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

Koen J.A. Martens, Sam P.B. van Beljouw, Simon van der Els, Jochem N.A. Vink, Sander Baas, George A. Vogelaar, Stan J.J. Brouns, Peter van Baarlen, Michiel Kleerebezem, Johannes Hohlbein

Research output: Contribution to journalArticleAcademicpeer-review

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.

Original languageEnglish
Article number3552
JournalNature Communications
Volume10
Issue number1
DOIs
Publication statusPublished - 7 Aug 2019

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Clustered Regularly Interspaced Short Palindromic Repeats
Microscopy
Microscopic examination
Screening
Plasmids
Visualization
microscopy
plasmids
screening
Pulse amplitude modulation
pulse amplitude modulation
editing
DNA
cleavage
Kinetics
deoxyribonucleic acid
optimization
kinetics

Cite this

@article{84c0813410374125a62a5f24dde558b6,
title = "Visualisation of dCas9 target search in vivo using an open-microscopy framework",
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.",
author = "Martens, {Koen J.A.} and {van Beljouw}, {Sam P.B.} and {van der Els}, Simon and Vink, {Jochem N.A.} and Sander Baas and Vogelaar, {George A.} and Brouns, {Stan J.J.} and {van Baarlen}, Peter and Michiel Kleerebezem and Johannes Hohlbein",
year = "2019",
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doi = "10.1038/s41467-019-11514-0",
language = "English",
volume = "10",
journal = "Nature Communications",
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Visualisation of dCas9 target search in vivo using an open-microscopy framework. / Martens, Koen J.A.; van Beljouw, Sam P.B.; van der Els, Simon; Vink, Jochem N.A.; Baas, Sander; Vogelaar, George A.; Brouns, Stan J.J.; van Baarlen, Peter; Kleerebezem, Michiel; Hohlbein, Johannes.

In: Nature Communications, Vol. 10, No. 1, 3552, 07.08.2019.

Research output: Contribution to journalArticleAcademicpeer-review

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T1 - Visualisation of dCas9 target search in vivo using an open-microscopy framework

AU - Martens, Koen J.A.

AU - van Beljouw, Sam P.B.

AU - van der Els, Simon

AU - Vink, Jochem N.A.

AU - Baas, Sander

AU - Vogelaar, George A.

AU - Brouns, Stan J.J.

AU - van Baarlen, Peter

AU - Kleerebezem, Michiel

AU - Hohlbein, Johannes

PY - 2019/8/7

Y1 - 2019/8/7

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AB - 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.

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