CRISPR-mediated direct mutation of cancer genes in the mouse liver
The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem cells. Here we describe a new method of cancer model generation using the CRISPR/Cas (clustered regularly interspaced short palindromic repea...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | en_US |
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Nature Publishing Group
2015
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| Online Access: | http://hdl.handle.net/1721.1/96801 https://orcid.org/0000-0001-5785-8911 https://orcid.org/0000-0003-1465-1691 https://orcid.org/0000-0001-9587-0233 https://orcid.org/0000-0002-7045-7837 https://orcid.org/0000-0001-5629-4798 https://orcid.org/0000-0001-6898-3793 https://orcid.org/0000-0003-3675-6961 https://orcid.org/0000-0002-0460-8246 |
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| author | Xue, Wen Chen, Sidi Yin, Hao Tammela, Tuomas Papagiannakopoulos, Thales Cai, Wenxin Bronson, Roderick T. Crowley, Denise G. Zhang, Feng Sharp, Phillip A. Joshi, Nik Yang, Gillian R. Anderson, Daniel Griffith Jacks, Tyler E |
| author2 | Massachusetts Institute of Technology. Institute for Medical Engineering & Science |
| author_facet | Massachusetts Institute of Technology. Institute for Medical Engineering & Science Xue, Wen Chen, Sidi Yin, Hao Tammela, Tuomas Papagiannakopoulos, Thales Cai, Wenxin Bronson, Roderick T. Crowley, Denise G. Zhang, Feng Sharp, Phillip A. Joshi, Nik Yang, Gillian R. Anderson, Daniel Griffith Jacks, Tyler E |
| author_sort | Xue, Wen |
| collection | MIT |
| description | The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem cells. Here we describe a new method of cancer model generation using the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system in vivo in wild-type mice. We used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs) to the liver that directly target the tumour suppressor genes Pten and p53 (also known as TP53 and Trp53), alone and in combination. CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre–LoxP technology. Simultaneous targeting of Pten and p53 induced liver tumours that mimicked those caused by Cre–loxP-mediated deletion of Pten and p53. DNA sequencing of liver and tumour tissue revealed insertion or deletion mutations of the tumour suppressor genes, including bi-allelic mutations of both Pten and p53 in tumours. Furthermore, co-injection of Cas9 plasmids harbouring sgRNAs targeting the β-catenin gene and a single-stranded DNA oligonucleotide donor carrying activating point mutations led to the generation of hepatocytes with nuclear localization of β-catenin. This study demonstrates the feasibility of direct mutation of tumour suppressor genes and oncogenes in the liver using the CRISPR/Cas system, which presents a new avenue for rapid development of liver cancer models and functional genomics. |
| first_indexed | 2024-09-23T13:54:33Z |
| format | Article |
| id | mit-1721.1/96801 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T13:54:33Z |
| publishDate | 2015 |
| publisher | Nature Publishing Group |
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| spelling | mit-1721.1/968012022-09-28T16:58:16Z CRISPR-mediated direct mutation of cancer genes in the mouse liver Xue, Wen Chen, Sidi Yin, Hao Tammela, Tuomas Papagiannakopoulos, Thales Cai, Wenxin Bronson, Roderick T. Crowley, Denise G. Zhang, Feng Sharp, Phillip A. Joshi, Nik Yang, Gillian R. Anderson, Daniel Griffith Jacks, Tyler E Massachusetts Institute of Technology. Institute for Medical Engineering & Science Harvard University--MIT Division of Health Sciences and Technology Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Chemical Engineering Koch Institute for Integrative Cancer Research at MIT Xue, Wen Chen, Sidi Yin, Hao Tammela, Tuomas Papagiannakopoulos, Thales Joshi, Nik Cai, Wenxin Yang, Gillian R. Crowley, Denise G. Anderson, Daniel Griffith Sharp, Phillip A. Jacks, Tyler E. The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem cells. Here we describe a new method of cancer model generation using the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system in vivo in wild-type mice. We used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs) to the liver that directly target the tumour suppressor genes Pten and p53 (also known as TP53 and Trp53), alone and in combination. CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre–LoxP technology. Simultaneous targeting of Pten and p53 induced liver tumours that mimicked those caused by Cre–loxP-mediated deletion of Pten and p53. DNA sequencing of liver and tumour tissue revealed insertion or deletion mutations of the tumour suppressor genes, including bi-allelic mutations of both Pten and p53 in tumours. Furthermore, co-injection of Cas9 plasmids harbouring sgRNAs targeting the β-catenin gene and a single-stranded DNA oligonucleotide donor carrying activating point mutations led to the generation of hepatocytes with nuclear localization of β-catenin. This study demonstrates the feasibility of direct mutation of tumour suppressor genes and oncogenes in the liver using the CRISPR/Cas system, which presents a new avenue for rapid development of liver cancer models and functional genomics. National Institutes of Health (U.S.) (Grant 2-PO1-CA42063) National Institutes of Health (U.S.) (Grant RO1-EB000244) National Institutes of Health (U.S.) (Grant RO1-CA115527) National Institutes of Health (U.S.) (Grant RO1-CA132091) National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051) National Institutes of Health (U.S.) (Grant R01-CA133404) David H. Koch Institute for Integrative Cancer Research at MIT (Marie D. and Pierre Casimir-Lambert Fund) National Institutes of Health (U.S.) (Centers for Cancer Nanotechnology Excellence 5-U54-CA151884-04) MIT-Harvard Center of Cancer Nanotechnology Excellence Damon Runyon Cancer Research Foundation (Fellowship DRG-2117-12) American Association for Cancer Research (Fellowship) Leukemia & Lymphoma Society of America (Fellowship) National Institutes of Health (U.S.) (1K99CA169512) 2015-04-24T18:46:42Z 2015-04-24T18:46:42Z 2014-08 2014-02 Article http://purl.org/eprint/type/JournalArticle 0028-0836 1476-4687 http://hdl.handle.net/1721.1/96801 Xue, Wen, Sidi Chen, Hao Yin, Tuomas Tammela, Thales Papagiannakopoulos, Nikhil S. Joshi, Wenxin Cai, et al. “CRISPR-Mediated Direct Mutation of Cancer Genes in the Mouse Liver.” Nature 514, no. 7522 (August 6, 2014): 380–384. https://orcid.org/0000-0001-5785-8911 https://orcid.org/0000-0003-1465-1691 https://orcid.org/0000-0001-9587-0233 https://orcid.org/0000-0002-7045-7837 https://orcid.org/0000-0001-5629-4798 https://orcid.org/0000-0001-6898-3793 https://orcid.org/0000-0003-3675-6961 https://orcid.org/0000-0002-0460-8246 en_US http://dx.doi.org/10.1038/nature13589 Nature 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 | Xue, Wen Chen, Sidi Yin, Hao Tammela, Tuomas Papagiannakopoulos, Thales Cai, Wenxin Bronson, Roderick T. Crowley, Denise G. Zhang, Feng Sharp, Phillip A. Joshi, Nik Yang, Gillian R. Anderson, Daniel Griffith Jacks, Tyler E CRISPR-mediated direct mutation of cancer genes in the mouse liver |
| title | CRISPR-mediated direct mutation of cancer genes in the mouse liver |
| title_full | CRISPR-mediated direct mutation of cancer genes in the mouse liver |
| title_fullStr | CRISPR-mediated direct mutation of cancer genes in the mouse liver |
| title_full_unstemmed | CRISPR-mediated direct mutation of cancer genes in the mouse liver |
| title_short | CRISPR-mediated direct mutation of cancer genes in the mouse liver |
| title_sort | crispr mediated direct mutation of cancer genes in the mouse liver |
| url | http://hdl.handle.net/1721.1/96801 https://orcid.org/0000-0001-5785-8911 https://orcid.org/0000-0003-1465-1691 https://orcid.org/0000-0001-9587-0233 https://orcid.org/0000-0002-7045-7837 https://orcid.org/0000-0001-5629-4798 https://orcid.org/0000-0001-6898-3793 https://orcid.org/0000-0003-3675-6961 https://orcid.org/0000-0002-0460-8246 |
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