Section: New Results

Genome Engineering of Mammalian Cells: Targeted and Efficient Integration of Multi-unit Genetic Payloads

Participants : Grégory Batt, Xavier Duportet.

Targeted integration of multi-unit genetic payloads would greatly benefit elucidating complex cellular mechanisms and implementing new functions in mammalian cells. Current technologies are however time-consuming and require tedious post-integration controls. To address this problem, we propose a modular framework to assemble large multi-unit genetic payloads and target their integration into either one or both alleles of a chromosomal locus of choice. To achieve this, we combine in a two-step process the customizable targeting properties of homing endonucleases with the efficiency and specificity of a large serine recombinase. We have demonstrated that an optimized version of BxB1 recombinase allows the targeted integration of large genetic circuits (up to 7 transcription units, 60kb) into a preintegrated landing pad in the AAVS1 locus, with a significant increase in efficiency compared to other site-specific recombination systems (integration in 10% of transfected cells without selection) . By reducing the time and efforts to generate large populations of isogenic stable cell lines adapted to study multi-component genetic systems, our framework is a valuable tool for mammalian synthetic biology and offers great potential for a broad range of biotechnology and therapeutic applications.