Harnessing Non-Canonical crRNAs to Improve Functionality of Cas12a Orthologs

There is a broad diversity among Cas12a endonucleases that possess nucleic acid detection and gene editing capabilities, but few are studied extensively. Here we present an exhaustive investigation of 23 Cas12a orthologs, with a focus on their cis- and trans-cleavage activities in combination with non-canonical crRNAs. Through biochemical assays, we observe that some non-canonical crRNA:Cas12a effector complexes outperform their corresponding wild-type crRNA:Cas12a. Cas12a can recruit crRNA with modifications such as loop extensions and split scaffolds. Moreover, the tolerance of Cas12a to non-canonical crRNA is also observed in mammalian cells through formation of indels. We apply the adaptability of Cas12a:crRNA 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 and gene editing.