IDEAS home Printed from https://ideas.repec.org/a/nat/natcom/v10y2019i1d10.1038_s41467-019-11514-0.html
   My bibliography  Save this article

Visualisation of dCas9 target search in vivo using an open-microscopy framework

Author

Listed:
  • Koen J. A. Martens

    (Wageningen University and Research
    Wageningen University and Research)

  • Sam P. B. Beljouw

    (Wageningen University and Research)

  • Simon Els

    (Wageningen University and Research
    NIZO food research)

  • Jochem N. A. Vink

    (Delft University of Technology)

  • Sander Baas

    (Wageningen University and Research)

  • George A. Vogelaar

    (Wageningen University and Research)

  • Stan J. J. Brouns

    (Delft University of Technology)

  • Peter Baarlen

    (Wageningen University and Research)

  • Michiel Kleerebezem

    (Wageningen University and Research)

  • Johannes Hohlbein

    (Wageningen University and Research
    Wageningen University and Research)

Abstract

CRISPR-Cas9 is widely used in genomic editing, but the kinetics of target search and its relation to the cellular concentration of Cas9 have remained elusive. Effective target search requires constant screening of the protospacer adjacent motif (PAM) and a 30 ms upper limit for screening was recently found. To further quantify the rapid switching between DNA-bound and freely-diffusing states of dCas9, we developed an open-microscopy framework, the miCube, and introduce Monte-Carlo diffusion distribution analysis (MC-DDA). Our analysis reveals that dCas9 is screening PAMs 40% of the time in Gram-positive Lactoccous lactis, averaging 17 ± 4 ms per binding event. Using heterogeneous dCas9 expression, we determine the number of cellular target-containing plasmids and derive the copy number dependent Cas9 cleavage. Furthermore, we show that dCas9 is not irreversibly bound to target sites but can still interfere with plasmid replication. Taken together, our quantitative data facilitates further optimization of the CRISPR-Cas toolbox.

Suggested Citation

  • Koen J. A. Martens & Sam P. B. Beljouw & Simon Els & Jochem N. A. Vink & Sander Baas & George A. Vogelaar & Stan J. J. Brouns & Peter Baarlen & Michiel Kleerebezem & Johannes Hohlbein, 2019. "Visualisation of dCas9 target search in vivo using an open-microscopy framework," Nature Communications, Nature, vol. 10(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-11514-0
    DOI: 10.1038/s41467-019-11514-0
    as

    Download full text from publisher

    File URL: https://www.nature.com/articles/s41467-019-11514-0
    File Function: Abstract
    Download Restriction: no
    File URL: https://libkey.io/10.1038/s41467-019-11514-0?utm_source=ideas
    LibKey link: if access is restricted and if your library uses this service, LibKey will redirect you to where you can use your library subscription to access this item
    ---><---

    More about this item

    Statistics

    Access and download statistics

    Corrections

    All material on this site has been provided by the respective publishers and authors. You can help correct errors and omissions. When requesting a correction, please mention this item's handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-11514-0. See general information about how to correct material in RePEc.

    If you have authored this item and are not yet registered with RePEc, we encourage you to do it here. This allows to link your profile to this item. It also allows you to accept potential citations to this item that we are uncertain about.

    We have no bibliographic references for this item. You can help adding them by using this form .

    If you know of missing items citing this one, you can help us creating those links by adding the relevant references in the same way as above, for each refering item. If you are a registered author of this item, you may also want to check the "citations" tab in your RePEc Author Service profile, as there may be some citations waiting for confirmation.

    For technical questions regarding this item, or to correct its authors, title, abstract, bibliographic or download information, contact: Sonal Shukla or Springer Nature Abstracting and Indexing (email available below). General contact details of provider: http://www.nature.com .

    Please note that corrections may take a couple of weeks to filter through the various RePEc services.

    IDEAS is a RePEc service. RePEc uses bibliographic data supplied by the respective publishers.