Harnessing mutagenic homologous recombination for in vivo targeted mutagenesis by TaGTEAM

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(?). 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. TaGTEAM was applied to the problem of ethanol tolerance through multigene gTME in a lab strain of S. cerevisiae. While results indicate that the mutation rate achieved by TaGTEAM is not sufficient to realize novel gain of function mutations in this case, important lessons about how to deploy TaGTEAM in a more effective manner were learned. Conducting TaGTEAM in G2/M checkpoint arrested cells increased the mutation rate further to 6 x 10-4 cell- gen-1, relieving various rate limiting steps in point mutagenesis and suggesting that a sequential mutate and then select protocol will make best use of TaGTEAM's abilities for novel phenotype evolution. The insights gained in switching repair of targeted double strand breaks to error-prone HR at high rates opens up the possibility of using targeted endonucleases in diverse organisms for in vivo targeted mutagenesis.