Kan, Y. | Search bioRxiv

bioRxiv2019

Extensive Mammalian Germline Genome Engineering

Xenotransplantation, specifically the use of porcine organs for human transplantation, has long been sought after as an alternative for patients suffering from organ failure. However, clinical application of this approach has been impeded by two main hurdles: 1) risk of transmission of porcine endogenous retroviruses (PERVs) and 2) molecular incompatibilities between donor pigs and humans which culminate in rejection of the graft. We previously demonstrated that all 25 copies of the PERV elements in the pig genome could be inactivated and live pigs successfully generated. In this study, we improved the scale of porcine germline editing from targeting a single repetitive locus with CRISPR to engineering 13 different genes using multiple genome engineering methods. we engineered the pig genome at 42 alleles using CRISPR-Cas9 and transposon and produced PERVKO{middle dot}3KO{middle dot}9TG pigs which carry PERV inactivation, xeno-antigen KO and 9 effective human transgenes. The engineered pigs exhibit normal physiology, fertility, and germline transmission of the edited alleles. In vitro assays demonstrated that these pigs gain significant resistance to human humoral and cell mediated damage, and coagulation dysregulations, similar to that of allotransplantation. Successful creation of PERVKO{middle dot}3KO{middle dot}9TG pigs represents a significant step forward towards safe and effective porcine xenotransplantation, which also represents a synthetic biology accomplishment of engineering novel functions in a living organism. One Sentence SummaryExtensive genome engineering is applied to modify pigs for safe and immune compatible organs for human transplantation

synthetic biology↗

bioRxiv2018

Temporal modulation of host aerobic glycolysis determines the outcome of M. marinum infection

Macrophages are the first-line host defense where the invading Mycobacterium tuberculosis (Mtb) encounters. It has been recently reported that host aerobic glycolysis was elevated post the infection by a couple of virulent mycobacterial species. However, whether this metabolic transition is required for host defense against intracellular pathogens and the underlying mechanisms remain to be further investigated. By analyzing carbon metabolism, we found that macrophages infected by M. marinum, a surrogate mycobacterial specie to Mtb, showed a strong elevation of glycolysis. Next, three glycolysis inhibitors were examined for their ability to inhibit mycobacterial proliferation inside RAW264.7, a murine macrophage-like cell line. Among them, a glucose analog, 2-deoxyglucose (2-DG) displayed a protective effect on assisting host to resist mycobacterial infection, which was further validated in zebrafish-infection model. The phagocytosis of M. marinum was significantly decreased in macrophages pre-treated with 2-DG at concentrations of 0.5 and 1 mM, at which no inhibitory effect was posed on M. marinum growth in vitro. Moreover, 2-DG pre-treatment exerted a significant protective effect on zebrafish larvae to limit the proliferation of M. marinum, and such effect was correlated to tumor necrosis factor alpha (TNF-). On the contrary, the 2-DG treatment post infection did not restrain proliferation of M. marinum in WT zebrafish, and even accelerated bacterial replication in TNF--/- zebrafish. Together, modulation of glycolysis prior to infection boosts host immunity against M. marinum infection, indicating a potential intervention strategy to control mycobacterial infection.\n\nAuthor SummaryAs an intracellular pathogen, Mtb exploits multiple strategies to invade and hijack macrophages for its own advantages. Accordingly, recent investigations have shown that Mtb infection is accompanied with an alteration of host glucose metabolism. Macrophage and zebrafish infection models of M. marinum, facilitating our understanding towards mycobacterial pathogenesis, were applied in this study. We found that the pre-treatment of macrophages with a glucose analog, 2-DG, inhibited aerobic glycolysis and made host cells more inert to phagocytose the bud. In infected zebrafish larvae, bacterial load inside host pretreated with 2-DG remains at a significantly lower level compared to the untreated group. These findings imply that the modulation of host glycolysis regulates the fate of M. marinum infection, and indicate a promising metabolic target in TB intervention.

microbiology↗