Harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM
Author(s)Finney-Manchester, Shawn Pete; Maheshri, Narendra
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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.
DepartmentMassachusetts Institute of Technology. Department of Chemical Engineering; Massachusetts Institute of Technology. School of Engineering
Nucleic Acids Research
Oxford University Press
Finney-Manchester, S. P., and N. Maheshri. “Harnessing Mutagenic Homologous Recombination for Targeted Mutagenesis in Vivo by TaGTEAM.” Nucleic Acids Research (2013).
Final published version