Abstract
Safety and reliability of transgene integration in human genome continue to pose challenges for stem cell-based gene therapy. Here, we report a baculovirus-transcription activator-like effector nuclease system for AAVS1 locus-directed homologous recombination in human induced pluripotent stem cells (iPSCs). This viral system, when optimized in human U87 cells, provided a targeted integration efficiency of 95.21% in incorporating a Neo-eGFP cassette and was able to mediate integration of DNA insert up to 13.5 kb. In iPSCs, targeted integration with persistent transgene expression was achieved without compromising genomic stability. The modified iPSCs continued to express stem cell pluripotency markers and maintained the ability to differentiate into three germ lineages in derived embryoid bodies. Using a baculovirus-Cre/LoxP system in the iPSCs, the Neo-eGFP cassette at the AAVS1 locus could be replaced by a Hygro-mCherry cassette, demonstrating the feasibility of cassette exchange. Moreover, as assessed by measuring γ-H2AX expression levels, genome toxicity associated with chromosomal double-strand breaks was not detectable after transduction with moderate doses of baculoviral vectors expressing transcription activator-like effector nucleases. Given high targeted integration efficiency, flexibility in transgene exchange and low genome toxicity, our baculoviral transduction-based approach offers great potential and attractive option for precise genetic manipulation in human pluripotent stem cells.
Highlights
Human pluripotent stem cells, as accessible and renewable human cell sources, have great prospective applications in developmental biology research, drug development, regenerative medicine and gene therapy
To confirm the TALENinduced cleavage at the endogenous target AAVS1 site, the mismatch-sensitive nuclease assay using the T7E1 endonuclease was performed on genomic DNA from HEK293 cells transfected with transcription activator-like effector nuclease (TALEN)-expressing plasmid vectors
TALEN cleavage rate was not quantified, the intensity of the small fragments from the cells transfected with pFB-TALEN, an expression vector in which the two TALEN arms were inserted into one plasmid vector, appeared higher than that from the cells co-transfected with two plasmid vectors expressing left and right TALEN separately
Summary
As accessible and renewable human cell sources, have great prospective applications in developmental biology research, drug development, regenerative medicine and gene therapy. To realize the full potential of these stem cells, efficient and safe methods are required for precise genetic manipulation without affecting their self-renewal and differentiation capacities. Given low efficiency of conventional HR in mammalian cells [1], zinc finger nuclease (ZFN)-induced DNA double-strand breaks (DSBs) have been used to stimulate endogenous HR machinery [2]. Nucleases based on transcriptional activator-like effectors (TALEs) have rapidly emerged as an alternative to ZFNs [3]. TALEs are bacterial proteins characterized by a highly conserved central domain of tandem repeats that mediate binding to DNA. The 12th residue of a unit stabilizes the structural domain of protein backbone, whereas the 13th
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