Craspase is a CRISPR RNA-guided, RNA-activated protease

Hu, Chunyi; Beljouw, Sam P. B. van; Nam, Ki Hyun; Schuler, Gabriel; Ding, Fran; Cui, Yanru; Rodríguez-Molina, Alicia; Haagsma, Anna C.; Valk, M.; Pabst, Martin; Brouns, Stan J. J.; Ke, Ailong

doi:10.1126/science.add5064

Cited by 64 publications

(89 citation statements)

References 39 publications

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“…Furthermore, we provide a mechanism for self versus non-self RNA target discrimination governed by conformational change-induced activation of the TPR-CHAT protease in type III-E CRISPR-Cas antiviral immunity. While our manuscript was under review, several studies 42 – 47 reported the structural and functional insights of type III-E Craspase complexes; these independent studies are overall in agreement with our results.…”

Section: Discussionsupporting

confidence: 88%

Structural basis for the non-self RNA-activated protease activity of the type III-E CRISPR nuclease-protease Craspase

Cui

Zhang

et al. 2022

Nat Commun

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The RNA-targeting type III-E CRISPR-gRAMP effector interacts with a caspase-like protease TPR-CHAT to form the CRISPR-guided caspase complex (Craspase), but their functional mechanism is unknown. Here, we report cryo-EM structures of the type III-E gRAMPcrRNA and gRAMPcrRNA-TPR-CHAT complexes, before and after either self or non-self RNA target binding, and elucidate the mechanisms underlying RNA-targeting and non-self RNA-induced protease activation. The associated TPR-CHAT adopted a distinct conformation upon self versus non-self RNA target binding, with nucleotides at positions −1 and −2 of the CRISPR-derived RNA (crRNA) serving as a sensor. Only binding of the non-self RNA target activated the TPR-CHAT protease, leading to cleavage of Csx30 protein. Furthermore, TPR-CHAT structurally resembled eukaryotic separase, but with a distinct mechanism for protease regulation. Our findings should facilitate the development of gRAMP-based RNA manipulation tools, and advance our understanding of the virus-host discrimination process governed by a nuclease-protease Craspase during type III-E CRISPR-Cas immunity.

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Section: Discussionsupporting

confidence: 88%

Structural basis for the non-self RNA-activated protease activity of the type III-E CRISPR nuclease-protease Craspase

Cui

Zhang

et al. 2022

Nat Commun

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“…The major difference between these two structure is that the Cas11 domain rotates about 30 °, and the Insertion domain is not observed in our structure due to flexibility (Figure S6). Recently, Nishimasu’s group has resolved the cryo-EM structures of the DiCas7-11-crRNA-Csx29 complex with and without target RNA 11 . In the structure of DiCas7-11-crRNA-Csx29-tgRNA, the CTD of Csx29 (residues 75-751) is not observed.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, Nishimasu's group has resolved the cryo-EM structures of the DiCas7-11-crRNA-Csx29 complex with and without target RNA 11 . In the structure of DiCas7-11-crRNA-Csx29-tgRNA, the CTD of Csx29 (residues 75-751) is not observed.…”

Section: Discussionmentioning

confidence: 99%

Cryo-EM structure of the type III-E CRISPR-Cas effector gRAMP in complex with TPR-CHAT

Huang

Wang

Guo

et al. 2022

Preprint

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The type III-E CRISPR-Cas effector protein, named gRAMP, is the largest single-unit CRISPR-Cas effector. A caspase-like peptidase (TPR-CHAT) gene often co-occurs with gRAMP gene clusters. TPR-CHAT interacts directly with gRAMP, indicating a functional relation between the CRISPR-Cas system and caspase peptidase. However, the exact mechanism of the recognition and cleavage of target RNA of Sb-gRAMP, as well as the molecular architecture of the CRISPR-guided caspase complex, remains unclear. Here, we report the cryo-EM structures of the type III-E effector Sb-gRAMP-crRNA in a complex with TPR-CHAT, with and without target ssRNA at 3.0 and 2.9Å, respectively. The overall structure of the gRAMP-crRNA-ssRNA-TPR-CHAT complex adopts an L-shaped conformation, consisting of a copy of gRAMP, a copy of TPR-CHAT, a 37-nt crRNA, and an 18-nt target ssRNA. The interactions between gRAMP and crRNA: target ssRNA heteroduplex were mainly mediated by the sugar-phosphate backbone and the neighboring basic amino acids. TPR-CHAT is a 716-residue protein that consists of two domains. The N-terminal TPR domain displays a 16 α-helix bundle conformation. The C-terminal CHAT domain contains an 11-stranded antiparallel β-sheet flanked by four α-helices on one side and five α-helices on the other side. The data presented in this manuscript reveal the mechanism of recognition of crRNA and target ssRNA by gRAMP, also, this work reports the structure of the CRISPR type III-E effector in complex with the binding partner TPR-CHAT, which provides vital clues for elucidating the functional relation between the CRISPR-Cas system and caspase peptidase.

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“…Rather, it allosterically activates a different effector, the TPR-CHAT protease, in a 3’ PFS-dependent manner. Though 13 of the 15 5’-tag nucleotides are buried by the protein, the last two, C(–1) and A(–2), are partially solvent-accessible and can potentially participate in PFS-mediated activation of the TRP-CHAT activity ( Hu et al, 2022 ).…”

Section: Resultsmentioning

confidence: 99%

Molecular mechanism of active Cas7-11 in processing CRISPR RNA and interfering target RNA

Goswami

Rai

Das

2022

eLife

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Cas7-11 is a Type III-E CRISPR Cas effector that confers programmable RNA cleavage and has potential applications in RNA interference. Cas7-11 encodes a single polypeptide containing four Cas7- and one Cas11-like segments that obscures the distinction between the multi-subunit Class 1 and the single-subunit Class-2 CRISPR-Cas systems. We report a cryo-EM structure of the active Cas7-11 from Desulfonema ishimotonii (DiCas7-11) that reveals the molecular basis for RNA processing and interference activities. DiCas7-11 arranges its Cas7- and Cas11-like domains in an extended form that resembles the backbone made up by four Cas7 and one Cas11 subunits in the multi-subunit enzymes. Unlike the multi-subunit enzymes, however, the backbone of DiCas7-11 contains evolutionarily different Cas7 and Cas11 domains, giving rise to their unique functionality. The first Cas7-like domain nearly engulfs the last 15 direct repeat nucleotides in processing and recognition of the CRISPR RNA, and its free-standing fragment retains most of the activity. Both the second and the third Cas7-like domains mediate target RNA cleavage in a metal-dependent manner. The structure and mutational data indicate that the long variable insertion to the fourth Cas7 domain has little impact to RNA processing or targeting, suggesting the possibility for engineering a compact and programmable RNA interference tool.

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Craspase is a CRISPR RNA-guided, RNA-activated protease

Cited by 64 publications

References 39 publications

Structural basis for the non-self RNA-activated protease activity of the type III-E CRISPR nuclease-protease Craspase

Structural basis for the non-self RNA-activated protease activity of the type III-E CRISPR nuclease-protease Craspase

Cryo-EM structure of the type III-E CRISPR-Cas effector gRAMP in complex with TPR-CHAT

Molecular mechanism of active Cas7-11 in processing CRISPR RNA and interfering target RNA

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