CRISPR For COVID-19: Approaches To Speeding Therapeutic Discovery

By Allison Proffitt

March 3, 2021 | Neville Sanjana from the New York Genome Center and NYU turned his attention to COVID-19 early in the pandemic. For several years, his lab has focused on using CRISPR to identify which regions of the genome drive disease. Last year, he applied that expertise to using genome-wide CRISPR screens to look for COVID-19 risk factors.

While today the outlook is much brighter, there is still a real need for COVID-19 therapeutics, Sanjana told the audience at the virtual AGBT General Meeting this week. His team aimed to use CRISPR screens to understand the genes behind key host dependency and viral biology, translate those genetic findings into actionable results, and identify any common pathways and mechanisms underlying resistance.

In a collaboration with Ben tenOever’s lab at Mount Sinai, Sanjana’s lab started with GeCKOv2—a second generation CRISPR knock out library—and packaged it into a lentivirus and transduced human lung cells. Those cells were challenged with either SARS-CoV-2 or a control and looked for surviving guide RNAs.

“Coming out of the screen we get this ranked list of all the 20,000 genes… and we tried to place them within the life cycle of the virus,” Sanjana explained. ACE2 ranked highly—number 8 of all the genes—which was expected, but less expected were the pathways and complexes that emerged. “Initially we didn’t know too much about these genes,” Sanjana said, “but it was very reassuring to see multiple genes that worked together in pathways… that we would get either multiple members of the family or all members of the protein complex family.”

Most of the top-ranked genes were widely expressed across human tissues and interact directly with viral proteins. “This gave us further assurance that we were on to something here.” The team chose 30 interesting and highly ranked genes to dig into deeper with new CRISPRs.

“Of course, finding these gene hits and validating them is very important. It teaches us something about the required host genes and the virus hosts and interactions,” Sanjana said. But the real goal was to translate those genetic findings into therapeutics.

The team identified compounds that were already known to inhibit the genes on their short list. Remdesivir stood out, but several other known therapeutics also had significant function and Sanajana proposed that they could be included in a therapeutic cocktail similar to how HIV is treated.

For some genes—with no direct inhibitor and for which the mechanism isn’t well-known—the team used ECCITE-seq, a technique NYGC published in 2019 to identify shared mechanisms (DOI: 10.1038/s41592-019-0392-0). By looking at pathways, they saw that the top up-regulated pathway was cholesterol biosynthesis. Further CRISPR screen revealed that six genes believed to be unrelated all drove an increase in cellular cholesterol.

“Now we thought: forget about these genes in particular. Let’s target the phenotype!”

The team chose to test amlodipine, an older, FDA-approved calcium channel blocker that is used to lower blood cholesterol while increasing cellular cholesterol. They found that the drug prevented viral infection of cells. (The work was published in Cell last month. DOI: 10.1016/j.cell.2020.10.030)

The work revealed two complimentary approaches, Sanjana said. “One, we can take the top-ranked genes from these CRISPR screens and directly inhibit them. The second thing is, when we don’t have good guesses about a mechanism or we don’t have a particular inhibitor, we can use multi-dimensional phenotyping like ECCITE-seq and look for common mechanisms in cell-state alteration that can generate new hypotheses.”

The two approaches don’t need to be limited to small molecule inhibitors, Sanjana emphasized. They can also be used for gene therapies.

RNA-Targeting CRISPRs

Sanajana’s group is not only using Cas9s to edit DNA; they are also working with Cas13 to edit RNA. The lab has published on the work last March in Nature Biotechnology (DOI: 10.1038/s41587-020-0456-9), but Sanajana outlined several additional next steps in this area.

Cas13 can be used to profile non-coding RNAs and their function, identifying novel noncoding RNA therapeutic targets. They could be used to help develop chemically-modified guide RNAs for targeting difficult to target cells like immune cells. RNA-targeting CRISPRs could be used for in vivo delivery and transcript targeting, and Sanajana hopes to use Cas13 to understand transcript localization within the cell, to capture the micro-environment of proteins and perturb the localization of the transcript.

Sanajana’s lab has shared web-based and command line Cas13 design tools online for others looking to guide RNA scoring.