Harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM
Name
Finney-Manchester-2013-Harnessing mutagenic.pdf
Size
6.27 MB
Format
Adobe PDF
Checksum (MD5)
e42e27b490d5267d8128b773b00b286a
Author(s)
Finney-Manchester, Shawn Pete
Maheshri, Narendra
Date Issued
March 2013
Journal
Nucleic Acids Research
Publisher
Oxford University Press
Citation
Finney-Manchester, S. P., and N. Maheshri. “Harnessing Mutagenic Homologous Recombination for Targeted Mutagenesis in Vivo by TaGTEAM.” Nucleic Acids Research (2013).
Version
Final published version
Abstract
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.
MIT Department
Massachusetts Institute of Technology. Department of Chemical Engineering
Massachusetts Institute of Technology. School of Engineering
Terms of Use
Creative Commons Attribution Non-Commercial
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1093/nar/gkt150
