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

Abstract 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 o...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Pan P. Li, Russell L. Margolis
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/b8d48dc1c8e94fc1be92edab617e9721
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:b8d48dc1c8e94fc1be92edab617e9721
record_format	dspace
spelling	oai:doaj.org-article:b8d48dc1c8e94fc1be92edab617e97212021-12-02T14:35:47ZUse of single guided Cas9 nickase to facilitate precise and efficient genome editing in human iPSCs10.1038/s41598-021-89312-22045-2322https://doaj.org/article/b8d48dc1c8e94fc1be92edab617e97212021-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-89312-2https://doaj.org/toc/2045-2322Abstract 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.Pan P. LiRussell L. MargolisNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Pan P. Li Russell L. Margolis Use of single guided Cas9 nickase to facilitate precise and efficient genome editing in human iPSCs
description	Abstract 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.
format	article
author	Pan P. Li Russell L. Margolis
author_facet	Pan P. Li Russell L. Margolis
author_sort	Pan P. Li
title	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_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_sort	use of single guided cas9 nickase to facilitate precise and efficient genome editing in human ipscs
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/b8d48dc1c8e94fc1be92edab617e9721
work_keys_str_mv	AT panpli useofsingleguidedcas9nickasetofacilitatepreciseandefficientgenomeeditinginhumanipscs AT russelllmargolis useofsingleguidedcas9nickasetofacilitatepreciseandefficientgenomeeditinginhumanipscs
_version_	1718391094865035264

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

Ejemplares similares