Digital logic circuits in yeast with CRISPR-dCas9 NOR gates

Area of Science:

• Synthetic biology
• Molecular and cell biology
• Genetic engineering

Background:

• Natural genetic circuits allow cellular digital decision-making.
• Existing synthetic eukaryotic circuits face challenges like transcriptional leakage and limited interconnectivity.
• Developing complex synthetic gene circuits requires robust, digitally responsive components.

Purpose of the Study:

• To design and implement novel NOR gates for eukaryotic synthetic biology.
• To overcome limitations of existing synthetic gene circuit components, such as transcriptional leak.
• To enable the construction of large, arbitrarily interconnected genetic logic circuits in eukaryotes.

Main Methods:

• Engineered dCas9-Mxi1 fusion proteins to function as NOR gates in Saccharomyces cerevisiae.
• Developed a combinatorial library of NOR gates with guide RNA (gRNA) inputs and outputs for modularity.
• Constructed multi-layer repression cascades and logic circuits using the designed NOR gates.

Main Results:

• Achieved minimal transcriptional leak and digital responses using the dCas9-Mxi1 NOR gates.
• Successfully built and demonstrated logic circuits with up to seven gRNAs and seven layers.
• Experimental results align with modeling predictions of near-zero transcriptional leak.

Conclusions:

• The dCas9-Mxi1 NOR gates provide a robust platform for building large, complex eukaryotic gene circuits.
• This approach overcomes key limitations in synthetic circuit design, enabling arbitrary connectivity and digital responses.
• The developed system represents a significant advancement for creating sophisticated synthetic cellular decision-making systems.