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CRISPR Cas9 searches for a protospacer adjacent motif by one-dimensional diffusion

By Viktorija Globyte, Seung Hwan Lee, Taegun Bae, Jin-Soo Kim, Chirlmin Joo

Posted 13 Feb 2018
bioRxiv DOI: 10.1101/264879 (published DOI: 10.15252/embj.201899466)

Since its discovery, the CRISPR/Cas9 system has been at the focus of fundamental researchers, genome engineers, and the general public alike. Despite being in the spotlight for several years, aspects of the precise molecular mechanism of Cas9 activity remain ambiguous. We use single-molecule Foerster resonance energy transfer (smFRET) to reveal Cas9 target search mechanism with nanometer sensitivity. We have developed single-molecule assays to monitor transient interactions of Cas9 and DNA in real time. Our study shows that Cas9 interacts with the protospacer adjacent motif (PAM) sequence weakly, yet probing neighboring sequences via lateral 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 3-dimensional diffusion and thus might aid PAM finding and consequently on-target binding.

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