A CRISPRi-dCas9 System for Archaea and Its Use To Examine Gene Function during Nitrogen Fixation by Methanosarcina acetivorans

Abstract

CRISPR-based systems are emerging as the premier method to manipulate many cellular processes. In this study, a simple and efficient CRISPR interference (CRISPRi) system for targeted gene repression in archaea was developed. The Methanosarcina acetivorans CRISPR-Cas9 system was repurposed by replacing Cas9 with the catalytically dead Cas9 (dCas9) to generate a CRISPRi-dCas9 system for targeted gene repression. To test the utility of the system, genes involved in nitrogen (N2) fixation were targeted for dCas9-mediated repression. First, the nif operon (nifHI1I2DKEN) that encodes molybdenum nitrogenase was targeted by separate guide RNAs (gRNA), one targeting the promoter and the other nifD. Remarkably, growth of M. acetivorans with N2 was abolished by dCas9-mediated repression of the nif operon with each gRNA. The abundance of nif transcripts was >90% reduced in both strains expressing the gRNAs, and NifD was not detected in cell lysate. Next, we targeted NifB, which is required for nitrogenase cofactor biogenesis. Expression of a gRNA targeting the coding sequence of NifB decreased nifB transcript abundance >85% and impaired but did not abolish growth of M. acetivorans with N2. Finally, to ascertain the ability to study gene regulation using CRISPRi-dCas9, nrpR1 encoding a subunit of the repressor of the nif operon was targeted. The nrpR1 repression strain grew normally with N2 but had increased nif operon transcript abundance consistent with a NrpR1 as repressor. These results highlight the utility of the system, whereby a single gRNA when expressed with dCas9 can block transcription of targeted genes and operons in M. acetivorans. IMPORTANCE Genetic tools are needed to understand and manipulate the biology of archaea, which serve critical roles in the biosphere. Methanogenic archaea (methanogens) are essential for the biological production of methane, an intermediate in the global carbon cycle, an important greenhouse gas and a biofuel. The CRISPRi-dCas9 system in the model methanogen M. acetivorans is the first Cas9-based CRISPR interference system in archaea. Results demonstrate that the system is remarkably efficient in targeted gene repression and provide new insight into nitrogen fixation by methanogens, the only archaea with nitrogenase. Overall, the CRISPRi-dCas9 system provides a simple, yet powerful, genetic tool to control the expression of target genes and operons in methanogens.

Vht hydrogenase is required for hydrogen cycling during nitrogen fixation by the non-hydrogenotrophic methanogen Methanosarcina acetivorans

“…Expression of Vht hydrogenase is required for H 2 cycling during nitrogen fixation. To test the importance of each hydrogenase to diazotrophic growth by M. acetivorans, the transcription of the vht, vhx, and frh operons was targeted for repression by dCas9 using the recently developed CRISPRi-dCas9 system [18]. A gRNA was designed to target the coding strand just after the start codon of the first gene in each operon (Fig.…”

“…M. acetivorans strains were maintained in HS medium with 125 mM methanol and 2 µg/ml puromycin. M. acetivorans strain DJL72 harboring pDL730 without a gRNA was generated previously and used as a control for phenotypic comparisons [18]. Gene expression analysis.…”

“…The genome of M. acetivorans encodes molybdenum (Mo), vanadium (V), and Fe-only (Fe) nitrogenases, the enzymes required for growth using dinitrogen (N 2 ) as a nitrogen source (i.e., N 2 fixation or diazotrophy) [12, 16, 17]. We have recently shown that M. acetivorans can grow by fixing N 2 using Mo-nitrogenase [18]. N 2 fixation is an energy intensive process; it is estimated that two ATP are hydrolyzed for every electron used by nitrogenase.…”

“…Therefore, we hypothesized that M. acetivorans utilizes Frh and/or Vht/Vhx to recover energy lost to H 2 production during N 2 reduction. To test this hypothesis, we examined the expression and activity of hydrogenase in response to fixed nitrogen availability and used the recently developed CRISPRi-dCas9 system [18] to repress the transcription of the frh, vht, vhx , and hyp operons in M. acetivorans to determine the effect of the loss of hydrogenase expression on diazotrophy and H 2 metabolism.…”

Heavily Armed Ancestors: CRISPR Immunity and Applications in Archaea with a Comparative Analysis of CRISPR Types in Sulfolobales

“…Repair of the Cas9-induced double strand break without a repair template was only possible when co-expressing a nonhomologous end-joining pathway [222]. The nuclease deficient variant of Cas9 efficiently blocked transcription of desired genes by crRNA-guided binding to the DNA, achieving a silencing efficiency of up to 90% of the nif genes involved in nitrogen fixation [223].…”