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

Viktorija Globyte, Seung Hwan Lee, Taegeun Bae, Jin Soo Kim, Chirlmin Joo

Research output: Contribution to journalArticleScientificpeer-review

9 Citations (Scopus)

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 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.

Original languageEnglish
Article numbere99466
Number of pages12
JournalEMBO Journal
Volume38
Issue number1
DOIs
Publication statusPublished - 2018

Keywords

  • CRISPR/Cas9
  • lateral diffusion
  • single-molecule FRET
  • target search

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