Optimized electroporation of CRISPR-Cas9/gRNA ribonucleoprotein complex for selection-free homologous recombination in human pluripotent stem cells

Summary: Selection-free, scarless genome editing in human pluripotent stem cells (PSCs) by utilizing ribonucleoprotein (RNP) of CRISPR-Cas9 is a useful tool for a variety of applications. However, the process can be hampered by time-consuming subcloning steps and inefficient delivery of the RNP comp...

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Autores principales:	Huaigeng Xu, Yuto Kita, Uikyu Bang, Peter Gee, Akitsu Hotta
Formato:	article
Lenguaje:	EN
Publicado:	Elsevier 2021
Materias:	Cell isolation CRISPR Genetics Molecular Biology Stem Cells Science (General) Q1-390
Acceso en línea:	https://doaj.org/article/450590eb0003428ebdcbf99ca83667f2
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spelling	oai:doaj.org-article:450590eb0003428ebdcbf99ca83667f22021-11-18T04:51:44ZOptimized electroporation of CRISPR-Cas9/gRNA ribonucleoprotein complex for selection-free homologous recombination in human pluripotent stem cells2666-166710.1016/j.xpro.2021.100965https://doaj.org/article/450590eb0003428ebdcbf99ca83667f22021-12-01T00:00:00Zhttp://www.sciencedirect.com/science/article/pii/S2666166721006717https://doaj.org/toc/2666-1667Summary: Selection-free, scarless genome editing in human pluripotent stem cells (PSCs) by utilizing ribonucleoprotein (RNP) of CRISPR-Cas9 is a useful tool for a variety of applications. However, the process can be hampered by time-consuming subcloning steps and inefficient delivery of the RNP complex and ssDNA template. Here, we describe the optimized protocol to introduce a single nucleotide change or a loxP site insertion in feeder-free, xeno-free iPSCs by utilizing MaxCyte and 4D-Nucleofector electroporators.For complete details on the use and execution of this protocol, please refer to Kagita et al. (2021) and Xu et al. (2019).Huaigeng XuYuto KitaUikyu BangPeter GeeAkitsu HottaElsevierarticleCell isolationCRISPRGeneticsMolecular BiologyStem CellsScience (General)Q1-390ENSTAR Protocols, Vol 2, Iss 4, Pp 100965- (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Cell isolation CRISPR Genetics Molecular Biology Stem Cells Science (General) Q1-390
spellingShingle	Cell isolation CRISPR Genetics Molecular Biology Stem Cells Science (General) Q1-390 Huaigeng Xu Yuto Kita Uikyu Bang Peter Gee Akitsu Hotta Optimized electroporation of CRISPR-Cas9/gRNA ribonucleoprotein complex for selection-free homologous recombination in human pluripotent stem cells
description	Summary: Selection-free, scarless genome editing in human pluripotent stem cells (PSCs) by utilizing ribonucleoprotein (RNP) of CRISPR-Cas9 is a useful tool for a variety of applications. However, the process can be hampered by time-consuming subcloning steps and inefficient delivery of the RNP complex and ssDNA template. Here, we describe the optimized protocol to introduce a single nucleotide change or a loxP site insertion in feeder-free, xeno-free iPSCs by utilizing MaxCyte and 4D-Nucleofector electroporators.For complete details on the use and execution of this protocol, please refer to Kagita et al. (2021) and Xu et al. (2019).
format	article
author	Huaigeng Xu Yuto Kita Uikyu Bang Peter Gee Akitsu Hotta
author_facet	Huaigeng Xu Yuto Kita Uikyu Bang Peter Gee Akitsu Hotta
author_sort	Huaigeng Xu
title	Optimized electroporation of CRISPR-Cas9/gRNA ribonucleoprotein complex for selection-free homologous recombination in human pluripotent stem cells
title_short	Optimized electroporation of CRISPR-Cas9/gRNA ribonucleoprotein complex for selection-free homologous recombination in human pluripotent stem cells
title_full	Optimized electroporation of CRISPR-Cas9/gRNA ribonucleoprotein complex for selection-free homologous recombination in human pluripotent stem cells
title_fullStr	Optimized electroporation of CRISPR-Cas9/gRNA ribonucleoprotein complex for selection-free homologous recombination in human pluripotent stem cells
title_full_unstemmed	Optimized electroporation of CRISPR-Cas9/gRNA ribonucleoprotein complex for selection-free homologous recombination in human pluripotent stem cells
title_sort	optimized electroporation of crispr-cas9/grna ribonucleoprotein complex for selection-free homologous recombination in human pluripotent stem cells
publisher	Elsevier
publishDate	2021
url	https://doaj.org/article/450590eb0003428ebdcbf99ca83667f2
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Optimized electroporation of CRISPR-Cas9/gRNA ribonucleoprotein complex for selection-free homologous recombination in human pluripotent stem cells

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