Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting ∼100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses.

A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response

The Rab family of small GTPases regulates intracellular membrane trafficking by orchestrating the biogenesis, transport, tethering, and fusion of membrane-bound organelles and vesicles. Like other small GTPases, Rabs cycle between two states, an active (GTP-loaded) state and an inactive (GDP-loaded) state, and their cycling is catalyzed by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Because an active form of each Rab localizes on a specific organelle (or vesicle) and recruits various effector proteins to facilitate each step of membrane trafficking, knowing when and where Rabs are activated and what effectors Rabs recruit is crucial to understand their functions. Since the discovery of Rabs, they have been regarded as one of the central hubs for membrane trafficking, and numerous biochemical and genetic studies have revealed the mechanisms of Rab functions in recent years. The results of these studies have included the identification and characterization of novel GEFs, GAPs, and effectors, as well as post-translational modifications, for example, phosphorylation, of Rabs. Rab functions beyond the simple effector-recruiting model are also emerging. Furthermore, the recently developed CRISPR/Cas technology has enabled acceleration of knockout analyses in both animals and cultured cells and revealed previously unknown physiological roles of many Rabs. In this review article, we provide the most up-to-date and comprehensive lists of GEFs, GAPs, effectors, and knockout phenotypes of mammalian Rabs and discuss recent findings in regard to their regulation and functions.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/febs.15453

Rab family of small GTPases: an updated view on their regulation and functions.

The past 25 years of genomics research first revealed which genes are encoded by the human genome and then a detailed catalogue of human genome variation associated with many diseases. Despite this, the function of many genes and gene regulatory elements remains poorly characterized, which limits our ability to apply these insights to human disease. The advent of new CRISPR functional genomics tools allows for scalable and multiplexable characterization of genes and gene regulatory elements encoded by the human genome. These approaches promise to reveal mechanisms of gene function and regulation, and to enable exploration of how genes work together to modulate complex phenotypes. In this Review, Przybyla and Gilbert describe the latest approaches for CRISPR-based functional genomics screens, including the adoption of single-cell transcriptomic read-outs and applications in characterizing the non-coding genome and mapping genetic interactions at scale.

A new era in functional genomics screens.

Monoclonal antibody therapies targeting tumour antigens drive cancer cell elimination in large part by triggering macrophage phagocytosis of cancer cells1–7. However, cancer cells evade phagocytosis using mechanisms that are incompletely understood. Here we develop a platform for unbiased identification of factors that impede antibody-dependent cellular phagocytosis (ADCP) using complementary genome-wide CRISPR knockout and overexpression screens in both cancer cells and macrophages. In cancer cells, beyond known factors such as CD47, we identify many regulators of susceptibility to ADCP, including the poorly characterized enzyme adipocyte plasma membrane-associated protein (APMAP). We find that loss of APMAP synergizes with tumour antigen-targeting monoclonal antibodies and/or CD47-blocking monoclonal antibodies to drive markedly increased phagocytosis across a wide range of cancer cell types, including those that are otherwise resistant to ADCP. Additionally, we show that APMAP loss synergizes with several different tumour-targeting monoclonal antibodies to inhibit tumour growth in mice. Using genome-wide counterscreens in macrophages, we find that the G-protein-coupled receptor GPR84 mediates enhanced phagocytosis of APMAP-deficient cancer cells. This work reveals a cancer-intrinsic regulator of susceptibility to antibody-driven phagocytosis and, more broadly, expands our knowledge of the mechanisms governing cancer resistance to macrophage phagocytosis. CRISPR-mediated gene knockout and activation are used to identify molecules in cancer cells and macrophages that regulate antibody-dependent cancer cell phagocytosis.

Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis.

Ongoing social, political and ecological changes in the 21st century have placed more people at risk of life-threatening acute and chronic infections than ever before. The development of new diagnostic, prophylactic, therapeutic and curative strategies is critical to address this burden but is predicated on a detailed understanding of the immensely complex relationship between pathogens and their hosts. Traditional, reductionist approaches to investigate this dynamic often lack the scale and/or scope to faithfully model the dual and co-dependent nature of this relationship, limiting the success of translational efforts. With recent advances in large-scale, quantitative omics methods as well as in integrative analytical strategies, systems biology approaches for the study of infectious disease are quickly forming a new paradigm for how we understand and model host-pathogen relationships for translational applications. Here, we delineate a framework for a systems biology approach to infectious disease in three parts: discovery - the design, collection and analysis of omics data; representation - the iterative modelling, integration and visualization of complex data sets; and application - the interpretation and hypothesis-based inquiry towards translational outcomes.

A systems approach to infectious disease

https://escholarship.org/content/qt17t4s1kg/qt17t4s1kg.pdf?t=qxbb3a

Systematic Identification of Host Cell Regulators of Legionella pneumophila Pathogenesis Using a Genome-wide CRISPR Screen

Author(s): West, Patrick T; Peters, Samantha L; Olm, Matthew R; Yu, Feiqiao B; Gause, Haley; Lou, Yue Clare; Firek, Brian A; Baker, Robyn; Johnson, Alexander D; Morowitz, Michael J; Hettich, Robert L; Banfield, Jillian F | Abstract: BackgroundCandida parapsilosis is a common cause of invasive candidiasis, especially in newborn infants, and infections have been increasing over the past two decades. C. parapsilosis has been primarily studied in pure culture, leaving gaps in understanding of its function in a microbiome context.ResultsHere, we compare five unique C. parapsilosis genomes assembled from premature infant fecal samples, three of which are newly reconstructed, and analyze their genome structure, population diversity, and in situ activity relative to reference strains in pure culture. All five genomes contain hotspots of single nucleotide variants, some of which are shared by strains from multiple hospitals. A subset of environmental and hospital-derived genomes share variants within these hotspots suggesting derivation of that region from a common ancestor. Four of the newly reconstructed C. parapsilosis genomes have 4 to 16 copies of the gene RTA3, which encodes a lipid translocase and is implicated in antifungal resistance, potentially indicating adaptation to hospital antifungal use. Time course metatranscriptomics and metaproteomics on fecal samples from a premature infant with a C. parapsilosis blood infection revealed highly variable in situ expression patterns that are distinct from those of similar strains in pure cultures. For example, biofilm formation genes were relatively less expressed in situ, whereas genes linked to oxygen utilization were more highly expressed, indicative of growth in a relatively aerobic environment. In gut microbiome samples, C. parapsilosis co-existed with Enterococcus faecalis that shifted in relative abundance over time, accompanied by changes in bacterial and fungal gene expression and proteome composition.ConclusionsThe results reveal potentially medically relevant differences in Candida function in gut vs. laboratory environments, and constrain evolutionary processes that could contribute to hospital strain persistence and transfer into premature infant microbiomes. Video abstract.

/pdf/genetic-and-behavioral-adaptation-of-candida-parapsilosis-to-3dsxrkyz3d.pdf

Genetic and behavioral adaptation of Candida parapsilosis to the microbiome of hospitalized infants revealed by in situ genomics, transcriptomics, and proteomics.

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Systematic Identification of Host Cell Regulators of Legionella pneumophila Pathogenesis Using a Genome-wide CRISPR Screen

Genetic and behavioral adaptation of Candida parapsilosis to the microbiome of hospitalized infants revealed by in situ genomics, transcriptomics, and proteomics.