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Bio-Layer Interferometry Analysis of the Target Binding Activity of CRISPR-Cas Effector Complexes

CRISPR-Cas systems employ ribonucleoprotein complexes to identify nucleic acid targets with complementarity to bound CRISPR RNAs. Analyses of the high diversification of these effector complexes suggest that they can exhibit a wide spectrum of target requirements and binding affinities. Therefore, s...

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Autores principales: Müller-Esparza, Hanna, Osorio-Valeriano, Manuel, Steube, Niklas, Thanbichler, Martin, Randau, Lennart
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7266957/
https://www.ncbi.nlm.nih.gov/pubmed/32528975
http://dx.doi.org/10.3389/fmolb.2020.00098
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author Müller-Esparza, Hanna
Osorio-Valeriano, Manuel
Steube, Niklas
Thanbichler, Martin
Randau, Lennart
author_facet Müller-Esparza, Hanna
Osorio-Valeriano, Manuel
Steube, Niklas
Thanbichler, Martin
Randau, Lennart
author_sort Müller-Esparza, Hanna
collection PubMed
description CRISPR-Cas systems employ ribonucleoprotein complexes to identify nucleic acid targets with complementarity to bound CRISPR RNAs. Analyses of the high diversification of these effector complexes suggest that they can exhibit a wide spectrum of target requirements and binding affinities. Therefore, streamlined analysis techniques to study the interactions between nucleic acids and proteins are necessary to facilitate the characterization and comparison of CRISPR-Cas effector activities. Bio-layer Interferometry (BLI) is a technique that measures the interference pattern of white light that is reflected from a layer of biomolecules immobilized on the surface of a sensor tip (bio-layers) in real time and in solution. As streptavidin-coated sensors and biotinylated oligonucleotides are commercially available, this method enables straightforward measurements of the interaction of CRISPR-Cas complexes with different targets in a qualitative and quantitative fashion. Here, we present a general method to carry out binding assays with the Type I-Fv complex from Shewanella putrefaciens and the Type I-F complex from Shewanella baltica as model effectors. We report target specificities, dissociation constants and interactions with the Anti-CRISPR protein AcrF7 to highlight possible applications of this technique.
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spelling pubmed-72669572020-06-10 Bio-Layer Interferometry Analysis of the Target Binding Activity of CRISPR-Cas Effector Complexes Müller-Esparza, Hanna Osorio-Valeriano, Manuel Steube, Niklas Thanbichler, Martin Randau, Lennart Front Mol Biosci Molecular Biosciences CRISPR-Cas systems employ ribonucleoprotein complexes to identify nucleic acid targets with complementarity to bound CRISPR RNAs. Analyses of the high diversification of these effector complexes suggest that they can exhibit a wide spectrum of target requirements and binding affinities. Therefore, streamlined analysis techniques to study the interactions between nucleic acids and proteins are necessary to facilitate the characterization and comparison of CRISPR-Cas effector activities. Bio-layer Interferometry (BLI) is a technique that measures the interference pattern of white light that is reflected from a layer of biomolecules immobilized on the surface of a sensor tip (bio-layers) in real time and in solution. As streptavidin-coated sensors and biotinylated oligonucleotides are commercially available, this method enables straightforward measurements of the interaction of CRISPR-Cas complexes with different targets in a qualitative and quantitative fashion. Here, we present a general method to carry out binding assays with the Type I-Fv complex from Shewanella putrefaciens and the Type I-F complex from Shewanella baltica as model effectors. We report target specificities, dissociation constants and interactions with the Anti-CRISPR protein AcrF7 to highlight possible applications of this technique. Frontiers Media S.A. 2020-05-27 /pmc/articles/PMC7266957/ /pubmed/32528975 http://dx.doi.org/10.3389/fmolb.2020.00098 Text en Copyright © 2020 Müller-Esparza, Osorio-Valeriano, Steube, Thanbichler and Randau. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Molecular Biosciences
Müller-Esparza, Hanna
Osorio-Valeriano, Manuel
Steube, Niklas
Thanbichler, Martin
Randau, Lennart
Bio-Layer Interferometry Analysis of the Target Binding Activity of CRISPR-Cas Effector Complexes
title Bio-Layer Interferometry Analysis of the Target Binding Activity of CRISPR-Cas Effector Complexes
title_full Bio-Layer Interferometry Analysis of the Target Binding Activity of CRISPR-Cas Effector Complexes
title_fullStr Bio-Layer Interferometry Analysis of the Target Binding Activity of CRISPR-Cas Effector Complexes
title_full_unstemmed Bio-Layer Interferometry Analysis of the Target Binding Activity of CRISPR-Cas Effector Complexes
title_short Bio-Layer Interferometry Analysis of the Target Binding Activity of CRISPR-Cas Effector Complexes
title_sort bio-layer interferometry analysis of the target binding activity of crispr-cas effector complexes
topic Molecular Biosciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7266957/
https://www.ncbi.nlm.nih.gov/pubmed/32528975
http://dx.doi.org/10.3389/fmolb.2020.00098
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