CRISPR/Cas12a toolbox for genomic manipulation inMethanosarcina acetivorans

Abstract

Methanogenic archaea play an important role in the global carbon cycle and are regarded as promising host organisms for the biotechnological generation of fuels and chemicals from one-carbon substrates. Methanosarcina acetivorans is extensively studied as a model methanogen due to the availability of genetic tools and its versatile substrate range. Although genome editing in M. acetivorans via CRISPR/Cas9 has already been demonstrated, we now describe a user-friendly CRISPR/Cas12a toolbox that recognizes a T-rich (5′-TTTV) PAM sequence. This new system can manage deletions of 3500 bp (i.e., knockout of the entire frhADGB operon) and heterologous gene insertions with 80% efficiency observed in ten PurR transformants. Our CRISPR/Cas12a system also enables multiplex genome editing at high efficiency, which helps speed up genetic engineering. Deletions of 100 bp generated on two separate sites of the genome yielded 8/8 correctly edited transformants. Simultaneous gene deletion (100 bp) and replacement (100-bp region replaced by the 2400-bp uidA expression cassette) at a separate site was achieved, with 3/6 of transformants being edited correctly. In combination with the Cas9-based system, our CRISPR/Cas12a toolbox enables targeted genome editing at two sites (guanine-rich and thymine-rich, respectively) and, in so doing, hastens the overall genetic engineering of the Methanosarcinales species.

Methane and ethane production rates by methyl-coenzyme M reductase in cell extracts from different methanogens

“…marburgensis under different assay conditions (data not shown). Nevertheless, multiple genetic modification tools for methanogens have been developed [20,21] along with expression of uncultured archaea Mcr [22] which may allow better conversion of longer alkyl-CoM (C3 and above) in the near future. To conclude, employing our cell-free lysate Mcr assay approach to investigate this enzyme family is a rapid and efficient assay to test the enzyme activity of these recombinant enzymes in order to provide a first milestone for further engineering of artificial cultivable alkanogens.…”

“…marburgensis under different assay conditions (data not shown). Nevertheless, multiple genetic modification tools for methanogens have been developed [20,21] along with expression of uncultured archaea…”

Nature AND Nurture: Enabling formate-dependent growth inMethanosarcina acetivorans

“…Liposome-mediated methods and polyethylene glycol-mediated methods were used to transform M. acetivorans (12). An optimized CRISPR/Cas9 genome editing toolbox was used to delete genes (11, 13). The media composition, strains, primers and gRNA, and plasmids used in this study were listed in SI Appendix Table S7, S8, S9, and S10 respectively.…”

“…Methanosarcina acetivorans is one of the most versatile methanogens in its substrate utilization (methanol, methyl amines, methyl sulfides, carbon monoxide, acetate) (6)(7)(8)(9). It is also genetically tractable, with various genetic tools available, making it a model organism for future applications in biofuel production and even methane oxidation (10)(11)(12)(13). Versatility of M. acetivorans spans all major methanogenesis pathways summarized in Fig.…”

Teaching old dogs new tricks: genetic engineering methanogens

“…A shuttle vector (PWM307) that would replicate in multiple Methanosarcina species was used to develop a selection marker for pseudomonic acid resistance in M. barkeri Fusaro ( 37 , 41 ). Multiple shuttle vectors have since been developed for M. acetivorans using puromycin resistance as a selection marker ( 38 , 39 , 80 , 81 ). The shuttle vectors, pJK89 and pMCp4, include 8ADP selection ( 38 , 81 ), and one vector, pPB35, includes pseudomonic acid selection ( 39 ).…”