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Cas12a is an RNA‐guided DNA endonuclease of the type V‐A CRISPR‐Cas system that has evolved convergently with the type II Cas9 protein. We previously showed that proline substitutions in the bridge helix (BH) impart target DNA cleavage selectivity inStreptococcus pyogenes(Spy) Cas9. Here, we examined a BH variant of Cas12a fromFrancisella novicida(FnoCas12aKD2P) to test mechanistic conservation. Our results show that for RNA‐guided DNA cleavage (cis‐activity), FnoCas12aKD2Paccumulates nicked products while cleaving supercoiled DNA substrates with mismatches, with certain mismatch positions being more detrimental for linearization. FnoCas12aKD2Palso possess reducedtrans‐single‐stranded DNA cleavage activity. These results implicate the BH in substrate selectivity in bothcis‐andtrans‐cleavages and show its conserved role in target discrimination among Cas nucleases.
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Unidirectional trans -cleaving behavior of CRISPR-Cas12a unlocks for an ultrasensitive assay using hybrid DNA reporters containing a 3′ toehold
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.
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- Award ID(s):
- 1944167
- PAR ID:
- 10451638
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 51
- Issue:
- 18
- ISSN:
- 0305-1048
- Format(s):
- Medium: X Size: p. 9894-9904
- Size(s):
- p. 9894-9904
- Sponsoring Org:
- National Science Foundation
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