A versatile Cas12k-based genetic engineering toolkit (C12KGET) for metabolic engineering in genetic manipulation-deprived strains.

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.