Genome-wide CRISPR screen for Zika virus resistance in human neural cells

Li, Yun; Muffat, Julien; Javed, Attya Omer; Keys, Heather R.; Lungjangwa, Tenzin; Bosch, Irene; Khan, Mehreen Ali; Virgilio, Maria; Gehrke, Lee; Sabatini, David M.; Jaenisch, Rudolf

doi:10.1073/pnas.1900867116

Cited by 104 publications

(96 citation statements)

References 45 publications

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“…Using the dSMADi strategy ( Figure 6A), we rapidly generated NSCs sufficient for 184 plates (384-well format) enabling the knockdown of 21,584 genes (three unique siRNAs for each target gene). These high-throughput screens confirmed previously reported targets (Li et al, 2019) (Cahan and Daley, 2013;Choi et al, 2015;Sharma et al, 2020). Another challenge is continuous passage of self-renewing hPSCs, while cell differentiation experiments are initiated in parallel.…”

Section: Zika Virus Infection Of Robotically Generated Cardiomyocytessupporting

confidence: 84%

Robotic High-Throughput Biomanufacturing and Functional Differentiation of Human Pluripotent Stem Cells

Tristan

Ormanoglu

Slamecka

et al. 2020

Preprint

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Efficient translation of human induced pluripotent stem cells (hiPSCs) depends on implementing scalable cell manufacturing strategies that ensure optimal self-renewal and functional differentiation. Currently, manual culture of hiPSCs is highly variable and labor-intensive posing significant challenges for high-throughput applications. Here, we established a robotic platform and automated all essential steps of hiPSC culture and differentiation under chemically defined conditions. This streamlined approach allowed rapid and standardized manufacturing of billions of hiPSCs that can be produced in parallel from up to 90 different patient-and disease-specific cell lines. Moreover, we established automated multi-lineage differentiation to generate primary embryonic germ layers and more mature phenotypes such as neurons, cardiomyocytes, and hepatocytes. To validate our approach, we carefully compared robotic and manual cell culture and performed molecular and functional cell characterizations (e.g. bulk culture and single-cell transcriptomics, mass cytometry, metabolism, electrophysiology, Zika virus experiments) in order to benchmark industrial-scale cell culture operations towards building an integrated platform for efficient cell manufacturing for disease modeling, drug screening, and cell therapy. Combining stem cell-based models and non-stop robotic cell culture may become a powerful strategy to increase scientific rigor and productivity, which are particularly important during public health emergencies (e.g. opioid crisis, COVID-19 pandemic).

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Section: Zika Virus Infection Of Robotically Generated Cardiomyocytessupporting

confidence: 84%

Robotic High-Throughput Biomanufacturing and Functional Differentiation of Human Pluripotent Stem Cells

Tristan

Ormanoglu

Slamecka

et al. 2020

Preprint

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“…ZIKV is known to be neurotropic and mainly infects neuronal cells within the central nervous system to cause microcephaly 2 . Recent studies have used iPSC-derived human neural progenitor cells (NPCs) for their studies as a proxy to physiologically relevant primary cells 3,45–47 . Therefore, we decided to use iPSC-derived NPCs to validate our key findings by manipulating sphingolipid levels in these cells using myriocin and FB1.…”

Section: Resultsmentioning

confidence: 99%

A global lipid map defines a network essential for Zika virus replication

Leier

Weinstein

Kyle

et al. 2020

Preprint

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26Zika virus (ZIKV), an arbovirus of global concern, remodels intracellular membranes to form 27 replication sites. How ZIKV dysregulates lipid networks to allow this, and consequences for 28 disease, is poorly understood. Here, we performed comprehensive lipidomics to create a lipid 29 network map during ZIKV infection. We found that ZIKV significantly alters host lipid 30 composition, with the most striking changes seen within subclasses of sphingolipids. Ectopic 31 expression of ZIKV NS4B protein resulted in similar changes, demonstrating a role for NS4B in 32 modulating sphingolipid pathways. Disruption of sphingolipid biosynthesis in various cell types, 33including human neural progenitor cells, blocked ZIKV infection. Additionally, the sphingolipid 34 ceramide redistributes to ZIKV replication sites and increasing ceramide levels by multiple 35 pathways sensitizes cells to ZIKV infection. Thus, we identify a sphingolipid metabolic network 36 with a critical role in ZIKV replication and show that ceramide flux is a key mediator of ZIKV 37 infection. Results 112 ZIKV alters the lipid landscape of host cells 113To understand how cellular lipid metabolism is altered by ZIKV infection, we carried out 114 a global lipidomic survey of the model Huh7 human hepatic carcinoma cell line 20 infected for 24 115 or 48 hours with Asian lineage ZIKV strain FSS13025 (Fig. 1a). Lipids extracted from 116 populations of mock and infected cells (n = 5 biological replicates per condition) were analyzed 117 with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) (Supplementary Data 118 1 and Supplementary Fig. 1a-f). We identified 340 lipid species spanning the phospholipid, 119 sphingolipid, glycerolipid, and sterol classes for which pairwise comparisons of normalized 120 abundance between mock and infected cells could be made (Supplementary Data 1). Of these, 121 80 species (23.5%) showed significant changes in abundance by 24 hours post-infection (hpi) 122 and 172 species (50.6%) were significantly altered by 48 hpi (P < 0.05, ANOVA or g-test) 123( Supplementary Data 1). Principal component analysis (PCA) of these observations confirmed 124 that infection status (mock or ZIKV) and timepoint (24 or 48 hpi) accounted most of these 125 changes, with changes in lipid composition between mock and infected cells increasing over 126 time (Fig. 1b). 127Next, we examined how ZIKV-induced changes in host lipid composition broke down by 128 subclass and species (Fig. 1c, d). A map of the pairwise correlations of all 340 species at 48 hpi 129 ( Supplementary Fig. 2a) revealed that lipid subclasses largely fell into two groups of species 130 that were either enriched or depleted in abundance ( Supplementary Fig. 2b), suggesting that 131 individual metabolic pathways are up-or down-regulated to create a specific lipid milieu around 132 the events of the viral replication cycle. Supporting this, many of the trends we observed were 133 consistent with earlier reports of functional roles for lipids during flavivirus infection. In ...

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“…Genes required for uptake of Ebola, influenza A, and Zika viruses were obtained from published CRISPR screens [31][32][33] . Since the Ebola data were also analysed with MAGeCK, we defined genes required for viral uptake using an unadjusted p-value threshold of 0.01; this information was not available for the influenza A and Zika studies, so we defined genes required for viral uptake as having an adjusted p-value < 0.05.…”

Section: Discussionmentioning

confidence: 99%

“…The host factors or genes that regulate the entry and life cycle of several viruses have been comprehensively studied using RNAi and CRISPR screens. These include the neurotropic Zika virus 31 and Influenza A 32 , which enter cells via receptor-mediated endocytosis, and Ebola 33 , which uses micropinocytosis.…”

Section: Neuronal Uptake Of Extracellular Tau Shares Functional Similmentioning

confidence: 99%

Whole genome CRISPR screens identify LRRK2-regulated endocytosis as a major mechanism for extracellular tau uptake by human neurons

Evans¹,

Strano²,

Campbell³

et al. 2020

Preprint

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Pathological protein aggregation in Alzheimer's disease and other dementias is proposed to spread through the nervous system by a process of intercellular transfer of pathogenic forms of tau protein. Defining the cellular mechanisms of tau entry to human neurons is essential for understanding dementia pathogenesis and the rational design of disease-modifying therapeutics. Using whole genome CRISPR knockout screens in human iPSC-derived excitatory neurons, the primary cell type affected in these diseases, we identified genes and pathways required specifically for uptake of monomeric and aggregated tau. Monomeric and aggregated tau are both taken up by human neurons by receptor-mediated endocytosis, with the low-density lipoprotein LRP1 a significant surface receptor for both forms of tau. Perturbations of the endolysosome and autophagy systems at many levels, and specifically endosome sorting and receptor recycling, greatly reduced tau uptake. Of particular therapeutic interest is that loss of function of the endocytosis and autophagy regulator LRRK2, as well as acute inhibition of its kinase activity, reduced neuronal uptake of monomeric and aggregated tau. Kinase-activating mutations in LRRK2 are a cause of Parkinson's disease accompanied by neuronal tau aggregation, suggesting that LRRK2 mediates tau spreading in vivo and that LRRK2 inhibition has the potential to inhibit interneuronal spread of tau pathology, slowing disease progression. Overall, pathways for tau entry share significant similarity with those required for virus entry by receptor-mediated endocytosis, suggesting that tau spreading is a quasi-infectious process.

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Genome-wide CRISPR screen for Zika virus resistance in human neural cells

Cited by 104 publications

References 45 publications

Robotic High-Throughput Biomanufacturing and Functional Differentiation of Human Pluripotent Stem Cells

Robotic High-Throughput Biomanufacturing and Functional Differentiation of Human Pluripotent Stem Cells

A global lipid map defines a network essential for Zika virus replication

Whole genome CRISPR screens identify LRRK2-regulated endocytosis as a major mechanism for extracellular tau uptake by human neurons

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