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Harnessing Non-Canonical crRNAs to Improve Functionality of Cas12a Orthologs

48 Pages Posted: 24 Jul 2023 Publication Status: Published

See all articles by Long T. Nguyen

Long T. Nguyen

University of Florida - Department of Chemical Engineering

Nicolas C. Macaluso

Johns Hopkins University - Department of Chemical and Biomolecular Engineering

Noah R. Rakestraw

University of Florida - Department of Molecular Genetics and Microbiology; University of Florida - Department of Graduate Education

Dylan R. Carman

University of Florida - Department of Chemical Engineering

Brianna L.M. Pizzano

University of Florida - Department of Chemical Engineering; University of Florida - Department of Agricultural and Biological Engineering

Raymond C. Hautamaki

University of Florida - Department of Microbiology and Cell Science

Piyush K. Jain

University of Florida - Department of Chemical Engineering

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Abstract

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.

Note:
Funding Information: This work was supported by funds in part from the University of Florida (UF), the UF Herbert Wertheim College of Engineering, Dinesh O. Shah endowed professorship, Florida Breast Cancer Foundation (AGR00018466), the United States India Science & Technology Endowment Fund (USISTEF/COVID-I/247/2020), and the National Institute of General Medical Sciences of the National Institutes of Health (R35GM147788).

Declaration of Interests: P.K.J. and L.T.N. are listed as inventors on patent applications related to the content of this work. P.K.J. is a co-founder of Genable Biosciences, Par Biosciences, and CRIPSR, LLC. The remaining authors declare no competing interests.

Ethical Approval Statement: Nasopharyngeal swabs and saliva samples were obtained from the University of Florida (UF) Clinical and Translational Science Institute adhering to the guidelines approved by UF Institutional Review Board (protocol# IRB202000781).

Keywords: Cas12a, CRISPR/Cas12a, Trans-cleavage, Cis-cleavage, cCRISPR, Non-canonical crRNAs, Scaffold engineering, gene editing, Diagnostics, COVID-19, SARS-CoV-2

Suggested Citation

Nguyen, Long T. and Macaluso, Nicolas C. and Rakestraw, Noah R. and Carman, Dylan R. and Pizzano, Brianna L.M. and Hautamaki, Raymond C. and Jain, Piyush K., Harnessing Non-Canonical crRNAs to Improve Functionality of Cas12a Orthologs. Available at SSRN: https://ssrn.com/abstract=4515211 or http://dx.doi.org/10.2139/ssrn.4515211
This version of the paper has not been formally peer reviewed.

Long T. Nguyen

University of Florida - Department of Chemical Engineering ( email )

Gainesville, FL
United States

Nicolas C. Macaluso

Johns Hopkins University - Department of Chemical and Biomolecular Engineering ( email )

Noah R. Rakestraw

University of Florida - Department of Molecular Genetics and Microbiology ( email )

University of Florida - Department of Graduate Education ( email )

Dylan R. Carman

University of Florida - Department of Chemical Engineering ( email )

Brianna L.M. Pizzano

University of Florida - Department of Chemical Engineering ( email )

University of Florida - Department of Agricultural and Biological Engineering ( email )

Gainesville, FL
United States

Raymond C. Hautamaki

University of Florida - Department of Microbiology and Cell Science ( email )

Piyush K. Jain (Contact Author)

University of Florida - Department of Chemical Engineering ( email )

Gainesville, FL
United States

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