Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks

Abstract

Genetic circuits require many regulatory parts in order to implement signal processing or execute algorithms in cells. A potentially scalable approach is to use dCas9, which employs small guide RNAs (sgRNAs) to repress genetic loci via the programmability of RNA:DNA base pairing. To this end, we use dCas9 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[subscript 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).United States. Defense Advanced Research Projects Agency (CLIO N66001‐12‐C‐4016)National Institutes of Health (U.S.) (GM095765)National Institute of General Medical Sciences (U.S.) (Grant P50 GMO98792)Synthetic Biology Engineering Research Center (EEC0540879)United States. Defense Advanced Research Projects Agency (Ginkgo BioWorks. CLIO N66001‐12‐C‐4018)United States. Office of Naval Research. Multidisciplinary University Research Initiative (Grant N00014‐13‐1‐0074)United States. Office of Naval Research. Multidisciplinary University Research Initiative (Boston University. Award 4500000552)United States. Air Force Office of Scientific Research (FA9550‐11‐C‐0028)American Society for Engineering Education. National Defense Science and Engineering Graduate Fellowship (32 CFR 168a