A precise gene delivery approach for human induced pluripotent stem cells using Cas9 RNP complex and recombinant AAV6 donor vectors.
Genome editing in human induced pluripotent stem cells (hiPSCs) offers a potential tool for studying gene functions in disease models and correcting genetic mutations for cell-based therapy. Precise transgene insertion in hiPSCs represents a significant challenge. In the past decade, viral transduct...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2022-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0270963 |
| _version_ | 1818041131596775424 |
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| author | Koollawat Chupradit Nontaphat Thongsin Chatchai Tayapiwatana Methichit Wattanapanitch |
| author_facet | Koollawat Chupradit Nontaphat Thongsin Chatchai Tayapiwatana Methichit Wattanapanitch |
| author_sort | Koollawat Chupradit |
| collection | DOAJ |
| description | Genome editing in human induced pluripotent stem cells (hiPSCs) offers a potential tool for studying gene functions in disease models and correcting genetic mutations for cell-based therapy. Precise transgene insertion in hiPSCs represents a significant challenge. In the past decade, viral transduction has been widely used due to its high transduction efficiency; however, it can result in random transgene integration and variable transgene copy numbers. Non-viral-based strategies are generally safer but limited by their low transfection efficiency in hiPSCs. Recently, genome engineering using adeno-associated virus (AAV) vectors has emerged as a promising gene delivery approach due to AAVs' low immunogenicity, toxicity, and ability to infect a broad range of cells. The following protocol describes the workflow for genome editing in hiPSCs using the CRISPR/Cas9 ribonucleoprotein (RNP) complex combined with the recombinant AAV serotype 6 (AAV6) donor vectors to introduce a gene of interest (GOI) fused with mCherry fluorescent reporter gene into the AAVS1 safe harbor site. This approach leads to efficient transgene insertion and is applicable to precise genome editing of hiPSCs or other types of stem cells for research purposes. |
| first_indexed | 2024-12-10T08:25:33Z |
| format | Article |
| id | doaj.art-500251c425cd4c6f84a1aef45a97ec28 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-10T08:25:33Z |
| publishDate | 2022-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-500251c425cd4c6f84a1aef45a97ec282022-12-22T01:56:14ZengPublic Library of Science (PLoS)PLoS ONE1932-62032022-01-01177e027096310.1371/journal.pone.0270963A precise gene delivery approach for human induced pluripotent stem cells using Cas9 RNP complex and recombinant AAV6 donor vectors.Koollawat ChupraditNontaphat ThongsinChatchai TayapiwatanaMethichit WattanapanitchGenome editing in human induced pluripotent stem cells (hiPSCs) offers a potential tool for studying gene functions in disease models and correcting genetic mutations for cell-based therapy. Precise transgene insertion in hiPSCs represents a significant challenge. In the past decade, viral transduction has been widely used due to its high transduction efficiency; however, it can result in random transgene integration and variable transgene copy numbers. Non-viral-based strategies are generally safer but limited by their low transfection efficiency in hiPSCs. Recently, genome engineering using adeno-associated virus (AAV) vectors has emerged as a promising gene delivery approach due to AAVs' low immunogenicity, toxicity, and ability to infect a broad range of cells. The following protocol describes the workflow for genome editing in hiPSCs using the CRISPR/Cas9 ribonucleoprotein (RNP) complex combined with the recombinant AAV serotype 6 (AAV6) donor vectors to introduce a gene of interest (GOI) fused with mCherry fluorescent reporter gene into the AAVS1 safe harbor site. This approach leads to efficient transgene insertion and is applicable to precise genome editing of hiPSCs or other types of stem cells for research purposes.https://doi.org/10.1371/journal.pone.0270963 |
| spellingShingle | Koollawat Chupradit Nontaphat Thongsin Chatchai Tayapiwatana Methichit Wattanapanitch A precise gene delivery approach for human induced pluripotent stem cells using Cas9 RNP complex and recombinant AAV6 donor vectors. PLoS ONE |
| title | A precise gene delivery approach for human induced pluripotent stem cells using Cas9 RNP complex and recombinant AAV6 donor vectors. |
| title_full | A precise gene delivery approach for human induced pluripotent stem cells using Cas9 RNP complex and recombinant AAV6 donor vectors. |
| title_fullStr | A precise gene delivery approach for human induced pluripotent stem cells using Cas9 RNP complex and recombinant AAV6 donor vectors. |
| title_full_unstemmed | A precise gene delivery approach for human induced pluripotent stem cells using Cas9 RNP complex and recombinant AAV6 donor vectors. |
| title_short | A precise gene delivery approach for human induced pluripotent stem cells using Cas9 RNP complex and recombinant AAV6 donor vectors. |
| title_sort | precise gene delivery approach for human induced pluripotent stem cells using cas9 rnp complex and recombinant aav6 donor vectors |
| url | https://doi.org/10.1371/journal.pone.0270963 |
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