CRISPR/Cas9 searches for a protospacer adjacent motif by lateral diffusion.
CRISPR/Cas9
lateral diffusion
single‐molecule FRET
target search
Journal
The EMBO journal
ISSN: 1460-2075
Titre abrégé: EMBO J
Pays: England
ID NLM: 8208664
Informations de publication
Date de publication:
15 02 2019
15 02 2019
Historique:
received:
19
03
2018
revised:
09
11
2018
accepted:
15
11
2018
pubmed:
24
12
2018
medline:
7
1
2020
entrez:
22
12
2018
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 30573670
pii: embj.201899466
doi: 10.15252/embj.201899466
pmc: PMC6376262
pii:
doi:
Substances chimiques
DNA
9007-49-2
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Commentaires et corrections
Type : CommentIn
Informations de copyright
© 2018 The Authors.
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