Section for Synthetic Biology, Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads 223, 2800 Kongens Lyngby, Denmark.
FEMS Yeast Res. 2024 Jan 9;24. doi: 10.1093/femsyr/foae026.
Komagataella phaffii (Pichia pastoris) is a methylotrophic yeast that is favored by industry and academia mainly for expression of heterologous proteins. However, its full potential as a host for bioproduction of valuable compounds cannot be fully exploited as genetic tools are lagging behind those that are available for baker's yeast. The emergence of CRISPR-Cas9 technology has significantly improved the efficiency of gene manipulations of K. phaffii, but improvements in gene-editing methods are desirable to further accelerate engineering of this yeast. In this study, we have developed a versatile vector-based CRISPR-Cas9 method and showed that it works efficiently at different genetic loci using linear DNA fragments with very short targeting sequences including single-stranded oligonucleotides. Notably, we performed site-specific point mutations and full gene deletions using short (90 nt) single-stranded oligonucleotides at very high efficiencies. Lastly, we present a strategy for transient inactivation of nonhomologous end-joining (NHEJ) pathway, where KU70 gene is disrupted by a visual marker (uidA gene). This system enables precise CRISPR-Cas9-based editing (including multiplexing) and facilitates simple reversion to NHEJ-proficient genotype. In conclusion, the tools presented in this study can be applied for easy and efficient engineering of K. phaffii strains and are compatible with high-throughput automated workflows.
毕赤酵母（Komagataella phaffii）是一种甲醇营养型酵母，主要因其能够表达异源蛋白而受到工业界和学术界的青睐。然而，由于遗传工具的落后，其作为生物生产有价值化合物的宿主的潜力尚未得到充分开发。CRISPR-Cas9 技术的出现极大地提高了毕赤酵母基因操作的效率，但仍需要改进基因编辑方法，以进一步加速该酵母的工程改造。在本研究中，我们开发了一种基于载体的通用 CRISPR-Cas9 方法，并证明其可使用具有非常短靶向序列（包括单链寡核苷酸）的线性 DNA 片段在不同遗传位点上高效工作。值得注意的是，我们使用非常短的（90nt）单链寡核苷酸在很高的效率下进行了定点突变和全基因缺失。最后，我们提出了一种瞬时失活非同源末端连接（NHEJ）途径的策略，该策略通过一个可视化标记（uidA 基因）破坏 KU70 基因。该系统可实现精确的基于 CRISPR-Cas9 的编辑（包括多重编辑），并有助于简单地恢复到 NHEJ 高效基因型。总之，本研究中提供的工具可用于轻松高效地工程改造毕赤酵母菌株，并且与高通量自动化工作流程兼容。
