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Updated: Aug 21, 2026
Ubiquitous and Tissue-specific RNA Targeting in Drosophila Melanogaster using CRISPR/CasRx
Published on: February 5, 2021
Structural basis for target discrimination and activation by Cas13d
Chia-Wei Chou1, Selma Sinan1, Hung-Che Kuo1
1Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA.
Abstract:
CRISPR-Cas13d is increasingly used for RNA knockdowns, but off-target cleavage of near-cognate RNAs hinders its broader adoption. Here, we solve seven cryo-electron microscopy structures of wild-type Cas13d in complex with matched and mismatched targets. These structures reveal active, intermediate, and inactive states that illustrate a detailed activation mechanism. Upon target RNA binding, the CRISPR RNA undergoes marked conformational changes. The Helical-1 domain transitions from a docked state with the amino-terminal domain to an allosterically switched conformation that stabilizes the RNA duplex. Quantitative kinetics show that a single proximal mismatch preserves the binding rate constant but abolishes nuclease activity by trapping Cas13d in an inactive state. We also identify an active site loop in the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains that regulates substrate accessibility and can be mutated to generate both hypo- and hyperactivated variants. These findings establish the structural basis for Cas13d mismatch surveillance and provide a framework for engineering HEPN nuclease specificity and activity.
Insights
CRISPR-Cas13d
Area of Science:
- Molecular Biology
- Structural Biology
- Biochemistry
Background:
- CRISPR-Cas13d is a key tool for RNA knockdown.
- Off-target cleavage of near-cognate RNAs limits its application.
- Understanding Cas13d's mechanism is crucial for improving specificity.
Purpose of the Study:
- To elucidate the structural basis of CRISPR-Cas13d activation and mismatch surveillance.
- To provide a framework for engineering Cas13d variants with enhanced specificity and activity.
Main Methods:
- Cryo-electron microscopy (cryo-EM) to solve seven structures of wild-type Cas13d.
- Structural analysis of active, intermediate, and inactive states.
- Quantitative kinetic analysis of RNA binding and cleavage.
Main Results:
- Detailed mechanism of Cas13d activation upon target RNA binding.
- Identification of conformational changes in CRISPR RNA and Helical-1 domain.
- Demonstration that proximal mismatches trap Cas13d in an inactive state, preventing cleavage.
- Characterization of an active site loop in HEPN domains regulating substrate access.
Conclusions:
- Structural insights reveal Cas13d's mismatch surveillance mechanism.
- Engineering of Cas13d's HEPN nuclease domain can modulate specificity and activity.
- Findings facilitate the development of more precise RNA-targeting tools.
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