Kinetic analysis of Cas12a and Cas13a RNA-Guided nucleases for development of improved CRISPR-Based diagnostics
Eric A. Nalefski,
Nidhi Patel,
Philip J.Y. Leung,
Zeba Islam,
Remy M. Kooistra,
Ishira Parikh,
Estelle Marion,
Gavin J. Knott,
Jennifer A. Doudna,
Anne-Laure M. Le Ny,
Damian Madan
Affiliations
Eric A. Nalefski
Global Health Labs, Bellevue, WA 98007, USA; Center for In Vitro Diagnostics, Intellectual Ventures Global Good Fund, Bellevue, WA 98007, USA; Corresponding author
Nidhi Patel
Global Health Labs, Bellevue, WA 98007, USA
Philip J.Y. Leung
Global Health Labs, Bellevue, WA 98007, USA; Center for In Vitro Diagnostics, Intellectual Ventures Global Good Fund, Bellevue, WA 98007, USA
Zeba Islam
Global Health Labs, Bellevue, WA 98007, USA
Remy M. Kooistra
Global Health Labs, Bellevue, WA 98007, USA; Center for In Vitro Diagnostics, Intellectual Ventures Global Good Fund, Bellevue, WA 98007, USA
Ishira Parikh
Global Health Labs, Bellevue, WA 98007, USA
Estelle Marion
Inserm, Université d'Angers, Angers, France
Gavin J. Knott
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94704, USA; Monash Biomedicine Discovery Institute, Department of Chemistry & Molecular Biology, Monash University, Melbourne, VIC 3800, Australia; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA
Jennifer A. Doudna
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94704, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94704, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94704, USA; Gladstone Institute of Data Science and Biotechnology, Gladstone Institutes, San Francisco, CA 94158, USA
Anne-Laure M. Le Ny
Global Health Labs, Bellevue, WA 98007, USA; Center for In Vitro Diagnostics, Intellectual Ventures Global Good Fund, Bellevue, WA 98007, USA
Damian Madan
Global Health Labs, Bellevue, WA 98007, USA; Center for In Vitro Diagnostics, Intellectual Ventures Global Good Fund, Bellevue, WA 98007, USA; Corresponding author
Summary: Bacterial CRISPR systems provide acquired immunity against invading nucleic acids by activating RNA-programmable RNases and DNases. Cas13a and Cas12a enzymes bound to CRISPR RNA (crRNA) recognize specific nucleic acid targets, initiating cleavage of the targets as well as non-target (trans) nucleic acids. Here, we examine the kinetics of single-turnover target and multi-turnover trans-nuclease activities of both enzymes. High-turnover, non-specific Cas13a trans-RNase activity is coupled to rapid binding of target RNA. By contrast, low-turnover Cas12a trans-nuclease activity is coupled to relatively slow cleavage of target DNA, selective for DNA over RNA, indifferent to base identity, and preferential for single-stranded substrates. Combining multiple crRNA increases detection sensitivity of targets, an approach we use to quantify pathogen DNA in samples from patients suspected of Buruli ulcer disease. Results reveal that these enzymes are kinetically adapted to play distinct roles in bacterial adaptive immunity and show how kinetic analysis can be applied to CRISPR-based diagnostics.
