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Ectopic expression of proteins in human pluripotent stem cells (hPSCs) is highly desirable as a research tool and important for clinical translation. However, genetically engineering hPSCs for long-term overexpression of proteins remains inefficient, labor-intensive, and plagued by epigenetic silencing, necessitating dedication of significant resources, and entailing laborious workflows. To address these limitations, we report the development of XPRESSO (expedited persistent and robust engineering of stem cells with sleeping beauty for overexpression), a modular "anti-silencing" transposon vector, which we have combined with a highly efficient and accessible methodology for the rapid generation of genetically modified hPSC lines in a gene-independent manner. Using this method, we successfully generated dozens of stable hPSC lines with robust and continuous functional expression of optogenetic proteins, Cas9, shRNA, and a calcium indicator in both undifferentiated and differentiated (cardiomyocyte and neuronal) cells.
Wexler et al. (Thu,) studied this question.