Efficient CRISPR/Cas12a-based genome editing toolbox for metabolic engineering in Methanococcus maripaludis

Abstract

AbstractMethanococcus maripaludis is a fast-growing and genetically tractable methanogen. To become a useful host organism for the biotechnological conversion of CO2 and renewable hydrogen to fuels and value-added products, its product scope needs to be extended. Metabolic engineering requires reliable and efficient genetic tools, in particular for genome editing related to the primary metabolism that may affect cell growth.We have constructed a genome editing toolbox by utilizing Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) in combination with the homology-directed repair machinery natively present in M. maripaludis.The toolbox enables gene knock-out with a positive rate typically above 89%, despite M. maripaludis being hyper-polyploid. We have replaced the flagellum operon (around 8.9kb) by a β-glucuronidase gene to demonstrate a larger deletion, and to enable quantification of promotor strengths.The CRISPR/LbCas12a toolbox presented here is currently perhaps the most reliable and fastest method for genome editing in a methanogen.