Dynamics of CRISPR-Cas9 genome interrogation in living cells

Spencer C Knight; Liangqi Xie; Wulan Deng; Benjamin Guglielmi; Lea B Witkowsky; Lana Bosanac; Elisa T Zhang; Mohamed El Beheiry; Jean-Baptiste Masson; Maxime Dahan; Zhe Liu; Jennifer A Doudna; Robert Tjian

doi:10.1126/science.aac6572

Dynamics of CRISPR-Cas9 genome interrogation in living cells

Science. 2015 Nov 13;350(6262):823-6. doi: 10.1126/science.aac6572.

Authors

Affiliations

¹ Department of Chemistry, University of California, Berkeley, CA, USA.
² Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
³ Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA. Transcriptional Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.
⁴ Laboratoire Physico-Chimie Curie, Institut Curie, Centre National de la Recherche Scientifique UMR 168, Paris, France.
⁵ Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.
⁶ 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.
⁷ 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.
⁸ 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.
⁹ 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.

PMID: 26564855
DOI: 10.1126/science.aac6572

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.

Publication types

Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

3T3 Cells
Animals
Bacterial Proteins / chemistry
Bacterial Proteins / metabolism*
CRISPR-Associated Protein 9
CRISPR-Cas Systems*
Chromatin / chemistry
Chromatin / metabolism*
Chromatin / ultrastructure
Clustered Regularly Interspaced Short Palindromic Repeats
DNA Cleavage*
Endonucleases / chemistry
Endonucleases / metabolism*
Genetic Engineering*
Genome
Mice
Single-Cell Analysis

Substances

Bacterial Proteins
Chromatin
CRISPR-Associated Protein 9
Cas9 protein, Francisella novicida
Endonucleases

Grants and funding

Howard Hughes Medical Institute/United States