Protocol for high-efficiency generation of iPSCs stably expressing Cas9-EGFP using the selection by essential gene exon knockin method
Summary: CRISPR-Cas9 is widely used for genome editing. However, Cas9 silencing occurs during the directed differentiation of induced pluripotent stem cells (iPSCs), even when it is inserted into the safe harbor locus. Here, we generate iPSC-Cas9-EGFP using selection by essential gene exon knockin t...
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| Format: | Article |
| Language: | English |
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Elsevier
2025-09-01
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| Series: | STAR Protocols |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S266616672500334X |
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| author | Yao Zhang Hao Yang Yuan Yang Zijun Lu Lokyu Cheng Hongmei Tan Joe Z. Zhang |
| author_facet | Yao Zhang Hao Yang Yuan Yang Zijun Lu Lokyu Cheng Hongmei Tan Joe Z. Zhang |
| author_sort | Yao Zhang |
| collection | DOAJ |
| description | Summary: CRISPR-Cas9 is widely used for genome editing. However, Cas9 silencing occurs during the directed differentiation of induced pluripotent stem cells (iPSCs), even when it is inserted into the safe harbor locus. Here, we generate iPSC-Cas9-EGFP using selection by essential gene exon knockin technology. We describe steps for inserting the Cas9-EGFP into exon 9 of GAPDH, bypassing Cas9 silencing. We then detail procedures for Cas9 function validation.For complete details on the use and execution of this protocol, please refer to Zhang et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| format | Article |
| id | doaj-art-3e3f2db01e144fc48d6db81a991e5642 |
| institution | DOAJ |
| issn | 2666-1667 |
| language | English |
| publishDate | 2025-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj-art-3e3f2db01e144fc48d6db81a991e56422025-08-20T03:15:20ZengElsevierSTAR Protocols2666-16672025-09-016310392810.1016/j.xpro.2025.103928Protocol for high-efficiency generation of iPSCs stably expressing Cas9-EGFP using the selection by essential gene exon knockin methodYao Zhang0Hao Yang1Yuan Yang2Zijun Lu3Lokyu Cheng4Hongmei Tan5Joe Z. Zhang6Institute of Neurological and Psychiatric Disorders, Shenzhen Bay Laboratory, Shenzhen 518132, China; School of Medicine, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen 518107, ChinaInstitute of Neurological and Psychiatric Disorders, Shenzhen Bay Laboratory, Shenzhen 518132, China; Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong 999077, ChinaInstitute of Neurological and Psychiatric Disorders, Shenzhen Bay Laboratory, Shenzhen 518132, China; Regenerative Medicine Program, Bellvitge Institute for Biomedical Research (IDIBELL) and Program for Clinical Translation of Regenerative Medicine in Catalonia (P-CMRC), 08908 Barcelona, SpainInstitute of Neurological and Psychiatric Disorders, Shenzhen Bay Laboratory, Shenzhen 518132, China; School of Life Science and Technology, Harbin Institute of Technology, Harbin 150001, ChinaInstitute of Neurological and Psychiatric Disorders, Shenzhen Bay Laboratory, Shenzhen 518132, ChinaSchool of Medicine, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen 518107, ChinaInstitute of Neurological and Psychiatric Disorders, Shenzhen Bay Laboratory, Shenzhen 518132, China; Corresponding authorSummary: CRISPR-Cas9 is widely used for genome editing. However, Cas9 silencing occurs during the directed differentiation of induced pluripotent stem cells (iPSCs), even when it is inserted into the safe harbor locus. Here, we generate iPSC-Cas9-EGFP using selection by essential gene exon knockin technology. We describe steps for inserting the Cas9-EGFP into exon 9 of GAPDH, bypassing Cas9 silencing. We then detail procedures for Cas9 function validation.For complete details on the use and execution of this protocol, please refer to Zhang et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S266616672500334XCell BiologyCRISPRMolecular BiologyStem Cells |
| spellingShingle | Yao Zhang Hao Yang Yuan Yang Zijun Lu Lokyu Cheng Hongmei Tan Joe Z. Zhang Protocol for high-efficiency generation of iPSCs stably expressing Cas9-EGFP using the selection by essential gene exon knockin method STAR Protocols Cell Biology CRISPR Molecular Biology Stem Cells |
| title | Protocol for high-efficiency generation of iPSCs stably expressing Cas9-EGFP using the selection by essential gene exon knockin method |
| title_full | Protocol for high-efficiency generation of iPSCs stably expressing Cas9-EGFP using the selection by essential gene exon knockin method |
| title_fullStr | Protocol for high-efficiency generation of iPSCs stably expressing Cas9-EGFP using the selection by essential gene exon knockin method |
| title_full_unstemmed | Protocol for high-efficiency generation of iPSCs stably expressing Cas9-EGFP using the selection by essential gene exon knockin method |
| title_short | Protocol for high-efficiency generation of iPSCs stably expressing Cas9-EGFP using the selection by essential gene exon knockin method |
| title_sort | protocol for high efficiency generation of ipscs stably expressing cas9 egfp using the selection by essential gene exon knockin method |
| topic | Cell Biology CRISPR Molecular Biology Stem Cells |
| url | http://www.sciencedirect.com/science/article/pii/S266616672500334X |
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