High-throughput single-cell transfection system for gene editing and isogenic iPS cell line generation

InfinitesimalLLCSBIR Phase I ProposalProject Summary Induced pluripotent stem cellsiPSCshave become a prominent model for the understanding of genetic diseasesThe introduction of specific point mutations using the CRISPR Cassystem in iPSCs having the same genetic background results in isogenic cell lines that are invaluable to study disease mechanisms and for future personalized therapiesHowevercurrent methods for iPSC gene editing and cell line generation based on bulk electroporationrequire a lot of manual stepsare lengthyhave poor efficiencyrequire large amount of starting material of both cells and transfectantand are not amenable to multiplexingThereforenew 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 automationtransfection precisionand minute amounts of reagent necesary per experimental condition are highly neededTo achieve this we propose the creation of the CellFabTM systeman integrated platform that will enable highthroughput single cell transfection of iPSCs using awell plate formatAfter transfection and growtheach colonyresulting from a single iPSC per wellwill be easily picked for screening and expansionThe CellFabTM system will include an inverted microscope with fluorescence and phase contrast capabilitiesa motorized stage and an image recognition algorithm for automated localization of individual iPSCs plated in awell plateCells will be transfected automatically using a vertical glass micropipettecontaining the transfectant and a platinum electrodeand a proprietary contact detection algorithmIn Phase IIthe system will include automated micropipette change and cleaning of the pipette housing and embedded electrodeThe specific aims for this proposal are the followingDesign and fabrication of the integrated system for high throughput single cell electroporationImage recognition software and contact algorithm will allow automated cell localization and transfection using a vertically positioned glass micropipetteGeneration of iPSC isogenic cell lines using the newly fabricated instrumentTwo gene engineering strategiescommonly used in cell line generationwill be assayed and comparedone using the CRISP Cassystem to knock out and knock in a specific geneand the other integrating a reporter gene employing a plasmid DNAGenerated cell lines will be expanded for analysis and the precision and efficiency of the CellFabTM system will be evaluatedThe successful completion of these aims will lead to the first integrated system for high throughput isogenic cell line generationwith multiplexed gene editingfor disease modeling and pharmacological studiesThis system is designed to interface with commercially available multiplexed systemsallowing automation of the whole procedurefrom dispensing to subculture InfinitesimalLLC SBIR Phase I ProposalProject Narrative The project is aimed at the development of the CellFabTM systeman integrated platform that will enable high throughput and automated single cell transfection of induced pluripotent stem cells and other adherent cells for biomedical studiesThe CellFabTM system is designed and engineered to achieve a paradigm shift in efficiency during multiplexed gene editingisogenic cell line generationand pharmacological studies necessary for the development of new therapiesBeing designed to interface with commercially available multiplexed systemsthe CellFabTM will allow unprecedented levels of automation of the protocols used in cell line generationfrom single cell dispensing to subculture DNA profiling of the genetically modified colonies