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-CRISPR protein AcrF7 to highlight possible applications of this technique.
Highlights
CRISPR-Cas systems are adaptive immune systems found in Archaea and Bacteria
The kinetic values obtained can be corroborated with complementary techniques, such as MicroScale Thermophoresis (MST), as each platform has different levels of accuracy depending on the experimental design
BLI offers a practical alternative for studying the interaction between CRISPR-Cas complexes and nucleic acids
Summary
CRISPR-Cas systems are adaptive immune systems found in Archaea and Bacteria. They are widespread and diverse, with 6 types (I–VI) and 33 different subtypes described so far (Makarova et al, 2020). They are able to establish immunity against invading genetic material through ribonucleoprotein (RNP) complexes, formed by a CRISPR RNA (crRNA) and CRISPR-associated (Cas) proteins, in a process termed CRISPR interference (Barrangou et al, 2007). CRISPR-Cas types are classified by unique Cas proteins, which are often part of distinct RNP complexes and convey specific features and mechanistic variations. Type I, II, and V complexes scan for DNA targets and first recognize a 2–5 bp short motif next to the region with complementarity to the crRNA (protospacer), termed protospacer adjacent motif (PAM) (Anders et al, 2014; Hayes et al, 2016; Yang et al, 2016)
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