Premium
CRISPR /Cas9 searches for a protospacer adjacent motif by lateral diffusion
Author(s) -
Globyte Viktorija,
Lee Seung Hwan,
Bae Taegeun,
Kim JinSoo,
Joo Chirlmin
Publication year - 2018
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.15252/embj.201899466
Subject(s) - biology , crispr , cas9 , computational biology , genetics , gene
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 (sm FRET ) 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.