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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...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
eLife Sciences Publications, Ltd
2022
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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. |
format | Online Article Text |
id | pubmed-9584603 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | eLife Sciences Publications, Ltd |
record_format | MEDLINE/PubMed |
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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