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Broad Institute Researchers Develop CRISPR-Based Diagnostic, Antiviral Platform


NEW YORK – Broad Institute researchers have developed a CRISPR-based platform that uses the Cas13 nuclease to diagnose and then destroy single-stranded RNA (ssRNA) viruses. The end-to-end diagnostic/antiviral system has potential utility as a research tool for those studying RNA-based viruses, and could also be used in the future to diagnose and treat viruses in the field and the clinic.

Broad researchers in the lab of Pardis Sabeti described the platform — called Cas13-assisted restriction of viral expression and readout (CARVER) — in a study published today in Molecular Cell. CARVER is based on the SHERLOCK (Specific High sensitivity Enzymatic Reporter unLOCKing) platform developed by Broad researchers in Feng Zhang's lab.

SHERLOCK, which was first described in April 2017, uses Cas13a to target, bind, and cleave RNA. In March 2018, the Zhang team described improvements it had made to the technology, including four-channel single-reaction multiplexing using orthogonal CRISPR enzymes, quantitative measurement of input down to 2-attomolar concentration, a 3.5-fold increase in signal sensitivity through a combination of Cas13 with auxiliary CRISPR-associated enzyme Csm6, and a lateral flow readout.

In a study published in Science in April 2018, Sabeti's team demonstrated that SHERLOCK could be used to detect Zika virus in the field. The researchers demonstrated that SHERLOCK could detect Zika (ZIKV) and dengue virus (DENV) in patient samples at concentrations down to 1 copy per μl, and that the platform could distinguish between the four DENV serotypes as well as region-specific strains of ZIKV from the 2015-2016 pandemic.

With CARVER, the researchers are taking the technology one step further, adding a virus destruction component to the diagnostic function. Many common or deadly human pathogens, such as Ebola, Zika, or influenza, are RNA-based viruses.

Most of these diseases have no treatments approved by the US Food and Drug Administration — in fact, according to the Broad, only 16 viruses have FDA-approved vaccines. Further, viral pathogens tend to evolve resistance to treatment.

While Cas9 targets DNA-based bacteriophages, about two-thirds of viruses that can infect humans have ssRNA genomes, and only 2.5 percent of those viruses have DNA intermediates that could be targeted with Cas9, the researchers wrote in their study. They speculated that Cas13 could be an effective antiviral for ssRNA viruses because it programmably cleaves RNAs complementary to its CRISPR RNA (crRNA). For the study, they computationally identified thousands of potential Cas13 crRNA target sites in hundreds of ssRNA viral species that can potentially infect humans. They then demonstrated Cas13's activity against three distinct ssRNA viruses: lymphocytic choriomeningitis virus (LCMV), influenza A virus (IAV), and vesicular stomatitis virus (VSV). The researchers also screened hundreds of crRNAs along the LCMV genome to evaluate how conservation and target RNA nucleotide content influence Cas13's antiviral activity.

"The way we started thinking about it is that Cas13 has this unique activity that's much different than most other CRISPR effector proteins and much different than Cas9. Our group as well as a few other groups have demonstrated that Cas13 can be harnessed for diagnostics because it has this collateral cleavage activity outside of cells that can be harnessed for diagnostics," said co-first author Catherine Freije, a graduate student at Harvard University in the Sabeti lab. "We started thinking about how to pair that diagnostic capability with Cas13's actual original purpose, which in bacteria is … to defend bacteria against bacteriophages." 

Recent studies have shown that certain Cas13 orthologs have minimal off-target effects on the host transcriptome in mammalian cells. The team started by analyzing Cas13 target sites in the genomes of human-associated viruses (HAVs), noting that 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.

They generated an alignment for each genome segment of each of the 396 species and found a set of antiviral crRNAs that could be multiplexed in a combinatorial fashion. For computational screening and experimental validation, the researchers used Cas13a from Leptotrichia wadei (LwaCas13a) and Cas13b from Prevotella sp. P5-125 (PspCas13b), both of which efficiently knock down messenger RNA (mRNA) in mammalian cells.

The researchers found that 94.6 percent of the 396 ssRNA HAVs had 10 or more putative Cas13a target sites and that 86.3 percent had 10 or more putative Cas13b target sites. Further, 86 percent of ssRNA HAVs had 100 or more Cas13a target sites and 76 percent had 100 or more Cas13b target sites.

"One of the great things about Cas13 is that simply by changing the CRISPR RNA sequence you can direct it to go after different viral species," said co-first author Cameron Myhrvold, a postdoc also in the Sabeti lab. "When we do a bioinformatic search across lots of different viral genomes, there are many highly conserved sites that you could potentially target with Cas13. So, I think there's a lot of potential there in the future to go after lots of different single-stranded RNA viruses."

Specifically, the researchers found that LwaCas13a efficiently reduced the levels of LCMV viral RNA, and that LwaCas13a's antiviral activity persisted even at a low multiplicity of infection (MOI). Further, LwaCas13a targeting of LCMV infection did not lead to changes in cell viability.

The researchers also designed a set of crRNAs tiling the entire LCMV genome. The genome-wide screen identified 124 active crRNA target sites, many of which had high-nucleotide sequence conservation. They hypothesized that PspCas13b could have increased knockdown efficiency if directed toward conserved regions of the LCMV genome, and designed 23 crRNAs targeting viral RNA in regions with high- or low-nucleotide-level sequence conservation.

Indeed, they found that when crRNAs targeted highly conserved regions, a higher proportion were active target sites compared to poorly conserved regions, and that crRNAs designed against regions conserved among all strains of LCMV were more likely to be active than those targeting conserved regions specific to the LCMV strain being tested.

Using these analyses to develop the CARVER platform, the researchers paired Cas13-based targeting with a SHERLOCK assay that measured LCMV RNA levels in cell culture supernatant following Cas13 treatment. They then isolated LCMV RNA in the supernatant and used SHERLOCK for RNA detection and targeted LCMV with a selection of crRNAs identified in the LCMV full-genome screen. This led to rapid detection of LCMV levels.

"The speed of the diagnostic was actually one of the things that we're excited about," Freije said. "Most people that are working on SHERLOCK are continually trying to make even faster. In our study, all the things that we have done work within one to two hours. So, it's very, very fast."

Cas13's highly specific targeting ability could also mean that CARVER has potential utility as a treatment response test. Because Cas13 is active even in the presence of a low number of nucleotide mismatches, CARVER could also be used to detect single-nucleotide changes in viral populations that are present after treatment. To achieve this, the researchers developed a SHERLOCK assay that identified the F260L mutation associated with the switch from acute to persistent infection by LCMV — such an assay could provide rapid feedback about the effectiveness of treatment with Cas13 and information about specific viral mutations.

"We first wanted to show that Cas13 could work as an inhibitor of RNA viruses in mammalian cells, and then we thought that we could pair that activity with the diagnostic," Freije said. "The way we like to think about it is that it could be used as an iterative technology in which you use the Cas13-based diagnostic to see which virus is making someone sick, [which] would help inform what type of Cas13-based antiviral you would need, and then you can retest a sample to see how well the antiviral activity is working."

The ability to use Cas13 to detect single-nucleotide mismatches could also be helpful for finding mutations that inhibit the activity of specific crRNAs, or to identify drug resistance mutations, which would signal to a clinician not to use a specific antiviral therapy in a given patient.

The development of CARVER is still in the early stages, so the researchers still have a lot of work to do before they achieve their long-term goal of making the platform usable in the field or the clinic. For example, no one has yet been able to show that Cas13 can be delivered, even in mice.

"There are a few different approaches that are current gold standard approaches for delivery [of CRISPR-based therapeutics]," Myhrvold said. "But I think people are doing a lot of active research to try and make better ones, specifically for delivering all sorts of different CRISPR proteins. So, that's what we're hoping will be happening over the next few years, that that will continue to improve and that we can use some of the same tools that other people are using to try and deliver Cas9 to all different parts of the body, [but] for Cas13."

He noted that Cas9 and Cas13 are a similar size, which would make breakthroughs in Cas9 delivery applicable to Cas13.

Despite the challenges and the nascent stage of the research, however, both Freije and Myhrvold are excited about the possibilities for the platform's use.

"An outbreak scenario is something where [CARVER] could be very useful, because most other drugs take a really long time to develop whereas here, if you had some knowledge of the viral genome sequence, you could design and quickly prototype a set of CRISPR RNAs that might be effective [for treatment]," Myhrvold said. "Actually getting them to work in a person is much more challenging obviously. But if there were a real need for it, you could probably do it faster than with other types of [antiviral] drugs."

The ability to target multiple viruses using Cas13 just by changing the crRNA creates a long list of viruses that might respond to Cas13-based therapeutics, Freije added. "You can imagine once Cas13 has been vetted and has a good delivery strategy [that there would be] multiple scenarios in which this could be helpful," she said, such as applications in the field, in hospitals, or even in doctor's offices.

And because CARVER is based on SHERLOCK, the platform could easily be used with a paper strip-based readout, making it highly useful in situations where patients can't necessarily get to a hospital for testing.

Further, Freije said, CARVER has given researchers the ability to more easily study how viruses respond to Cas13 therapy and targeting, and look at how resistance to crRNAs can develop over time. "We can use Cas13 as a tool for looking at particular viruses and functional components of the viral genome, which we do a little bit by tiling the full LCMV genome in this work," she said. "That's something we're really excited about going forward — Cas13 really gives us a system to look at multiple parts of an RNA virus and get a better understanding of functional importance."