Simultaneously introducing diverse genomic edits remains a challenge in crop genome engineering. Here we describe a twin prime editing-based knockout (TKO) system that installs stop codon clusters (SCCs) for precise translational termination with minimal in-frame mutations. TKO achieves knockout efficiencies of up to 70.5%, 58.6% and 75.1% in rice, maize and wheat protoplasts, respectively, and produces heritable knockout alleles in 96.8% of regenerated rice plants. In hexaploid wheat, TKO outperforms Cas9 4.2-fold in generating triple-homolog knockouts, largely by reducing in-frame mutations. Orthogonal TKO editors with sequence-divergent SCCs enable simultaneous knockout of up to ten genes without cross-interference. Integration of TKO with conventional prime editing establishes TRIM1 (TKO editor-enabled gene rupture and development of integrated multitype genome modification system) for simultaneous knockout and precise editing, achieving a 22.8% coediting of four genes in rice. TRIM2 extends this capacity to kilobase-scale modifications through a prime editor–recombinase system, enabling a 4.9-kb insertion (1.2% efficiency) and gene knockout (up to 79.8%) in protoplasts. Plant genome editing is multiplexed with twin prime editing.

Power analysis is a critical step in designing a microbiome study. Existing power calculation tools for microbiome studies mainly rely on parametric models of the sequencing counts, which underestimate the complexity of microbiome data and could produce overly optimistic power estimates. In this work, we present a new simulation-based power analysis tool, mPower, for microbiome study design. The tool uses a real data-based semi-parametric simulation framework to generate realistic microbiome data, upon which the power assessment is performed. Coupled with a select differential analysis tool, our power tool supports different study designs, including cross-sectional, case-control, and matched-pair studies, with or without confounders. It allows power analysis for both community-level and taxon-level testing. By using microbiome reference datasets from different environments, the users could perform power calculation based on the environment of interest. The mPower is primarily designed for 16S amplicon sequencing data, and it also incorporates a parametric simulation framework that enables power analysis for shotgun metagenomic data. We showcase the application of mPower with several real-world examples. The web interface of mPower is available at https://microbiomestat.shinyapps.io/mPower/. Video Abstract

The plant microbiome plays a crucial role in enhancing disease resistance, yet microbiome-based plant protection strategies remain limited by an incomplete understanding of how host selection, microbial interactions, and rhizosphere chemistry jointly shape pathogen suppression. Here, we adopt a “learning from nature” approach to design synthetic microbial communities (SynComs) that recapitulate naturally evolved disease-suppressive interactions, using banana Fusarium wilt as a model system. High-throughput profiling revealed that both bacterial and fungal communities contribute to varietal resistance. Resistance-associated microbial taxa were identified and isolated to assemble bacterial, fungal, and cross-kingdom SynComs representative of resistant versus susceptible hosts. SynComs derived from resistant varieties suppressed pathogen growth more effectively than those from susceptible hosts, with cross-kingdom SynComs exhibiting the strongest effects. Cross-kingdom SynCom inoculation significantly reduced disease severity and restructured both the composition and functional potential of the rhizosphere microbiome. Integrative transcriptomic and metabolomic analyses revealed coordinated host metabolic reprogramming, characterized by increased accumulation of diverse metabolites, including alkaloids, amino acids, and flavonoids. Notably, supplementation with resistance-associated rhizosphere metabolites, such as stearic acid and shikimic acid, further enhanced disease suppression. Together, our findings establish a mechanistic framework in which host-guided microbiome assembly and metabolite-mediated interactions jointly enable effective cross-kingdom SynComs for disease suppression, providing ecological principles for microbiome-based plant protection strategies.

Since its discovery, the CRISPR-Cas system has ushered in a transformative era in biodetection, leveraging its simplicity and efficiency to enable Cas protein-based signaling systems for applications in early tumor screening, viral detection, and molecular logic circuits. However, the constrained compatibility of CRISPR/Cas-based signaling systems with diverse input types limits their versatility, primarily due to the restricted activation mechanisms of the Cas protein. Herein, we developed the Cas12a and Cas13a Integrated Targeting (CACIT) system, which harnesses DNA/RNA strand displacement reactions to integrate the enzymatic capabilities of Cas12a and Cas13a. This system supports simultaneous DNA and RNA inputs, offering exceptional programmability and cost-effectiveness. By employing strand displacement reactions, the CACIT system achieves synchronized activation of Cas12a and Cas13a. We have demonstrated that the CACIT system excels in single-nucleotide-variant (SNV) detection, viral RNA detection, machine learning-driven nucleic acid concentration response modeling, logic operations, and intracellular imaging. As a streamlined and versatile signaling platform, the CACIT system expands the scope of CRISPR/Cas activation strategies. With its inherent simplicity and compatibility, this system facilitates integration with diverse nanodevices. Further, this system provides a highly programmable, multifunctional computational module for molecular networks, heralding new possibilities for artificial signaling systems.

Cardioprotective effects of current therapies for mitigating ischaemia/reperfusion (I/R) injury have had limited success. The major challenge is to effectively control oxidative stress while preserving mitochondrial function in a timely manner. Hydrogen (H2) selectively reduces cytotoxic oxygen radicals, aiding in the regulation of physiological and pathological functions. However, the efficacy of H2 therapy is highly dependent on the amount and rate of H2 release, making it critically important to develop rapid, simple and efficient techniques for evolving therapeutic H2. Here we encapsulate H2-producing photosynthetic bacteria (PSB) in an injectable porcine dermal extracellular matrix (ECM) hydrogel to facilitate cardiac I/R injury repair. Upon light exposure, sustained and high H2 production from PSB hydrogel preserves mitochondrial homeostasis and essential functions. In a porcine model of cardiac I/R injury, PSB hydrogel treatment effectively mitigates myocardial damage and salvages jeopardized myocardium. We anticipate that this bacterial therapy for photosynthetic H2 production could provide an improved treatment for I/R-related diseases. Photosynthetic bacteria embedded in an injectable hydrogel produce ROS-scavenging species for mitigating myocardial damage and improving heart repair in a porcine model.

Protease-mediated activation of immune effectors is an evolutionarily conserved mechanism. This study identifies a widespread trypsin–MBL (metallo-β-lactamase) module as a core effector in diverse antiviral bacterial immune systems, such as Hachiman, AVAST and Argonaute. Focusing on the Hachiman-associated trypsin–MBL system, we show that trypsin•HamAB protease activity is inhibited by ATP, while MBL is an autoinhibited DNase with two insertion loops obstructing its catalytic site. Upon infection, trypsin•HamAB senses foreign DNA and hydrolyzes ATP, activating trypsin-like activity, which specifically cleaves MBL at the insertion loops to release repression. The activated MBL depletes DNA and arrests host cell growth. Cryo-electron microscopy structures of trypsin•HamAB–DNA reveal that DNA binding and ATP hydrolysis trigger HamAB oligomerization and trypsin-like domain release, enabling its activation. Our work elucidates a conserved immune mechanism wherein proteolytic activation of a nuclease enables robust immunity against phage while multilayered controls prevent self-toxicity, expanding the repertoire of immune processes governed by regulatory proteolysis. This study identifies and characterizes a conserved trypsin–MBL (metallo-β-lactamase) pair in bacterial immunity; upon infection, trypsin is activated through inhibitory ATP hydrolysis, subsequently activating MBL through site-specific proteolysis, which depletes DNA and restricts cell growth.

Gene expression is controlled by transcription factors (TFs), whose genome binding is shaped by chromatin accessibility and histone modifications, yet mapping these interactions, particularly those with weak affinity or a transient nature, in single cells remains technically challenging. To address this gap, we developed docking and deamination followed by sequencing (D&D-seq), a single-cell immuno-tethering technology for profiling DNA-protein interactions. D&D-seq couples an antibody-binding nanobody to a cytosine base editor, a combination that enables detection of weak or transient factor binding through targeted cytosine-to-uracil editing at protein-bound genomic sites. This approach is compatible with standard single-cell multi-omic workflows and therefore allows integrated analyses of gene regulation. Using assay for transposase-accessible chromatin using sequencing (ATAC-seq) and single-cell ATAC-seq (scATAC-seq), we assessed chromatin accessibility as a functional readout of TF activity, and by coupling D&D-seq with whole-genome sequencing, we captured CTCF binding in both active and inactive chromatin compartments.