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

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 s...

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Autores principales: Xu, Huaigeng, Kita, Yuto, Bang, Uikyu, Gee, Peter, Hotta, Akitsu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2021
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8605105/
https://www.ncbi.nlm.nih.gov/pubmed/34825222
http://dx.doi.org/10.1016/j.xpro.2021.100965
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author Xu, Huaigeng
Kita, Yuto
Bang, Uikyu
Gee, Peter
Hotta, Akitsu
author_facet Xu, Huaigeng
Kita, Yuto
Bang, Uikyu
Gee, Peter
Hotta, Akitsu
author_sort Xu, Huaigeng
collection PubMed
description 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).
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spelling pubmed-86051052021-11-24 Optimized electroporation of CRISPR-Cas9/gRNA ribonucleoprotein complex for selection-free homologous recombination in human pluripotent stem cells Xu, Huaigeng Kita, Yuto Bang, Uikyu Gee, Peter Hotta, Akitsu STAR Protoc Protocol 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). Elsevier 2021-11-16 /pmc/articles/PMC8605105/ /pubmed/34825222 http://dx.doi.org/10.1016/j.xpro.2021.100965 Text en © 2021 The Author(s) https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Protocol
Xu, Huaigeng
Kita, Yuto
Bang, Uikyu
Gee, Peter
Hotta, Akitsu
Optimized electroporation of CRISPR-Cas9/gRNA ribonucleoprotein complex for selection-free homologous recombination in human pluripotent stem cells
title 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_short 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
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8605105/
https://www.ncbi.nlm.nih.gov/pubmed/34825222
http://dx.doi.org/10.1016/j.xpro.2021.100965
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