A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines

Abstract CRISPR prime-editors are emergent tools for genome editing and offer a versatile alternative approach to HDR-based genome engineering or DNA base-editors. However, sufficient prime-editor expression levels and availability of optimized transfection protocols may affect editing efficiencies,...

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Autores principales:	Reto Eggenschwiler, Thomas Gschwendtberger, Christian Felski, Christopher Jahn, Florian Langer, Jared Sterneckert, Andreas Hermann, Jonathan Lühmann, Doris Steinemann, Alexandra Haase, Ulrich Martin, Susanne Petri, Tobias Cantz
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/0135844e7f1c4db4843fcf46ee926ef6
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spelling	oai:doaj.org-article:0135844e7f1c4db4843fcf46ee926ef62021-11-14T12:19:37ZA selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines10.1038/s41598-021-01689-22045-2322https://doaj.org/article/0135844e7f1c4db4843fcf46ee926ef62021-11-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-01689-2https://doaj.org/toc/2045-2322Abstract CRISPR prime-editors are emergent tools for genome editing and offer a versatile alternative approach to HDR-based genome engineering or DNA base-editors. However, sufficient prime-editor expression levels and availability of optimized transfection protocols may affect editing efficiencies, especially in hard-to-transfect cells like hiPSC. Here, we show that piggyBac prime-editing (PB-PE) allows for sustained expression of prime-editors. We demonstrate proof-of-concept for PB-PE in a newly designed lentiviral traffic light reporter, which allows for estimation of gene correction and defective editing resulting in indels, based on expression of two different fluorophores. PB-PE can prime-edit more than 50% of hiPSC cells after antibiotic selection. We also show that improper design of pegRNA cannot simply be overcome by extended expression, but PB-PE allows for estimation of effectiveness of selected pegRNAs after few days of cultivation time. Finally, we implemented PB-PE for efficient editing of an amyotrophic lateral sclerosis-associated mutation in the SOD1-gene of patient-derived hiPSC. Progress of genome editing can be monitored by Sanger-sequencing, whereas PB-PE vectors can be removed after editing and excised cells can be enriched by fialuridine selection. Together, we present an efficient prime-editing toolbox, which can be robustly used in a variety of cell lines even when non-optimized transfection-protocols are applied.Reto EggenschwilerThomas GschwendtbergerChristian FelskiChristopher JahnFlorian LangerJared SterneckertAndreas HermannJonathan LühmannDoris SteinemannAlexandra HaaseUlrich MartinSusanne PetriTobias CantzNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-14 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Reto Eggenschwiler Thomas Gschwendtberger Christian Felski Christopher Jahn Florian Langer Jared Sterneckert Andreas Hermann Jonathan Lühmann Doris Steinemann Alexandra Haase Ulrich Martin Susanne Petri Tobias Cantz A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines
description	Abstract CRISPR prime-editors are emergent tools for genome editing and offer a versatile alternative approach to HDR-based genome engineering or DNA base-editors. However, sufficient prime-editor expression levels and availability of optimized transfection protocols may affect editing efficiencies, especially in hard-to-transfect cells like hiPSC. Here, we show that piggyBac prime-editing (PB-PE) allows for sustained expression of prime-editors. We demonstrate proof-of-concept for PB-PE in a newly designed lentiviral traffic light reporter, which allows for estimation of gene correction and defective editing resulting in indels, based on expression of two different fluorophores. PB-PE can prime-edit more than 50% of hiPSC cells after antibiotic selection. We also show that improper design of pegRNA cannot simply be overcome by extended expression, but PB-PE allows for estimation of effectiveness of selected pegRNAs after few days of cultivation time. Finally, we implemented PB-PE for efficient editing of an amyotrophic lateral sclerosis-associated mutation in the SOD1-gene of patient-derived hiPSC. Progress of genome editing can be monitored by Sanger-sequencing, whereas PB-PE vectors can be removed after editing and excised cells can be enriched by fialuridine selection. Together, we present an efficient prime-editing toolbox, which can be robustly used in a variety of cell lines even when non-optimized transfection-protocols are applied.
format	article
author	Reto Eggenschwiler Thomas Gschwendtberger Christian Felski Christopher Jahn Florian Langer Jared Sterneckert Andreas Hermann Jonathan Lühmann Doris Steinemann Alexandra Haase Ulrich Martin Susanne Petri Tobias Cantz
author_facet	Reto Eggenschwiler Thomas Gschwendtberger Christian Felski Christopher Jahn Florian Langer Jared Sterneckert Andreas Hermann Jonathan Lühmann Doris Steinemann Alexandra Haase Ulrich Martin Susanne Petri Tobias Cantz
author_sort	Reto Eggenschwiler
title	A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines
title_short	A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines
title_full	A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines
title_fullStr	A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines
title_full_unstemmed	A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines
title_sort	selectable all-in-one crispr prime editing piggybac transposon allows for highly efficient gene editing in human cell lines
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/0135844e7f1c4db4843fcf46ee926ef6
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A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines

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