Methods for the intracellular delivery of gene editing tools

The goal of the summer research project is to develop methods for the co-delivery of gene editing enzymes and guide RNAs into mammalian cells. The research position will involve the engineering of gene editing enzymes to promote electrostatic interactions with synthetic polyelectrolytes. Specifically, the enzymes will be “supercharged” through the introduction of a charged polypeptide tag. These engineered enzymes will be produced in E. coli and purified using standard protein purification techniques. The self-assembly of the engineered enzymes with synthetic block copolymers will be characterized by dynamic light scattering and transmission electron microscopy as a function of protein concentration, solution pH and ionic strength, and competing biomolecules. Following successful demonstration of the formation of well-defined micelles, the ability of the micelles to enter cells and deliver the gene editing tools to the nucleus will be evaluated. Finally, the activity of the delivered enzyme will be tested using a fluorescent reporter assay to evaluate gene editing efficiency.