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.

中文翻译：

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升级高效和可扩展的 CRISPR-Cas 介导的嗜热真菌嗜热毁丝霉基因工程技术。

背景 嗜热丝状真菌嗜热毁丝霉具有很强的生物质降解能力，是从植物生物质直接生产酶和化学品的有吸引力的系统。它的工业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，此后在嗜热真菌基因工程方面取得了一些进展，但仍存在许多限制。它们包括需要复杂的独立表达盒来靶向多重基因组基因座和有限数量的可用选择标记基因。结果在这项研究中，我们开发了一种酸氨基球菌。基于 Cas12a 的 CRISPR 系统用于高效的多重基因组编辑，在 M. thermophila 中使用单阵列方法。这些 CRISPR-Cas12a 盒在同时进行多个基因缺失/插入时效果很好。我们还开发了一种新的简单标记回收方法，该方法依靠 CRISPR-Cas12a 系统的新切割活性来使选定标记中的 DNA 断裂。我们通过三轮转化，使用两个可选择的标记 neo 和 bar，靶向涉及嗜热分枝杆菌中纤维素酶生产途径的九个基因，证明了它的性能。我们获得了非双突变体 M9，其中蛋白质生产力和木质纤维素酶活性比野生型高 9.0 倍和 18.5 倍。我们使用我们的瞬态 CRISPR-Cas9 系统进行了平行调查，发现这两种技术是互补的。我们一起称它们为 CRISPR-Cas 辅助标记回收技术（Camr 技术）。结论 我们的研究描述了新方法（Camr 技术），该方法允许使用新建立的 CRISPR-Cas12a 系统或已建立的 CRISPR-Cas9 系统在同一嗜热真菌菌株中轻松有效地进行标记回收和性状迭代堆叠。从理论上讲，这种 Camr 技术将成为一种多功能且高效的工具，可用于设计无限数量的真菌基因。我们预计这一进步将加速以生物技术为导向的真菌工程过程。真菌中无限数量的基因。我们预计这一进步将加速以生物技术为导向的真菌工程过程。真菌中无限数量的基因。我们预计这一进步将加速以生物技术为导向的真菌工程过程。