Broad takes RNA CRISPR into viral infections

Cas13 continues to prove its worth as the latest Broad CRISPR study points to antiviral applications

The latest CRISPR study from the Broad Institute takes gene editing into infections caused by RNA viruses, using Cas13 -- which is proving to be one of the most versatile of the CRISPR-Cas enzyme family.

In a paper published in Molecular Cell on Thursday, a Broad Institute of MIT and Harvard team led by Pardis Sabeti showed that CRISPR-Cas13 can kill RNA viruses in infected cells.

Sabeti is an institute member at the Broad and a Harvard University professor. The study also included the Broad’s Feng Zhang, one of the pioneers of CRISPR.

The standard-bearer of the CRISPR family is Cas9, on which the foundational IP is based. Cas9 cuts DNA and makes permanent changes to a cell’s genome. Behind it are at least three other families of Cas enzymes, which vary in their potential target sites, structures and cutting properties, and which offer more freedom to operate for drug developers.

Cas13 is a family that naturally targets RNA rather than DNA, meaning the changes operate at a transcriptional level and don’t tinker with cell’s genome. It also means they have the potential to eliminate viruses with RNA genomes, which represent about two thirds of viruses that infect humans, ranging from the common cold to Ebola and HCV.

For proof of concept, the Broad group used lymphocytic choriomeningitis virus (LCMV), influenza A virus and vesicular stomatitis virus (VSV). The group infected human or canine cell lines with the viruses, and delivered Cas13 plus a CRISPR RNA (crRNA) to guide the enzyme to the right site.

CRISPR-Cas13 decreased viral RNA levels in a human cell line from 2- to 14-fold after 48 hours for LCMV, 7- to 22-fold after 24 hours for influenza A, and 7.8- to 43.3-fold after 48 hours for VSV (p-values not disclosed).

Multiplexing -- or targeting multiple sites at once -- decreased viral replication of LCMV more than targeting individual sites. One of the advantages of Cas13 is that it releases individual crRNAs after they are processed, allowing multiplexed targeting with a single enzyme.

Another benefit is that when Cas13 encounters its target, it can become activated to indiscriminately cleave surrounding single-stranded RNA.

That has a greater impact on diagnostic then therapeutic applications because very little, if any, collateral cleavage occurs in mammalian cells, according to study authors Catherine Freije and Cameron Myhrvold.

However, in diagnostic uses, the collateral cleavage can be harnessed to activate a collateral cleavage-triggered RNA reporter.

Taking advantage of that property, the team combined Cas13’s diagnostic and therapeutic applications into a single end-to-end system for viral detection, treatment and monitoring, dubbed Cas13-Assisted Restriction of Viral Expression and Readout (CARVER).

CARVER incorporates the SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) Cas13-based diagnostic system, which was created at the Broad and is being developed by Sherlock Biosciences Inc.

Although the study suggests CRISPR-Cas13 can reduce viral RNA in vitro, delivering enough of the gene editing therapy into every infected cell in vivo will be a challenge.

Before tackling that, the team plans to continue evaluating how different RNA viruses respond to Cas13.

Freije and Myhrvold said they are considering acute and chronic viral infections. Although it’s been difficult to develop antivirals that act quickly enough to change the course of acute infections, the researchers think Cas13 could work because they saw a greater than 300-fold decrease in infectivity of influenza in eight hours.

Unlike most other antiviral strategies for acute and chronic infections, Cas13-based antivirals are readily programmable, allowing rapid design and testing of modified therapies when resistance evolves, they said.

Last year, Vir Biotechnology Inc. acquired Agenovir Corp., which was developing CRISPR-Cas9 based antivirals targeting viral DNA. Vir declined to disclose whether it is pursuing the programs.

Mammoth Biosciences, a University of California Berkeley spinout developing CRISPR-based diagnostics, is using Cas13 for diagnostics applications. Base editing company Beam Therapeutics Inc. is using the enzyme to make therapeutic RNA edits.

While the paper’s authors include co-founders of Sherlock and Beam, the researchers have not disclosed whether any company has licensed the IP covering the technology for treating viral infections.

Freije is a Ph.D. candidate in Sabeti’s lab, and Myhrvold is a postdoctoral fellow.

Targets

Cas9 - CRISPR-associated protein 9

Cas13 - CRISPR-associated protein 13

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