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 then tested the results in the lab. When both regions were altered, the enzyme was a little too sensitive and would signal even if the RNA target wasn’t present. But when just one region was altered, sensitivity went up 100 fold without any loss of stability.
“We tested the sensitive — but not too sensitive — engineered Cas13a against a synthetic, noninfectious HIV sample and found that it worked in our detection assay with a 100-fold performance improvement,” Oladeji said. “It was exciting and a good reminder to be careful not to overengineer a protein.”
While the team’s test detected HIV, the benefit of using a CRISPR system is its flexibility. The Cas13a enzyme is paired with a guide that is programmed to target a specific sequence of RNA. It’s easy to change the programmed guide — the team could, for example, use the same Cas13a enzyme and reprogram its guide to detect SARS-CoV-2, influenza virus, Ebola virus, Zika virus, respiratory syncytial virus or hepatitis C virus instead of HIV.
“In a CRISPR system, the enzyme is paired with a guide that targets a particular sequence of RNA,” Gao said. “The guide is easy to program, so the critical part of the system is having an enzyme that’s compatible with your assays. Our version of Cas13a is optimized for sensitive and stable RNA detection assays and could potentially be used in commercial assays like RT-qPCR and lateral flow.”
This work was funded by the National Science Foundation (CBET-2431018, CBET-2431019, CBET-2431020), Welch Foundation (C-2033-20200401), the National Institutes of Health (R61AI167037) and the Diana McSherry and Patrick Poe Research Award (AY-23-24).
Cell Chemical Biology
10.1016/j.chembiol.2026.09.012
Experimental study
Not applicable
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