Scarless Genome Editing of Human Pluripotent Stem Cells via Transient Puromycin Selection
Summary: Genome-edited human pluripotent stem cells (hPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. We present and characterize a robust method for rapid, scarless introduction or correction of disease-associated variants in hPSCs using CRISPR/Cas9. Ut...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2018-02-01
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| Series: | Stem Cell Reports |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2213671117305520 |
| _version_ | 1818911089196269568 |
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| author | Benjamin Steyer Qian Bu Evan Cory Keer Jiang Stella Duong Divya Sinha Stephanie Steltzer David Gamm Qiang Chang Krishanu Saha |
| author_facet | Benjamin Steyer Qian Bu Evan Cory Keer Jiang Stella Duong Divya Sinha Stephanie Steltzer David Gamm Qiang Chang Krishanu Saha |
| author_sort | Benjamin Steyer |
| collection | DOAJ |
| description | Summary: Genome-edited human pluripotent stem cells (hPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. We present and characterize a robust method for rapid, scarless introduction or correction of disease-associated variants in hPSCs using CRISPR/Cas9. Utilizing non-integrated plasmid vectors that express a puromycin N-acetyl-transferase (PAC) gene, whose expression and translation is linked to that of Cas9, we transiently select for cells based on their early levels of Cas9 protein. Under optimized conditions, co-delivery with single-stranded donor DNA enabled isolation of clonal cell populations containing both heterozygous and homozygous precise genome edits in as little as 2 weeks without requiring cell sorting or high-throughput sequencing. Edited cells isolated using this method did not contain any detectable off-target mutations and displayed expected functional phenotypes after directed differentiation. We apply the approach to a variety of genomic loci in five hPSC lines cultured using both feeder and feeder-free conditions. : In this article, Saha and colleagues describe a workflow for isolation of hPSC clones containing scarless, HDR-mediated, genome edits without the use of FACS or high-throughput sequencing technologies. They demonstrate that transient expression of a puromycin-resistance gene, from a non-integrated CRISPR/Cas9 plasmid, enables stringent selection for clones precisely edited with a single-stranded donor DNA template. Keywords: genome editing, human pluripotent stem cells, disease modeling, CRISPR/Cas9, scarless, transient selection, puromycin |
| first_indexed | 2024-12-19T22:53:09Z |
| format | Article |
| id | doaj.art-b72ef3c7118243a0acd3d5ac148b1fbd |
| institution | Directory Open Access Journal |
| issn | 2213-6711 |
| language | English |
| last_indexed | 2024-12-19T22:53:09Z |
| publishDate | 2018-02-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Stem Cell Reports |
| spelling | doaj.art-b72ef3c7118243a0acd3d5ac148b1fbd2022-12-21T20:02:44ZengElsevierStem Cell Reports2213-67112018-02-01102642654Scarless Genome Editing of Human Pluripotent Stem Cells via Transient Puromycin SelectionBenjamin Steyer0Qian Bu1Evan Cory2Keer Jiang3Stella Duong4Divya Sinha5Stephanie Steltzer6David Gamm7Qiang Chang8Krishanu Saha9Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USAWaisman Center, University of Wisconsin-Madison, Madison, WI 53705, USAWisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USAWaisman Center, University of Wisconsin-Madison, Madison, WI 53705, USAWaisman Center, University of Wisconsin-Madison, Madison, WI 53705, USAWaisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, WI 53705, USAWisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USAWaisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Ophthalmology & Visual Sciences, University of Wisconsin-Madison, Madison, WI 53705, USAWaisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Medical Genetics, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA; Department of Neurology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA; Corresponding authorWisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA; Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA; Corresponding authorSummary: Genome-edited human pluripotent stem cells (hPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. We present and characterize a robust method for rapid, scarless introduction or correction of disease-associated variants in hPSCs using CRISPR/Cas9. Utilizing non-integrated plasmid vectors that express a puromycin N-acetyl-transferase (PAC) gene, whose expression and translation is linked to that of Cas9, we transiently select for cells based on their early levels of Cas9 protein. Under optimized conditions, co-delivery with single-stranded donor DNA enabled isolation of clonal cell populations containing both heterozygous and homozygous precise genome edits in as little as 2 weeks without requiring cell sorting or high-throughput sequencing. Edited cells isolated using this method did not contain any detectable off-target mutations and displayed expected functional phenotypes after directed differentiation. We apply the approach to a variety of genomic loci in five hPSC lines cultured using both feeder and feeder-free conditions. : In this article, Saha and colleagues describe a workflow for isolation of hPSC clones containing scarless, HDR-mediated, genome edits without the use of FACS or high-throughput sequencing technologies. They demonstrate that transient expression of a puromycin-resistance gene, from a non-integrated CRISPR/Cas9 plasmid, enables stringent selection for clones precisely edited with a single-stranded donor DNA template. Keywords: genome editing, human pluripotent stem cells, disease modeling, CRISPR/Cas9, scarless, transient selection, puromycinhttp://www.sciencedirect.com/science/article/pii/S2213671117305520 |
| spellingShingle | Benjamin Steyer Qian Bu Evan Cory Keer Jiang Stella Duong Divya Sinha Stephanie Steltzer David Gamm Qiang Chang Krishanu Saha Scarless Genome Editing of Human Pluripotent Stem Cells via Transient Puromycin Selection Stem Cell Reports |
| title | Scarless Genome Editing of Human Pluripotent Stem Cells via Transient Puromycin Selection |
| title_full | Scarless Genome Editing of Human Pluripotent Stem Cells via Transient Puromycin Selection |
| title_fullStr | Scarless Genome Editing of Human Pluripotent Stem Cells via Transient Puromycin Selection |
| title_full_unstemmed | Scarless Genome Editing of Human Pluripotent Stem Cells via Transient Puromycin Selection |
| title_short | Scarless Genome Editing of Human Pluripotent Stem Cells via Transient Puromycin Selection |
| title_sort | scarless genome editing of human pluripotent stem cells via transient puromycin selection |
| url | http://www.sciencedirect.com/science/article/pii/S2213671117305520 |
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