Analysis of single-cell CRISPR perturbations indicates that enhancers predominantly act multiplicatively.

A single gene may have multiple enhancers, but how they work in concert to regulate transcription is poorly understood. To analyze enhancer interactions throughout the genome, we developed a generalized linear modeling framework, GLiMMIRS, for interrogating enhancer effects from single-cell CRISPR experiments. We applied GLiMMIRS to a published dataset and tested for interactions between 46,166 enhancer pairs and corresponding genes, including 264 "high-confidence" enhancer pairs. We found that enhancer effects combine multiplicatively but with limited evidence for further interactions. Only 31 enhancer pairs exhibited significant interactions (false discovery rate <0.1), none of which came from the high-confidence set, and 20 were driven by outlier expression values. Additional analyses of a second CRISPR dataset and in silico enhancer perturbations with Enformer both support a multiplicative model of enhancer effects without interactions. Altogether, our results indicate that enhancer interactions are uncommon or have small effects that are difficult to detect.

Dynamic convergence of autism disorder risk genes across neurodevelopment.

Over a hundred risk genes underlie risk for autism spectrum disorder (ASD) but the extent to which they converge on shared downstream targets to increase ASD risk is unknown. To test the hypothesis that cellular context impacts the nature of convergence, here we apply a pooled CRISPR approach to target 29 ASD loss-of-function genes in human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells, glutamatergic neurons, and GABAergic neurons. Two distinct approaches (gene-level and network-level analyses) demonstrate that convergence is greatest in mature glutamatergic neurons. Convergent effects are dynamic, varying in strength, composition, and biological role between cell types, increasing with functional similarity of the ASD genes examined, and driven by cell-type-specific gene co-expression patterns. Stratification of ASD genes yield targeted drug predictions capable of reversing gene-specific convergent signatures in human cells and ASD-related behaviors in zebrafish. Altogether, convergent networks downstream of ASD risk genes represent novel points of individualized therapeutic intervention.

In vivo CRISPR screens identify Mga as an immunotherapy target in triple-negative breast cancer.

Immune evasion is not only critical for tumor initiation and progression, but also determines the efficacy of immunotherapies. Through iterative in vivo CRISPR screens with seven syngeneic tumor models, we identified core and context-dependent immune evasion pathways across cancer types. This valuable high-confidence dataset is available for the further understanding of tumor intrinsic immunomodulators, which may lead to the discovery of effective anticancer therapeutic targets. With a focus on triple-negative breast cancer (TNBC), we found that Mga knock-out significantly enhances antitumor immunity and inhibits tumor growth. Transcriptomics and single-cell RNA sequencing analyses revealed that Mga influences various immune-related pathways in the tumor microenvironment. Our findings suggest that Mga may play a role in modulating the tumor immune landscape, though the precise mechanisms require further investigation. Interestingly, we observed that low MGA expression in breast cancer patients correlates with a favorable prognosis, particularly in those with active interferon-γ signaling. These observations provide insights into tumor immune escape mechanisms and suggest that further exploration of MGA's function could potentially lead to effective therapeutic strategies in TNBC.

Expression and biological significance of topoisomerase II α (TOP2A) in oral squamous cell carcinoma.

BACKGROUND: Topoisomerase II α(TOP2A) is usually highly expressed in rapidly proliferating cells, and its expression is regulated by cell cycle. The relationship between TOP2A and oral squamous cell carcinoma (OSCC) needs further study. METHODS: TOP2A immunoreactivity was analyzed using immunohistochemical (IHC) staining analysis in specimens from 20 OSCC patients. Based on the high-throughput sequencing and gene microarray database, the expression of TOP2A mRNA in OSCC was calculated and its ability to identify OSCC tissues was evaluated by diagnostic analysis. CRISPR screen was used to select the genes necessary for OSCC cell growth, and the gene set was analyzed for function enrichment. Single-cell RNA sequencing analysis was conducted to evaluate the expression level of TOP2A mRNA in OSCC cells. RESULTS: Compared with normal oral tissues, the expression of TOP2A protein was up-regulated in OSCC tissues. A total of 1240 OSCC and 428 non-OSCC oral tissue samples were included based on high-throughput dataset retrieval, and it was confirmed that TOP2A mRNA was highly expressed in OSCC tissues [SMD = 1.51 (95% CI 0.94-2.07), sROC AUC = 0.96 (95% CI 0.94-0.98)]. As an essential gene for OSCC cell growth, single-cell RNA sequencing data also confirmed that TOP2A mRNA expression was up-regulated in OSCC cells. CONCLUSION: The up-regulation of TOP2A may play a pivotal role in OSCC.

A combination of four Toxoplasma gondii nuclear-targeted effectors protects against interferon gamma-driven human host cell death.

In both mice and humans, Type II interferon gamma (IFNγ) is crucial for the regulation of Toxoplasma gondii (T. gondii) infection, during acute or chronic phases. To thwart this defense, T. gondii secretes protein effectors hindering the host's immune response. For example, T. gondii relies on the MYR translocon complex to deploy soluble dense granule effectors (GRAs) into the host cell cytosol or nucleus. Recent genome-wide loss-of-function screens in IFNγ-primed primary human fibroblasts identified MYR translocon components as crucial for parasite resistance against IFNγ-driven vacuole clearance. However, these screens did not pinpoint specific MYR-dependent GRA proteins responsible for IFNγ signaling blockade, suggesting potential functional redundancy. Our study reveals that T. gondii depends on the MYR translocon complex to prevent parasite premature egress and host cell death in human cells stimulated with IFNγ post-infection, a unique phenotype observed in various human cell lines but not in murine cells. Intriguingly, inhibiting parasite egress did not prevent host cell death, indicating this mechanism is distinct from those described previously. Genome-wide loss-of-function screens uncovered TgIST, GRA16, GRA24, and GRA28 as effectors necessary for a complete block of IFNγ response. GRA24 and GRA28 directly influenced IFNγ-driven transcription, GRA24's action depended on its interaction with p38 MAPK, while GRA28 disrupted histone acetyltransferase activity of CBP/p300. Given the intricate nature of the immune response to T. gondii, it appears that the parasite has evolved equally elaborate mechanisms to subvert IFNγ signaling, extending beyond direct interference with the JAK/STAT1 pathway, to encompass other signaling pathways as well.IMPORTANCEToxoplasma gondii, an intracellular parasite, affects nearly one-third of the global human population, posing significant risks for immunocompromised patients and infants infected in utero. In murine models, the core mechanisms of IFNγ-mediated immunity against T. gondii are consistently preserved, showcasing a remarkable conservation of immune defense mechanisms. In humans, the recognized restriction mechanisms vary among cell types, lacking a universally applicable mechanism. This difference underscores a significant variation in the genes employed by T. gondii to shield itself against the IFNγ response in human vs murine cells. Here, we identified a specific combination of four parasite-secreted effectors deployed into the host cell nucleus, disrupting IFNγ signaling. This disruption is crucial in preventing premature egress of the parasite and host cell death. Notably, this phenotype is exclusive to human cells, highlighting the intricate and unique mechanisms T. gondii employs to modulate host responses in the human cellular environment.

Characterization and bioinformatic filtering of ambient gRNAs in single-cell CRISPR screens using CLEANSER.

Recent technological developments in single-cell RNA-seq CRISPR screens enable high-throughput investigation of the genome. Through transduction of a gRNA library to a cell population followed by transcriptomic profiling by scRNA-seq, it is possible to characterize the effects of thousands of genomic perturbations on global gene expression. A major source of noise in scRNA-seq CRISPR screens are ambient gRNAs, which are contaminating gRNAs that likely originate from other cells. If not properly filtered, ambient gRNAs can result in an excess of false positive gRNA assignments. Here, we utilize CRISPR barnyard assays to characterize ambient gRNA noise in single-cell CRISPR screens. We use these datasets to develop and train CLEANSER, a mixture model that identifies and filters ambient gRNA noise. This model takes advantage of the bimodal distribution between native and ambient gRNAs and includes both gRNA and cell-specific normalization parameters, correcting for confounding technical factors that affect individual gRNAs and cells. The output of CLEANSER is the probability that a gRNA-cell assignment is in the native distribution over the ambient distribution. We find that ambient gRNA filtering methods impact differential gene expression analysis outcomes and that CLEANSER outperforms alternate approaches by increasing gRNA-cell assignment accuracy.

Identification of Cables1 as a critical host factor that promotes ALV-J replication via genome-wide CRISPR/Cas9 gene knockout screening.

Avian leukosis virus subgroup J (ALV-J), a member of the genus Alpharetrovirus, possesses a small genome and exploits a vast array of host factors during its replication cycle. To identify host factors required for ALV-J replication and potentially guide the development of key therapeutic targets for ALV-J prevention, we employed a chicken genome-wide CRISPR/Cas9 knockout library to screen host factors involved in ALV-J infection within DF-1 cells. This screening revealed 42 host factors critical for ALV-J infection. Subsequent knockout assays showed that the absence of the genes encoding cycle-regulatory proteins, namely Cables1, CDK1, and DHFR, significantly inhibited ALV-J replication. Notably, Cables1 knockout cell lines displayed the most pronounced inhibitory effect. Conversely, overexpression assays confirmed that Cables1 significantly promotes ALV-J replication. Immunoprecipitation assays further indicated that Cables1 specifically interacts with the viral protein p15 (viral protease) among all ALV-J proteins, enhancing ALV-J p15 polyubiquitination. Additionally, we identified 26 lysine residues of ALV-J p15 as key sites for ubiquitination, and their replacement with arginine attenuated the replication ability of ALV-J in both in vitro and in vivo assays. This study demonstrates that Cables1 is a critical replication-dependent host factor of ALV-J by enhancing p15 ubiquitination and thereby promoting viral replication. Overall, these findings contribute to a deeper understanding of the ALJ-V replication mechanism and offer a potential target for the prevention and control of ALV-J infection.

Rapid iPSC inclusionopathy models shed light on formation, consequence, and molecular subtype of α-synuclein inclusions.

The heterogeneity of protein-rich inclusions and its significance in neurodegeneration is poorly understood. Standard patient-derived iPSC models develop inclusions neither reproducibly nor in a reasonable time frame. Here, we developed screenable iPSC "inclusionopathy" models utilizing piggyBac or targeted transgenes to rapidly induce CNS cells that express aggregation-prone proteins at brain-like levels. Inclusions and their effects on cell survival were trackable at single-inclusion resolution. Exemplar cortical neuron α-synuclein inclusionopathy models were engineered through transgenic expression of α-synuclein mutant forms or exogenous seeding with fibrils. We identified multiple inclusion classes, including neuroprotective p62-positive inclusions versus dynamic and neurotoxic lipid-rich inclusions, both identified in patient brains. Fusion events between these inclusion subtypes altered neuronal survival. Proteome-scale α-synuclein genetic- and physical-interaction screens pinpointed candidate RNA-processing and actin-cytoskeleton-modulator proteins like RhoA whose sequestration into inclusions could enhance toxicity. These tractable CNS models should prove useful in functional genomic analysis and drug development for proteinopathies.

CRISPR screens identify genes essential for in vivo virulence among proteins of hyperLOPIT-unassigned subcellular localization in Toxoplasma.

The research field to identify and characterize genes essential for in vivo virulence in Toxoplasma gondii has been dramatically advanced by a series of in vivo clustered regularly interspaced short palindromic repeats (CRISPR) screens. Although subcellular localizations of thousands of proteins were predicted by the spatial proteomic method called hyperLOPIT, those of more than 1,000 proteins remained unassigned, and their essentiality in virulence was also unknown. In this study, we generated two small-scale gRNA libraries targeting approximately 600 hyperLOPIT-unassigned proteins and performed in vivo CRISPR screens. As a result, we identified several genes essential for in vivo virulence that were previously unreported. We further characterized two candidates, TgGTPase and TgRimM, which are localized in the cytoplasm and the apicoplast, respectively. Both genes are essential for parasite virulence and widely conserved in the phylum Apicomplexa. Collectively, our current study provides a resource for estimating the in vivo essentiality of Toxoplasma proteins with previously unknown localizations.IMPORTANCEToxoplasma gondii is a protozoan parasite that causes severe infection in immunocompromised patients or newborns. Toxoplasma possesses more than 8,000 genes; however, the genes essential for in vivo virulence were not fully identified. The apicomplexan parasites, including Toxoplasma, developed unique organelles that do not exist in other model organisms; thus, determining the subcellular location of parasite proteins is important for understanding their functions. Here, we used in vivo genetic screens that enabled us to investigate hundreds of genes in Toxoplasma during mouse infection. We screened approximately 600 parasite proteins with previously unknown subcellular localizations. We identified many novel genes that confer parasite virulence in mice. Among the top hits, we characterized two genes essential for in vivo virulence, TgGTPase and TgRimM, which are widely conserved in the phylum Apicomplexa. Our findings will contribute to understanding how apicomplexans adapt to the host environment and cause disease.

A large-scale CRISPR screen reveals context-specific genetic regulation of retinal ganglion cell regeneration.

Many genes are known to regulate retinal regeneration after widespread tissue damage. Conversely, genes controlling regeneration after limited cell loss, as per degenerative diseases, are undefined. As stem/progenitor cell responses scale to injury levels, understanding how the extent and specificity of cell loss impact regenerative processes is important. Here, transgenic zebrafish enabling selective retinal ganglion cell (RGC) ablation were used to identify genes that regulate RGC regeneration. A single cell multiomics-informed screen of 100 genes identified seven knockouts that inhibited and 11 that promoted RGC regeneration. Surprisingly, 35 out of 36 genes known and/or implicated as being required for regeneration after widespread retinal damage were not required for RGC regeneration. The loss of seven even enhanced regeneration kinetics, including the proneural factors neurog1, olig2 and ascl1a. Mechanistic analyses revealed that ascl1a disruption increased the propensity of progenitor cells to produce RGCs, i.e. increased 'fate bias'. These data demonstrate plasticity in the mechanism through which Müller glia convert to a stem-like state and context specificity in how genes function during regeneration. Increased understanding of how the regeneration of disease-relevant cell types is specifically controlled will support the development of disease-tailored regenerative therapeutics.

CCAR1 promotes DNA repair via alternative splicing.

DNA repair is directly performed by hundreds of core factors and indirectly regulated by thousands of others. We massively expanded a CRISPR inhibition and Cas9-editing screening system to discover factors indirectly modulating homology-directed repair (HDR) in the context of ∼18,000 individual gene knockdowns. We focused on CCAR1, a poorly understood gene that we found the depletion of reduced both HDR and interstrand crosslink repair, phenocopying the loss of the Fanconi anemia pathway. CCAR1 loss abrogated FANCA protein without substantial reduction in the level of its mRNA or that of other FA genes. We instead found that CCAR1 prevents inclusion of a poison exon in FANCA. Transcriptomic analysis revealed that the CCAR1 splicing modulatory activity is not limited to FANCA, and it instead regulates widespread changes in alternative splicing that would damage coding sequences in mouse and human cells. CCAR1 therefore has an unanticipated function as a splicing fidelity factor.

Genome-scale exon perturbation screens uncover exons critical for cell fitness.

CRISPR-Cas technology has transformed functional genomics, yet understanding of how individual exons differentially shape cellular phenotypes remains limited. Here, we optimized and conducted massively parallel exon deletion and splice-site mutation screens in human cell lines to identify exons that regulate cellular fitness. Fitness-promoting exons are prevalent in essential and highly expressed genes and commonly overlap with protein domains and interaction interfaces. Conversely, fitness-suppressing exons are enriched in nonessential genes, exhibiting lower inclusion levels, and overlap with intrinsically disordered regions and disease-associated mutations. In-depth mechanistic investigation of the screen-hit TAF5 alternative exon-8 revealed that its inclusion is required for assembly of the TFIID general transcription initiation complex, thereby regulating global gene expression output. Collectively, our orthogonal exon perturbation screens established a comprehensive repository of phenotypically important exons and uncovered regulatory mechanisms governing cellular fitness and gene expression.

Identification of WNK1 as a therapeutic target to suppress IgH/MYC expression in multiple myeloma.

Multiple myeloma (MM) remains an incurable hematological malignancy demanding innovative therapeutic strategies. Targeting MYC, the notorious yet traditionally undruggable oncogene, presents an appealing avenue. Here, using a genome-scale CRISPR-Cas9 screen, we identify the WNK lysine-deficient protein kinase 1 (WNK1) as a regulator of MYC expression in MM cells. Genetic and pharmacological inhibition of WNK1 reduces MYC expression and, further, disrupts the MYC-dependent transcriptional program. Mechanistically, WNK1 inhibition attenuates the activity of the immunoglobulin heavy chain (IgH) enhancer, thus reducing MYC transcription when this locus is translocated near the MYC locus. WNK1 inhibition profoundly impacts MM cell behaviors, leading to growth inhibition, cell-cycle arrest, senescence, and apoptosis. Importantly, the WNK inhibitor WNK463 inhibits MM growth in primary patient samples as well as xenograft mouse models and exhibits synergistic effects with various anti-MM compounds. Collectively, our study uncovers WNK1 as a potential therapeutic target in MM.

Massively parallel in vivo Perturb-seq reveals cell-type-specific transcriptional networks in cortical development.

Leveraging AAVs' versatile tropism and labeling capacity, we expanded the scale of in vivo CRISPR screening with single-cell transcriptomic phenotyping across embryonic to adult brains and peripheral nervous systems. Through extensive tests of 86 vectors across AAV serotypes combined with a transposon system, we substantially amplified labeling efficacy and accelerated in vivo gene delivery from weeks to days. Our proof-of-principle in utero screen identified the pleiotropic effects of Foxg1, highlighting its tight regulation of distinct networks essential for cell fate specification of Layer 6 corticothalamic neurons. Notably, our platform can label >6% of cerebral cells, surpassing the current state-of-the-art efficacy at <0.1% by lentivirus, to achieve analysis of over 30,000 cells in one experiment and enable massively parallel in vivo Perturb-seq. Compatible with various phenotypic measurements (single-cell or spatial multi-omics), it presents a flexible approach to interrogate gene function across cell types in vivo, translating gene variants to their causal function.

Targeting tumor cell-to-macrophage communication by blocking Vtn-C1qbp interaction inhibits tumor progression via enhancing macrophage phagocytosis.

Background: Cancer cells are capable of evading clearance by macrophages through overexpression of anti-phagocytic surface proteins known as "don't eat me" signals. Monoclonal antibodies that antagonize the "don't-eat-me" signaling in macrophages and tumor cells by targeting phagocytic checkpoints have shown therapeutic promises in several cancer types. However, studies on the responses to these drugs have revealed the existence of other unknown "don't eat me" signals. Moreover, identification of key molecules and interactions regulating macrophage phagocytosis is required for tumor therapy. Methods: CRISPR screen was used to identify genes that impede macrophage phagocytosis. To explore the function of Vtn and C1qbp in phagocytosis, knockdown and subsequent functional experiments were conducted. Flow cytometry were performed to explore the phagocytosis rate, polarization of macrophage, and immune microenvironment of mouse tumor. To explore the underlying molecular mechanisms, RNA sequencing, immunoprecipitation, mass spectrometry, and immunofluorescence were conducted. Then, in vivo experiments in mouse models were conducted to explore the probability of Vtn knockdown combined with anti-CD47 therapy in breast cancer. Single-cell sequencing data from the Gene Expression Omnibus from The Cancer Genome Atlas database were analyzed. Results: We performed a genome-wide CRISPR screen to identify genes that impede macrophage phagocytosis, followed by analysis of cell-to-cell interaction databases. We identified a ligand-receptor pair of Vitronectin (Vtn) and complement C1Q binding protein (C1qbp) in tumor cells or macrophages, respectively. We demonstrated tumor cell-secreted Vtn interacts with C1qbp localized on the cell surface of tumor-associated macrophages, inhibiting phagocytosis of tumor cells and shifting macrophages towards the M2-like subtype in the tumor microenvironment. Mechanistically, the Vtn-C1qbp axis facilitated FcγRIIIA/CD16-induced Shp1 recruitment, which reduced the phosphorylation of Syk. Furthermore, the combination of Vtn knockdown and anti-CD47 antibody effectively enhanced phagocytosis and infiltration of macrophages, resulting in a reduction of tumor growth in vivo. Conclusions: This work has revealed that the Vtn-C1qbp axis is a new anti-phagocytic signal in tumors, and targeting Vtn and its interaction with C1qbp may sensitize cancer to immunotherapy, providing a new molecular target for the treatment of triple-negative breast cancer.

CRISPR Screen Reveals PACT as a Pro-Viral Factor for Dengue Viral Replication.

The dengue virus is a single-stranded, positive-sense RNA virus that infects ~400 million people worldwide. Currently, there are no approved antivirals available. CRISPR-based screening methods have greatly accelerated the discovery of host factors that are essential for DENV infection and that can be targeted in host-directed antiviral interventions. In the present study, we performed a focused CRISPR (Clustered Regularly Interspaced Palindromic Repeats) library screen to discover the key host factors that are essential for DENV infection in human Huh7 cells and identified the Protein Activator of Interferon-Induced Protein Kinase (PACT) as a novel pro-viral factor for DENV. PACT is a double-stranded RNA-binding protein generally known to activate antiviral responses in virus-infected cells and block viral replication. However, in our studies, we observed that PACT plays a pro-viral role in DENV infection and specifically promotes viral RNA replication. Knockout of PACT resulted in a significant decrease in DENV RNA and protein abundances in infected cells, which was rescued upon ectopic expression of full-length PACT. An analysis of global gene expression changes indicated that several ER-associated pro-viral genes such as ERN1, DDIT3, HERPUD1, and EIF2AK3 are not upregulated in DENV-infected PACT knockout cells as compared to infected wildtype cells. Thus, our study demonstrates a novel role for PACT in promoting DENV replication, possibly through modulating the expression of ER-associated pro-viral genes.

CRISPR Screen Identifies the RNA-Binding Protein Eef1a1 as a Key Regulator of Myogenesis.

Skeletal muscle myogenesis hinges on gene regulation, meticulously orchestrated by molecular mechanisms. While the roles of transcription factors and non-coding RNAs in myogenesis are widely known, the contribution of RNA-binding proteins (RBPs) has remained unclear until now. Therefore, to investigate the functions of post-transcriptional regulators in myogenesis and uncover new functional RBPs regulating myogenesis, we employed CRISPR high-throughput RBP-KO (RBP-wide knockout) library screening. Through this approach, we successfully identified Eef1a1 as a novel regulatory factor in myogenesis. Using CRISPR knockout (CRISPRko) and CRISPR interference (CRISPRi) technologies, we successfully established cellular models for both CRISPRko and CRISPRi. Our findings demonstrated that Eef1a1 plays a crucial role in promoting proliferation in C2C12 myoblasts. Through siRNA inhibition and overexpression methods, we further elucidated the involvement of Eef1a1 in promoting proliferation and suppressing differentiation processes. RIP (RNA immunoprecipitation), miRNA pull-down, and Dual-luciferase reporter assays confirmed that miR-133a-3p targets Eef1a1. Co-transfection experiments indicated that miR-133a-3p can rescue the effect of Eef1a1 on C2C12 myoblasts. In summary, our study utilized CRISPR library high-throughput screening to unveil a novel RBP, Eef1a1, involved in regulating myogenesis. Eef1a1 promotes the proliferation of myoblasts while inhibiting the differentiation process. Additionally, it acts as an antagonist to miR-133a-3p, thus modulating the process of myogenesis.

Long-term expandable mouse and human-induced nephron progenitor cells enable kidney organoid maturation and modeling of plasticity and disease.

Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here, manipulation of p38 and YAP activity allowed for long-term clonal expansion of primary mouse and human NPCs and induced NPCs (iNPCs) from human pluripotent stem cells (hPSCs). Molecular analyses demonstrated that cultured iNPCs closely resemble primary human NPCs. iNPCs generated nephron organoids with minimal off-target cell types and enhanced maturation of podocytes relative to published human kidney organoid protocols. Surprisingly, the NPC culture medium uncovered plasticity in human podocyte programs, enabling podocyte reprogramming to an NPC-like state. Scalability and ease of genome editing facilitated genome-wide CRISPR screening in NPC culture, uncovering genes associated with kidney development and disease. Further, NPC-directed modeling of autosomal-dominant polycystic kidney disease (ADPKD) identified a small-molecule inhibitor of cystogenesis. These findings highlight a broad application for the reported iNPC platform in the study of kidney development, disease, plasticity, and regeneration.

Systematic review and meta-analysis of genome-wide pooled CRISPR screens to identify host factors involved in influenza A virus infection.

The host-virus interactome is increasingly recognized as an important research field to discover new therapeutic targets to treat influenza. Multiple pooled genome-wide CRISPR-Cas screens have been reported to identify new pro- and antiviral host factors of the influenza A virus. However, at present, a comprehensive summary of the results is lacking. We performed a systematic review of all reported CRISPR studies in this field in combination with a meta-analysis using the algorithm of meta-analysis by information content (MAIC). Two ranked gene lists were generated based on evidence in 15 proviral and 4 antiviral screens. Enriched pathways in the proviral MAIC results were compared to those of a prior array-based RNA interference (RNAi) meta-analysis. The top 50 proviral MAIC list contained genes whose role requires further elucidation, such as the endosomal ion channel TPCN1 and the kinase WEE1. Moreover, MAIC indicated that ALYREF, a component of the transcription export complex, has antiviral properties, whereas former knockdown experiments attributed a proviral role to this host factor. CRISPR-Cas-pooled screens displayed a bias toward early-replication events, whereas the prior RNAi meta-analysis covered early and late-stage events. RNAi screens led to the identification of a larger fraction of essential genes than CRISPR screens. In summary, the MAIC algorithm points toward the importance of several less well-known pathways in host-influenza virus interactions that merit further investigation. The results from this meta-analysis of CRISPR screens in influenza A virus infection may help guide future research efforts to develop host-directed anti-influenza drugs. IMPORTANCE: Viruses rely on host factors for their replication, whereas the host cell has evolved virus restriction factors. These factors represent potential targets for host-oriented antiviral therapies. Multiple pooled genome-wide CRISPR-Cas screens have been reported to identify pro- and antiviral host factors in the context of influenza virus infection. We performed a comprehensive analysis of the outcome of these screens based on the publicly available gene lists, using the recently developed algorithm meta-analysis by information content (MAIC). MAIC allows the systematic integration of ranked and unranked gene lists into a final ranked gene list. This approach highlighted poorly characterized host factors and pathways with evidence from multiple screens, such as the vesicle docking and lipid metabolism pathways, which merit further exploration.

Functional CRISPR screens in T cells reveal new opportunities for cancer immunotherapies.

T cells are fundamental components in tumour immunity and cancer immunotherapies, which have made immense strides and revolutionized cancer treatment paradigm. However, recent studies delineate the predicament of T cell dysregulation in tumour microenvironment and the compromised efficacy of cancer immunotherapies. CRISPR screens enable unbiased interrogation of gene function in T cells and have revealed functional determinators, genetic regulatory networks, and intercellular interactions in T cell life cycle, thereby providing opportunities to revamp cancer immunotherapies. In this review, we briefly described the central roles of T cells in successful cancer immunotherapies, comprehensively summarised the studies of CRISPR screens in T cells, elaborated resultant master genes that control T cell activation, proliferation, fate determination, effector function, and exhaustion, and highlighted genes (BATF, PRDM1, and TOX) and signalling cascades (JAK-STAT and NF-κB pathways) that extensively engage in multiple branches of T cell responses. In conclusion, this review bridged the gap between discovering element genes to a specific process of T cell activities and apprehending these genes in the global T cell life cycle, deepened the understanding of T cell biology in tumour immunity, and outlined CRISPR screens resources that might facilitate the development and implementation of cancer immunotherapies in the clinic.