Genome manipulation using l-integrase-mediated recombination

• Main Author: Harshyaa Makhija
• Other Authors: Peter Droge
• Format: Theses and Dissertations
• Language: English
• Published: 2017
• Subjects: DRNTU::Science::Biological sciences
• Online Access: http://hdl.handle.net/10356/70616
• Institution: Nanyang Technological University

Precise and safe genome engineering of mammalian cells plays an important role in biotechnology and molecular medicine. Several random (viral, transposon, plasmid-based) and site-specific (endonuclease-based) genome-editing tools have been previously employed to meet the scientific and translational needs. They could, for example, help to solve fundamental biological problems and be employed for therapeutic purposes. However, inherent safety issues such as genotoxicity, insertional mutagenesis, off-target site activities and other unforeseen risks make existing methods suboptimal for clinical applications. Hence, improved genome manipulation tools are needed. In this context, we have developed a novel site-specific transgene insertion tool for the human genome. Our system is derived from phage lambda integrase {f.. -lnt) that not only exhibits high target site specificity, but also integrates large DNA molecules into a safe harbor site of a bacterial genome. To this end, a novel, highly active lnt variant (lnt-C3) has been jointly developed with collaborators, which catalyzes site-specific transgene insertion into a particular sequence (attH4X) found in a subset of human Long INterspersed Elements (LJNE-1). We have validated our system for single-copy integration oftransgenes (~8kb) in various cell lines including human embryonic stem cells (hESCs). The integrated transgenes remain stable and functional , perhaps due to a more permissive chromatin structure at the targeted LINE-1 elements. In addition, our safety profiling data indicates that expression of tnt is safe with respect to cell toxicity and genomic integrity. Transcriptome analysis of the targeted clones revealed 20-40/20,000 (~0 . 2%) differentially expressed genes, suggesting that the global cellular RNA profile remains largely undisturbed. It has also been known that LINE-1 elements are more prevalent in ATrich, low-recombining and sparse gene regions of the genome. Thus, based on our studies, at least a subset of attH4X can be considered as putative human genome ' safe harbor sites' . Therefore, predetermined targeting using our system will reduce or even eliminate the problems associated with other currently used transgenesis tools. Our tool would thus be an addition to the existing genome editing toolbox. It will find broad applications in stem cellrelated therapies, bio-production of therapeutic proteins, CAR-T cell engineering and other human genome engineering applications that require multi-transgenes.