Shree Harsha Vijaya Chandra, Harshyaa Makhija, Sabrina Peter, Cho Mar Myint Wai, Jinming Li, Jindong Zhu, Zhonglu Ren, Martina Stagno D'Alcontres, Jia Wei Siau, Sharon Chee, Farid John Ghadessy, Peter Dröge, Conservative site-specific and single-copy transgenesis in human LINE-1 elements, Nucleic Acids Research, Volume 44, Issue 6, 7 April 2016, Page e55, https://doi.org/10.1093/nar/gkv1345
Genome engineering of human cells plays an impor- tant role in biotechnology and molecular medicine. In particular, insertions of functional multi-transgene cassettes into suitable endogenous sequences will lead to novel applications. Although several tools have been exploited in this context, safety issues such as cytotoxicity, insertional mutagenesis and off-target cleavage together with limitations in cargo size/expression often compromise utility. Phage integrase (Int) is a transgenesis tool that mediates conservative site-specific integration of 48 kb DNA into a safe harbor site of the bacterial genome. Here, we show that an Int variant precisely recombines large episomes into a sequence, termed attH4X, found in 1000 human Long INterspersed Elements-1 (LINE-1). We demonstrate single-copy transgenesis through attH4X-targeting in various cell lines includ- ing hESCs, with the flexibility of selecting clones ac- cording to transgene performance and downstream applications. This is exemplified with pluripotency reporter cassettes and constitutively expressed pay- loads that remain functional in LINE1-targeted hESCs and differentiated progenies. Furthermore, LINE-1 targeting does not induce DNA damage-response or chromosomal aberrations, and neither global nor lo- calized endogenous gene expression is substantially affected. Hence, this simple transgene addition tool should become particularly useful for applications that require engineering of the human genome with multi-transgenes.
This work was supported by Exploit Pte Ltd [ETPL/11-R15COT-0006]; Nanyang Technological University PhD
Scholarship [to H.M.]; Singapore Ministry of Education Academic Research Fund Tier 3 [MOE2012-T3-1-001]; National Natural Science Foundation of China [31371290 to J.L.]; Southern Medical University (to J.L.). Funding for open access charge: Singapore Ministry of Education Academic Research Fund Tier 3 [MOE2012-T3-1-001].