Precise Editing at DNA Replication Forks Enables Multiplex Genome Engineering in Eukaryotes

We describe a multiplex genome engineering technology in Saccharomyces cerevisiae based on annealing synthetic oligonucleotides at the lagging strand of DNA replication. The mechanism is independent of Rad51-directed homologous recombination and avoids the creation of double-strand DNA breaks, enabling precise chromosome modifications at single base-pair resolution with an efficiency of >40%, without unintended mutagenic changes at the targeted genetic loci. We observed the simultaneous incorporation of up to 12 oligonucleotides with as many as 60 targeted mutations in one transformation. Iterative transformations of a complex pool of oligonucleotides rapidly produced large combinatorial genomic diversity >105. This method was used to diversify a heterologous β-carotene biosynthetic pathway that produced genetic variants with precise mutations in promoters, genes, and terminators, leading to altered carotenoid levels. Our approach of engineering the conserved processes of DNA replication, repair, and recombination could be automated and establishes a general strategy for multiplex combinatorial genome engineering in eukaryotes. [Display omitted] •Yeast multiplex genome engineering technology that avoids DNA double-strand breaks•Rad51-independent mechanism of gene editing by ssODN annealing at DNA replication•Silencing DNA repair and slowing replication enhances multiplex editing by ssODNs•Generation of combinatorial genetic variants at base-pair precision in eukaryotes Replication forks can be co-opted to introduce multisite mutations in eukaryotic genomes without the need for DNA double-strand break.