CRISPR/Cas9-Based Protocol for Precise Genome Editing in Induced Pluripotent Stem Cells
The advent of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing has marked a significant advancement in genetic engineering technology. However, the editing of induced pluripotent stem cells (iPSCs) with CRISPR presents notable challenges in ensuring cell s...
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Bio-protocol LLC
2024-12-01
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| author | Avinash Singh Swathy Babu Marcus Phan Shauna Yuan |
| author_facet | Avinash Singh Swathy Babu Marcus Phan Shauna Yuan |
| author_sort | Avinash Singh |
| collection | DOAJ |
| description | The advent of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing has marked a significant advancement in genetic engineering technology. However, the editing of induced pluripotent stem cells (iPSCs) with CRISPR presents notable challenges in ensuring cell survival and achieving high editing efficiency. These challenges become even more complex when considering the specific target site. P53 activation as a result of traditional CRISPR editing can lead to apoptosis, potentially worsening cell health or even resulting in cell death. Mitigating this apoptotic response can enhance cell survival post-CRISPR editing, which will ultimately increase editing efficiency. In our study, we observed that combining p53 inhibition with pro-survival small molecules yields a homologous recombination rate of over 90% when using CRISPR in human iPSCs. This protocol significantly streamlines the editing process and reduces the time and resources necessary for creating isogenic lines. |
| format | Article |
| id | doaj-art-babd1999fc38467ea6170b04d9d939e2 |
| institution | Kabale University |
| issn | 2331-8325 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Bio-protocol LLC |
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| spelling | doaj-art-babd1999fc38467ea6170b04d9d939e22025-02-07T08:16:22ZengBio-protocol LLCBio-Protocol2331-83252024-12-01142410.21769/BioProtoc.5141CRISPR/Cas9-Based Protocol for Precise Genome Editing in Induced Pluripotent Stem CellsAvinash Singh0Swathy Babu1Marcus Phan2Shauna Yuan3Department of Neurology, University of Minnesota, Twin Cities, Minneapolis, MN, USAMinneapolis Veterans Administration Health Care System, Minneapolis, MN, USADepartment of Neurology, University of Minnesota, Twin Cities, Minneapolis, MN, USAMinneapolis Veterans Administration Health Care System, Minneapolis, MN, USADepartment of Neurology, University of Minnesota, Twin Cities, Minneapolis, MN, USAGraduate Program in Neuroscience, University of Minnesota, Twin Cities, Minneapolis, MN, USADepartment of Neurology, University of Minnesota, Twin Cities, Minneapolis, MN, USAGraduate Program in Neuroscience, University of Minnesota, Twin Cities, Minneapolis, MN, USA, Minneapolis Veterans Administration Health Care System, Minneapolis, MN, USAThe advent of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing has marked a significant advancement in genetic engineering technology. However, the editing of induced pluripotent stem cells (iPSCs) with CRISPR presents notable challenges in ensuring cell survival and achieving high editing efficiency. These challenges become even more complex when considering the specific target site. P53 activation as a result of traditional CRISPR editing can lead to apoptosis, potentially worsening cell health or even resulting in cell death. Mitigating this apoptotic response can enhance cell survival post-CRISPR editing, which will ultimately increase editing efficiency. In our study, we observed that combining p53 inhibition with pro-survival small molecules yields a homologous recombination rate of over 90% when using CRISPR in human iPSCs. This protocol significantly streamlines the editing process and reduces the time and resources necessary for creating isogenic lines.https://bio-protocol.org/en/bpdetail?id=5141&type=0 |
| spellingShingle | Avinash Singh Swathy Babu Marcus Phan Shauna Yuan CRISPR/Cas9-Based Protocol for Precise Genome Editing in Induced Pluripotent Stem Cells Bio-Protocol |
| title | CRISPR/Cas9-Based Protocol for Precise Genome Editing in Induced Pluripotent Stem Cells |
| title_full | CRISPR/Cas9-Based Protocol for Precise Genome Editing in Induced Pluripotent Stem Cells |
| title_fullStr | CRISPR/Cas9-Based Protocol for Precise Genome Editing in Induced Pluripotent Stem Cells |
| title_full_unstemmed | CRISPR/Cas9-Based Protocol for Precise Genome Editing in Induced Pluripotent Stem Cells |
| title_short | CRISPR/Cas9-Based Protocol for Precise Genome Editing in Induced Pluripotent Stem Cells |
| title_sort | crispr cas9 based protocol for precise genome editing in induced pluripotent stem cells |
| url | https://bio-protocol.org/en/bpdetail?id=5141&type=0 |
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