The construction and validation of a set of Yarrowia lipolytica CRISPR/Cas9 vectors containing six different markers that allows virtually any genetic background to be edited, including those of wild-type strains. Using the Golden Gate method, we assembled a set of six CRISPR/Cas9 vectors, each containing a different selection marker, to be used for editing the genome of the industrial yeast Y. lipolytica. This vector set is available via Addgene. Any guide RNA (gRNA) sequence can be easily and rapidly introduced in any of these vectors using Golden Gate assembly. We successfully edited six different genes in a variety of genetic backgrounds, including those of wild-type strains, with five of the six vectors. Use of these vectors strongly improved homologous recombination and cassette integration at a specific locus. We have created a versatile and modular set of CRISPR/Cas9 vectors that will allow any Y. lipolytica strain to be rapidly edited; this tool will facilitate experimentation with any prototroph wild-type strains displaying interesting features.

中文翻译：

---

一组用于开发野生型菌株多样性的解脂耶氏酵母 CRISPR/Cas9 载体

一组包含六种不同标记的解脂耶氏酵母 CRISPR/Cas9 载体的构建和验证，几乎可以编辑任何遗传背景，包括野生型菌株的遗传背景。使用 Golden Gate 方法，我们组装了一组 6 个 CRISPR/Cas9 载体，每个载体包含不同的选择标记，用于编辑工业酵母 Y. lipolytica 的基因组。该载体集可通过 Addgene 获得。使用 Golden Gate 组装，任何向导 RNA (gRNA) 序列都可以轻松快速地引入任何这些载体中。我们使用六个载体中的五个成功编辑了各种遗传背景中的六个不同基因，包括野生型菌株的基因。这些载体的使用极大地改善了特定基因座的同源重组和盒式整合。我们创建了一套通用的模块化 CRISPR/Cas9 载体，可以快速编辑任何解脂耶氏酵母菌株；该工具将有助于对任何具有有趣特征的原养野生型菌株进行实验。