Optimizing approaches for targeted integration of transgenic cassettes by integrase mediated cassette exchange in mouse and human stem cells More about Open Access at the Crick
Authors list
Phalguni Rath Philipp Kramer Daniel Biggs Chris Preece Nicole Hortin Rebeca Diaz Marta Perez-Alcantara Xiang Li Arnaud Bolard Nicola Beer Mark McCarthy Ben DaviesAbstract
To enable robust expression of transgenes in stem cells, recombinase mediated cassette exchange at safe harbour 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 three 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 two 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 down-regulation of target genes was demonstrated.
Journal details
Journal
Stem Cells
Volume
43
Issue number
1
Pages
sxae092
Available online
Publication date
Full text links
Publisher website (DOI)
10.1093/stmcls/sxae092
Europe PubMed Central
39777513
Pubmed
39777513
Keywords
Related topics
Type of publication
