A CRISPRi-dCas9 system 1 for archaea and its use to examine gene function during nitrogen fixation by Methanosarcina acetivorans.
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| Title: | A CRISPRi-dCas9 system 1 for archaea and its use to examine gene function during nitrogen fixation by Methanosarcina acetivorans. |
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| Authors: | Dhamad, Ahmed E.1,2, Lessner, Daniel J.2 dlessner@uark.edu |
| Source: | Applied & Environmental Microbiology. Nov2020, Vol. 86 Issue 21, p1-35. 35p. |
| Subjects: | Nitrogen fixation, CRISPRs, Genes, Genetic regulation, Nitrogenases, Operons |
| 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. [ABSTRACT FROM AUTHOR] |
| Copyright of Applied & Environmental Microbiology is the property of American Society for Microbiology and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.) | |
| Database: | Engineering Source |
| FullText | Text: Availability: 0 |
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| Header | DbId: egs DbLabel: Engineering Source An: 146523615 AccessLevel: 6 PubType: Academic Journal PubTypeId: academicJournal PreciseRelevancyScore: 0 |
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| Items | – Name: Title Label: Title Group: Ti Data: A CRISPRi-dCas9 system 1 for archaea and its use to examine gene function during nitrogen fixation by Methanosarcina acetivorans. – Name: Author Label: Authors Group: Au Data: <searchLink fieldCode="AR" term="%22Dhamad%2C+Ahmed+E%2E%22">Dhamad, Ahmed E.</searchLink><relatesTo>1,2</relatesTo><br /><searchLink fieldCode="AR" term="%22Lessner%2C+Daniel+J%2E%22">Lessner, Daniel J.</searchLink><relatesTo>2</relatesTo><i> dlessner@uark.edu</i> – Name: TitleSource Label: Source Group: Src Data: <searchLink fieldCode="JN" term="%22Applied+%26+Environmental+Microbiology%22">Applied & Environmental Microbiology</searchLink>. Nov2020, Vol. 86 Issue 21, p1-35. 35p. – Name: Subject Label: Subjects Group: Su Data: <searchLink fieldCode="DE" term="%22Nitrogen+fixation%22">Nitrogen fixation</searchLink><br /><searchLink fieldCode="DE" term="%22CRISPRs%22">CRISPRs</searchLink><br /><searchLink fieldCode="DE" term="%22Genes%22">Genes</searchLink><br /><searchLink fieldCode="DE" term="%22Genetic+regulation%22">Genetic regulation</searchLink><br /><searchLink fieldCode="DE" term="%22Nitrogenases%22">Nitrogenases</searchLink><br /><searchLink fieldCode="DE" term="%22Operons%22">Operons</searchLink> – Name: Abstract Label: Abstract Group: Ab Data: 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. [ABSTRACT FROM AUTHOR] – Name: AbstractSuppliedCopyright Label: Group: Ab Data: <i>Copyright of Applied & Environmental Microbiology is the property of American Society for Microbiology and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.) |
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| RecordInfo | BibRecord: BibEntity: Identifiers: – Type: doi Value: 10.1128/AEM.01402-20 Languages: – Code: eng Text: English PhysicalDescription: Pagination: PageCount: 35 StartPage: 1 Subjects: – SubjectFull: Nitrogen fixation Type: general – SubjectFull: CRISPRs Type: general – SubjectFull: Genes Type: general – SubjectFull: Genetic regulation Type: general – SubjectFull: Nitrogenases Type: general – SubjectFull: Operons Type: general Titles: – TitleFull: A CRISPRi-dCas9 system 1 for archaea and its use to examine gene function during nitrogen fixation by Methanosarcina acetivorans. Type: main BibRelationships: HasContributorRelationships: – PersonEntity: Name: NameFull: Dhamad, Ahmed E. – PersonEntity: Name: NameFull: Lessner, Daniel J. IsPartOfRelationships: – BibEntity: Dates: – D: 01 M: 11 Text: Nov2020 Type: published Y: 2020 Identifiers: – Type: issn-print Value: 00992240 Numbering: – Type: volume Value: 86 – Type: issue Value: 21 Titles: – TitleFull: Applied & Environmental Microbiology Type: main |
| ResultId | 1 |
