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
Abstract
-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.
Topics & Concepts
Trans-activating crRNARNACRISPRNucleaseNucleic acidRibonucleaseDNARNase PBiologyComputational biologyGenome editingChemistryBiochemistryGeneCRISPR and Genetic EngineeringCytomegalovirus and herpesvirus researchMycobacterium research and diagnosis