RE-GENESis · GENome Editing and delivery Strategies for REcoding the mammalian genome

GENome Editing and delivery Strategies for REcoding the mammalian genome

The last few decades have witnessed the deciphering of a large number of genomes, including those of several extinct organisms. Such progress was made possible by the development of highly efficient DNA reading and sequencing technologies. Pioneering studies used this information for DNA writing, to create synthetic genes, mitochondrial and chloroplast DNA, viral and prokaryotic genomes, opening up the age of synthetic genomics. What constitutes a minimal cellular genome? How did the genetic code originate? What is the function of the “dark genome”? The field of synthetic genomics offers the opportunity to answer fundamental questions in life development and evolution, to recreate extinct life forms (e.g. mammoths), to render precision medicine more affordable and to engineer new pharmaceuticals, vaccines and cells. Synthetic genomics has generated remarkable results in viruses and bacteria, but the de novo assembly of eukaryotic genomes is still limited to some small chromosomes, while a fully synthetic yeast genome remains to be completed. Although DNA engineering technologies have not yet been applied to large scale genome writing of mammalian cells, a few relevant examples have been reported, including the multiplex inactivation of endogenous retroviruses, potential guide lines for homology-mediated replacement of 10kbp-size genes and the humanisation of mouse immune repertoire. However, efficient delivery and genome engineering strategies for systematic genome replacement and assembly in higher eukaryotic cells are still missing. The project RE-GENESis objectives were to demonstrate the feasibility of synthetic genomics and genetic code reprogramming on the Mb-scale in mammalian cells, using mESC to identify efficient delivery vectors, genome editing strategies and recoding schemes to rewrite mammalian genomes. RE-GENESis used CRISPR-Cas in combination with large vectors to find genome surgery strategies for genome recoding and gene correction and exploit them to replace the largest cellular genes as a landmark application. RE-GENESis was a complex and interdisciplinary project combining and contributing to several areas of the life science research, including genome editing and repair, stem cell engineering and differentiation, gene therapy, mRNA processing, protein translation and folding, molecular biology and vectors engineering. While the development of the futuristic vision upon which RE-GENESis was built took longer than foreseable in a period of time heavily impacted by the pandemic, promising results have been obtained regarding the delivery of large DNA vectors their use for the engineering of the mESC genome.

Data: CORDIS, © European Union