New RNA-Targeting CRISPR Technology Useful for Coronavirus

CRISPR-based genetic screens have helped scientists identify genes that are key players in sickle-cell anemia, cancer immunotherapy, lung cancer metastasis, and many other diseases. However, these genetic screens are limited in scope: They can only edit or target DNA. For many regions of the human genome, targeting DNA may not be effective, and RNA viruses like coronavirus or flu cannot be targeted at all with existing DNA-targeting CRISPR screens. But in a study published today in Nature Biotechnology, researchers have developed a new kind of CRISPR screen technology to target RNA.

Using Cas13, a CRISPR enzyme that targets RNA instead of DNA, the researchers engineered an optimized platform for massively parallel genetic screens at the RNA level in human cells. Additionally, the researchers developed a machine learning–based predictive model to expedite identification of the most effective Cas13 guide RNAs. The new technology is available to researchers through an interactive website.

The researchers developed a suite of new Cas13-based tools and conducted a transcript tiling and permutation screen in mammalian cells. In total, the researchers gathered information for more than 24,000 RNA-targeting guides. “We tiled guide RNAs across many different transcripts, including several human genes where we could easily measure transcript knock-down via antibody staining and flow cytometry,” says first author Hans-Hermann Wessels. “Along the way, we uncovered some interesting biological insights that may expand the application of RNA-targeting Cas13 enzymes.”

Among the team’s findings, for example, are insights about which regions of the guide RNA are more important for recognition of a target RNA. Using thousands of guide RNAs with one, two, or three single-letter mismatches to their target RNA, the researchers identified a critical “seed” region that is exquisitely sensitive to mismatches between the CRISPR guide and the target. This discovery will aid scientists in designing guide RNAs to avoid off-target activity on unintended target RNAs.

The team recently leveraged their guide RNA predictive model for a particularly critical analysis: The COVID-19 public health emergency is due to a coronavirus, which contains an RNA genome. Using the model derived from their massively parallel screens, the researchers have identified optimal guide RNAs that could be used for future detection and therapeutic applications. Predictions for Cas13 guide RNAs for a strain of SARS-CoV-2 isolated in New York have been made available online.