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    CRISPR/Cas9 searches for a protospacer adjacent motif by lateral diffusion (2018)
    Springer Nature
    Details
    Publication Date: 2018
    Description: 〈sec〉〈st〉Synopsis〈/st〉〈p〉〈textbox textbox-type="graphic"〉〈p〉〈inline-fig〉〈/inline-fig〉〈/p〉〈/textbox〉〈/p〉 〈p〉Single-molecule FRET studies illustrate that lateral diffusion contributes to protospacer adjacent motif (PAM) and target site search by the CRISPR/Cas9 nuclease, expanding the Cas9 search mode beyond random 3D diffusion and emphasizing the importance of neighboring sequences for genetic engineering approaches.〈/p〉 〈p〉 〈l type="unord"〉〈li〉〈p〉Single-molecule FRET allows real-time monitoring of transient Cas9-DNA interactions.〈/p〉〈/li〉 〈li〉〈p〉Weak PAM binding allows Cas9 to probe neighboring sequences by facilitated diffusion.〈/p〉〈/li〉 〈li〉〈p〉Cas9 can laterally diffuse between individual target sites.〈/p〉〈/li〉 〈li〉〈p〉Multiplicity of PAM sequences can interfere with efficient on-target binding.〈/p〉〈/li〉〈/l〉 〈/p〉〈/sec〉
    Print ISSN: 0261-4189
    Electronic ISSN: 1460-2075
    Topics: Biology , Medicine
    Published by Springer Nature on behalf of The European Molecular Biology Organization (EMBO).
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  • 2
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    CRISPR/Cas9 searches for a protospacer adjacent motif by lateral diffusion (2019)
    Springer Nature
    Details
    Publication Date: 2019
    Description: 〈p〉The Streptococcus pyogenes CRISPR/Cas9 (SpCas9) nuclease has been widely applied in genetic engineering. Despite its importance in genome editing, aspects of the precise molecular mechanism of Cas9 activity remain ambiguous. In particular, because of the lack of a method with high spatio-temporal resolution, transient interactions between Cas9 and DNA could not be reliably investigated. It therefore remains controversial how Cas9 searches for protospacer adjacent motif (PAM) sequences. We have developed single-molecule Förster resonance energy transfer (smFRET) assays to monitor transient interactions of Cas9 and DNA in real time. Our study shows that Cas9 interacts with the PAM sequence weakly, yet probing neighboring sequences via facilitated diffusion. This dynamic mode of interactions leads to translocation of Cas9 to another PAM nearby and consequently an on-target sequence. We propose a model in which lateral diffusion competes with three-dimensional diffusion and thus is involved in PAM finding and consequently on-target binding. Our results imply that the neighboring sequences can be very important when choosing a target in genetic engineering applications.〈/p〉
    Print ISSN: 0261-4189
    Electronic ISSN: 1460-2075
    Topics: Biology , Medicine
    Published by Springer Nature on behalf of The European Molecular Biology Organization (EMBO).
    Location Call Number Expected Availability
    BibTip Others were also interested in ...
    PAPER CURRENT
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