Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining
Methods to introduce targeted double-strand breaks (DSBs) into DNA enable precise genome editing by increasing the rate at which externally supplied DNA fragments are incorporated into the genome through homologous recombination. The efficiency of these methods is limited by nonhomologous end joinin...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | en_US |
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Nature Publishing Group
2017
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| Online Access: | http://hdl.handle.net/1721.1/106223 https://orcid.org/0000-0002-1090-6071 |
| _version_ | 1826191460079239168 |
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| author | Maruyama, Takeshi Dougan, Stephanie K Truttmann, Matthias C Bilate, Angelina M Ingram, Jessica R Ploegh, Hidde |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Maruyama, Takeshi Dougan, Stephanie K Truttmann, Matthias C Bilate, Angelina M Ingram, Jessica R Ploegh, Hidde |
| author_sort | Maruyama, Takeshi |
| collection | MIT |
| description | Methods to introduce targeted double-strand breaks (DSBs) into DNA enable precise genome editing by increasing the rate at which externally supplied DNA fragments are incorporated into the genome through homologous recombination. The efficiency of these methods is limited by nonhomologous end joining (NHEJ), an alternative DNA repair pathway that competes with homology-directed repair (HDR). To promote HDR at the expense of NHEJ, we targeted DNA ligase IV, a key enzyme in the NHEJ pathway, using the inhibitor Scr7. Scr7 treatment increased the efficiency of HDR-mediated genome editing, using Cas9 in mammalian cell lines and in mice for all four genes examined, up to 19-fold. This approach should be applicable to other customizable endonucleases, such as zinc finger nucleases and transcription activator–like effector nucleases, and to nonmammalian cells with sufficiently conserved mechanisms of NHEJ and HDR. |
| first_indexed | 2024-09-23T08:56:12Z |
| format | Article |
| id | mit-1721.1/106223 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T08:56:12Z |
| publishDate | 2017 |
| publisher | Nature Publishing Group |
| record_format | dspace |
| spelling | mit-1721.1/1062232022-09-26T09:20:19Z Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining Maruyama, Takeshi Dougan, Stephanie K Truttmann, Matthias C Bilate, Angelina M Ingram, Jessica R Ploegh, Hidde Massachusetts Institute of Technology. Department of Biology Whitehead Institute for Biomedical Research Ploegh, Hidde Methods to introduce targeted double-strand breaks (DSBs) into DNA enable precise genome editing by increasing the rate at which externally supplied DNA fragments are incorporated into the genome through homologous recombination. The efficiency of these methods is limited by nonhomologous end joining (NHEJ), an alternative DNA repair pathway that competes with homology-directed repair (HDR). To promote HDR at the expense of NHEJ, we targeted DNA ligase IV, a key enzyme in the NHEJ pathway, using the inhibitor Scr7. Scr7 treatment increased the efficiency of HDR-mediated genome editing, using Cas9 in mammalian cell lines and in mice for all four genes examined, up to 19-fold. This approach should be applicable to other customizable endonucleases, such as zinc finger nucleases and transcription activator–like effector nucleases, and to nonmammalian cells with sufficiently conserved mechanisms of NHEJ and HDR. National Institutes of Health (U.S.) (RO1 Grant AI087879-01) Pancreatic Cancer Action Network 2017-01-06T14:34:36Z 2017-01-06T14:34:36Z 2015-03 2014-07 Article http://purl.org/eprint/type/JournalArticle 1087-0156 1546-1696 http://hdl.handle.net/1721.1/106223 Maruyama, Takeshi et al. “Increasing the Efficiency of Precise Genome Editing with CRISPR-Cas9 by Inhibition of Nonhomologous End Joining.” Nature Biotechnology 33.5 (2015): 538–542. https://orcid.org/0000-0002-1090-6071 en_US http://dx.doi.org/10.1038/nbt.3190 Nature Biotechnology Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf Nature Publishing Group PMC |
| spellingShingle | Maruyama, Takeshi Dougan, Stephanie K Truttmann, Matthias C Bilate, Angelina M Ingram, Jessica R Ploegh, Hidde Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining |
| title | Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining |
| title_full | Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining |
| title_fullStr | Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining |
| title_full_unstemmed | Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining |
| title_short | Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining |
| title_sort | increasing the efficiency of precise genome editing with crispr cas9 by inhibition of nonhomologous end joining |
| url | http://hdl.handle.net/1721.1/106223 https://orcid.org/0000-0002-1090-6071 |
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