Developing genetic tools in Methanococcus maripaludis

Highly sophisticated genetic tools have been deployed in many eukaryotes and bacteria, yet those available in the third domain of life: the archaea, remain comparatively limited. The constraints this imposes must be overcome if the unique capabilities of this domain are to be tapped. One biological pathway found solely within members of the archaea is methanogenesis: the conversion of simple molecules into methane. The manipulation of methane production through genetic modification of methanogens is highly desirable, since in addition to its potential use as a biofuel, methane contributes significantly to the greenhouse effect. Among the methanogens, Methanococcus maripaludis (M. maripaludis) is one of the best developed as a model, however many of its available genetic tools are outdated. The CRISPR/Cas9 system is a relatively recently developed and highly versatile genetic technique, and at the outset of this work had not been deployed in any archaeal system. The aim of this project was therefore to provide a proof of principle that the CRISPR/Cas9 system could be deployed successfully in this organism. The experimental approach selected was a plasmid invader assay, in which the activity of the system could be demonstrated by Cas9 mediated plasmid destruction resulting in reduced M. maripaludis cell viability. A scheme by which this assay could be conducted using existing genetic tools in two different M. maripaludis strains was designed, and the full series of required plasmids was produced. Attempts to use these plasmids to produce the M. maripaludis strains required for the invader assay were unsuccessful, and it could therefore not be carried out. However, it is anticipated that the assay system designed and plasmids produced here should enable the rapid testing of this system were this work continued, which should enable the addition of the CRISPR/Cas9 system to the M. maripaludis genetic toolbox.