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.
['Barbieri|Edward M|EM|', 'Muir|Paul|P|', 'Akhuetie-Oni|Benjamin O|BO|', 'Yellman|Christopher M|CM|', 'Isaacs|Farren J|FJ|']
D004261:DNA Replication D004926:Escherichia coli D000072669:Gene Editing D005818:Genetic Engineering D009841:Oligonucleotides D012441:Saccharomyces cerevisiae
DNA replication multiplex genome engineering homologous recombination Rad51 genome editing metabolic engineering natural products ssDNA oligodeoxynucleotides