To identify new potential therapeutic targets for SARS-CoV-2, a research team at the New York Genome Center, New York University and the Icahn School of Medicine at Mount Sinai performed a large-scale CRISPR loss of function. screen to systematically knock out all genes in the human genome. The team investigated which genetic modifications made human lung cells more resistant to SARS-CoV-2 infection. Their results revealed individual genes and gene regulatory networks in the human genome required by SARS-CoV-2 that confer resistance to viral infection when repressed. The collaborative study described a wide range of genes that have not previously been considered as therapeutic targets for SARS-CoV-2. Their study was published online by Cell on October 24th.
To better understand the complex relationships between host and virus genetic dependencies, the team used a wide range of analytical and experimental methods to validate their results. This integrative approach included genome editing, single cell sequencing, confocal image processing, and computational analyzes of gene expression and proteomic data sets. The researchers found that these new gene targets, when inhibited by small molecules (drugs), significantly reduced the viral load and with some drugs up to 1000 times. Their results provide insight into new therapies that may be effective in treating COVID-19 and reveal the underlying molecular goals of these therapies.
?? Given the tragic impact of COVID-19 here in New York and around the world, we felt we could use the high-throughput CRISPR gene editing tools we have applied to other diseases to understand what are the major human genes required by The SARS-CoV-2 virus, ?? said the study’s co-author, Dr. Neville Sanjana, Core Faculty Member at the New York Genome Center, Assistant Professor of Biology, New York University and Assistant Professor of Neuroscience and Physiology at NYU Grossman School of Medicine. In the past, Dr Sanjana has applied comprehensive CRISPR monitors to identify the genetic drivers of various diseases, including drug resistance in melanoma, immunotherapy failure, lung cancer metastasis, congenital immunity, congenital metabolic disorders and muscular dystrophy.
For this project, through editing was only half of the equation. We have previously developed a series of human cell models for coronavirus infection in our work to understand the immune response to the virus. It was great to work with Neville’s group to understand and fully profile host genes from a new angle, ?? said co-author Dr. Benjamin tenOever, Fishberg Professor of Medicine, Icahn Scholar and Professor of Microbiology, Icahn School of Medicine at Mount Sinai.
Gene clusters lead the way
The team discovered that the highest ranked genes ?? those whose loss reduces viral infection significantly ?? grouped into a handful of protein complexes, including vacuolar ATPases, Retromer, Commander, Arp2 / 3 and PI3K. Many of these protein complexes are involved in the trade in proteins to and from the cell membrane.
We were very pleased to see several genes within the same family as top-ranked hits on our screen. This gave us a high degree of confidence that these protein families were crucial to the life cycle of the virus, either to enter human cells or successful viral replication, ?? says Dr. Zharko Daniloski, a postdoctoral fellow in the Sanjana Lab and co-author of the study.
While researchers performed the CRISPR screen using human lung cells, the team also investigated whether the expression of required host genes was lung-specific or more broadly expressed. Among the top-ranked genes, only ACE2, the receptor known to be responsible for binding the SARS-CoV-2 viral protein Spike, showed tissue-specific expression, with the rest of the top gene hitting many tissues, suggesting that these mechanisms may function independently of cell or tissue type. Using proteomic data, they found that several of the top-ranked host genes interact directly with the virus’ own proteins, highlighting their central role in the viral life cycle. The team also analyzed common host genes required for other viral pathogens, such as Zika or H1N1 pandemic influenza.
Mechanical insights: Cholesterol and viral receptors
After completing the primary screen, the research team used several different techniques to validate the role of many of the highest ranked genes in viral infection. Using human cell lines derived from the lung and other organs susceptible to SARS-CoV-2 infection, they measured viral infection after gene knockout by CRISPR, gene suppression by RNA interference, or drug inhibition. After confirming that these manipulations reduced viral infection, they then sought to understand the mechanisms by which loss of these genes blocks coronavirus infection.
Using a newly developed technique that links large-scale CRISPR editing with single-cell RNA sequencing (ECCITE-seq), the team identified that loss of several top-ranked genes results in upregulation of cholesterol biosynthetic pathways and an increase in cellular cholesterol. Using this insight, they studied the effects of amlodipine, a drug that alters cholesterol levels.
We found that amlodipine, a calcium channel antagonist, regulates cellular cholesterol levels and blocks SARS-CoV-2 infection. Since new clinical studies have also suggested that patients taking calcium channel blockers have a reduced mortality rate on COVID-19 cases, an important future direction of research will be to further elucidate the link between cholesterol synthesis pathways and SARS-CoV-2, ?? says Dr. Tristan Jordan, a postdoctoral fellow at tenOever Lab and co-author of the study.
Based on previous work on mutations in the Spike protein and virus entry through the ACE2 receptor, the research team also asked whether loss of certain genes can confer resistance to coronavirus by lowering ACE2 levels. In particular, they identified a gene, RAB7A, which has a major impact on the trade in ACE2 to the cell membrane. Using a combination of flow cytometry and confocal microscopy, the team demonstrated that RAB7A loss prevents viral entry by sequestering ACE2 receptors within cells.
?? Current treatments for SARS-CoV-2 infection are currently based on the virus itself, but this study provides a better understanding of how host genes affect viral entry and will enable new avenues for therapeutic detection and hopefully accelerate recovery for susceptible populations, ?? said Dr. Sanjana.
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About the New York Genome Center
The New York Genome Center (NYGC) is an independent, non-profit academic research institution that serves as a multinational genome research center. By utilizing our strengths in whole genome sequencing and genome analysis, our mission is to develop genomics and its application to drive new biomedical discoveries. NYGC’s focus areas include the development of computational and experimental genome methods and disease-focused research to better understand the genetic basis of cancer, neurodegenerative disease, and neuropsychiatric disease. By 2020, NYGC has also directed its expertise to COVID-19 research.
NYGC utilizes the expertise and builds on the combined strength of our faculty, human resources researchers, member institutions, scientific working groups, affiliates and industry partners to promote genomic discovery. Central to our scientific mission is an outstanding faculty that leads independent research laboratories based on NYGC and has joint tenure with one of our member institutions.
Institutional founding members of NYGC are: Cold Spring Harbor Laboratory, Columbia University, Albert Einstein College of Medicine, The Jackson Laboratory, Memorial Sloan Kettering Cancer Center, Icahn School of Medicine at Mount Sinai, New York-Presbyterian Hospital, New York University, Northwell Health , The Rockefeller University, Stony Brook University and Weill Cornell Medicine. Institutional associate members are: American Museum of Natural History, Georgetown Lombardi Comprehensive Cancer Center, Hackensack Meridian Health, Hospital for Special Surgery, The New York Stem Cell Foundation, Princeton University, Roswell Park Cancer Institute and Rutgers Cancer Institute of New Jersey.
For more information about NYGC, visit: http://www.nygenome.org.
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