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1. Proteomic and genetic analyses of influenza A viruses identify pan-viral host targets

Author

Haas, Kelsey M, McGregor, Michael J, Bouhaddou, Mehdi, Polacco, Benjamin J, Kim, Eun-Young, Nguyen, Thong T, Newton, Billy W, Urbanowski, Matthew, Kim, Heejin, Williams, Michael AP, Rezelj, Veronica V, Hardy, Alexandra, Fossati, Andrea, Stevenson, Erica J, Sukerman, Ellie, Kim, Tiffany, Penugonda, Sudhir, Moreno, Elena, Braberg, Hannes, Zhou, Yuan, Metreveli, Giorgi, Harjai, Bhavya, Tummino, Tia A, Melnyk, James E, Soucheray, Margaret, Batra, Jyoti, Pache, Lars, Martin-Sancho, Laura, Carlson-Stevermer, Jared, Jureka, Alexander S, Basler, Christopher F, Shokat, Kevan M, Shoichet, Brian K, Shriver, Leah P, Johnson, Jeffrey R, Shaw, Megan L, Chanda, Sumit K, Roden, Dan M, Carter, Tonia C, Kottyan, Leah C, Chisholm, Rex L, Pacheco, Jennifer A, Smith, Maureen E, Schrodi, Steven J, Albrecht, Randy A, Vignuzzi, Marco, Zuliani-Alvarez, Lorena, Swaney, Danielle L, Eckhardt, Manon, Wolinsky, Steven M, White, Kris M, Hultquist, Judd F, Kaake, Robyn M, García-Sastre, Adolfo, and Krogan, Nevan J

Subjects
Microbiology, Medical Microbiology, Biomedical and Clinical Sciences, Biological Sciences, Infectious Diseases, Prevention, Emerging Infectious Diseases, Vaccine Related, Influenza, Pneumonia & Influenza, Biodefense, Biotechnology, 2.1 Biological and endogenous factors, 2.2 Factors relating to the physical environment, Aetiology, Infection, Humans, Influenza A virus, Influenza, Human, Influenza A Virus, H5N1 Subtype, Influenza A Virus, H3N2 Subtype, Proteomics, Virus Replication, COVID-19, SARS-CoV-2, Antiviral Agents, Host-Pathogen Interactions
Abstract

Influenza A Virus (IAV) is a recurring respiratory virus with limited availability of antiviral therapies. Understanding host proteins essential for IAV infection can identify targets for alternative host-directed therapies (HDTs). Using affinity purification-mass spectrometry and global phosphoproteomic and protein abundance analyses using three IAV strains (pH1N1, H3N2, H5N1) in three human cell types (A549, NHBE, THP-1), we map 332 IAV-human protein-protein interactions and identify 13 IAV-modulated kinases. Whole exome sequencing of patients who experienced severe influenza reveals several genes, including scaffold protein AHNAK, with predicted loss-of-function variants that are also identified in our proteomic analyses. Of our identified host factors, 54 significantly alter IAV infection upon siRNA knockdown, and two factors, AHNAK and coatomer subunit COPB1, are also essential for productive infection by SARS-CoV-2. Finally, 16 compounds targeting our identified host factors suppress IAV replication, with two targeting CDK2 and FLT3 showing pan-antiviral activity across influenza and coronavirus families. This study provides a comprehensive network model of IAV infection in human cells, identifying functional host targets for pan-viral HDT.

Published
2023

2. Viral E protein neutralizes BET protein-mediated post-entry antagonism of SARS-CoV-2

Author

Chen, Irene P, Longbotham, James E, McMahon, Sarah, Suryawanshi, Rahul K, Khalid, Mir M, Taha, Taha Y, Tabata, Takako, Hayashi, Jennifer M, Soveg, Frank W, Carlson-Stevermer, Jared, Gupta, Meghna, Zhang, Meng Yao, Lam, Victor L, Li, Yang, Yu, Zanlin, Titus, Erron W, Diallo, Amy, Oki, Jennifer, Holden, Kevin, Krogan, Nevan, Fujimori, Danica Galonić, and Ott, Melanie

Subjects
Biological Sciences, Lung, Emerging Infectious Diseases, Vaccine Related, Pneumonia, Pneumonia & Influenza, Biodefense, Prevention, Infectious Diseases, Development of treatments and therapeutic interventions, 5.1 Pharmaceuticals, Infection, Good Health and Well Being, Angiotensin-Converting Enzyme 2, Animals, Antiviral Agents, COVID-19, Interferons, Mice, Nuclear Proteins, SARS-CoV-2, Transcription Factors, Viral Proteins, BET inhibitors, BET proteins, BRD2, BRD3, BRD4, CP: Microbiology, antiviral response, histone mimetic, viral replication, Biochemistry and Cell Biology, Medical Physiology, Biological sciences
Abstract

Inhibitors of bromodomain and extraterminal domain (BET) proteins are possible anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prophylactics as they downregulate angiotensin-converting enzyme 2 (ACE2). Here we show that BET proteins should not be inactivated therapeutically because they are critical antiviral factors at the post-entry level. Depletion of BRD3 or BRD4 in cells overexpressing ACE2 exacerbates SARS-CoV-2 infection; the same is observed when cells with endogenous ACE2 expression are treated with BET inhibitors during infection and not before. Viral replication and mortality are also enhanced in BET inhibitor-treated mice overexpressing ACE2. BET inactivation suppresses interferon production induced by SARS-CoV-2, a process phenocopied by the envelope (E) protein previously identified as a possible "histone mimetic." E protein, in an acetylated form, directly binds the second bromodomain of BRD4. Our data support a model where SARS-CoV-2 E protein evolved to antagonize interferon responses via BET protein inhibition; this neutralization should not be further enhanced with BET inhibitor treatment.

Published
2022

3. Identification of DAXX as a restriction factor of SARS-CoV-2 through a CRISPR/Cas9 screen.

Author

Mac Kain, Alice, Maarifi, Ghizlane, Aicher, Sophie-Marie, Arhel, Nathalie, Baidaliuk, Artem, Munier, Sandie, Donati, Flora, Vallet, Thomas, Tran, Quang, Hardy, Alexandra, Chazal, Maxime, Porrot, Françoise, OhAinle, Molly, Carlson-Stevermer, Jared, Oki, Jennifer, Holden, Kevin, Zimmer, Gert, Simon-Lorière, Etienne, Bruel, Timothée, Schwartz, Olivier, van der Werf, Sylvie, Jouvenet, Nolwenn, Nisole, Sébastien, Vignuzzi, Marco, and Roesch, Ferdinand

Subjects
COVID-19, CRISPR-Cas Systems, Co-Repressor Proteins, Humans, Interferons, Molecular Chaperones, Proteasome Endopeptidase Complex, SARS-CoV-2
Abstract

Interferon restricts SARS-CoV-2 replication in cell culture, but only a handful of Interferon Stimulated Genes with antiviral activity against SARS-CoV-2 have been identified. Here, we describe a functional CRISPR/Cas9 screen aiming at identifying SARS-CoV-2 restriction factors. We identify DAXX, a scaffold protein residing in PML nuclear bodies known to limit the replication of DNA viruses and retroviruses, as a potent inhibitor of SARS-CoV-2 and SARS-CoV replication in human cells. Basal expression of DAXX is sufficient to limit the replication of SARS-CoV-2, and DAXX over-expression further restricts infection. DAXX restricts an early, post-entry step of the SARS-CoV-2 life cycle. DAXX-mediated restriction of SARS-CoV-2 is independent of the SUMOylation pathway but dependent on its D/E domain, also necessary for its protein-folding activity. SARS-CoV-2 infection triggers the re-localization of DAXX to cytoplasmic sites and promotes its degradation. Mechanistically, this process is mediated by the viral papain-like protease (PLpro) and the proteasome. Together, these results demonstrate that DAXX restricts SARS-CoV-2, which in turn has evolved a mechanism to counteract its action.

Published
2022

4. BRD2 inhibition blocks SARS-CoV-2 infection by reducing transcription of the host cell receptor ACE2

Author

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, and Kampmann, Martin

Subjects
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
Abstract

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.

Published
2022

5. Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses

Author

Wang, Ruofan, Simoneau, Camille R, Kulsuptrakul, Jessie, Bouhaddou, Mehdi, Travisano, Katherine A, Hayashi, Jennifer M, Carlson-Stevermer, Jared, Zengel, James R, Richards, Christopher M, Fozouni, Parinaz, Oki, Jennifer, Rodriguez, Lauren, Joehnk, Bastian, Walcott, Keith, Holden, Kevin, Sil, Anita, Carette, Jan E, Krogan, Nevan J, Ott, Melanie, and Puschnik, Andreas S

Subjects
Medical Microbiology, Biomedical and Clinical Sciences, Biological Sciences, Pneumonia & Influenza, Emerging Infectious Diseases, Pneumonia, Infectious Diseases, Prevention, Vaccine Related, Biodefense, Lung, 2.1 Biological and endogenous factors, Aetiology, 2.2 Factors relating to the physical environment, Infection, Good Health and Well Being, A549 Cells, Animals, Biosynthetic Pathways, COVID-19, Cell Line, Chlorocebus aethiops, Cholesterol, Cluster Analysis, Clustered Regularly Interspaced Short Palindromic Repeats, Common Cold, Coronavirus, Coronavirus Infections, Gene Knockout Techniques, Genome-Wide Association Study, Host-Pathogen Interactions, Humans, Mice, Phosphatidylinositols, SARS-CoV-2, Vero Cells, Virus Internalization, Virus Replication, 229E, CRISPR, OC43, coronavirus, genetic screen, host factors, host-targeted antivirals, virus-host interactions, Medical and Health Sciences, Developmental Biology, Biological sciences, Biomedical and clinical sciences
Abstract

The Coronaviridae are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E), and glycosaminoglycans (for OC43). Additionally, we identified phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies.

Published
2021

6. Long term rescue of Alzheimer deficits in vivo by one-time gene-editing of App C-terminus.

Author

Aulston, Brent, primary, Gimse, Kristen, additional, Bazick, Hannah O, additional, Kramar, Eniko A, additional, Pizzo, Donald P, additional, Parra-Rivas, Leonardo A, additional, Sun, Jichao, additional, Branes-Guerrero, Kristen, additional, Cheka, Nidhi, additional, Bagheri, Neda, additional, Satyadev, Nihal, additional, Carlson-Stevermer, Jared, additional, Saito, Takashi, additional, Saido, Takaomi C, additional, Audhya, Anjon, additional, Wood, Marcelo A, additional, Zylka, Mark J, additional, Saha, Krishanu, additional, and Roy, Subhojit, additional

Published
2024
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16. Identification of DAXX As A Restriction Factor Of SARS-CoV-2 Through A CRISPR/Cas9 Screen

Author

Mac Kain, Alice, primary, Maarifi, Ghizlane, additional, Aicher, Sophie-Marie, additional, Arhel, Nathalie, additional, Baidaliuk, Artem, additional, Munier, Sandie, additional, Donati, Flora, additional, Vallet, Thomas, additional, Tran, Quang Dinh, additional, Hardy, Alexandra, additional, Chazal, Maxime, additional, Porrot, Françoise, additional, OhAinle, Molly, additional, Carlson-Stevermer, Jared, additional, Oki, Jennifer, additional, Holden, Kevin, additional, Simon-Lorière, Etienne, additional, Bruel, Timothée, additional, Schwartz, Olivier, additional, van der Werf, Sylvie, additional, Jouvenet, Nolwenn, additional, Nisole, Sébastien, additional, Vignuzzi, Marco, additional, and Roesch, Ferdinand, additional

Published
2021
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17. Comparative host-coronavirus protein interaction networks reveal pan-viral disease mechanisms

Author

Gordon, David E., Hiatt, Joseph, Bouhaddou, Mehdi, Rezelj, Veronica V., Ulferts, Svenja, Braberg, Hannes, Jureka, Alexander S., Obernier, Kirsten, Guo, Jeffrey Z., Batra, Jyoti, Kaake, Robyn M., Weckstein, Andrew R., Owens, Tristan W., Gupta, Meghna, Pourmal, Sergei, Titus, Erron W., Cakir, Merve, Soucheray, Margaret, McGregor, Michael, Cakir, Zeynep, Jang, Gwendolyn, O’Meara, Matthew J., Tummino, Tia A., Zhang, Ziyang, Foussard, Helene, Rojc, Ajda, Zhou, Yuan, Kuchenov, Dmitry, Hüttenhain, Ruth, Xu, Jiewei, Eckhardt, Manon, Swaney, Danielle L., Fabius, Jacqueline M., Ummadi, Manisha, Tutuncuoglu, Beril, Rathore, Ujjwal, Modak, Maya, Haas, Paige, Haas, Kelsey M., Naing, Zun Zar Chi, Pulido, Ernst H., Shi, Ying, Barrio-Hernandez, Inigo, Memon, Danish, Petsalaki, Eirini, Dunham, Alistair, Marrero, Miguel Correa, Burke, David, Koh, Cassandra, Vallet, Thomas, Silvas, Jesus A., Azumaya, Caleigh M., Billesbølle, Christian, Brilot, Axel F., Campbell, Melody G., Diallo, Amy, Dickinson, Miles Sasha, Diwanji, Devan, Herrera, Nadia, Hoppe, Nick, Kratochvil, Huong T., Liu, Yanxin, Merz, Gregory E., Moritz, Michelle, Nguyen, Henry C., Nowotny, Carlos, Puchades, Cristina, Rizo, Alexandrea N., Schulze-Gahmen, Ursula, Smith, Amber M., Sun, Ming, Young, Iris D., Zhao, Jianhua, Asarnow, Daniel, Biel, Justin, Bowen, Alisa, Braxton, Julian R., Chen, Jen, Chio, Cynthia M., Chio, Un Seng, Deshpande, Ishan, Doan, Loan, Faust, Bryan, Flores, Sebastian, Jin, Mingliang, Kim, Kate, Lam, Victor L., Li, Fei, Li, Junrui, Li, Yen-Li, Li, Yang, Liu, Xi, Lo, Megan, Lopez, Kyle E., Melo, Arthur A., Moss, Frank R., Nguyen, Phuong, Paulino, Joana, Pawar, Komal Ishwar, Peters, Jessica K., Pospiech, Thomas H., Safari, Maliheh, Sangwan, Smriti, Schaefer, Kaitlin, Thomas, Paul V., Thwin, Aye C., Trenker, Raphael, Tse, Eric, Tsui, Tsz Kin Martin, Wang, Feng, Whitis, Natalie, Yu, Zanlin, Zhang, Kaihua, Zhang, Yang, Zhou, Fengbo, Saltzberg, Daniel, Hodder, Anthony J., Shun-Shion, Amber S., Williams, Daniel M., White, Kris M., Rosales, Romel, Kehrer, Thomas, Miorin, Lisa, Moreno, Elena, Patel, Arvind H., Rihn, Suzannah, Khalid, Mir M., Vallejo-Gracia, Albert, Fozouni, Parinaz, Simoneau, Camille R., Roth, Theodore L., Wu, David, Karim, Mohd Anisul, Ghoussaini, Maya, Dunham, Ian, Berardi, Francesco, Weigang, Sebastian, Chazal, Maxime, Park, Jisoo, Logue, James, McGrath, Marisa, Weston, Stuart, Haupt, Robert, Hastie, C. James, Elliott, Matthew, Brown, Fiona, Burness, Kerry A., Reid, Elaine, Dorward, Mark, Johnson, Clare, Wilkinson, Stuart G., Geyer, Anna, Giesel, Daniel M., Baillie, Carla, Raggett, Samantha, Leech, Hannah, Toth, Rachel, Goodman, Nicola, Keough, Kathleen C., Lind, Abigail L., Klesh, Reyna J., Hemphill, Kafi R., Carlson-Stevermer, Jared, Oki, Jennifer, Holden, Kevin, Maures, Travis, Pollard, Katherine S., Sali, Andrej, Agard, David A., Cheng, Yifan, Fraser, James S., Frost, Adam, Jura, Natalia, Kortemme, Tanja, Manglik, Aashish, Southworth, Daniel R., Stroud, Robert M., Alessi, Dario R., Davies, Paul, Frieman, Matthew B., Ideker, Trey, Abate, Carmen, Jouvenet, Nolwenn, Kochs, Georg, Shoichet, Brian, Ott, Melanie, Palmarini, Massimo, Shokat, Kevan M., García-Sastre, Adolfo, Rassen, Jeremy A., Grosse, Robert, Rosenberg, Oren S., Verba, Kliment A., Basler, Christopher F., Vignuzzi, Marco, Peden, Andrew A., Beltrao, Pedro, Krogan, Nevan J., Trinidad, Donovan, Damas, Joana, Hughes, Graham M., Painter, Corrie A., Persky, Nicole S., Corbo, Marco, Kirilenko, Bodgan, Hiller, Michael, Koepfli, Klaus-Peter, Kaplow, Irene, Wirthlin, Morgan, Pfenning, Andreas R., Zhao, Huabin, Genereux, Diane P., Swofford, Ross, Lind, Abigail, Ryderq, Oliver A., Nweeia, Martin T., Meadows, Jennifer, Dong, Michael, Wallerman, Ola, Marinescu, Vikki, Lindblad-Toh, Kerstin, Ray, David A., Power, Sarahjane, Teeling, Emma C., Chauhan, Gaurav, Li, Shirley Xue, Karlsson, Elinor K., Lewin, Harris A., Centre National de la Recherche Scientifique (CNRS), Quantitative Biosciences Institute [UC San Francisco, USA] (QBI), University of California [San Francisco] (UC San Francisco), University of California (UC)-University of California (UC), Populations virales et Pathogenèse - Viral Populations and Pathogenesis, and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Infectious Medicine, Middle East respiratory syndrome coronavirus, Viral protein, viruses, [SDV]Life Sciences [q-bio], Infektionsmedicin, medicine.disease_cause, Conserved sequence, Microbiology in the medical area, 03 medical and health sciences, Mitochondrial membrane transport protein, 0302 clinical medicine, Protein structure, Pandemic, medicine, Mikrobiologi inom det medicinska området, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Coronavirus, 0303 health sciences, Multidisciplinary, biology, virus diseases, Virology, 3. Good health, biology.protein, Viral disease, 030217 neurology & neurosurgery
Abstract

INTRODUCTION The emergence of three lethal coronaviruses in Jennifer Meadows, Michael Dong, Ola Wallerman, Vikki Marinescu & Kerstin Lindblad-Toh ingår i gruppen Zoonomia Consortium

Published
2020
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18. BRD2 inhibition blocks SARS-CoV-2 infection by reducing transcription of the host cell receptor ACE2

Author

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

Published
2021
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20. Functional genomic screens identify human host factors for SARS-CoV-2 and common cold coronaviruses

Author

Wang, Ruofan, primary, Simoneau, Camille R., additional, Kulsuptrakul, Jessie, additional, Bouhaddou, Mehdi, additional, Travisano, Katherine, additional, Hayashi, Jennifer M., additional, Carlson-Stevermer, Jared, additional, Oki, Jennifer, additional, Holden, Kevin, additional, Krogan, Nevan J., additional, Ott, Melanie, additional, and Puschnik, Andreas S., additional

Published
2020
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22. A CRISPR/Cas9 based strategy to manipulate the Alzheimer’s amyloid pathway

Author

Sun, Jichao, primary, Carlson-Stevermer, Jared, additional, Das, Utpal, additional, Shen, Minjie, additional, Delenclos, Marion, additional, Snead, Amanda M., additional, Wang, Lina, additional, Loi, Jonathan, additional, Petersen, Andrew J, additional, Stockton, Michael, additional, Bhattacharyya, Anita, additional, Jones, Mathew V., additional, Sproul, Andrew A., additional, McLean, Pamela J., additional, Zhao, Xinyu, additional, Saha, Krishanu, additional, and Roy, Subhojit, additional

Published
2018
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33. Long term rescue of Alzheimer's deficits in vivo by one-time gene-editing of App C-terminus.

Author

Aulston BD, Gimse K, Bazick HO, Kramar EA, Pizzo DP, Parra-Rivas LA, Sun J, Branes-Guerrero K, Checka N, Bagheri N, Satyadev N, Carlson-Stevermer J, Saito T, Saido TC, Audhya A, Wood MA, Zylka MJ, Saha K, and Roy S

Abstract

Gene-editing technologies promise to create a new class of therapeutics that can achieve permanent correction with a single intervention. Besides eliminating mutant alleles in familial disease, gene-editing can also be used to favorably manipulate upstream pathophysiologic events and alter disease-course in wider patient populations, but few such feasible therapeutic avenues have been reported. Here we use CRISPR-Cas9 to edit the last exon of amyloid precursor protein ( App ), relevant for Alzheimer's disease (AD). Our strategy effectively eliminates an endocytic (YENPTY) motif at APP C-terminus, while preserving the N-terminus and compensatory APP-homologues. This manipulation favorably alters events along the amyloid-pathway - inhibiting toxic APP-β-cleavage fragments (including Aβ) and upregulating neuroprotective APP-α-cleavage products. AAV-driven editing ameliorates neuropathologic, electrophysiologic, and behavioral deficits in an AD knockin mouse model. Effects persist for many months, and no abnormalities are seen in WT mice even after germline App -editing; underlining overall efficacy and safety. Pathologic alterations in the glial-transcriptome of App -KI mice, as seen by single nuclei RNA-sequencing (sNuc-Seq), are also normalized by App C-terminus editing. Our strategy takes advantage of innate transcriptional rules that render terminal exons insensitive to nonsense-decay, and the upstream manipulation is expected to be effective for all forms of AD. These studies offer a path for a one-time disease-modifying treatment for AD., Competing Interests: Competing interest declaration: S.R. is the scientific co-founder and owns equity in CRISPRAlz.

Published
2024
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34. Viral E Protein Neutralizes BET Protein-Mediated Post-Entry Antagonism of SARS-CoV-2.

Author

Chen IP, Longbotham JE, McMahon S, Suryawanshi RK, Carlson-Stevermer J, Gupta M, Zhang MY, Soveg FW, Hayashi JM, Taha TY, Lam VL, Li Y, Yu Z, Titus EW, Diallo A, Oki J, Holden K, Krogan N, Galonić Fujimori D, and Ott M

Abstract

Inhibitors of Bromodomain and Extra-terminal domain (BET) proteins are possible anti-SARS-CoV-2 prophylactics as they downregulate angiotensin-converting enzyme 2 (ACE2). Here, we show that BET proteins should not be inactivated therapeutically as they are critical antiviral factors at the post-entry level. Knockouts of BRD3 or BRD4 in cells overexpressing ACE2 exacerbate SARS-CoV-2 infection; the same is observed when cells with endogenous ACE2 expression are treated with BET inhibitors during infection, and not before. Viral replication and mortality are also enhanced in BET inhibitor-treated mice overexpressing ACE2. BET inactivation suppresses interferon production induced by SARS-CoV-2, a process phenocopied by the envelope (E) protein previously identified as a possible "histone mimetic." E protein, in an acetylated form, directly binds the second bromodomain of BRD4. Our data support a model where SARS-CoV-2 E protein evolved to antagonize interferon responses via BET protein inhibition; this neutralization should not be further enhanced with BET inhibitor treatment.

Published
2021
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35. BRD2 inhibition blocks SARS-CoV-2 infection by reducing transcription of the host cell receptor ACE2.

Author

Samelson AJ, Tran QD, Robinot R, Carrau L, Rezelj VV, Mac Kain A, Chen M, Ramadoss GN, Guo X, Lim SA, Lui I, Nunez J, Rockwood SJ, Wang J, Liu N, Carlson-Stevermer J, Oki J, Maures T, Holden K, Weissman JS, Wells JA, Conklin BR, TenOever BR, Chakrabarti LA, Vignuzzi M, Tian R, and Kampmann M

Abstract

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. We found 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 novel therapeutic target for COVID-19., Competing Interests: Competing Interests: JCS, JO, TM and KH are employees and shareholders of Synthego Corporation.

Published
2021
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36. Functional genomic screens identify human host factors for SARS-CoV-2 and common cold coronaviruses.

Author

Wang R, Simoneau CR, Kulsuptrakul J, Bouhaddou M, Travisano K, Hayashi JM, Carlson-Stevermer J, Oki J, Holden K, Krogan NJ, Ott M, and Puschnik AS

Abstract

The Coronaviridae are a family of viruses that causes disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors that are common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted parallel genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E) and glycosaminoglycans (for OC43). Additionally, we discovered phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle as well as the potential development of host-directed therapies.

Published
2020
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37. Genome Editing in Human Pluripotent Stem Cells.

Author

Carlson-Stevermer J and Saha K

Subjects
CRISPR-Cas Systems genetics, Cell Line, Electroporation methods, Gene Editing methods, Genetic Engineering methods, Genome, Human genetics, Humans, Transcription Activator-Like Effector Nucleases genetics, Zinc Finger Nucleases genetics, Pluripotent Stem Cells physiology
Abstract

Genome editing in human pluripotent stem cells (hPSCs) enables the generation of reporter lines and knockout cell lines. Zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9 technology have recently increased the efficiency of proper gene editing by creating double strand breaks (DSB) at defined sequences in the human genome. These systems typically use plasmids to transiently transcribe nucleases within the cell. Here, we describe the process for preparing hPSCs for transient expression of nucleases via electroporation and subsequent analysis to create genetically modified stem cell lines.

Published
2017
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