iScience
Volume 24, Issue 9, 24 September 2021, 102996
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Article
Kinetic analysis of Cas12a and Cas13a RNA-Guided nucleases for development of improved CRISPR-Based diagnostics

https://doi.org/10.1016/j.isci.2021.102996Get rights and content
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open access

Highlights

  • Cas13a HEPN trans-RNase activation is directly coupled to rapid target RNA binding

  • Cas12a RuvC trans-nuclease activity is coupled to slow target DNA cleavage

  • Individual crRNA generate widely varying levels of targeted trans-cleavage

  • Pooling multiple crRNA allows pathogen quantification without target amplification

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.

Subject areas

Chemical reaction kinetics
Biochemistry
Molecular biology

Data and code availability

The published article includes all datasets generated or analyzed during this study.

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