Mar 1, 2016

Robust digital logic circuits in eukaryotic cells with CRISPR/dCas9 NOR gates

BioRxiv : the Preprint Server for Biology
Miles W GanderEric Klavins

Abstract

Natural genetic circuits enable cells to make sophisticated digital decisions. Building equally complex synthetic circuits in eukaryotes remains difficult, however, because commonly used genetic components leak transcriptionally, do not allow arbitrary interconnections, or do not have digital responses. Here, we designed a new dCas9-Mxi1 based NOR gate architecture in S. cerevisiae that allows arbitrary connectivity and large genetic circuits. Because we used the strong chromatin remodeler Mxi1, our system showed very little leak and exhibits a highly digital response. In particular, we built a combinatorial library of NOR gates that each directly convert guide RNA (gRNA) input signals into gRNA output signals, enabling NOR gates to be ″wired″ together. We constructed and characterized logic circuits with up to seven independent gRNAs, including repression cascades with up to seven layers. Modeling predicted that the NOR gates have Hill Coefficients of approximately 1.71±0.09, explaining the minimal signal degradation we observed in these deeply layered circuits. Our approach enables the construction of the largest, eukaryotic gene circuits to date and will form the basis for large, synthetic, decision making systems in living ...Continue Reading

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Mentioned in this Paper

Anatomical Layer
MXI1 gene
Chromatin Remodeling Factor
Chromatin Location
Clustered Regularly Interspaced Short Palindromic Repeats
Catabolism
Saccharomyces cerevisiae
Gene Circuits
Enzyme Repression
MAX Interacting Protein 1

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