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Bright fluorescent nucleic acid detection with CRISPR-Cas12a and poly(thymine) templated copper nanoparticles

Fluorescence-based diagnostic tools are attractive and versatile tests with multiple advantages: ease of use, sensitivity and rapid results. The advent of CRISPR-Cas technology has created new avenues for the development of diagnostic testing tools. In this study, by effectively combining the specif...

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Autores principales: Bogers, Janna F M, Berghuis, Nicole F, Busker, Ruud W, van Booma, Angelo, Paauw, Armand, van Leeuwen, Hans C
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891128/
https://www.ncbi.nlm.nih.gov/pubmed/33628946
http://dx.doi.org/10.1093/biomethods/bpaa020
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author Bogers, Janna F M
Berghuis, Nicole F
Busker, Ruud W
van Booma, Angelo
Paauw, Armand
van Leeuwen, Hans C
author_facet Bogers, Janna F M
Berghuis, Nicole F
Busker, Ruud W
van Booma, Angelo
Paauw, Armand
van Leeuwen, Hans C
author_sort Bogers, Janna F M
collection PubMed
description Fluorescence-based diagnostic tools are attractive and versatile tests with multiple advantages: ease of use, sensitivity and rapid results. The advent of CRISPR-Cas technology has created new avenues for the development of diagnostic testing tools. In this study, by effectively combining the specific functions of two enzymes, CRISPR-Cas12a and terminal deoxynucleotidyl transferase (TdT), we developed a DNA detection assay that generates copper nanoparticles (CuNPs) that are easily visible to the naked eye under UV-light; we named this detection assay Cas12a Activated Nuclease poly-T Reporter Illuminating Particles (CANTRIP). Upon specific target DNA recognition by Cas12a, single-stranded DNA (ssDNA) reporter oligos with blocked 3′-ends are cut into smaller ssDNA fragments, thereby generating neo 3′-hydroxyl moieties. TdT subsequently elongates these newly formed ssDNA fragments, incorporating only dTTP nucleotides, and these poly(thymine)-tails subsequently function as scaffolds for the formation of CuNPs. These CuNPs produce a bright fluorescent signal upon UV excitation, and thus, this bright orange signal indicates the presence of target DNA, which in this proof-of-concept study consisted of anthrax lethal factor plasmid DNA. CANTRIP, which combines two detection platforms consisting of CRISPR-Cas12a and fluorescent CuNPs into a single reaction, appears to be a robust, low-cost and simple diagnostic tool.
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spelling pubmed-78911282021-02-23 Bright fluorescent nucleic acid detection with CRISPR-Cas12a and poly(thymine) templated copper nanoparticles Bogers, Janna F M Berghuis, Nicole F Busker, Ruud W van Booma, Angelo Paauw, Armand van Leeuwen, Hans C Biol Methods Protoc Methods Article Fluorescence-based diagnostic tools are attractive and versatile tests with multiple advantages: ease of use, sensitivity and rapid results. The advent of CRISPR-Cas technology has created new avenues for the development of diagnostic testing tools. In this study, by effectively combining the specific functions of two enzymes, CRISPR-Cas12a and terminal deoxynucleotidyl transferase (TdT), we developed a DNA detection assay that generates copper nanoparticles (CuNPs) that are easily visible to the naked eye under UV-light; we named this detection assay Cas12a Activated Nuclease poly-T Reporter Illuminating Particles (CANTRIP). Upon specific target DNA recognition by Cas12a, single-stranded DNA (ssDNA) reporter oligos with blocked 3′-ends are cut into smaller ssDNA fragments, thereby generating neo 3′-hydroxyl moieties. TdT subsequently elongates these newly formed ssDNA fragments, incorporating only dTTP nucleotides, and these poly(thymine)-tails subsequently function as scaffolds for the formation of CuNPs. These CuNPs produce a bright fluorescent signal upon UV excitation, and thus, this bright orange signal indicates the presence of target DNA, which in this proof-of-concept study consisted of anthrax lethal factor plasmid DNA. CANTRIP, which combines two detection platforms consisting of CRISPR-Cas12a and fluorescent CuNPs into a single reaction, appears to be a robust, low-cost and simple diagnostic tool. Oxford University Press 2020-10-08 /pmc/articles/PMC7891128/ /pubmed/33628946 http://dx.doi.org/10.1093/biomethods/bpaa020 Text en © The Author(s) 2020. Published by Oxford University Press. https://creativecommons.org/licenses/by-nc/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/ (https://creativecommons.org/licenses/by-nc/4.0/) ), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Methods Article
Bogers, Janna F M
Berghuis, Nicole F
Busker, Ruud W
van Booma, Angelo
Paauw, Armand
van Leeuwen, Hans C
Bright fluorescent nucleic acid detection with CRISPR-Cas12a and poly(thymine) templated copper nanoparticles
title Bright fluorescent nucleic acid detection with CRISPR-Cas12a and poly(thymine) templated copper nanoparticles
title_full Bright fluorescent nucleic acid detection with CRISPR-Cas12a and poly(thymine) templated copper nanoparticles
title_fullStr Bright fluorescent nucleic acid detection with CRISPR-Cas12a and poly(thymine) templated copper nanoparticles
title_full_unstemmed Bright fluorescent nucleic acid detection with CRISPR-Cas12a and poly(thymine) templated copper nanoparticles
title_short Bright fluorescent nucleic acid detection with CRISPR-Cas12a and poly(thymine) templated copper nanoparticles
title_sort bright fluorescent nucleic acid detection with crispr-cas12a and poly(thymine) templated copper nanoparticles
topic Methods Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891128/
https://www.ncbi.nlm.nih.gov/pubmed/33628946
http://dx.doi.org/10.1093/biomethods/bpaa020
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