Gassler Thomas, Heistinger Lina, Mattanovich Diethard, Gasser Brigitte, Prielhofer Roland
Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria.
Austrian Centre of Industrial Biotechnology (acib), Vienna, Austria.
Methods Mol Biol. 2019;1923:211-225. doi: 10.1007/978-1-4939-9024-5_9.
State-of-the-art strain engineering techniques for the methylotrophic yeast Pichia pastoris (syn. Komagataella spp.) include overexpression of endogenous and heterologous genes and deletion of host genes. For efficient gene deletion, methods such as the split-marker technique have been established. However, synthetic biology trends move toward building up large and complex reaction networks, which often require endogenous gene knockouts and simultaneous overexpression of individual genes or whole pathways. Realization of such engineering tasks by conventional approaches employing subsequent steps of transformations and marker recycling is very time- and labor-consuming. Other applications require tagging of certain genes/proteins or promoter exchange approaches, which are hard to design and construct with conventional methods. Therefore, efficient systems are required that allow precise manipulations of the P. pastoris genome, including simultaneous overexpression of multiple genes. To meet this challenge, we have developed a CRISPR/Cas9-based kit for gene insertions, deletions, and replacements, which paves the way for precise genomic modifications in P. pastoris. In this chapter, the versatile method for performing these modifications without the integration of a selection marker is described. A ready-to-use plasmid kit for performing CRISPR/Cas9-mediated genome editing in P. pastoris based on the GoldenPiCS modular cloning vectors is available at Addgene as CRISPi kit (#1000000136).
用于甲基营养型酵母巴斯德毕赤酵母（同义词：Komagataella spp.）的先进菌株工程技术包括内源性和异源基因的过表达以及宿主基因的缺失。为了实现高效的基因缺失，已经建立了诸如分裂标记技术等方法。然而，合成生物学的发展趋势是构建大型和复杂的反应网络，这通常需要内源性基因敲除以及单个基因或整个途径的同时过表达。通过采用转化和标记回收后续步骤的传统方法来实现此类工程任务非常耗时且费力。其他应用需要对某些基因/蛋白质进行标记或启动子交换方法，而用传统方法很难设计和构建。因此，需要高效的系统来精确操纵巴斯德毕赤酵母基因组，包括多个基因的同时过表达。为了应对这一挑战，我们开发了一种基于CRISPR/Cas9的试剂盒，用于基因插入、缺失和替换，这为巴斯德毕赤酵母中的精确基因组修饰铺平了道路。在本章中，将描述一种无需整合选择标记即可进行这些修饰的通用方法。基于GoldenPiCS模块化克隆载体的用于在巴斯德毕赤酵母中进行CRISPR/Cas9介导的基因组编辑的即用型质粒试剂盒可在Addgene公司作为CRISPi试剂盒（#1000000136）获得。
