CRISPR interference as low burden logic inverters in synthetic circuits: characterization and tuning

The rational design of complex biological systems through the interconnection of single functional building blocks is hampered by many unpredictability sources; this is mainly due to the tangled context-dependency behavior of those parts once placed into an intrinsically complex living system. Among others, the finite amount of translational resources in prokaryotic cells leads to load effects in heterologous protein expression. As a result, hidden interactions among protein synthesis rates arise, leading to unexpected and counterintuitive behaviors. To face this issue in rational design of synthetic circuits in bacterial cells, CRISPR interference is here evaluated as genetic logic inverters with low translational resource usage, compared with traditional transcriptional regulators. This system has been studied and characterized in several circuit configurations. Each module composing the circuit architecture has been optimized in order to meet the desired specifications, and its reduced metabolic load has been eventually demonstrated via in-vivo assays. ### Competing Interest Statement The authors have declared no competing interest. * CRISPRi : Clustered Regularly Interspaced Short Palindromic Repeats interference GFP : Green Fluorescent Protein HSL : N-oxohexanoyl-L-homoserine lactone IPTG : Isopropyl-β-D-1-thiogalactopyranoside OD600 : optical density at 600 nm PCR : polymerase chain reaction RBS : ribosome binding site RFP : Red Fluorescent Protein rpm : rotation per minute