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Abstract Parasitic diseases, particularly malaria (caused by Plasmodium falciparum) and theileriosis (caused by Theileria spp.), profoundly impact global health and the socioeconomic well-being of lower-income countries. Despite recent advances, identifying host metabolic proteins essential for these auxotrophic pathogens remains challenging. Here, we generate a novel metabolic model of human hepatocytes infected with P. falciparum and integrate it with a genome-wide CRISPR knockout screen targeting Theileria-infected cells to pinpoint shared vulnerabilities. We identify key host metabolic enzymes critical for the intracellular survival of both of these lethal hemoparasites. Remarkably, among the metabolic proteins identified by our synergistic approach, we find that host purine and heme biosynthetic enzymes are essential for the intracellular survival of P. falciparum and Theileria, while other host enzymes are only essential under certain metabolic conditions, highlighting P. falciparum’s adaptability and ability to scavenge nutrients selectively. Unexpectedly, host porphyrins emerge as being essential for both parasites. The shared vulnerabilities open new avenues for developing more effective therapies against these debilitating diseases, with the potential for broader applicability in combating apicomplexan infections.

Abstract Reducing disparities is vital for equitable access to precision treatments in cancer. Socioenvironmental factors are a major driver of disparities, but differences in genetic variation likely also contribute. The impact of genetic ancestry on prioritization of cancer targets in drug discovery pipelines has not been systematically explored due to the absence of pre-clinical data at the appropriate scale. Here, we analyze data from 611 genome-scale CRISPR/Cas9 viability experiments in human cell line models to identify ancestry-associated genetic dependencies essential for cell survival. Surprisingly, we find that most putative associations between ancestry and dependency arise from artifacts related to germline variants. Our analysis suggests that for 1.2-2.5% of guides, germline variants in sgRNA targeting sequences reduce cutting by the CRISPR/Cas9 nuclease, disproportionately affecting cell models derived from individuals of recent African descent. We propose three approaches to mitigate this experimental bias, enabling the scientific community to address these disparities.

Abstract Genetic interactions mediate the emergence of phenotype from genotype, but technologies for combinatorial genetic perturbation in mammalian cells are challenging to scale. Here, we identify background-independent paralog synthetic lethals from previous CRISPR genetic interaction screens, and find that the Cas12a platform provides superior sensitivity and assay replicability. We develop the in4mer Cas12a platform that uses arrays of four independent guide RNAs targeting the same or different genes. We construct a genome-scale library, Inzolia, that is ~30% smaller than a typical CRISPR/Cas9 library while also targeting ~4000 paralog pairs. Screens in cancer cells demonstrate discrimination of core and context-dependent essential genes similar to that of CRISPR/Cas9 libraries, as well as detection of synthetic lethal and masking/buffering genetic interactions between paralogs of various family sizes. Importantly, the in4mer platform offers a fivefold reduction in library size compared to other genetic interaction methods, substantially reducing the cost and effort required for these assays.

Abstract CAR-T therapy is a promising, novel treatment modality for B-cell malignancies and yet many patients relapse through a variety of means, including loss of CAR-T cells and antigen escape. To investigate leukemia-intrinsic CAR-T resistance mechanisms, we performed genome-wide CRISPR-Cas9 loss-of-function screens in an immunocompetent murine model of B-cell acute lymphoblastic leukemia (B-ALL) utilizing a modular guide RNA library. We identified IFNγR/JAK/STAT signaling and components of antigen processing and presentation pathway as key mediators of resistance to CAR-T therapy in vivo; intriguingly, loss of this pathway yielded the opposite effect in vitro (sensitized leukemia to CAR-T cells). Transcriptional characterization of this model demonstrated upregulation of these pathways in tumors relapsed after CAR-T treatment, and functional studies showed a surprising role for natural killer (NK) cells in engaging this resistance program. Finally, examination of data from B-ALL patients treated with CAR-T revealed an association between poor outcomes and increased expression of JAK/STAT and MHC-I in leukemia cells. Overall, our data identify an unexpected mechanism of resistance to CAR-T therapy in which tumor cell interaction with the in vivo tumor microenvironment, including NK cells, induces expression of an adaptive, therapy-induced, T-cell resistance program in tumor cells.

Efferocytosis describes the engulfment and clearance of apoptotic cells by phagocytes. Here the authors identify in primary mouse macrophage WDFY3 as a regulator for efferocytosis, in which c-terminal WDFY3 is sufficient to modulate degradation while full-length WDFY3 is required to modulate the uptake of apoptotic cells.

LSD1 inhibition blocks the neuroendocrine phenotype of some small cell lung cancers (SCLCs). Here, a genome-wide CRISPR/Cas9 LSD1 inhibitor resistance screen identifies the mRNA-binding protein ZFP36L1 as a gene repressed by LSD1 that when restored inhibits SCLC neuroendocrine differentiation.

Summary: It is challenging to apply traditional mutational scanning to voltage-gated sodium channels (NaVs) and functionally annotate the large number of coding variants in these genes. Using a cytosine base editor and a pooled viability assay, we screen a library of 368 guide RNAs (gRNAs) tiling NaV1.2 to identify more than 100 gRNAs that change NaV1.2 function. We sequence base edits made by a subset of these gRNAs to confirm specific variants that drive changes in channel function. Electrophysiological characterization of these channel variants validates the screen results and provides functional mechanisms of channel perturbation. Most of the changes caused by these gRNAs are classifiable as loss of function along with two missense mutations that lead to gain of function in NaV1.2 channels. This two-tiered strategy to functionally characterize ion channel protein variants at scale identifies a large set of loss-of-function mutations in NaV1.2.

Numerous rationally-designed and directed-evolution variants of SpCas9 have been reported to expand the utility of CRISPR technology. Here the authors make comparisons of numerous Cas9 variants, nominate options for base editing screens with denser coverage with A>G and C>T base editing screens and identify loss-of-function mutations in BRCA1 and Venetoclax-resistant mutations in BCL2.

CRISPR-based knockout screens in cancer cells have suggested the existence of proliferation suppressor genes (PSG). Here, the authors develop an approach to systematically identify them, and reveal a PSG module involved in fatty acid synthesis and tumour suppression in acute myeloid leukemia cell lines.

Mitochondrial oxidative phosphorylation produces ATP and is an important source for cellular energy equivalents. Here the authors perform a cell-based screen to identify genes that alleviate perturbed mitochondrial complex I function and identify malic enzyme (ME-1), which promotes survival by production of NADPH and glutathione.

Here, Li et al. perform a genome-wide CRISPR screen to identify host dependency factors for influenza A virus infection and show that the host mRNA cap methyltransferase CMTR1 is important for viral cap snatching and that it affects expression of antiviral genes.

Summary: Metastasis is a complex and poorly understood process. In pancreatic cancer, loss of the transforming growth factor (TGF)-β/BMP effector SMAD4 is correlated with changes in altered histopathological transitions, metastatic disease, and poor prognosis. In this study, we use isogenic cancer cell lines to identify SMAD4 regulated genes that contribute to the development of metastatic colonization. We perform an in vivo screen identifying FOSL1 as both a SMAD4 target and sufficient to drive colonization to the lung. The targeting of these genes early in treatment may provide a therapeutic benefit.

Integrating independent large-scale pharmacogenomic screens can enable unprecedented characterization of genetic vulnerabilities in cancers. Here, the authors show that the two largest independent CRISPR-Cas9 gene-dependency screens are concordant, paving the way for joint analysis of the data sets.

Objective: Impaired expansion of peripheral fat contributes to the pathogenesis of insulin resistance and Type 2 Diabetes (T2D). We aimed to identify novel disease–gene interactions during adipocyte differentiation. Methods: Genes in disease-associated loci for T2D, adiposity and insulin resistance were ranked according to expression in human adipocytes. The top 125 genes were ablated in human pre-adipocytes via CRISPR/CAS9 and the resulting cellular phenotypes quantified during adipocyte differentiation with high-content microscopy and automated image analysis. Morphometric measurements were extracted from all images and used to construct morphologic profiles for each gene. Results: Over 107 morphometric measurements were obtained. Clustering of the morphologic profiles accross all genes revealed a group of 14 genes characterized by decreased lipid accumulation, and enriched for known lipodystrophy genes. For two lipodystrophy genes, BSCL2 and AGPAT2, sub-clusters with PLIN1 and CEBPA identifed by morphological similarity were validated by independent experiments as novel protein–protein and gene regulatory interactions. Conclusions: A morphometric approach in adipocytes can resolve multiple cellular mechanisms for metabolic disease loci; this approach enables mechanistic interrogation of the hundreds of metabolic disease loci whose function still remains unknown. Keywords: Gene discovery, Functional genomics, Metabolic syndrome, Insulin resistance, Type 2 diabetes, Genetic screen, High content imaging, Lipodystrophy

Clear-cell carcinomas are aggressive tumours characterised by high accumulation of lipids and glycogen. Here, the authors report that these cancers have a common vulnerability to GPX4 inhibition-induced ferroptosis and using CRISPR screen and lipodomic profiling, they identify HIF-2α- HILPDA axis promotes ferroptosis via enrichment of PUFA lipids.

Summary: The invasion of Chlamydia trachomatis, an obligate intracellular bacterium, into epithelial cells is driven by a complex interplay of host and bacterial factors. To comprehensively define the host genes required for pathogen invasion, we undertook a fluorescence-activated cell sorting (FACS)-based CRISPR screen in human cells. A genome-wide loss-of-function library was infected with fluorescent C. trachomatis and then sorted to enrich for invasion-deficient mutants. The screen identified heparan sulfate, a known pathogen receptor, as well as coatomer complex I (COPI). We found that COPI, through a previously unappreciated role, promotes heparan sulfate cell surface presentation, thereby facilitating C. trachomatis attachment. The heparan sulfate defect does not fully account for the resistance of COPI mutants. COPI also promotes the activity of the pathogen's type III secretion system. Together, our findings establish the requirement for COPI in C. trachomatis invasion and the utility of FACS-based CRISPR screening for the elucidation of host factors required for pathogen invasion. : Molecular Mechanism of Behavior; Medical Microbiology; Methodology in Biological Sciences; Cell Biology; Host-pathogen Interactions; Molecular Microbiology; Genetic Engineering; Genetic Screens Subject Areas: Molecular Mechanism of Behavior, Medical Microbiology, Methodology in Biological Sciences, Cell Biology, Host-pathogen Interactions, Molecular Microbiology, Genetic Engineering, Genetic Screens

Genome-wide libraries for CRISPR knockout, interference, and activation have allowed the systemic interrogation of gene function. Here, the authors evaluate the Brunello CRISPRko library and introduce Dolcetto and Calabrese for CRISPRi and CRISPRa, respectively.

Summary: CD40 has major roles in B cell development, activation, and germinal center responses. CD40 hypoactivity causes immunodeficiency whereas its overexpression causes autoimmunity and lymphomagenesis. To systematically identify B cell autonomous CD40 regulators, we use CRISPR/Cas9 genome-scale screens in Daudi B cells stimulated by multimeric CD40 ligand. These highlight known CD40 pathway components and reveal multiple additional mechanisms regulating CD40. The nuclear ubiquitin ligase FBXO11 supports CD40 expression by targeting repressors CTBP1 and BCL6. FBXO11 knockout decreases primary B cell CD40 abundance and impairs class-switch recombination, suggesting that frequent lymphoma monoallelic FBXO11 mutations may balance BCL6 increase with CD40 loss. At the mRNA level, CELF1 controls exon splicing critical for CD40 activity, while the N6-adenosine methyltransferase WTAP negatively regulates CD40 mRNA abundance. At the protein level, ESCRT negatively regulates activated CD40 levels while the negative feedback phosphatase DUSP10 limits downstream MAPK responses. These results serve as a resource for future studies and highlight potential therapeutic targets. : CD40 is critical for B cell development, germinal center formation, somatic hypermutation, and class-switch recombination. Increased CD40 abundance is associated with autoimmunity and cancer, whereas CD40 hypoactivity causes immunodeficiency. Jiang et al. performed a genome-wide CRISPR/Cas9 screen to reveal key B cell factors that control CD40 abundance and that regulate CD40 responses. Keywords: B cell activation, NF-kappaB, MAP kinase, CRISPR screen, TNF receptor superfamily, humoral immunity, immunodeficiency, N6-Methyladenosine, ESCRT, germinal center

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors—a type III secretion system (T3SS) and Shiga toxins (Stxs)—that are required for the pathogen to colonize the intestine and cause diarrheal disease. Here, we carried out a genome-wide CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats with Cas9) loss-of-function screen to identify host loci that facilitate EHEC infection of intestinal epithelial cells. Many of the guide RNAs identified targeted loci known to be associated with sphingolipid biosynthesis, particularly for production of globotriaosylceramide (Gb3), the Stx receptor. Two loci (TM9SF2 and LAPTM4A) with largely unknown functions were also targeted. Mutations in these loci not only rescued cells from Stx-mediated cell death, but also prevented cytotoxicity associated with the EHEC T3SS. These mutations interfered with early events associated with T3SS and Stx pathogenicity, markedly reducing entry of T3SS effectors into host cells and binding of Stx. The convergence of Stx and T3SS onto overlapping host targets provides guidance for design of new host-directed therapeutic agents to counter EHEC infection. IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors—a type III secretion system (T3SS) and Shiga toxins (Stxs)—that are required for colonizing the intestine and causing diarrheal disease. We screened a genome-wide collection of CRISPR mutants derived from intestinal epithelial cells and identified mutants with enhanced survival following EHEC infection. Many had mutations that disrupted synthesis of a subset of lipids (sphingolipids) that includes the Stx receptor globotriaosylceramide (Gb3) and hence protect against Stx intoxication. Unexpectedly, we found that sphingolipids also mediate early events associated with T3SS pathogenicity. Since antibiotics are contraindicated for the treatment of EHEC, therapeutics targeting sphingolipid biosynthesis are a promising alternative, as they could provide protection against both of the pathogen’s key virulence factors.

Differentiation of erythroblasts to mature red blood cells involves dynamic changes of the membrane and cytoskeleton networks that are not fully characterized. Using a mouse fetal liver erythroblast culture system and a targeted shRNA functional screening strategy, we identified a critical role of pleckstrin-2 in actin dynamics and protection of early stage terminal erythroblasts from oxidative damage. Knockdown of pleckstrin-2 in the early stage of terminal erythropoiesis disrupted the actin cytoskeleton and led to differentiation inhibition and apoptosis. This pro-survival and differentiation function of pleckstrin-2 was mediated through its interaction with cofilin, by preventing cofilin’s mitochondrial entry when the intracellular level of reactive oxygen species was higher in the early stage of terminal erythropoiesis. Treatment of the cells with a scavenger of reactive oxygen species rescued cofilin’s mitochondrial entry and differentiation inhibition induced by pleckstrin-2 knockdown. In contrast, pleckstrin-2 knockdown in late stage terminal erythroblasts had no effect on survival or differentiation but blocked enucleation due to disorganized actin cytoskeleton. Thus, our study identified a dual function of pleckstrin-2 in the early and late stages of terminal erythropoiesis through its regulations of actin dynamics and cofilin’s mitochondrial localization, which reflects intracellular level of reactive oxygen species in different developmental stages.