Gene Repression in Haloarchaea Using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas I-B System

Stachler, Aris-Edda; Marchfelder, Anita

doi:10.1074/jbc.m116.724062

Cited by 68 publications

(67 citation statements)

References 46 publications

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“…We conclude from these findings that the two archaeal HARPs are basically capable of acting as RNases P. On the other hand, the ease with which HARP gene deletions could be obtained in the two archaea and the lack of considerable growth defects of the deletion strains suggests that the RNA-based RNase P, expressed in all archaea that encode HARPs (19), is the major archaeal RNase P activity. This notion receives support from our observation that the RNase P RNA subunit gene could not be deleted in H. volcanii and repression of the RNase P RNA gene caused severe defects in tRNA biogenesis (25). It is a possibility that archaeal HARPs exert specialised functions in tRNA biogenesis, possibly under extreme conditions not yet identified.…”

Section: Discussionmentioning

confidence: 60%

Homologs of aquifex aeolicus protein‐only RNase P are not the major RNase P activities in the archaea haloferax volcanii and methanosarcina mazei

et al. 2019

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The mature 5′‐ends of tRNAs are generated by RNase P in all domains of life. The ancient form of the enzyme is a ribonucleoprotein consisting of a catalytic RNA and one or more protein subunits. However, in the hyperthermophilic bacterium Aquifex aeolicus and close relatives, RNase P is a protein‐only enzyme consisting of a single type of polypeptide (Aq_880, ~23 kDa). In many archaea, homologs of Aq_880 were identified (termed HARPs for Homologs of Aquifex RNase P) in addition to the RNA‐based RNase P, raising the question about the functions of HARP and the classical RNase P in these archaea. Here we investigated HARPs from two euryarchaeotes, Haloferax volcanii and Methanosarcina mazei. Archaeal strains with HARP gene knockouts showed no growth phenotypes under standard conditions, temperature and salt stress (H. volcanii) or nitrogen deficiency (M. mazei). Recombinant H. volcanii and M. mazei HARPs were basically able to catalyse specific tRNA 5′‐end maturation in vitro. Furthermore, M. mazei HARP was able to rescue growth of an Escherichia coli RNase P depletion strain with comparable efficiency as Aq_880, while H. volcanii HARP was unable to do so. In conclusion, both archaeal HARPs showed the capacity (in at least one functional assay) to act as RNases P. However, the ease to obtain knockouts of the singular HARP genes and the lack of growth phenotypes upon HARP gene deletion contrasts with the findings that the canonical RNase P RNA gene cannot be deleted in H. volcanii, and a knockdown of RNase P RNA in H. volcanii results in severe tRNA processing defects. We conclude that archaeal HARPs do not make a major contribution to global tRNA 5′‐end maturation in archaea, but may well exert a specialised, yet unknown function in (t)RNA metabolism. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1109–1116, 2019

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Section: Discussionmentioning

confidence: 60%

Homologs of aquifex aeolicus protein‐only RNase P are not the major RNase P activities in the archaea haloferax volcanii and methanosarcina mazei

et al. 2019

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“…This Cas3 variant has a D444A mutation that results in failure of interference (22). Using this Cas3 variant in the assay did not generate white colonies, confirming that self-targeting DNA degradation in our system requires an active Cas3.…”

Section: Resultsmentioning

confidence: 99%

High tolerance to self-targeting of the genome by the endogenous CRISPR-Cas system in an archaeon

Stachler

Turgeman-Grott

Shtifman-Segal

et al. 2017

Nucleic Acids Research

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CRISPR-Cas systems allow bacteria and archaea to acquire sequence-specific immunity against selfish genetic elements such as viruses and plasmids, by specific degradation of invader DNA or RNA. However, this involves the risk of autoimmunity if immune memory against host DNA is mistakenly acquired. Such autoimmunity has been shown to be highly toxic in several bacteria and is believed to be one of the major costs of maintaining these defense systems. Here we generated an experimental system in which a non-essential gene, required for pigment production and the reddish colony color, is targeted by the CRISPR-Cas I-B system of the halophilic archaeon Haloferax volcanii. We show that under native conditions, where both the self-targeting and native crRNAs are expressed, self-targeting by CRISPR-Cas causes no reduction in transformation efficiency of the plasmid encoding the self-targeting crRNA. Furthermore, under such conditions, no effect on organismal growth rate or loss of the reddish colony phenotype due to mutations in the targeted region could be observed. In contrast, in cells deleted for the pre-crRNA processing gene cas6, where only the self-targeting crRNA exists as mature crRNA, self-targeting leads to moderate toxicity and the emergence of deletion mutants. Sequencing of the deletions caused by CRISPR-Cas self targeting indicated DNA repair via microhomology-mediated end joining.

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“…Then the Cascade-bound R-loop is more stable than that of Cas9 [31, 32], enabling greater control over regulation. Additionally, dCas9 does not function properly in some Achaea, and thus an endogenous system is required for regulation purposes [33]. …”

Section: Discussionmentioning

confidence: 99%

“…Spacers targeting the promoter region of both strands repressed GFP expression tightly, while targeting the non-promoter region on the non-template strand generally shows more repression effect than targeting the other strand [11, 16, 17, 33]. Interestingly, while transcription occurred on the template strand, targeting the non-template strand should cause a stronger repression effect.…”

Section: Discussionmentioning

confidence: 99%

Easy regulation of metabolic flux in Escherichia coli using an endogenous type I-E CRISPR-Cas system

Chang¹,

Su²,

Qi³

et al. 2016

Microb Cell Fact

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BackgroundClustered regularly interspaced short palindromic repeats interference (CRISPRi) is a recently developed powerful tool for gene regulation. In Escherichia coli, the type I CRISPR system expressed endogenously shall be easy for internal regulation without causing metabolic burden in compared with the widely used type II system, which expressed dCas9 as an additional plasmid.ResultsBy knocking out cas3 and activating the expression of CRISPR-associated complex for antiviral defense (Cascade), we constructed a native CRISPRi system in E. coli. Downregulation of the target gene from 6 to 82% was demonstrated using green fluorescent protein. Regulation of the citrate synthase gene (gltA) in the TCA cycle affected host metabolism. The effect of metabolic flux regulation was demonstrated by the poly-3-hydroxbutyrate (PHB) accumulation in vivo.ConclusionBy regulating native gltA in E. coli using an engineered endogenous type I-E CRISPR system, we redirected metabolic flux from the central metabolic pathway to the PHB synthesis pathway. This study demonstrated that the endogenous type I-E CRISPR-Cas system is an easy and effective method for regulating internal metabolic pathways, which is useful for product synthesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0594-4) contains supplementary material, which is available to authorized users.

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Gene Repression in Haloarchaea Using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas I-B System

Cited by 68 publications

References 46 publications

Homologs of aquifex aeolicus protein‐only RNase P are not the major RNase P activities in the archaea haloferax volcanii and methanosarcina mazei

Homologs of aquifex aeolicus protein‐only RNase P are not the major RNase P activities in the archaea haloferax volcanii and methanosarcina mazei

High tolerance to self-targeting of the genome by the endogenous CRISPR-Cas system in an archaeon

Easy regulation of metabolic flux in Escherichia coli using an endogenous type I-E CRISPR-Cas system

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