonstrated effective and target-specific gene expression regulation.
• Achieved superior performance compared to existing CRISPRa systems.
• Validated the system's capability for precise control of endogenous gene transcription.

Conclusions:

• The eCWCas12f-VPR system represents a significant advancement in CRISPRa technology.
• This system offers substantial potential for controlling transcription in living organisms.
• Provides a foundation for future applications in gene therapy and biological research.