Crystal structure of a CRISPR RNA–guided surveillance complex bound to a ssDNA target

Mulepati, Sabin; Héroux, A.; Bailey, Scott

doi:10.1126/science.1256996

Cited by 222 publications

(353 citation statements)

References 47 publications

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“…Our results also invite comparisons to the mechanisms of RNP assembly and DNA targeting in other CRISPR-Cas systems, particularly the type I-E Cascade interference complex. Although Cascade is composed of 11 distinct subunits, none of which possesses nuclease activity, the guide RNA (crRNA) plays a similarly critical role in scaffolding the assembly of distinct domains into a structure that is primed to engage DNA targets (19)(20)(21). Similar principles are likely to govern the assembly and activity of other CRISPR RNA-guided DNA-targeting complexes (2).…”

Section: Discussionmentioning

confidence: 99%

Rational design of a split-Cas9 enzyme complex

Wright

Sternberg²,

Taylor

et al. 2015

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

273

207

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Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.CRISPR-Cas9 | genome engineering | split enzyme B acteria use RNA-guided adaptive immune systems encoded by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) genomic loci to defend against invasive DNA (1, 2). In type II CRISPR-Cas systems, a single enzyme called Cas9 is responsible for targeting and cleavage of foreign DNA (3). The ability to program Cas9 for DNA cleavage at sites defined by engineered single-guide RNAs (sgRNAs) (4) has led to its adoption as a robust and versatile platform for genome engineering (for recent reviews, see refs. 5-7).Cas9 contains two nuclease active sites that function together to generate DNA double-strand breaks (DSBs) at sites complementary to the 20-nt guide RNA sequence and adjacent to a protospacer adjacent motif (PAM). Structural studies of the Streptococcus pyogenes Cas9 showed that the protein exhibits a bilobed architecture comprising the catalytic nuclease lobe and the α-helical lobe of the enzyme (8). Electron microscopy (EM) studies and comparisons with X-ray crystal structures with and without a bound guide RNA and target DNA revealed a largescale conformational rearrangement of the two lobes relative to each other upon nucleic acid binding (8, 9). Strikingly, RNA binding induces the nuclease lobe to rotate ∼100°relative to the α-helical lobe, generating a nucleic-acid binding cleft that can accommodate DNA, and interactions between the two lobes seem to be mediated primarily through contacts with the bound nucleic acid rather than direct protein-protein contacts (8, 9). These observations suggested that the two structural lobes of Cas9 might be separable into independent polypeptides that retain the ability to assemble into an active enzyme complex. Such a system would enable analysis of the functionally distinct properties of each Cas9 structural region and might offer a unique mechanism for control...

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Section: Discussionmentioning

confidence: 99%

Rational design of a split-Cas9 enzyme complex

Wright

Sternberg²,

Taylor

et al. 2015

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

273

207

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“…In the case of Cmr-associated crRNAs, both the 45-and 39-nt forms are functional, suggesting that the precise 3 ′ terminus is not critical for activity (Hale et al 2009(Hale et al , 2012. Structural studies reveal that each Cmr crRNP accommodates a single crRNA species (Zhang et al 2012;Spilman et al 2013;Staals et al 2013;Ramia et al 2014), and this is very likely the case for the Csa and Cst complexes based on recent structural information from other Type I systems Mulepati et al 2014;Zhao et al 2014). Further experiments are required to determine if all or a subset of the multiple size forms of crRNAs within the Csa and Cst crRNPs are functional or if crRNA 3 ′ trimming leads to functional activation for these specific complexes.…”

Section: Discussion Three Distinct Crrnps Coexist In Pyrococcus Furiosusmentioning

confidence: 99%

“…The black boxes at the top correspond to CRISPR repeats interspersed by guide sequences shown as black lines. Jackson et al 2014;Mulepati et al 2014;Zhao et al 2014), including in the Cmr complex where the Cmr3 protein is implicated in direct 5 ′ tag binding (Spilman et al 2013;Ramia et al 2014). Presumably, the Cas5 superfamily proteins of the Csa and Cst complex (Cas5a and Cas5t, respectively) recognize the 5 ′ tag sequences in the context of Csa and Cst crRNPs.…”

Section: Discussion Three Distinct Crrnps Coexist In Pyrococcus Furiosusmentioning

confidence: 99%

Three CRISPR-Cas immune effector complexes coexist in Pyrococcus furiosus

Majumdar

Zhao

Pfister

et al. 2015

RNA

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CRISPR-Cas immune systems function to defend prokaryotes against potentially harmful mobile genetic elements including viruses and plasmids. The multiple CRISPR-Cas systems (Types I, II, and III) each target destruction of foreign nucleic acids via structurally and functionally diverse effector complexes (crRNPs). CRISPR-Cas effector complexes are comprised of CRISPR RNAs (crRNAs) that contain sequences homologous to the invading nucleic acids and Cas proteins specific to each immune system type. We have previously characterized a crRNP in Pyrococcus furiosus (Pfu) that contains Cmr (Type III-B) Cas proteins associated with one of two size classes of crRNAs and cleaves complementary target RNAs. Here, we have isolated and characterized two additional native Pfu crRNPs containing either Csa (Type I-A) or Cst (Type I-G) Cas proteins and distinct profiles of associated crRNAs. For each complex, the Cas proteins were identified by mass spectrometry and immunoblotting and the crRNAs by RNA sequencing and Northern blot analysis. The crRNAs associated with both the Csa and Cst complexes originate from all seven Pfu CRISPR loci and contain identical 5 ′ ends (8-nt repeat-derived 5 ′ tag sequences) but heterogeneous 3 ′ ends (containing variable amounts of downstream repeat sequences). These crRNA forms are distinct from Cmr-associated crRNAs, indicating different 3 ′ end processing pathways following primary cleavage of common pre-crRNAs. Like other previously characterized Type I CRISPR-Cas effector complexes, we predict that the newly identified Pfu Csa and Cst crRNPs each function to target invading DNA, adding an additional layer of protection beyond that afforded by the previously characterized RNA targeting Cmr complex.

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“…For both the Cas9 and the class 1 Cascade complex, a remarkable feature of the transition from the pre-target-bound binary state to the target-bound ternary state involves the formation of a preordered A-form crR-NA, either only within the seed region (Cas9) [14] or throughout the entire guide region (Cascade complex) [31][32][33]. This strategy is also employed by the eukaryotic Agronaute complexes during the transition from guide-RNA bound form to target transcript recognition [28][29][30], representing a convergent evolution of this mechanism [14].…”

Section: Disordered Seed Sequence In Pre-target-bound Binary Cpf1 Commentioning

confidence: 99%

Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition

et al. 2016

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CRISPR-Cas9 and CRISPR-Cpf1 systems have been successfully harnessed for genome editing. In the CRIS-PR-Cas9 system, the preordered A-form RNA seed sequence and preformed protein PAM-interacting cleft are essential for Cas9 to form a DNA recognition-competent structure. Whether the CRISPR-Cpf1 system employs a similar mechanism for target DNA recognition remains unclear. Here, we have determined the crystal structure of Acidaminococcus sp. Cpf1 (AsCpf1) in complex with crRNA and target DNA. Structural comparison between the AsCpf1-crRNA-DNA ternary complex and the recently reported Lachnospiraceae bacterium Cpf1 (LbCpf1)-crRNA binary complex identifies a unique mechanism employed by Cpf1 for target recognition. The seed sequence required for initial DNA interrogation is disordered in the Cpf1-cRNA binary complex, but becomes ordered upon ternary complex formation. Further, the PAM interacting cleft of Cpf1 undergoes an "open-to-closed" conformational change upon target DNA binding, which in turn induces structural changes within Cpf1 to accommodate the ordered A-form seed RNA segment. This unique mechanism of target recognition by Cpf1 is distinct from that reported previously for Cas9.

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Crystal structure of a CRISPR RNA–guided surveillance complex bound to a ssDNA target

Cited by 222 publications

References 47 publications

Rational design of a split-Cas9 enzyme complex

Rational design of a split-Cas9 enzyme complex

Three CRISPR-Cas immune effector complexes coexist in Pyrococcus furiosus

Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition

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