The genetic modification of microorganisms is conducive to the selection of high-yield producers of high-value-added chemicals, but a lack of genetic tools hinders the industrialization of most wild species. Therefore, it is crucial to develop host-independent gene editing tools that can be used for genetic manipulation-deprived strains. The Tn7-like transposon from Scytonema hofmanni has been shown to mediate homologous recombination-independent genomic integration after heterologous expression in Escherichia coli, but the integration efficiency of heterologous sequences larger than 5 kb remains suboptimal. Here, we constructed a versatile Cas12k-based genetic engineering toolkit (C12KGET) that can achieve genomic integration of fragments up to 10 kb in size with up to 100% efficiency in challenging strains. Using C12KGET, we achieved the first example of highly efficient genome editing in Sinorhizobium meliloti, which successfully solved the problem that industrial strains are difficult to genetically modify, and increased vitamin B12 production by 25%. In addition, Cas12k can be directly used for transcriptional regulation of genes with up to 92% efficiency due to its naturally inactivated nuclease domain. The C12KGET established in this study is a versatile and efficient marker-free tool for gene integration as well as transcriptional regulation that can be used for challenging strains with underdeveloped genetic toolkits.

中文翻译：

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一种基于 Cas12k 的多功能基因工程工具包 (C12KGET)，用于基因操作剥夺菌株的代谢工程


微生物的基因改造有利于选择高附加值化学品的高产生产者，但缺乏遗传工具阻碍了大多数野生物种的产业化。因此，开发可用于缺乏基因操作的菌株的宿主独立基因编辑工具至关重要。来自 Scytonema hofmanni 的 Tn7 样转座子已被证明在大肠杆菌中异源表达后可介导同源重组独立的基因组整合，但大于 5 kb 的异源序列的整合效率仍然不理想。在这里，我们构建了一个基于 Cas12k 的多功能基因工程工具包 (C12KGET)，可以实现大小高达 10 kb 的片段的基因组整合，在挑战性菌株中效率高达 100%。利用C12KGET，我们在苜蓿中华根瘤菌中实现了高效基因组编辑的第一个例子，成功解决了工业菌株难以进行基因改造的问题，并将维生素B12产量提高了25%。此外，由于Cas12k具有天然失活的核酸酶结构域，因此可以直接用于基因的转录调控，效率高达92%。本研究中建立的 C12KGET 是一种多功能、高效的无标记工具，用于基因整合和转录调控，可用于具有不发达遗传工具包的挑战性菌株。