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CRISPR/Cas9 searches for a protospacer adjacent motif by lateral diffusion

Cited 8 time in Web of Science Cited 9 time in Scopus
Authors
Globyte, Viktorija; Lee, Seung Hwan; Bae, Taegeun; Kim, Jin-Soo; Joo, Chirlmin
Issue Date
2019-02
Citation
EMBO Journal, Vol.38 No.4, p. e99466
Keywords
CRISPR/Cas9lateral diffusionsingle-molecule FRETtarget search
Abstract
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 Forster 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.
ISSN
0261-4189
URI
http://hdl.handle.net/10371/165713
DOI
https://doi.org/10.15252/embj.201899466
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