In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex

Abstract

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems of type I use a Cas ribonucleoprotein complex for antiviral defense (Cascade) to mediate the targeting and degradation of foreign DNA. To address molecular features of the archaeal type I-A Cascade interference mechanism, we established the in vitro assembly of the Thermoproteus tenax Cascade from six recombinant Cas proteins, synthetic CRISPR RNAs (crRNAs) and target DNA fragments. RNA-Seq analyses revealed the processing pattern of crRNAs from seven T. tenax CRISPR arrays. Synthetic crRNA transcripts were matured by hammerhead ribozyme cleavage. The assembly of type I-A Cascade indicates that Cas3′ and Cas3′′ are an integral part of the complex, and the interference activity was shown to be dependent on the crRNA and the matching target DNA. The reconstituted Cascade was used to identify sequence motifs that are required for efficient DNA degradation and to investigate the role of the subunits Cas7 and Cas3′′ in the interplay with other Cascade subunits.

Three CRISPR-Cas immune effector complexes coexist in Pyrococcus furiosus

“…The first experimental evidence for the existence of native Csa crRNPs came from work with the thermophilic archaeaon Sulfolobus solfataricus, where affinity purifications performed with tagged Csa2 (Cas7) provided evidence for a stable interaction of Csa2 (Cas7) with Cas5a (Cas5) and crRNA with possible additional (less stable) interactions with Csa5 (Cas11 or small subunit protein), Csa4 (Cas8), and Cas6 (Lintner et al 2011b). Similar to our findings, Type I-A (Csa) crRNPs reconstituted with recombinant proteins in Thermoproteus tenax contained Cas3 ′′ and Cas3 ′ as stable components of the complex (Plagens et al 2012(Plagens et al , 2014. The recombinant T. tenax Csa crRNP targets DNA, and Cas3 ′′ (HD-nuclease protein) is the effector nuclease (Plagens et al 2014).…”

“…CRISPR RNAs (crRNAs) are produced by processing of CRISPR locus transcripts that arise from promoter elements in the leader region (Jansen et al 2002;Tang et al 2002). Each crRNA contains a guide region comprised of invader-derived sequences that allow crRNA-Cas protein effector complexes to recognize and destroy invader nucleic acids (Barrangou et al 2007;Marraffini and Sontheimer 2008;Hale et al 2009Hale et al , 2012Garneau et al 2010;Jore et al 2011;Lintner et al 2011b;Wiedenheft et al 2011a;Fischer et al 2012;Gasiunas et al 2012;Jinek et al 2012;Westra et al 2012a;Elmore et al 2013;Maier et al 2013;Mulepati and Bailey 2013;Peng et al 2013Peng et al , 2015Sinkunas et al 2013;Plagens et al 2014).…”

“…While the Csa and Cst complexes have not been extensively studied, Cas5, Cas7, Cas8 (large subunit), and Cas11 (small subunit) superfamily proteins comprise other characterized Type I crRNPs (the latter sometimes fused to the large subunit as a domain) (Brouns et al 2008;Lintner et al 2011b;Wiedenheft et al 2011a,b;Nam et al 2012;Plagens et al 2012;van Duijn et al 2012;Brendel et al 2014). The signature Cas3 (HD nuclease-DExH helicase) proteins of Type I systems interact with the crRNPs and cleave invader DNA (Cady and O'Toole 2011;Westra et al 2012a;Mulepati and Bailey 2013;Sinkunas et al 2013;Hochstrasser et al 2014;Plagens et al 2014). The cas gene clusters in Pfu also encode predicted adaptation proteins (Cas1, Cas2, Cas4) and a crRNA biogenesis protein (Cas6) (Fig.…”

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

“…Recombinant Cas7 proteins from T. pendens and T. tenax were cloned and expressed as described elsewhere [24,31] and the polyU (20) RNA was synthesized by Purimex. For cross-linking with polyU (20) , 1 nmol protein was mixed with 1 nmol polyU (20) in a total volume of 200 ll in a buffer containing 20 mM HEPES (pH 7.5), 10 mM NaCl, 4 mM MgCl 2 and 2 mM DTT.…”

“…In all Cas7 proteins characterized to date both these domains are defined by insertions within the secondary structure elements of the central RRM domain (insertion domain 1 is b1-a1, b2-b3, a2-b4, and insertion domain 2 is a1-b2). Using polyU and crRNA substrates, we were able to point to the potential RNA interacting regions of three Cas7 family proteins: a to date uncharacterized Cas7 protein from T. tenax [31] and two structurally and functionally characterized homologs from T. pendens and T. thermophilus [24,30].…”