CRISPR/Cas9 ribonucleoprotein-mediated knockin generation in hTERT-RPE1 cells

Summary: hTERT-RPE1 cells are genetically stable near diploid cells widely used to model cell division, DNA repair, or ciliogenesis in a non-transformed context. However, poor transfectability and limited homology-directed repair capacity hamper their amenability to gene editing. Here, we describe a...

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Main Authors:	Sabrina Ghetti, Matteo Burigotto, Alessia Mattivi, Giovanni Magnani, Antonio Casini, Andrea Bianchi, Anna Cereseto, Luca L. Fava
Format:	Article
Language:	English
Published:	Elsevier 2021-06-01
Series:	STAR Protocols
Subjects:	Cell Biology CRISPR
Online Access:	http://www.sciencedirect.com/science/article/pii/S2666166721001143

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spelling	doaj-294fe2aa56894733919f4f9a45f9d3c42021-06-21T04:25:00ZengElsevierSTAR Protocols2666-16672021-06-0122100407CRISPR/Cas9 ribonucleoprotein-mediated knockin generation in hTERT-RPE1 cellsSabrina Ghetti0Matteo Burigotto1Alessia Mattivi2Giovanni Magnani3Antonio Casini4Andrea Bianchi5Anna Cereseto6Luca L. Fava7Armenise-Harvard Laboratory of Cell Division, Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento 38123, ItalyArmenise-Harvard Laboratory of Cell Division, Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento 38123, ItalyArmenise-Harvard Laboratory of Cell Division, Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento 38123, ItalyArmenise-Harvard Laboratory of Cell Division, Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento 38123, ItalyAlia Therapeutics, Trento 38123, ItalyLaboratory of Molecular Virology, Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento 38123, ItalyLaboratory of Molecular Virology, Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento 38123, ItalyArmenise-Harvard Laboratory of Cell Division, Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento 38123, Italy; Corresponding authorSummary: hTERT-RPE1 cells are genetically stable near diploid cells widely used to model cell division, DNA repair, or ciliogenesis in a non-transformed context. However, poor transfectability and limited homology-directed repair capacity hamper their amenability to gene editing. Here, we describe a protocol for rapid and efficient generation of diverse homozygous knockins. In contrast to other approaches, this strategy bypasses the need for molecular cloning. Our approach can also be applied to a variety of cell types including cancer and induced pluripotent stem cells (iPSCs).http://www.sciencedirect.com/science/article/pii/S2666166721001143Cell BiologyCRISPR
collection	DOAJ
language	English
format	Article
sources	DOAJ
author	Sabrina Ghetti Matteo Burigotto Alessia Mattivi Giovanni Magnani Antonio Casini Andrea Bianchi Anna Cereseto Luca L. Fava
spellingShingle	Sabrina Ghetti Matteo Burigotto Alessia Mattivi Giovanni Magnani Antonio Casini Andrea Bianchi Anna Cereseto Luca L. Fava CRISPR/Cas9 ribonucleoprotein-mediated knockin generation in hTERT-RPE1 cells STAR Protocols Cell Biology CRISPR
author_facet	Sabrina Ghetti Matteo Burigotto Alessia Mattivi Giovanni Magnani Antonio Casini Andrea Bianchi Anna Cereseto Luca L. Fava
author_sort	Sabrina Ghetti
title	CRISPR/Cas9 ribonucleoprotein-mediated knockin generation in hTERT-RPE1 cells
title_short	CRISPR/Cas9 ribonucleoprotein-mediated knockin generation in hTERT-RPE1 cells
title_full	CRISPR/Cas9 ribonucleoprotein-mediated knockin generation in hTERT-RPE1 cells
title_fullStr	CRISPR/Cas9 ribonucleoprotein-mediated knockin generation in hTERT-RPE1 cells
title_full_unstemmed	CRISPR/Cas9 ribonucleoprotein-mediated knockin generation in hTERT-RPE1 cells
title_sort	crispr/cas9 ribonucleoprotein-mediated knockin generation in htert-rpe1 cells
publisher	Elsevier
series	STAR Protocols
issn	2666-1667
publishDate	2021-06-01
description	Summary: hTERT-RPE1 cells are genetically stable near diploid cells widely used to model cell division, DNA repair, or ciliogenesis in a non-transformed context. However, poor transfectability and limited homology-directed repair capacity hamper their amenability to gene editing. Here, we describe a protocol for rapid and efficient generation of diverse homozygous knockins. In contrast to other approaches, this strategy bypasses the need for molecular cloning. Our approach can also be applied to a variety of cell types including cancer and induced pluripotent stem cells (iPSCs).
topic	Cell Biology CRISPR
url	http://www.sciencedirect.com/science/article/pii/S2666166721001143
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CRISPR/Cas9 ribonucleoprotein-mediated knockin generation in hTERT-RPE1 cells

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