| First Author | Rath P | Year | 2025 |
| Journal | Stem Cells | Volume | 43 |
| Issue | 1 | PubMed ID | 39777513 |
| Mgi Jnum | J:363465 | Mgi Id | MGI:7868384 |
| Doi | 10.1093/stmcls/sxae092 | Citation | Rath P, et al. (2025) Optimizing approaches for targeted integration of transgenic cassettes by integrase-mediated cassette exchange in mouse and human stem cells. Stem Cells 43(1) |
| abstractText | 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 Ef1alpha (EIF1A), were tested for their suitability in driving expression of the integrated transgenic cargo. AAVS1-integrated Ef1alpha 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. |
