Selectively eradicating target cells on the basis of their genetic or transcriptional identity remains important in basic research, medicine, biotechnology and agriculture. For applications involving bacteria, CRISPR nucleases offer promising options due to their ability to enact RNA-guided counterselection; however, using these same nucleases for counterselection in eukaryotes has proven much more restrictive. Here we show that Cas12a2, a recently discovered type V CRISPR nuclease, exhibits RNA-triggered DNA shredding and enables programmable and sequence-specific elimination of yeast and human cells expressing a target transcript. Triggering Cas12a2 elicits rampant double-stranded DNA breaks in trans, leading to cell death. Cell killing can be activated by a wide range of target transcripts, with no observed off-target activation. Leveraging thist approach, we selectively eliminate cells that harbor human papillomavirus, cells that failed to undergo gene editing, or cells that encode a prevalent oncogenic point mutation in KRAS. These findings expand the CRISPR toolbox to allow the selective elimination of eukaryotic cells on the basis of their transcriptional profile. Cas12a2 enables RNA-triggered, sequence-specific killing of eukaryotic cells via widespread DNA shredding, allowing selective elimination of cells on the basis of gene expression, including virus-infected or mutation-bearing cells.

Identifying genomic regions bound by individual proteins such as transcription factors is essential to understanding bacterial gene regulation; however, comprehensive understanding of the effect of protein occupancy on gene regulation would be prohibitively laborious and expensive to achieve using methods such as chromatin immunoprecipitation with sequencing (ChIP-seq) and ChIP with exonuclease treatment (ChIP-exo) for every protein and condition of interest. Here we describe a protocol for performing in vivo protein occupancy display–high resolution (IPOD-HR), a powerful method for genome-wide profiling of protein-bound DNA in prokaryotic systems. Although assay for transposase-accessible chromatin with sequencing (ATAC-seq) is the method of choice for assaying general protein occupancy in eukaryotic systems, bacterial nucleoid-associated proteins can affect ATAC-seq, rendering it unsuitable for use in bacteria. In contrast, IPOD-HR can be used to identify regions of bacterial genomes that are highly bound by proteins, regardless of the identity of the proteins bound, allowing the identification of condition- and genotype-dependent changes in protein occupancy associated with changes in gene regulation. The technique is coupled to RNA polymerase ChIP, followed by sequencing of the extracted samples and downstream analysis using open-source, automated software that we provide and actively maintain. Once cross-linked samples are obtained, the core DNA selection portion of the IPOD-HR protocol takes 3 calendar days to perform. The resulting DNA extracts are subjected to high-throughput sequencing, resulting in sequencing data that are analyzed, which typically requires a few additional days, depending on the number of samples and computing resources. The IPOD-HR experimental method requires familiarity with standard molecular biology techniques suitable for preparing Illumina sequencing inputs, and the computational post-processing pipeline requires basic knowledge of the Linux command line environment. This protocol presents IPOD-HR, a high-resolution approach for profiling genome-wide protein occupancy in bacteria, supported by a computational pipeline for streamlined downstream analysis.

The clinical success of gene therapy depends critically on the development of delivery platforms capable of achieving precise, efficient and tissue-specific delivery of genetic payloads in vivo. A diverse array of carriers, including viral, non-viral, synthetic and natural vectors, have been explored to address this challenge. Among them, adeno-associated viruses, lipid nanoparticles and extracellular vesicles have emerged as leading candidates, each offering distinct advantages and translational hurdles. Here we provide a comparative analysis of these delivery modalities, highlighting their respective design principles, targeting capabilities, immunogenicity profiles and clinical progress. We survey preclinical and clinically adopted delivery strategies and explore how the three delivery platforms can be tailored for gene therapeutics in different diseases. Finally, we discuss emerging strategies to overcome current limitations and outline future directions for the rational design of next-generation gene delivery platforms that combine safety, scalability and functional precision. This Review offers a comparative analysis of platforms for the tissue-specific delivery of genetic payloads, discusses how carriers such as adeno-associated viruses, lipid nanoparticles and extracellular vesicles can be tailored for use in different diseases, and charts a path for engineering improved platforms for clinical use.

Animal venoms constitute a rich source of bioactive peptides and proteins with high target specificity, representing valuable scaffolds for therapeutic development. However, the biotechnological exploitation of venom-derived toxins is limited by challenges in achieving efficient, scalable, and reproducible production. Native venom extraction is constrained by low yields and biological variability, making recombinant platforms essential. Yet, most venom toxins are cysteine-rich peptides with complex disulfide bond architectures and stringent structure–function relationships, posing significant challenges to heterologous expression. Inefficient folding, proteolysis, and secretion bottlenecks frequently compromise functional yield. Among microbial hosts, Komagataella phaffii has emerged as a robust system combining eukaryotic protein processing with high cell-density fermentation and cost-effective cultivation. Its oxidative secretory pathway, strong and regulatable promoters, and suitability for strain engineering make it particularly attractive for producing disulfide-rich toxins. This review provides a critical analysis of recombinant venom toxin production in K. phaffii, focusing on molecular and bioprocess determinants of expression performance. We discuss post-translational modifications, yields, and bioactivity, as well as promoter selection and secretion signal optimization. By integrating data across toxin families, we identify recurring technical bottlenecks and highlight engineering approaches to enhance venom biomanufacturing within microbial biotechnology frameworks.

The human gut microbiome is shaped by diverse selective forces that originate from host and environmental factors and it substantially influences health and disease. Whereas the association of microbial lineages with various health conditions has been shown at different taxonomic levels, the extent to which unifying adaptive mechanisms sort microbial lineages into ecologically differentiated populations remains poorly understood. Here we show that genome-wide selective sweeps are a pervasive mechanism that differentiates bacteria in the microbiome. This mechanism leads to population structures akin to global epidemics across geographically and ethnically diverse human populations. Such sweeps arise when an adaptation allows a clone to outcompete others in its niche followed by rediversification, and they manifest as clusters of closely related genomes on long branches in phylogenetic trees. This structure is revealed by excluding recombination events that mask the clonal descent of the genomes. Indeed, we show that genome-wide sweeps originate under a wide range of recombination rates in at least 66 taxa from 25 bacterial families. Estimated ages of divergence suggest that sweep clusters can spread globally within decades and that this process has occurred throughout human history. Sweep clusters are associated with different host conditions—such as age, colorectal cancer, inflammatory bowel diseases and type 2 diabetes—as an indication of their ecological differentiation. Our results reveal an evolutionary mechanism for the observation of stably inherited strains with differential associations and provide a theoretical foundation for analysing adaptation among microbial populations. Genome-wide selective sweeps commonly occur in the human gut microbiome and can spread across the world within decades to produce epidemic-like population structures.