A stress-free strategy to correct point mutations in patient iPS cells
When studying patient specific induced pluripotent stem cells (iPS cells) as a disease model, the ideal control is an isogenic line that has corrected the point mutation, instead of iPS cells from siblings or other healthy subjects. However, repairing a point mutation in iPS cells even with the newl...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2021-05-01
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| Series: | Stem Cell Research |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S1873506121001781 |
| _version_ | 1818647225042993152 |
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| author | Jingli Cai Elizabeth Kropf Ya-Ming Hou Lorraine Iacovitti |
| author_facet | Jingli Cai Elizabeth Kropf Ya-Ming Hou Lorraine Iacovitti |
| author_sort | Jingli Cai |
| collection | DOAJ |
| description | When studying patient specific induced pluripotent stem cells (iPS cells) as a disease model, the ideal control is an isogenic line that has corrected the point mutation, instead of iPS cells from siblings or other healthy subjects. However, repairing a point mutation in iPS cells even with the newly developed CRISPR-Cas9 technique remains difficult and time-consuming. Here we report a strategy that makes the Cas9 “knock-in” methodology both hassle-free and error-free. Instead of selecting a Cas9 recognition site close to the point mutation, we chose a site located in the nearest intron. We constructed a donor template with the fragment containing the corrected point mutation as one of the homologous recombination arms flanking a PGK-PuroR cassette. After selection with puromycin, positive clones were identified and further transfected with a CRE vector to remove the PGK-PuroR cassette. Using this methodology, we successfully repaired the point mutation G2019S of the LRRK2 gene in a Parkinson Disease (PD) patient iPS line and the point mutation R329H of the AARS1 gene in a Charcot-Marie-Tooth disease (CMT) patient iPS line. These isogenic iPS lines are ideal as a control in future studies. |
| first_indexed | 2024-12-17T00:59:09Z |
| format | Article |
| id | doaj.art-66ac66f6087e41d182a92fca6e547270 |
| institution | Directory Open Access Journal |
| issn | 1873-5061 |
| language | English |
| last_indexed | 2024-12-17T00:59:09Z |
| publishDate | 2021-05-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Stem Cell Research |
| spelling | doaj.art-66ac66f6087e41d182a92fca6e5472702022-12-21T22:09:32ZengElsevierStem Cell Research1873-50612021-05-0153102332A stress-free strategy to correct point mutations in patient iPS cellsJingli Cai0Elizabeth Kropf1Ya-Ming Hou2Lorraine Iacovitti3Department of Neuroscience Vickie & Jack Farber Institute for Neuroscience, Sidney Kimmel Medical College, Thomas Jefferson University, 900 Walnut Street, JHN Suite 461, Philadelphia, PA 19107, USADepartment of Neuroscience Vickie & Jack Farber Institute for Neuroscience, Sidney Kimmel Medical College, Thomas Jefferson University, 900 Walnut Street, JHN Suite 461, Philadelphia, PA 19107, USADepartment of Biochemistry and Molecular Biology, Sidney Kimmel Medical College, Thomas Jefferson University, 233 South 10th Street, BLSB Suite 220, Philadelphia, PA 19107, USADepartment of Neuroscience Vickie & Jack Farber Institute for Neuroscience, Sidney Kimmel Medical College, Thomas Jefferson University, 900 Walnut Street, JHN Suite 461, Philadelphia, PA 19107, USA; Corresponding author.When studying patient specific induced pluripotent stem cells (iPS cells) as a disease model, the ideal control is an isogenic line that has corrected the point mutation, instead of iPS cells from siblings or other healthy subjects. However, repairing a point mutation in iPS cells even with the newly developed CRISPR-Cas9 technique remains difficult and time-consuming. Here we report a strategy that makes the Cas9 “knock-in” methodology both hassle-free and error-free. Instead of selecting a Cas9 recognition site close to the point mutation, we chose a site located in the nearest intron. We constructed a donor template with the fragment containing the corrected point mutation as one of the homologous recombination arms flanking a PGK-PuroR cassette. After selection with puromycin, positive clones were identified and further transfected with a CRE vector to remove the PGK-PuroR cassette. Using this methodology, we successfully repaired the point mutation G2019S of the LRRK2 gene in a Parkinson Disease (PD) patient iPS line and the point mutation R329H of the AARS1 gene in a Charcot-Marie-Tooth disease (CMT) patient iPS line. These isogenic iPS lines are ideal as a control in future studies.http://www.sciencedirect.com/science/article/pii/S1873506121001781CRISPR-Cas9Gene targetingPoint mutationIsogenic iPS cells |
| spellingShingle | Jingli Cai Elizabeth Kropf Ya-Ming Hou Lorraine Iacovitti A stress-free strategy to correct point mutations in patient iPS cells Stem Cell Research CRISPR-Cas9 Gene targeting Point mutation Isogenic iPS cells |
| title | A stress-free strategy to correct point mutations in patient iPS cells |
| title_full | A stress-free strategy to correct point mutations in patient iPS cells |
| title_fullStr | A stress-free strategy to correct point mutations in patient iPS cells |
| title_full_unstemmed | A stress-free strategy to correct point mutations in patient iPS cells |
| title_short | A stress-free strategy to correct point mutations in patient iPS cells |
| title_sort | stress free strategy to correct point mutations in patient ips cells |
| topic | CRISPR-Cas9 Gene targeting Point mutation Isogenic iPS cells |
| url | http://www.sciencedirect.com/science/article/pii/S1873506121001781 |
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