KSEF RDE: Development of Hyperactive DNA Transposases by Directed Evolution

Transposons are a proven, efficient and cost-effective technology for genome modification. Transposon technology has great commercial potential in areas such as gene therapy, therapeutic protein production, and transgenesis. However, in order for transposon technology to be commercially viable in these areas, it will be necessary to create “hyperactive” transposases, enzymes that can mobilize transposons with extreme efficiency. We propose to apply directed evolution (DE) technology to create hyperactive transposases. DE comprises two discrete components: first, genetic diversity is created through the production of a library of genetic variants, and second, the library is evaluated through genetic selection and/or high-throughput screens to identify variants with the desired function(s). DE differs from natural evolution in two key aspects: i) DE is performed under controlled selection pressure for predetermined functions and ii) in DE, “non-natural” functions of practical use may be derived through appropriate selection schemes. Therefore, DE is a powerful tool that is ideal for the creation of hyperactive transposases. For this proposed project we will engineer a newly characterized transponsase, PiggyBac (PB). One major advantage of using PB for transgenesis is that it can carry a very large cargo, able to easily deliver sequences in the 10-15 kB. High quality random or targeted mutant libraries will be generated and screened using flow cytometry-based transposition assays. Selected variants will serve as parents for subsequent rounds of evolution in which positive mutations will be recombined. This project promotes and capitalizes on interactions and collaborations between scientists from University of Kentucky and a Kentucky-based biotech company, Transposagen Biopharmaceuticals, Inc. One of the research focuses of the PI’s is the DE of DNA-binding proteins, while Transposagen is a company commercializing mobile DNA technologies and a leader in the functional manipulation of transposons including PB. The breadth of interests of the collaborative team provides experience and expertise in areas including protein engineering by DE, high throughput screening, and functional characterization of hyperactive transposases.