Koplik, S. E. | Search bioRxiv

bioRxiv2022

Expanding the scope of bacterial CRISPR activation with PAM-flexible dCas9 variants

CRISPR-Cas transcriptional tools have been widely applied for programmable regulation of complex biological networks. In comparison to eukaryotic systems, bacterial CRISPR activation (CRISPRa) has stringent target site requirements for effective gene activation. While genes may not always have an NGG PAM at the appropriate position, PAM-flexible dCas9 variants can expand the range of targetable sites. Here we systematically evaluate a panel of PAM-flexible dCas9 variants for their ability to activate bacterial genes. We observe that dxCas9-NG provides a high dynamic range of gene activation for sites with NGN PAMs while dSpRY permits modest activity across almost any PAM. Similar trends were observed for heterologous and endogenous promoters. For all variants tested, improved PAM-flexibility comes with the tradeoff that CRISPRi-mediated gene repression becomes less effective. Weaker CRISPRi gene repression can be partially rescued by expressing multiple sgRNAs to target many sites in the gene of interest. Our work provides a framework to choose the most effective dCas9 variant for a given set of gene targets, which will further expand the utility of CRISPRa/i gene regulation in bacterial systems.

synthetic biology↗

bioRxiv2022

Programmable Mixed-Signal Biocomputers in Mammalian Cells

Living cells perform sophisticated computations that guide them toward discrete states. Synthetic genetic circuits are powerful tools for programing these computations, where transcription-regulatory networks and DNA recombination are the two dominant paradigms for implementing these systems. While each strategy exhibits unique strengths and weaknesses, integrating both into one seamless design framework would enable advanced gene circuit designs intractable with either approach alone. Here, we present Computation via Recombinase Assisted Transcriptional Effectors (CREATE), which leverages site-specific recombination to perform robust logic on discreet computational layers and programmable transcription factors that connect these layers, allowing individual calculations to contribute toward larger operations. We demonstrate the functionality of CREATE by producing sophisticated circuits using a simple plug- and-play framework, including 189 2-input-3-output circuits, modular digital-to-analog signal converters, a 2-bit multiplier circuit, and a digital and analog mixed-signal generator. This work establishes CREATE as a versatile platform for programming complex signal processing systems capable of high-fidelity logic computation and tunable control over circuit output levels. One-Sentence SummaryWe present a minimal and robust genetic circuit platform for programming cells with sophisticated signal processing capabilities.

synthetic biology↗