Oda Arisa H, Yasukawa Taishi, Tamura Miki, Sano Ayumu, Masuo Naohisa, Ohta Kunihiro
Department of Life Sciences, Graduate School of Arts & Sciences, the University of Tokyo, Tokyo, Japan.
Collaborative Research Institute for Innovative Microbiology, Tokyo, Japan.
Genes Cells. 2025 Mar;30(2):e70010. doi: 10.1111/gtc.70010.
We previously developed a genome engineering method (TAQing2.0) based on the direct delivery of DNA endonucleases into living cells, which induces genome rearrangements even in non-sporulating nonconventional yeasts without introducing foreign DNA. Using TAQing2.0 and conventional mutagenesis (by nitrosoguanidine), we obtained mutant asexual Candida utilis strains capable of growing under highly acidic conditions (pH 1.8). Whole genome resequencing revealed that the genomic sequences of mutants generated by both methods contain a negligible small population of unmappable sequences, suggesting that both types of mutants can be regarded as equivalent to naturally occurring mutants. TAQing2.0 mutants exhibit multiple genome rearrangements with few point mutations, whereas conventional mutagenesis produces numerous point mutations. This feature enabled us to easily identify candidate genes (e.g., LYP1 homolog) responsible for acid resistance. TAQing2.0 is a powerful and versatile tool for mutant production and gene hunting without invasion of foreign DNA.
我们之前开发了一种基因组工程方法（TAQing2.0），该方法基于将DNA核酸内切酶直接导入活细胞，即使在不产孢的非常规酵母中也能诱导基因组重排，且不会引入外源DNA。使用TAQing2.0和传统诱变方法（通过亚硝基胍），我们获得了能够在高酸性条件（pH 1.8）下生长的突变型无性解脂耶氏酵母菌株。全基因组重测序显示，两种方法产生的突变体的基因组序列都包含数量可忽略不计的少量无法映射的序列，这表明这两种类型的突变体都可被视为等同于自然发生的突变体。TAQing2.0突变体表现出多个基因组重排，点突变很少，而传统诱变则产生大量点突变。这一特性使我们能够轻松鉴定出负责耐酸性的候选基因（例如LYP1同源物）。TAQing2.0是一种强大且通用的工具，可用于在不侵入外源DNA的情况下进行突变体生产和基因搜寻。
