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.