Biology subjects

Li, S.

Publications and source records attributed to Li, S..

At least 1,117 records · Page 62Linked to original sources

bioRxiv2020

Binding of guide piRNA triggers methylation of the unstructured N-terminal region of Aub leading to assembly of the piRNA amplification complex

Piwi proteins use guide piRNAs to repress selfish genomic elements, protecting the genomic integrity of gametes and ensuring the fertility of animal species. Efficient transposon repression depends on amplification of piRNA guides in the ping-pong cycle, which in Drosophila entails tight cooperation between two Piwi proteins, Aub and Ago3. Here we show that post-translational modification, symmetric dimethylarginine (sDMA), of Aub is essential for piRNA biogenesis, transposon silencing and fertility. Methylation is triggered by loading of a piRNA guide into Aub, which exposes its unstructured N-terminal region to the PRMT5 methylosome complex. Thus, sDMA modification is a signal that Aub is loaded with piRNA guide. Amplification of piRNA in the ping-pong cycle requires assembly of a tertiary complex scaffolded by Krimper, which simultaneously binds the N-terminal regions of Aub and Ago3. To promote generation of new piRNA, Krimp uses its two Tudor domains to bind Aub and Ago3 in opposite modification and piRNA-loading states. Our results reveal that post-translational modifications in unstructured regions of Piwi proteins and their binding by Tudor domains that are capable of discriminating between modification states is essential for piRNA biogenesis and silencing. HighlightsO_LIsDMA modification of Aub is essential for ping-pong cycle, transposon silencing and fertility. C_LIO_LIpiRNA loading triggers Aub sDMA modification by exposing its unstructured N terminal region to the methylosome complex. C_LIO_LIA single molecule of Krimp simultaneously binds piRNA-bound Aub and empty Ago3 to assemble ping-pong complex. C_LIO_LIThe distinct structure of two Tudor domains of Krimp ensures binding of Ago3 and Aub in opposite modification and piRNA-loading states. C_LI

biochemistry↗

bioRxiv2020

Astrocytes express DMT1 and transferrin receptors, which transport iron thus activating Ca2+ signalling: possible role in neuroprotection against iron overload?

ABSTRACTIron is the fundamental element for numerous physiological functions. Reduced ferrous (Fe2+) and oxidized ferric (Fe3+) are the two ionized iron states in the living organisms. In the cell membrane, divalent metal ion transporter 1 (DMT1) is responsible for cellular uptake of Fe2+, whereas transferrin receptors (TFR) carry transferrin (TF)-bound Fe3+. In this study we performed, for the first time, detailed analysis of the action of Fe ions on cytoplasmic free calcium ion concentration ([Ca2+]i) in astrocytes. Using qPCR and immunocytochemistry we identified DMT1 and TFR in astrocytes in primary cultures, in acutely isolated astrocytes and in brain tissue preparations; in situ both DMT1 and TFR are concentrated in astroglial perivascular endfeet. Administration of Fe2+ or Fe3+ in low μM concentrations evoked Ca2+ signals in astrocytes in vitro and in vivo. Iron ions triggered increase in [Ca2+]i by acting through two distinct molecular cascades. Uptake of Fe2+ by DMT1 inhibited astroglial Na+-K+-ATPase (NKA), which led to an elevation in cytoplasmic Na+ concentration (as measured by SBFI probe), thus reversing Na+/Ca2+ exchanger (NCX) thereby generating Ca2+ influx. Uptake of Fe3+ by TF-TFR stimulated phospholipase C to produce inositol 1,4,5-trisphosphate (InsP3), thus trigering InsP3 receptor-mediated Ca2+ release from the endoplasmic reticulum. Iron-induced Ca2+ signals promote astroglial release of arachidonic acid and prostaglandin E2 cytokines by activating cytosolic phospholipase A2 (cPLA2) and NF-κB signalling cascade. In summary, these findings reveal new mechanisms of iron-induced astrocytic signalling operational in conditions of iron overload, in response to which astrocytes actively accumulate excessive iron and activate neuroprotective pathways.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2020

Next generation of anti-PD-L1 Atezolizumab with better anti-tumor efficacy in vivo

Some cancer patients treated with Atezolizumab, PD-L1 antibody drug launched by Genentech, quickly developed anti-drug antibody (ADA), led to loss of efficacy. This was likely due to the heavy aggregation of Atezolizumab, caused by mutation of N297A for removing unwanted antibody-dependent cytotoxicity (ADCC) of IgG1 antibody drug. Here, we developed a new version of Atezolizumab (Maxatezo), which was demonstrated better anti-tumor efficacy in vivo. In Atezolizumab, we mutated 297A to 297N back to bring back the glycosylation, and inserted a short sequence GGGS between G237 and G238 in the hinge region of the IgG1 heavy chain. Our data shown that insertion of GGGS, without altering the anti-PD-L1 antibody affinity and inhibitory activity, completely abolished the ADCC activity, as same as Atezolizumab. Moreover, the insertion of GGGS, without altering the glycosylation profile of IgG1, increased the yields of anti-PD-L1 antibody considerately. Additionally, glycosylation improved the stability yet reduced the amounts of aggregations in the antibody solutions. In turn, the level of ADA in animals treated with Maxatezo was 70% lower than the ones treated with Atezolizumab. Most importantly, at the same 10mg/kg dose, the anti-tumor activity of Maxatezo had attained 98% compared to that of Atezolizumab at 68%.Competing Interest StatementThe authors declare that they are affiliated with four commercial entities with stakes in the data. Patent of De-ADCC pending.View Full Text

bioengineering↗

bioRxiv2020

Potassium starvation induces autophagy in yeast

ABSTRACTAutophagy is a conserved process that recycles cellular contents to promote survival. Although nitrogen starvation is the canonical inducer of autophagy, recent studies have revealed several other nutrients important to this process. In this study, we used a quantitative, high-throughput assay to identify potassium starvation as a new and potent inducer of autophagy. We found that potassium-dependent autophagy requires the core pathway kinases Atg1, Atg5, Vps34, as well as other components of Phosphatidylinositol 3-kinase Complex I. Transmission electron microscopy revealed abundant autophagosome formation in response to both stimuli. RNA sequencing indicated distinct transcriptional responses – nitrogen affects transport of ions such as copper while potassium targets the organization of other cellular components. Thus, nitrogen and potassium share the ability to influence metabolic supply and demand but do so in different ways. Both inputs promote catabolism through bulk autophagy, but inhibit cellular anabolism through distinct mechanisms.Competing Interest StatementThe authors have declared no competing interest.ABBREVIATIONSPIPhosphatidylinositolATGAutophagy-related geneSCDSynthetic Complete medium with DextroseYNBYeast Nitrogen BaseTEMTransmission Electron MicroscopyMAPKMitogen-Activated Protein KinaseDEGDifferentially Expressed Gene.View Full Text

biochemistry↗

bioRxiv2020

Double-strand breaks can induce DNA replication and damage amplification in G2 phase-like oocytes of mice

Break-induced DNA replication (BIR) have been detected not only in the genome of rare disease patients but also in cancer cells, however, the mechanisms of BIR formation havent been explained in details. In the late G2 phase-like mouse oocytes, we found DNA double-strand breaks (DSBs) could induce Rad51 dependent small-scale DNA replication. In addition, we also found the DSBs could be amplified in mouse oocytes, and the amplification could be inhibited by Rad51 inhibitor IBR2 and DNA replication inhibitor ddATP. Lastly, we found the DSB repair was relatively inefficiency in hybrid mouse oocytes compared with that of the purebred mouse oocytes. We found DSBs could induce BIR more easier in hybrid mouse oocytes, indicating the DNA repair in oocytes could be affected by the sequence differences between homologous chromatids. In summary, our results indicated that the condensed chromatin configuration in late G2 phase and the sequence similarity between broken DNA and template DNA are causing factors of BIR in mammalian genome, and the DNA damage could be amplified in late G2 phase cells.

genetics↗

bioRxiv2020

Topographic profiling of memory-related pattern separation processes in humans

AO_SCPLOWBSTRACTC_SCPLOWEpisodic memory is our ability to remember past events accurately. Pattern separation, the process of of orthogonalizing similar aspects of external information into nonoverlapping representations, is one of its mechanisms. Converging evidence suggests a pivotal role of the hippocampus, in concert with neocortical areas, in this process. The current study aimed to identify principal dimensions of functional activation associated with pattern separation in hippocampal and neocortical areas, in both healthy individuals and patients with lesions to the hippocampus. Administering a pattern separation fMRI paradigm to a group of healthy adults, we detected task-related activation in bilateral hippocampal and distributed neocortical areas. Capitalizing on manifold learning techniques applied to parallel resting-state fMRI data, we could identify that hippocampal and neocortical activity patterns were efficiently captured by their principal gradients of intrinsic functional connectivity, which follows the hippocampal long axis and sensory-fugal cortical organization. Functional activation patterns and their alignment with these principal dimensions were altered in patients. Notably, inter-individual differences in the concordance between task-related activity and intrinsic functional gradients were correlated with pattern separation performance in both patients and controls. Our work outlines a parsimonious approach to capture the functional underpinnings of episodic memory processes at the systems level, and to decode functional reorganization in clinical populations.

neuroscience↗

bioRxiv2020

Construction of High Content Nanobody Library in Mammalian Cells by Linear-double-stranded DNA Based Strategies

Here, we developed two novel methods to construct high content nanobody library in mammalian cells. For the first method, we employed a linear-double-stranded DNA based AND-gate (LBAG) strategy. Upstream- and downstream-linear-double-stranded DNAs (ldsDNAs), containing identical overlapping sequences, were co-transfected into cultured cell lines to conduct AND-gate calculation to form intact nanobody expression cassette. For the second method, we generated full-length nanobody expression ldsDNA by in vitro ligation of restrict cut up- and down-stream ldsDNAs. Then the ldsDNAs were directly transfected into mammalian cells to express nanobody library. Both methods generated over a million different nanobody sequences as revealed by high-throughput sequencing (HTS).

immunology↗

bioRxiv2020

The Profiling of Bisecting N-acetylglucosamine (GlcNAc) Modification in Human Amniotic Membrane by Glycomic and Glycoproteomic Analyses

It is acknowledged that the bisecting N-acetylglucosamine (GlcNAc) structure, a GlcNAc linked to the core {beta}-mannose residue via a {beta}1,4 linkage, represents a special type of N-glycosylated modification and has been reported to be involved in various biological processes, such as cell adhesion and fetal development. Clark et al. has found that the majority of N-glycans in human trophoblasts bearing a bisecting GlcNAc. This type of glycan has been reported to help trophoblasts get resistant to natural killer (NK) cell-mediated cytotoxicity, and this would provide a possible explanation for the question how could the mother nourish a fetus within herself without rejection. Herein, we hypothesized that human amniotic membrane which is the last barrier for the fetus may also express bisecting type glycans to protect the fetus. To test this hypothesis, glycomic analysis of human amniotic membrane was performed, and the bisecting N-glycans with high abundance were detected. In addition, we re-analyzed our proteomic data with high fractionation and amino acid sequence coverage from human amniotic membrane, which had been released for the exploration of human missing proteins. The presence of bisecting GlcNAc peptides was revealed and confirmed. A total of 41 glycoproteins with 43 glycopeptides were found to possess a bisecting GlcNAc, 25 of which are for the first time to be reported to have this type of modification. These results provide the profiling of bisecting GlcNAc modification in human amniotic membrane and benefit to the function studies of glycoproteins with bisecting GlcNAc modification and the function studies in immune suppression of human placenta. The mass spectrometry placenta data are available via ProteomeXchange (PXD010630).

cell biology↗

bioRxiv2020

Nutritional combinatorial impact on the gut microbiota and plasma short-chain fatty acids levels in the prevention of mammary cancer in Her2/neu estrogen receptor-negative transgenic mice

Breast cancer is the second leading cause of cancer-related mortality in women. Various nutritional compounds possess anti-carcinogenic properties which may be mediated through their effects on the gut microbiota and its production of short-chain fatty acids (SCFAs) for the prevention of breast cancer. We evaluated the impact of broccoli sprouts (BSp), green tea polyphenols (GTPs) and their combination on the gut microbiota and SCFAs metabolism from the microbiota in Her2/neu transgenic mice that spontaneously develop estrogen receptor-negative [ER (-)] mammary tumors. The mice were grouped based on the dietary treatment: control, BSp, GTPs or their combination from beginning in early life (BE) or life-long from conception (LC). We found that the combination group showed the strongest inhibiting effect on tumor growth volume and a significant increase in tumor latency. BSp treatment was integrally more efficacious than the GTPs group when compared to the control group. There was similar clustering of microbiota of BSp-fed mice with combination-fed mice, and GTPs-fed mice with control-fed mice at pre-tumor and post-tumor in both BE and LC groups. The mice on all dietary treatment groups incurred a significant increase of Adlercreutzia genus and S24-7 family in the both BE and LC groups. We found no change in SCFAs levels in the plasma of BSp-fed, GTPs-fed and combination-fed mice of the BE group. Marked changes were observed in the mice of the LC group consisting of significant increases in propionate and isobutyrate in GTPs-fed and combination-fed mice. These studies indicate that nutrients such as BSp and GTPs differentially affect the gut microbial composition in both the BE and LC groups and the key metabolites (SCFAs) levels in the LC group. The findings also suggest that temporal factors related to different time windows of consumption during the life-span can have a promising influence on the gut microbial composition, SCFAs profiles and ER (-) breast cancer prevention.

microbiology↗

bioRxiv2020

Assessing Attention Orienting in Mice: A Novel Touchscreen Adaptation of the Posner-Style Cueing Task

Atypical attention orienting has been found to be impaired in many neuropsychological disorders, but the underlying neural mechanism remains unclear. Attention can be oriented exogenously (i.e., driven by salient stimuli) or endogenously (i.e., driven by one’s goals or intentions). Genetic mouse models are useful tools to investigate the neurobiology of cognition, but a well-established assessment of attention orienting in mice is missing. This study aimed to adapt the Posner task, a widely used attention orienting task in humans, for use in mice using touchscreen technology and to test the effects of two attention-modulating drugs, methylphenidate (MPH) and atomoxetine (ATX), on the performance of mice during this task. In accordance with human performance, mice responded more quickly and more accurately to validly cued targets compared to invalidly cued targets, thus supporting mice as a valid animal model to study the neural mechanisms of attention orienting. This is the first evidence that mice can be trained to voluntarily maintain their nose-poke on a touchscreen and to complete attention orienting tasks using exogenous peripheral cues and endogenous symbolic cues. The results also showed no significant effects of MPH and ATX on attention orienting, although MPH improved overall response times in mice during the exogenous orienting task. In summary, the current study provides a critical translational task for assessing attention orienting in mice and to investigate the effects of attention-modulating drugs on attention orienting.Competing Interest StatementThe authors have declared no competing interest.View Full Text

animal behavior and cognition↗

bioRxiv2020

Prefrontal and Parietal Attractor Networks Mediate Working Memory Judgments

The dorsolateral prefrontal cortex plays a critical role in spatial working memory and its activity predicts behavioral responses in delayed response tasks. Here we addressed whether this predictive ability extends to categorical judgments based on information retained in working memory, and is present in other brain areas. We trained monkeys in a novel, Match-Stay, Nonmatch-Go task, which required them to observe two stimuli presented in sequence with an intervening delay period between them. If the two stimuli were different, the monkeys had to saccade to the location of the second stimulus; if they were the same, they held fixation. Neurophysiological recordings were performed in areas 8a and 46 of the dlPFC and 7a and lateral intraparietal cortex (LIP) of the PPC. We hypothesized that random drifts causing the peak activity of the network to move away from the first stimulus location and towards the location of the second stimulus would result in categorical errors. Indeed, for both areas, when the first stimulus appeared in a neurons preferred location, the neuron showed significantly higher firing rates in correct than in error trials. When the first stimulus appeared at a nonpreferred location and the second stimulus at a preferred, activity in error trials was higher than in correct. The results indicate that the activity of both dlPFC and PPC neurons is predictive of categorical judgments of information maintained in working memory, and the magnitude of neuronal firing rate deviations is revealing of the contents of working memory as it determines performance. SIGNIFICANCE STATEMENTThe neural basis of working memory and the areas mediating this function is a topic of controversy. Persistent activity in the prefrontal cortex has traditionally been thought to be the neural correlate of working memory, however recent studies have proposed alternative mechanisms and brain areas. Here we show that persistent activity in both the dorsolateral prefrontal cortex and posterior parietal cortex predicts behavior in a working memory task that requires a categorical judgement. Our results offer support to the idea that a network of neurons in both areas act as an attractor network that maintains information in working memory, which informs behavior.

neuroscience↗

bioRxiv2020

Cascade Diversification Directs the Generation of Neuronal Diversity in Hypothalamus

The hypothalamus contains an astounding heterogeneity of neurons to achieve its role in regulating endocrine, autonomic and behavioral functions. Despite previous progress in deciphering the gene regulatory programs linked to hypothalamus development, its molecular developmental trajectory and origin of neuronal diversity remain largely unknown. Here we combine transcriptomic profiling of 43,261 cells derived from Rax+ hypothalamic neuroepithelium with lineage tracing to map a developmental landscape of mouse hypothalamus and delineate the developmental trajectory of radial glial cells (RGCs), intermediate progenitor cells (IPCs), nascent neurons and peptidergic neurons in the lineage hierarchy. We show that RGCs adopt a conserved strategy for multipotential differentiation but generate both Ascl1+ and Neurog2+ IPCs, which display regionally differential origins in telencephalon. As transit-amplifying cells, Ascl1+ IPCs differ from their telencephalic counterpart by displaying fate bifurcation to produce both glutamatergic and GABAergic neurons. After classifying the developing neurons into 29 subtypes coded by diverse transcription factors, neurotransmitters and neuropeptides, we identified their molecular determinants via regulon analysis and further found that postmitotic neurons at nascent state possess the potential to resolve into more diverse subtypes of peptidergic neurons. Together, our study offers a single-cell framework for hypothalamus development and reveals that multiple cell types along the order of lineage hierarchy contribute to the fate diversification of hypothalamic neurons in a stepwise fashion, suggesting that a cascade diversifying model can deconstruct the origin of neuronal diversity.

developmental biology↗

bioRxiv2020

An enormous Paris polyphylla genome sheds light on genome size evolution and polyphyllin biogenesis

The monocot family Melanthiaceae with varying genome sizes in a range of 230-fold is an ideal model to study the genome size fluctuation in plants. Its family member Paris genus demonstrates an evolutionary trend of bearing huge genomes characterized by an average c-value of 49.22 pg. Here, we report a 70.18 Gb genome assembly out of the 82.55 Gb genome of Paris polyphylla var. yunnanensis (PPY), which represents the biggest sequenced genome to date. We annotate 69.53% repetitive sequences in this genome and 62.50% of which are long-terminal repeat (LTR) transposable elements. Further evolution analysis indicates that the giant genome likely results from the joint effect of common and species-specific expansion of different LTR superfamilies, which might contribute to the environment adaptation after speciation. Moreover, we identify the candidate pathway genes for the biogenesis of polyphyllins, the PPY-specific medicinal saponins, by complementary approaches including genome mining, comprehensive analysis of 31 next-generation RNA-seq data and 55.23 Gb single-molecule circular consensus sequencing (CCS) RNA-seq reads, and correlation of the transcriptome and phytochemical data of five different tissues at four growth stages. This study not only provides significant insights into plant genome size evolution, but also paves the way for the following polyphyllin synthetic biology.

plant biology↗

bioRxiv2020

SHP2 Inhibition Abrogates Adaptive Resistance to KRASG12C-Inhibition and Remodels the Tumor Microenvironment of KRAS-Mutant Tumors

KRAS is the most frequently mutated oncogene in human cancer, and KRAS inhibition has been a longtime therapeutic goal. Recently, inhibitors (G12C-Is) that bind KRASG12C-GDP and react with Cys-12 were developed. Using new affinity reagents to monitor KRASG12C activation and inhibitor engagement, we found that, reflecting its action upstream of SOS1/2, SHP2 inhibitors (SHP2-Is) increased KRAS-GDP occupancy, enhancing G12C-I efficacy. SHP2-Is abrogated feedback signaling by multiple RTKs and blocked adaptive resistance to G12C-Is in vitro, in xenografts, and in syngeneic KRASG12C-mutant pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC) models. Biochemical analysis revealed enhanced suppression of ERK-, MYC-, anti-apoptotic-, and cell-cycle genes, and increased pro-apoptotic gene expression in tumors from combination-treated mice. SHP2-I/G12C-I also evoked favorable changes in the immune microenvironment, decreasing myeloid suppressor cells, increasing CD8+ T cells, and sensitizing tumors to PD-1 blockade. Experiments using cells expressing inhibitor-resistant SHP2 showed that SHP2 inhibition in PDAC cells is required for tumor regression and remodeling of the immune microenvironment, but also revealed direct inhibitory effects on angiogenesis resulting in decreased tumor vascularity. Our results demonstrate that SHP2-I/G12C-I combinations confer a substantial survival benefit in PDAC and NSCLC and identify additional combination strategies for enhancing the efficacy of G12C-Is.

cancer biology↗

bioRxiv2020

Using high abundance proteins as guides for fast and effective peptide/protein identification from metaproteomic data

BackgroundA few recent large efforts significantly expanded the collection of human-associated bacterial genomes, which now contains thousands of entities including reference complete/draft genomes and metagenome assembled genomes (MAGs). These genomes provide useful resource for studying the functionality of the human-associated microbiome and their relationship with human health and diseases. One application of these genomes is to provide a universal reference for database search in metaproteomic studies, when matched metagenomic/metatranscriptomic data are unavailable. However, a greater collection of reference genomes may not necessarily result in better peptide/protein identification because the increase of search space often leads to fewer spectrum-peptide matches, not to mention the drastic increase of computation time. MethodsHere, we present a new approach that uses two steps to optimize the use of the reference genomes and MAGs as the universal reference for human gut metaproteomic MS/MS data analysis. The first step is to use only the High Abundance Proteins (HAPs) (i.e., ribosomal proteins and elongation factors) for metaproteomic MS/MS database search and, based on the identification results, to derive the taxonomic composition of the underlying microbial community. The second step is to expand the search database by including all proteins from identified abundant species. We call our approach HAPiID (HAPs guided metaproteomics IDentification). ResultsWe tested our approach using human gut metaproteomic datasets from a previous study and compared it to the state-of-the-art reference database search method MetaPro-IQ for metaproteomic identification in studying human gut microbiota. Our results show that our two-steps method not only performed significantly faster but also was able to identify more peptides. We further demonstrated the application of HAPiID to revealing protein profiles of individual human-associated bacterial species, one or a few species at a time, using metaproteomic data. ConclusionsThe HAP guided profiling approach presents a novel effective way for constructing target database for metaproteomic data analysis. The HAPiID pipeline built upon this approach provides a universal tool for analyzing human gut-associated metaproteomic data.

bioinformatics↗

bioRxiv2020

Massively parallel quantification of CRISPR editing in cells by TRAP-seq enables better design of Cas9, ABE, CBE gRNAs of high efficiency and accuracy

The CRISPR RNA-guided endonucleases Cas9, and Cas9-derived adenine/cytosine base editors (ABE/CBE), have been used in both research and therapeutic applications. However, broader use of this gene editing toolbox is hampered by the great variability of efficiency among different target sites. Here we present TRAP-seq, a versatile and scalable approach in which the CRISPR gRNA expression cassette and the corresponding surrogate site are captured by Targeted Reporter Anchored Positional Sequencing in cells. TRAP-seq can faithfully recapitulate the CRISPR gene editing outcomes introduced to the corresponding endogenous genome site and most importantly enables massively parallel quantification of CRISPR gene editing in cells. We demonstrate the utility of this technology for high-throughput quantification of SpCas9 editing efficiency and indel outcomes for 12,000 gRNAs in human embryonic kidney cells. Using this approach, we also showed that TRAP-seq enables high throughput quantification of both ABE and CBE efficiency at 12,000 sites in cells. This rich amount of ABE/CBE outcome data enable us to reveal several novel nucleotide features (e.g. preference of flanking bases, nucleotide motifs, STOP recoding types) affecting base editing efficiency, as well as designing improved machine learning-based prediction tools for designing SpCas9, ABE and CBE gRNAs of high efficiency and accuracy (>70%). We have integrated all the 12,000 CRISPR gene editing outcomes for SpCas9, ABE and CBE into a CRISPR-centered portal: The Human CRISPR Atlas. This study extends our knowledge on CRISPR gene and base editing, and will facilitate the application and development of CRISPR in both research and therapy.

systems biology↗

bioRxiv2020

Using targeted genome integration for virus-free genome-wide mammalian CRISPR screen

Pooled CRISPR screens have been widely applied in mammalian cells to discover genes regulating various phenotypes of interest. In such screens, CRISPR components are generally delivered with a lentivirus. However, lentiviral CRISPR screens are limited by unpredictable genome insertion, the requirement of biosafety level II lab facilities and personnel trained to work with viruses. Here we established a virus-free (VF) genome-wide CRISPR screening platform for Chinese hamster ovary (CHO) cells with 74,617 gRNAs targeting 18,353 genes. Each gRNA expression cassette in the library is precisely integrated into a genomic landing pad thus reducing the clonal variation. Using this VF CRISPR screening platform, 338 genes are identified as essential for CHO cell growth and 76 genes were found to be involved in the unfolded protein response (UPR) induced endoplasmic reticulum (ER) stress. Extensive validation of the candidate genes further demonstrated the robustness of this novel non-viral CRISPR screen method.

genomics↗

bioRxiv2020

PSGL-1 inhibits HIV-1 infection by restricting actin dynamics and sequestering HIV envelope proteins

PSGL-1 has recently been identified as an HIV restriction factor that inhibits HIV DNA synthesis and more potently, virion infectivity. But the underlying mechanisms of these inhibitions are unknown. Here we show that PSGL-1 directly binds to cellular actin filaments (F-actin) and restricts actin dynamics, which leads to inhibition of HIV DNA synthesis. PSGL-1 is incorporated into nascent virions and restricts actin dynamics in the virions, which partially accounts for the inhibition of virion infectivity. More potently, PSGL-1 inhibits incorporation of Env proteins into nascent virions, leading to loss of envelope spikes on the virions as shown by Cryo-electron microscopy and super-resolution imaging. This loss is associated with a profound defect in viral entry. Mechanistically, PSGL-1 binds gp41 and sequesters gp41 at the plasma membrane, explaining the inhibition of Env incorporation in nascent virions. PSGL-1s dual anti-HIV mechanisms represent novel strategies of human cells to defend against HIV infection.

microbiology↗