Enhancement of CRISPR/Cas12a <i>trans</i>-cleavage activity using hairpin DNA reporters-Reference-Cited by-同舟云学术

Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reporters

Published:2022-07-13 Issue:14 Volume:50 Page:8377-8391
ISSN:0305-1048
Container-title:Nucleic Acids Research
language:en
Short-container-title:

Author:

Rossetti Marianna¹,Merlo Rosa²,Bagheri Neda¹,Moscone Danila¹,Valenti Anna²,Saha Aakash³,Arantes Pablo R³,Ippodrino Rudy⁴,Ricci Francesco¹^ORCID,Treglia Ida⁵,Delibato Elisabetta⁵,van der Oost John⁶^ORCID,Palermo Giulia³^ORCID,Perugino Giuseppe²⁷,Porchetta Alessandro¹^ORCID

Affiliation:

1. Department of Chemistry, University of Rome , Tor Vergata, Via della Ricerca Scientifica 00133 , Rome , Italy

2. Institute of Biosciences and BioResources , National Research Council of Italy, Via Pietro Castellino 111, 80131 Naples, Italy

3. Department of Bioengineering and Department of Chemistry, University of California Riverside, 900 University Avenue , Riverside , CA 52512 USA

4. Ulisse BioMed S.r.l. Area Science Park , 34149 Trieste, Italy

5. Department of Food Safety, Nutrition and Veterinary Public Health, Istituto Superiore di Sanità , Viale Regina Elena 299, Rome , Italy

6. Laboratory of Microbiology, Wageningen University , Stippeneng 4, 6708 WE Wageningen, The Netherlands

7. Department of Biology, University of Naples “Federico II” , Complesso Universitario di Monte Sant’Angelo, Ed. 7, Via Cintia 26, 80126 Naples, Italy

Abstract

Abstract The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (Km) for hairpin DNA structures, and provide mechanistic insights of our findings through Molecular Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clinically relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.

Funder

European Union

Italian Ministry of University

National Institutes of Health

National Science Foundation

XSEDE

NERSC

Publisher

Oxford University Press (OUP)

Subject

Genetics

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