Genetic manipulation of bacteriophages is essential for interrogating phage biology and advancing antimicrobial therapies. However, current genome editing approaches can be inefficient, require multiple steps, or drastically reduce phage titers. Here, we show that targeted DNA nicking enables template-mediated editing of phage genomes in one step without reducing phage titers. Using T7 phage, we show that Cas9-mediated nicking achieved up to 100% recombination across multiple loci, including substitutions and deletions of up to 200 bp and insertions of up to 500 bp, all while preserving phage titers. Editing in T7 was RecA-independent and extended to other phages. Leveraging high titers, we engineered a T7 library of over 440,000 tail-fiber mutants, with isolated mutants restoring infection of two LPS-deficient E. coli hosts by shifting recognition to core LPS components. Overall, DNA nicking is a simple and distinct editing strategy that can advance phage genome engineering, genetic interrogation, and antimicrobial development.

Many bacterial antiphage defence systems are encoded by mobile genetic elements, yet much of this antiviral repertoire remains undiscovered. Antimicrobial-resistance plasmids are well known for disseminating antibiotic-resistance genes, but their contribution to bacterial immunity remains largely unexplored. Using phenotype-guided interrogation of a naturally occurring methicillin-resistant Staphylococcus aureus plasmid, we identify Evangelion, a widespread family of single-gene anti-phage defence systems in which a conserved DUF4062 core is coupled to a diversified auxiliary region required for defence activity. Evangelion systems are enriched on antimicrobial-resistance plasmids but are distributed across diverse bacterial hosts and mobile genetic elements, revealing an evolutionarily conserved defence architecture that has diversified through horizontal gene transfer and adaptation to distinct phage environments. Genetic, structural and evolutionary analyses support a model in which Eva01 senses intracellular phage replication-associated processes and couples activation of a DUF4062 effector to NAD⁺ depletion and abortive infection. Together, our findings define a previously unrecognised family of mobile anti-phage defence systems, establish DUF4062 proteins as a new component of the bacterial anti-phage repertoire, and demonstrate that phenotype-guided interrogation of mobile genetic elements provides a powerful strategy for discovering defence systems beyond the reach of current computational approaches.

The basidiomycete Ustilago maydis is a well-characterized model organism for studying pathogen-host interactions and of great interest for a broad spectrum of biotechnological applications. We set here to develop light inducible molecular tools to enable dynamic studies on signaling networks and fungi-host communication, and for metabolic engineering approaches. In particular, light-controlled, optogenetic switches provide quantitative, spatio-temporal control capabilities, are minimal invasive and reversible. We engineered two blue light-inducible LOV-domain-based gene expression switches, to up- (Blue-ON) and down-regulate (Blue-OFF) gene expression, and performed a functional characterization in sporidia and hyphae of U. maydis. Profiting from the dynamic control ranges and rapid kinetics, we implemented the optogenetic switches to control cell morphology by initiating the transition from a haploid sporidial cellular morphotype to filaments upon regulation of the levels of the polarity factor Rac1 and its constitutive active mutant Q61L. In addition to showing how expression level of effectors can be precisely regulated as an approach to understand fungi-plants interaction, we show in two proof-of-principle applications targeted control over U. maydis filamentous fungal invasion of plant tissue and the mechanisms of tumor formation. For this we placed under Blue-ON and Blue-OFF control two U. maydis effectors, See1 (Seedling efficient effector 1) and TIN2 (Tumor inducing 2), and tumor formation was assayed on maize leaves. Taken together, this study established blue-light switches as effective tools to control morphogenesis and pathogenesis in U. maydis.

Cas12a2 CRISPR nucleases, including SuCas12a2, have been shown to have extensive collateral activity towards RNA, ssDNA, and dsDNA. This collateral activity results in targeted cell elimination and has applications across biotechnology, agriculture, and human health. We explored the natural genetic diversity of Cas12a2 nucleases and characterized nine novel orthologs in a DNA damage kinetic assay in E. coli. Three new Cas12a2 orthologs (RsCas12a2, SdCas12a2, and HmCas12a2) were shown to have high collateral activity towards DNA. These nucleases are highly divergent from SuCas12a2, have conserved core RuvC catalytic residues, and have sequence diversity in the previously reported aromatic clamp residues required for nucleic acid positioning in the active site. We defined PFS preferences and mismatch tolerance for each high-activity Cas12a2 nuclease, expanding the available Cas12a2 toolbox, and discovered functional differences with obvious impacts on downstream applications.

Sustainable hydrogen production from microalgae remains limited by intrinsic physiological constraints and the need to preserve biomass value for food and feed applications. Transgenic approaches to overcome these limitations were proven successful, yet result in genetically modified (GMO) strains that face major regulatory and deployment barriers. Here, we present a non-GMO experimental platform that enables systematic isolation of hydrogen-producing phenotypes through high-throughput UV mutagenesis pipline coupled with targeted physiological screening. Applying this approach across phylogenetically distinct algal species, including the industrial strain Chlorella vulgaris and the extremophile Chlorella ohadii, we achieve high discovery efficiency, recovering 0.4-0.6% validated hydrogen-producing mutants and achieving 6.7-25% validation rates among screen-positive candidates, indicating strong enrichment at the primary screening stage. We show that sustained hydrogen production represents a physiologically accessible state emerging across diverse genetic backgrounds. This state is consistently associated with reorganization of photosynthetic electron partitioning, yet arises through multiple distinct configurations that differentially balance hydrogen production, oxygen metabolism, and carbon fixation. This framework provides a scalable route to identify hydrogen-producing strains in industrially relevant algae without introducing foreign DNA and expands the accessible design space for photobiological hydrogen production.