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

Frontiers in Molecular Biosciences
Hanna Müller-EsparzaLennart Randau

Abstract

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...Continue Reading

References

Mar 24, 2007·Science·Rodolphe BarrangouPhilippe Horvath
Jan 15, 2010·Nature·Luciano A Marraffini, Erik J Sontheimer
Mar 17, 2010·BMC Bioinformatics·Jessica S Reuter, David H Mathews
Jan 31, 2014·Nature·Samuel H SternbergJennifer A Doudna
Apr 16, 2014·Current Protocols in Molecular Biology·Lynn C ThomasonDonald L Court
Aug 19, 2014·Nature Structural & Molecular Biology·Yanwu HuoAilong Ke
Dec 10, 2014·Nucleic Acids Research·Natalia BeloglazovaAlexander F Yakunin
Feb 3, 2015·Current Protocols in Protein Science·Azmiri Sultana, Jeffrey E Lee
Feb 11, 2015·Nucleic Acids Research·MaryClare F RollinsBlake Wiedenheft
Sep 10, 2015·Nucleic Acids Research·Srivatsa DwarakanathLennart Randau
Sep 30, 2015·Nature·Joseph Bondy-DenomyAlan R Davidson
Nov 21, 2015·Nucleic Acids Research·Chaoyou XueDipali G Sashital
May 25, 2016·Nucleic Acids Research·Daniel GleditzschLennart Randau
Aug 31, 2016·Nature Microbiology·April PawlukAlan R Davidson
Jul 15, 2017·Science Advances·Jiyung ShinJennifer A Doudna
Aug 7, 2017·Molecular Cell·Patrick PauschGert Bange
Aug 7, 2018·Molecular Cell·Isabel StrohkendlRick Russell
Feb 7, 2019·EcoSal Plus·Chaoyou Xue, Dipali G Sashital
Dec 21, 2019·Nature Reviews. Microbiology·Kira S MakarovaEugene V Koonin

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Citations

Nov 26, 2020·Analytical and Bioanalytical Chemistry·Ross D OverackerSandra Loesgen
Oct 23, 2020·Sensors·Donggee RhoSeunghyun Kim
Mar 20, 2021·Emerging Topics in Life Sciences·Mahalakshmi Harish, Prasanna Venkatraman
May 8, 2021·Scientific Reports·Asheley P ChapmanJason M Goldstein
Feb 3, 2022·PloS One·John K Barrows, Michael W Van Dyke

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Methods Mentioned

BETA
Assay
Bio-layer Interferometry
biosensor
biosensors
PCR
size exclusion chromatography

Software Mentioned

BLItz
GraphPad Prism
BLItz Pro
RNAstructure

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