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Optimised insert design for improved single-molecule imaging and quantification through CRISPR-Cas9 mediated knock-in

Khan, Abdullah O.; White, Carl W.; Pike, Jeremy A.; Yule, Jack; Slater, Alexandre; Hill, Stephen J.; Poulter, Natalie S.; Thomas, Steven G.; Morgan, Neil V.

Authors

Abdullah O. Khan

Carl W. White

Jeremy A. Pike

Jack Yule

Alexandre Slater

STEPHEN HILL steve.hill@nottingham.ac.uk
Professor of Molecular Pharmacology

Natalie S. Poulter

Steven G. Thomas

Neil V. Morgan



Abstract

© 2019, The Author(s). The use of CRISPR-Cas9 genome editing to introduce endogenously expressed tags has the potential to address a number of the classical limitations of single molecule localisation microscopy. In this work we present the first systematic comparison of inserts introduced through CRISPR-knock in, with the aim of optimising this approach for single molecule imaging. We show that more highly monomeric and codon optimised variants of mEos result in improved expression at the TubA1B locus, despite the use of identical guides, homology templates, and selection strategies. We apply this approach to target the G protein-coupled receptor (GPCR) CXCR4 and show a further insert dependent effect on expression and protein function. Finally, we show that compared to over-expressed CXCR4, endogenously labelled samples allow for accurate single molecule quantification on ligand treatment. This suggests that despite the complications evident in CRISPR mediated labelling, the development of CRISPR-PALM has substantial quantitative benefits.

Citation

Khan, A. O., White, C. W., Pike, J. A., Yule, J., Slater, A., Hill, S. J., …Morgan, N. V. (2019). Optimised insert design for improved single-molecule imaging and quantification through CRISPR-Cas9 mediated knock-in. Scientific Reports, 9(1), https://doi.org/10.1038/s41598-019-50733-9

Journal Article Type Article
Acceptance Date Sep 18, 2019
Online Publication Date Oct 2, 2019
Publication Date Dec 1, 2019
Deposit Date Jan 13, 2020
Journal Scientific Reports
Print ISSN 2045-2322
Electronic ISSN 2045-2322
Publisher Nature Publishing Group
Peer Reviewed Peer Reviewed
Volume 9
Issue 1
Article Number 14219
DOI https://doi.org/10.1038/s41598-019-50733-9
Public URL https://nottingham-repository.worktribe.com/output/3110129
Publisher URL https://www.nature.com/articles/s41598-019-50733-9
Additional Information Received: 5 April 2019; Accepted: 18 September 2019; First Online: 2 October 2019; : The authors declare no competing interests.

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