(236a) A Combinatorial Approach Towards the Adaptability of 22 Functional Cas12a Orthologs for Nucleic Acid Detection and Gene Editing

CRISPR/Cas systems have been widely used for gene editing and, more recently, for nucleic acid diagnostics. There is a broad diversity among Cas12a nucleases with immense gene editing and detection capability, but relatively 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, toleration of non-canonical crRNAs, and gene editing activity in human cells. We observed that some non-canonical crRNA:Cas12a effector complexes outperform the corresponding wild-type crRNA:Cas12a complex in terms of nucleic acid detection. 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. Moreover, Cas12a was observed to have the ability to recruit segments of truncated crRNAs providing insights into the crRNA:Cas12a catalytic complex with potential for further applications. Lastly, we identified non-canonical crRNA:Cas12a effector pairings that had significantly improved gene editing efficiency compared to the wild-type counterparts. Our findings further expand the toolbox for next-generation CRISPR-based diagnostics and genome editing.