Cargando…
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...
Autores principales: | , , , , , , , , |
---|---|
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 |
_version_ | 1782400568699912192 |
---|---|
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. |
format | Online Article Text |
id | pubmed-4643799 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | American Association for the Advancement of Science |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT hanxin crisprcas9deliverytohardtotransfectcellsviamembranedeformation AT liuzongbin crisprcas9deliverytohardtotransfectcellsviamembranedeformation AT jomyeongchan crisprcas9deliverytohardtotransfectcellsviamembranedeformation AT zhangkai crisprcas9deliverytohardtotransfectcellsviamembranedeformation AT liying crisprcas9deliverytohardtotransfectcellsviamembranedeformation AT zengzihua crisprcas9deliverytohardtotransfectcellsviamembranedeformation AT linan crisprcas9deliverytohardtotransfectcellsviamembranedeformation AT zuyouli crisprcas9deliverytohardtotransfectcellsviamembranedeformation AT qinlidong crisprcas9deliverytohardtotransfectcellsviamembranedeformation |