This article has been reviewed according to Science X's editorial process and policies. Editors have highlighted the following attributes while ensuring the content's credibility: Scientists have long used a powerful gene-editing tool called CRISPR to make precise changes to DNA. But a different version of the technology, known as Cas13a, targets RNA, the genetic material found in viruses such as HIV.
Now, researchers at Rice University have developed an improved version of Cas13a that could make it easier to detect HIV and other RNA viruses in blood and environmental samples. The study was published in Cell Chemical Biology. "Any enzyme used in an assay needs to be both sensitive and stable," said Yang Gao, an associate professor of biosciences and the corresponding author on this paper.
"It needs to be able to detect very small amounts of RNA while enduring harsh conditions, like high temperatures. Here, we took a stable Cas13a and engineered it to have the desired sensitivity." In a detection assay, an enzyme like Cas13a will bind to its target—say, a strand of RNA from a virus like HIV or SARS-CoV-2—and produce a signal, like a colored light or color change, that can be read by a human or machine. In most assays, the path to producing a signal requires harsh conditions.
While the team started with a version of Cas13a that could withstand those harsh conditions while still performing its job, it wasn't able to detect very small amounts of the target RNA. "The issue we had was with sensitivity," said Seye Oladeji, a graduate student at Rice and the first author on this paper. "Ideally, a detection assay will tell you if there's as little as one strand of RNA per microliter of liquid—around a drop of blood.
Our stable Cas13a variant was just not sensitive enough: a perfect problem for an engineering approach." To engineer this stable Cas13a into a more sensitive version, Oladeji first had to understand how it worked. This required resolving the structure of the protein, or collecting enough information to accurately draw out the enzyme's building blocks and how they fold together. Cas13a is a protein that moves a lot as it binds, making it hard to capture it in the crisp detail needed for resolution.
Oladeji designed a way to hold the CRISPR/Cas13a and its target RNA in place while collecting information on its structure. Once Cas13a's structure was resolved, the team was able to identify regions of the enzyme they believed would increase sensitivity. They selected two regions to engineer and then tested the results in the lab.
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