Engineering activatable promoters for scalable and multi-input CRISPRa/i circuits.

Dynamic, multi-input gene regulatory networks (GRNs) are ubiquitous in nature. Multilayer CRISPR-based genetic circuits hold great promise for building GRNs akin to those found in naturally occurring biological systems. We develop an approach for creating high-performing activatable promoters that can be assembled into deep, wide, and multi-input CRISPR-activation and -interference (CRISPRa/i) GRNs. By integrating sequence-based design and in vivo screening, we engineer activatable promoters that achieve up to 1,000-fold dynamic range in an Escherichia coli-based cell-free system. These components enable CRISPRa GRNs that are six layers deep and four branches wide. We show the generalizability of the promoter engineering workflow by improving the dynamic range of the light-dependent EL222 optogenetic system from 6-fold to 34-fold. Additionally, high dynamic range promoters enable CRISPRa systems mediated by small molecules and protein-protein interactions. We apply these tools to build input-responsive CRISPRa/i GRNs, including feedback loops, logic gates, multilayer cascades, and dynamic pulse modulators. Our work provides a generalizable approach for the design of high dynamic range activatable promoters and enables classes of gene regulatory functions in cell-free systems.

Membrane Augmented Cell-Free Systems: A New Frontier in Biotechnology.

Membrane proteins are present in a wide array of cellular processes from primary and secondary metabolite synthesis to electron transport and single carbon metabolism. A key barrier to applying membrane proteins industrially is their difficult functional production. Beyond expression, folding, and membrane insertion, membrane protein activity is influenced by the physicochemical properties of the associated membrane, making it difficult to achieve optimal membrane protein performance outside the endogenous host. In this review, we highlight recent work on production of membrane proteins in membrane augmented cell-free systems (CFSs) and applications thereof. CFSs lack membranes and can thus be augmented with user-specified, tunable, mimetic membranes to generate customized environments for production of functional membrane proteins of interest. Membrane augmented CFSs would enable the synthesis of more complex plant secondary metabolites, the growth and division of synthetic cells for drug delivery and cell therapeutic applications, as well as enable green energy applications including methane capture and artificial photosynthesis.

A Micropatterned Multielectrode Shell for 3D Spatiotemporal Recording from Live Cells.

Microelectrode arrays (MEAs) have proved to be useful tools for characterizing electrically active cells such as cardiomyocytes and neurons. While there exist a number of integrated electronic chips for recording from small populations or even single cells, they rely primarily on the interface between the cells and 2D flat electrodes. Here, an approach that utilizes residual stress-based self-folding to create individually addressable multielectrode interfaces that wrap around the cell in 3D and function as an electrical shell-like recording device is described. These devices are optically transparent, allowing for simultaneous fluorescence imaging. Cell viability is maintained during and after electrode wrapping around the cel and chemicals can diffuse into and out of the self-folding devices. It is further shown that 3D spatiotemporal recordings are possible and that the action potentials recorded from cultured neonatal rat ventricular cardiomyocytes display significantly higher signal-to-noise ratios in comparison with signals recorded with planar extracellular electrodes. It is anticipated that this device can provide the foundation for the development of new-generation MEAs where dynamic electrode-cell interfacing and recording substitutes the traditional method using static electrodes.