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Highly efficient generation of isogenic pluripotent stem cell models using prime editing

The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multicomponent editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-R...

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Autores principales: Li, Hanqin, Busquets, Oriol, Verma, Yogendra, Syed, Khaja Mohieddin, Kutnowski, Nitzan, Pangilinan, Gabriella R, Gilbert, Luke A, Bateup, Helen S, Rio, Donald C, Hockemeyer, Dirk, Soldner, Frank
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9584603/
https://www.ncbi.nlm.nih.gov/pubmed/36069759
http://dx.doi.org/10.7554/eLife.79208
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author Li, Hanqin
Busquets, Oriol
Verma, Yogendra
Syed, Khaja Mohieddin
Kutnowski, Nitzan
Pangilinan, Gabriella R
Gilbert, Luke A
Bateup, Helen S
Rio, Donald C
Hockemeyer, Dirk
Soldner, Frank
author_facet Li, Hanqin
Busquets, Oriol
Verma, Yogendra
Syed, Khaja Mohieddin
Kutnowski, Nitzan
Pangilinan, Gabriella R
Gilbert, Luke A
Bateup, Helen S
Rio, Donald C
Hockemeyer, Dirk
Soldner, Frank
author_sort Li, Hanqin
collection PubMed
description The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multicomponent editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site. Here, we systematically compared the editing efficiencies of PE to conventional gene editing methods in hPSCs. This analysis revealed that PE is overall more efficient and precise than homology-directed repair of site-specific nuclease-induced double-strand breaks. Specifically, PE is more effective in generating heterozygous editing events to create autosomal dominant disease-associated mutations. By stably integrating the nCas9-RT into hPSCs we achieved editing efficiencies equal to those reported for cancer cells, suggesting that the expression of the PE components, rather than cell-intrinsic features, limit PE in hPSCs. To improve the efficiency of PE in hPSCs, we optimized the delivery modalities for the PE components. Delivery of the nCas9-RT as mRNA combined with synthetically generated, chemically-modified pegRNAs and nicking guide RNAs improved editing efficiencies up to 13-fold compared with transfecting the PE components as plasmids or ribonucleoprotein particles. Finally, we demonstrated that this mRNA-based delivery approach can be used repeatedly to yield editing efficiencies exceeding 60% and to correct or introduce familial mutations causing Parkinson’s disease in hPSCs.
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spelling pubmed-95846032022-10-21 Highly efficient generation of isogenic pluripotent stem cell models using prime editing Li, Hanqin Busquets, Oriol Verma, Yogendra Syed, Khaja Mohieddin Kutnowski, Nitzan Pangilinan, Gabriella R Gilbert, Luke A Bateup, Helen S Rio, Donald C Hockemeyer, Dirk Soldner, Frank eLife Genetics and Genomics The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multicomponent editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site. Here, we systematically compared the editing efficiencies of PE to conventional gene editing methods in hPSCs. This analysis revealed that PE is overall more efficient and precise than homology-directed repair of site-specific nuclease-induced double-strand breaks. Specifically, PE is more effective in generating heterozygous editing events to create autosomal dominant disease-associated mutations. By stably integrating the nCas9-RT into hPSCs we achieved editing efficiencies equal to those reported for cancer cells, suggesting that the expression of the PE components, rather than cell-intrinsic features, limit PE in hPSCs. To improve the efficiency of PE in hPSCs, we optimized the delivery modalities for the PE components. Delivery of the nCas9-RT as mRNA combined with synthetically generated, chemically-modified pegRNAs and nicking guide RNAs improved editing efficiencies up to 13-fold compared with transfecting the PE components as plasmids or ribonucleoprotein particles. Finally, we demonstrated that this mRNA-based delivery approach can be used repeatedly to yield editing efficiencies exceeding 60% and to correct or introduce familial mutations causing Parkinson’s disease in hPSCs. eLife Sciences Publications, Ltd 2022-09-07 /pmc/articles/PMC9584603/ /pubmed/36069759 http://dx.doi.org/10.7554/eLife.79208 Text en © 2022, Li, Busquets et al https://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Genetics and Genomics
Li, Hanqin
Busquets, Oriol
Verma, Yogendra
Syed, Khaja Mohieddin
Kutnowski, Nitzan
Pangilinan, Gabriella R
Gilbert, Luke A
Bateup, Helen S
Rio, Donald C
Hockemeyer, Dirk
Soldner, Frank
Highly efficient generation of isogenic pluripotent stem cell models using prime editing
title Highly efficient generation of isogenic pluripotent stem cell models using prime editing
title_full Highly efficient generation of isogenic pluripotent stem cell models using prime editing
title_fullStr Highly efficient generation of isogenic pluripotent stem cell models using prime editing
title_full_unstemmed Highly efficient generation of isogenic pluripotent stem cell models using prime editing
title_short Highly efficient generation of isogenic pluripotent stem cell models using prime editing
title_sort highly efficient generation of isogenic pluripotent stem cell models using prime editing
topic Genetics and Genomics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9584603/
https://www.ncbi.nlm.nih.gov/pubmed/36069759
http://dx.doi.org/10.7554/eLife.79208
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