Structures of an active type III-A CRISPR effector complex

Smith, Eric M.; Ferrell, Sé; Tokars, Valerie; Mondragón, Alfonso

doi:10.1016/j.str.2022.05.013

Cited by 6 publications

(18 citation statements)

References 66 publications

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“…The comparison of the SisCmr complex shown in Figure 1A was edited to exclude the quite unusual Cmr7 subunit (35), which is present as 13 dimers decorating the crRNP particle (36). In contrast to the SsoCmr complex in negative staining (37), and other multi-subunit CRISPR systems such as he Type III-A S. epidermidis Csm complex (21), the SsoCsm complex did not undergo disassembly, showing that it is a rather stable assembly. The analysis of the local resolution of the map (Figure 1B), showing local resolutions in the range 3.411 to 6.101 Å, suggested that SsoCsm has some inherent flexibility, particularly with respect to the movement of the Cas10 catalytic subunit, which may be relevant for its activity (26).…”

Section: Resultsmentioning

confidence: 89%

“…In the past ten years, multiple structural studies have confirmed the overall “boot-shaped” structure of type III effectors, with Cas10 at the toe, Cas5 at the heel and helical constellations of Cas7 and Cas 11 subunits making up the shaft (or backbone) (13–21). The overall organisation resembles that of type I systems, and the two are sometimes known by the collective name “Cascade” (CRISPR-associated complex for antiviral defence) (22).…”

Section: Introductionmentioning

confidence: 98%

“…Many groups have contributed to our current understanding of the target RNA recognition and activation of these effectors (reviewed in (3,23)), and type III effectors have been repurposed to develop sensitive new diagnostic assays (10,24,25). Despite this, the full diversity of type III systems has not been sampled at a structural level, and fundamental questions remain about the mechanism of activation of the Cas10 subunit on target RNA binding (21,26).…”

Section: Introductionmentioning

confidence: 99%

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Structure of theSaccharolobus solfataricustype III-D CRISPR effector

Cannone

Kompaniiets

Graham

et al. 2022

Preprint

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CRISPR-Cas is a prokaryotic adaptive immune system, classified into six different types, each characterised by a signature protein. Type III systems, classified based on the presence of a Cas10 subunit, are rather diverse multi-subunit assemblies with a range of enzymatic activities and downstream ancillary effectors. The broad array of current biotechnological CRISPR applications is mainly based on proteins classified as Type II, however recent developments established the feasibility and efficacy of multi-protein Type III CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes. The crenarchaeon Saccharolobus solfataricus has two type III system subtypes (III-B and III-D). Here, we report the cryo-EM structure of the Csm Type III-D complex from S. solfataricus (SsoCsm), which uses CRISPR RNA to bind target RNA molecules, activating the Cas10 subunit for antiviral defence. The structure reveals the complex organisation, subunit/subunit connectivity and protein/guide RNA interactions of the SsoCsm complex, one of the largest CRISPR effectors known.

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Section: Resultsmentioning

confidence: 89%

Section: Introductionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Structure of theSaccharolobus solfataricustype III-D CRISPR effector

Cannone

Kompaniiets

Graham

et al. 2022

Preprint

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“…While this manuscript was in preparation, high-resolution structures of S. epidermidis Cas10-Csm also were reported; however, in these structures, the cognate target RNA is not intact [24]. Thus, the confirmation of the complex in the active pre-cleavage state and corresponding contacts with the target RNA remain unclear.…”

Section: Introductionmentioning

confidence: 99%

“…An L. lactis structure was reported further enabling structural biologists ability to compare and contrast across the available structures to formulate hypotheses regarding the mechanisms of target RNA sensing and activation [21]. While this manuscript was in preparation, high-resolution structures of S. epidermidis Cas10-Csm also were reported; however, in these structures, the cognate target RNA is not intact [24]. Thus, the confirmation of the complex in the active pre-cleavage state and corresponding contacts with the target RNA remain unclear.…”

Section: Introductionmentioning

confidence: 99%

The structure of a Type III-A CRISPR-Cas effector complex reveals conserved and idiosyncratic contacts to target RNA and crRNA among Type III-A systems

Paraan

Nasef

Chou-Zheng

et al. 2022

Preprint

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Type III CRISPR-Cas systems employ multiprotein effector complexes bound to small CRISPR RNAs (crRNAs) to detect foreign RNA transcripts and elicit a complex immune response that leads to the destruction of invading RNA and DNA. Type III systems are among the most widespread in nature, and emerging interest in harnessing these systems for biotechnology applications highlights the need for detailed structural analyses of representatives from diverse organisms. We performed cryo-EM reconstructions of the Type III-A Cas10-Csm effector complex from S. epidermidis bound to an intact, cognate target RNA and identified two oligomeric states, a 276 kDa complex and a 318 kDa complex. 3.1 angstrom density for the well-ordered 276 kDa complex allowed construction of atomic models for the Csm2, Csm3, Csm4 and Csm5 subunits within the complex along with the crRNA and target RNA. We also collected small-angle X-ray scattering data which was consistent with the 276 kDa Cas10-Csm architecture we identified. Detailed comparisons between the S. epidermidis Cas10-Csm structure and the well-resolved bacterial (S. thermophilus) and archaeal (T. onnurineus) Cas10-Csm structures reveal differences in how the complexes interact with target RNA and crRNA which are likely to have functional ramifications. These structural comparisons shed light on the unique features of Type III-A systems from diverse organisms and will assist in improving biotechnologies derived from Type III-A effector complexes.

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