Automated CRISPR Screening Using the CellRaft Technology

Project SummaryGenome wide functional genetics has been recently made possible by pooled library based CRISPR Casgene editing technologyBy randomly knocking out thousands of geneslarge scale screens can be conducted for functional geneticsdrug sensitivity and other phenotypesCurrently howeverthis forward genetic approach is largely relegated to live dead screens in mammalian cellsImproving the sophistication ofphenotypes of interestin these screens beyond live dead would significantly broaden the utility of guide RNAgRNAlibrary screensHereCell Microsystems proposes the use of its proprietary CellRaftTechnology to automate imaging based CRISPR CasscreeningThe CellRaft Technology comprises the CytoSortArraywhich containss of thousands of microwells where cells randomly distributeOnce seededcells can be can be transfected with Casand gRNA librariesAfter undergoing CRISPR Casbased gene editingcells can be screened using the AIRSysteman instrument designed to imagesort and isolate cells from the CytoSort ArrayCells exhibiting the phenotype of interest can then be collected individually for either molecular analysis or clonal propagationTo pursue these goalsCell Microsystems will collaborate with William BuchserPhD of Washington UniversityStLouisHis core facility has extensive expertise in genome wide screening and has identified the CellRaft Technology as a solution for both accelerating gRNA based screening and employing sophisticated phenotype targetsFor Phase I feasibility testingAimof the CellRaft Technology for gRNA panel screeningwe will employ a CRISPR screen against cells expressing various fluorescent proteinsCells will be transfected with Casand a pool of gRNAs against each transgenic fluorescent protein and control gRNAsThis method allows a quantitative assessment of both CRISPR Casefficiency and imaging based sorting of cells prior to isolation using the CellRaft TechnologyIn a parallel programCell Microsystems has already demonstrated successful CRISPR Casgenome editing using the CellRaft Technologywhich integrates the entire genome editing workflow including transfectionimaging based screening of cellsharvestingand clonal colony growthThese capabilities are all essential to streamlining gRNA screening methods and enable investigators to sort cells for relatively subtle phenotypesThe imaging capabilities of the AIRSystem are sufficient for both cellular morphology analysissub cellular localization of fluorescent signalsand cell cell interactions all key criteria for screening sophisticated cellular phenotypesThe proposed program also includes the development of appropriate image analysis and cell sorting software to monitor the growth of clonal coloniesTailoring several features of the CellRaft Technologyalready a staple for single cell isolation in many labswill address the currently unmet needs of large scale pooled gRNA CRISPR screening workflows Project NarrativeForward genetic screening using CRISPR Casis a powerful new toolbut it is generally limited to growth based screening of single perturbationsCell Microsystems proposes the use of the CellRaft Technology and automated AIRSystem to both accelerate CRISPR Casscreening and increase the sophistication ofphenotypes of interestbeyond live deadeven for multiple perturbationsIn addition to automated imagingthe technology allows post screening isolation of single cells for molecular analysisbiochemistry or clonal colony propagation