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

Nguyen, L. T.; Macaluso, N. C.; Jain, P. K.

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.