Stojković Lazar, Gligorovski Vojislav, Geramimanesh Mahsa, Labagnara Marco, Rahi Sahand Jamal
Laboratory of the Physics of Biological Systems, Institute of Physics, École polytechnique fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.
G3 (Bethesda). 2025 Mar 18;15(3). doi: 10.1093/g3journal/jkae297.
Scarless genome editing in budding yeast with elimination of the selection marker has many advantages. Some markers such as URA3 and TRP1 can be recycled through counterselection. This permits seamless genome modification with pop-in/pop-out, in which a DNA construct first integrates in the genome and, subsequently, homologous regions recombine and excise undesired sequences. Popular approaches for creating such constructs use oligonucleotides and PCR. However, the use of oligonucleotides has many practical disadvantages. With the rapid reduction in price, synthesizing custom DNA sequences in specific plasmid backbones has become an appealing alternative. For designing plasmids for seamless pop-in/pop-out gene tagging or deletion, there are a number of factors to consider. To create only the shortest DNA sequences necessary, avoid errors in manual design, specify the amount of homology desired, and customize restriction sites, we created the computational tool PIPOline. Using it, we tested the ratios of homology that improve pop-out efficiency when targeting the genes HTB2 or WHI5. We supply optimal pop-in/pop-out plasmid sequences for tagging or deleting almost all S288C budding yeast open reading frames. Finally, we demonstrate how the histone variant Htb2 marked with a red fluorescent protein can be used as a cell-cycle stage marker, alternative to superfolder GFP, reducing light toxicity. We expect PIPOline to streamline genome editing in budding yeast.
在芽殖酵母中进行无疤痕基因组编辑并消除选择标记具有许多优势。一些标记，如URA3和TRP1，可以通过反选择进行循环利用。这允许通过“插入/弹出”实现无缝基因组修饰，即DNA构建体首先整合到基因组中，随后同源区域重组并切除不需要的序列。创建此类构建体的常用方法是使用寡核苷酸和PCR。然而，使用寡核苷酸存在许多实际缺点。随着价格的迅速降低，在特定质粒骨架中合成定制DNA序列已成为一种有吸引力的替代方法。为设计用于无缝“插入/弹出”基因标签或缺失的质粒，有许多因素需要考虑。为了仅创建所需的最短DNA序列、避免手动设计中的错误、指定所需的同源性量并定制限制酶切位点，我们创建了计算工具PIPOline。使用它，我们测试了靶向HTB2或WHI5基因时提高“弹出”效率的同源性比例。我们提供了用于标记或删除几乎所有S288C芽殖酵母开放阅读框的最佳“插入/弹出”质粒序列。最后，我们展示了用红色荧光蛋白标记的组蛋白变体Htb2如何可以用作细胞周期阶段标记，替代超级折叠绿色荧光蛋白，降低光毒性。我们期望PIPOline能简化芽殖酵母中的基因组编辑。
