title: Cas9-mediated genome editing in the methanogenic archaeonMethanosarcina acetivorans.
abstract: Although Cas9-mediated genome editing has proven to be a powerful genetic tool in eukaryotes, its application in Bacteria has been limited because of inefficient targeting or repair; and its application to Archaea has yet to be reported. Here we describe the development of a Cas9-mediated genome-editing tool that allows facile genetic manipulation of the slow-growing methanogenic archaeonMethanosarcina acetivoransIntroduction of both insertions and deletions by homology-directed repair was remarkably efficient and precise, occurring at a frequency of approximately 20% relative to the transformation efficiency, with the desired mutation being found in essentially all transformants examined. Off-target activity was not observed. We also observed that multiple single-guide RNAs could be expressed in the same transcript, reducing the size of mutagenic plasmids and simultaneously simplifying their design. Cas9-mediated genome editing reduces the time needed to construct mutants by more than half (3 vs. 8 wk) and allows simultaneous construction of double mutants with high efficiency, exponentially decreasing the time needed for complex strain constructions. Furthermore, coexpression the nonhomologous end-joining (NHEJ) machinery from the closely related archaeon,Methanocella paludicola, allowed efficient Cas9-mediated genome editing without the need for a repair template. The NHEJ-dependent mutations included deletions ranging from 75 to 2.7 kb in length, most of which appear to have occurred at regions of naturally occurring microhomology. The combination of homology-directed repair-dependent and NHEJ-dependent genome-editing tools comprises a powerful genetic system that enables facile insertion and deletion of genes, rational modification of gene expression, and testing of gene essentiality.
authors.affiliation: Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801.
authors.forename: Dipti D
authors.identifier: 0000-0002-8390-7251
authors.initials: DD
authors.lastname: Nayak
authors.affiliation: Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801 metcalf@illinois.edu.
authors.forename: William W
authors.identifier: 0000-0002-0182-0671
authors.initials: WW
authors.lastname: Metcalf
mesh_terms: D064113:CRISPR-Cas Systems; D004260:DNA Repair; D000072669:Gene Editing; D019848:Gene Expression Regulation, Archaeal; D020043:Genes, Essential; D005822:Genetic Vectors / Q000235:genetics; D016678:Genome; D023281:Genomics / Q000379:methods; D017020:Methanosarcina / Q000235:genetics*; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems
keywords: Archaea; Cas9; Methanosarcina; genetics; methanogens