CRISPR Interference Modules as Low-Burden Logic Inverters in Synthetic Circuits

Area of Science:

• Synthetic Biology
• Molecular Engineering
• Bacterial Genetics

Background:

• CRISPR interference (CRISPRi) systems, utilizing dCas9-sgRNA complexes, function as programmable logic inverters for gene regulation.
• CRISPRi has emerged as a powerful tool in bacterial synthetic biology, offering an alternative to traditional transcriptional regulators for information processing tasks.

Purpose of the Study:

• To investigate and modulate the transfer function of CRISPRi logic inverters, focusing on minimizing cellular burden.
• To rationally design an optimal expression cassette for dCas9 to balance low burden and high repression.
• To evaluate the performance of CRISPRi NOT gates in various synthetic circuits and their application in upgrading existing genetic logic gates.

Main Methods:

• Rational design of a dCas9 expression cassette for optimized performance.
• Systematic study of CRISPRi NOT gates targeting tet, lac, and lux promoters at different DNA copy numbers and varying dCas9/sgRNA levels.
• Application of CRISPRi modules to repair a resource-intensive circuit and construct a 2-input NOR gate.

Main Results:

• An optimized dCas9 expression cassette was developed, achieving a low-burden, high-repression trade-off.
• CRISPRi NOT gates demonstrated low-burden characteristics, successfully rectifying a non-functional, high-resource-consuming circuit.
• CRISPRi modules were effectively utilized to upgrade a transcriptional regulator-based NOT gate into a 2-input NOR gate.

Conclusions:

• CRISPRi-based modules serve as effective low-burden components for constructing synthetic circuits for information processing.
• The study highlights the potential of CRISPRi systems in advancing bacterial synthetic biology applications.
• Optimized CRISPRi components enable the development of more efficient and robust genetic logic circuits.