Advantages of Mutant Generation by Genome Rearrangements of Non‐Conventional Yeast via Direct Nuclease Transfection

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.