Efficient CRISPR/Cas12a-Based Genome-Editing Toolbox for Metabolic Engineering in .

The rapid-growing and genetically tractable methanogen is a promising host organism for the biotechnological conversion of carbon dioxide and renewable hydrogen to fuels and value-added products. Expansion of its product scope through metabolic engineering necessitates reliable and efficient genetic tools, particularly for genome edits that affect the primary metabolism and cell growth. Here, we have designed a genome-editing toolbox by utilizing Cas12a from ND2006 (LbCas12a) in combination with the homology-directed repair machinery endogenously present in . This toolbox can delete target genes with a success rate of up to 95%, despite the hyperpolyploidy of . For the purpose of demonstrating a large deletion, the flagellum operon (∼8.9 kbp) was replaced by the β-glucuronidase gene. To facilitate metabolic engineering and flux balancing in , the relative strength of 15 different promoters was quantified in the presence of two common growth substrates, either formate or carbon dioxide and hydrogen. This CRISPR/LbCas12a toolbox can be regarded as a reliable and quick method for genome editing in a methanogen.