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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...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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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. |
format | Online Article Text |
id | pubmed-7288465 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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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