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 the biotechnology industry. However, stably integrated transgene is subject to epigenetic silencing, which represents a major bottleneck in this field. Numerous regulatory elements (e.g., cHS4, UCOE, S/MARs) have been adopted to overcome transgene silencing, yet they are associated with various limitations such as reduced viral titer and context-dependent activity. Therefore, there are continuing efforts to discover novel anti-silencing elements with better functionalities. 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 together (CRISPR and serine integrase) to achieve targeted insertion of large DNA fragments (5-10kb) at highly compact heterochromatin loci with high efficiency. Compared with the classical barrier assay which takes up to 40~60 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 feature of SHIELD, polyclonal cells can be directly used for downstream analysis, circumventing the need for clonal isolation or the control of integrant copy number. Using SHIELD, we demonstrated the screening of >20 candidates from in silico prediction and identified 3 elements with activity equal or higher than the positive control. Taken together, we anticipate the SHIELD platform would accelerate the discovery of novel anti-silencing DNA elements as well as the functional assessment of engineered variants in human cells.