Institute of Molecular Biotechnology, Graz University of Technology, Graz, Austria.
Bisy e.U., Wetzawinkel, Hofstätten/Raab, Austria.
J Cell Biochem. 2018 Apr;119(4):3183-3198. doi: 10.1002/jcb.26474. Epub 2017 Dec 26.
Komagataella phaffii (syn. Pichia pastoris) is one of the most commonly used host systems for recombinant protein expression. Achieving targeted genetic modifications had been hindered by low frequencies of homologous recombination (HR). Recently, a CRISPR/Cas9 genome editing system has been implemented for P. pastoris enabling gene knockouts based on indels (insertion, deletions) via non-homologous end joining (NHEJ) at near 100% efficiency. However, specifically integrating homologous donor cassettes via HR for replacement studies had proven difficult resulting at most in ∼20% correct integration using CRISPR/Cas9. Here, we demonstrate the CRISPR/Cas9 mediated integration of markerless donor cassettes at efficiencies approaching 100% using a ku70 deletion strain. The Ku70p is involved in NHEJ repair and lack of the protein appears to favor repair via HR near exclusively. While the absolute number of transformants in the Δku70 strain is reduced, virtually all surviving transformants showed correct integration. In the wildtype strain, markerless donor cassette integration was also improved up to 25-fold by placing an autonomously replicating sequence (ARS) on the donor cassette. Alternative strategies for improving donor cassette integration using a Cas9 nickase variant or reducing off targeting associated toxicity using a high fidelity Cas9 variant were so far not successful in our hands in P. pastoris. Furthermore we provide Cas9/gRNA expression plasmids with a Geneticin resistance marker which proved to be versatile tools for marker recycling. The reported CRSIPR-Cas9 tools can be applied for modifying existing production strains and also pave the way for markerless whole genome modification studies in P. pastoris.
毕赤酵母（Komagataella phaffii）（又名巴斯德毕赤酵母（Pichia pastoris））是用于重组蛋白表达的最常用宿主系统之一。由于同源重组（HR）的频率较低，因此实现靶向基因修饰受到了阻碍。最近，已经为毕赤酵母实施了 CRISPR/Cas9 基因组编辑系统，能够通过非同源末端连接（NHEJ）实现基于插入/缺失（indels）的基因敲除，效率接近 100％。然而，通过 HR 特异性地整合同源供体盒用于替换研究已被证明很困难，使用 CRISPR/Cas9 最多只能实现约 20％的正确整合。在这里，我们展示了使用 ku70 缺失株接近 100％的效率通过 CRISPR/Cas9 介导的无标记供体盒的整合。Ku70p 参与 NHEJ 修复，并且缺乏该蛋白似乎特别有利于 HR 修复。尽管Δku70 菌株中的转化子绝对数量减少，但几乎所有存活的转化子都显示出正确的整合。在野生型菌株中，通过在供体盒上放置自主复制序列（ARS），无标记供体盒的整合也提高了 25 倍。迄今为止，在我们的实验中，使用 Cas9 切口酶变体提高供体盒整合或使用高保真 Cas9 变体降低脱靶相关毒性的替代策略在毕赤酵母中均未成功。此外，我们还提供了带有遗传霉素抗性标记的 Cas9/gRNA 表达质粒，这被证明是回收标记的多功能工具。所报道的 CRISPR-Cas9 工具可用于修饰现有的生产菌株，也为毕赤酵母无标记全基因组修饰研究铺平了道路。
