(234b) Shield: A Platform for Fast and Simple Screening of Anti-Silencing DNA Elements in Human Cells

Transgene expression in mammalian cells plays an essential role in both fundamental research and biotechnology industry. However, stably integrated transgene is subject to epigenetic silencing, which represents a major bottleneck in this field. Numerous DNA elements have been identified to overcome transgene silencing, yet the choice is limited. Therefore, there remains a great need to discover novel elements with better functionality. Here we report SHIELD (Site-specific Heterochromatin Insertion of DNA Elements at Lamina-associated Domains), a platform for fast and simple screening of anti-silencing DNA elements in human cells. SHIELD combines two distinct genome engineering technologies (CRISPR and serine integrase) to achieve targeted insertion of large DNA fragments (5-10 kb) at highly compact heterochromatin loci with high efficiency. Compared with the classical barrier assay which takes up to 60 days to fully observe the silencing effect, SHIELD expedites the process by placing the reporter cassette at a highly repressive heterochromatin locus, thus significantly shortening the time frame to ~14 days. Moreover, due to the high efficiency and single-copy insertion nature of SHIELD, polyclonal cells can be directly used for analysis, thus circumventing the tedious clonal isolation step as required by classical barrier assay. Using SHIELD, we demonstrated the screening of >20 candidates from previous in silico predictions and identified several promising elements with activity equal or higher than the positive control. Taken together, we anticipate the SHIELD platform would greatly accelerate the discovery of novel anti-silencing DNA elements in human cells.