Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 13 (Cas13) has been rapidly developed for nucleic-acid-based diagnostics by using its characteristic collateral activity. Despite the recent progress in optimizing the Cas13 system for the detection of nucleic acids, engineering Cas13 protein with enhanced collateral activity has been challenging, mostly because of its complex structural dynamics. Here we successfully employed a novel strategy to engineer the Leptotrichia wadei (Lwa)Cas13a by inserting different RNA-binding domains into a unique active-site-proximal loop within its higher eukaryotes and prokaryotes nucleotide-binding domain. Two LwaCasl3a variants showed enhanced collateral activity and improved sensitivity over the wild type in various buffer conditions. By combining with an electrochemical method, our variants detected the SARS-CoV-2 genome at attomolar concentrations from both inactive viral and unextracted clinical samples, without target preamplification. Our engineered LwaCasl3a enzymes with enhanced collateral activity are ready to be integrated into other Cas13a-based platforms for ultrasensitive detection of nucleic acids.

Automated summary

In this study, we successfully engineered the Leptotrichia wadei (Lwa) Cas13a protein by inserting different RNA-binding domains, resulting in enhanced collateral activity and improved sensitivity. By combining with an electrochemical method, our engineered variants showed the capability to detect the SARS-CoV-2 genome at extremely low concentrations without target preamplification. This study provides a highly sensitive approach for nucleic acid detection.