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Significance Methanogenic archaea play a central role in the global carbon cycle, with profound implications for climate change, yet our knowledge regarding the biology of these important organisms leaves much to be desired. A key bottleneck that hinders the study of methanogenic archaea, especially those within the genusMethanosarcina , results from the time-consuming and often cumbersome tools that are currently available for genetic analysis of these microbes. The Cas9-mediated genome-editing approach forMethanosarcina acetivorans described in this study addresses this major constraint by streamlining the mutagenic process and enabling simultaneous introduction of multiple mutations. This work also sheds light on the distinct properties of homology-dependent repair and nonhomologous end-joining machinery in Archaea.

Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans