Widespread RNA-based cas regulation monitors crRNA abundance and anti-CRISPR proteins.

CRISPR RNAs (crRNAs) and Cas proteins work together to provide prokaryotes with adaptive immunity against genetic invaders like bacteriophages and plasmids. However, the coordination of crRNA production and cas expression remains poorly understood. Here, we demonstrate that widespread modulatory mini-CRISPRs encode cas-regulating RNAs (CreRs) that mediate autorepression of type I-B, I-E, and V-A Cas proteins, based on their limited complementarity to cas promoters. This autorepression not only reduces autoimmune risks but also responds to changes in the abundance of canonical crRNAs that compete with CreR for Cas proteins. Furthermore, the CreR-guided autorepression of Cas proteins can be alleviated or even subverted by diverse bacteriophage anti-CRISPR (Acr) proteins that inhibit Cas effectors, which, in turn, promotes the generation of new Cas proteins. Our findings reveal a general RNA-guided autorepression paradigm for diverse Cas effectors, shedding light on the intricate self-coordination of CRISPR-Cas and its transcriptional counterstrategy against Acr proteins.

Associate toxin-antitoxin with CRISPR-Cas to kill multidrug-resistant pathogens.

CreTA, CRISPR-regulated toxin-antitoxin (TA), safeguards CRISPR-Cas immune systems by inducing cell dormancy/death upon their inactivation. Here, we characterize a bacterial CreTA associating with the I-F CRISPR-Cas in Acinetobacter. CreT is a distinct bactericidal small RNA likely targeting several essential RNA molecules that are required to initiate protein synthesis. CreA guides the CRISPR effector to transcriptionally repress CreT. We further demonstrate a proof-of-concept antimicrobial strategy named ATTACK, which AssociaTes TA and CRISPR-Cas to Kill multidrug resistant (MDR) pathogens. In this design, CRISPR-Cas is programed to target antibiotic resistance gene(s) to selectively kill MDR pathogens or cure their resistance, and when CRISPR-Cas is inactivated or suppressed by unwanted genetic or non-genetic events/factors, CreTA triggers cell death as the last resort. Our data highlight the diversity of RNA toxins coevolving with CRISPR-Cas, and illuminate a combined strategy of CRISPR and TA antimicrobials to 'ATTACK' MDR pathogens.

De novo design of an intercellular signaling toolbox for multi-channel cell-cell communication and biological computation.

Intercellular signaling is indispensable for single cells to form complex biological structures, such as biofilms, tissues and organs. The genetic tools available for engineering intercellular signaling, however, are quite limited. Here we exploit the chemical diversity of biological small molecules to de novo design a genetic toolbox for high-performance, multi-channel cell-cell communications and biological computations. By biosynthetic pathway design for signal molecules, rational engineering of sensing promoters and directed evolution of sensing transcription factors, we obtain six cell-cell signaling channels in bacteria with orthogonality far exceeding the conventional quorum sensing systems and successfully transfer some of them into yeast and human cells. For demonstration, they are applied in cell consortia to generate bacterial colony-patterns using up to four signaling channels simultaneously and to implement distributed bio-computation containing seven different strains as basic units. This intercellular signaling toolbox paves the way for engineering complex multicellularity including artificial ecosystems and smart tissues.

CRISPRi/dCpf1-mediated dynamic metabolic switch to enhance butenoic acid production in Escherichia coli.

Butenoic acid is a short-chain unsaturated fatty acid and important precursor for pharmaceutical and other applications. Heterologous thioesterases are able to convert a fatty acid biosynthesis intermediate in Escherichia coli to butenoic acid. In order to acquire high titer and yield of the product, dynamically switching the metabolic flux from fatty acid biosynthesis pathway to butenoic acid is critical after achieving enough cell mass of the host. A previous developed switch for butenoic acid fermentation is based on triclosan molecule as the FabI inhibitor in the fatty acid biosynthesis cycle. However, triclosan is toxic to human, which may limit its pharmaceutical application. Alternatively, we here purposed a nontoxic switch of carbon flux by harnessing recently developed CRISPR interference (CRISPRi) approach. In our work, we constructed a CRISPRi/dCpf1-mediated dynamic metabolic switch to separate the host growth and production phase via switching the expression of the fabI gene in fatty acid biosynthesis pathway. After optimizing the programmable targets, the CRISPRi-based switch boosted the titer of butenoic acid by 6-fold (1.41 g/L) in fed-batch fermentation. Our work supported that the CRISPRi/dCpf1 switch could replace triclosan-based switch as a nontoxic switch for butenoic acid production, and outcompeted the later switch in the biomass accumulation of the host cell. Moreover, the CRISPRi/dCpf1 system was integrated into the chromosome of the host to improve its genetic stability for long-term fermentation and other applications.Key Points• A programmable metabolic switch was developed to replace the toxic chemical switch to separate the growth phase and production phase of the butenoic acid.• The programmable CRISPRi/dCpf1 switch was efficiently and stably integrated into the host genome to increase their genetic stability during fermentation.• The optimized metabolic switch simultaneously increased the host biomass and butenoic acid titer, and solved the paradox of the competition between growth and production.

The effectiveness of nanobiochar for reducing phytotoxicity and improving soil remediation in cadmium-contaminated soil.

There is growing concern that Cd in soils can be transferred to plants, resulting in phytotoxicity and threats to human health via the food chain. Biochar has been reported to be a soil amendment capable of reducing the bioavailability of metals in soil by electrostatic interactions, ionic exchange and the specific binding of metal ions by surface ligands. To determine the effects of Cd contamination and nanobiochar on the growth characteristics of plants, the dynamics of Cd in soil were explored in Petri dish and pot experiments (0%, 0.2%, 0.5% and 1% nanobiochar), respectively. The diversity, distribution and composition of the bacterial community in treated soil were monitored by high-throughput sequencing. The results showed that the germination potential and height and weight of plants were significantly decreased in Cd-treated soil samples (P < 0.05). The Cd content of Brassica chinensis L. in the treated soil groups was lower than that in the untreated soil groups (P < 0.05) after nanobiochar application. The application of biochar significantly improved the microbial biomass, microorganism abundance and diversity of Actinobacteria and Bacteroidetes in Cd-contaminated soil and reduced the diversity of Proteobacteria, which was relatively more persistent than in the contaminated sites without biochar application. The results of this study provide theoretical and technical support for understanding the environmental behavior of nanopassivators, thus enhancing the role of biochar in the remediation of soil pollution.

Systematically investigating the key features of the DNase deactivated Cpf1 for tunable transcription regulation in prokaryotic cells.

With a unique crRNA processing capability, the CRISPR associated Cpf1 protein holds great potential for multiplex gene regulation. Unlike the well-studied Cas9 protein, however, conversion of Cpf1 to a transcription regulator and its related properties have not been systematically explored yet. In this study, we investigated the mutation schemes and crRNA requirements for the DNase deactivated Cpf1 (dCpf1). By shortening the direct repeat sequence, we obtained genetically stable crRNA co-transcripts and improved gene repression with multiplex targeting. A screen of diversity-enriched PAM library was designed to investigate the PAM-dependency of gene regulation by dCpf1 from Francisella novicida and Lachnospiraceae bacterium. We found novel PAM patterns that elicited strong or medium gene repressions. Using a computational algorithm, we predicted regulatory outputs for all possible PAM sequences, which spanned a large dynamic range that could be leveraged for regulatory purposes. These newly identified features will facilitate the efficient design of CRISPR-dCpf1 based systems for tunable multiplex gene regulation.

[Advances in synthetic physiology of artificial genetic parts].

Artificial genetic parts should be modularized and can be predictably scaled up via assembly or reused in other contexts. Under intracellular physiological conditions, however, the functions of the assembled parts are severely impeded by multi-level physiological interference, i.e., most artificial assembled systems cannot be functional as predicted. Here we proposed a concept of synthetic physiology, defining it as the branch of synthetic biology to investigate and control interferences between artificial genetic parts and intracellular physiological system. Under such framework, we describe the part-host interactions and review the methods and strategies used to characterize and address these interactions.