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.

中文翻译：

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DNA复制叉的精确编辑可在真核生物中实现多重基因组工程。

我们在酿酒酵母中基于在DNA复制的滞后链上退火合成寡核苷酸，描述了酿酒酵母中的多元基因组工程技术。该机制独立于Rad51定向的同源重组，避免了双链DNA断裂的产生，从而能够在单碱基对的分辨率下以> 40％的效率进行精确的染色体修饰，而不会在目标遗传基因座上引起意想不到的诱变变化。我们观察到在一次转化中同时掺入了多达12个具有多达60个靶向突变的寡核苷酸。复杂的寡核苷酸池的迭代转化迅速产生了大于10 ^5的大组合基因组多样性。该方法用于使异源β-胡萝卜素生物合成途径多样化，该途径产生了在启动子，基因和终止子上具有精确突变的遗传变异，从而导致类胡萝卜素水平改变。我们对DNA复制，修复和重组的保守过程进行工程改造的方法可以实现自动化，并为真核生物的多重组合基因组工程建立通用策略。