Kinetic analysis of Cas12a and Cas13a RNA-Guided nucleases for development of improved CRISPR-Based diagnostics

Nalefski, Eric A.; Patel, Nidhi; Leung, Philip J. Y.; Islam, Zeba; Kooistra, Remy M.; Parikh, Ishira; Marion, Estelle; Knott, Gavin J.; Doudna, Jennifer A.; Le Ny, Anne-Laure M.; Madan, Damian

doi:10.1016/j.isci.2021.102996

Kinetic analysis of Cas12a and Cas13a RNA-Guided nucleases for development of improved CRISPR-Based diagnostics

Journal Article · Wed Aug 18 00:00:00 EDT 2021 · iScience

DOI:https://doi.org/10.1016/j.isci.2021.102996· OSTI ID:2470743

Nalefski, Eric A. ^[1]; Patel, Nidhi ^[2]; Leung, Philip J. Y. ^[1]; Islam, Zeba ^[2]; ^[1]; Parikh, Ishira ^[2]; Marion, Estelle ^[3]; ^[4]; Doudna, Jennifer A. ^[5]; ^[1]; ^[1]

Global Health Labs, Bellevue, WA (United States); Intellectual Ventures Global Good Fund, Bellevue, WA (United States)
Global Health Labs, Bellevue, WA (United States)
Université d'Angers, Angers (France)
Univ. of California, Berkeley, CA (United States); Monash Univ., Melbourne, VIC (Australia)
Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); Gladstone Institutes, San Francisco, CA (United States)

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.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: National Institutes of Health (NIH); National Science Foundation (NSF); USDOE

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 2470743

Journal Information:: iScience, Journal Name: iScience Journal Issue: 9 Vol. 24; ISSN 2589-0042

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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