RT Journal Article
SR Electronic
T1 Robust digital logic circuits in eukaryotic cells with CRISPR/dCas9 NOR gates
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 041871
DO 10.1101/041871
A1 Miles W. Gander
A1 Justin D. Vrana
A1 William E. Voje, Jr.
A1 James M. Carothers
A1 Eric Kalvins
YR 2016
UL http://biorxiv.org/content/early/2016/03/02/041871.abstract
AB 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 cells.