Harnessing noncanonical 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 noncanonical crRNAs. Through biochemical assays, we observe that some noncanonical 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 noncanonical crRNA is also observed in mammalian cells through the formation of indels. We apply the adaptability of Cas12a:crRNA complexes to detect SARS-CoV-2 in clinical nasopharyngeal swabs, saliva samples, and tracheal aspirates. Our findings further expand the toolbox for next-generation CRISPR-based diagnostics and gene editing. [Display omitted] • Engineered crRNAs with variable stem-loop designs demonstrate adaptability with Cas12a • cCRISPR broadens the gene editing toolbox by screening 207 combinations of Cas12a and crRNAs • cCRISPR detects SARS-CoV-2 in saliva, nasopharyngeal swabs, and tracheal aspirates Nguyen et al. explore the diversity and adaptability of 23 Cas12a orthologs in combination with their canonical and 8 noncanonical crRNAs. A total of 207 combinations of Cas12a and crRNAs were applied for clinical nucleic acid detection and gene editing in cells. The data expand the toolbox for next-generation CRISPR-based diagnostics and genome engineering. [ABSTRACT FROM AUTHOR]