Open AccessMulti‐input CRISPR / C as genetic circuits that interface host regulatory networks
Author(s) -
Nielsen Alec AK,
Voigt Christopher A
Publication year - 2014
Publication title -
molecular systems biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 8.523
H-Index - 148
ISSN - 1744-4292
DOI - 10.15252/msb.20145735
Subject(s) - synthetic biology , biology , crispr , electronic circuit , computational biology , promoter , genetics , guide rna , cas9 , computer science , gene , physics , gene expression , quantum mechanics
Genetic circuits require many regulatory parts in order to implement signal processing or execute algorithms in cells. A potentially scalable approach is to use dC as9, which employs small guide RNA s (sgRNAs) to repress genetic loci via the programmability of RNA : DNA base pairing. To this end, we use dC as9 and designed sgRNAs to build transcriptional logic gates and connect them to perform computation in living cells. We constructed a set of NOT gates by designing five synthetic Escherichia coli σ 70 promoters that are repressed by corresponding sgRNAs, and these interactions do not exhibit crosstalk between each other. These sgRNAs exhibit high on‐target repression (56‐ to 440‐fold) and negligible off‐target interactions (< 1.3‐fold). These gates were connected to build larger circuits, including the Boolean‐complete NOR gate and a 3‐gate circuit consisting of four layered sgRNAs. The synthetic circuits were connected to the native E. coli regulatory network by designing output sgRNAs to target an E. coli transcription factor ( malT ). This converts the output of a synthetic circuit to a switch in cellular phenotype (sugar utilization, chemotaxis, phage resistance).