Abstract Electrotransfer (ET) or electroporation has been widely used to deliver molecular cargo into cells for genome and epigenome editing, cell engineering, and DNA vaccination in treatment and prevention of diseases (e.g., the COVID-19 pandemic). To enhance ET efficiency, different chemicals have been combined with electric pulses in treatment of cells. However, a challenge with this strategy is how to screen for chemicals. In most studies, the screening process is accomplished empirically in vitro, but this approach is impractical or prohibitive in vivo. Furthermore, a chemical treatment that works well for ET in cultured cells may not necessarily work for ET in the body. To this end, the proposed study will explore a new approach to chemical screening. Since mechanisms governing molecular transport are conserved across different cells and tissues, can chemical treatments that assist DNA transport be used to ubiquitously improve ET in vitro and in vivo? To answer this question, the Yuan lab will build upon their previous successes to deepen the understanding of transport mechanisms. One area of focus is to investigate cytoplasmic transport. Specifically, the study will determine mechanisms of vesicular transport and the escape of DNA from vesicles prior to nuclear entry. The second area of focus is to investigate the nuclear entry of DNA in non-dividing cells. The study will address key questions such as: How does DNA travel through the nuclear pore complex? How does the transport depend on the size and structures of DNA? How does the amount of nuclear localization signal or DNA nuclear targeting sequence per DNA molecule influence the transport? In addition to the mechanistic studies, the Yuan lab will develop new techniques to screen for nontoxic compounds and nanoparticles that can be used to improve cytoplasmic transport and nuclear entry. The third area of focus is to explore different combinations of strategies to simultaneously improve intracellular and extracellular transport of DNA in tissues. The study will demonstrate that the combination can synergistically enhance ET efficiency and prolong the transgene expression in vivo. The Yuan lab has extensive experiences in the analysis of molecular transport in cells and tissues, especially in the context of ET research. This R35 will give the lab the flexibility and power to advance the understanding of transport mechanisms, and open new avenues for research and applications. Findings from the studies mentioned above will lead to the development of common strategies to enhance DNA transport that in turn will improve ET in vitro and in vivo.