Harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM
A major hurdle to evolutionary engineering approaches for multigenic phenotypes is the ability to simultaneously modify multiple genes rapidly and selectively. Here, we describe a method for in vivo-targeted mutagenesis in yeast, targeting glycosylases to embedded arrays for mutagenesis (TaGTEAM). B...
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| Format: | Article |
| Language: | en_US |
| Published: |
Oxford University Press
2013
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| Online Access: | http://hdl.handle.net/1721.1/78588 |
| _version_ | 1826197567268978688 |
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| author | Finney-Manchester, Shawn Pete Maheshri, Narendra |
| author2 | Massachusetts Institute of Technology. Department of Chemical Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Chemical Engineering Finney-Manchester, Shawn Pete Maheshri, Narendra |
| author_sort | Finney-Manchester, Shawn Pete |
| collection | MIT |
| description | A major hurdle to evolutionary engineering approaches for multigenic phenotypes is the ability to simultaneously modify multiple genes rapidly and selectively. Here, we describe a method for in vivo-targeted mutagenesis in yeast, targeting glycosylases to embedded arrays for mutagenesis (TaGTEAM). By fusing the yeast 3-methyladenine DNA glycosylase MAG1 to a tetR DNA-binding domain, we are able to elevate mutation rates >800 fold in a specific ∼20-kb region of the genome or on a plasmid that contains an array of tetO sites. A wide spectrum of transitions, transversions and single base deletions are observed. We provide evidence that TaGTEAM generated point mutations occur through error-prone homologous recombination (HR) and depend on resectioning and the error-prone polymerase Pol ζ. We show that HR is error-prone in this context because of DNA damage checkpoint activation and base pair lesions and use this knowledge to shift the primary mutagenic outcome of targeted endonuclease breaks from HR-independent rearrangements to HR-dependent point mutations. The ability to switch repair in this way opens up the possibility of using targeted endonucleases in diverse organisms for in vivo-targeted mutagenesis. |
| first_indexed | 2024-09-23T10:49:37Z |
| format | Article |
| id | mit-1721.1/78588 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T10:49:37Z |
| publishDate | 2013 |
| publisher | Oxford University Press |
| record_format | dspace |
| spelling | mit-1721.1/785882022-09-27T15:16:18Z Harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM Finney-Manchester, Shawn Pete Maheshri, Narendra Massachusetts Institute of Technology. Department of Chemical Engineering Massachusetts Institute of Technology. School of Engineering Finney-Manchester, Shawn Pete Maheshri, Narendra A major hurdle to evolutionary engineering approaches for multigenic phenotypes is the ability to simultaneously modify multiple genes rapidly and selectively. Here, we describe a method for in vivo-targeted mutagenesis in yeast, targeting glycosylases to embedded arrays for mutagenesis (TaGTEAM). By fusing the yeast 3-methyladenine DNA glycosylase MAG1 to a tetR DNA-binding domain, we are able to elevate mutation rates >800 fold in a specific ∼20-kb region of the genome or on a plasmid that contains an array of tetO sites. A wide spectrum of transitions, transversions and single base deletions are observed. We provide evidence that TaGTEAM generated point mutations occur through error-prone homologous recombination (HR) and depend on resectioning and the error-prone polymerase Pol ζ. We show that HR is error-prone in this context because of DNA damage checkpoint activation and base pair lesions and use this knowledge to shift the primary mutagenic outcome of targeted endonuclease breaks from HR-independent rearrangements to HR-dependent point mutations. The ability to switch repair in this way opens up the possibility of using targeted endonucleases in diverse organisms for in vivo-targeted mutagenesis. National Institute of Environmental Health Sciences (Pilot P30-ES002109) 2013-04-24T16:03:28Z 2013-04-24T16:03:28Z 2013-03 2013-02 Article http://purl.org/eprint/type/JournalArticle 0305-1048 1362-4962 http://hdl.handle.net/1721.1/78588 Finney-Manchester, S. P., and N. Maheshri. “Harnessing Mutagenic Homologous Recombination for Targeted Mutagenesis in Vivo by TaGTEAM.” Nucleic Acids Research (2013). en_US http://dx.doi.org/10.1093/nar/gkt150 Nucleic Acids Research Creative Commons Attribution Non-Commercial http://creativecommons.org/licenses/by-nc/3.0 application/pdf Oxford University Press Oxford University Press |
| spellingShingle | Finney-Manchester, Shawn Pete Maheshri, Narendra Harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM |
| title | Harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM |
| title_full | Harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM |
| title_fullStr | Harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM |
| title_full_unstemmed | Harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM |
| title_short | Harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM |
| title_sort | harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by tagteam |
| url | http://hdl.handle.net/1721.1/78588 |
| work_keys_str_mv | AT finneymanchestershawnpete harnessingmutagenichomologousrecombinationfortargetedmutagenesisinvivobytagteam AT maheshrinarendra harnessingmutagenichomologousrecombinationfortargetedmutagenesisinvivobytagteam |