Strucko Tomas, Andersen Niklas L, Mahler Mikkel R, Martínez José L, Mortensen Uffe H
Department of Biotechnology and Biomedicine, Section for Synthetic Biology, Technical University of Denmark, Kongens Lyngby, Hovedstaden, Denmark.
Synth Biol (Oxf). 2021 Oct 12;6(1):ysab031. doi: 10.1093/synbio/ysab031. eCollection 2021.
Halophilic and osmotolerant yeast has a high potential for cell factory applications due to its resistance to harsh environmental factors and compatibility with a wide substrate range. However, currently available genetic techniques do not allow the full potential of as a cell factory to be harnessed. Moreover, most of the currently available tools rely on the use of auxotrophic markers that are not suitable in wild-type prototrophic strains. In addition, the preferred non-homologous end-joining (NHEJ) DNA damage repair mechanism poses further challenges when precise gene targeting is required. In this study, we present a novel plasmid-based CRISPR/Cas9 method for easy and efficient gene editing of the prototrophic strains of . Our toolset design is based on a dominant marker and facilitates quick assembly of the vectors expressing Cas9 and single or multiple single-guide RNAs (sgRNAs) that provide the possibility for multiplex gene engineering even in prototrophic strains. Moreover, we have constructed NHEJ-deficient that enable our CRISPR/Cas9 tools to support the highly efficient introduction of point mutations and single/double gene deletions. Importantly, we also demonstrate that 90-nt single-stranded DNA oligonucleotides are sufficient for direct repair of DNA breaks induced by sgRNA-Cas9, resulting in precise edits reaching 100% efficiencies. In conclusion, tools developed in this study will greatly advance basic and applied research in In addition, we envision that our tools can be rapidly adapted for gene editing of other non-conventional yeast species including the ones belonging to the CUG clade.
嗜盐和耐渗透压酵母因其对恶劣环境因素的抗性以及与广泛底物范围的兼容性，在细胞工厂应用方面具有很高的潜力。然而，目前可用的遗传技术无法充分发挥其作为细胞工厂的全部潜力。此外，目前大多数可用工具依赖于营养缺陷型标记的使用，而这些标记不适用于野生型原养型菌株。此外，当需要精确的基因靶向时，首选的非同源末端连接（NHEJ）DNA损伤修复机制带来了进一步的挑战。在本研究中，我们提出了一种基于质粒的新型CRISPR/Cas9方法，用于对原养型菌株进行简单高效的基因编辑。我们的工具集设计基于显性标记，便于快速组装表达Cas9和单个或多个单向导RNA（sgRNA）的载体，这甚至为原养型菌株中的多重基因工程提供了可能性。此外，我们构建了NHEJ缺陷型菌株，使我们的CRISPR/Cas9工具能够支持高效引入点突变和单/双基因缺失。重要的是，我们还证明90个核苷酸的单链DNA寡核苷酸足以直接修复由sgRNA-Cas9诱导的DNA断裂，从而实现100%效率的精确编辑。总之，本研究中开发的工具将极大地推动相关领域的基础研究和应用研究。此外，我们设想我们的工具可以迅速适用于其他非传统酵母物种的基因编辑，包括属于CUG进化枝的那些物种。
