Exploiting CRISPR/Cas9 to engineer precise segmental deletions in mouse embryonic stem cells
Summary: In this protocol, we use CRISPR/Cas9 to generate large deletions of the entire coding region of a gene of interest, generating a hemizygous cell line. Next, we systematically engineer precise in-frame deletions within the intact wild-type allele, facilitating study of multi-domain proteins....
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2022-09-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166722004312 |
| _version_ | 1818498107831222272 |
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| author | Rajula Elango Arvind Panday Nicholas A. Willis Ralph Scully |
| author_facet | Rajula Elango Arvind Panday Nicholas A. Willis Ralph Scully |
| author_sort | Rajula Elango |
| collection | DOAJ |
| description | Summary: In this protocol, we use CRISPR/Cas9 to generate large deletions of the entire coding region of a gene of interest, generating a hemizygous cell line. Next, we systematically engineer precise in-frame deletions within the intact wild-type allele, facilitating study of multi-domain proteins. The optimized protocol described here allows us to rapidly screen for effective sgRNA pairs and to engineer either an in-frame deletion or a frameshift mutation in high frequencies in mouse embryonic stem cells.For complete details on the use and execution of this protocol, please refer to Panday et al. (2021). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| first_indexed | 2024-12-10T18:53:50Z |
| format | Article |
| id | doaj.art-cf04c21040b54664ad092286e10ec344 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-12-10T18:53:50Z |
| publishDate | 2022-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-cf04c21040b54664ad092286e10ec3442022-12-22T01:37:14ZengElsevierSTAR Protocols2666-16672022-09-0133101551Exploiting CRISPR/Cas9 to engineer precise segmental deletions in mouse embryonic stem cellsRajula Elango0Arvind Panday1Nicholas A. Willis2Ralph Scully3Department of Medicine, Division of Hematology-Oncology and Cancer Research Institute, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA; Corresponding authorDepartment of Medicine, Division of Hematology-Oncology and Cancer Research Institute, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USADepartment of Medicine, Division of Hematology-Oncology and Cancer Research Institute, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USADepartment of Medicine, Division of Hematology-Oncology and Cancer Research Institute, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA; Corresponding authorSummary: In this protocol, we use CRISPR/Cas9 to generate large deletions of the entire coding region of a gene of interest, generating a hemizygous cell line. Next, we systematically engineer precise in-frame deletions within the intact wild-type allele, facilitating study of multi-domain proteins. The optimized protocol described here allows us to rapidly screen for effective sgRNA pairs and to engineer either an in-frame deletion or a frameshift mutation in high frequencies in mouse embryonic stem cells.For complete details on the use and execution of this protocol, please refer to Panday et al. (2021). : 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/S2666166722004312GeneticsMolecular BiologyCRISPRStem Cells |
| spellingShingle | Rajula Elango Arvind Panday Nicholas A. Willis Ralph Scully Exploiting CRISPR/Cas9 to engineer precise segmental deletions in mouse embryonic stem cells STAR Protocols Genetics Molecular Biology CRISPR Stem Cells |
| title | Exploiting CRISPR/Cas9 to engineer precise segmental deletions in mouse embryonic stem cells |
| title_full | Exploiting CRISPR/Cas9 to engineer precise segmental deletions in mouse embryonic stem cells |
| title_fullStr | Exploiting CRISPR/Cas9 to engineer precise segmental deletions in mouse embryonic stem cells |
| title_full_unstemmed | Exploiting CRISPR/Cas9 to engineer precise segmental deletions in mouse embryonic stem cells |
| title_short | Exploiting CRISPR/Cas9 to engineer precise segmental deletions in mouse embryonic stem cells |
| title_sort | exploiting crispr cas9 to engineer precise segmental deletions in mouse embryonic stem cells |
| topic | Genetics Molecular Biology CRISPR Stem Cells |
| url | http://www.sciencedirect.com/science/article/pii/S2666166722004312 |
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