Abstract
Natural genetic circuits enable cells to make sophisticated digital decisions. Building equally complex synthetic circuits in eukaryotes remains difficult, however, because commonly used components leak transcriptionally, do not arbitrarily interconnect or do not have digital responses. Here, we designed dCas9-Mxi1-based NOR gates in Saccharomyces cerevisiae that allow arbitrary connectivity and large genetic circuits. Because we used the chromatin remodeller Mxi1, our gates showed minimal leak and digital responses. We built a combinatorial library of NOR gates that directly convert guide RNA (gRNA) inputs into gRNA outputs, enabling the gates to be ‘wired’ together. We constructed logic circuits with up to seven gRNAs, including repression cascades with up to seven layers. Modelling predicted the NOR gates have effectively zero transcriptional leak explaining the limited signal degradation in the circuits. Our approach enabled the largest, eukaryotic gene circuits to date and will form the basis for large, synthetic, cellular decision-making systems.
Highlights
Natural genetic circuits enable cells to make sophisticated digital decisions
Libraries of DNA-binding domains (DBDs)-based parts have been shown in prokaryotes, but extensive part characterization and computer-aided design (CAD) was necessary to identify part combinations that yielded functional logic circuits[22]
We show experimentally that we can build a variety of digital logic circuits composed of up to five NOR gates and seven internal guide RNA (gRNA) wires, as well as cascades of gates with up to seven layers that still have digital responses according to our specifications
Summary
Natural genetic circuits enable cells to make sophisticated digital decisions. Building complex synthetic circuits in eukaryotes remains difficult, because commonly used components leak transcriptionally, do not arbitrarily interconnect or do not have digital responses. Programmable and orthogonal CRISPR-dCas[9] transcription factors have been employed[18,20,30,31,32,33,34] to build up to five component circuits using dCas9-mediated repression in prokaryotes[18]. Site-specific recombinases have been employed in genetic circuits as a means to reduce leak[35,36,37], but there are a limited number of such enzymes restricting the scalability of this approach We address these issues, advancing the art of engineering living digital circuits by focusing on two main engineering goals. The strong and consistent ‘OFF’ behaviour we observe with our NOR gates is a key factor that allows them to be a ri
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