Digital logic circuits in yeast with CRISPR-dCas9 NOR gates

TLDR

Natural genetic circuits enable cells to make sophisticated digital decisions, yet constructing equally complex synthetic circuits in eukaryotes is hindered by transcriptional leak, lack of arbitrary interconnectivity, and non‑digital responses. The study designs dCas9‑Mxi1‑based NOR gates in *Saccharomyces cerevisiae* to allow arbitrary connectivity and the construction of large genetic circuits. The authors built a combinatorial library of dCas9‑Mxi1 NOR gates that convert guide RNA inputs into outputs, enabling them to be wired together into logic circuits with up to seven gRNAs and cascades of seven layers. The gates showed minimal leak and digital responses, modeling predicted effectively zero transcriptional leak, and the approach produced the largest eukaryotic gene circuits to date, establishing a foundation for large synthetic cellular decision‑making systems.

Abstract

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.