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Updated: Jul 15, 2025
09:16
In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
Published on: March 25, 2020
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Programmable RNA-guided DNA endonucleases are widespread in eukaryotes and their viruses
Kaiyi Jiang1,2, Justin Lim1, Samantha Sgrizzi1
1McGovern Institute for Brain Research at MIT Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Science Advances
|September 27, 2023
Summary
Fanzor nucleases are newly characterized RNA-guided DNA endonucleases found in eukaryotes. These enzymes can be engineered for genome editing in human cells, offering new biotechnology tools.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Programmable RNA-guided DNA nucleases are vital in prokaryotes, but their presence and function in eukaryotes are largely unknown.
- Fanzors, eukaryotic homologs of prokaryotic TnpB proteins, have been identified but their activities remain uncharacterized.
Purpose of the Study:
- To biochemically and cellularly characterize Fanzors as functional RNA-guided DNA endonucleases in eukaryotes.
- To explore the evolutionary history and functional adaptations of Fanzors in eukaryotic systems.
- To assess the potential of Fanzors for genome editing applications in human cells.
Main Methods:
- Biochemical assays to determine nuclease activity.
- Cellular studies to investigate Fanzor function and localization.
- Phylogenetic analysis to reconstruct Fanzor evolution.
- Demonstration of Fanzor-based genome editing in human cells.
Main Results:
- Fanzors were confirmed as RNA-programmable DNA endonucleases with a rearranged RuvC catalytic site and no collateral cleavage activity.
- Diverse Fanzors were found associated with eukaryotic transposases, indicating co-option into eukaryotic mobile element systems.
- Evidence of intron capture and nuclear localization signals suggests long-term adaptation to eukaryotic cellular environments.
- Fanzors were successfully utilized for genome editing in human cells.
Conclusions:
- Fanzors represent a widespread class of eukaryotic RNA-guided DNA endonucleases with unique evolutionary adaptations.
- These nucleases possess distinct biochemical properties compared to prokaryotic counterparts.
- Fanzors hold significant potential as novel tools for genome editing and biotechnology applications in eukaryotes.
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