Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

Area of Science:

• Synthetic biology
• Genetic engineering
• Molecular biology

Background:

• CRISPR-Cas systems and anti-CRISPR proteins (Acrs) offer novel tools for in vivo gene digital circuit design.
• Previous work has laid the foundation for using gene circuits in various applications, including medical diagnostics and environmental monitoring.

Purpose of the Study:

• To describe a protocol for creating synthetic gene Boolean gates and transcriptional networks in Saccharomyces cerevisiae using dCas9/dCas12a and Acrs.
• To characterize the properties of dCas9/dCas12a as transcription factors for gene expression activation.
• To demonstrate the construction of CRISPR-dCas-Acr-based Boolean logic gates responsive to specific inputs.

Main Methods:

• Utilized the "Design-Build-Test-Learn" biological engineering cycle.
• Employed dCas9 and dCas12a nucleases, along with their corresponding anti-CRISPR proteins (Acrs).
• Engineered transcriptional networks in Saccharomyces cerevisiae, including scaffold RNAs and fusion proteins with activation domains (ADs).

Main Results:

• Maximal gene expression activation with dSpCas9 required an engineered scaffold RNA with multiple VP64 ADs.
• dCas12a required fusion to the VP64-p65-Rta (VPR) AD for maximal activation.
• CRISPR-dCas-Acr interactions formed functional Boolean gates: a NOT gate responsive to β-estradiol (using AcrIIA4) and YES/NOT gates responsive to galactose (using AcrVA5 with dLbCas12a).

Conclusions:

• dCas9 and dCas12a exhibit distinct properties as transcription factors, influenced by activation domains and guide RNA concentrations.
• CRISPR-dCas-Acr systems provide a versatile platform for constructing programmable biological logic gates in yeast.
• The developed protocol facilitates the design and implementation of sophisticated synthetic gene circuits for diverse applications.