Structure of an RNA Silencing Complex of the CRISPR-Cas Immune System

Spilman, Michael; Cocozaki, A.; Hale, Caryn; Shao, Yaming; Ramia, Nancy; Terns, Rebeca; Terns, Michael P.; Li, Hong; Stagg, Scott M.

doi:10.1016/j.molcel.2013.09.008

Cited by 123 publications

(191 citation statements)

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“…Vestergaard et al (2014) also classified the cas subtypes based on cas-protein occurrences for archaea. Phylogenetic analysis of this study also revealed that many functions of the identified cas proteins could be derived from distantly related genomes, plasmids and phages that was agreed to earlier findings (Nelson et al 1999;Zivanovic et al 2002;Boucher et al 2003;Marraffini and Sontheimer 2009;Chellapandi and Ranjani 2011;Spilman et al 2013). Euryarchaea are more diverse phylogenetically than crenarchaea because of they are generally heterotophic in nature and frequent a wide range of natural environments (Vestergaard et al 2014).…”

Section: Discussionsupporting

confidence: 91%

Knowledge-based discovery for designing CRISPR-CAS systems against invading mobilomes in thermophiles

Chellapandi

Ranjani

2015

Syst Synth Biol

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Clustered regularly interspaced short palindromic repeats (CRISPRs) are direct features of the prokaryotic genomes involved in resistance to their bacterial viruses and phages. Herein, we have identified CRISPR loci together with CRISPR-associated sequences (CAS) genes to reveal their immunity against genome invaders in the thermophilic archaea and bacteria. Genomic survey of this study implied that genomic distribution of CRISPR-CAS systems was varied from strain to strain, which was determined by the degree of invading mobiloms. Direct repeats found to be equal in some extent in many thermopiles, but their spacers were differed in each strain. Phylogenetic analyses of CAS superfamily revealed that genes cmr, csh, csx11, HD domain, devR were belonged to the subtypes of cas gene family. The members in cas gene family of thermophiles were functionally diverged within closely related genomes and may contribute to develop several defense strategies. Nevertheless, genome dynamics, geological variation and host defense mechanism were contributed to share their molecular functions across the thermophiles. A thermophilic archaean, Thermococcus gammotolerans and thermophilic bacteria, Petrotoga mobilis and Thermotoga lettingae have shown superoperons-like appearance to cluster cas genes, which were typically evolved for their defense pathways. A cmr operon was identified with a specific promoter in a thermophilic archaean, Caldivirga maquilingensis. Overall, we concluded that knowledge-based genomic survey and phylogeny-based functional assignment have suggested for designing a reliable genetic regulatory circuit naturally from CRISPR-CAS systems, acquired defense pathways, to thermophiles in future synthetic biology.

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Section: Discussionsupporting

confidence: 91%

Knowledge-based discovery for designing CRISPR-CAS systems against invading mobilomes in thermophiles

Chellapandi

Ranjani

2015

Syst Synth Biol

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“…Type II CRISPR systems are characterized by Cas9, a multidomain protein with a bilobed architecture harboring two nuclease sites that together catalyze cleavage of invading DNA (12, 21-24). The Csm system in CRISPR type IIIA and a related Cmr complex in type IIIB cleave DNA and RNA, respectively, forming a helical structure around crRNA that is structurally similar to Cascade (14,17,18,25,26).Whereas Cas9 and Csm/Cmr effector complexes directly recognize and nucleolytically degrade invading genes (12, 14, 16, 17, 21), Cascade complex by itself is not a nuclease for degradation of invader DNA (13). In Escherichia coli K-12 (type IE), Cascade recognizes and binds crRNA to complimentary sequence in target DNA, generating an RNA mediated displacement loop (R-loop) of single-stranded (ss) DNA and RNA-DNA hybrid within double-stranded (ds) target DNA (13).…”

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confidence: 99%

Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3

Gong

Shin

Sun

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Mobile genetic elements in bacteria are neutralized by a system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. Type I CRISPR-Cas systems use a "Cascade" ribonucleoprotein complex to guide RNA specifically to complementary sequence in invader double-stranded DNA (dsDNA), a process called "interference." After target recognition by Cascade, formation of an R-loop triggers recruitment of a Cas3 nuclease-helicase, completing the interference process by destroying the invader dsDNA. To elucidate the molecular mechanism of CRISPR interference, we analyzed crystal structures of Cas3 from the bacterium Thermobaculum terrenum, with and without a bound ATP analog. The structures reveal a histidine-aspartate (HD)-type nuclease domain fused to superfamily-2 (SF2) helicase domains and a distinct C-terminal domain. Binding of ATP analog at the interface of the SF2 helicase RecA-like domains rearranges a motif V with implications for the enzyme mechanism. The HDnucleolytic site contains two metal ions that are positioned at the end of a proposed nucleic acid-binding tunnel running through the SF2 helicase structure. This structural alignment suggests a mechanism for 3′ to 5′ nucleolytic processing of the displaced strand of invader DNA that is coordinated with ATP-dependent 3′ to 5′ translocation of Cas3 along DNA. In agreement with biochemical studies, the presented Cas3 structures reveal important mechanistic details on the neutralization of genetic invaders by type I CRISPR-Cas systems.M any bacteria and archaea can eliminate invading phages and plasmids by activating a defense system that is based on processing of clustered regularly interspaced short palindromic repeats (CRISPRs) by CRISPR-associated (Cas) proteins and various additional proteins. These CRISPR-Cas systems are diverse in structure and function, but common principles of action allow their classification into three major types (I, II, and III) with some further divisions into subtypes (e.g., type IA-F) (1-6).Common CRISPR-mediated immunity processes are usually defined into three stages: (i) acquisition of a short DNA segment (protospacer) from an invading virus or plasmid, and its insertion at the leader-proximal end of a CRISPR locus (7, 8), (ii) generation of small mature CRISPR RNAs (crRNAs) from a longer transcript of a CRISPR locus (9-11), and (iii) interference of foreign nucleic acid invaders by a crRNA-containing ribonucleoprotein effector complex (12-18).Different interference effector complexes characterize the three major CRISPR types. Cascade (Cas complex for antiviral defense) complexes are synonymous with interference in type I CRISPRs, comprising multiple proteins in a helical structure around crRNA that targets invader DNA (13,19, 20). Type II CRISPR systems are characterized by Cas9, a multidomain protein with a bilobed architecture harboring two nuclease sites that together catalyze cleavage of invading DNA (12, 21-24). The Csm system in CRISPR type IIIA and a related Cmr ...

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“…In vivo, several copies of Cas7 proteins are wrapped around crRNA in a sequence-unspecific helical fashion [5,30,50,51]. Crystal structures from single and complex-bound Cas7 proteins show two composite RNA-binding surfaces: a central cleft and a structurally variable insertion domain [5,23,24,52].…”

Section: Mapping the Rna Binding Interface In Cas7 Proteinsmentioning

confidence: 99%

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

Sharma

Hrle

Kramer

et al. 2015

Methods

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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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Structure of an RNA Silencing Complex of the CRISPR-Cas Immune System

Cited by 123 publications

References 50 publications

Knowledge-based discovery for designing CRISPR-CAS systems against invading mobilomes in thermophiles

Knowledge-based discovery for designing CRISPR-CAS systems against invading mobilomes in thermophiles

Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3

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

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