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

Alison Fanton^{1

2

3}, Liam J Bartie¹, Juliana Q Martins^{1

4}, Vincent Q Tran^{1

5}, Laine Goudy^{1

6

7}, Courtney Kernick^{1

8}, Matthew G Durrant¹, Jingyi Wei^{1

9}, Zev Armour-Garb⁶, April Pawluk¹, Silvana Konermann^{1

10}, Alexander Marson^{6

11}, Luke A Gilbert^{1

11

12}, Theodore L Roth^{1

8}, Patrick D Hsu^{13

14

15}

Affiliations

¹ Arc Institute, Palo Alto, CA, USA.
² Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA.
³ University of California, Berkeley - University of California, San Francisco, Graduate Program in Bioengineering, Berkeley, CA, USA.
⁴ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
⁵ Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA.
⁶ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA.
⁷ Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
⁸ Department of Pathology, Stanford University, Stanford, CA, USA.
⁹ Department of Bioengineering, Stanford University, Stanford, CA, USA.
¹⁰ Department of Biochemistry, Stanford University, Stanford, CA, USA.
¹¹ Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA.
¹² Department of Urology, University of California, San Francisco, San Francisco, CA, USA.
¹³ Arc Institute, Palo Alto, CA, USA. patrick@arcinstitute.org.
¹⁴ Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA. patrick@arcinstitute.org.
¹⁵ Center for Computational Biology, University of California, Berkeley, Berkeley, CA, USA. patrick@arcinstitute.org.

PMID: 41199024
DOI: 10.1038/s41587-025-02895-3

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

Alison Fanton et al. Nat Biotechnol. 2025.

. 2025 Nov 6.

doi: 10.1038/s41587-025-02895-3. Online ahead of print.

Authors

Affiliations

¹ Arc Institute, Palo Alto, CA, USA.
² Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA.
³ University of California, Berkeley - University of California, San Francisco, Graduate Program in Bioengineering, Berkeley, CA, USA.
⁴ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
⁵ Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA.
⁶ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA.
⁷ Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
⁸ Department of Pathology, Stanford University, Stanford, CA, USA.
⁹ Department of Bioengineering, Stanford University, Stanford, CA, USA.
¹⁰ Department of Biochemistry, Stanford University, Stanford, CA, USA.
¹¹ Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA.
¹² Department of Urology, University of California, San Francisco, San Francisco, CA, USA.
¹³ Arc Institute, Palo Alto, CA, USA. patrick@arcinstitute.org.
¹⁴ Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA. patrick@arcinstitute.org.
¹⁵ Center for Computational Biology, University of California, Berkeley, Berkeley, CA, USA. patrick@arcinstitute.org.

PMID: 41199024
DOI: 10.1038/s41587-025-02895-3

Abstract

Insertions of large DNA sequences into the genome are broadly enabling for research and therapeutic applications. Large serine recombinases (LSRs) can mediate direct, site-specific genomic integration of multi-kilobase DNA sequences without a pre-installed landing pad, albeit with low insertion rates and high off-target activity. Here we present an engineering roadmap for jointly optimizing their DNA recombination efficiency and specificity. We combine directed evolution, structural analysis and computational models to rapidly identify additive mutational combinations. We further enhance performance through donor DNA optimization and dCas9 fusions, enabling simultaneous target and donor recruitment. Our top engineered LSR variants, superDn29-dCas9, goldDn29-dCas9 and hifiDn29-dCas9, achieve up to 53% integration efficiency and 97% genome-wide specificity at an endogenous human locus and effectively integrate large DNA cargoes up to 12 kb for stable expression in non-dividing cells, stem cells and primary human T cells. Rational engineering of DNA recombinases enables precise and efficient single-step genome insertion for diverse applications across gene and cell therapies.

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Conflict of interest statement

Competing interests: P.D.H. acknowledges outside interest as a co-founder of Monet AI, Terrain Biosciences and Stylus Medicine; board of directors at Stylus Medicine; board observer at Terrain Biosciences; scientific advisory board member at Veda Bio; and venture partner at Thrive Capital. A.F. and M.G.D. acknowledge outside interest in Stylus Medicine. A.F., L.J.B., M.G.D. and P.D.H. are inventors on patents relating to this work. A.M. is a co-founder of Site Tx, Arsenal Biosciences, Spotlight Therapeutics and Survey Genomics; serves on the boards of directors at Site Tx and Survey Genomics; is a member of the scientific advisory boards of Network.bio, Site Tx, Arsenal Biosciences, Cellanome, Survey Genomics, NewLimit, Amgen and Tenaya; owns stock in Network.bio, Arsenal Biosciences, Site Tx, Cellanome, Spotlight Therapeutics, NewLimit, Survey Genomics, Tenaya and Lightcast; and has received fees from Network.bio, Site Tx, Arsenal Biosciences, Cellanome, Spotlight Therapeutics, NewLimit, AbbVie, Gilead, Pfizer, 23andMe, PACT Pharma, Juno Therapeutics, Tenaya, Lightcast, Trizell, Vertex, Merck, Amgen, Genentech, GLG, ClearView Healthcare, AlphaSights, Rupert Case Management, Bernstein and ALDA. A.M. is an investor in and informal advisor to Offline Ventures and a client of EPIQ. The Marson laboratory has received research support from the Parker Institute for Cancer Immunotherapy, CZI, the Emerson Collective, the Arc Institute, Juno Therapeutics, Epinomics, Sanofi, GlaxoSmithKline, Gilead and Anthem and reagents from Genscript, Illumina, Ultima and 10x Genomics. L.A.G. has filed patents on CRISPR tools and CRISPR functional genomics, is a co-founder of nChroma Bio and is a consultant for nChroma Bio. T.L.R. is a co-founder of Arsenal Biosciences, owns stock in Arsenal Biosciences and has received fees from Arsenal Biosciences, NewLimit and Alector. The Roth laboratory has received research support from the Parker Institute for Cancer Immunotherapy and Northpond Ventures. The other authors declare no competing interests.

References

1. 1000 Genomes Project Consortium. A global reference for human genetic variation. Nature 526, 68–74 (2015). - DOI
1. Gogol-Döring, A. et al. Genome-wide profiling reveals remarkable parallels between insertion site selection properties of the MLV retrovirus and the piggyBac transposon in primary human CD4⁺ T cells. Mol. Ther. 24, 592–606 (2016). - PubMed - PMC - DOI
1. Chaikind, B., Bessen, J. L., Thompson, D. B., Hu, J. H. & Liu, D. R. A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells. Nucleic Acids Res. 44, 9758–9770 (2016). - PubMed - PMC
1. Standage-Beier, K. et al. RNA-guided recombinase-Cas9 fusion targets genomic DNA deletion and integration. CRISPR J. 2, 209–222 (2019). - PubMed - PMC - DOI
1. Hew, B. E., Sato, R., Mauro, D., Stoytchev, I. & Owens, J. B. RNA-guided transposition in human cells. Synth. Biol. 4, ysz018 (2019). - DOI

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2019284848/National Science Foundation (NSF)

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

Affiliations

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

Authors

Affiliations

Abstract

Conflict of interest statement

References

Grants and funding

LinkOut - more resources

Full Text Sources

Research Materials