Rapid Generation of Human Genetic Loss-of-Function iPSC Lines by Simultaneous Reprogramming and Gene Editing
Specifically ablating genes in human induced pluripotent stem cells (iPSCs) allows for studies of gene function as well as disease mechanisms in disorders caused by loss-of-function (LOF) mutations. While techniques exist for engineering such lines, we have developed and rigorously validated a metho...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2017-09-01
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| Series: | Stem Cell Reports |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2213671117303132 |
| _version_ | 1818928294133760000 |
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| author | Andrew M. Tidball Louis T. Dang Trevor W. Glenn Emma G. Kilbane Daniel J. Klarr Joshua L. Margolis Michael D. Uhler Jack M. Parent |
| author_facet | Andrew M. Tidball Louis T. Dang Trevor W. Glenn Emma G. Kilbane Daniel J. Klarr Joshua L. Margolis Michael D. Uhler Jack M. Parent |
| author_sort | Andrew M. Tidball |
| collection | DOAJ |
| description | Specifically ablating genes in human induced pluripotent stem cells (iPSCs) allows for studies of gene function as well as disease mechanisms in disorders caused by loss-of-function (LOF) mutations. While techniques exist for engineering such lines, we have developed and rigorously validated a method of simultaneous iPSC reprogramming while generating CRISPR/Cas9-dependent insertions/deletions (indels). This approach allows for the efficient and rapid formation of genetic LOF human disease cell models with isogenic controls. The rate of mutagenized lines was strikingly consistent across experiments targeting four different human epileptic encephalopathy genes and a metabolic enzyme-encoding gene, and was more efficient and consistent than using CRISPR gene editing of established iPSC lines. The ability of our streamlined method to reproducibly generate heterozygous and homozygous LOF iPSC lines with passage-matched isogenic controls in a single step provides for the rapid development of LOF disease models with ideal control lines, even in the absence of patient tissue. |
| first_indexed | 2024-12-20T03:26:37Z |
| format | Article |
| id | doaj.art-5242b95b4a4946f5a8fdb5aab5823ec7 |
| institution | Directory Open Access Journal |
| issn | 2213-6711 |
| language | English |
| last_indexed | 2024-12-20T03:26:37Z |
| publishDate | 2017-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Stem Cell Reports |
| spelling | doaj.art-5242b95b4a4946f5a8fdb5aab5823ec72022-12-21T19:55:05ZengElsevierStem Cell Reports2213-67112017-09-019372573110.1016/j.stemcr.2017.07.003Rapid Generation of Human Genetic Loss-of-Function iPSC Lines by Simultaneous Reprogramming and Gene EditingAndrew M. Tidball0Louis T. Dang1Trevor W. Glenn2Emma G. Kilbane3Daniel J. Klarr4Joshua L. Margolis5Michael D. Uhler6Jack M. Parent7Department of Neurology, University of Michigan Medical School, 5021 BSRB, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200, USADepartment of Pediatrics, University of Michigan Medical School, Ann Arbor, MI 48109, USADepartment of Neurology, University of Michigan Medical School, 5021 BSRB, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200, USADepartment of Neurology, University of Michigan Medical School, 5021 BSRB, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200, USADepartment of Neurology, University of Michigan Medical School, 5021 BSRB, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200, USADepartment of Neurology, University of Michigan Medical School, 5021 BSRB, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200, USADepartment of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USADepartment of Neurology, University of Michigan Medical School, 5021 BSRB, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200, USASpecifically ablating genes in human induced pluripotent stem cells (iPSCs) allows for studies of gene function as well as disease mechanisms in disorders caused by loss-of-function (LOF) mutations. While techniques exist for engineering such lines, we have developed and rigorously validated a method of simultaneous iPSC reprogramming while generating CRISPR/Cas9-dependent insertions/deletions (indels). This approach allows for the efficient and rapid formation of genetic LOF human disease cell models with isogenic controls. The rate of mutagenized lines was strikingly consistent across experiments targeting four different human epileptic encephalopathy genes and a metabolic enzyme-encoding gene, and was more efficient and consistent than using CRISPR gene editing of established iPSC lines. The ability of our streamlined method to reproducibly generate heterozygous and homozygous LOF iPSC lines with passage-matched isogenic controls in a single step provides for the rapid development of LOF disease models with ideal control lines, even in the absence of patient tissue.http://www.sciencedirect.com/science/article/pii/S2213671117303132CRISPR/Cas9gene editinginduced pluripotent stem cellsreprogramminggenetic epilepsyepileptic encephalopathy |
| spellingShingle | Andrew M. Tidball Louis T. Dang Trevor W. Glenn Emma G. Kilbane Daniel J. Klarr Joshua L. Margolis Michael D. Uhler Jack M. Parent Rapid Generation of Human Genetic Loss-of-Function iPSC Lines by Simultaneous Reprogramming and Gene Editing Stem Cell Reports CRISPR/Cas9 gene editing induced pluripotent stem cells reprogramming genetic epilepsy epileptic encephalopathy |
| title | Rapid Generation of Human Genetic Loss-of-Function iPSC Lines by Simultaneous Reprogramming and Gene Editing |
| title_full | Rapid Generation of Human Genetic Loss-of-Function iPSC Lines by Simultaneous Reprogramming and Gene Editing |
| title_fullStr | Rapid Generation of Human Genetic Loss-of-Function iPSC Lines by Simultaneous Reprogramming and Gene Editing |
| title_full_unstemmed | Rapid Generation of Human Genetic Loss-of-Function iPSC Lines by Simultaneous Reprogramming and Gene Editing |
| title_short | Rapid Generation of Human Genetic Loss-of-Function iPSC Lines by Simultaneous Reprogramming and Gene Editing |
| title_sort | rapid generation of human genetic loss of function ipsc lines by simultaneous reprogramming and gene editing |
| topic | CRISPR/Cas9 gene editing induced pluripotent stem cells reprogramming genetic epilepsy epileptic encephalopathy |
| url | http://www.sciencedirect.com/science/article/pii/S2213671117303132 |
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