| Summary: | 碩士 === 國立臺灣大學 === 生化科學研究所 === 107 === Natural Killer (NK) cell is an effective arsenal in immune system against malignant cells, and is highly regarded as a promising cell type for immunotherapy. Activation of NK cell is an intricate process that involves the interaction between a series of NK cell surface receptors and target cell ligands, providing positive and negative signaling to ultimately determine the activation status of NK cells. A robust and precise genetic tool will be useful to dissect this complex mechanism and to allow engineering of NK cells with enhanced cytotoxicity against malignant cells. Currently, the genetic modification of NK cells relies largely on retroviral transduction, which is time-consuming, inefficient and unsafe due to random viral integration. This thesis aimed to develop a CRISPR-Cas9 gene editing platform for NK cells. By electroporation of pre-assembled Cas9:guide RNA ribonucleoprotein (Cas9 RNP) complexes, the gene knockout process in NK-92 cell line was optimized to achieve editing efficiency up to 90% while maintaining the viability up to 80%. Cas9 RNP method also enabled site-specific knock-in of attP39B sequence, a common landing pad for phiC31-mediated recombination. Collectively, this thesis established a protocol for NK-92 genome editing that can be adopted for primary NK cells to facilitate biological research and therapeutic engineering.
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