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CRISPR-based nucleic acid detection is used in a platform that can simultaneously detect 169 human-associated viruses in multiple samples, providing scalable, multiplexed pathogen detection aimed at routine surveillance for public health.

The great majority of globally circulating pathogens go undetected, undermining patient care and hindering outbreak preparedness and response. To enable routine surveillance and comprehensive diagnostic applications, there is a need for detection technologies that can scale to test many samples(1-3)while simultaneously testing for many pathogens(4-6). Here, we develop Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection. In the CARMEN platform, nanolitre droplets containing CRISPR-based nucleic acid detection reagents(7)self-organize in a microwell array(8)to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN-Cas13) enables robust testing of more than 4,500 crRNA-target pairs on a single array. Using CARMEN-Cas13, we developed a multiplexed assay that simultaneously differentiates all 169 human-associated viruses with at least 10 published genome sequences and rapidly incorporated an additional crRNA to detect the causative agent of the 2020 COVID-19 pandemic. CARMEN-Cas13 further enables comprehensive subtyping of influenza A strains and multiplexed identification of dozens of HIV drug-resistance mutations. The intrinsic multiplexing and throughput capabilities of CARMEN make it practical to scale, as miniaturization decreases reagent cost per test by more than 300-fold. Scalable, highly multiplexed CRISPR-based nucleic acid detection shifts diagnostic and surveillance efforts from targeted testing of high-priority samples to comprehensive testing of large sample sets, greatly benefiting patients and public health(9-11).

The production of pore-forming toxins that disrupt the plasma membrane of host cells is a common virulence strategy for bacterial pathogens such as methicillin-resistant Staphylococcus aureus (MRSA)(1-3). It is unclear, however, whether host species possess innate immune mechanisms that can neutralize pore-forming toxins during infection. We previously showed that the autophagy protein ATG16L1 is necessary for protection against MRSA strains encoding alpha-toxin(4)-a pore-forming toxin that binds the metalloprotease ADAM10 on the surface of a broad range of target cells and tissues(2,5,6). Autophagy typically involves the targeting of cytosolic material to the lysosome for degradation. Here we demonstrate that ATG16L1 and other ATG proteins mediate protection against alpha-toxin through the release of ADAM10 on exosomes-extracellular vesicles of endosomal origin. Bacterial DNA and CpG DNA induce the secretion of ADAM10-bearing exosomes from human cells as well as in mice. Transferred exosomes protect host cells in vitro by serving as scavengers that can bind multiple toxins, and improve the survival of mice infected with MRSA in vivo. These findings indicate that ATG proteins mediate a previously unknown form of defence in response to infection, facilitating the release of exosomes that serve as decoys for bacterially produced toxins.

Elucidating elementary mechanisms that underlie bacterial diversity is central to ecology(1,2) and microbiome research(3). Bacteria are known to coexist by metabolic specialization(4), cooperation(5) and cyclic warfare(6-8). Many species are also motile(9), which is studied in terms of mechanism(10,11), benefit(12,13), strategy(14,15), evolution(16,17) and ecology(18,19). Indeed, bacteria often compete for nutrient patches that become available periodically or by random disturbances(2,20,21). However, the role of bacterial motility in coexistence remains unexplored experimentally. Here we show that-for mixed bacterial populations that colonize nutrient patches-either population outcompetes the other when low in relative abundance. This inversion of the competitive hierarchy is caused by active segregation and spatial exclusion within the patch: a small fast-moving population can outcompete a large fast-growing population by impeding its migration into the patch, while a small fast-growing population can outcompete a large fast-moving population by expelling it from the initial contact area. The resulting spatial segregation is lost for weak growth-migration trade-offs and a lack of virgin space, but is robust to population ratio, density and chemotactic ability, and is observed in both laboratory and wild strains. These findings show that motility differences and their trade-offs with growth are sufficient to promote diversity, and suggest previously undescribed roles for motility in niche formation and collective expulsion-containment strategies beyond individual search and survival.

In mixed bacterial populations that colonize nutrient patches, a growth-migration trade-off can lead to spatial exclusion that provides an advantage to populations that become rare, thereby stabilizing the community.