努南氏症(Noonan syndrome, NS)是一種體染色體顯性和遺傳異質性疾病,其特徵有身材矮小,發育遲緩和先天性心臟異常。我們已經透過外顯子體定序在努南氏症的病人裡找到 RASIP1 的突變。努南氏症病人帶有 RASIP1 的突變會有較少見的心臟缺陷表現型,像是主動脈根部擴張症、主動脈縮窄、大動脈瘤。這些表現型有可能只發生在帶有 RASIP1 突變的努南氏症病人身上。為了研究 RASIP1 突變而導致努南氏症的分子機制,我們的目標在透過基因組編輯產生帶有努南氏症 RASIP1 突變誘導性多功能幹細胞(iPS細胞)。因為iPS細胞有能力分化成多種心血管細胞,因此我們可以透過iPS細胞的分化來研究突變在功能上的影響。先前的研究已經成功的運用群聚且有規律間隔的短迴文重複序列(CRISPR)系統在iPS細胞上。然而 CRISPR 系統在iPS細胞所造成的基因敲入之效率只有2~3%。在這個研究裡,我目標在改善iPS細胞同源性定向修復(Homology directed repair, HDR)的頻率並產生帶有病人突變。我用RNA定序的方式在iPS細胞建立雙股DNA斷裂相關基因的表現量檔案。此外也做出了雙股DNA模板並送到人胚胎腎細胞293(Human Embryonic Kidney Cells 293）和iPS細胞，也比較了單股模板和雙股模板HDR的效率。此外，我用了RASIP1敲除的iPS細胞分化到不同心血管細胞，發現RASIP1敲除的iPS細胞分化能力和功能下降，磷酸化的GSK3β表現量也下降。

Noonan syndrome (NS), an autosomal dominant and genetically heterogeneous disorder, is characterized by short stature, developmental delay and congenital heart diseases. Previously, we identified candidate NS-associated variants in RASIP1 through exome sequencing. Patients carrying RASIP1 variants showed atypical cardiac phenotypes for NS, such as dilation of aortic root, coarctation of aorta and aortic aneurysms, which raise the possibility that these cardiac phenotypes occurred specifically to NS patients carrying RASIP1. To investigate the molecular mechanisms through which RASIP1 mutations lead to NS, we aim to generate cell models that carry patient-specific RASIP1 mutations through genome editing in the induced pluripotent stem cells (iPSCs). Because iPSCs have the ability to differentiate into many cell lineages of cardiovascular cells, we can study the impact of mutations on the function of cardiovascular cells. Previous studies have successfully utilized CRISPR system in iPSCs, however, the efficiency of CRISPR/Cas9 mediated knock-in was only 2-3% in iPSCs. In this study, I aim to improve the homology-direct repair (HDR) rates in iPSCs and generate iPSCs carrying patient-specific mutations. I used RNA-sequencing (RNA-seq) to establish double strand break-related gene profile in iPSCs. I also generated a reporter system in iPSCs. In addition, I compared the HDR rate in cells supplied with long dsDNA donor and that with single strand oligodeoxy nucleotides. Moreover, I differentiated RASIP1 knock-out (RKO) iPSCs into cardiac cell lineages and found that the differentiation ability of RKO iPSCs was decreased. The ability of forming plexus like vascular structures was also decrease. Furthermore, results of RNA-seq suggested that several signaling pathways were altered in RKO cells. Lastly, I found that phosphorylated-GSK3β level was altered in RKO cells. Taken together, these results revealed the possible mechanisms through which RASIP1 mutation affect the cardiovascular cell differentiation and function.

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