Genomic Researchers Modify CRISPR Technology To Fight Viruses in Human

Cas13, a common gene-editing enzyme could be used to disable RNA viruses such as flu, Zika  or Ebola. CRISPR is usually thought of as a laboratory tool to edit DNA in order to fix genetic defects or enhance certain traits but in reality, the mechanism originally evolved in bacteria as a way to fend off viruses called bacteriophages.



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 treat a viral infection, including infections caused by new and emerging viruses. Their system, called CARVER (Cas13-Assisted Restriction of Viral Expression and Readout), is published in the  Molecular Cell Journal.
 
The work was co-led by senior author Pardis Sabeti, institute member at the Broad Institute and professor at Harvard University, and co-first authors Catherine Freije, a graduate student at Harvard University in the Sabeti lab, and Cameron Myhrvold, a postdoc also in the Sabeti lab.
 
In a genomic study, the team 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 reengineered 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 st udies 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.
 
Dr Freije, who is also  doctoral student in virology at Harvard commented in an interview with Thailand Medical News, “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. 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.”
 
Dr 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.
 
To assess how effective Cas13 was at destroying the viruses, the researchers also used it as a diagnostic tool to see how much viral RNA was being released from infected cells. They saw a twofold to 44-fold reduction in RNA, depending on which virus they were looking at and the time point. They also looked at how well the released RNA was able to go on and infect new cells. In most cases, they saw a 100-fold reduction in infectivity and in some cases, more than 300-fold, according to Freije. 
 
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.
 
"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," says 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.
 
As with any approach, there are limitations. One is the question of how to deliver the Cas13 to target a virus in a living person, The researchers have not yet done any animal studies. Another is the fact that viruses will eventually develop resistance. But Cas13 has an advantage here: when Cas9 cuts viral DNA, mammalian cells repair it and can cause mutations that make the virus more resistant. Yet with Cas13, these cells do not have the mechanism to repair the RNA and introduce errors that would help the virus escape being destroyed. Even if a virus does evolve resistance, or if a new virus is encountered, the method could be quickly adapted. 
 
Dr Freije agrees. “We are definitely excited about future prospects of optimizing the system and trying it out in mouse models,” she says. Beyond therapeutics, the team hopes to understand more about how viruses operate how they replicate and what parts of their genomes are most important. Using approaches like this, “you can really start to get a better picture of what parts of these viruses are and, most importantly, what really makes them tick.”
 
The team further explored Cas13's effect on virus infectivity, in other words, how much of the remaining virus could actually continue to infect human cells. The data indicated that eight hours after viral exposure, Cas13 had reduced the infectivity of the flu virus by more than 300-fold.

To add a diagnostic component, the researchers also incorporated the Cas13-based nucleic acid detection technology SHERLOCK. The resulting CARVER system could rapidly measure remaining levels of viral RNA in a sample.
 
Dr  Freije further commented, "We envision Cas13 as a research tool to explore many aspects of viral biology in human cells. It could also potentially be a clinical tool, where these systems could be used to diagnose a sample, treat a viral infection, and measure the effectiveness of the treatment  all with the ability to adapt CARVER quickly to deal with new or drug-resistant viruses as they emerge."

The team is still pursuing further research using the newly developed CARVER platform and wil be updating the medical community of new developments.
 
Reference: Catherine A. Freije, Cameron Myhrvold, Chloe K. Boehm, Aaron E. Lin, Nicole L. Welch, Amber Carter, Hayden C. Metsky, Cynthia Y. Luo, Omar O. Abudayyeh, Jonathan S. Gootenberg, Nathan L. Yozwiak, Feng Zhang, Pardis C. Sabeti. Programmable Inhibition and Detection of RNA Viruses Using Cas13. Molecular Cell, 2019; DOI: 10.1016/j.molcel.2019.09.013