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 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.

Keywords: Archaea; Cas9; Methanosarcina; genetics; methanogens.

Fig. S1.

An overview of Cas9-mediated genome editing. Heterologous expression of Cas9 from Streptococcus pyogenes (gray) and a chimeric sgRNA, containing a 80-bp scaffold sequence to facilitate Cas9 binding (in pink) and a 20-bp spacer identical to a region on the host chromosome (in blue) flanked by a 3′ NGG PAM (in yellow) generates a DSB. In Archaea, HDR (in orange) is the prevalent mechanism for DSB repair and can be leveraged for genome editing by providing appropriate repair templates for targeted insertions, deletions, or allelic replacements.

Fig. 1.

Cas9-mediated genome editing in M. acetivorans. (A) Key elements of the pDN_CRISPR plasmid series include the Cas9 ORF from S. pyogenes fused to the tetracycline-inducible PmcrB(tetO1) promoter (in green), sgRNA(s) fused to the methanol-inducible PmtaCB1 promoter (in pink), a homology repair template (in orange), and the entire pC2A plasmid replicon containing an autonomous Methanosarcina origin of replication (in gray). The puromycin transacetylase (pac) marker enables selection of puromycin resistant (Pur^R) transformants and the hypoxanthine phosphoribosyltransferase (hpt) marker facilitates plasmid curing by counter selection on medium containing 8ADP. Note: The E. coli replicon and resistance marker genes have not been shown. (B) Expression of sgRNA with a 20-bp target sequence identical to a region of the WT ssuC locus (in blue) flanked by a 3′ NGG PAM (in red) with Cas9 generates a DSB at the ssuC locus. A region of the plasmid pDN211 contains a homology repair (HR) template to abolish the target site by generating a 34-bp deletion and simultaneously introducing a diagnostic NotI restriction endonuclease site in the ssuC ORF (in orange). (C) The chromosomal ssuC locus amplified from 20 Pur^R transformants containing pDN211 as well as the parent strain (WWM60) and subjected to restriction digest with NotI. Upon digestion, 1.1-kbp and 1.3-kbp fragments are observed for all Pur^R transformants (lanes 2–21), whereas a single 2.4-kbp fragment corresponding to the WT locus is observed for WWM60 (lane 22).

Fig. S2.

Using the counter-selectable hpt marker to cure plasmids containing the genome editing machinery. (A) Growth curves for three independent puromycin-resistant (Pur^R) transformants (blue) and an 8ADP^R isolate derived from each PurR parent (red) in liquid medium containing TMA hydrochloride as the growth substrate and 2 μg/mL puromycin. Pur^R transformants contain pDN211 in the WWM60 strain background. A 1:10 dilution of stationary-phase cultures grown in liquid medium containing TMA as the growth substrate was inoculated for growth measurement. (B) Primers to amplify the repA gene in pC2A (approximately 1 kbp) were used to screen for the presence of the plasmid containing the genome editing machinery in each of the three Pur^R parents (lanes 2–4) and 8ADP^R isolates (lanes 5–7). The plasmid pDN211 was used as a positive control (lane 8) and the parent strain WWM60 was used as a negative control (lane 9).

Fig. 2.

Optimization of Cas9-mediated genome editing in M. acetivorans. (A) A dose–response curve showing the relative transformation efficiency of pDN211 for different expression levels of Cas9 and the sgRNA. Transformants were plated on solid medium containing either TMA hydrochloride (sgRNA uninduced; in blue) or methanol (sgRNA induced; in green) as the growth substrate with tetracycline concentrations ranging from 0 to 64 μg/mL, as indicated. (B) Mean transformation efficiencies of pDN211 and pDN207 (a control vector that lacks the sgRNA targeting ssuC). (C) Mean transformation efficiencies of plasmids containing repair templates placed at variable distance from the sgRNA-directed DSB for ssuC. Values above each column represent the fraction of transformants for the corresponding plasmid that tested positive for the desired mutation by a PCR-based screen. The error bars represent one SD of the mean transformation efficiency for three independent transformation reactions. All transformations were plated on medium lacking tetracycline with TMA as the growth substrate.

Fig. 3.

Simultaneous expression of multiple sgRNAs and generation of multiple mutations in M. acetivorans. (A) Two configurations for the expression of multiple sgRNAs were tested: in configuration one each sgRNA contains an individual promoter, whereas in configuration two a single promoter drives the expression of multiple sgRNAs separated by a 30-bp linker sequence. (B) Mean transformation efficiency of plasmids with sgRNAs in configuration one (light gray) or two (dark gray) configurations to delete either mtmCB1 or mtmCB2. Note: two independent transformation reactions were performed per plasmid. (C) Mean transformation efficiencies of plasmids to generate either ΔmtmCB1 (green), ΔmtmCB2 (blue), or ΔmtmCB1ΔmtmCB2 (purple) simultaneously. The error bars represent one SD of the mean transformation efficiency for three independent transformation reactions. All transformations were plated on medium lacking tetracycline with methanol as the growth substrate.

Fig. S3.

Genomic context of the isozymes encoding the monomethylamnie specific methyltransferases (mtmCB1 and mtmCB2) in Methanosarcina acetivorans. The genes encoding the corrinoid proteins MtmC1 and MtmC2 share 89% amino acid identity, whereas the genes encoding the methyltransferase MtmB1 and MtmB2 share 95% amino acid identity. The orange and pink arrows indicate the location of the two sgRNAs used to generate an in-frame deletion.

Fig. S4.

Plasmid map of pDN237. The plasmid pDN237 contains the appropriate sgRNAs and homology repair templates to generate in-frame deletions in mtmCB1 and mtmCB2 simultaneously. The plasmid map was generated using Geneious version R9.

Fig. S5.

Homology between target sequences for mtmCB1 and mtmCB2. (A) Alignment of the spacer and the PAM (underlined) for mtmB1 and mtmC1 with the corresponding regions in the mtmB2 and mtmC2 CDS, respectively. (B) Alignment of the spacer and the PAM (underlined) for mtmB2 and mtmC2 with the corresponding regions in the mtmB1 and mtmC1 CDS.

Fig. S6.

Design of repair templates for Cas9-mediated gene insertions at the ssuC locus in M. acetivorans. (A) Homology repair template to insert the mtmCB1 operon (green) and an 840-bp region upstream (likely to contain the putative promoter) within the ssuC CDS. (B) Homology repair template to insert the mtmCB2 operon (blue) and a 390-bp region upstream (likely to contain the putative promoter) within the ssuC CDS.

Fig. 4.

Coexpression of NHEJ genes with the Cas9-sgRNA complex in M. acetivorans. (A) Transformation efficiency of plasmids with a sgRNA targeting the ssuC locus containing either a repair template for HDR-mediated DSB repair, the NHEJ genes, or no repair template. The error bars represent one SD of the mean transformation efficiency for three independent transformation reactions. All transformations were plated on medium lacking tetracycline with TMA as the growth substrate. (B) Regions of naturally occurring microhomology surrounding the ssuC locus at which NHEJ-mediated deletions were observed in Pur^R transformants.

Fig. S7.

Heterlogous expression of the NHEJ genes from Methanocella paludicola. Design of a 3.25-kbp artificial operon with the NHEJ polymerase (Mcp_2125), DNA ligase (Mcp_2126), phosphoesterase (Mcp_2127), and Ku (Mcp_0581) genes from M. paludicola SANAE fused to the Methanosarcina barkeri Fusaro serC promoter and followed by the M. acetivorans Mcr terminator.