Orthogonal Modular Gene Repression in Escherichia coli Using Engineered CRISPR/Cas9.

Area of Science:

• Synthetic biology
• Molecular biology
• Bioengineering

Background:

• Synthetic gene circuit development is limited by transcription factor availability.
• CRISPR/Cas9 systems have expanded transcription factor options but suffer from imperfect DNA specificity and context-dependent regulation.
• These limitations hinder the specificity and modularity of Cas9-based transcription factors, impacting synthetic circuit design.

Purpose of the Study:

• To develop a computational approach for selecting highly orthogonal Cas9/gRNA transcription factor/promoter pairs.
• To address specificity and modularity issues in CRISPR/Cas9-mediated gene regulation for synthetic biology applications.
• To design and experimentally validate orthogonal regulatory components for robust synthetic gene circuits.

Main Methods:

• Computational design of Cas9/gRNA transcription factor/promoter pairs for maximal orthogonality.
• Experimental validation of four orthogonal promoter/repressor pairs using the strong promoter PL from phage lambda.
• Construction of double and triple inverter circuits to demonstrate promoter interfacability.
• Development and validation of a scheme for predictable incorporation of orthogonal CRISPR/Cas9 regulation into natural promoters.

Main Results:

• Successfully designed and experimentally validated four orthogonal CRISPR/Cas9-based promoter/repressor pairs.
• Demonstrated the interfacability of these orthogonal promoters by constructing functional double and triple inverter synthetic gene circuits.
• Validated a method to predictably integrate orthogonal CRISPR/Cas9 regulatory elements into diverse natural promoters, enhancing modularity.

Conclusions:

• The developed computational approach effectively enhances the specificity and modularity of CRISPR/Cas9-based synthetic gene circuits.
• Orthogonal CRISPR/Cas9 regulatory components can be reliably designed and implemented, paving the way for more complex and robust synthetic biological systems.
• This work provides a framework for overcoming key limitations in CRISPR/Cas9 gene regulation, advancing the field of synthetic biology.