Computational design of CRISPR guide RNAs to enable strain-specific control of microbial consortia.

Microbes naturally coexist in complex, multistrain communities. However, extracting individual microbes from and specifically manipulating the composition of these consortia remain challenging. The sequence-specific nature of CRISPR guide RNAs can be leveraged to accurately differentiate microorganisms and facilitate the creation of tools that can achieve these tasks. We developed a computational program, ssCRISPR, which designs strain-specific CRISPR guide RNA sequences with user-specified target strains, protected strains, and guide RNA properties. We experimentally verify the accuracy of the strain specificity predictions in both Escherichia coli and Pseudomonas spp. and show that up to three nucleotide mismatches are often required to ensure perfect specificity. To demonstrate the functionality of ssCRISPR, we apply computationally designed CRISPR-Cas9 guide RNAs to two applications: the purification of specific microbes through one- and two-plasmid transformation workflows and the targeted removal of specific microbes using DNA-loaded liposomes. For strain purification, we utilize gRNAs designed to target and kill all microbes in a consortium except the specific microbe to be isolated. For strain elimination, we utilize gRNAs designed to target only the unwanted microbe while protecting all other strains in the community. ssCRISPR will be of use in diverse microbiota engineering applications.

Genetically stable CRISPR-based kill switches for engineered microbes.

Microbial biocontainment is an essential goal for engineering safe, next-generation living therapeutics. However, the genetic stability of biocontainment circuits, including kill switches, is a challenge that must be addressed. Kill switches are among the most difficult circuits to maintain due to the strong selection pressure they impart, leading to high potential for evolution of escape mutant populations. Here we engineer two CRISPR-based kill switches in the probiotic Escherichia coli Nissle 1917, a single-input chemical-responsive switch and a 2-input chemical- and temperature-responsive switch. We employ parallel strategies to address kill switch stability, including functional redundancy within the circuit, modulation of the SOS response, antibiotic-independent plasmid maintenance, and provision of intra-niche competition by a closely related strain. We demonstrate that strains harboring either kill switch can be selectively and efficiently killed inside the murine gut, while strains harboring the 2-input switch are additionally killed upon excretion. Leveraging redundant strategies, we demonstrate robust biocontainment of our kill switch strains and provide a template for future kill switch development.

Engineering ligand-specific biosensors for aromatic amino acids and neurochemicals.

Microbial biosensors have diverse applications in metabolic engineering and medicine. Specific and accurate quantification of chemical concentrations allows for adaptive regulation of enzymatic pathways and temporally precise expression of diagnostic reporters. Although biosensors should differentiate structurally similar ligands with distinct biological functions, such specific sensors are rarely found in nature and challenging to create. Using E. coli Nissle 1917, a generally regarded as safe microbe, we characterized two biosensor systems that promiscuously recognize aromatic amino acids or neurochemicals. To improve the sensors' selectivity and sensitivity, we applied rational protein engineering by identifying and mutagenizing amino acid residues and successfully demonstrated the ligand-specific biosensors for phenylalanine, tyrosine, phenylethylamine, and tyramine. Additionally, our approach revealed insights into the uncharacterized structure of the FeaR regulator, including critical residues in ligand binding. These results lay the groundwork for developing kinetically adaptive microbes for diverse applications. A record of this paper's transparent peer review process is included in the supplemental information.

Engineering microbial diagnostics and therapeutics with smart control.

Microbes have become an increasingly powerful chassis for developing diagnostic and therapeutic technologies. While many of the earlier engineering efforts used microbes that expressed relevant proteins constitutively, more microbes are being engineered to express them with region-selectivity and disease-responsiveness through biosensors. Such 'smart' microbes have been developed to diagnose and treat a wide range of disorders and diseases, including bacterial infections, cancers, inflammatory disorders, and metabolic disorders. In this review, we discuss synthetic biology technologies that have been applied to engineer microbes for biomedical applications, focusing on recent reports that demonstrate microbial sensing by using animal models or clinical samples. Advances in synthetic biology will enable engineered microbes to significantly improve the medical field.

Biosensing in Smart Engineered Probiotics.

Engineered microbes are exciting alternatives to current diagnostics and therapeutics. Researchers have developed a wide range of genetic tools and parts to engineer probiotic and commensal microbes. Among these tools and parts, biosensors allow the microbes to sense and record or to sense and respond to chemical and environmental signals in the body, enabling them to report on health conditions of the animal host and/or deliver therapeutics in a controlled manner. This review focuses on how biosensing is applied to engineer "smart" microbes for in vivo diagnostic, therapeutic, and biocontainment goals. Hurdles that need to be overcome when transitioning from high-throughput in vitro systems to low-throughput in vivo animal models, new technologies that can be implemented to alleviate this experimental gap, and areas where future advancements can be made to maximize the utility of biosensing for medical applications are also discussed. As technologies for engineering microbes continue to be developed, these engineered organisms will be used to address many medical challenges.

Molecular Toolkit for Gene Expression Control and Genome Modification in Rhodococcus opacus PD630.

Rhodococcus opacus PD630 is a non-model Gram-positive bacterium that possesses desirable traits for lignocellulosic biomass conversion. In particular, it has a relatively rapid growth rate, exhibits genetic tractability, produces high quantities of lipids, and can tolerate and consume toxic lignin-derived aromatic compounds. Despite these unique, industrially relevant characteristics, R. opacus has been underutilized because of a lack of reliable genetic parts and engineering tools. In this work, we developed a molecular toolbox for reliable gene expression control and genome modification in R. opacus. To facilitate predictable gene expression, a constitutive promoter library spanning ∼45-fold in output was constructed. To improve the characterization of available plasmids, the copy numbers of four heterologous and nine endogenous plasmids were determined using quantitative PCR. The molecular toolbox was further expanded by screening a previously unreported antibiotic resistance marker (HygR) and constructing a curable plasmid backbone for temporary gene expression (pB264). Furthermore, a system for genome modification was devised, and three neutral integration sites were identified using a novel combination of transcriptomic data, genomic architecture, and growth rate analysis. Finally, the first reported system for targeted, tunable gene repression in Rhodococcus was developed by utilizing CRISPR interference (CRISPRi). Overall, this work greatly expands the ability to manipulate and engineer R. opacus, making it a viable new chassis for bioproduction from renewable feedstocks.

Engineering Modular Biosensors to Confer Metabolite-Responsive Regulation of Transcription.

Efforts to engineer microbial factories have benefitted from mining biological diversity and high throughput synthesis of novel enzymatic pathways, yet screening and optimizing metabolic pathways remain rate-limiting steps. Metabolite-responsive biosensors may help to address these persistent challenges by enabling the monitoring of metabolite levels in individual cells and metabolite-responsive feedback control. We are currently limited to naturally evolved biosensors, which are insufficient for monitoring many metabolites of interest. Thus, a method for engineering novel biosensors would be powerful, yet we lack a generalizable approach that enables the construction of a wide range of biosensors. As a step toward this goal, we here explore several strategies for converting a metabolite-binding protein into a metabolite-responsive transcriptional regulator. By pairing a modular protein design approach with a library of synthetic promoters and applying robust statistical analyses, we identified strategies for engineering biosensor-regulated bacterial promoters and for achieving design-driven improvements of biosensor performance. We demonstrated the feasibility of this strategy by fusing a programmable DNA binding motif (zinc finger module) with a model ligand binding protein (maltose binding protein), to generate a novel biosensor conferring maltose-regulated gene expression. This systematic investigation provides insights that may guide the development of additional novel biosensors for diverse synthetic biology applications.