Unidirectional trans-cleaving behavior of CRISPR-Cas12a unlocks for an ultrasensitive assay using hybrid DNA reporters containing a 3′ toehold

• Published in: Nucleic acids research Vol. 51; no. 18; pp. 9894 - 9904
• Main Authors: Mohammad, Noor, Talton, Logan, Hetzler, Zach, Gongireddy, Megha, Wei, Qingshan
• Format: Journal Article
• Language: English
• Published: Oxford University Press 13-10-2023
• Subjects: Nucleic Acid Enzymes
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkad715

Abstract
CRISPR-Cas12a can induce nonspecific trans-cleavage of dsDNA substrate, including long and stable λ DNA. However, the mechanism behind this is still largely undetermined. In this study, we observed that while trans-activated Cas12a didn’t cleave blunt-end dsDNA within a short reaction time, it could degrade dsDNA reporters with a short overhang. More interestingly, we discovered that the location of the overhang also affected the susceptibility of dsDNA substrate to trans-activated Cas12a. Cas12a trans-cleaved 3′ overhang dsDNA substrates at least 3 times faster than 5′ overhang substrates. We attributed this unique preference of overhang location to the directional trans-cleavage behavior of Cas12a, which may be governed by RuvC and Nuc domains. Utilizing this new finding, we designed a new hybrid DNA reporter as nonoptical substrate for the CRISPR-Cas12a detection platform, which sensitively detected ssDNA targets at sub picomolar level. This study not only unfolded new insight into the trans-cleavage behavior of Cas12a but also demonstrated a sensitive CRISPR-Cas12a assay by using a hybrid dsDNA reporter molecule.
Graphical Abstract
Graphical Abstract