Harnessing noncanonical crRNAs to improve functionality of Cas12a orthologs
Long T. Nguyen,
Nicolas C. Macaluso,
Noah R. Rakestraw,
Dylan R. Carman,
Brianna L.M. Pizzano,
Raymond C. Hautamaki,
Santosh R. Rananaware,
Isabel E. Roberts,
Piyush K. Jain
Affiliations
Long T. Nguyen
Department of Chemical Engineering, University of Florida, Gainesville, FL, USA
Nicolas C. Macaluso
Department of Chemical and Biomolecular Engineering, Whiting School of Engineering, Johns Hopkins University, Baltimore, MD, USA
Noah R. Rakestraw
Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL, USA
Dylan R. Carman
Department of Chemical Engineering, University of Florida, Gainesville, FL, USA
Brianna L.M. Pizzano
Department of Chemical Engineering, University of Florida, Gainesville, FL, USA
Raymond C. Hautamaki
Department of Microbiology and Cell Science, College of Agricultural and Life Sciences, University of Florida, Gainesville, FL, USA
Santosh R. Rananaware
Department of Chemical Engineering, University of Florida, Gainesville, FL, USA
Isabel E. Roberts
Department of Chemical Engineering, John and Marcia Price College of Engineering, University of Utah, Salt Lake City, UT, USA
Piyush K. Jain
Department of Chemical Engineering, University of Florida, Gainesville, FL, USA; Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL, USA; Health Cancer Center, University of Florida, Gainesville, FL, USA; Corresponding author
Summary: 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.
