当前医学生物学领域的核心问题之一，是如何高通量、高效率地确定在特定生理、病理等情况下发挥关键作用的基因产物和通路。最近几年基因打靶技术，特别是ZFN、TALEN、CRISPR/Cas9系统等的出现和发展为科研人员研究高等生物的基因功能、生理病理机制提供极大方便。我们前期在TALE相关技术包括重复模块组装及新DNA碱基识别RVD等方面有许多积累，在利用CRISPR/Cas9系统构建基于基因敲除的功能性筛选平台也有突破性进展（Nature 2014）。我们计划对真核基因定点修饰技术进行优化，特别是建立更加高效的全基因组真核基因敲除文库用于高通量功能性筛选。我们将主要利用这一新型强大的正向遗传筛选平台研究艰难梭菌毒素蛋白A及B的宿主细胞靶位点，以及和丙型肝炎病毒入胞以及在细胞间传递途径中起重要作用的宿主蛋白。真核基因敲除特别是高通量功能性筛选的应用，必将极大地推动广泛的生物医学相关领域的发展。

One of the core questions in the field of biomedical research is the effective and massive association of genes’ function and certain biological, developmental or disease-related process. The recent emergence of gene targeting technology in eukaryotes, especially ZFN, TALENs and the CRSIPR/Cas9 systems have enabled researchers across broad-spectrum of fields to study gene and its function in an unprecedented fashion and efficiency. We have been involved in TALE-related technique development, i.e. the fast assembly methodology of TALE arrays and the complete decoding of TALE RVDs for DNA recognition preference (PLoS One, 2013; Cell Research 2014). We have recently generated a CRISPR/Cas9-mediated gene knockout library in human cells, and developed the complete strategy for functional genomics using this powerful platform (Nature 2014). We propose to upgrade the gene targeting technology, especially the development of the whole genome gene knockout library in eukaryotic cells through refined CRISPR/Cas9 system. With the establishment of the collection of sgRNA libraries, we are determined to screen for host targets essential for the toxicity of both TcdA and TcdB of Clostridium difficile, as well as for the host cell surface proteins important for HCV infection, especially for viral entry and cell-to-cell transmission. The broad application of CRISPR/Cas9 library in functional genomics will undoubtedly promote the fast progress of biological and biomedical research studies.