Virus World

CRISPR is usually thought of as a laboratory tool to edit DNA in order to fix genetic defects or enhance certain traits—but the mechanism originally evolved in bacteria as a way to fend off viruses called bacteriophages. Now, scientists have found a way to adapt this ability to fight viruses in human cells. In a recent study, Catherine Freije and Cameron Myhrvold of the Broad Institute of MIT and Harvard and their colleagues programmed a CRISPR-related enzyme to target three different single-stranded RNA viruses in human embryonic kidney cells (as well as human lung cancer cells and dog kidney cells) grown in vitro, and chop them up, rendering them largely unable to infect additional cells. If further experiments show this process works in living animals, it could eventually lead to new antiviral therapies for diseases such as Ebola or Zika in humans. 

Viruses come in many forms, including DNA and RNA, double-stranded and single-stranded. About two thirds of the ones that infect humans are RNA viruses, and many have no approved treatment. Existing therapies often use a small molecule that interferes with viral replication—but this approach does not work for newly emerging viruses or ones that are evolving rapidly. “CRISPR” refers to a series of DNA sequences in bacterial genomes that were left behind from previous bacteriophage infections. When the bacteria encounter these pathogens again, enzymes called CRISPR-associated (Cas) proteins recognize and bind to these sequences in the virus and destroy them. In recent years, researchers (including study co-author Feng Zhang) have re-engineered one such enzyme, called Cas9, to cut and paste DNA in human cells. The enzyme binds to a short genetic tag called a guide RNA, which directs the enzyme to a particular part of the genome to make cuts. Previous studies have used Cas9 to prevent replication of double-stranded DNA viruses, or of single-stranded RNA viruses that produce DNA in an intermediate step during replication. Only a small fraction of RNA viruses that infect humans produce such DNA intermediates—but another CRISPR enzyme, called Cas13, can be programmed to cleave single-stranded RNA viruses.

“The nice thing about CRISPR systems and systems like Cas13 is that their initial purpose in bacteria was to defend against viral infection of bacteria, and so we sort of wanted to bring Cas13 back to its original function—and apply this to mammalian viruses in mammalian cells,” says Freije, who is a doctoral student in virology at Harvard University. “Because CRISPR systems rely on guide RNAs to specifically guide the CRISPR protein to a target, we saw this as a great opportunity to use it as a programmable antiviral.”  Freije and her colleagues programmed Cas13 to target three different viruses: lymphocytic choriomeningitis virus (LCMV); influenza A virus (IAV); and vesicular stomatitis virus (VSV). LCMV is an RNA virus that mostly infects mice—but it is in the same family as the virus that causes Lassa fever, which is found in West Africa and is much more dangerous to study in the lab. IAV is a flu virus; although some antiviral medications for flu already exist, such viruses evolve rapidly, so there is a need for better options. Finally, VSV is a model for many other single-stranded RNA viruses.

Published in Molecular Cell, October 10, 2019:

https://doi.org/10.1016/j.molcel.2019.09.013:

Researchers have turned a CRISPR enzyme into an antiviral that can be programmed to detect and destroy RNA-based viruses in human cells. Many of the world’s most common or most deadly human pathogens are RNA-based viruses — Ebola, Zika, and flu, for example — and most have no FDA-approved treatments. A team led by researchers at the Broad Institute of MIT and Harvard has now turned a  CRISPR RNA-cutting enzyme into an antiviral agent that can be programmed to detect and destroy RNA-based viruses in human cells.

Researchers have previously adapted the  Cas13 enzyme as a tool to cut and edit human RNA and as a diagnostic to detect the presence of viruses, bacteria, or other targets. This study is one of the first to harness Cas13, or any CRISPR system, as an antiviral in cultured human cells. The researchers combined Cas13’s antiviral activity with its diagnostic capability to create a single system that may one day be used to both diagnose and a viral infections, including infections caused by new and emerging viruses. Their system, called CARVER (Cas13-Assisted Restriction of Viral Expression and Readout), is described today in Molecular Cell.

The work was co-led by senior authorPardis Sabeti, institute member at the Broad Institute and professor at Harvard University, and co-first authors Catherine Freije, a graduate student at Harvard University, and Cameron Myhrvold, a Graduate School of Arts and Sciences postdoc. Freije and Myhrvold both work in the Sabeti lab. “Human viral pathogens are extremely diverse and constantly adapting to their environment, even within a single species of virus, which underscores both the challenge and need for flexible antiviral platforms,” said Sabeti, who is also a Howard Hughes Medical Institute investigator. “Our work establishes CARVER as a powerful and rapidly programmable diagnostic and antiviral technology for a wide variety of these viruses.”

The need for new antiviral approaches is urgent. In the past 50 years, 90 clinically approved antiviral drugs have been produced, but they treat only nine diseases — and viral pathogens can rapidly evolve resistance to treatment. Only 16 viruses have FDA-approved vaccines. To explore new antiviral strategies, the team focused on Cas13, which naturally targets viral RNA in bacteria. The enzyme can be programmed to target specific sequences of RNA with few limitations, is relatively easy to get into cells, and has been well-studied in mammalian cells by researchers including Broad Institute core member FEng  Zhang. The team first screened a suite of RNA-based viruses in search of viral RNA sequences that Cas13 could efficiently target. They primarily looked for pieces that are both least likely to mutate and most likely, when cut, to disable a virus. “In theory, you could program Cas13 to attack virtually any part of a virus,” explained Myhrvold. “But there’s huge diversity within and among species, and much of the genome changes rapidly as a virus evolves. If you’re not careful, you could be going after a target that will ultimately have no effect.” The researchers computationally identified thousands of sites, in hundreds of viral species, which could be effective targets for Cas13.

With a list of potential viral RNA targets in hand, the team could then program Cas13 to seek out and cut any of these nucleic acid sequences by engineering the enzyme’s guide RNA. The researchers experimentally tested Cas13’s activity in human cells infected with one of three distinct RNA-based viruses: lymphocytic choriomeningitis virus (LCMV), influenza A virus (IAV), and vesicular stomatitis virus (VSV). They introduced the Cas13 gene and an engineered guide RNA into the cells, and 24 hours later, exposed the cells to the virus. After another 24 hours, the Cas13 enzymes had reduced the level of viral RNA in the cell cultures by up to 40-fold....

Findings published in Molecular Cell on October 10, 2019 (Open Access):

https://doi.org/10.1016/j.molcel.2019.09.013