Scientists have been using an advanced gene-editing tool known as CRISPR to make targeted changes to DNA for quite some time. Recently, a different version of this technology called Cas13a has gained attention for its ability to target RNA, the genetic material found in viruses like HIV. A team from Rice University has made significant strides by creating an enhanced version of Cas13a, which may simplify the detection of HIV and other RNA viruses in blood and environmental samples. Their findings were shared in the journal Cell Chemical Biology.
Yang Gao, an associate professor of biosciences and the lead author on the study, emphasized the importance of enzyme stability and sensitivity in detection activities. He explained, “Any enzyme used in an assay needs to be both sensitive and stable. It must detect very small amounts of RNA while also enduring harsh conditions, such as high temperatures. In our research, we took a stable version of Cas13a and improved its sensitivity.”
In a typical detection process, an enzyme like Cas13a attaches to a target, such as a viral RNA strand, and creates a signal—this could be a color change or light that a person or a machine can read. Many assays often require tough conditions to produce these signals. Although the Rice team began with a version of Cas13a that was robust enough for these harsh environments, it struggled to detect extremely low quantities of target RNA.
Seye Oladeji, a graduate student and the first author of the study, noted, “Our main challenge was increasing sensitivity. Ideally, a detection system should signal the presence of just one RNA strand per microliter of fluid, which is about the amount found in a drop of blood. However, our stable Cas13a variant lacked the required sensitivity, which presented an engineering challenge.”
To create a more sensitive version of stable Cas13a, Oladeji first needed to understand how it functioned. This involved determining the structure of the protein and gathering detailed information on how its components fit together. Since Cas13a is quite dynamic when binding, it was tricky to achieve the clarity needed for structural resolution. Oladeji devised a method to keep the CRISPR/Cas13a complex and its target RNA steady while they collected structural information.
After determining the structure, the researchers identified specific areas of the enzyme that could enhance sensitivity. They modified two areas of the protein and ran various tests. When adjustments were made to both areas, the enzyme became overly sensitive and signaled even in the absence of the target RNA. However, modifying just one region increased sensitivity one hundredfold without sacrificing stability.
Oladeji expressed excitement about the results: “We tested our engineered Cas13a against a synthetic HIV sample, and it showed a remarkable improvement in detection performance, being 100 times better.” He added, “It was a valuable lesson to not overengineer such proteins.”
While the experiments confirmed the ability to detect HIV, the versatile nature of the CRISPR system is a significant advantage. The Cas13a enzyme pairs with a programmable guide that targets specific RNA sequences. This means switching the guide to target different viruses—like SARS-CoV-2, Ebola, influenza, Zika, respiratory syncytial virus, or hepatitis C—is straightforward.
Gao summed it up by saying, “In a CRISPR system, the enzyme is matched with a guide that focuses on a specific RNA sequence. The guide can be easily programmed, which is crucial for any assay. Our version of Cas13a is fine-tuned for both sensitive and stable RNA detection and could eventually be integrated into commercial tests like RT-qPCR and lateral flow assays.”
This exciting research was supported by grants from the National Science Foundation, the Welch Foundation, the National Institutes of Health, and the Diana McSherry and Patrick Poe Research Award.
