title: Conservative site-specific and single-copy transgenesis in human LINE-1 elements.
abstract: Genome engineering of human cells plays an important 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 λ 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, term edattH4X, found in 1000 human Long INterspersed Elements-1 (LINE-1). We demonstrate single-copy transgenesis through attH4X-targeting in various cell lines including hESCs, with the flexibility of selecting clones according to transgene performance and downstream applications. This is exemplified with pluripotency reporter cassettes and constitutively expressed payloads 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 localized 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.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551.
authors.forename: Shree Harsha
authors.initials: SH
authors.lastname: Vijaya Chandra
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551.
authors.forename: Harshyaa
authors.initials: H
authors.lastname: Makhija
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Peter
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551.
authors.forename: Cho Mar
authors.initials: CM
authors.lastname: Myint Wai
authors.affiliation: Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Tonghe GuangZhou 510515, People's Republic of China State Key Laboratory of Organ Failure Research, Division of Nephrology, Nanfang Hospital, Tonghe, Guangzhou 510515, People's Republic of China.
authors.forename: Jinming
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Tonghe GuangZhou 510515, People's Republic of China State Key Laboratory of Organ Failure Research, Division of Nephrology, Nanfang Hospital, Tonghe, Guangzhou 510515, People's Republic of China.
authors.forename: Jindong
authors.initials: J
authors.lastname: Zhu
authors.affiliation: Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Tonghe GuangZhou 510515, People's Republic of China State Key Laboratory of Organ Failure Research, Division of Nephrology, Nanfang Hospital, Tonghe, Guangzhou 510515, People's Republic of China.
authors.forename: Zhonglu
authors.initials: Z
authors.lastname: Ren
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551.
authors.forename: Martina Stagno
authors.initials: MS
authors.lastname: D'Alcontres
authors.affiliation: p53Lab, Agency for Science Technology and Research, Singapore 138673.
authors.forename: Jia Wei
authors.initials: JW
authors.lastname: Siau
authors.affiliation: p53Lab, Agency for Science Technology and Research, Singapore 138673.
authors.forename: Sharon
authors.initials: S
authors.lastname: Chee
authors.affiliation: p53Lab, Agency for Science Technology and Research, Singapore 138673.
authors.forename: Farid John
authors.initials: FJ
authors.lastname: Ghadessy
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 pdroge@ntu.edu.sg.
authors.forename: Peter
authors.initials: P
authors.lastname: Dröge
mesh_terms: D010582:Bacteriophage lambda / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D045744:Cell Line, Tumor; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D015894:Genome, Human; D006801:Humans; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D020084:Long Interspersed Nucleotide Elements; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D019076:Transgenes; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D014764:Viral Proteins; D019426:Integrases