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Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors

In contrast to CRISPR/Cas9 nucleases, CRISPR base editors (BE) and prime editors (PE) enable predefined nucleotide exchanges in genomic sequences without generating DNA double strand breaks. Here, we employed BE and PE mRNAs in conjunction with chemically synthesized sgRNAs and pegRNAs for efficient...

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Autores principales: Sürün, Duran, Schneider, Aksana, Mircetic, Jovan, Neumann, Katrin, Lansing, Felix, Paszkowski-Rogacz, Maciej, Hänchen, Vanessa, Lee-Kirsch, Min Ae, Buchholz, Frank
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7288465/
https://www.ncbi.nlm.nih.gov/pubmed/32384610
http://dx.doi.org/10.3390/genes11050511
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author Sürün, Duran
Schneider, Aksana
Mircetic, Jovan
Neumann, Katrin
Lansing, Felix
Paszkowski-Rogacz, Maciej
Hänchen, Vanessa
Lee-Kirsch, Min Ae
Buchholz, Frank
author_facet Sürün, Duran
Schneider, Aksana
Mircetic, Jovan
Neumann, Katrin
Lansing, Felix
Paszkowski-Rogacz, Maciej
Hänchen, Vanessa
Lee-Kirsch, Min Ae
Buchholz, Frank
author_sort Sürün, Duran
collection PubMed
description In contrast to CRISPR/Cas9 nucleases, CRISPR base editors (BE) and prime editors (PE) enable predefined nucleotide exchanges in genomic sequences without generating DNA double strand breaks. Here, we employed BE and PE mRNAs in conjunction with chemically synthesized sgRNAs and pegRNAs for efficient editing of human induced pluripotent stem cells (iPSC). Whereas we were unable to correct a disease-causing mutation in patient derived iPSCs using a CRISPR/Cas9 nuclease approach, we corrected the mutation back to wild type with high efficiency utilizing an adenine BE. We also used adenine and cytosine BEs to introduce nine different cancer associated TP53 mutations into human iPSCs with up to 90% efficiency, generating a panel of cell lines to investigate the biology of these mutations in an isogenic background. Finally, we pioneered the use of prime editing in human iPSCs, opening this important cell type for the precise modification of nucleotides not addressable by BEs and to multiple nucleotide exchanges. These approaches eliminate the necessity of deriving disease specific iPSCs from human donors and allows the comparison of different disease-causing mutations in isogenic genetic backgrounds.
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spelling pubmed-72884652020-06-17 Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors Sürün, Duran Schneider, Aksana Mircetic, Jovan Neumann, Katrin Lansing, Felix Paszkowski-Rogacz, Maciej Hänchen, Vanessa Lee-Kirsch, Min Ae Buchholz, Frank Genes (Basel) Article In contrast to CRISPR/Cas9 nucleases, CRISPR base editors (BE) and prime editors (PE) enable predefined nucleotide exchanges in genomic sequences without generating DNA double strand breaks. Here, we employed BE and PE mRNAs in conjunction with chemically synthesized sgRNAs and pegRNAs for efficient editing of human induced pluripotent stem cells (iPSC). Whereas we were unable to correct a disease-causing mutation in patient derived iPSCs using a CRISPR/Cas9 nuclease approach, we corrected the mutation back to wild type with high efficiency utilizing an adenine BE. We also used adenine and cytosine BEs to introduce nine different cancer associated TP53 mutations into human iPSCs with up to 90% efficiency, generating a panel of cell lines to investigate the biology of these mutations in an isogenic background. Finally, we pioneered the use of prime editing in human iPSCs, opening this important cell type for the precise modification of nucleotides not addressable by BEs and to multiple nucleotide exchanges. These approaches eliminate the necessity of deriving disease specific iPSCs from human donors and allows the comparison of different disease-causing mutations in isogenic genetic backgrounds. MDPI 2020-05-06 /pmc/articles/PMC7288465/ /pubmed/32384610 http://dx.doi.org/10.3390/genes11050511 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Sürün, Duran
Schneider, Aksana
Mircetic, Jovan
Neumann, Katrin
Lansing, Felix
Paszkowski-Rogacz, Maciej
Hänchen, Vanessa
Lee-Kirsch, Min Ae
Buchholz, Frank
Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors
title Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors
title_full Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors
title_fullStr Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors
title_full_unstemmed Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors
title_short Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors
title_sort efficient generation and correction of mutations in human ips cells utilizing mrnas of crispr base editors and prime editors
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7288465/
https://www.ncbi.nlm.nih.gov/pubmed/32384610
http://dx.doi.org/10.3390/genes11050511
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