Optimizing approaches for targeted integration of transgenic cassettes by integrase-mediated cassette exchange in mouse and human stem cells
Summary
To enable robust expression of transgenes in stem cells, recombinase-mediated cassette exchange at safe harbor loci is frequently adopted. The choice of recombinase enzyme is a critical parameter to ensure maximum efficiency and accuracy of the integration event. We have explored the serine recombinase family of site-specific integrases and have directly compared the efficiency of PhiC31, W-beta, and Bxb1 integrase for targeted transgene integration at the Gt(ROSA)26Sor locus in mouse embryonic stem cells. All 3 integrases were found to be suitable for efficient engineering and long-term expression of each integrase was compatible with pluripotency, as evidenced by germline transmission. Bxb1 integrase was found to be 2-3 times more efficient than PhiC31 and W-beta. The Bxb1 system was adapted for cassette exchange at the AAVS1 locus in human induced pluripotent stem (iPS) cells, and the 2 commonly used ubiquitous promoters, CAG and Ef1α (EIF1A), were tested for their suitability in driving expression of the integrated transgenic cargo. AAVS1-integrated Ef1α promoter led to a very mosaic pattern of expression in targeted hiPS cells, whereas the AAVS1-integrated CAG promoter drove consistent and stable expression. To validate the system for the integration of functional machinery, the Bxb1 integrase system was used to integrate CAG-driven CRISPR-activation and CRISPR-inhibition machinery in human iPS cells and robust sgRNA-induced up- and downregulation of target genes was demonstrated. © The Author(s) 2025. Published by Oxford University Press.
| Authors | Rath P, Kramer P, Biggs D, Preece C, Hortin N, Diaz R, Perez-Alcantara M, Li X, Bolard A, Beer N, McCarthy M, Davies B |
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| Journal | Stem cells (Dayton, Ohio) |
| Publication Date | 2025 Jan 17;43(1) |
| PubMed | 39777513 |
| PubMed Central | PMC11740728 |
| DOI | 10.1093/stmcls/sxae092 |