Todokoro Takehiko, Hata Yoji, Ishida Hiroki
Research Institute, Gekkeikan Sake Co., Ltd., 101 Shimotoba-koyanagi-cho, Fushimi-ku, Kyoto, 612-8385, Japan.
Biotechnol Notes. 2024 Apr 4;5:58-63. doi: 10.1016/j.biotno.2024.03.002. eCollection 2024.
is an important fungus in food and industrial enzyme production. In , targeted knock-in transformation is primarily limited to homologous recombination (HR)-based systems, in which non-homologous end-joining (NHEJ)-disruptant hosts are required. However, preparation of hosts and transformation templates for such systems is laborious, in addition to other disadvantages. In the present study, we examined alternative targeted knock-in mediated by CRISPR/Cas9, in which a microhomology-mediated end-joining (MMEJ) and single-strand annealing (SSA) repair system was employed. This approach enabled the efficient development of targeted knock-in transformants without host preparation using only a short homology template. We conclude that this new method could be applied to facilitate the transformation of , and will make it easier to acquire targeted knock-in transformants, especially from industrially important non-model strains.
是食品和工业酶生产中的一种重要真菌。在[具体情境未提及]中，靶向敲入转化主要限于基于同源重组（HR）的系统，在该系统中需要非同源末端连接（NHEJ）破坏的宿主。然而，除了其他缺点外，此类系统的宿主和转化模板的制备非常费力。在本研究中，我们研究了由CRISPR/Cas9介导的替代靶向敲入，其中采用了微同源性介导的末端连接（MMEJ）和单链退火（SSA）修复系统。这种方法仅使用短同源模板就能够在无需制备宿主的情况下高效开发靶向敲入转化体。我们得出结论，这种新方法可用于促进[具体对象未提及]的转化，并将使获取靶向敲入转化体变得更容易，特别是从具有工业重要性的非模式菌株中获取。
