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。先前研究指出，努南氏症基因突變在全身性細胞都會表現，在特定的心臟族群細胞會受到影響。此外，在努南氏症患者中，不同基因型對於心臟的表現型會以不同的形式呈現。由於誘導型多功能幹細胞（iPS細胞）有潛力分化為組成心臟系統的多種細胞，我將利用群聚且有規律間隔的短迴文重複序列(CRISPR)系統建構攜帶病患特定突變的誘導型多功能幹細胞調查努南氏症突變與其影響心臟缺陷之間的關聯性。目前CRISPR系統在真核細胞中被用來做基因體修改，並且未來會將誘導型多功能幹細胞分化成多種心肌細胞進而評估它們的生理功能。我已經構建攜帶努南氏症患者特定性突變的細胞。而經由基因組切割檢測法檢測CRISPR的作用效率。接著利用次世代定序發現CRISPR核酸酶和切口酶有專一性的切割點位。利用CRISPR與ssODN轉染到細胞中進行同源定向修復產生單個細胞群落，之後將單個細胞群落的gDNA藉由次世代定序法來確認努南氏症患者特定性突變。努南氏症患者特定性RASA2突變的穩定細胞可以成功建構出來，並且特定突變細胞會活化的RAS-ERK途徑。除此之外，在利用CRISPR編輯RASIP1突變的誘導型多功能幹細胞會影響其分化成內皮細胞的能力。總而言之，CRISPR編輯的細胞提供一個優秀的模型體系來研究努南氏症相關基因對細胞生理調節的影響。

　Noonan syndrome (NS) is an autosomal dominant and genetically heterogeneous disease, is characterized by short stature, developmental delay, and congenital cardiac abnormalities. Previous studies showed that NS mutations, although occurs in germline, only affected very specific populations of cells in the heart. Moreover, different genotypes had different manifestation on cardiac phenotypes in NS patients. Because induced pluripotent stem cells (iPSCs) have potentials to differentiate to cells in many lineages that made up the cardiac system, I aim to investigate the origin of the cardiac defects associated with specific NS mutations using iPSCs carrying patient-specific mutation generated by clustered regularly interspaced short palindromic repeats (CRISPR) system, which has been used to facilitate genome editing in eukaryotic cells, and will differentiate iPSCs into cardiac lineage cells and evaluated on their physiological functions. I have generated cells carrying NS patient-specific mutation. The efficiency of CRISPR was evaluated with Genomic cleavage detection assay. Targeted sequencing revealed specific cutting sites with CRISPR nuclease and nickase. Cells transfected with CRISPR and single strand oligo deoxy nucleotides (ssODN) for homology directed repair were allowed to form single colonies. Genomic DNAs from single colony were sequenced through next-generation sequencing (NGS) to confirm the NS patient-specific mutation. Stable cell lines carrying NS patient-specific RASA2 mutations were constructed successfully and activation of RAS-ERK pathway was confirmed in the cells. Moreover, iPSC carrying RASIP1 mutation affected the differentiation of endothelial cells. In conclusion, CRISPR engineered cells provide excellent modeling system for studying the impact of NS associated mutation in the physiological condition.

Aoki, Y., et al.
2016 Recent advances in RASopathies. J Hum Genet 61(1):33-9.
Araki, T., et al.
2009 Noonan syndrome cardiac defects are caused by PTPN11 acting in endocardium to enhance endocardial-mesenchymal transformation. Proc Natl Acad Sci U S A 106(12):4736-41.
Chen, G., et al.
2011 Chemically defined conditions for human iPSC derivation and culture. Nat Methods 8(5):424-9.
Chen, P. C., et al.
2010 Activation of multiple signaling pathways causes developmental defects in mice with a Noonan syndrome-associated Sos1 mutation. J Clin Invest 120(12):4353-65.
Chen, P. C., et al.
2014 Next-generation sequencing identifies rare variants associated with Noonan syndrome. Proc Natl Acad Sci U S A 111(31):11473-8.
Chetty, S., et al.
2013 A simple tool to improve pluripotent stem cell differentiation. Nat Methods 10(6):553-6.
Chu, V. T., et al.
2015 Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol 33(5):543-8.
Cong, L., et al.
2013 Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819-23.
Fu, Yanfang, et al.
2014 Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotech 32(3):279-284.
Gelb, B. D., and M. Tartaglia
2006 Noonan syndrome and related disorders: dysregulated RAS-mitogen activated protein kinase signal transduction. Hum Mol Genet 15 Spec No 2:R220-6.
Hou, Z., et al.
2013 Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc Natl Acad Sci U S A 110(39):15644-9.
Hsu, Patrick D., et al.
2013 DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotech 31(9):827-832.
Hunt, Michelle A., et al.
2010 Optimizing Transfection of Primary Human Umbilical Vein Endothelial Cells Using Commercially Available Chemical Transfection Reagents. Journal of Biomolecular Techniques : JBT 21(2):66-72.
Jinek, M., et al.
2012 A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816-21.
Kim, T. Y., et al.
2009 Role of DLC-1, a tumor suppressor protein with RhoGAP activity, in regulation of the cytoskeleton and cell motility. Cancer Metastasis Rev 28(1-2):77-83.
Kreitzer, F. R., et al.
2013 A robust method to derive functional neural crest cells from human pluripotent stem cells. Am J Stem Cells 2(2):119-31.
Lan, F., et al.
2013 Abnormal calcium handling properties underlie familial hypertrophic cardiomyopathy pathology in patient-specific induced pluripotent stem cells. Cell Stem Cell 12(1):101-13.
Lian, X., et al.
2015 Chemically defined, albumin-free human cardiomyocyte generation. Nat Methods 12(7):595-6.
Liu, J., et al.
2016 CRISPR/Cas9 facilitates investigation of neural circuit disease using human iPSCs: mechanism of epilepsy caused by an SCN1A loss-of-function mutation. Transl Psychiatry 6:e703.
Marraffini, Luciano A., and Erik J. Sontheimer
2010 CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nature reviews. Genetics 11(3):181-190.
Maruyama, T., et al.
2015 Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol 33(5):538-42.
Mitin, Natalia, Stephen F. Konieczny, and Elizabeth J. Taparowsky
2006 RAS and the RAIN/RasIP1 Effector. 407:322-335.
Moretti, A., et al.
2010 Patient-specific induced pluripotent stem-cell models for long-QT syndrome. N Engl J Med 363(15):1397-409.
Muller, M., et al.
2016 Streptococcus thermophilus CRISPR-Cas9 Systems Enable Specific Editing of the Human Genome. Mol Ther 24(3):636-44.
Musunuru, Kiran
2013 Genome editing of human pluripotent stem cells to generate human cellular disease models. Disease Models and Mechanisms 6(4):896-904.
Nakamura, T., et al.
2007 Mediating ERK 1/2 signaling rescues congenital heart defects in a mouse model of Noonan syndrome. J Clin Invest 117(8):2123-32.
Nathwani, A. C., et al.
1994 Efficient gene transfer into human umbilical vein endothelial cells allows functional analysis of the human tissue factor gene promoter. Br J Haematol 88(1):122-8.
Nishimasu, Hiroshi, et al.
2014 Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell 156(5):935-949.
Niu, X., et al.
2016 Combining single-strand oligodeoxynucleotides and CRISPR/Cas9 to correct gene mutations in Beta-thalassemia-induced Pluripotent Stem Cells. J Biol Chem.
Paquet, D., et al.
2016 Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533(7601):125-9.
Person, A. D., S. E. Klewer, and R. B. Runyan
2005 Cell biology of cardiac cushion development. Int Rev Cytol 243:287-335.
Ran, F. A., et al.
2013 Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8(11):2281-308.
Rath, D., et al.
2015 The CRISPR-Cas immune system: biology, mechanisms and applications. Biochimie 117:119-28.
Ratner, N., and S. J. Miller
2015 A RASopathy gene commonly mutated in cancer: the neurofibromatosis type 1 tumour suppressor. Nat Rev Cancer 15(5):290-301.
Roberts, Amy E., et al.
2013 Noonan syndrome. The Lancet 381(9863):333-342.
Romano, Alicia A., et al.
2010 Noonan Syndrome: Clinical Features, Diagnosis, and Management Guidelines. Pediatrics 126(4):746-759.
Sander, J. D., and J. K. Joung
2014 CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32(4):347-55.
Sapranauskas, R., et al.
2011 The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res 39(21):9275-82.
Schneider-Poetsch, Tilman, et al.
2010 Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin. Nat Chem Biol 6(3):209-217.
Shen, Bin, et al.
2014 Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat Meth 11(4):399-402.
Spater, D., et al.
2014 How to make a cardiomyocyte. Development 141(23):4418-31.
Staton, Carolyn A., Malcolm W. R. Reed, and Nicola J. Brown
2009 A critical analysis of current in vitro and in vivo angiogenesis assays. International Journal of Experimental Pathology 90(3):195-221.
Stewart, Rodney A., et al.
2010 Phosphatase-Dependent and -Independent Functions of Shp2 in Neural Crest Cells Underlie LEOPARD Syndrome Pathogenesis. Developmental Cell 18(5):750-762.
Stolfi, Alberto, et al.
2014 Tissue-specific genome editing in Ciona embryos by CRISPR/Cas9. Development 141(21):4115-4120.
Sun, N., et al.
2012 Patient-specific induced pluripotent stem cells as a model for familial dilated cardiomyopathy. Sci Transl Med 4(130):130ra47.
Takahashi, K., et al.
2007 Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861-72.
Tang, S., et al.
2016 Patient-Specific Induced Pluripotent Stem Cells for Disease Modeling and Phenotypic Drug Discovery. J Med Chem 59(1):2-15.
Tidyman, W. E., and K. A. Rauen
2009 The RASopathies: developmental syndromes of Ras/MAPK pathway dysregulation. Curr Opin Genet Dev 19(3):230-6.
Vissers, L. E., et al.
2015 Heterozygous germline mutations in A2ML1 are associated with a disorder clinically related to Noonan syndrome. Eur J Hum Genet 23(3):317-24.
Wang, G., et al.
2014 Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent stem cell and heart-on-chip technologies. Nat Med 20(6):616-23.
Ward, R. J., E. Alvarez-Curto, and G. Milligan
2011 Using the Flp-In T-Rex system to regulate GPCR expression. Methods Mol Biol 746:21-37.
Watanabe, K., et al.
2007 A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol 25(6):681-6.
Wilson, C. W., et al.
2013 Rasip1 regulates vertebrate vascular endothelial junction stability through Epac1-Rap1 signaling. Blood 122(22):3678-90.
Wu, Y. T., et al.
2015 Defining minimum essential factors to derive highly pure human endothelial cells from iPS/ES cells in an animal substance-free system. Sci Rep 5:9718.
Xu, K., et al.
2009 Rasip1 is required for endothelial cell motility, angiogenesis and vessel formation. Dev Biol 329(2):269-79.
Xue, W., et al.
2014 CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 514(7522):380-4.
Yamagishi, T., K. Ando, and H. Nakamura
2009 Roles of TGFbeta and BMP during valvulo-septal endocardial cushion formation. Anat Sci Int 84(3):77-87.
Yang, D., et al.
2016 Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases. Sci Rep 6:21264.
Yang, Wen-Yu, et al.
2006 Kinase domain insert containing receptor promoter controlled suicide gene system selectively kills human umbilical vein endothelial cells. World Journal of Gastroenterology : WJG 12(33):5331-5335.
Zhang, X. H., et al.
2015 Off-target Effects in CRISPR/Cas9-mediated Genome Engineering. Mol Ther Nucleic Acids 4:e264.