title: Dynamics of CRISPR-Cas9 genome interrogation in living cells.
abstract: The RNA-guided CRISPR-associated protein Cas9 is used for genome editing, transcriptional modulation, and live-cell imaging. Cas9-guide RNA complexes recognize and cleave double-stranded DNA sequences on the basis of 20-nucleotide RNA-DNA complementarity, but the mechanism of target searching in mammalian cells is unknown. Here, we use single-particle tracking to visualize diffusion and chromatin binding of Cas9 in living cells. We show that three-dimensional diffusion dominates Cas9 searching in vivo, and off-target binding events are, on average, short-lived (<1 second). Searching is dependent on the local chromatin environment, with less sampling and slower movement within heterochromatin. These results reveal how the bacterial Cas9 protein interrogates mammalian genomes and navigates eukaryotic chromatin structure.
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA, USA.
authors.forename: Spencer C
authors.initials: SC
authors.lastname: Knight
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
authors.forename: Liangqi
authors.initials: L
authors.lastname: Xie
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA. Transcriptional Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.
authors.forename: Wulan
authors.initials: W
authors.lastname: Deng
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Guglielmi
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
authors.forename: Lea B
authors.initials: LB
authors.lastname: Witkowsky
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
authors.forename: Lana
authors.initials: L
authors.lastname: Bosanac
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
authors.forename: Elisa T
authors.initials: ET
authors.lastname: Zhang
authors.affiliation: Laboratoire Physico-Chimie Curie, Institut Curie, Centre National de la Recherche Scientifique UMR 168, Paris, France.
authors.forename: Mohamed
authors.initials: M
authors.lastname: El Beheiry
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.
authors.forename: Jean-Baptiste
authors.initials: JB
authors.lastname: Masson
authors.affiliation: Transcriptional Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA. Laboratoire Physico-Chimie Curie, Institut Curie, Centre National de la Recherche Scientifique UMR 168, Paris, France.
authors.forename: Maxime
authors.initials: M
authors.lastname: Dahan
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA. Transcriptional Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA. liuz11@janelia.hhmi.org doudna@berkeley.edu jmlim@berkeley.edu.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA, USA. Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA. Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. Innovative Genomics Initiative, University of California, Berkeley, CA, USA. liuz11@janelia.hhmi.org doudna@berkeley.edu jmlim@berkeley.edu.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Doudna
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA. Transcriptional Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA. Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA. Li Ka Shing Biomedical and Health Sciences Center, University of California, Berkeley, CA, USA. liuz11@janelia.hhmi.org doudna@berkeley.edu jmlim@berkeley.edu.
authors.forename: Robert
authors.initials: R
authors.lastname: Tjian
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D053837:DNA Cleavage; D004720:Endonucleases / Q000737:chemistry / Q000378:metabolism*; D005818:Genetic Engineering; D016678:Genome; D051379:Mice; D059010:Single-Cell Analysis
chemical_list: D001426:Bacterial Proteins; D002843:Chromatin; D000076987:CRISPR-Associated Protein 9; C000600049:Cas9 protein, Francisella novicida; D004720:Endonucleases