Upgrading of efficient and scalable CRISPR-Cas-mediated technology for genetic engineering in thermophilic fungus Myceliophthora thermophila
Upgrading of efficient and scalable CRISPR-Cas-mediated technology for genetic engineering in thermophilic fungus Myceliophthora thermophila
复制标题
高效且可扩展的 CRISPR-Cas 介导技术的升级，用于嗜热真菌嗜热毁丝霉的基因工程
DOI：
10.1186/s13068-019-1637-y
复制
发表时间：
2019-12-23
影响因子：
6.3
通讯作者：
Tian, Chaoguang
中科院分区：
文献类型：
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作者：
Liu, Qian;Zhang, Yongli;Tian, Chaoguang
嗜热丝状真菌菌丝疫霉具有很强的生物质降解能力，是一种有吸引力的从植物生物质中直接生产酶和化学物质的系统。它在工业上的重要性激发了我们开发基因组编辑工具来加速这种真菌的基因工程。第一代CRISPR-Cas9技术于2017年开发，自那时以来，在嗜热真菌基因工程方面取得了一些进展，但仍然存在一些局限性。它们包括需要复杂的独立表达盒来靶向多重基因组位点和有限数量的可用可选择标记基因。在这项研究中，我们开发了一种基于酸胺球菌cas12的CRISPR系统，利用单阵列方法对嗜热分枝杆菌进行高效的多重基因组编辑。这些CRISPR-Cas12a磁带可以很好地用于同时进行多个基因缺失/插入。我们还开发了一种新的简单的标记回收方法，该方法依赖于CRISPR-Cas12a系统的新型切割活性，在选定的标记中制造DNA断裂。我们利用两个选择性标记neo和bar，通过三轮转化，靶向了嗜热分枝杆菌纤维素酶生产途径中涉及的9个基因，证明了它的性能。我们获得了蛋白质产量和木质纤维素酶活性分别比野生型高9.0倍和18.5倍的非单克隆突变体M9。我们使用我们的瞬时CRISPR-Cas9系统进行了平行调查，发现这两种技术是互补的。我们把它们统称为crispr - cas辅助标记回收技术（Camr技术）。我们的研究描述了新的方法（Camr技术），可以使用新建立的CRISPR-Cas12a系统或CRISPR-Cas9系统，在同一嗜热真菌菌株中实现简单有效的标记循环和性状的迭代堆叠。这种Camr技术将是一种多功能和高效的工程工具，理论上，真菌中无限数量的基因。我们期望这一进展能够加速真菌生物技术导向的工程过程。
Background Thermophilic filamentous fungus Myceliophthora thermophila has great capacity for biomass degradation and is an attractive system for direct production of enzymes and chemicals from plant biomass. Its industrial importance inspired us to develop genome editing tools to speed up the genetic engineering of this fungus. First-generation CRISPR-Cas9 technology was developed in 2017 and, since then, some progress has been made in thermophilic fungi genetic engineering, but a number of limitations remain. They include the need for complex independent expression cassettes for targeting multiplex genomic loci and the limited number of available selectable marker genes. Results In this study, we developed an Acidaminococcus sp. Cas12a-based CRISPR system for efficient multiplex genome editing, using a single-array approach in M. thermophila. These CRISPR-Cas12a cassettes worked well for simultaneous multiple gene deletions/insertions. We also developed a new simple approach for marker recycling that relied on the novel cleavage activity of the CRISPR-Cas12a system to make DNA breaks in selected markers. We demonstrated its performance by targeting nine genes involved in the cellulase production pathway in M. thermophila via three transformation rounds, using two selectable markers neo and bar. We obtained the nonuple mutant M9 in which protein productivity and lignocellulase activity were 9.0- and 18.5-fold higher than in the wild type. We conducted a parallel investigation using our transient CRISPR-Cas9 system and found the two technologies were complementary. Together we called them CRISPR-Cas-assisted marker recycling technology (Camr technology). Conclusions Our study described new approaches (Camr technology) that allow easy and efficient marker recycling and iterative stacking of traits in the same thermophilic fungus strain either, using the newly established CRISPR-Cas12a system or the established CRISPR-Cas9 system. This Camr technology will be a versatile and efficient tool for engineering, theoretically, an unlimited number of genes in fungi. We expect this advance to accelerate biotechnology-oriented engineering processes in fungi.