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.

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), Article 14219. 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
• Publicly Available Date: Jan 14, 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.