Sulfate-reducing bacteria (SRB) are key functional microorganisms in the bioremediation of acid mine drainage (AMD), simultaneously removing sulfate, generating alkalinity, and precipitating metal sulfides via dissimilatory sulfate reduction. AMD is typically characterized by low pH, high sulfate and metal concentrations, and limited organic carbon, imposing persistent stress on SRB growth, metabolism, and function. Environmental stressors include acidic conditions, heavy metal toxicity, low-temperature stress, and electron-donor limitation. These stressors impair membrane stability, enzymatic activity, and cellular energy conservation. Their combined effects increase maintenance requirements while limiting energy acquisition, ultimately reducing the range of environmental conditions under which SRB can sustain sulfate reduction. At the community level, SRB function is further modulated by complex microbial networks. Various functional groups, including fermenters, methanogens, and sulfur- and iron-cycling microorganisms, interact with SRB through cooperative, competitive, and regulatory processes that influence electron transfer, carbon turnover, and iron–sulfur transformations. Sulfate reduction in AMD depends on these community-level interactions, which can be disrupted under environmental stress and may reduce the stability of sulfate-reducing communities. Engineering strategies such as slow-release carbon supplementation, pH microenvironment optimization, conductive material amendment, mineral–microbe interface regulation, and immobilized reactor design can enhance SRB persistence by stabilizing the extracellular microenvironment, regulating electron flow, and spatially decoupling metabolic and mineralization interfaces. This review highlights how environmental stressors and microbial networks jointly regulate SRB function, emphasizes the roles of metabolic niche constraints and community resilience, and provides mechanistic insights for improving the stability and practical performance of SRB-based AMD treatment systems.

Targeted drug delivery in the gastrointestinal tract remains challenging because therapeutics must overcome multiple hierarchical barriers before reaching diseased tissue. Here, we present a multistage delivery platform that integrates magnetic microrobots, a pH-responsive protective coating, and platelet membrane–coated nanoparticles (PNPs) in one platform. A fillable design enables the formation of an internal magnetic layer for microrobot actuation, while the pH-responsive coating protects the cargo during transit and selectively degrades upon pH change, releasing cancer cell–targeting PNPs. In an in vitro colon cancer model that reproduces key gastrointestinal features, including flow, pH variation, and villi-like structures, this strategy increased nanoparticle retention and enhanced cancer cell cytotoxicity compared to nanoparticles administered alone. Ex vivo studies in porcine stomach and intestine further demonstrated robust locomotion on compliant and folded tissue surfaces. These results establish an environment-responsive hierarchical delivery strategy for more precise oral delivery in complex gastrointestinal settings.

Since the development of CRISPR-based genome editing tools, a number of novel technologies have emerged. This includes Prime-Editing that acts as a search and replace genome editing tool. Prime-Editing has been deployed across multiple clades, including in a few flowering plants. Here, we report on the development of an efficient Prime Editor (PE) for the model bryophyte Marchantia. Initial tests were conducted on Acetolactate Synthase as a target and revealed an average efficiency above 40%. The system has been developed in the GoldenGate cloning system, facilitating construct design. The development of PE in Marchantia expands the Genome-Editing tools available for this emerging model in plant biology.

The gastrointestinal tract is rich in metabolic, immune, and microbiome-derived signals that can inform the design of live biotherapeutics and diagnosis of intestinal disorders. Engineered cell-based biosensors can tap into this molecular information and report on their environment, yet their development in gut-resident symbionts has been limited by a lack of validated sensor systems. Here, we present a generalizable pipeline that leverages bacterial transcriptional profiling to identify environment-responsive systems for biosensor engineering. Candidate Sensors Systems (CSSs) mined from healthy, disease, and in vitro transcriptomes were assembled into a barcoded library in Bacteroidaceae chassis and screened in high-throughput in vivo to identify responsive promoters. A unique Bacteroidales ECF-type sigma factor operon with ties to sphingolipid metabolism and flux was highly responsive in chemically-induced colitis models. The biosensor responded robustly to disease and returned to baseline upon recovery, establishing an in vivo-driven strategy for discovering functional biosensors in non-model gut-resident bacteria.

Chromatin organization plays a critical role in regulating gene expression. Chromatin compaction represses gene expression by physically restricting the access of the transcriptional machinery to DNA, while spatial proximity between enhancers and promoters, often mediated by chromatin loops, is essential for gene activation. To investigate the regulatory mechanisms underlying loop formation and chromatin compaction, as well as their effects on gene expression, we developed CRISPR-FOIL (utilizing CRISPR to FOld and ILluminate chromosomal DNA), a novel programmable platform for engineering chromatin loops and inducing chromatin compaction in live cells. CRISPR-FOIL anchors pairs of genomic loci in proximity by engineered single-guide RNAs (sgRNAs), resulting in an artificial chromatin loop. The fused two CRISPR-Sirius gRNAs enable genomic loci to be visualized through fluorescent RNA coat proteins in various colors. In addition, multiple CRISPR-FOIL complexes can act cooperatively to drive chromatin compaction. These results establish CRISPR-FOIL as a powerful tool for engineering chromatin organization in live cells and highlight its potential as a therapeutic platform for gene regulation and disease control.

CRISPR-based molecular diagnostics have emerged as powerful and programmable platforms that enable sensitive and specific detection for disease management and epidemiological surveillance. Advances in CRISPR engineering and assay design are driving the emergence of next-generation detection platforms that are highly sensitive, rapid, and amenable to field deployment. These engineering breakthroughs have the potential to reshape point-of-care tests (POCT) and transform how emerging and persistent health threats are monitored in decentralized and resource-limited settings. Herein, we systematically review the recent advancements in CRISPR engineering strategies aimed at improving detection sensitivity and specificity, eliminating the dependence on preamplification, and enabling robust POC deployment. The discussed strategies encompass both the rational engineering of CRISPR ribonucleoproteins (RNPs) and the optimization of downstream signaling modules for molecular diagnostic applications. We further highlight key challenges and future perspectives that may inspire impactful research directions and accelerate the advancement of CRISPR engineering strategies toward robust, field-deployable POCT platforms.

The therapeutic potential of CRISPR-Cas9 genome editing is fundamentally constrained by the requirement for specific short DNA sequences (PAMs) flanking the target site, limiting access to many clinically relevant genomic loci. This stringent PAM requirement is particularly problematic in applications which require precise positioning, such as base editing and allele-specific editing. Although PAM-relaxed variants have expanded the targetable genome, they incur trade-offs in on-target activity, off-target editing, and cleavage kinetics. This highlights an unmet need for variants that are re-targeted to alternative PAMs in order to maintain the specificity and enzymatic performance inherent to stringent dinucleotide PAM recognition. To overcome these limitations, we developed a yeast selection platform capable of engineering SpCas9 variants with re-specified PAM recognition. Using a clinically relevant Huntington's disease gene (HTT) SNP as a proof-of-concept target, we engineered variants with reciprocal NGC and NGT PAM selectivity, as a step toward allele-specific editing in a large percentage of Huntington's disease patients. These yeast-selected SpCas9 variants retained their modified activity across multiple endogenous HEK293T loci, demonstrating that this specificity is robust across diverse genomic contexts. The variants surpassed PAM-broadened variants on their respective on-target PAM while displaying broad loss of activity across alternative PAMs, effectively re-specifying PAM recognition toward a single dinucleotide sequence. Retargeted variants recovered on-target cleavage kinetics approaching wild-type SpCas9, even under competing substrate conditions, demonstrating that PAM re-specification can simultaneously restore catalytic efficiency and improve specificity. Beyond NGC and NGT, we leveraged our high-throughput platform to engineer Cas9 with re-specified activity across multiple additional non-canonical PAMs in yeast, further demonstrating its utility as a general and programmable framework for expanding the therapeutic reach of precision genome editing.

Selenocysteine (Sec), the 21st amino acid, is a rare non-canonical amino acid that represents an attractive target for protein engineering due to its desirable chemical properties such as high affinity for metals, strong nucleophilicity, and reversible covalent bond formation. To bypass the natural constraints on Sec placement within proteins, several strategies have been developed to rewire the native translational machinery to enable site-specific incorporation. However, these usually abolish the quality control mechanism that excludes the serine-charged selenocysteinyl-tRNA (Ser-tRNASec), the immediate biosynthetic precursor, from translation resulting in heterogenous protein species. This challenge is confounded by a lack of genetic tools to accurately report the selenylation state of the tRNA pool as most are blind to competing process of Ser incorporation, which can only be observed using analytical methods. To resolve this issue, we have developed a new fluorescent reporter, Selenocysteine Adjusted Ratiometric Chromophore (SeARCh), which exhibits two distinct spectral outputs dependent on the incorporation of either Ser (red) or Sec (green). Using SeARCh, we define several factors which influence the observed Sec:Ser ratio and construct a new hybrid biosynthetic pathway with improved performance, achieving 90% Sec incorporation. Furthermore, SeARCh displays unusually complex mass spectra due to the isotope distribution of selenium and heterogenous nature of the protein in solution and we report specific methods to account for this behavior and precisely quantify the rare Ser-containing species found at high Sec incorporation efficiencies. Our findings suggest that the equilibrium between selenoprotein and tRNASec expression levels is a key driver of incorporation efficiency and implies a process that is broadly biosynthetically constrained. Collectively these tools represent a significant advance in the metrology of selenocysteine biosynthesis and incorporation and can be used to inform and standardize future engineering efforts.

Many peptides often do not have a single dominant structure. Instead, many remain disordered in water and fold when they encounter membranes or other chemical environments, a property that underlies diverse biological functions but is difficult to predict. Here we introduce ApexFold, a machine-learning framework that predicts how peptide secondary structure change across environments. ApexFold uses peptide sequence and features together with physicochemical descriptors of the surrounding medium to estimate the fractions of helical, β-like and disordered structure expected in each condition. Trained on circular dichroism measurements from 1,187 peptides assayed in water, co-solvents and membrane-mimicking micelles, ApexFold predicted solvent-induced structural shifts in independent peptide panels and outperformed static structure predictors that return a single conformation. These results show that peptide structural plasticity can be learned from sequence and environment, providing a way to prioritize peptides and experimental conditions before synthesis and structural characterization.

Cooperative behaviors among microorganisms, such as biofilm formation, are widespread and play a key role in the emergence and evolution of antibiotic resistance by promoting genetic exchange and environmental adaptability. Bacterial cooperation can involve the secretion of metabolically costly “public goods” that benefit neighboring cells. These include β-lactamases, chloramphenicol acetyltransferase, outer membrane vesicles, metabolites, and signaling molecules, which can protect susceptible bacteria by reducing local antibiotic concentrations and thereby attenuating selection pressure. Importantly, genes encoding these extracellular products are often subject to horizontal gene transfer, facilitating cooperative interactions across species within microbial communities. In this review, we summarize current knowledge of these extracellular products, highlight their roles in the development and evolution of cooperative antibiotic resistance, examine their potential implications for antibiotic therapy, and identify key gaps in current research. We also examine future research directions and consider how integrating microbial social interactions and community dynamics in antimicrobial strategies could offer new ways to reduce the emergence and spread of antibiotic resistance.