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Improved and Flexible HDR Editing by Targeting Introns in iPSCs

Highly efficient gene knockout (KO) editing of CRISPR–Cas9 has been achieved in iPSCs, whereas homology-directed repair (HDR)-mediated precise gene knock-in (KI) and high-level expression are still bottlenecks for the clinical applications of iPSCs. Here, we developed a novel editing strategy that t...

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Detalles Bibliográficos
Autores principales: Fu, Juan, Fu, Ya-Wen, Zhao, Juan-Juan, Yang, Zhi-Xue, Li, Si-Ang, Li, Guo-Hua, Quan, Zi-Jun, Zhang, Feng, Zhang, Jian-Ping, Zhang, Xiao-Bing, Sun, Chang-Kai
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer US 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9209395/
https://www.ncbi.nlm.nih.gov/pubmed/35089463
http://dx.doi.org/10.1007/s12015-022-10331-1
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author Fu, Juan
Fu, Ya-Wen
Zhao, Juan-Juan
Yang, Zhi-Xue
Li, Si-Ang
Li, Guo-Hua
Quan, Zi-Jun
Zhang, Feng
Zhang, Jian-Ping
Zhang, Xiao-Bing
Sun, Chang-Kai
author_facet Fu, Juan
Fu, Ya-Wen
Zhao, Juan-Juan
Yang, Zhi-Xue
Li, Si-Ang
Li, Guo-Hua
Quan, Zi-Jun
Zhang, Feng
Zhang, Jian-Ping
Zhang, Xiao-Bing
Sun, Chang-Kai
author_sort Fu, Juan
collection PubMed
description Highly efficient gene knockout (KO) editing of CRISPR–Cas9 has been achieved in iPSCs, whereas homology-directed repair (HDR)-mediated precise gene knock-in (KI) and high-level expression are still bottlenecks for the clinical applications of iPSCs. Here, we developed a novel editing strategy that targets introns. By targeting the intron before the stop codon, this approach tolerates reading frameshift mutations caused by nonhomologous end-joining (NHEJ)-mediated indels, thereby maintaining gene integrity without damaging the non-HDR-edited allele. Furthermore, to increase the flexibility and screen for the best intron-targeting sgRNA, we designed an HDR donor with an artificial intron in place of the endogenous intron. The presence of artificial introns, particularly an intron that carries an enhancer element, significantly increased the reporter expression levels in iPSCs compared to the intron-deleted control. In addition, a combination of the small molecules M3814 and trichostatin A (TSA) significantly improves HDR efficiency by inhibiting NHEJ. These results should find applications in gene therapy and basic research, such as creating reporter cell lines. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12015-022-10331-1.
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spelling pubmed-92093952022-06-22 Improved and Flexible HDR Editing by Targeting Introns in iPSCs Fu, Juan Fu, Ya-Wen Zhao, Juan-Juan Yang, Zhi-Xue Li, Si-Ang Li, Guo-Hua Quan, Zi-Jun Zhang, Feng Zhang, Jian-Ping Zhang, Xiao-Bing Sun, Chang-Kai Stem Cell Rev Rep Article Highly efficient gene knockout (KO) editing of CRISPR–Cas9 has been achieved in iPSCs, whereas homology-directed repair (HDR)-mediated precise gene knock-in (KI) and high-level expression are still bottlenecks for the clinical applications of iPSCs. Here, we developed a novel editing strategy that targets introns. By targeting the intron before the stop codon, this approach tolerates reading frameshift mutations caused by nonhomologous end-joining (NHEJ)-mediated indels, thereby maintaining gene integrity without damaging the non-HDR-edited allele. Furthermore, to increase the flexibility and screen for the best intron-targeting sgRNA, we designed an HDR donor with an artificial intron in place of the endogenous intron. The presence of artificial introns, particularly an intron that carries an enhancer element, significantly increased the reporter expression levels in iPSCs compared to the intron-deleted control. In addition, a combination of the small molecules M3814 and trichostatin A (TSA) significantly improves HDR efficiency by inhibiting NHEJ. These results should find applications in gene therapy and basic research, such as creating reporter cell lines. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12015-022-10331-1. Springer US 2022-01-28 2022 /pmc/articles/PMC9209395/ /pubmed/35089463 http://dx.doi.org/10.1007/s12015-022-10331-1 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Fu, Juan
Fu, Ya-Wen
Zhao, Juan-Juan
Yang, Zhi-Xue
Li, Si-Ang
Li, Guo-Hua
Quan, Zi-Jun
Zhang, Feng
Zhang, Jian-Ping
Zhang, Xiao-Bing
Sun, Chang-Kai
Improved and Flexible HDR Editing by Targeting Introns in iPSCs
title Improved and Flexible HDR Editing by Targeting Introns in iPSCs
title_full Improved and Flexible HDR Editing by Targeting Introns in iPSCs
title_fullStr Improved and Flexible HDR Editing by Targeting Introns in iPSCs
title_full_unstemmed Improved and Flexible HDR Editing by Targeting Introns in iPSCs
title_short Improved and Flexible HDR Editing by Targeting Introns in iPSCs
title_sort improved and flexible hdr editing by targeting introns in ipscs
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9209395/
https://www.ncbi.nlm.nih.gov/pubmed/35089463
http://dx.doi.org/10.1007/s12015-022-10331-1
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