Abstract

Viral-based systems are a popular delivery method for introducing exogenous genetic material (e.g., plasmids or shRNA) into mammalian cells. In particular, virus-like particles have shown to be efficient, in packaging large vectors and complexes (e.g., Cas 9) and in their delivery into cells via entry mechanisms of an enveloped virus particle. Unfortunately, the preparation and packaging of virus-based particles containing the machinery to edit the cells is labour-intensive, with many steps requiring optimization and sensitive handling. Furthermore, following packaging, is delivering the viral particles efficiently into the desired cell line, which can vary significantly between cell lines since different cells uptake the virus at different rates. In recognition to these challenges, we introduce the first microfluidic method that integrates lentiviral generation, packaging, and transduction. The new method allows for the production of viral titers between 10^6-10^7 (similar to macroscale production) and high transduction efficiency for hard-to-transfect cell lines. To extend the technique to be useful for gene-editing applications, we show how this technique can be used to knockout and knockdown estrogen receptor gene – a gene prominently responsible for 70% of breast cancer cases. This new technique is automated with multiplexing capabilities, which have the potential to standardize the methods for viral-based genome engineering.