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 σ70 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).

Design principles for robust oscillatory behavior

“…In the last years, a significant number of ''orthogonal'' (non cross-interacting) transcriptional activators and repressors have been identified and used to construct basic synthetic biological circuits (Rhodius et al 2013;Stanton et al 2014;Nielsen and Voigt 2014). It would therefore be interesting to perform the analysis presented here with transcriptional regulation based on Hill functions.…”

An analytical approach to bistable biological circuit discrimination using real algebraic geometry

“…A second MBN of particular interest, herein referred to as the monomerdimer (MD) toggle, is a double-negative switch variant in which one of the repressors functions as a monomer (figure 1a, bottom). While to the best of our knowledge no MD toggle circuit has been constructed, the components necessary for its implementation exist in the form of monomeric singlechain transcriptional repressor proteins [17], as well as in transcription-activator-like effectors (TALEs) and CRISPR/Cas nucleases that have been engineered as repressors [18,19]. Other exotic toggle-like circuit topologies have also been proposed and/or built [13,18,[20][21][22].…”

Benchmarking of TALE- and CRISPR/dCas9-Based Transcriptional Regulators in Mammalian Cells for the Construction of Synthetic Genetic Circuits

“…It has been demonstrated that even logic circuits comprising three layers retained a clear separation between active and inactive states 11 . CRISPR/dCas9-based transcription factors have recently been implemented to perform simple logic functions in bacterial 20 and mammalian cells [21][22][23] . The advantage of CRISPR-based multiplexing based on several gRNAs represents an attractive opportunity, as several strategies have been proposed to generate multiple gRNAs in a single transcript 22,24 .…”