Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina 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 archaeon Methanosarcina acetivorans. Introduction 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 NHEJdependent 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 homologydirected 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.he CRISPR (clustered regularly interspaced palindromic repeats) array and associated cas genes are widespread in microbial genomes (1), where they confer acquired immunity to phage and foreign DNA elements (2). The type IIA system from Streptococcus pyogenes is especially well characterized and has been widely applied as a remarkably effective genome-editing tool (3). During genome editing, heterologous expression of the RNA-guided DNA endonuclease Cas9 and a chimeric singleguide (sg) RNA, comprised of a 20-bp spacer that targets the chromosome and an 80-bp scaffold that binds Cas9, leads to a lethal double-strand break (DSB) at all target sites within the genome that are flanked by a 3′ NGG protospacer adjacent motif (PAM) (4) (Fig. S1). In eukaryotes, the nonhomologous endjoining (NHEJ) repair pathway can mend the DSB by generating simple insertions or deletions at the sgRNA target site, thus preventing additional rounds of Cas9-mediated cleavage (3, 5). Alternatively, the native homology-dependent repair (HDR) pathway can repair the fatal DSB, so long as a repair template that modifies or removes the sgRNA target site is provided, again preventing additional rounds of Cas9-mediated cleavage (3, 5) (Fig. S1). Appropriately ...

A Membrane-Bound Cytochrome EnablesMethanosarcina acetivoransto Conserve Energy to Support Growth from Extracellular Electron Transfer

“…The methanol oxidation coupled with AQDS reduction in the presence of BES described here is the first demonstration of a methanogen conserving energy to support growth with electron transfer to an external electron acceptor as the sole means of energy conservation. The ability of M. acetivorans to grow in this manner, and the availability of tools for genetic manipulation (25–27) provide the opportunity for functional analysis of extracellular electron transfer in an archaeon.…”

“…Tools are available for genetic manipulation of the methanogen Methanosarcina acetivorans (25–27). A methyl-coenzyme M reductase from an uncultured ANME was introduced into M. acetivorans to generate a strain that could convert methane to acetate with simultaneous reduction of Fe(III) (28).…”

Functional interactions between post-translationally modified amino acids of methyl-coenzyme M reductase inMethanosarcina acetivorans

“…To test the hypothesis that MA4545 is responsible for S- methylation of Cys472 in McrA, we deleted the gene in M. acetivorans using recently developed Cas9-based genome editing tools (34). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) showed that the McrA tryptic peptide containing the Cys residue of interest was 14 Da lighter in the mutant than in the corresponding peptide from wild-type, consistent with loss of a methyl group in the mutant (Figure 3C).…”

“…All mutations in M. acetivorans were introduced using a Cas9-based genome editing technique (34). Mutagenic plasmids were derived from pDN201, a base vector containing Cas9 from Streptococcus pyogenes under the control of the tetracycline-inducible pMcrB(tetO1) promoter.…”

Modern methods for laboratory diversification of biomolecules

“…The elucidation of CRISPR-Cas biology has rendered this system convenient for engineering of prokaryotic [67], archaeal [68], and eukaryotic [23] genomes. The simplest and most commonly used implementation of CRISPR-Cas systems consists of only two components: an endonuclease (e.g.…”