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Genome editing techniques have revolutionized the way that researchers study genes and their functions in eukaryotic systems and model species.Here we report the development of the whole genome CRISPR/Cas knockout library and its application in the identification of new drug targets through function-based screening and high-throughput sequencing analysis.Representative screenings will be presented, especially for the identification of host components important for Clostridium difficile infections and HCV infection.Further optimization and application of genome editing techniques will also be discussed.The application of this powerful genetic screening strategy will have broad application in the discovery of disease mechanisms and drug targets.