Functional and Phylogenetic Diversity of Cas10 Proteins

Wiegand, Tanner; Wilkinson, Royce A.; Santiago-Frangos, Andrew; Lynes, Mackenzie M.; Hatzenpichler, Roland; Wiedenheft, Blake

doi:10.1089/crispr.2022.0085

Cited by 5 publications

(4 citation statements)

References 68 publications

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“…The current view of the Type III defense mechanism implies three arms of the immune response (target transcript degradation, followed by target-dependent DNA cleavage, and cOA-activated nonspecific RNA cleavage). Recent systematic analysis shows that more than half of Type III-B Cas10 proteins lack the HD domain while many others have it inactivated [62], which implies the lack of transcription-dependent DNase activity. Whatever the target of this activity, our data as well as the data by others show that it is necessary for optimal Type III-A immunity.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Interference Requirements of Type III CRISPR-Cas Systems fromThermus thermophilus

Karneyeva,

Kolesnik,

Livenskyi

et al. 2023

Preprint

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Among the diverse prokaryotic adaptive immunity mechanisms, the Type III CRISPR-Cas systems are the most complex. The multisubunit Type III effectors recognize RNA targets complementary to CRISPR RNAs (crRNAs). Target recognition causes synthesis of cyclic oligoadenylates that activate downstream auxiliary effectors, which affect cell physiology in complex and poorly understood ways. Here, we studied the ability of III-A and III-B CRISPR-Cas subtypes fromThermus thermophilusto interfere with plasmid transformation. We find that for both systems, requirements for crRNA-target complementarity sufficient for interference depend on the target transcript abundance, with more abundant targets requiring shorter complementarity segments. This result and thermodynamic calculations indicate that Type III effectors bind their targets in a simple bimolecular reaction with more extensive crRNA-target base pairing compensating for lower target abundance. Since the targeted RNA used in our work is non-essential for either the host or the plasmid, the results also establish that a certain number of target-bound effector complexes must be present in the cell to interfere with plasmid establishment. For the more active III-A system, we determine the minimal length of RNA-duplex sufficient for interference and show that the position of this minimal duplex can vary within the effector. Finally, we show that the III-A immunity is dependent on the HD nuclease domain of the Cas10 subunit. Since this domain is absent from the III-B system the result implies that theT. thermophilusIII-B system must elicit a more efficient cyclic oligoadenylate dependent response to provide the immunity.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Interference Requirements of Type III CRISPR-Cas Systems fromThermus thermophilus

Karneyeva,

Kolesnik,

Livenskyi

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…SthCas10 and SthCsm2 were cloned into pACYCDuet1 (pACYCDuet1-SthCas10-SthCsm2) using Nco I and Sac I restriction sites. SthCas6 and CRISPR array containing five repeats and four identical spacers, designed to target the N-gene of SARS-CoV-2, were expressed in the pMA vector (pMA-SthCas6-4xcrRNA-N-gene, GenScript) ( 39 ). Cas10 gene with 15HD>HA and 573GGDD>GGAA mutations was synthesized and cloned in pACYCDuet1-SthCas10-SthCsm2 plasmid using Nco I and Not I restriction sites.…”

Section: Methodsmentioning

confidence: 99%

CRISPR-based engineering of RNA viruses

Nemudryi,

Nemudraia,

Nichols

et al. 2023

Sci. Adv.

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“…This highlights an auto-inhibition mechanism whereby the 5′ end of a cleaved RNA target remains stably associated with the crRNA while the 3′ end dissociates, which would allow Cas10 to return to an inactive conformation that ceases cOA production and prevents initiation of host dormancy or death. Phylogenetic analyses revealed that most Cas10 subunits from type III-D systems are predicted to not have an HD domain (Makarova et al 10 ; Weigand et al 50 ). Indeed, a large truncation was observed in our type III-Dv Cas10 when compared to a type III-A Cas10 with known HD nuclease activity 13 (Supplementary Fig.…”

Section: Programmable Target Rna Cleavage By the Type Iii-dv Complexmentioning

confidence: 99%

RNA targeting and cleavage by the type III-Dv CRISPR effector complex

Schwartz,

Bravo,

Ahsan

et al. 2024

Nat Commun

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CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction1–5. Target RNA cleavage at regular intervals is characteristic of type III effector complexes6–8. Here, we determine the structures of the Synechocystis type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors9,10, in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2′-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes.

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Functional and Phylogenetic Diversity of Cas10 Proteins

Cited by 5 publications

References 68 publications

Interference Requirements of Type III CRISPR-Cas Systems fromThermus thermophilus

Interference Requirements of Type III CRISPR-Cas Systems fromThermus thermophilus

CRISPR-based engineering of RNA viruses

RNA targeting and cleavage by the type III-Dv CRISPR effector complex

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