Structural rearrangements allow nucleic acid discrimination by type I-D Cascade

Schwartz, Eric M.; McBride, Tess M.; Bravo, Jack P.K.; Wrapp, Daniel; Fineran, Peter C.; Fagerlund, Robert D.; Taylor, David W.

doi:10.1038/s41467-022-30402-8

Cited by 27 publications

(53 citation statements)

References 73 publications

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“…For type I-D, Cas8 is replaced with a Cas10 subunit, also in orange, and bound target RNA is also present in the structure. Structures shown are: type I-A ( 7 ), type I-C ( 8 ), type I-D ( 10 ), type I-E ( 14 ), type I-F ( 49 ) and type I-G (this study).…”

Section: Discussionmentioning

confidence: 80%

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Structure and mechanism of the type I-G CRISPR effector

Shangguan

Graham

Sundaramoorthy

et al. 2022

Nucleic Acids Research

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Type I CRISPR systems are the most common CRISPR type found in bacteria. They use a multisubunit effector, guided by crRNA, to detect and bind dsDNA targets, forming an R-loop and recruiting the Cas3 enzyme to facilitate target DNA destruction, thus providing immunity against mobile genetic elements. Subtypes have been classified into families A-G, with type I-G being the least well understood. Here, we report the composition, structure and function of the type I-G Cascade CRISPR effector from Thioalkalivibrio sulfidiphilus, revealing key new molecular details. The unique Csb2 subunit processes pre-crRNA, remaining bound to the 3′ end of the mature crRNA, and seven Cas7 subunits form the backbone of the effector. Cas3 associates stably with the effector complex via the Cas8g subunit and is important for target DNA recognition. Structural analysis by cryo-Electron Microscopy reveals a strikingly curved backbone conformation with Cas8g spanning the belly of the structure. These biochemical and structural insights shed new light on the diversity of type I systems and open the way to applications in genome engineering.

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Section: Discussionmentioning

confidence: 80%

“…High resolution crystal or cryo-EM structures are available for type I-A ( 7 ), I-C ( 8 , 9 ), I-D ( 10 ), I-E ( 11–14 ), I-F ( 15 ), I-Fv ( 16 ) and type I-F-TniQ ( 17 ) complexes. However, within this broad family there are many variations.…”

Section: Introductionmentioning

confidence: 99%

Structure and mechanism of the type I-G CRISPR effector

Shangguan

Graham

Sundaramoorthy

et al. 2022

Nucleic Acids Research

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“…Overall, our findings highlight the importance of type III-Dv in the evolution of type III CRISPR systems. Previously, we reported on the structure of the Synechocystis type I-D complex 29,30 The structures of the type III-Dv and III-E effectors provide insights into engineering multi-subunit class 1 complexes into single polypeptide effectors that may be more suitable for biotechnological applications. These systems utilize flexible linkers that hold different domains together, aiding their assembly into a CRISPR-Cas effector.…”

Section: Discussionmentioning

confidence: 99%

“…Overall, our findings highlight the importance of type III-Dv in the evolution of type III CRISPR systems. Previously, we reported on the structure of the Synechocystis type I-D complex 29,30 . Our structural work highlighting subunit fusions and insertions in the type III-Dv complex indicate that that I-D and III-Dv likely diverged from a type III-A or III-B ancestor.…”

Section: Discussionmentioning

confidence: 99%

Assembly of multi-subunit fusion proteins into the RNA-targeting type III-D CRISPR-Cas effector complex

Schwartz

Bravo

Macias

et al. 2022

Preprint

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CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems are a type of adaptive immune response in bacteria and archaea that utilize crRNA (CRISPR RNA)-guided effector complexes to target complementary RNA or DNA for destruction. The prototypical type III-A and III-B CRISPR-Cas systems utilize multi-subunit effector complexes composed of individual proteins to cleave ssRNA targets at 6-nt intervals, as well as non-specifically degrading ssDNA and activating cyclic oligoadenylate (cOA) synthesis. Recent studies have shown that type III systems can contain subunit fusions yet maintain canonical type III RNA-targeting capabilities. To understand how a multi-subunit fusion effector functions, we determine structures of a variant type III-D effector and biochemically characterize how it cleaves RNA targets. These findings provide insights into how multi-subunit fusion proteins are tethered together and assemble into an active and programmable RNA endonuclease, how the effector utilizes a novel mechanism for target RNA seeding, and the structural basis for the evolution of type III effector complexes. Furthermore, our results provide a blueprint for fusing subunits in class 1 effectors for design of user-defined effector complexes with disparate activities.

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“…High resolution crystal or cryo-EM structures are available for type I-A (7), I-C (8,9), I-D (10), I-E (11)(12)(13)(14), I-F (15), I-Fv (16) and type I-F-TniQ (17) complexes. However, within this broad family there are many variations.…”

Section: Introductionmentioning

confidence: 99%

Structure and mechanism of the type I-G CRISPR effector

Shangguan

Graham

Sundaramoorthy

et al. 2022

Preprint

View full text Add to dashboard Cite

Type I CRISPR systems are the most common CRISPR type found in bacteria. They use a multisubunit effector, guided by crRNA, to detect and bind dsDNA targets, forming an R-loop and recruiting the Cas3 enzyme to facilitate target DNA destruction, thus providing immunity against mobile genetic elements. Subtypes have been classified into families A-G, with type I-G being the least well understood. Here, we report the composition, structure and function of the type I-G Cascade CRISPR effector from Thioalkalivibrio sulfidiphilus, revealing key new molecular details. The unique Csb2 subunit processes pre-crRNA, remaining bound to the 3-prime end of the mature crRNA, and seven Cas7 subunits form the backbone of the effector. Cas3 associates stably with the effector complex via the Cas8g subunit and is important for target DNA recognition. Structural analysis by cryo-Electron Microscopy reveals a strikingly curved backbone conformation with Cas8g spanning the belly of the structure. Type I-G Cascade is one of the most streamlined Class 1 CRISPR effectors. These biochemical and structural insights shed new light on the diversity of type I systems and open the way to applications in genome engineering.

show abstract

Structural rearrangements allow nucleic acid discrimination by type I-D Cascade

Cited by 27 publications

References 73 publications

Structure and mechanism of the type I-G CRISPR effector

Structure and mechanism of the type I-G CRISPR effector

Assembly of multi-subunit fusion proteins into the RNA-targeting type III-D CRISPR-Cas effector complex

Structure and mechanism of the type I-G CRISPR effector

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