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Use of single guided Cas9 nickase to facilitate precise and efficient genome editing in human iPSCs

Cas9 nucleases permit rapid and efficient generation of gene-edited cell lines. However, in typical protocols, mutations are intentionally introduced into the donor template to avoid the cleavage of donor template or re-cleavage of the successfully edited allele, compromising the fidelity of the iso...

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Autores principales: Li, Pan P., Margolis, Russell L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8110799/
https://www.ncbi.nlm.nih.gov/pubmed/33972655
http://dx.doi.org/10.1038/s41598-021-89312-2
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author Li, Pan P.
Margolis, Russell L.
author_facet Li, Pan P.
Margolis, Russell L.
author_sort Li, Pan P.
collection PubMed
description Cas9 nucleases permit rapid and efficient generation of gene-edited cell lines. However, in typical protocols, mutations are intentionally introduced into the donor template to avoid the cleavage of donor template or re-cleavage of the successfully edited allele, compromising the fidelity of the isogenic lines generated. In addition, the double-stranded breaks (DSBs) used for editing can introduce undesirable “on-target” indels within the second allele of successfully modified cells via non-homologous end joining (NHEJ). To address these problems, we present an optimized protocol for precise genome editing in human iPSCs that employs (1) single guided Cas9 nickase to generate single-stranded breaks (SSBs), (2) transient overexpression of BCL-XL to enhance survival post electroporation, and (3) the PiggyBac transposon system for seamless removal of dual selection markers. We have used this method to modify the length of the CAG repeat contained in exon 7 of PPP2R2B. When longer than 43 triplets, this repeat causes the neurodegenerative disorder spinocerebellar ataxia type 12 (SCA12); our goal was to seamlessly introduce the SCA12 mutation into a human control iPSC line. With our protocol, ~ 15% of iPSC clones selected had the desired gene editing without “on target” indels or off-target changes, and without the deliberate introduction of mutations via the donor template. This method will allow for the precise and efficient editing of human iPSCs for disease modeling and other purposes.
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spelling pubmed-81107992021-05-12 Use of single guided Cas9 nickase to facilitate precise and efficient genome editing in human iPSCs Li, Pan P. Margolis, Russell L. Sci Rep Article Cas9 nucleases permit rapid and efficient generation of gene-edited cell lines. However, in typical protocols, mutations are intentionally introduced into the donor template to avoid the cleavage of donor template or re-cleavage of the successfully edited allele, compromising the fidelity of the isogenic lines generated. In addition, the double-stranded breaks (DSBs) used for editing can introduce undesirable “on-target” indels within the second allele of successfully modified cells via non-homologous end joining (NHEJ). To address these problems, we present an optimized protocol for precise genome editing in human iPSCs that employs (1) single guided Cas9 nickase to generate single-stranded breaks (SSBs), (2) transient overexpression of BCL-XL to enhance survival post electroporation, and (3) the PiggyBac transposon system for seamless removal of dual selection markers. We have used this method to modify the length of the CAG repeat contained in exon 7 of PPP2R2B. When longer than 43 triplets, this repeat causes the neurodegenerative disorder spinocerebellar ataxia type 12 (SCA12); our goal was to seamlessly introduce the SCA12 mutation into a human control iPSC line. With our protocol, ~ 15% of iPSC clones selected had the desired gene editing without “on target” indels or off-target changes, and without the deliberate introduction of mutations via the donor template. This method will allow for the precise and efficient editing of human iPSCs for disease modeling and other purposes. Nature Publishing Group UK 2021-05-10 /pmc/articles/PMC8110799/ /pubmed/33972655 http://dx.doi.org/10.1038/s41598-021-89312-2 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Li, Pan P.
Margolis, Russell L.
Use of single guided Cas9 nickase to facilitate precise and efficient genome editing in human iPSCs
title Use of single guided Cas9 nickase to facilitate precise and efficient genome editing in human iPSCs
title_full Use of single guided Cas9 nickase to facilitate precise and efficient genome editing in human iPSCs
title_fullStr Use of single guided Cas9 nickase to facilitate precise and efficient genome editing in human iPSCs
title_full_unstemmed Use of single guided Cas9 nickase to facilitate precise and efficient genome editing in human iPSCs
title_short Use of single guided Cas9 nickase to facilitate precise and efficient genome editing in human iPSCs
title_sort use of single guided cas9 nickase to facilitate precise and efficient genome editing in human ipscs
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8110799/
https://www.ncbi.nlm.nih.gov/pubmed/33972655
http://dx.doi.org/10.1038/s41598-021-89312-2
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