한빛사 논문
Viktorija Globyte1,†, Seung Hwan Lee1,2,† , Taegeun Bae2, Jin-Soo Kim2,3,* & Chirlmin Joo 1,**
1 Department of BioNanoScience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands
2 Center for Genome Engineering, Institute for Basic Science, Seoul, Korea
3 Department of Chemistry, Seoul National University, Seoul, Korea
*Corresponding author.
**Corresponding author.
†These authors contributed equally to this work as first authors
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.
Keywords CRISPR/Cas9; lateral diffusion; single-molecule FRET; target search
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