About two-thirds of the viruses that can infect humans, including deadly Ebola, Zika, and flu, have a single-stranded RNA genome, and for most of these types of virus there is no FDA-approved therapy. Scientists led by a team at the Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard now report on the development of a CRISPR-Cas13 enzyme-based technology called CARVER (Cas13-assisted restriction of viral expression and readout), that can be programmed both to detect and to destroy RNA-based viruses in human cells. The study is claimed to represent one of the first to harness Cas13, or any CRISPR system, as an antiviral in cultured human cells.
“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,” commented research lead Pardis Sabeti, PhD, institute member at the Broad Institute, a professor at Harvard University, and 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.” Sabeti, together with colleagues including co-first authors Catherine Freije, a graduate student in the Sabeti lab at Harvard University, and Cameron Myhrvold, PhD, a postdoc in the Sabeti lab, reported their findings in Molecular Cell, in a paper titled, “Programmable Inhibition and Detection of RNA Viruses using Cas13.”
CRISPR-Cas technologies have “revolutionized” our ability to precisely edit genes and target gene expression,” the authors stated. In nature, CRISPR systems have evolved as a bacterial defense mechanism against invading bacteriophages and other foreign nucleic acids. “This suggests that CRISPR effectors could be repurposed to aid in defending mammalian cells against both DNA and RNA viruses,” the researchers continued. Scientists have made progress with CRISPR-based technologies that harness Cas9 to inhibit replication of double-stranded DNA viruses, but most of the viruses that infect humans have single-stranded RNA genomes, which can’t be targeted effectively using Cas9-based strategies.
Adding perspective to difficulties in generating effective treatments against human viruses, and in particular ssRNA pathogens, the authors noted that while, over the past 50 years, 90 clinically approved antiviral drugs have been produced, they treat just nine viral diseases, only four of which are ssRNA viruses. “Vaccines have emerged as the predominant approach to combating viral diseases, but only 16 viruses have FDA-approved vaccines.” Developing antiviral approaches is particularly challenging, the team stated, as “human viral pathogens are quite diverse and evolve rapidly,” which highlights both “the challenge of developing and the great need for adaptable antiviral therapeutic platforms.”
Researchers have, more recently, adapted Cas13—which naturally targets viral RNA in bacteria—as a tool for cutting and editing human RNA, and as a diagnostic to detect the presence of viruses, bacteria, or other targets. The enzyme has been well-studied in mammalian cells by researchers, including Broad Institute core member and co-author Feng Zhang, PhD, and can be directed to target specific sequences in RNA. Cas13 is also relatively easy to deliver into cells.
For their newly reported work the investigators combined the antiviral activity of Cas13 with its diagnostic capability—using a technique called specific high-sensitivity enzymatic reporter unlocking, or SHERLOCK—to create the single CARVER platform that could ultimately be developed to both diagnose and treat ssRNA viral infections, including infections caused by new and emerging viruses.
The scientists first screened a suite of more than 350 human-associated viral (HAV) genomes to identify viral RNA sequences that could be effectively targeted by Cas13. “HAVs are defined as viral species that can infect humans or with close relatives that infect humans,” the authors explained. “There are 396 ssRNA viral species annotated as human associated in the NCBI genome neighbors database, at least 20 of which are high-risk human pathogens.” For their search, the team looked primarily for targets that would be both least likely to mutate, and most likely, when cut, to disable the 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 potential sites, in hundreds of viral species, which might represent effective targets for Cas13. Armed with this list of potential viral RNA targets they then engineered the Cas13 enzyme’s guide RNA so that the resulting system could find and cut any of the chosen target nucleic acid sequences.
They next experimentally tested Cas13 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). The three viruses were chosen as “optimal test cases,” at least in part because they demonstrate distinct sequence diversity and replication strategies. “LCMV is a model viral system for studying mammarenaviruses, many of which cause hemorrhagic fever in humans, including Lassa fever virus … Similarly, IAV is a very diverse viral pathogen with high seasonal prevalence and pandemic potential. IAV’s rapid evolution and propensity for recombination are obstacles for the development of both antivirals and vaccines and readily allow for the emergence of drug resistance … Lastly, we highlight Cas13’s utility against an additional ssRNA virus, VSV, which could enable high-throughput studies of crRNA targeting properties because productive VSV infection results in cell death.”
The team first introduced the Cas13 gene and an engineered guide RNA into the cells. Then, 24 hours later, they exposed the cells to one of the three viruses. The results showed that after another 24 hours the Cas13 enzymes had reduced the level of viral RNA in the cell cultures by up to 40-fold. Encouragingly, further experiments indicated that eight hours after viral exposure, Cas13 had reduced infectivity of the flu virus by more than 300-fold.
To add a diagnostic component, the researchers then incorporated the Cas13-based nucleic acid detection technology, SHERLOCK to the system. The resulting combined diagnostic and antiviral CARVER system could rapidly measure remaining levels of viral RNA in a sample. “Unlike other nucleic-acid-based approaches and quite remarkably, Cas13 can be applied in both viral detection and knock down contexts, creating a potential for an end-to-end platform for diagnosis and treatment of infectious diseases,” the authors explained. “… we developed CARVER as a platform for measuring viral RNA levels following Cas13 targeting in real time using the Cas13-based nucleic acid detection technology SHERLOCK.”
The researchers say their studies demonstrate how Cas13 can effectively target multiple distinct mammalian ssRNA viruses. “With CARVER, the CRISPR-Cas effector Cas13 can target multiple mammalian viruses as well as measure the effects of targeting and the viral response. Such technological flexibility is unprecedented for a single protein, and it underscores the power and promise of programmable nucleases, such as Cas13.”
“We envision Cas13 as a research tool to explore many aspects of viral biology in human cells,” stated Freije. “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.”