المؤلفون: Sunshine, Sara, Puschnik, Andreas, Replogle, Joseph, Laurie, Matthew, Liu, Jamin, Zha, Beth, Nuñez, James, Byrum, Janie, McMorrow, Aidan, Frieman, Matthew, Winkler, Juliane, Qiu, Xiaojie, Rosenberg, Oren, Leonetti, Manuel, Weissman, Jonathan, DeRisi, Joseph, Hein, Marco, Ye, Chun

المصدر: Nature Communications. 14(1)

الوصف: Genomic and proteomic screens have identified numerous host factors of SARS-CoV-2, but efficient delineation of their molecular roles during infection remains a challenge. Here we use Perturb-seq, combining genetic perturbations with a single-cell readout, to investigate how inactivation of host factors changes the course of SARS-CoV-2 infection and the host response in human lung epithelial cells. Our high-dimensional data resolve complex phenotypes such as shifts in the stages of infection and modulations of the interferon response. However, only a small percentage of host factors showed such phenotypes upon perturbation. We further identified the NF-κB inhibitor IκBα (NFKBIA), as well as the translation factors EIF4E2 and EIF4H as strong host dependency factors acting early in infection. Overall, our study provides massively parallel functional characterization of host factors of SARS-CoV-2 and quantitatively defines their roles both in virus-infected and bystander cells.

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/4f4875ddTest

المؤلفون: Samelson, Avi J, Tran, Quang Dinh, Robinot, Rémy, Carrau, Lucia, Rezelj, Veronica V, Kain, Alice Mac, Chen, Merissa, Ramadoss, Gokul N, Guo, Xiaoyan, Lim, Shion A, Lui, Irene, Nuñez, James K, Rockwood, Sarah J, Wang, Jianhui, Liu, Na, Carlson-Stevermer, Jared, Oki, Jennifer, Maures, Travis, Holden, Kevin, Weissman, Jonathan S, Wells, James A, Conklin, Bruce R, TenOever, Benjamin R, Chakrabarti, Lisa A, Vignuzzi, Marco, Tian, Ruilin, Kampmann, Martin

المصدر: Nature Cell Biology. 24(1)

مصطلحات موضوعية: Biochemistry and Cell Biology, Biological Sciences, Pneumonia, Emerging Infectious Diseases, Cancer, Biodefense, Vaccine Related, Infectious Diseases, Prevention, Lung, 2.1 Biological and endogenous factors, 5.1 Pharmaceuticals, Development of treatments and therapeutic interventions, Aetiology, Infection, Good Health and Well Being, Angiotensin-Converting Enzyme 2, Antiviral Agents, COVID-19, Cell Line, Epithelial Cells, Humans, Membrane Glycoproteins, SARS-CoV-2, Transcription Factors, COVID-19 Drug Treatment, Medical and Health Sciences, Developmental Biology, Biochemistry and cell biology

الوصف: SARS-CoV-2 infection of human cells is initiated by the binding of the viral Spike protein to its cell-surface receptor ACE2. We conducted a targeted CRISPRi screen to uncover druggable pathways controlling Spike protein binding to human cells. Here we show that the protein BRD2 is required for ACE2 transcription in human lung epithelial cells and cardiomyocytes, and BRD2 inhibitors currently evaluated in clinical trials potently block endogenous ACE2 expression and SARS-CoV-2 infection of human cells, including those of human nasal epithelia. Moreover, pharmacological BRD2 inhibition with the drug ABBV-744 inhibited SARS-CoV-2 replication in Syrian hamsters. We also found that BRD2 controls transcription of several other genes induced upon SARS-CoV-2 infection, including the interferon response, which in turn regulates the antiviral response. Together, our results pinpoint BRD2 as a potent and essential regulator of the host response to SARS-CoV-2 infection and highlight the potential of BRD2 as a therapeutic target for COVID-19.

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/9m5387j8Test

المؤلفون: Nuñez, James K, Chen, Jin, Pommier, Greg C, Cogan, J Zachery, Replogle, Joseph M, Adriaens, Carmen, Ramadoss, Gokul N, Shi, Quanming, Hung, King L, Samelson, Avi J, Pogson, Angela N, Kim, James YS, Chung, Amanda, Leonetti, Manuel D, Chang, Howard Y, Kampmann, Martin, Bernstein, Bradley E, Hovestadt, Volker, Gilbert, Luke A, Weissman, Jonathan S

المصدر: Cell. 184(9)

مصطلحات موضوعية: Biological Sciences, Genetics, Human Genome, Biotechnology, Stem Cell Research, Underpinning research, 1.1 Normal biological development and functioning, Generic health relevance, CRISPR-Cas Systems, Cell Differentiation, Cellular Reprogramming, CpG Islands, DNA Methylation, Epigenesis, Genetic, Epigenome, Gene Editing, Gene Silencing, Histone Code, Humans, Induced Pluripotent Stem Cells, Neurons, Protein Processing, Post-Translational, CRISPR, DNA methylation, cell therapy, dCas9, epigenetics, Medical and Health Sciences, Developmental Biology, Biological sciences, Biomedical and clinical sciences

الوصف: A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/4ph8w0v1Test
https://escholarship.org/content/qt4ph8w0v1/qt4ph8w0v1.pdfTest

المؤلفون: Chen, Jin, Brunner, Andreas-David, Cogan, J Zachery, Nuñez, James K, Fields, Alexander P, Adamson, Britt, Itzhak, Daniel N, Li, Jason Y, Mann, Matthias, Leonetti, Manuel D, Weissman, Jonathan S

المصدر: Science. 367(6482)

مصطلحات موضوعية: Biochemistry and Cell Biology, Biological Sciences, Genetics, Prevention, Biodefense, Vaccine Related, Human Genome, Biotechnology, 1.1 Normal biological development and functioning, Underpinning research, Generic health relevance, CRISPR-Cas Systems, Humans, Open Reading Frames, Operon, Peptides, Protein Biosynthesis, RNA, Messenger, Ribosomes, Transcriptome, General Science & Technology

الوصف: Ribosome profiling has revealed pervasive but largely uncharacterized translation outside of canonical coding sequences (CDSs). In this work, we exploit a systematic CRISPR-based screening strategy to identify hundreds of noncanonical CDSs that are essential for cellular growth and whose disruption elicits specific, robust transcriptomic and phenotypic changes in human cells. Functional characterization of the encoded microproteins reveals distinct cellular localizations, specific protein binding partners, and hundreds of microproteins that are presented by the human leukocyte antigen system. We find multiple microproteins encoded in upstream open reading frames, which form stable complexes with the main, canonical protein encoded on the same messenger RNA, thereby revealing the use of functional bicistronic operons in mammals. Together, our results point to a family of functional human microproteins that play critical and diverse cellular roles.

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/96m1n5z5Test

المؤلفون: Baskin, David, Buehler, Jim, Carter, Deirdre, Connelly, Brandon, Cummings, John, Davies, Evelyn, Elliott, John, Etherington, Kelley, Fulton, Brie, Jelinski, John, Kievit, Kenneth, Larson, Jennifer, Malvania, Kushal, Mulholland, Brendan, Nunez, James, Palladino, Carl, Philliber, Jeff, Robertson, Samantha, Saadeh, Joseph, Salvini, Karen, Santos, Bernadette, Sossikian, Leana, Tanouye, Patricia, Xu, Suying

الوصف: The annual Site Environmental Report documents Lawrence Berkeley National Laboratory’s performance in reducing its environmental impacts, progress toward cleaning up groundwater contamination, and compliance with applicable Department of Energy, federal, state, and local environmental regulations.

وصف الملف: application/pdf

العلاقة: qt9pv896tj; https://escholarship.org/uc/item/9pv896tjTest

الإتاحة: https://escholarship.org/uc/item/9pv896tjTest

المؤلفون: Adamson, Britt, Norman, Thomas M, Jost, Marco, Cho, Min Y, Nuñez, James K, Chen, Yuwen, Villalta, Jacqueline E, Gilbert, Luke A, Horlbeck, Max A, Hein, Marco Y, Pak, Ryan A, Gray, Andrew N, Gross, Carol A, Dixit, Atray, Parnas, Oren, Regev, Aviv, Weissman, Jonathan S

المصدر: Cell. 167(7)

مصطلحات موضوعية: Biochemistry and Cell Biology, Bioinformatics and Computational Biology, Genetics, Biological Sciences, Human Genome, Vaccine Related, Biotechnology, Underpinning research, 2.1 Biological and endogenous factors, 1.1 Normal biological development and functioning, Aetiology, Animals, Clustered Regularly Interspaced Short Palindromic Repeats, Endoribonucleases, Feedback, Humans, Models, Molecular, Protein Serine-Threonine Kinases, RNA, Guide, Kinetoplastida, Sequence Analysis, RNA, Single-Cell Analysis, Transcription, Genetic, Unfolded Protein Response, CRIPSRi, CRISPR, Single-cell RNA-seq, cell-to-cell heterogeneity, genome-scale screening, single-cell genomics, unfolded protein response, Medical and Health Sciences, Developmental Biology, Biological sciences, Biomedical and clinical sciences

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

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/9961h6m4Test

المؤلفون: Samelson, Avi, J, Tran, Quang Dinh, Robinot, Rémy, Carrau, Lucia, Rezelj, Veronica, V, Kain, Alice Mac, Chen, Merissa, Ramadoss, Gokul, N, Guo, Xiaoyan, Lim, Shion, A, Lui, Irene, Nuñez, James, K, Rockwood, Sarah, J, Wang, Jianhui, Liu, Na, Carlson-Stevermer, Jared, Oki, Jennifer, Maures, Travis, Holden, Kevin, Weissman, Jonathan, S, Wells, James, A, Conklin, Bruce, R, Tenoever, Benjamin, R, Chakrabarti, Lisa, A, Vignuzzi, Marco, Tian, Ruilin, Kampmann, Martin

المساهمون: University of California San Francisco (UC San Francisco), University of California (UC), Chan Zuckerberg BioHub San Francisco, CA, Populations virales et Pathogenèse - Viral Populations and Pathogenesis, Institut Pasteur Paris (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), École Doctorale Bio Sorbonne Paris Cité Paris (ED562 - BioSPC), Université Sorbonne Paris Cité (USPC)-Université Paris Cité (UPCité), Virus et Immunité - Virus and immunity (CNRS-UMR3569), NYU Langone Health New York, Gladstone Institutes San Francisco, Genentech, Inc., Genentech, Inc. San Francisco, Howard Hughes Medical Institute (HHMI), Southern University of Science and Technology (SUSTech), Synthego Redwood City, CA, Whitehead Institute, Massachusetts Institute of Technology (MIT), A.J.S. is supported by NIH grant F32AG063487. G.N.R. is supported by the NSF Graduate Research Fellowship Program (GRFP) and UCSF Discovery Fellowship. S.A.L. was a Merck Fellow of the Helen Hay Whitney Foundation. I.L. was supported by an NSF GRFP award. M.K. is a Chan Zuckerberg Biohub Investigator., We thank members of the Kampmann, Vignuzzi and Conklin laboratories, as well as V. Ramani, D. Ruggero, M. Ott and her laboratory and other members of the UCSF QBI Coronavirus Research Group (QCRG) for helpful discussions. We thank K. Leng for feedback on the manuscript. We acknowledge the Gladstone Stem Cell Core for help with cardiomyocyte production.

المصدر: ISSN: 1465-7392.

مصطلحات موضوعية: [SDV]Life Sciences [q-bio]

الوصف: International audience ; SARS-CoV-2 infection of human cells is initiated by the binding of the viral Spike protein to its cell-surface receptor ACE2. We conducted a targeted CRISPRi screen to uncover druggable pathways controlling Spike protein binding to human cells. Here we show that the protein BRD2 is required for ACE2 transcription in human lung epithelial cells and cardiomyocytes, and BRD2 inhibitors currently evaluated in clinical trials potently block endogenous ACE2 expression and SARS-CoV-2 infection of human cells, including those of human nasal epithelia. Moreover, pharmacological BRD2 inhibition with the drug ABBV-744 inhibited SARS-CoV-2 replication in Syrian hamsters. We also found that BRD2 controls transcription of several other genes induced upon SARS-CoV-2 infection, including the interferon response, which in turn regulates the antiviral response. Together, our results pinpoint BRD2 as a potent and essential regulator of the host response to SARS-CoV-2 infection and highlight the potential of BRD2 as a therapeutic target for COVID-19.

العلاقة: info:eu-repo/semantics/altIdentifier/pmid/35027731; pasteur-04133563; https://pasteur.hal.science/pasteur-04133563Test; https://pasteur.hal.science/pasteur-04133563/documentTest; https://pasteur.hal.science/pasteur-04133563/file/samelson_kampmann_nihms-1759623.pdfTest; PUBMED: 35027731; PUBMEDCENTRAL: PMC8820466

الإتاحة: https://doi.org/10.1038/s41556-021-00821-8Test
https://pasteur.hal.science/pasteur-04133563Test
https://pasteur.hal.science/pasteur-04133563/documentTest
https://pasteur.hal.science/pasteur-04133563/file/samelson_kampmann_nihms-1759623.pdfTest

المؤلفون: Nuñez, James K, Chen, Jin, Pommier, Greg C, Cogan, J Zachery, Replogle, Joseph M, Adriaens, Carmen, Ramadoss, Gokul N, Shi, Quanming, Hung, King L, Samelson, Avi J, Pogson, Angela N, Kim, James YS, Chung, Amanda, Leonetti, Manuel D, Chang, Howard Y, Kampmann, Martin, Bernstein, Bradley E, Hovestadt, Volker, Gilbert, Luke A, Weissman, Jonathan S

المساهمون: Whitehead Institute for Biomedical Research

المصدر: PMC

الوصف: A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.

وصف الملف: application/octet-stream

العلاقة: http://dx.doi.org/10.1016/J.CELL.2021.03.025Test; Cell; https://hdl.handle.net/1721.1/142534.2Test; Nuñez, James K, Chen, Jin, Pommier, Greg C, Cogan, J Zachery, Replogle, Joseph M et al. 2021. "Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing." Cell, 184 (9).

الإتاحة: https://doi.org/10.1016/J.CELL.2021.03.025Test
https://hdl.handle.net/1721.1/142534.2Test

المؤلفون: Baskin, David, Buehler, Jim, Carter, Deirdre, Connelly, Brandon, Cummings, John, Davies, Evelyn, Elliot, John, Etherington, Kelley, Fulton, Brie, Jelinski, John, Kievit, Ken, Larson, Jennifer, Mulholland, Brendan, Nunez, James, Palladino, Carl, Philliber, Jeff, Robertson, Samantha, Saadeh, Joseph, Salvini, Karen, Santos, Bernadette, Sossikian, Leana, Tanouye, Amy, Torkelson, Mike, Xu, Suying

الوصف: The annual Site Environmental Report documents Lawrence Berkeley National Laboratory’s performance in reducing its environmental impacts, progress toward cleaning up groundwater contamination, and compliance with applicable Department of Energy, federal, state, and local environmental regulations.

وصف الملف: application/pdf

العلاقة: qt2rb2w57h; https://escholarship.org/uc/item/2rb2w57hTest; https://escholarship.org/content/qt2rb2w57h/qt2rb2w57h.pdfTest

الإتاحة: https://escholarship.org/uc/item/2rb2w57hTest
https://escholarship.org/content/qt2rb2w57h/qt2rb2w57h.pdfTest

المؤلفون: Nuñez, James K, Harrington, Lucas B, Kranzusch, Philip J, Engelman, Alan N, Doudna, Jennifer A

المصدر: Nature. 527(7579)

مصطلحات موضوعية: Biological Sciences, Genetics, Adaptive Immunity, Bacteriophage M13, Base Sequence, CRISPR-Associated Proteins, Catalytic Domain, Clustered Regularly Interspaced Short Palindromic Repeats, Crystallography, X-Ray, DNA, Viral, Escherichia coli, Integrases, Models, Molecular, Virus Integration, General Science & Technology

الوصف: Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30-40-base-pair lengths into clustered regularly interspaced short palindromic repeat (CRISPR) loci as spacer segments. The universally conserved Cas1-Cas2 integrase complex catalyses spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases. How the Cas1-Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1-Cas2 complex bound to cognate 33-nucleotide protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3'-OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo. Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1-Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci.

الوصول الحر: https://escholarship.org/uc/item/618388d8Test