Key result
CRISPR-Cas9 targeted transgenes at the AAVS1 locus in human induced pluripotent stem cells exhibited variable expression and silencing during mesodermal differentiation, indicating it is not a true safe harbour.
Why the study?
Human induced pluripotent stem cell reporter lines are useful for disease modelling in conditions like HCM, and the AAVS1 locus is favored as a safe harbour for stable transgene expression.
Population
24 hiPSC clones targeted with pCAG-R-GECO1.0 and HOXA9-T2A-mScarlet hiPSC reporter lines
Comparison
Transgene expression compared between clones before, during, and/or after directed differentiation to mesodermal lineages
Design
In vitro experimental study
Authors
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AAVS1 integration risks transgene silencing in hiPSC-mesoderm differentiation; leaves open need for validated safe harbours in stem cell research.
Targeted knock-ins to the AAVS1 safe harbour locus in hiPSCs are subject to significant variability and transgene silencing upon differentiation to mesodermal lineages, requiring careful clone selection and maintenance strategies.
Bhagwan et al. (2020) studied Hypertrophic cardiomyopathy (in vitro model). CRISPR-Cas9 targeted transgenes at AAVS1 locus vs. Untargeted hiPSCs or different clones was evaluated on Transgene expression and silencing. CRISPR-Cas9 targeted transgenes at the AAVS1 locus in human induced pluripotent stem cells exhibited variable expression and silencing during mesodermal differentiation, indicating it is not a true safe harbour.
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