Classification and functional characterization of regulators of intracellular STING trafficking identified by genome-wide optical pooled screening.

Stimulator of interferon genes (STING) traffics across intracellular compartments to trigger innate responses. Mutations in factors regulating this process lead to inflammatory disorders. To systematically identify factors involved in STING trafficking, we performed a genome-wide optical pooled screen (OPS).

Catalase activity deficiency sensitizes multidrug-resistant Mycobacterium tuberculosis to the ATP synthase inhibitor bedaquiline.

Multidrug-resistant tuberculosis (MDR-TB), defined as resistance to the first-line drugs isoniazid and rifampin, is a growing source of global mortality and threatens global control of tuberculosis disease. The diarylquinoline bedaquiline has recently emerged as a highly efficacious drug against MDR-TB and kills Mycobacterium tuberculosis by inhibiting mycobacterial ATP synthase. However, the mechanisms underlying bedaquiline's efficacy against MDR-TB remain unknown.

Scalable, compressed phenotypic screening using pooled perturbations.

High-throughput phenotypic screens using biochemical perturbations and high-content readouts are constrained by limitations of scale. To address this, we establish a method of pooling exogenous perturbations followed by computational deconvolution to reduce required sample size, labor and cost. We demonstrate the increased efficiency of compressed experimental designs compared to conventional approaches through benchmarking with a bioactive small-molecule library and a high-content imaging readout.

Classification and functional characterization of regulators of intracellular STING trafficking identified by genome-wide optical pooled screening.

STING is an innate immune sensor that traffics across many cellular compartments to carry out its function of detecting cyclic di-nucleotides and triggering defense processes. Mutations in factors that regulate this process are often linked to STING-dependent human inflammatory disorders. To systematically identify factors involved in STING trafficking, we performed a genome-wide optical pooled screen and examined the impact of genetic perturbations on intracellular STING localization.

Single-cell image-based genetic screens systematically identify regulators of Ebola virus subcellular infection dynamics.

Ebola virus (EBOV) is a high-consequence filovirus that gives rise to frequent epidemics with high case fatality rates and few therapeutic options. Here, we applied image-based screening of a genome-wide CRISPR library to systematically identify host cell regulators of Ebola virus infection in 39,085,093 million single cells. Measuring viral RNA and protein levels together with their localization in cells identified over 998 related host factors and provided detailed information about the role of each gene across the virus replication cycle.

Massively parallel combination screen reveals small molecule sensitization of antibiotic-resistant Gram-negative ESKAPE pathogens.

Antibiotic resistance, especially in multidrug-resistant ESKAPE pathogens, remains a worldwide problem. Combination antimicrobial therapies may be an important strategy to overcome resistance and broaden the spectrum of existing antibiotics. However, this strategy is limited by the ability to efficiently screen large combinatorial chemical spaces.

CROPseq-multi: a versatile solution for multiplexed perturbation and decoding in pooled CRISPR screens.

Forward genetic screens seek to dissect complex biological systems by systematically perturbing genetic elements and observing the resulting phenotypes. While standard screening methodologies introduce individual perturbations, multiplexing perturbations improves the performance of single-target screens and enables combinatorial screens for the study of genetic interactions. Current tools for multiplexing perturbations are incompatible with pooled screening methodologies that require mRNA-embedded barcodes, including some microscopy and single cell sequencing approaches.

mutations mediate phenotypic switching in an opportunistic human lung pathogen.

Bacteria evolving within human hosts encounter selective tradeoffs that render mutations adaptive in one context and deleterious in another. Here, we report that the cystic fibrosis-associated pathogen overcomes in-human selective tradeoffs by acquiring successive point mutations that alternate phenotypes. We sequenced the whole genomes of 931 respiratory isolates from two recently infected patients and an epidemiologically-linked, chronically-infected patient.

Vaginal fatty acid response mechanisms reveal a novel strategy for bacterial vaginosis treatment.

Bacterial vaginosis (BV), a common syndrome characterized by -deficient vaginal microbiota, is associated with adverse health outcomes. BV often recurs after standard antibiotic therapy in part because antibiotics promote microbiota dominance by instead of , which has more beneficial health associations. Strategies to promote and inhibit are thus needed.

A genome-wide atlas of human cell morphology.

A key challenge of the modern genomics era is developing data-driven representations of gene function. Here, we present the first unbiased morphology-based genome-wide perturbation atlas in human cells, containing three genome-scale genotype-phenotype maps comprising >20,000 single-gene CRISPR-Cas9-based knockout experiments in >30 million cells. Our optical pooled cell profiling approach (PERISCOPE) combines a de-stainable high-dimensional phenotyping panel (based on Cell Painting) with optical sequencing of molecular barcodes and a scalable open-source analysis pipeline to facilitate massively parallel screening of pooled perturbation libraries.

Human STING is a proton channel.

Proton leakage from organelles is a common signal for noncanonical light chain 3B (LC3B) lipidation and inflammasome activation, processes induced upon stimulator of interferon genes (STING) activation. On the basis of structural analysis, we hypothesized that human STING is a proton channel. Indeed, we found that STING activation induced a pH increase in the Golgi and that STING reconstituted in liposomes enabled transmembrane proton transport.

A genome-wide optical pooled screen reveals regulators of cellular antiviral responses.

The infection of mammalian cells by viruses and innate immune responses to infection are spatiotemporally organized processes. Cytosolic RNA sensors trigger nuclear translocation of the transcription factor interferon regulatory factor 3 (IRF3) and consequent induction of host immune responses to RNA viruses. Previous genetic screens for factors involved in viral sensing did not resolve changes in the subcellular localization of host or viral proteins.

Genome-wide tiled detection of circulating Mycobacterium tuberculosis cell-free DNA using Cas13.

Detection of microbial cell-free DNA (cfDNA) circulating in the bloodstream has emerged as a promising new approach for diagnosing infection. Microbial diagnostics based on cfDNA require assays that can detect rare and highly fragmented pathogen nucleic acids. We now report WATSON (Whole-genome Assay using Tiled Surveillance Of Nucleic acids), a method to detect low amounts of pathogen cfDNA that couples pooled amplification of genomic targets tiled across the genome with pooled CRISPR/Cas13-based detection of these targets.

Microscale combinatorial stimulation of human myeloid cells reveals inflammatory priming by viral ligands.

Cells sense a wide variety of signals and respond by adopting complex transcriptional states. Most single-cell profiling is carried out today at cellular baseline, blind to cells' potential spectrum of functional responses. Exploring the space of cellular responses experimentally requires access to a large combinatorial perturbation space.

Compressed phenotypic screens for complex multicellular models and high-content assays.

High-throughput phenotypic screens leveraging biochemical perturbations, high-content readouts, and complex multicellular models could advance therapeutic discovery yet remain constrained by limitations of scale. To address this, we establish a method for compressing screens by pooling perturbations followed by computational deconvolution. Conducting controlled benchmarks with a highly bioactive small molecule library and a high-content imaging readout, we demonstrate increased efficiency for compressed experimental designs compared to conventional approaches.

Pooled genetic screens with image-based profiling.

Spatial structure in biology, spanning molecular, organellular, cellular, tissue, and organismal scales, is encoded through a combination of genetic and epigenetic factors in individual cells. Microscopy remains the most direct approach to exploring the intricate spatial complexity defining biological systems and the structured dynamic responses of these systems to perturbations. Genetic screens with deep single-cell profiling via image features or gene expression programs have the capacity to show how biological systems work in detail by cataloging many cellular phenotypes with one experimental assay.

The phenotypic landscape of essential human genes.

Understanding the basis for cellular growth, proliferation, and function requires determining the roles of essential genes in diverse cellular processes, including visualizing their contributions to cellular organization and morphology. Here, we combined pooled CRISPR-Cas9-based functional screening of 5,072 fitness-conferring genes in human HeLa cells with microscopy-based imaging of DNA, the DNA damage response, actin, and microtubules. Analysis of >31 million individual cells identified measurable phenotypes for >90% of gene knockouts, implicating gene targets in specific cellular processes.

Epidemiology and genomics of a slow outbreak of methicillin-resistant (MRSA) in a neonatal intensive care unit: Successful chronic decolonization of MRSA-positive healthcare personnel.

Objective: To describe the genomic analysis and epidemiologic response related to a slow and prolonged methicillin-resistant (MRSA) outbreak.

Design: Prospective observational study.

Setting: Neonatal intensive care unit (NICU).

Multiplexed detection of bacterial nucleic acids using Cas13 in droplet microarrays.

Rapid and accurate diagnosis of infections is fundamental to individual patient care and public health management. Nucleic acid detection methods are critical to this effort, but are limited either in the breadth of pathogens targeted or by the expertise and infrastructure required. We present here a high-throughput system that enables rapid identification of bacterial pathogens, bCARMEN, which utilizes: (1) modular CRISPR-Cas13-based nucleic acid detection with enhanced sensitivity and specificity; and (2) a droplet microfluidic system that enables thousands of simultaneous, spatially multiplexed detection reactions at nanoliter volumes; and (3) a novel preamplification strategy that further enhances sensitivity and specificity.

Designing sensitive viral diagnostics with machine learning.

Design of nucleic acid-based viral diagnostics typically follows heuristic rules and, to contend with viral variation, focuses on a genome's conserved regions. A design process could, instead, directly optimize diagnostic effectiveness using a learned model of sensitivity for targets and their variants. Toward that goal, we screen 19,209 diagnostic-target pairs, concentrated on CRISPR-based diagnostics, and train a deep neural network to accurately predict diagnostic readout.

Multiplexed CRISPR-based microfluidic platform for clinical testing of respiratory viruses and identification of SARS-CoV-2 variants.

The coronavirus disease 2019 (COVID-19) pandemic has demonstrated a clear need for high-throughput, multiplexed and sensitive assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses and their emerging variants. Here, we present a cost-effective virus and variant detection platform, called microfluidic Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (mCARMEN), which combines CRISPR-based diagnostics and microfluidics with a streamlined workflow for clinical use. We developed the mCARMEN respiratory virus panel to test for up to 21 viruses, including SARS-CoV-2, other coronaviruses and both influenza strains, and demonstrated its diagnostic-grade performance on 525 patient specimens in an academic setting and 166 specimens in a clinical setting.