CRISPR-Cas9 Mediated Labelling Allows for Single Molecule Imaging and Resolution

Abstract Single molecule imaging approaches like dSTORM and PALM resolve structures at 10–20 nm, and allow for unique insights into protein stoichiometry and spatial relationships. However, key obstacles remain in developing highly accurate quantitative single molecule approaches. The genomic taggin...

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Autores principales:	Abdullah O. Khan, Victoria A. Simms, Jeremy A. Pike, Steven G. Thomas, Neil V. Morgan
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/ffd5b73c61df47d892c81639af4e4a98
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spelling	oai:doaj.org-article:ffd5b73c61df47d892c81639af4e4a982021-12-02T15:18:54ZCRISPR-Cas9 Mediated Labelling Allows for Single Molecule Imaging and Resolution10.1038/s41598-017-08493-x2045-2322https://doaj.org/article/ffd5b73c61df47d892c81639af4e4a982017-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-08493-xhttps://doaj.org/toc/2045-2322Abstract Single molecule imaging approaches like dSTORM and PALM resolve structures at 10–20 nm, and allow for unique insights into protein stoichiometry and spatial relationships. However, key obstacles remain in developing highly accurate quantitative single molecule approaches. The genomic tagging of PALM fluorophores through CRISPR-Cas9 offers an excellent opportunity for generating stable cell lines expressing a defined single molecule probe at endogenous levels, without the biological disruption and variability inherent to transfection. A fundamental question is whether these comparatively low levels of expression can successfully satisfy the stringent labelling demands of super-resolution SMLM. Here we apply CRISPR-Cas9 gene editing to tag a cytoskeletal protein (α-tubulin) and demonstrate a relationship between expression level and the subsequent quality of PALM imaging, and that spatial resolutions comparable to dSTORM can be achieved with CRISPR-PALM. Our approach shows a relationship between choice of tag and the total expression of labelled protein, which has important implications for the development of future PALM tags. CRISPR-PALM allows for nanoscopic spatial resolution and the unique quantitative benefits of single molecule localization microscopy through endogenous expression, as well as the capacity for super-resolved live cell imaging.Abdullah O. KhanVictoria A. SimmsJeremy A. PikeSteven G. ThomasNeil V. MorganNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-9 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Abdullah O. Khan Victoria A. Simms Jeremy A. Pike Steven G. Thomas Neil V. Morgan CRISPR-Cas9 Mediated Labelling Allows for Single Molecule Imaging and Resolution
description	Abstract Single molecule imaging approaches like dSTORM and PALM resolve structures at 10–20 nm, and allow for unique insights into protein stoichiometry and spatial relationships. However, key obstacles remain in developing highly accurate quantitative single molecule approaches. The genomic tagging of PALM fluorophores through CRISPR-Cas9 offers an excellent opportunity for generating stable cell lines expressing a defined single molecule probe at endogenous levels, without the biological disruption and variability inherent to transfection. A fundamental question is whether these comparatively low levels of expression can successfully satisfy the stringent labelling demands of super-resolution SMLM. Here we apply CRISPR-Cas9 gene editing to tag a cytoskeletal protein (α-tubulin) and demonstrate a relationship between expression level and the subsequent quality of PALM imaging, and that spatial resolutions comparable to dSTORM can be achieved with CRISPR-PALM. Our approach shows a relationship between choice of tag and the total expression of labelled protein, which has important implications for the development of future PALM tags. CRISPR-PALM allows for nanoscopic spatial resolution and the unique quantitative benefits of single molecule localization microscopy through endogenous expression, as well as the capacity for super-resolved live cell imaging.
format	article
author	Abdullah O. Khan Victoria A. Simms Jeremy A. Pike Steven G. Thomas Neil V. Morgan
author_facet	Abdullah O. Khan Victoria A. Simms Jeremy A. Pike Steven G. Thomas Neil V. Morgan
author_sort	Abdullah O. Khan
title	CRISPR-Cas9 Mediated Labelling Allows for Single Molecule Imaging and Resolution
title_short	CRISPR-Cas9 Mediated Labelling Allows for Single Molecule Imaging and Resolution
title_full	CRISPR-Cas9 Mediated Labelling Allows for Single Molecule Imaging and Resolution
title_fullStr	CRISPR-Cas9 Mediated Labelling Allows for Single Molecule Imaging and Resolution
title_full_unstemmed	CRISPR-Cas9 Mediated Labelling Allows for Single Molecule Imaging and Resolution
title_sort	crispr-cas9 mediated labelling allows for single molecule imaging and resolution
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/ffd5b73c61df47d892c81639af4e4a98
work_keys_str_mv	AT abdullahokhan crisprcas9mediatedlabellingallowsforsinglemoleculeimagingandresolution AT victoriaasimms crisprcas9mediatedlabellingallowsforsinglemoleculeimagingandresolution AT jeremyapike crisprcas9mediatedlabellingallowsforsinglemoleculeimagingandresolution AT stevengthomas crisprcas9mediatedlabellingallowsforsinglemoleculeimagingandresolution AT neilvmorgan crisprcas9mediatedlabellingallowsforsinglemoleculeimagingandresolution
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CRISPR-Cas9 Mediated Labelling Allows for Single Molecule Imaging and Resolution

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