Automated plasmid design for marker-free genome editing in budding yeast

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.