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Updated: Jul 18, 2026
09:16
In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
Published on: March 25, 2020
A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences
Julianne Smith1, Sylvestre Grizot, Sylvain Arnould
1CELLECTIS S.A., 102 route de Noisy, 93235 Romainville, France.
Nucleic Acids Research
|November 30, 2006
Summary
Researchers engineered artificial homing endonucleases (HEs) with redesigned specificity for targeted gene editing. This breakthrough in protein engineering offers new tools for genome engineering and gene therapy applications.
Area of Science:
- Molecular Biology
- Protein Engineering
- Genomics
Background:
- Homing endonucleases (HEs) are sequence-specific enzymes crucial for gene targeting.
- Existing HEs have limited recognition sites, hindering broad genome engineering applications.
- Designing artificial HEs with novel specificities is essential for advancing gene editing technologies.
Purpose of the Study:
- To engineer artificial homing endonucleases (HEs) with entirely redesigned specificities.
- To demonstrate the feasibility of creating novel HEs for targeted DNA cleavage.
- To provide a general method for tailoring HE specificity for genome engineering.
Main Methods:
- Derived novel endonucleases with altered substrate specificity from the I-CreI LAGLIDADG family.
- Identified distinct DNA-binding subdomains within the HE protein structure.
- Assembled mutations into heterodimeric endonucleases to cleave specific target sequences, including a human RAG1 gene sequence.
Main Results:
- Successfully redesigned the specificity of HEs to cleave naturally occurring DNA sequences.
- Demonstrated the extensive engineering potential of LAGLIDADG endonucleases.
- Created functional artificial HEs targeting both model DNA and a human gene sequence.
Conclusions:
- The plasticity of LAGLIDADG HEs enables significant protein engineering.
- Developed a generalizable method for creating novel HEs with customized DNA specificities.
- These engineered HEs hold promise for advanced genome engineering and therapeutic applications.
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