Current antibiotics tend to be broad spectrum, leading to indiscriminate killing of commensal bacteria and accelerated evolution of drug resistance. Here, we use CRISPR-Cas technology to create antimicrobials whose spectrum of activity is chosen by design. RNA-guided nucleases (RGNs) targeting specific DNA sequences are delivered efficiently to microbial populations using bacteriophage or bacteria carrying plasmids transmissible by conjugation. The DNA targets of RGNs can be undesirable genes or polymorphisms, including antibiotic resistance and virulence determinants in carbapenem-resistant Enterobacteriaceae and enterohemorrhagic Escherichia coli. Delivery of RGNs significantly improves survival in a Galleria mellonella infection model. We also show that RGNs enable modulation of complex bacterial populations by selective knockdown of targeted strains based on genetic signatures. RGNs constitute a class of highly discriminatory, customizable antimicrobials that enact selective pressure at the DNA level to reduce the prevalence of undesired genes, minimize off-target effects and enable programmable remodeling of microbiota.

Synthetic biology involves the engineering of biological organisms by using modular and generalizable designs with the ultimate goal of developing useful solutions to real-world problems. One such problem involves bacterial biofilms, which are crucial in the pathogenesis of many clinically important infections and are difficult to eradicate because they exhibit resistance to antimicrobial treatments and removal by host immune systems. To address this issue, we engineered bacteriophage to express a biofilm-degrading enzyme during infection to simultaneously attack the bacterial cells in the biofilm and the biofilm matrix, which is composed of extracellular polymeric substances. We show that the efficacy of biofilm removal by this two-pronged enzymatic bacteriophage strategy is significantly greater than that of nonenzymatic bacteriophage treatment. Our engineered enzymatic phage substantially reduced bacterial biofilm cell counts by Ϸ4.5 orders of magnitude (Ϸ99.997% removal), which was about two orders of magnitude better than that of nonenzymatic phage. This work demonstrates the feasibility and benefits of using engineered enzymatic bacteriophage to reduce bacterial biofilms and the applicability of synthetic biology to an important medical and industrial problem.phage therapy ͉ synthetic biology

Synthetic gene networks can be constructed to emulate digital circuits and devices, giving one the ability to program and design cells with some of the principles of modern computing, such as counting. A cellular counter would enable complex synthetic programming and a variety of biotechnology applications. Here we report two complementary synthetic genetic counters in E. coli that can count up to three induction events, the first comprised of a riboregulated transcriptional cascade and the second of a recombinase-based cascade of memory units. These modular devices permit counting of varied user-defined inputs over a range of frequencies and can be expanded to count higher numbers.A counter is a key component in digital circuits and computing that retains memory of events or objects, representing each number of such as a distinct state. Counters would also be useful in cells, which often must have accurate accounting of tightly controlled processes or biomolecules to effectively maintain metabolism and growth. Counting mechanisms have been reportedly found in telomere length regulation (1,2) and cell aggregation (3). These system behaviors appear to be the result of a thresholding effect in which some critical molecule number or density must be reached for the observed phenotypic change.In this study, we first develop a counter, termed the Riboregulated Transcriptional Cascade (RTC) Counter, which is based on a transcriptional cascade with additional translational regulation. Figs. 1A and 1C illustrate two such cascades that can count up to 2 and 3, respectively (hence, the designations RTC 2-Counter and RTC 3-Counter). For the RTC 2-Counter, the constitutive promoter P Ltet0-1 drives transcription of T7 RNA polymerase (RNAP), whose protein binds the T7 promoter and transcribes the downstream gene, in this case Green Fluorescent Protein (GFP). Both genes are additionally regulated by riboregulators (4), whose cis and trans elements silence and activate post-transcriptional gene expression, respectively. The cis-repressor sequence (cr) is placed between the transcription start site and the ribosome binding site (RBS), and its complementarity with the RBS causes a stem-loop structure to form upon transcription. This secondary structure prevents binding of the 30S ribosomal subunit to the RBS, inhibiting translation. A short, trans-activating, noncoding RNA (taRNA) driven by the arabinose promoter P BAD binds to the cis-repressor in trans, relieving * This manuscript has been accepted for publication in Science. This version has not undergone final editing. Please refer to the complete version of record at http://www.sciencemag.org/. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the Copyright Act without the prior, written permission of AAAS. * These authors contributed equally to this work. 1A). With cis-repressed T7 RNAP mRNAs in the cell, the first pulse of arabinose drives a short burst of taRNA production and consequently expression of...

Logic and memory are essential functions of circuits that generate complex, state-dependent responses. Here we describe a strategy for efficiently assembling synthetic genetic circuits that use recombinases to implement Boolean logic functions with stable DNA-encoded memory of events. Application of this strategy allowed us to create all 16 two-input Boolean logic functions in living Escherichia coli cells without requiring cascades comprising multiple logic gates. We demonstrate long-term maintenance of memory for at least 90 cell generations and the ability to interrogate the states of these synthetic devices with fluorescent reporters and PCR. Using this approach we created two-bit digital-to-analog converters, which should be useful in biotechnology applications for encoding multiple stable gene expression outputs using transient inputs of inducers. We envision that this integrated logic and memory system will enable the implementation of complex cellular state machines, behaviors and pathways for therapeutic, diagnostic and basic science applications.

A central goal of synthetic biology is to achieve multi-signal integration and processing in living cells for diagnostic, therapeutic and biotechnology applications. Digital logic has been used to build small-scale circuits, but other frameworks may be needed for efficient computation in the resource-limited environments of cells. Here we demonstrate that synthetic analog gene circuits can be engineered to execute sophisticated computational functions in living cells using just three transcription factors. Such synthetic analog gene circuits exploit feedback to implement logarithmically linear sensing, addition, ratiometric and power-law computations. The circuits exhibit Weber's law behaviour as in natural biological systems, operate over a wide dynamic range of up to four orders of magnitude and can be designed to have tunable transfer functions. Our circuits can be composed to implement higher-order functions that are well described by both intricate biochemical models and simple mathematical functions. By exploiting analog building-block functions that are already naturally present in cells, this approach efficiently implements arithmetic operations and complex functions in the logarithmic domain. Such circuits may lead to new applications for synthetic biology and biotechnology that require complex computations with limited parts, need wide-dynamic-range biosensing or would benefit from the fine control of gene expression.

Many natural underwater adhesives harness hierarchically assembled amyloid nanostructures to achieve strong and robust interfacial adhesion under dynamic and turbulent environments. Despite recent advances, our understanding of the molecular design, self-assembly, and structure-function relationship of those natural amyloid fibers remains limited. Thus, designing biomimetic amyloid-based adhesives remains challenging. Here, we report strong and multi-functional underwater adhesives obtained from fusing mussel foot proteins (Mfps) of Mytilus galloprovincialis with CsgA proteins, the major subunit of Escherichia coli amyloid curli fibers. These hybrid molecular materials hierarchically self-assemble into higher-order structures, in which, according to molecular dynamics simulations, disordered adhesive Mfp domains are exposed on the exterior of amyloid cores formed by CsgA. Our fibers have an underwater adhesion energy approaching 20.9 mJ/m2, which is 1.5 times greater than the maximum of bio-inspired and bio-derived protein-based underwater adhesives reported thus far. Moreover, they outperform Mfps or curli fibers taken on their own at all pHs and exhibit better tolerance to auto-oxidation than Mfps at pH ≥7.0. This work establishes a platform for engineering multi-component self-assembling materials inspired by nature.

Summary RNA-based regulation and CRISPR/Cas transcription factors (CRISPR-TFs) have yet to be integrated for multiplexed and tunable modulation of gene networks. Moreover, guide RNAs (gRNAs) for CRISPR-TFs have only been expressed from RNA polymerase III promoters in human cells, which limits their use for conditional gene regulation. To overcome these challenges, we combined multiple RNA regulatory strategies, including RNA-triple-helix structures, introns, microRNAs, and ribozymes, with Cas9-based CRISPR-TFs and Cas6/Csy4-based RNA processing in human cells. We describe three strategies for expressing functional gRNAs from RNA polymerase II promoters, which enable multiplexed production of proteins and multiple gRNAs from a single transcript. We used these tools to efficiently modulate endogenous promoters and implement tunable synthetic circuits, including multi-stage cascades and RNA-dependent networks that could be rewired with Csy4 to achieve complex behaviors. This multiplexable toolkit will be valuable for programming scalable gene circuits and perturbing endogenous networks for biology, therapeutic, and synthetic-biology applications.

Transcriptional regulation is central to the complex behavior of natural biological systems and synthetic gene circuits. Platforms for the scalable, tunable, and simple modulation of transcription would enable new abilities to study natural systems and implement artificial capabilities in living cells. Previous approaches to synthetic transcriptional regulation have relied on engineering DNA-binding proteins, which necessitate multistep processes for construction and optimization of function. Here, we show that the CRISPR/Cas system of Streptococcus pyogenes can be programmed to direct both activation and repression to natural and artificial eukaryotic promoters through the simple engineering of guide RNAs with base-pairing complementarity to target DNA sites. We demonstrate that the activity of CRISPR-based transcription factors (crisprTFs) can be tuned by directing multiple crisprTFs to different positions in natural promoters and by arraying multiple crisprTF-binding sites in the context of synthetic promoters in yeast and human cells. Furthermore, externally controllable regulatory modules can be engineered by layering gRNAs with small molecule-responsive proteins. Additionally, single nucleotide substitutions within promoters are sufficient to render them orthogonal with respect to the same gRNA-guided crisprTF. We envision that CRISPR-based eukaryotic gene regulation will enable the facile construction of scalable synthetic gene circuits and open up new approaches for mapping natural gene networks and their effects on complex cellular phenotypes.