Conditional guide RNA deactivation by mRNA and small molecule triggers in Saccharomyces cerevisiae

Area of Science:

• Synthetic Biology
• Molecular Biology
• Bioengineering

Background:

• CRISPR interference (CRISPRi) offers precise gene expression modulation but lacks dynamic control.
• Existing CRISPRi systems are largely static, limiting real-time gene regulation.
• Dynamic control is crucial for complex biological applications like metabolic engineering.

Purpose of the Study:

• To develop switchable guide RNAs (gRNAs) for dynamic and tunable gene expression control using CRISPRi.
• To enable CRISPRi regulation in response to external signals like mRNA or small molecules.
• To create a modular platform for gRNA-based biocomputing and reversible gene control.

Main Methods:

• Engineered gRNAs with 5' or 3' extensions to block CRISPRi function.
• Utilized toehold-mediated strand displacement and aptazymes for signal-responsive gRNA deactivation.
• Demonstrated gRNA silencing and restoration of function using mRNA and small molecule triggers.
• Implemented modular gRNAs in Saccharomyces cerevisiae for genetic logic gate construction.

Main Results:

• Achieved complete silencing of gRNA activity via designed extensions.
• Successfully restored gRNA function through signal-induced sequestration or cleavage.
• Demonstrated deactivation of engineered gRNAs by specific mRNA and small molecule signals.
• Established gRNA-based biocomputing with multi-input genetic logic gates in yeast.

Conclusions:

• Developed a novel strategy for dynamic and reversible gene expression control using engineered gRNAs.
• The switchable gRNA system provides tunable CRISPRi regulation in response to cellular or environmental signals.
• This methodology expands the toolkit for advanced synthetic biology and biocomputing in eukaryotic systems.