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CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation

The CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) nuclease system represents an efficient tool for genome editing and gene function analysis. It consists of two components: single-guide RNA (sgRNA) and the enzyme Cas9. Typical sgRNA and Cas9 intracellular...

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Autores principales: Han, Xin, Liu, Zongbin, Jo, Myeong chan, Zhang, Kai, Li, Ying, Zeng, Zihua, Li, Nan, Zu, Youli, Qin, Lidong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Association for the Advancement of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4643799/
https://www.ncbi.nlm.nih.gov/pubmed/26601238
http://dx.doi.org/10.1126/sciadv.1500454
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author Han, Xin
Liu, Zongbin
Jo, Myeong chan
Zhang, Kai
Li, Ying
Zeng, Zihua
Li, Nan
Zu, Youli
Qin, Lidong
author_facet Han, Xin
Liu, Zongbin
Jo, Myeong chan
Zhang, Kai
Li, Ying
Zeng, Zihua
Li, Nan
Zu, Youli
Qin, Lidong
author_sort Han, Xin
collection PubMed
description The CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) nuclease system represents an efficient tool for genome editing and gene function analysis. It consists of two components: single-guide RNA (sgRNA) and the enzyme Cas9. Typical sgRNA and Cas9 intracellular delivery techniques are limited by their reliance on cell type and exogenous materials as well as their toxic effects on cells (for example, electroporation). We introduce and optimize a microfluidic membrane deformation method to deliver sgRNA and Cas9 into different cell types and achieve successful genome editing. This approach uses rapid cell mechanical deformation to generate transient membrane holes to enable delivery of biomaterials in the medium. We achieved high delivery efficiency of different macromolecules into different cell types, including hard-to-transfect lymphoma cells and embryonic stem cells, while maintaining high cell viability. With the advantages of broad applicability across different cell types, particularly hard-to-transfect cells, and flexibility of application, this method could potentially enable new avenues of biomedical research and gene targeting therapy such as mutation correction of disease genes through combination of the CRISPR-Cas9–mediated knockin system.
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spelling pubmed-46437992015-11-23 CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation Han, Xin Liu, Zongbin Jo, Myeong chan Zhang, Kai Li, Ying Zeng, Zihua Li, Nan Zu, Youli Qin, Lidong Sci Adv Research Articles The CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) nuclease system represents an efficient tool for genome editing and gene function analysis. It consists of two components: single-guide RNA (sgRNA) and the enzyme Cas9. Typical sgRNA and Cas9 intracellular delivery techniques are limited by their reliance on cell type and exogenous materials as well as their toxic effects on cells (for example, electroporation). We introduce and optimize a microfluidic membrane deformation method to deliver sgRNA and Cas9 into different cell types and achieve successful genome editing. This approach uses rapid cell mechanical deformation to generate transient membrane holes to enable delivery of biomaterials in the medium. We achieved high delivery efficiency of different macromolecules into different cell types, including hard-to-transfect lymphoma cells and embryonic stem cells, while maintaining high cell viability. With the advantages of broad applicability across different cell types, particularly hard-to-transfect cells, and flexibility of application, this method could potentially enable new avenues of biomedical research and gene targeting therapy such as mutation correction of disease genes through combination of the CRISPR-Cas9–mediated knockin system. American Association for the Advancement of Science 2015-08-14 /pmc/articles/PMC4643799/ /pubmed/26601238 http://dx.doi.org/10.1126/sciadv.1500454 Text en Copyright © 2015, The Authors http://creativecommons.org/licenses/by-nc/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license (http://creativecommons.org/licenses/by-nc/4.0/) , which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.
spellingShingle Research Articles
Han, Xin
Liu, Zongbin
Jo, Myeong chan
Zhang, Kai
Li, Ying
Zeng, Zihua
Li, Nan
Zu, Youli
Qin, Lidong
CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation
title CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation
title_full CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation
title_fullStr CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation
title_full_unstemmed CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation
title_short CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation
title_sort crispr-cas9 delivery to hard-to-transfect cells via membrane deformation
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4643799/
https://www.ncbi.nlm.nih.gov/pubmed/26601238
http://dx.doi.org/10.1126/sciadv.1500454
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