CRISPR screen identifies genes, drug targets to protect against SARS-
CoV-2 infection

Date:
October 26, 2020
Source:
New York Genome Center
Summary:
A new study demonstrates how changes in human genes can reduce
SARS-CoV- 2 infection and describes a wide array of genes that
have not previously been considered as therapeutic targets for
SARS-CoV-2.



FULL STORY ==========================================================================
To identify new potential therapeutic targets for SARS-CoV-2, a team
of scientists at the New York Genome Center, New York University, and
the Icahn School of Medicine at Mount Sinai, performed a genome-scale, loss-of-function CRISPR screen to systematically knockout all genes in
the human genome. The team examined which genetic modifications made
human lung cells more resistant to SARS-CoV-2 infection. Their findings revealed individual genes and gene regulatory networks in the human
genome that are required by SARS-CoV-2 and that confer resistance to viral infection when suppressed. The collaborative study described a wide array
of genes that have not previously been considered as therapeutic targets
for SARS-CoV-2. Their study was published online by Cell on October 24.


==========================================================================
In order to better understand the complex relationships between host and
virus genetic dependencies, the team used a broad range of analytical and experimental methods to validate their results. This integrative approach included genome editing, single-cell sequencing, confocal imaging, and computational analyses of gene expression and proteomic datasets. The researchers found that these new gene targets, when inhibited using
small molecules (drugs), significantly reduced viral load, and with
some drugs, up to 1,000-fold. Their findings offer insight into novel
therapies that may be effective in treating COVID-19 and reveal the
underlying molecular targets of those therapies.

"Seeing the tragic impact of COVID-19 here in New York and across the
world, we felt that we could use the high-throughput CRISPR gene editing
tools that we have applied to other diseases to understand what are
the key human genes required by the SARS-CoV-2 virus," said the study's co-senior 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. Previously, Dr.

Sanjana has applied genome-wide CRISPR screens to identify the genetic
drivers of diverse diseases, including drug resistance in melanoma, immunotherapy failure, lung cancer metastasis, innate immunity, inborn metabolic disorders, and muscular dystrophy.

For this project, genome editing was only one-half of the equation. "We previously developed a series of human cell models for coronavirus
infection in our work to understand immune responses to the virus. It was
great to team up with Neville's group to understand and comprehensively
profile host genes from a new angle," said co-senior 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 top-ranked genes
-- those whose loss reduces viral infection substantially -- clustered
into a handful of protein complexes, including vacuolar ATPases, Retromer, Commander, Arp2/3, and PI3K. Many of these protein complexes are involved
in trafficking proteins to and from the cell membrane.



==========================================================================
"We were very pleased to see multiple genes within the same family as
top- ranked hits in our genome-wide screen. This gave us a high degree
of confidence that these protein families were crucial to the virus
lifecycle, either for getting into human cells or successful viral replication," said Dr. Zharko Daniloski, a postdoctoral fellow in the
Sanjana Lab and co-first author of the study.

While researchers performed the CRISPR screen using human lung cells,
the team also explored 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 hits broadly expressed across many tissues,
suggesting that these mechanisms may function independent of cell
or tissue type. Using proteomic data, they found that several of the
top-ranked host genes directly interact with the virus's own proteins, highlighting their central role in the viral lifecycle. The team also
analyzed common host genes required for other viral pathogens, such as
Zika or H1N1 pandemic influenza.

Mechanistic insights: Cholesterol and viral receptors After completing
the primary screen, the group of researchers used several different
techniques to validate the role of many of the top-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 using RNA interference,
or drug inhibition. After validating that these manipulations reduced
viral infection, they next sought to understand the mechanisms by which
loss of these genes block coronavirus infection.

Using a recently-developed technology that couples 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 biosynthesis 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, upregulates
cellular cholesterol levels and blocks SARS-CoV-2 infection. Since recent clinical studies have also suggested that patients taking calcium-channel blockers have a reduced COVID-19 case fatality rate, an important future research direction will be to further illuminate the relationship between cholesterol synthesis pathways and SARS-CoV-2," said Dr. Tristan Jordan, a postdoctoral fellow in the tenOever Lab and co-first author of the study.

Building on previous work on mutations in the Spike protein and viral
entry through the ACE2 receptor, the research team also asked whether loss
of some genes might confer resistance to the coronavirus by lowering ACE2 levels. They identified one gene in particular, RAB7A, that has a large
impact on ACE2 trafficking to the cell membrane. Using a combination of
flow cytometry and confocal microscopy, the team showed that RAB7A loss prevents viral entry by sequestering ACE2 receptors inside cells.

"Current treatments for SARS-CoV-2 infection currently go after the
virus itself, but this study offers a better understanding of how host
genes influence viral entry and will enable new avenues for therapeutic discovery and hopefully accelerate recovery for susceptible populations,"
said Dr. Sanjana.


========================================================================== Story Source: Materials provided by New_York_Genome_Center. Note:
Content may be edited for style and length.


========================================================================== Journal Reference:
1. Zharko Daniloski, Tristan X. Jordan, Hans-Hermann Wessels, Daisy A.

Hoagland, Silva Kasela, Mateusz Legut, Silas Maniatis, Eleni
P. Mimitou, Lu Lu, Evan Geller, Oded Danziger, Brad R. Rosenberg,
Hemali Phatnani, Peter Smibert, Tuuli Lappalainen, Benjamin
R. tenOever, Neville E.

Sanjana. Identification of required host factors for
SARS-CoV-2 infection in human cells. Cell, 2020; DOI:
10.1016/j.cell.2020.10.030 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2020/10/201026114216.htm

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