Rice University Boosts Cas13a Sensitivity for HIV RNA Detection
Rice University Researchers Engineer Stable Cas13a for Enhanced RNA Virus Detection
Researchers at Rice University announced an improved version of the Cas13a CRISPR enzyme designed to detect human immunodeficiency virus (HIV) and other RNA viruses in blood and environmental samples with a hundredfold performance improvement, according to a study published in Cell Chemical Biology.
The Tech TL;DR:
- Rice University bioengineers modified a stable Cas13a enzyme to target RNA strands from viruses like HIV, SARS-CoV-2, influenza, Ebola, Zika, respiratory syncytial virus, and hepatitis C.
- By resolving the structural protein movements of Cas13a and altering a single targeted region, the team boosted test sensitivity one hundredfold without losing enzyme stability.
- The engineered protein successfully identified synthetic, noninfectious HIV samples in detection assays, pointing toward potential integration into commercial diagnostics such as RT-qPCR and lateral flow tests.
Cas13a Must Remain Stable Under Harsh Conditions
Scientists have long utilized CRISPR tools for precise DNA alterations, but Cas13a targets ribonucleic acid instead. Constructing a reliable diagnostic assay requires an enzyme capable of operating under punishing physical parameters while maintaining extreme precision. “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 the paper, in a report. “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.”
Standard detection assays require an enzyme to bind with a target RNA strand—such as viral material from HIV or SARS-CoV-2—and generate an observable indicator like a color change or a colored light. While initial Cas13a variants endured high heat and harsh chemical environments, they lacked the low-threshold detection required for clinical utility. “The issue we had was with sensitivity,” said Seye Oladeji, a graduate student at Rice and first author on the study. “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.”
Resolving Protein Conformations for 100-Fold Sensitivity
Because Cas13a is a highly dynamic protein that shifts continuously during binding operations, mapping its exact configuration presented a major hurdle for the research team. Oladeji developed a custom technique to immobilize the CRISPR/Cas13a complex alongside its target RNA strand to capture the structural folding in high resolution. Following this resolution, the team identified two specific protein regions suspected of governing sensitivity performance.
Altering both regions simultaneously produced an over-calibrated enzyme that triggered false-positive signals even in the absence of target RNA. Restricting the modification to just one region achieved the target threshold, increasing sensitivity one hundredfold while preserving thermal and structural 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 noted. “It was exciting and a good reminder to be careful not to overengineer a protein.”
Customizable Guide Molecules Target Various Viral Pathogens
The operational flexibility of CRISPR-based diagnostic architectures stems from customizable guide molecules. By swapping out the paired guide sequence, researchers can redirect the exact same Cas13a enzyme to identify alternative viral pathogens, including influenza, Ebola, Zika, respiratory syncytial virus, and hepatitis C. “In a CRISPR system, the enzyme is paired with a guide that targets a particular sequence of RNA,” Gao explained. “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.”
Funding for the development and structural characterization work was provided by the National Science Foundation under grants CBET-2431018, CBET-2431019, and CBET-2431020, alongside support from the Welch Foundation under award C-2033-20200401, the National Institutes of Health under grant R61AI167037, and the Diana McSherry and Patrick Poe Research Award under identifier AY-23-24.