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Sequence Engineering of Guide DNA for Precise RNA Targeting by Cas12a
Yan Shan Ang1, Lin-Yue Lanry Yung1
1Department of Chemical & Biomolecular Engineering, National University of Singapore, Singapore117585, Singapore.
ACS Nano
|July 18, 2026
Summary
Researchers reprogrammed Cas12a (CRISPR-associated protein 12a) into a precise RNA-targeting tool. A novel guide DNA (gDNA) design framework enhances RNA targeting efficiency and single-nucleotide discrimination for RNA-targeting applications.
Area of Science:
- Molecular Biology
- Biotechnology
- Gene Editing
Background:
- Cas12a is a CRISPR-associated enzyme typically guided by DNA.
- This enzyme can be engineered to target RNA, offering new possibilities in molecular biology and biotechnology.
- Understanding Cas12a's interaction with guide RNA (gRNA) is crucial for optimizing its RNA-targeting capabilities.
Purpose of the Study:
- To engineer Cas12a as a DNA-guided RNA-targeting effector with high efficiency and specificity.
- To identify optimal guide DNA (gDNA) sequence motifs for RNA targeting.
- To develop a framework for precise RNA targeting using reprogrammed Cas12a.
Main Methods:
- Sequence engineering approach to identify desirable gDNA motifs.
- Introduction of a split gDNA design concept to probe key spacer features.
- Comparative analysis of single gDNA versus split gDNA designs for RNA targeting.
- Evaluation of Cas12a's engagement with different gDNA regions.
Main Results:
- Identified gDNA sequence motifs enabling Cas12a RNA targeting comparable to canonical RNA-guided Cas12a.
- Demonstrated that Cas12a actively engages the scaffold-proximal region of gDNA, while the scaffold-distal region is hybridization-driven.
- The split gDNA design significantly enhanced sequence selectivity, achieving up to 21-fold improvement over single gDNA designs.
- Achieved single-nucleotide discrimination among let-7 family members, showcasing high precision.
Conclusions:
- Established a gDNA sequence design framework to reprogram Cas12a into a precise RNA targeting platform.
- The split gDNA design is a powerful strategy for enhancing sequence selectivity and enabling single-nucleotide discrimination.
- This work provides a foundation for developing advanced RNA-targeting tools based on Cas12a.
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