Development of Site-Specific Recombinase Technology for Targeted Integration with Marker Removal

Recombinase-mediated genetic engineering provides a favorable direction for enhancing the precision of biotechnological approaches. Technology is rapidly expanding the way genetic engineering can be accomplished, the questions asked and the applications that can be attained. With these possibilities in genetic manipulation the interest in metabolic engineering for products such as Astaxanthin or Tocopherols that requires the addition of many genes into the host cell for proper expression has been renewed. However, the combination of required genes and control elements is often empirically determined and thus requires multiple rounds DNA manipulation. While genetic engineering can be accomplished by nuclease or host cell mediated homologous recombination, the efficiencies for precisely inserting large DNA sequences is low and tends to contain errors. Recombinase-mediated engineering offers a solution for sequential rounds of DNA stacking at high rates of integration and low (to nonexistent) levels of error introduction. Another issue is the limitation of available selectable markers for multiple rounds of engineering. Recombinase technology, when properly designed allows the removal of selection marker genes from the system while integrating the next gene(s) of interest. This in turn allows marker gene recycling between successive rounds of DNA insertion. Due to concerns over the presence of antibiotic resistance genes in the food supply and their escape into the environment, the ability to efficiently remove marker genes prior to marketing or release is highly desirable. Previous studies have documented how site-specific recombination can produce transgenics with stable gene expression over multiple generations and also resolve multicopy transgene inserts, initially silenced for expression, to a single functional genomic copy. Research in this lab addresses the need for publicly available recombinase technology. Our team has developed a series of novel recombinases and strategies for precise genetic manipulation. Data for a practical combinatorial approach for transgene integration and DNA stacking combined with subsequent excision of the selectable marker will be presented.