Megabase delivery via engineered endosymbionts

Multiple technologies have been developed to deliver DNA into mammalian cells. However, they are all of limited size capacity with deliverable DNA ranging in length from a few kb to ~100 kb. Our inability to deliver very large synthetic DNA molecules (1 mb) to mammalian cells is a key technological barrier to genome-scale engineering. To solve this challenge, we aim to develop a novel method for directly delivering megabase scale synthetic DNA – assembled in E. coli – into the nucleus of target mammalian cells utilizing an engineered endosymbiont approach.

Existing delivery strategies have critical limitations. Despite high efficiency, virus transduction is limited by the low foreign DNA packing capacity of viral particles (~10 kb). Direct microinjection passes DNA through a very fine needle (tip diameter ~0.5 µm) into the target mammalian cell or nucleus. Physical shearing caused by pushing DNA through a small aperture limits the size of deliverable DNA molecule that can withstand this process (e.g. 100-kb BAC can be broken by manual pipetting with tip diameter ~1 mm). Chemical and physical methods such as lipofection, calcium phosphate co-precipitation or DEAE-dextran mediated transfection, particle bombardment (i.e. gene gun), electroporation, and laserfection facilitate the crossing of DNA through the cell membrane into the cytoplasm, but the DNA has to subsequently translocate into the nucleus in a separate step. The overall success rate is the product of each step's individual success rate. Consequently, this fundamentally limits the overall efficiency of DNA delivery into the nucleus by these methods. The delivery efficiency also quickly decreases as the size of transfected DNA gets bigger, with the reported upper limit ~100 kb (typically much lower in a few kb range). Delivery of larger DNA would be fundamentally challenging due to the intrinsic limits of these methods and the difficulty in handling purified DNA 1 mb without physical shearing. Cell fusion is capable of transferring megabase scale DNA, artificial chromosomes, or even an entire wildtype chromosome from the inside of one cell to another. Nonetheless, cell fusion requires DNA to initially be inside a mammalian cell and thus still faces the aforementioned delivery challenges.

To solve the delivery challenge, we propose to develop an engineered endosymbiotic bacterial delivery system that can effectively enter the cytoplasm of the mammalian cell to deliver megabase scale DNA pre-assembled inside the bacterium. By performing megabase assembly and subsequent delivery from the same bacterial cell, the synthetic DNA never needs to be purified out of the cell into its naked form during the delivery process. Thus, the synthetic DNA is always nicely wrapped and packaged inside the shuttle endosymbiotic bacteria, sheltered away from chemical and/or physical damages. This strategy thus eliminates the intrinsic barrier of physical shearing for very large DNA beyond 100 kb, which is a critical hurdle in all currently available methods. This novel design should thus be capable of unprecedented size capacity at megabase scale or higher.