Journal article
Optimizing approaches for targeted integration of transgenic cassettes by integrase-mediated cassette exchange in mouse and human stem cells
- Abstract:
- 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.
- Publication status:
- Published
- Peer review status:
- Peer reviewed
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(Preview, Version of record, pdf, 1.3MB, Terms of use)
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- Publisher copy:
- 10.1093/stmcls/sxae092
Authors
+ Wellcome Trust
More from this funder
- Funder identifier:
- https://ror.org/029chgv08
- Grant:
- 203141/Z/16/Z
- 090532/Z/09/Z
+ European Commission
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- Funder identifier:
- https://ror.org/00k4n6c32
- Grant:
- 115439
- Publisher:
- Oxford University Press
- Journal:
- Stem Cells More from this journal
- Volume:
- 43
- Issue:
- 1
- Article number:
- sxae092
- Place of publication:
- England
- Publication date:
- 2025-01-08
- Acceptance date:
- 2024-12-17
- DOI:
- EISSN:
-
1549-4918
- ISSN:
-
1066-5099
- Pmid:
-
39777513
- Language:
-
English
- Keywords:
- Pubs id:
-
2076878
- Local pid:
-
pubs:2076878
- Deposit date:
-
2025-03-19
- ARK identifier:
Terms of use
- Copyright holder:
- Rath et al
- Copyright date:
- 2025
- Rights statement:
- © The Author(s) 2025. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
- Licence:
- CC Attribution (CC BY)
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