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Related Experiment Video

Updated: Apr 3, 2026

Site-Directed φC31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria

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S-SELeCT: a human-evolved serine integrase system for efficient large-cargo genome integration.

Alfonso P Farruggio¹, Lin Jiang¹, Karen Duong¹

¹Research and Development, Applied StemCell Inc., Milpitas, CA 95035, United States.

Nucleic Acids Research

|April 2, 2026

Summary

Large-fragment knock-in using novel gene-editing enzymes facilitates gene therapy for large genetic mutations. These engineered serine integrases enable efficient DNA integration at safe harbor loci in human cells.

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Area of Science:

Molecular Biology
Gene Therapy
Biotechnology

Background:

Many genetic diseases stem from mutations in large genes, posing challenges for conventional gene therapy.
Large-fragment knock-in offers a unified approach to treat diverse mutations by integrating full-length coding sequences at safe harbor loci.

Purpose of the Study:

To develop novel gene-editing enzymes for efficient large-fragment knock-in at a specific safe harbor locus in human cells.
To create the first serine integrase enzymes evolved entirely within human cells for therapeutic applications.

Main Methods:

Engineering and expression of S-SELeCT (Site-Specific Large Cargo Targeting) integrase fusions in human cells.
Assessing large DNA fragment (10 kb plasmid) integration efficiency in stable cell lines and via transient transfection.

Main Results:

S-SELeCT integrase fusions facilitated 10 kb plasmid integration at frequencies up to 32% in stable cell lines.
Transient delivery via plasmid transfection achieved knock-in frequencies of up to 13%.

Conclusions:

The developed S-SELeCT integrase fusions represent a significant advancement in gene-editing technology for large-fragment knock-in.
These engineered serine integrases are the first to recognize an endogenous, symmetric, non-pseudosite, establishing a true human serine integrase attachment site for gene therapy.