McKetta Department of Chemical Engineering, The University of Texas at Austin, Austin, TX, USA.
Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA.
Methods Mol Biol. 2021;2307:1-24. doi: 10.1007/978-1-0716-1414-3_1.
A mutant excision/integration piggyBac transposase can be used to seamlessly excise a chromosomally integrated, piggyBac-compatible selection marker cassette from the Yarrowia lipolytica genome. This piggyBac transposase-based genome engineering process allows for both positive selection of targeted homologous recombination events and scarless or footprint-free genome modifications after precise marker recovery. Residual non-native sequences left in the genome after marker excision can be minimized (0-4 nucleotides) or customized (user-defined except for a TTAA tetranucleotide). Both of these options reduce the risk of unintended homologous recombination events in strains with multiple genomic edits. A suite of dual positive/negative selection marker pairs flanked by piggyBac inverted terminal repeats (ITRs) have been constructed and are available for precise genome engineering in Y. lipolytica using this method. This protocol specifically describes the split marker homologous recombination-based disruption of Y. lipolytica ADE2 with a piggyBac ITR-flanked URA3 cassette, followed by piggyBac transposase-mediated excision of the URA3 marker to leave a 50 nucleotide synthetic barcode at the ADE2 locus. The resulting ade2 strain is auxotrophic for adenine, which enables the use of ADE2 as a selectable marker for further strain engineering.
一种突变的切除/整合 piggyBac 转座酶可用于从解脂耶氏酵母基因组中无缝切除染色体整合的、与 piggyBac 兼容的选择标记盒。这种基于 piggyBac 转座酶的基因组工程过程允许对靶向同源重组事件进行正向选择，并在精确回收标记后进行无疤痕或无足迹的基因组修饰。标记切除后留在基因组中的残留非天然序列可以最小化（0-4 个核苷酸）或定制（用户定义，除 TTAA 四核苷酸外）。这两种选择都降低了在具有多个基因组编辑的菌株中发生意外同源重组事件的风险。已经构建了一系列带有 piggyBac 反向末端重复序列（ITR）的双正/负选择标记对，可用于使用该方法在解脂耶氏酵母中进行精确的基因组工程。本方案特别描述了基于分裂标记同源重组的解脂耶氏酵母 ADE2 的破坏，带有 piggyBac ITR 侧翼的 URA3 盒，然后通过 piggyBac 转座酶介导的 URA3 标记切除，在 ADE2 基因座留下 50 个核苷酸的合成条形码。所得 ade2 菌株对腺嘌呤是营养缺陷型的，这使得 ADE2 能够用作进一步菌株工程的可选择标记。
