2020-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/654624癌症是多基因突變的疾病。利用CRISPR/Cas9基因編修技術在體細胞進行突變的方法為新的癌症動物模式產生方法。之前，本實驗室開發了Inducible Cas9 Effector/CRIspr Mutagen（ICE CRIM）的策略，並利用該策略在Trp53被破壞的情況下觀察到如果DNA錯配修復機制同時發生問題會導致造血系統腫瘤的加速惡化。在本計畫中我們希望延續先前的成果，將ICE CRIM應用到大腸直腸癌小鼠模式相關研究，藉以討論MSI標的基因及腫瘤分子機制在人與小鼠間的差別。其次是希望將一個傾向出錯的DNA聚合酶系統帶入小鼠中，該系統可在基因體中產生核苷酸鹼基取代導致的點突變，其中包含錯義突變，這類突變在一般CRISPR/Cas9破壞時非同源末端連接（NHEJ）引起的插入/缺失中幾乎找不到，因此可以與當前所用之體細胞突變技術互補。若能將此技術引進哺乳動物細胞，將對於許多不同病患腫瘤中重複出現的顯性、增能、致癌或造成抗藥性之錯義突變的相關研究有幫助。最後，我們也希望引進其他Cas9變異體，來對此系統進行修改，期許能更進一步拓展其可標的的基因體區域以增加其應用時的彈性。 Cancer is a multigenic disease. CRISPR/Cas9-mediated somatic mutagenesis is a new way to generate cancer animal models. In the past, our laboratory developed an Inducible Cas9 Effector/CRIspr Mutagen (ICE CRIM) strategy, which proved DNA mismatch repair (MMR) deficiency can accelerate hematopoietic maliganacy in Trp53-deficient mice. This project includes three parts. Firstly we want to continue the previous achievement and further applied the ICE CRIM strategy for colorectal cancer model related research focusing on the discussion of microsatellite instability (MSI)-target conservation between human and murine. Secondly, we want to bring an error-prone DNA polymerase system (EvolvR) causing nucleotide base substitution resulting in point (missense) mutations into the mouse. This subtype of mutations is rarely seen in CRISPR-disrupted, nonhomologus end joining (NHEJ)-caused, indels, which can compensate the current strategies applied in mouse somatic mutagenesis. This new strategy will be useful for in vivo screenings on dominant, gain-of-function, oncogenic, or drug-resistant mutants in the mammalian cells. Thirdly we want to incorporate the Cas9 variants to the EvolvR system and aim to expand the target genomic territory for the system that it will be even more flexible for the future experiments.CRISPR/Cas9體細胞突變微衛星不穩定性錯義突變癌症somatic mutagenesismicrosatellite instabilitymissense mutationcancer