Evolution and classification of the CRISPR–Cas systems

Makarova, Kira S.; Haft, Daniel H.; Barrangou, Rodolphe; Brouns, Stan J. J.; Charpentier, Emmanuelle; Horvath, Philippe; Moineau, Sylvain; Mojica, Francisco J. M.; Wolf, Yuri I.; Yakunin, Alexander F.; Oost, John van der; Koonin, Eugene V.

doi:10.1038/nrmicro2577

Cited by 2,059 publications

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“…We mined our microbial genomes for the presence of CRISPRCas systems (clustered regularly interspaced short palindromic repeat-CRISPR associated), which act as an acquired immunity to viruses 28 (Supplementary Table 8). CRISPR-Cas frequency estimates range from 81% of archaeal and 40% of bacterial genomes in cultivated microbes 29 , to 10% of archaeal and bacterial genomes in metagenomic data sets 30 . In contrast, 100% of the three archaeal and 84% of the 31 bacterial genome bins of the Marcellus samples had evidence of a CRISPR-Cas system, with type 1 being the most prevalent (Supplementary Table 8).…”

Section: Active Viral Predation In the Deep Subsurfacementioning

confidence: 99%

Microbial metabolisms in a 2.5-km-deep ecosystem created by hydraulic fracturing in shales

et al. 2016

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Hydraulic fracturing is the industry standard for extracting hydrocarbons from shale formations. Attention has been paid to the economic benefits and environmental impacts of this process, yet the biogeochemical changes induced in the deep subsurface are poorly understood. Recent single-gene investigations revealed that halotolerant microbial communities were enriched after hydraulic fracturing. Here, the reconstruction of 31 unique genomes coupled to metabolite data from the Marcellus and Utica shales revealed that many of the persisting organisms play roles in methylamine cycling, ultimately supporting methanogenesis in the deep biosphere. Fermentation of injected chemical additives also sustains long-term microbial persistence, while thiosulfate reduction could produce sulfide, contributing to reservoir souring and infrastructure corrosion. Extensive links between viruses and microbial hosts demonstrate active viral predation, which may contribute to the release of labile cellular constituents into the extracellular environment. Our analyses show that hydraulic fracturing provides the organismal and chemical inputs for colonization and persistence in the deep terrestrial subsurface. S hale gas accounts for one-third of natural gas energy resources worldwide. It has been estimated that shale gas will provide half of the natural gas in the USA, annually, by 2040, with the Marcellus shale in the Appalachian Basin projected to produce three times more than any other formation 1 . Recovery of these hydrocarbons is dependent on hydraulic fracturing technologies, where the high-pressure injection of water and chemical additives generates extensive fractures in the shale matrix. Hydrocarbons trapped in tiny pore spaces are subsequently released and collected at the wellpad surface, together with a portion of the injected fluids that have reacted with the shale formation. The mixture of injected fluids and hydrocarbons collected is referred to as 'produced fluids'.Microbial metabolism and growth in hydrocarbon reservoirs has both positive and negative impacts on energy recovery. Whereas stimulation of methanogens in coal beds enhances energy recovery 2 , bacterial hydrogen sulfide production ('reservoir souring') decreases profits and contributes to corrosion and the risk of environmental contamination 3 . Additionally, biomass accumulation within newly generated fractures may reduce their permeability, decreasing natural gas recovery. Despite these potential microbial impacts, little is known about the function and activity of microorganisms in hydraulically fractured shale.Initial work by our group and others 4-9 used single marker gene analyses to identify microorganisms from several geographically distinct shale formations. These analyses showed similar halotolerant taxa in produced fluids several months after hydraulic fracturing. To assign functional roles to these organisms, we conducted metagenomic and metabolite analyses on input and produced fluids up to a year after hydraulic fracturing (HF) from two Appala...

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Section: Active Viral Predation In the Deep Subsurfacementioning

confidence: 99%

Microbial metabolisms in a 2.5-km-deep ecosystem created by hydraulic fracturing in shales

et al. 2016

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“…Locus geometry reconstruction allowed us to separate the effects of proliferation of spacers due to increase in relative abundance of host strains from high spacer abundance due to insertion of the same spacer in multiple incorporation events. We conducted two independent experiments with phage:host ratios (multiplicity of infection, MOI) of 2:1 and 10:1, and targeted the two active CRISPR loci, CRISPR1 and CRISPR3, both type II-A CRISPR-Cas systems 22 . The repeatable unexpected bias in the spacer acquisition observed in this work combined with the large amount of data processed reveals further insight into this CRISPR-Cas system step.…”

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Strong bias in the bacterial CRISPR elements that confer immunity to phage

et al. 2013

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Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems provide adaptive immunity against phage via spacer-encoded CRISPR RNAs that are complementary to invasive nucleic acids. Here, we challenge Streptococcus thermophilus with a bacteriophage, and used PCR-based metagenomics to monitor phage-derived spacers daily for 15 days in two experiments. Spacers that target the host chromosome are infrequent and strongly selected against, suggesting autoimmunity is lethal. In experiments that recover over half a million spacers, we observe early dominance by a few spacer sub-populations and rapid oscillations in sub-population abundances. In two CRISPR systems and in replicate experiments, a few spacers account for the majority of spacer sequences. Nearly all phage locations targeted by the acquired spacers have a proto-spacer adjacent motif (PAM), indicating PAMs are involved in spacer acquisition. We detect a strong and reproducible bias in the phage genome locations from which spacers derive. This may reflect selection for specific spacers based on location and effectiveness.

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“…In this system Cas proteins are guided by a CRISPR RNA (crRNA) to target and degrade complementary foreign nucleic acids in a manner that is functionally reminiscent of the eukaryotic RNA interference mechanism [27]. Type I, II and III CRISPR-Cas systems are classified based on their signature Cas genes (cas3, cas9 and cas10 respectively) that are further classified into different subtypes based on the presence of other Cas genes [28]. Type I and subtypes III-A and III-B form multiprotein RNPs together with different Cas proteins in addition to Cas3 or Cas10.…”

Section: Introductionmentioning

confidence: 99%

“…Some Cas proteins comprise nuclease domains, distinct helicase domains and also RRM domains that are typical for RNA-binding proteins [29]. The Cas7 family proteins, which form the backbone of the surveillance and effector complexes in Type I and Type III systems, consist of RRMs and belong to the RAMP (repeat associated mysterious proteins) superfamily [28]. Interestingly, most Cas proteins lack conserved amino-acid residues that account for RNA interaction.…”

Section: Introductionmentioning

confidence: 99%

Analysis of protein–RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry

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Kramer

et al. 2015

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a b s t r a c tRibonucleoprotein (RNP) complexes play important roles in the cell by mediating basic cellular processes, including gene expression and its regulation. Understanding the molecular details of these processes requires the identification and characterization of protein-RNA interactions. Over the years various approaches have been used to investigate these interactions, including computational analyses to look for RNA binding domains, gel-shift mobility assays on recombinant and mutant proteins as well as co-crystallization and NMR studies for structure elucidation. Here we report a more specialized and direct approach using UV-induced cross-linking coupled with mass spectrometry. This approach permits the identification of cross-linked peptides and RNA moieties and can also pin-point exact RNA contact sites within the protein. The power of this method is illustrated by the application to different singleand multi-subunit RNP complexes belonging to the prokaryotic adaptive immune system, CRISPR-Cas (CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR associated). In particular, we identified the RNA-binding sites within three Cas7 protein homologs and mapped the cross-linking results to reveal structurally conserved Cas7 -RNA binding interfaces. These results demonstrate the strong potential of UV-induced cross-linking coupled with mass spectrometry analysis to identify RNA interaction sites on the RNA binding proteins.

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Evolution and classification of the CRISPR–Cas systems

Cited by 2,059 publications

References 50 publications

Microbial metabolisms in a 2.5-km-deep ecosystem created by hydraulic fracturing in shales

Microbial metabolisms in a 2.5-km-deep ecosystem created by hydraulic fracturing in shales

Strong bias in the bacterial CRISPR elements that confer immunity to phage

Analysis of protein–RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry

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