Infinitesimal, LLC SBIR Phase I Proposal: Project Summary Induced pluripotent stem cells (iPSCs) have become a prominent model for the understanding of genetic diseases. The introduction of specific point mutations using the CRISPR/Cas9 system in iPSCs having the same genetic background results in isogenic cell lines that are invaluable to study disease mechanisms and for future personalized therapies. However, current methods for iPSC gene editing and cell line generation based on bulk electroporation34 require a lot of manual steps, are lengthy, have poor efficiency, require large amount of starting material of both cells and transfectant, and are not amenable to multiplexing. Therefore, new and automated multiplexed systems that will enable unprecedented levels of gene editing and monoclonal isogenic cell line generation by dramatically reducing the cost per cell line as a result of automation, transfection precision, and minute amounts of reagent necesary per experimental condition are highly needed. To achieve this we propose the creation of the CellFabTM system, an integrated platform that will enable high- throughput single-cell transfection of iPSCs using a 96-well plate format. After transfection and growth, each colony, resulting from a single iPSC per well, will be easily picked for screening and expansion. The CellFabTM system will include an inverted microscope with fluorescence and phase-contrast capabilities, a motorized stage and an image recognition algorithm for automated localization of individual iPSCs plated in a 96-well plate. Cells will be transfected automatically using a vertical glass micropipette, containing the transfectant and a platinum electrode, and a proprietary contact detection algorithm. In Phase II, the system will include automated micropipette change and cleaning of the pipette housing and embedded electrode. The specific aims for this proposal are the following: (1) Design and fabrication of the integrated system for high-throughput single cell electroporation. Image recognition software and contact algorithm will allow automated cell localization and transfection using a vertically-positioned glass micropipette. (2) Generation of iPSC isogenic cell lines using the newly fabricated instrument. Two gene-engineering strategies, commonly used in cell line generation, will be assayed and compared: one using the CRISP/Cas9 system to knock-out and knock-in a specific gene, and the other integrating a reporter gene employing a plasmid DNA. Generated cell lines will be expanded for analysis and the precision and efficiency of the CellFabTM system will be evaluated. The successful completion of these aims will lead to the first integrated system for high-throughput isogenic cell line generation, with multiplexed gene editing, for disease modeling and pharmacological studies. This system is designed to interface with commercially available multiplexed systems, allowing automation of the whole procedure, from dispensing to subculture.