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Massively parallel profiling of RNA-targeting CRISPR-Cas13d
Hung-Che Kuo1, Joshua Prupes2, Chia-Wei Chou2
1Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, 78712, USA. hckuo@utexas.edu.
Nature Communications
|January 12, 2024
Summary
CRISPR-Cas13d RNA targeting is sensitive to RNA secondary structure, not sequence alone. This discovery enables precise RNA detection, like differentiating SARS-CoV-2 variants using engineered CRISPR RNA (crRNA).
Area of Science:
- Molecular Biology
- Biotechnology
- Genetics
Background:
- CRISPR-Cas13d is a versatile tool for RNA manipulation and diagnostics.
- A comprehensive understanding of its RNA binding and cleavage specificity is currently limited.
Purpose of the Study:
- To systematically characterize the RNA binding and cleavage specificity of CRISPR-Cas13d.
- To develop a predictive model for Cas13d RNA targeting.
- To engineer Cas13d systems for specific RNA detection, such as differentiating viral variants.
Main Methods:
- Development and application of the RNA Chip-Hybridized Association-Mapping Platform (RNA-CHAMP) to profile binding affinities across thousands of RNA variants.
- Deep profiling of CRISPR-Cas13d interactions with diverse RNA targets.
- Biophysical modeling to elucidate RNA recognition mechanisms.
Main Results:
- CRISPR-Cas13d does not require a protospacer flanking sequence.
- Target RNA secondary structure significantly influences Cas13d binding and cleavage.
- Binding is sensitive to mismatches in the distal crRNA-target RNA region, while proximal alterations affect nuclease activity.
- Target recognition initiates at the distal end of the RNA.
Conclusions:
- CRISPR-Cas13d's RNA targeting is primarily dictated by RNA secondary structure rather than solely sequence.
- A biophysical model accurately predicts Cas13d RNA recognition.
- Engineered CRISPR RNA (crRNA) can be designed to differentiate between specific RNA targets, demonstrated by distinguishing SARS-CoV-2 variants.
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