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Site-specific DNA insertion into the human genome with engineered recombinases.

Alison Fanton^1,2,3, Liam J Bartie¹, Juliana Q Martins^1,4

¹Arc Institute, Palo Alto, CA, USA.

Nature Biotechnology

|November 6, 2025

Summary

Researchers engineered large serine recombinases (LSRs) for precise genome editing. Optimized LSR variants achieve high efficiency and specificity for large DNA insertions, advancing gene and cell therapies.

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Area of Science:

Genomics
Molecular Biology
Biotechnology

Background:

Large DNA sequence integration is crucial for research and therapeutics.
Large serine recombinases (LSRs) offer direct genomic integration but have low efficiency and specificity.

Purpose of the Study:

To engineer LSRs for improved DNA recombination efficiency and specificity.
To enable precise, large DNA insertions for gene and cell therapies.

Main Methods:

Combined directed evolution, structural analysis, and computational models.
Optimized donor DNA and utilized dCas9 fusions for target and donor recruitment.

Main Results:

Developed engineered LSR variants (superDn29-dCas9, goldDn29-dCas9, hifiDn29-dCas9).
Achieved up to 53% integration efficiency and 97% specificity at an endogenous human locus.
Successfully integrated large DNA cargoes (up to 12 kb) for stable expression in various cell types.

Conclusions:

Rational engineering of LSRs enables precise and efficient single-step genome insertion.
Optimized LSRs have broad applications in gene and cell therapies.