Engineering of LwaCas13a Protein with Enhanced Collateral Activity for Ultrasensitive Nucleic Acid Detection

CRISPR-Cas13 has been rapidly developed for nucleic acid-based diagnostics by utilizing its characteristic collateral activity, easy programmability, and equipment-free nature. Despite recent progress in CRISPR based diagnostics, engineering the Cas13 enzyme is challenging due to its complex structural dynamics, but promising for point-of-care detection from raw patient samples. Here, we successfully employed a novel strategy to engineer the Leptotrichia wadei (Lwa)Cas13a by inserting different RNA binding domains (RBDs) to increase enzyme-substrate binding affinity. Two LwaCas13a variants achieved up to 58-fold enhanced collateral activity over the wild-type (WT) in a fluorescence-based assay. When using an electrochemical detection method, our variants achieved 50,000-fold sensitivity over the WT, showing a clear detection of ~1 attomolar (0.6 copy/μL) of the SARS-CoV-2 genome within 30 minutes. We are currently integrating our engineered LwaCas13a enzymes into a new diagnostic test for HIV with lateral flow strips. Most importantly, our engineered LwaCas13a enzymes can be further integrated into other fields including biological assessments and environmental surveillance.