A conformational switch-controlled RNA sensor based on orthogonal dCas12a for RNA imaging in live cells.

RNA imaging technology is essential for understanding the complex RNA regulatory mechanisms and serves as a powerful tool for disease diagnosis. However, conventional RNA imaging methods often require multiple fluorescent tags for the specific labeling of individual targets, complicating both the imaging process and subsequent analysis. Herein, we develop an RNA sensor that integrates a blocked CRISPR RNA (crRNA)-based conformational switch with a controllable CRISPR activation (CRISPRa) system and apply for RNA imaging.

Programming ADAR-recruiting hairpin RNA sensor to detect endogenous molecules.

RNA editing leveraging ADARs (adenosine deaminases acting on RNA) shows promising potential for in vivo biosensing beyond gene therapy. However, current ADAR sensors sense only a single target of RNA transcripts, thus limiting their use in different biosensing scenarios. Here, we report a hairpin RNA sensor that exploits new mechanisms to generate intramolecular duplex substrates for efficient ADAR recruitment and editing and apply it to detection of various intracellular molecules, including messenger RNA, small molecules and proteins.

Genetically engineered bacteria as inflammatory bowel disease therapeutics.

Inflammatory bowel disease (IBD) is a chronic and recurrent disease caused by immune response disorders that disrupt the intestinal lumen symbiotic ecosystem and dysregulate mucosal immune functions. Current therapies available for IBD primarily focus on symptom management, making early diagnosis and prompt intervention challenging. The development of genetically engineered bacteria using synthetic biology presents a new strategy for addressing these challenges.

A dual program for CRP-mediated regulation in bacterial alarmone (p)ppGpp.

Gene expression and proper downstream cellular functions upon facing environmental shifts depend on the combined and cooperative regulation of genetic networks. Here, we identified cAMP receptor protein (CRP) as a master regulator of (p)ppGpp (guanosine tetra- and penta-phosphate) homeostasis. Via CRP-mediated direct transcriptional regulation of the (p)ppGpp synthetase/hydrolase RelA and SpoT, cAMP-CRP stimulates pervasive accumulation of (p)ppGpp under glucose-limiting conditions.

The microbiota-dependent tryptophan metabolite alleviates high-fat diet-induced insulin resistance through the hepatic AhR/TSC2/mTORC1 axis.

Type 2 diabetes (T2D) is potentially linked to disordered tryptophan metabolism that attributes to the intricate interplay among diet, gut microbiota, and host physiology. However, underlying mechanisms are substantially unknown. Comparing the gut microbiome and metabolome differences in mice fed a normal diet (ND) and high-fat diet (HFD), we uncover that the gut microbiota-dependent tryptophan metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) is present at lower concentrations in mice with versus without insulin resistance.

A network of acetyl phosphate-dependent modification modulates c-di-AMP homeostasis in .

Cyclic purine nucleotides are important signal transduction molecules across all domains of life. 3',5'-cyclic di-adenosine monophosphate (c-di-AMP) has roles in both prokaryotes and eukaryotes, while the signals that adjust intracellular c-di-AMP and the molecular machinery enabling a network-wide homeostatic response remain largely unknown. Here, we present evidence for an acetyl phosphate (AcP)-governed network responsible for c-di-AMP homeostasis through two distinct substrates, the diadenylate cyclase NA ntegrity canning protein (DisA) and its newly identified transcriptional repressor, DasR.

An Orthogonal CRISPR/dCas12a System for RNA Imaging in Live Cells.

CRISPR/Cas technology has made great progress in the field of live-cell imaging beyond genome editing. However, effective and easy-to-use CRISPR systems for labeling multiple RNAs of interest are still needed. Here, we engineered a CRISPR/dCas12a system that enables the specific recognition of the target RNA under the guidance of a PAM-presenting oligonucleotide (PAMmer) to mimic the PAM recognition mechanism for DNA substrates.

Universal DNA-Based Sensing Toolbox for Programming Cell Functions.

By recombining natural cell signaling systems and further reprogramming cell functions, use of genetically engineered cells and bacteria as therapies is an innovative emerging concept. However, the inherent properties and structures of the natural signal sensing and response pathways constrain further development. We present a universal DNA-based sensing toolbox on the cell surface to endow new signal sensing abilities for cells, control cell states, and reprogram multiple cell functions.

RNase H-Driven crRNA Switch Circuits for Rapid and Sensitive Detection of Various Analytical Targets.

The clustered regularly interspaced short palindromic repeats (CRISPR/Cas12a) system has exhibited great promise in the rapid and sensitive molecular diagnostics for its trans-cleavage property. However, most CRISPR/Cas system-based detection methods are designed for nucleic acids and require target preamplification to improve sensitivity and detection limits. Here, we propose a generic crRNA switch circuit-regulated CRISPR/Cas sensor for the sensitive detection of various targets.

A meet-up of acetyl phosphate and c-di-GMP modulates BldD activity for development and antibiotic production.

Actinobacteria are ubiquitous bacteria undergoing complex developmental transitions coinciding with antibiotic production in response to stress or nutrient starvation. This transition is mainly controlled by the interaction between the second messenger c-di-GMP and the master repressor BldD. To date, the upstream factors and the global signal networks that regulate these intriguing cell biological processes remain unknown.

Overcoming Multidrug Resistance by Base-Editing-Induced Codon Mutation.

Multidrug resistance (MDR) is the main obstacle in cancer chemotherapy. ATP binding cassette (ABC) transporters on the MDR cell membrane can transport a wide range of antitumor drugs out of cells, which is one of the main causes of MDR. Therefore, disturbing ABC transporters becomes the key to reversing MDR.

A DNA Nanodevice-Based Platform with Diverse Capabilities.

Social biotic colonies often perform intricate tasks by interindividual communication and cooperation. Inspired by these biotic behaviors, a DNA nanodevice community is proposed as a universal and scalable platform. The modular nanodevice as the infrastructure of platform contains a DNA origami triangular prism framework and a hairpin-swing arm machinery core.

Acylation driven by intracellular metabolites in host cells inhibits Cas9 activity used for genome editing.

CRISPR-Cas, the immune system of bacteria and archaea, has been widely harnessed for genome editing, including gene knockouts and knockins, single-base editing, gene activation, and silencing. However, the molecular mechanisms underlying fluctuations in the genome editing efficiency of crispr in various cells under different conditions remain poorly understood. In this work, we found that Cas9 can be ac(et)ylated by acetyl-phosphate or acyl-CoA metabolites both and .

Nano-Biohybrid DNA Engager That Reprograms the T-Cell Receptor.

Although engineered T cells with transgenic chimeric antigen receptors (CARs) have made a breakthrough in cancer therapeutics, this approach still faces many challenges in the specificity, efficacy, and self-safety of genetic engineering. Here, we developed a nano-biohybrid DNA engager-reprogrammed T-cell receptor (EN-TCR) system to improve the specificity and efficacy, mitigate the excessive activation, and shield against risks from transgenesis, thus achieving a diversiform and precise control of the T-cell response. Utilizing modular assembly, the EN-TCR system can graft different specificities on T cells via antibody assembly.

An miRISC-initiated DNA nanomachine for monitoring MicroRNA activity in living cells.

MicroRNAs (miRNAs) play an important role in post-transcriptional regulation of gene expression. However, methods to accurately detect miRNA activity in living cells are still limited. Here we developed a DNA nanomachine initiated by a miRNA-induced silencing complex (miRISC) for imaging miRNA activity in living cells.

Switching the Activity of CRISPR/Cas12a Using an Allosteric Inhibitory Aptamer for Biosensing.

The current CRISPR/Cas12a-based diagnostic techniques focus on designing the crRNA or substrate DNA elements to indirectly switch the trans-cleavage activity of Cas12a responsive to target information. Here, we propose the use of an allosteric DNA probe to directly regulate the trans-cleavage activity of Cas12a and present a method for sensing different types of analytes. An allosteric inhibitor probe is rationally designed to couple the target recognition sequence with the inhibitory aptamer of the CRISPR/Cas12a system and enables binding to a specific target to induce the change of conformation, which leads to the loss of its inhibitory function on Cas12a.

An Ultrasensitive, One-Pot RNA Detection Method Based on Rationally Engineered Cas9 Nickase-Assisted Isothermal Amplification Reaction.

RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR) have revolutionized molecular diagnostics by offering versatile Cas effectors. We previously developed an isothermal amplification reaction method using Cas9 nickase (Cas9 nAR) to detect genomic DNA. However, slow dissociation of Cas9n from nicked double-stranded DNA (dsDNA) substrates dramatically hampers the cooperation between Cas9n and DNA polymerase, leading to low amplification efficiency.

A programmable and sensitive CRISPR/Cas12a-based MicroRNA detection platform combined with hybridization chain reaction.

MicroRNAs (miRNAs) play an essential role in cancer diagnosis and prognosis. Developing a new method for sensitive detection of miRNA is constantly in demand. CRISPR/Cas12a system can nonspecifically cleave single-stranded DNA after specific recognition of target DNA, showing tremendous potential in molecular diagnostics.

Delivery of siRNA based on engineered exosomes for glioblastoma therapy by targeting STAT3.

Small interfering RNA (siRNA) therapy has been considered as a promising strategy for treatment of glioblastoma (GBM), which is an aggressive brain disease with poor prognosis. However, siRNA therapy for GBM is seriously hindered by a multitude of barriers including possible immunogenicity, poor cellular uptake, short blood circulation, poor blood stability and low blood-brain barrier (BBB) penetration. This paper reports Angiopep-2 (An2)-functionalized signal transducers and activators of transcription 3 (STAT3) siRNA-loaded exosomes (Exo-An2-siRNA) as potential therapeutic agents to improve GBM therapy.

Simultaneous imaging of cancer biomarkers in live cells based on DNA-engineered exosomes.

Cancer biomarkers are directly related to the development of cancers. Noninvasive identification of the location and expression levels of these biomarkers in live cancer cells offers great potential for accurate early-stage cancer diagnosis and cancer metastasis monitoring. Herein, we propose a DNA-engineered exosome (DNA-Exo) nanoplatform to image dual cancer biomarkers at the single-cell level, in which DNA probes were modified with the cholesterol group to facilely anchor on the exosomal membrane through hydrophobic interaction.

A lateral flow strip combined with Cas9 nickase-triggered amplification reaction for dual food-borne pathogen detection.

Nucleic acid-based detection methods are accurate and rapid, which are widely-used in food-borne pathogen detection. However, traditional nucleic acid-based detection methods usually rely on special instruments, weakening their practicality for on-site tests in resource-limited locations. In this work, we developed a convenient and affordable method for food-borne pathogen detection based on a lateral flow strip combined with Cas9 nickase-triggered isothermal DNA amplification, which allows instrument-free and dual target detection.

A Cas12a-mediated cascade amplification method for microRNA detection.

MicroRNAs (miRNAs) play a vital role in various biological processes and act as important biomarkers for clinical cancer diagnosis, prognosis, and therapy. Here, we took advantage of Cas12a trans-cleavage activity to develop an enzyme-assisted cascade amplification method for isothermal miRNA detection. A target miRNA-initiated ligation reaction would allow for the production of transcription templates that triggered the transcriptional amplification of RNA strands.

Catalytic-Hairpin-Assembly-Assisted DNA Tetrahedron Nanoprobe for Intracellular MicroRNA Imaging.

The aberrant expression levels of microRNAs (miRNAs) are tightly linked with the initiation and development of various diseases and genetic disorders. Here, we reported a catalytic-hairpin-assembly-assisted DNA tetrahedron nanoprobe for intracellular miRNA detection. The target miRNA initiated the catalytic-hairpin-assembly reaction between the tetrahedron nanoprobes to generate large tetrahedron clusters with an enhanced fluorescence resonance energy transfer between the Cy3 and Cy5 dyes.

A telomerase-responsive nanoprobe with theranostic properties in tumor cells.

Multidrug resistance (MDR) is the main cause of treatment failure in clinical cancer chemotherapy due to the presence of P-glycoproteins (P-gp), which widely exist in stubborn drug-resistant tumor membranes and actively pump drugs from inside the tumor cell to the outside. In this study, we report a novel telomerase-responsive nanoprobe with theranostic properties for inhibiting P-gp expression and reversing MDR by gene silencing. This nanoprobe is composed of an AuNP assembled with telomerase primer, antisense oligonucleotide (ASO), and doxorubicin (Dox).