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COVID-19 crisis brings CRISPR-Cas13 research into the spotlight

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It makes sense that CRISPR-Cas13 would enter the limelight during the COVID-19 crisis, given that the SARS-CoV-2 is an RNA virus, and this Cas enzyme targets RNA.

With diagnostics one of the most critical components of this outbreak, the CRISPR-based diagnostic system could offer a new dimension to the suite of tools that are too-slowly coming into play, at least in the U.S.

In the short term, CRISPR-based diagnostics can offer shorter run times and less complexity than the existing PCR tests. In the longer term, the technology could be used for point-of-care testing (see “CRISPR-Based Diagnostics are Poised to Make an Early Debut”).

The most recent activity includes a March 16 study in Nature Biotechnology from a team at the New York Genome Center (NYGC) and New York University that describes a machine-learning algorithm for identifying the most effective Cas13d guide RNA (gRNA) for a particular target, which could help CRISPR diagnostic developers quickly adapt assays to new sequences.

A separate Stanford University study published on the preprint server bioRxiv on March 14 described CRISPR-Cas13d as a potential therapeutic tool to fight the infection.

Most of the research into CRISPR gene editing has revolved around the Cas9 enzyme, which naturally targets DNA sequences. However, several more recently identified enzymes -- including Cas13 -- bind and cut RNA rather than DNA sites.

That’s an important distinction when you’re trying to target certain viruses like COVID-19, which have RNA rather than DNA as genetic material.

The gRNA is the nucleic acid segment used to direct the CRISPR machinery to its nucleic acid target. Selecting the right guide RNA is key because there are countless short sequences within each gene or transcript that could be used as the target, and they aren’t all created equal. How efficiently CRISPR knocks down, detects or edits a gene depends on the guide strand’s exact sequence and length.

In the first study, the group measured how efficiently 24,460 different RNA guides knocked down select targets in the human genome, and developed a set of rules for designing highly selective guide RNAs for a given target. The predictive algorithm that they developed, which is available to researchers on an interactive website, was validated by testing 3,979 gRNAs against 48 different human targets.

Neville Sanjana, principal investigator on the new study, applied the predictive algorithm to SARS-CoV-2 targets, and described the method on the interactive website. Sanjana is an NYGC core member and assistant investigator, as well as an assistant professor at NYU.

During the study, the team also found that a certain region of the gRNA, dubbed the seed region, is highly sensitive to sequence mismatches. That means the guide won’t bind a target site if there is even a minor mismatch between that part of its sequence and the complementary target site.

That’s an important feature that can be exploited for CRISPR-based diagnostic applications because it allows the system to discriminate between very similar sequences.

Rahul Dhanda, co-founder, president and CEO of CRISPR diagnostics developer Sherlock Biosciences Inc, previously told BioCentury the turnaround time to design an assay against a new viral sequence is about 24 hours, and it may be necessary to adapt a test to detect new sequences as the outbreak evolves. A method to predict the most effective gRNAs could help in that process.

The first CRISPR-based therapeutics are probably still years from the market, but the Stanford study is one of the earliest indications that the gene editing tool might also be useful for treating COVID-19.

In the preprint, the team showed that its antiviral Cas13d system dubbed PAC-MAN (Prophylactic Antiviral CRISPR in huMAN cells) targeted and degraded SARS-CoV-2 fragments in a human lung epithelial cell line.

The PAC-MAN system also inhibited viral replication of influenza A virus in the cell line. The team could not test the effects on replication of SARS-Cov-2 because it did not have access to the virus.

In the paper, the authors wrote that the technology could be used to develop a prophylactic or therapeutic agent against the coronavirus, but delivery challenges will need to be sorted out first.

Further analysis of the coronavirus crisis can be found at https://www.biocentury.com/coronavirus.

Targets

Cas13d - CRISPR-associated protein 13d

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