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Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reporters.
Rossetti, Marianna; Merlo, Rosa; Bagheri, Neda; Moscone, Danila; Valenti, Anna; Saha, Aakash; Arantes, Pablo R; Ippodrino, Rudy; Ricci, Francesco; Treglia, Ida; Delibato, Elisabetta; van der Oost, John; Palermo, Giulia; Perugino, Giuseppe; Porchetta, Alessandro.
  • Rossetti M; Department of Chemistry, University of Rome, Tor Vergata, Via della Ricerca Scientifica 00133, Rome, Italy.
  • Merlo R; Institute of Biosciences and BioResources, National Research Council of Italy, Via Pietro Castellino 111, 80131 Naples, Italy.
  • Bagheri N; Department of Chemistry, University of Rome, Tor Vergata, Via della Ricerca Scientifica 00133, Rome, Italy.
  • Moscone D; Department of Chemistry, University of Rome, Tor Vergata, Via della Ricerca Scientifica 00133, Rome, Italy.
  • Valenti A; Institute of Biosciences and BioResources, National Research Council of Italy, Via Pietro Castellino 111, 80131 Naples, Italy.
  • Saha A; Department of Bioengineering and Department of Chemistry, University of California Riverside, 900 University Avenue, Riverside, CA 52512 USA.
  • Arantes PR; Department of Bioengineering and Department of Chemistry, University of California Riverside, 900 University Avenue, Riverside, CA 52512 USA.
  • Ippodrino R; Ulisse BioMed S.r.l. Area Science Park, 34149 Trieste, Italy.
  • Ricci F; Department of Chemistry, University of Rome, Tor Vergata, Via della Ricerca Scientifica 00133, Rome, Italy.
  • Treglia I; Department of Food Safety, Nutrition and Veterinary Public Health, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome, Italy.
  • Delibato E; Department of Food Safety, Nutrition and Veterinary Public Health, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome, Italy.
  • van der Oost J; Laboratory of Microbiology, Wageningen University, Stippeneng 4, 6708 WE Wageningen, The Netherlands.
  • Palermo G; Department of Bioengineering and Department of Chemistry, University of California Riverside, 900 University Avenue, Riverside, CA 52512 USA.
  • Perugino G; Institute of Biosciences and BioResources, National Research Council of Italy, Via Pietro Castellino 111, 80131 Naples, Italy.
  • Porchetta A; Department of Biology, University of Naples "Federico II", Complesso Universitario di Monte Sant'Angelo, Ed. 7, Via Cintia 26, 80126 Naples, Italy.
Nucleic Acids Res ; 50(14): 8377-8391, 2022 08 12.
Article en En | MEDLINE | ID: mdl-35822842
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.
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Texto completo: 1 Banco de datos: MEDLINE Asunto principal: Proteínas Asociadas a CRISPR Idioma: En Año: 2022 Tipo del documento: Article
Texto completo
Imprimir
XML
PubMed Links
Search on Google

Texto completo: 1 Banco de datos: MEDLINE Asunto principal: Proteínas Asociadas a CRISPR Idioma: En Año: 2022 Tipo del documento: Article