Structural basis for self-cleavage prevention by tag:anti-tag pairing complementarity in type VI Cas13 CRISPR systems

Jan 20, 2021Molecular cell

How matching tag and anti-tag pairs prevent self-cutting in type VI Cas13 CRISPR systems

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Abstract

The cryo-EM structure of Leptotrichia shahii LshCas13a with target RNA reveals important conformational changes related to RNA cleavage activity.

Target RNAs with extended complementarity to repeat sequences can significantly reduce RNA cleavage by the Cas13a system.
Observed structural changes in LshCas13a provide a molecular explanation for the inactivation of its cleavage activity.
Key mutants were studied in both in vitro and in vivo assays to explore their effects on Cas13a's targeting capabilities.
Insights gained may help regulate Cas13a's activity, potentially impacting its use in biotechnological applications.

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Bacteria and archaea apply CRISPR-Cas surveillance complexes to defend against foreign invaders. These invading genetic elements are captured and integrated into the CRISPR array as spacer elements, guiding sequence-specific DNA/RNA targeting and cleavage. Recently, in vivo studies have shown that target RNAs with extended complementarity with repeat sequences flanking the target element (tag:anti-tag pairing) can dramatically reduce RNA cleavage by the type VI-A Cas13a system. Here, we report the cryo-EM structure of Leptotrichia shahii LshCas13ain complex with target RNA harboring tag:anti-tag pairing complementarity, with the observed conformational changes providing a molecular explanation for inactivation of the composite HEPN domain cleavage activity. These structural insights, together with in vitro biochemical and in vivo cell-based assays on key mutants, define the molecular principles underlying Cas13a's capacity to target and discriminate between self and non-self RNA targets. Our studies illuminate approaches to regulate Cas13a's cleavage activity, thereby influencing Cas13a-mediated biotechnological applications. crRNA

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Structural basis for self-cleavage prevention by tag:anti-tag pairing complementarity in type VI Cas13 CRISPR systems

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