| 1. |
- Das, Anuska, et al.
(författare)
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Structural principles of CRISPR-Cas enzymes used in nucleic acid detection
- 2022
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Ingår i: JOURNAL OF STRUCTURAL BIOLOGY. - : Elsevier BV. - 1047-8477 .- 1095-8657. ; 214:1
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Tidskriftsartikel (refereegranskat)abstract
- Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-based technology has revolutionized the field of biomedicine with broad applications in genome editing, therapeutics and diagnostics. While a majority of applications involve the RNA-guided site-specific DNA or RNA cleavage by CRISPR enzymes, recent successes in nucleic acid detection rely on their collateral and non-specific cleavage activated by viral DNA or RNA. Ranging in enzyme composition, the mechanism for distinguishing self-from foreign-nucleic acids, the usage of second messengers, and enzymology, the CRISPR enzymes provide a diverse set of diagnosis tools in further innovations. Structural biology plays an important role in elucidating the mechanisms of these CRISPR enzymes. Here we summarize and compare structures of three types of CRISPR enzymes used in nucleic acid detection captured in their respective functional forms and illustrate the current understanding of their activation mechanism.
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| 2. |
- Sridhara, Sagar, 1989, et al.
(författare)
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Structural and biochemical characterization of in vivo assembled Lactococcus lactis CRISPR-Csm complex
- 2022
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 5:1
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Tidskriftsartikel (refereegranskat)abstract
- The small RNA-mediated immunity in bacteria depends on foreign RNA-activated and self RNA-inhibited enzymatic activities. The multi-subunit Type III-A CRISPR-Cas effector complex (Csm) exemplifies this principle and is in addition regulated by cellular metabolites such as divalent metals and ATP. Recognition of the foreign or cognate target RNA (CTR) triggers its single-stranded deoxyribonuclease (DNase) and cyclic oligoadenylate (cOA) synthesis activities. The same activities remain dormant in the presence of the self or non-cognate target RNA (NTR) that differs from CTR only in its 3 '-protospacer flanking sequence (3 '-PFS). Here we employ electron cryomicroscopy (cryoEM), functional assays, and comparative cross-linking to study in vivo assembled mesophilic Lactococcus lactis Csm (LlCsm) at the three functional states: apo, the CTR- and the NTR-bound. Unlike previously studied Csm complexes, we observed binding of 3 '-PFS to Csm in absence of bound ATP and analyzed the structures of the four RNA cleavage sites. Interestingly, comparative crosslinking results indicate a tightening of the Csm3-Csm4 interface as a result of CTR but not NTR binding, reflecting a possible role of protein dynamics change during activation. This study presents Cryo-EM structures of the Lactococcus lactis Type III-A CRISPR-Cas effector complex in three functional states, including bound forms with cognate and non-cognate target RNA. The results suggest changing protein dynamics during effector complex activation.
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