A Combinatorial Approach towards Adaptability of 22 Functional Cas12a Orthologs for Nucleic Acid Detection in Clinical Samples

Reliable, efficient, and cheap detection of infectious diseases, especially in the wake of the SARS-CoV-2 pandemic, is of increasing importance. CRISPR/Cas systems have the capabilities to be optimized for this purpose. There is a broad diversity among Cas12a nucleases with immense detection capability, but only a few have been purified and studied biochemically. Here we present the investigation of 22 Cas12a orthologs, with a focus on their cis- and trans-cleavage activity, and thermal stability in combination with non-canonical crRNAs. We noticed that some non-canonical crRNA:Cas12a effector complexes outperformed its corresponding wild-type crRNA:Cas12a complex in trans-cleavage assays. In particular, TsCas12a, ErCas12a, and ArCas12a showed an increase in thermal stability in binary complex compared to its wild-type structure and apo form. Moreover, Cas12a was observed to have the ability to recruit segments of truncated crRNAs providing insights into crRNA:Cas12a catalytic complex with potential for further applications. We also discovered that ErCas12a, BsCas12a, BoCas12a, and TsCas12a possess robust trans-cleavage activity with a shorter PAM sequence requirement. Finally, we applied these effector complexes to discriminately detect SARS-CoV-2 and its B.1.1.7 lineage in clinical nasopharyngeal swabs, saliva samples, and tracheal aspirates. Our findings further expand the toolbox for next-generation CRISPR-based diagnostics.