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Schlagwörter:
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Zusammenfassung:
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 fromShewanella putrefaciensand the Type I-F
complex fromShewanella balticaas model effectors. We report target
specificities, dissociation constants and interactions with the
Anti-CRISPR protein AcrF7 to highlight possible applications of this
technique.
