Research

Development of site-specific genome integration system  site specific illu

CRISPR/Cas9 technology for genome editing offers great potentials for research, biotechnology, and clinical applications. Gene therapy is one major application to which CRISPR/Cas9 can significantly contribute. It is based on the genetic modification of patient’s cells, and can be achieved by correcting nucleotide mutations by homologous recombination or by inserting a copy of the therapeutic gene to restore proper expression and function of such damaged gene. To expand the tool kits based on CRISPR/Cas9system, we have recently developed a new method for stable transgenesis, named as “DirectIntegrate” system. This system is based on the reaction of retroviral integrase. We accordingly designed vectors, which is gateway cloning compatible, containing truncated LTR sequences that flank the desired transgene. The linearized cassette, or the transgene fragment, contains no viral components and will be inserted into the host genome with the help of integrase. We demonstrated this system is highly efficient to perform transgenesis both in mammalian cells and vertebrate animal, such as zebrafish. Importantly, given that most of the viral components have been removed, this system diminishes the safety concerns from other viral vectors, while retaining the high efficiency of transgene integration. Based on this system, we further develop a novel method for site-specific transgenesis in aim to be used in clinical applications. This new system uses the engineered LEDGF/p75 fused with dCas9 to tether integrase complex for site-directed gene insertion. With the promising preliminary results, our lab is currently revise the design and improve the overall efficiency of this method.

 

Gene functions in vascular development

Our lab has a long-term interests in the gene regulation for vascular development. We have identified several genes which may play important roles to regulate the proliferation and apoptosis of endothelial cells during the development. One particular gene is, Psip1, the gene codes LEDGF/p75, has been implicated in cancer, autoimmune diseases and HIV pathogenesis. For its role in HIV replication, psip1 tethers and targets HIV integrase to the bodies of active genes, which makes it a novel target for anti-HIV treatment. However, the basic biology of psip1 is not fully elucidated. Our results show that psip1a is essential for vascular development in zebrafish. Knockdown or knockout of psip1a caused structure disruption in the caudal vein plexus suggesting endothelial cell death. Interestingly, RNA-seq data revealed psip1a is a major regulator of p53 signaling. Our data showed that psip1a directly associates to the alternative p53 promoter and potentially regulates delta-113p53 expression to antagonize p53 pathway and exhibited an anti-apoptotic effect. We anticipate our study to provide a new insight into the function of psip1 in vascular development.