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

Viktorija Globyte, Seung Hwan Lee, Taegun Bae, Jin-Soo Kim, Chirlmin Joo
doi: https://doi.org/10.1101/264879
Viktorija Globyte
1Kavli Institute of Nanoscience and Department of BioNanoScience, Delft University of Technology, Delft, The Netherlands
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Seung Hwan Lee
1Kavli Institute of Nanoscience and Department of BioNanoScience, Delft University of Technology, Delft, The Netherlands
2Center for Genome Engineering, Institute for Basic Science, Seoul 08826, Republic of Korea
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Taegun Bae
2Center for Genome Engineering, Institute for Basic Science, Seoul 08826, Republic of Korea
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Jin-Soo Kim
2Center for Genome Engineering, Institute for Basic Science, Seoul 08826, Republic of Korea
4Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea
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  • For correspondence: jskim01@snu.ac.kr c.joo@tudelft.nl
Chirlmin Joo
1Kavli Institute of Nanoscience and Department of BioNanoScience, Delft University of Technology, Delft, The Netherlands
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  • For correspondence: jskim01@snu.ac.kr c.joo@tudelft.nl
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Abstract

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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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.
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Posted February 13, 2018.
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CRISPR Cas9 searches for a protospacer adjacent motif by one-dimensional diffusion
Viktorija Globyte, Seung Hwan Lee, Taegun Bae, Jin-Soo Kim, Chirlmin Joo
bioRxiv 264879; doi: https://doi.org/10.1101/264879
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CRISPR Cas9 searches for a protospacer adjacent motif by one-dimensional diffusion
Viktorija Globyte, Seung Hwan Lee, Taegun Bae, Jin-Soo Kim, Chirlmin Joo
bioRxiv 264879; doi: https://doi.org/10.1101/264879

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