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Mutational scanning of TnpB reveals latent activity for genome editing
Brittney W Thornton1,2, Rachel F Weissman1,2, Ryan V Tran1,2
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
Biorxiv : the Preprint Server for Biology
|February 24, 2025
Summary
Researchers enhanced RNA-guided endonuclease TnpB activity by discovering activating mutations in both its protein and RNA components. This breakthrough significantly boosts genome editing potential in human cells and plants.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- TnpB proteins are RNA-guided endonucleases found in prokaryotic transposons.
- Their small size and relation to Cas12 enzymes suggest potential for genome editing.
- Current TnpB variants often exhibit limited gene-editing efficiency.
Purpose of the Study:
- To map sequence-function landscapes of TnpB ribonucleoproteins.
- To identify mutations enhancing TnpB gene-editing activity.
- To explore regulatory elements influencing TnpB endonuclease function.
Main Methods:
- Comprehensive profiling of TnpB sequence-function relationships.
- Systematic mutagenesis of both TnpB protein and RNA components.
- Creation and screening of combinatorial protein variant libraries.
Main Results:
- Discovered numerous activating mutations in TnpB protein and RNA.
- Identified single-position RNA changes that significantly improve editing.
- Developed enhanced TnpB variants with over two-fold (human cells) and fifty-fold (plant cells) increased editing activity.
- Uncovered critical regulatory elements within TnpB and its RNA scaffold.
Conclusions:
- Systematic RNA mutagenesis is a powerful strategy for enhancing TnpB editing.
- Enhanced TnpB variants show significantly improved genome editing capabilities.
- TnpB possesses substantial latent activity, accessible through targeted mutations.
