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GRASP: a modular toolkit for building synthetic pentatricopeptide repeat RNA-binding proteins.
Michael Dennis1,2, Su Yi Low1,2, Amy Viljoen1,2,3
1School of Molecular Sciences, The University of Western Australia, Perth, WA 6009, Australia.
Nucleic Acids Research
|November 12, 2025
Summary
The GRASP kit simplifies the creation of synthetic Pentatricopeptide repeat (PPR) proteins for targeted RNA manipulation. This tool enables researchers to easily design and build custom PPR proteins for diverse RNA applications.
Area of Science:
- Molecular Biology
- RNA Biology
- Protein Engineering
Background:
- Pentatricopeptide repeat (PPR) proteins are crucial eukaryotic RNA-binding proteins involved in mitochondrial and chloroplast transcript processing.
- Their modular nature offers potential for synthetic biology tools, but construction is hindered by repetitive sequences.
Purpose of the Study:
- To present the GRASP kit, a novel system for the efficient assembly of synthetic PPR (sPPR) proteins.
- To enable rapid design and construction of sPPR proteins with specific RNA-targeting capabilities.
Main Methods:
- Development of the GRASP kit using S-variant PPR motifs and a library of 42 plasmids.
- Assembly of sPPR proteins with 9, 14, or 19 motifs targeting specific RNA sequences.
- Compatibility with the MoClo assembly standard for streamlined protein construction.
Main Results:
- Demonstrated GRASP kit's capability by assembling sPPR-RNA-editing proteins and variants.
- Tested 31 sPPR variants against 46 RNA targets, analyzing RNA editing levels via sequencing.
- Generated insights into PPR-RNA interactions through observed editing variations.
Conclusions:
- The GRASP kit facilitates the rapid and specific assembly of synthetic PPR proteins.
- This technology provides a foundation for advancing sPPR protein applications in RNA processing and gene expression control.
