One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering
The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Us...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | en_US |
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Elsevier
2017
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| Online Access: | http://hdl.handle.net/1721.1/109552 https://orcid.org/0000-0002-2905-0306 |
| _version_ | 1811080021430763520 |
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| author | Yang, Hui Wang, Haoyi Cheng, Albert W. Shi, Linyu Shivalila, Chikdu Shakti Jaenisch, Rudolf |
| author2 | Massachusetts Institute of Technology. Computational and Systems Biology Program |
| author_facet | Massachusetts Institute of Technology. Computational and Systems Biology Program Yang, Hui Wang, Haoyi Cheng, Albert W. Shi, Linyu Shivalila, Chikdu Shakti Jaenisch, Rudolf |
| author_sort | Yang, Hui |
| collection | MIT |
| description | The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances. |
| first_indexed | 2024-09-23T11:24:26Z |
| format | Article |
| id | mit-1721.1/109552 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T11:24:26Z |
| publishDate | 2017 |
| publisher | Elsevier |
| record_format | dspace |
| spelling | mit-1721.1/1095522022-10-01T03:24:53Z One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering Yang, Hui Wang, Haoyi Cheng, Albert W. Shi, Linyu Shivalila, Chikdu Shakti Jaenisch, Rudolf Massachusetts Institute of Technology. Computational and Systems Biology Program Massachusetts Institute of Technology. Department of Biology Whitehead Institute for Biomedical Research Shivalila, Chikdu Shakti Jaenisch, Rudolf The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances. United States. National Institutes of Health (HD 045022) United States. National Institutes of Health (R37CA084198) 2017-06-02T17:54:53Z 2017-06-02T17:54:53Z 2013-08 2013-08 Article http://purl.org/eprint/type/JournalArticle 0092-8674 1097-4172 http://hdl.handle.net/1721.1/109552 Yang, Hui; Wang, Haoyi; Shivalila, Chikdu S.; Cheng, Albert W.; Shi, Linyu and Jaenisch, Rudolf. "One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering." Cell 154, no.6 (September 2013): 1370–1379 © 2013 Elsevier Inc https://orcid.org/0000-0002-2905-0306 en_US http://dx.doi.org/10.1016/j.cell.2013.08.022 Cell Creative Commons Attribution-NonCommercial-NoDerivs License http://creativecommons.org/licenses/by-nc-nd/4.0/ application/pdf Elsevier PMC |
| spellingShingle | Yang, Hui Wang, Haoyi Cheng, Albert W. Shi, Linyu Shivalila, Chikdu Shakti Jaenisch, Rudolf One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering |
| title | One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering |
| title_full | One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering |
| title_fullStr | One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering |
| title_full_unstemmed | One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering |
| title_short | One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering |
| title_sort | one step generation of mice carrying reporter and conditional alleles by crispr cas mediated genome engineering |
| url | http://hdl.handle.net/1721.1/109552 https://orcid.org/0000-0002-2905-0306 |
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