請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/81061完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳佑宗(You-Tzung Chen) | |
| dc.contributor.author | Tzu-Chia Wang | en |
| dc.contributor.author | 王子家 | zh_TW |
| dc.date.accessioned | 2022-11-24T03:28:37Z | - |
| dc.date.available | 2022-02-18 | |
| dc.date.available | 2022-11-24T03:28:37Z | - |
| dc.date.copyright | 2022-02-18 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2022-02-08 | |
| dc.identifier.citation | 1. Platt, R. J. et al. CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling. Cell 159, 440–455 (2014). 2. An introduction to genetic analysis. https://www.cabdirect.org/cabdirect/abstract/19760126398. 3. Behringer, R., Gertsenstein, M., Nagy, K. V. Nagy, A. Manipulating the Mouse Embryo: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, 2014). 4. Dow, L. E. et al. Inducible in vivo genome editing with CRISPR-Cas9. Nat Biotechnol 33, 390–394 (2015). 5. Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S. Gregory, P. D. Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11, 636–646 (2010). 6. Joung, J. K. Sander, J. D. TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol 14, 49–55 (2013). 7. Mirgayazova, R. et al. Therapeutic Editing of the TP53 Gene: Is CRISPR/Cas9 an Option? Genes 11, 704 (2020). 8. Cong, L. et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339, 819–823 (2013). 9. Jinek, M. et al. A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science 337, 816–821 (2012). 10. Carroll, D. A CRISPR Approach to Gene Targeting. Molecular Therapy 20, 1658–1660 (2012). 11. Sapranauskas, R. et al. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Research 39, 9275–9282 (2011). 12. Wang, H. et al. One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Cell 153, 910–918 (2013). 13. Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 1–9 (2019) doi:10.1038/s41586-019-1711-4. 14. Anzalone, A. V., Koblan, L. W. Liu, D. R. Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors. Nat Biotechnol 38, 824–844 (2020). 15. Schene, I. F. et al. Prime editing for functional repair in patient-derived disease models. Nat Commun 11, 5352 (2020). 16. Jang, H. et al. Prime editing enables precise genome editing in mouse liver and retina. http://biorxiv.org/lookup/doi/10.1101/2021.01.08.425835 (2021) doi:10.1101/2021.01.08.425835. 17. Kim, Y. et al. Adenine base editing and prime editing of chemically derived hepatic progenitors rescue genetic liver disease. Cell Stem Cell S1934590921001673 (2021) doi:10.1016/j.stem.2021.04.010. 18. Liu, Y. et al. Efficient generation of mouse models with the prime editing system. Cell Discovery 6, 1–4 (2020). 19. Sürün, D. et al. Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors. Genes 11, 511 (2020). 20. Petri, K. et al. CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells. Nat Biotechnol (2021) doi:10.1038/s41587-021-00901-y. 21. Bosch, J. A., Birchak, G. Perrimon, N. Precise genome engineering in Drosophila using prime editing. 34. 22. Jin, S. et al. Genome-wide specificity of prime editors in plants. Nat Biotechnol (2021) doi:10.1038/s41587-021-00891-x. 23. Lin, Q. et al. High-efficiency prime editing with optimized, paired pegRNAs in plants. Nat Biotechnol (2021) doi:10.1038/s41587-021-00868-w. 24. Lin, Q. et al. Prime genome editing in rice and wheat. Nat Biotechnol 38, 582–585 (2020). 25. Evans, J. P. et al. From mice to men: Murine models of colorectal cancer for use in translational research. Critical Reviews in Oncology/Hematology 98, 94–105 (2016). 26. Bürtin, F., Mullins, C. S. Linnebacher, M. Mouse models of colorectal cancer: Past, present and future perspectives. WJG 26, 1394–1426 (2020). 27. Comprehensive molecular characterization of human colon and rectal cancer. Nature 487, 330–337 (2012). 28. Gatalica, Z., Vranic, S., Xiu, J., Swensen, J. Reddy, S. High microsatellite instability (MSI-H) colorectal carcinoma: a brief review of predictive biomarkers in the era of personalized medicine. Fam Cancer 15, 405–412 (2016). 29. Smith, G. et al. Mutations in APC, Kirsten-ras, and p53—alternative genetic pathways to colorectal cancer. PNAS 99, 9433–9438 (2002). 30. Rowan, A. J. et al. APC mutations in sporadic colorectal tumors: A mutational “hotspot” and interdependence of the “two hits”. PNAS 97, 3352–3357 (2000). 31. Cancer Browser. https://cancer.sanger.ac.uk/cosmic/browse/tissue?wgs=off sn=large_intestine ss=all hn=carcinoma sh=all in=t src=tissue all_data=n. 32. Imazeki, F., Omata, M., Nose, H., Ohto, M. Isono, K. p53 Gene mutations in gastric and esophageal cancers. Gastroenterology 103, 892–896 (1992). 33. Russo, A. et al. The TP53 Colorectal Cancer International Collaborative Study on the Prognostic and Predictive Significance of p53 Mutation: Influence of Tumor Site, Type of Mutation, and Adjuvant Treatment. JCO 23, 7518–7528 (2005). 34. Soussi, T. May, P. Structural Aspects of the p53 Protein in Relation to Gene Evolution: A Second Look. Journal of Molecular Biology 260, 623–637 (1996). 35. Børresen-Dale, A. L. et al. TP53 and long-term prognosis in colorectal cancer: mutations in the L3 zinc-binding domain predict poor survival. Clin Cancer Res 4, 203–210 (1998). 36. Béroud, C. Soussi, T. The UMD-p53 database: New mutations and analysis tools. Human Mutation 21, 176–181 (2003). 37. Tate, J. G. et al. COSMIC: the Catalogue Of Somatic Mutations In Cancer. Nucleic Acids Research 47, D941–D947 (2019). 38. Das, S. et al. Hzf Determines Cell Survival upon Genotoxic Stress by Modulating p53 Transactivation. Cell 130, 624–637 (2007). 39. Frebourg, T. et al. Germ-line p53 mutations in 15 families with Li-Fraumeni syndrome. Am J Hum Genet 56, 608–615 (1995). 40. Sjöblom, T. et al. The Consensus Coding Sequences of Human Breast and Colorectal Cancers. Science 314, 268–274 (2006). 41. Yoshimi, K. et al. Combi-CRISPR: combination of NHEJ and HDR provides efficient and precise plasmid-based knock-ins in mice and rats. Hum Genet 140, 277–287 (2021). 42. Kim, H. K. et al. Predicting the efficiency of prime editing guide RNAs in human cells. Nat Biotechnol 39, 198–206 (2021). 43. Kluesner, M. G. et al. EditR: A Method to Quantify Base Editing from Sanger Sequencing. The CRISPR Journal 1, 239–250 (2018). 44. Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat Biotechnol 37, 224–226 (2019). 45. Geurts, M. H. et al. Evaluating CRISPR-based Prime Editing for cancer modeling and CFTR repair in intestinal organoids. bioRxiv 2020.10.05.325837 (2020) doi:10.1101/2020.10.05.325837. 46. Chow, R. D., Chen, J. S., Shen, J. Chen, S. pegFinder: A pegRNA designer for CRISPR prime editing. 2020.05.06.081612 https://www.biorxiv.org/content/10.1101/2020.05.06.081612v1 (2020) doi:10.1101/2020.05.06.081612. 47. Morris, J. A., Rahman, J. A., Guo, X. Sanjana, N. E. Automated design of CRISPR prime editors for thousands of human pathogenic variants. 2020.05.07.083444 https://www.biorxiv.org/content/10.1101/2020.05.07.083444v1 (2020) doi:10.1101/2020.05.07.083444. 48. Hsu, J. Y. et al. PrimeDesign software for rapid and simplified design of prime editing guide RNAs. Nat Commun 12, 1034 (2021). | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/81061 | - |
| dc.description.abstract | CRISPR/Cas9系統是目前熱門的基因編修方式，這項技術於2020年贏得諾貝爾化學獎，且其不論是在各種模式動植物或臨床試驗中都被廣泛使用。而麻省理工大學David Liu 團隊改良CRISPR/Cas9系統，研發出'Prime editing (PE)'。這個新的基因編修版本，在符合一定設計條件下，能任意地進行核苷酸編修(序列置換、插入、刪除等)，不同於另一個編修版本”Base editor (BE)”，這個系統在設計上與使用上較具有彈性。與先前多種建立基因編修小鼠的方法比較，PE有潛力以更簡捷的步驟去精確地建立特定癌症小鼠模型，讓小鼠體細胞中這些特定點位突變更貼近臨床病人的基因突變狀況。因此，我在小鼠中的R26位點建立了PE2報導基因，未來希望能使用此R26R-PE2報導基因小鼠，與組織特異環化重組酶(tissue-specific cre)轉基因小鼠交配，以得到於特定組織啟動PE2編修系統的小鼠，來協助在體細胞中產生能精準模擬疾病基因突變組合的癌症模型。本論文聚焦於人類大腸直腸癌病患中的高頻突變等位基因TP53R175H所同源對應到小鼠之Trp53R172H (c.515G>A)突變，對此特定點位設計多種prime editor guideRNAs (pegRNAs)，來進行單個核苷酸G-to-A的基因編修。為進行pegRNAs之間的編修效率測試、比較，我於小鼠神經母細胞瘤細胞株與小鼠胚胎幹細胞中分別建立穩定表現PE2的系統，並透過桑格氏定序及次世代定序分析，嘗試去探討可能影響pegRNA編修效率之因素。未來希望能將效率較佳的pegRNA以腺相關病毒(AAV)載體送入小鼠特定體細胞內，以精確編修產生包含突變等位基因Trp53R172H的癌細胞，進而在不變動小鼠其他組織細胞之遺傳背景下，模擬病患疾病發生的狀況，以俾未來運用此系統去協助探討此特定突變等位基因在大腸癌症進程中所扮演的角色，抑或是藉此於活體內尋找或驗證目前尚未確立的多基因交互作用。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-24T03:28:37Z (GMT). No. of bitstreams: 1 U0001-2801202216463700.pdf: 2911366 bytes, checksum: 818eeb6ee730f790e50606387f7fe1f6 (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | "口試委員會審定書 I 致謝 II 中文摘要 III Abstract V Contents VII List of figures X List of tables XI Supplemental Information XII 1. Introduction 1 1.1 Use CRISPR/Cas9 technology to generate genetically engineered mouse model 1 1.2 Prime editing, a search-and-replace gene editing technology 3 1.3 Use prime editing to establish colorectal cancer mouse model with specific mutations 5 1.4 The role of TP53 R175H in human colorectal cancer 7 2. Materials and Methods 8 2.1 Establish PE2-expressing stable clone in Neuro-2a cell line 8 2.2 Establish PE2 system in mouse embryonic stem cells 9 2.3 Establish R26R-PE2 mice 10 2.4 Mouse genotyping 10 2.5 Protein extraction and Western blotting 11 2.6 Prime editing – pegRNA design 12 2.7 pegRNA cloning – Golden Gate Assembly 14 2.8 Cell culture 15 2.9 Transient transfection 16 2.10 Flow cytometry and cell sorting 17 2.11 Electroporate pegRNA into mouse embryonic stem cells 17 2.12 Cell genomic DNA extraction 18 2.13 Sanger sequencing 18 2.14 Analysis of Sanger sequencing data and pegRNA editing efficiency 19 2.15 Amplicon-based deep sequencing 20 3. Results 22 3.1 Design the PE2 system plasmids and validate the protein expression in cell line and mouse 22 3.2 The design and nomenclature of pegRNAs and the analyzed results from EditR 24 3.3 Repeat the pegRNF2+6 transfection to validate experimental processes. 25 3.4 The effect of PBS Tm value and GC content on editing efficiency 26 3.5 The effect of PAM site, PBS and RT template lengths on pegTrp53 editing efficiency 27 3.6 The editing efficiency of pegTrp53s in mouse embryonic stem cells 28 3.7 Use deep sequencing to analyze efficiencies of pegRNAs 29 4. Discussion 31 5. References 37 Figure 1. The overview of prime editing 44 Figure 2. Use restriction enzymes to verify the popping out event of loxP-stop- loxP cassette in pR26R-CAG-PE2 46 Figure 3. Validate PE2 system expression by Western blotting in different research models 47 Figure 4. The Southern blot evidence for R26R-PE2 mESCs and the PCR genotyping result of a R26R-PE2 mouse 49 Figure 5. The ranking of all pegTrp53s according to the times of significant editing observed in my experiment 50 Figure 6. The editing result of pegRNF2+6(15-14) in HEK293T cells 51 Figure 7. The correlations between PBS Tm value, GC content and pegRNA editing efficiency 53 Figure 8. The comparison of editing rates under different designed parameters 54 Figure 9. The editing results of Trp53 c.508G>C (p.V170L) and Trp53 c.515G>A (p.R172H) in mESCs 55 Figure 10. Deep sequencing results in HEK293T cells and mESCs 56 Table 1. R26R-PE2 mouse genotyping primers and editing target region primers 58 Table 2. Details of pegRNA designs used in this study 59 Supplemental Information 61 S1. Materials and Methods 62 S1.1 Transient transfect pegTrp53 to N2a cells and PCR target site 62 S2. Results 63 S2.1 The N2a cell editing results after the pegTrp53 transient transfection 63 S2.2 Analyze the editing results of three pegTrp53s in N2a cells 63 S2.3 Latest pegRNA design software for prime editing 64 S3. Figures 66 Figure S1. The schematic presentation of pU57-PE2 plasmid 66 Figure S2. The schematic presentation of pR26R-CAG-PE2 plasmid 67 Figure S3. The detailed oligo design for pegRNA and the final construction 68 Figure S4. The phenotype and sorting data of N2a cells after transfected with pegTrp53 and pCMV-PE2-P2A-GFP 69 S4. Tables 70 Table S1. The raw data of editing results for repeat testing in PE2-expreesing N2a cells 70 Table S3. Raw data of deep sequencing for Trp53 c.515G>A (p.R172H) in N2a PE2-expressing cells and mESCs 71 Table S2. The raw data of editing rate when targeting Trp53 (c.515 G>A) in N2a cells 73" | |
| dc.language.iso | en | |
| dc.subject | 大腸直腸癌 | zh_TW |
| dc.subject | Prime editing | zh_TW |
| dc.subject | 疾病小鼠模型 | zh_TW |
| dc.subject | pegRNA | zh_TW |
| dc.subject | disease mouse model | en |
| dc.subject | Prime editing | en |
| dc.subject | pegRNA | en |
| dc.subject | colorectal cancer | en |
| dc.title | 建構可經環化重組酶活化之PE2報導基因小鼠及優化可用於Trp53 c.515G>A (p.R172H)基因編修之pegRNA設計 | zh_TW |
| dc.title | Construction of a Cre-activable PE2 reporter mouse and optimization of pegRNA designs for Trp53 c.515G>A (p.R172H) prime editing | en |
| dc.date.schoolyear | 110-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳俊銘(Hsiu-Hsi Chen),黃祥博,游益興,周祐吉 | |
| dc.subject.keyword | Prime editing,pegRNA,疾病小鼠模型,大腸直腸癌, | zh_TW |
| dc.subject.keyword | Prime editing,pegRNA,disease mouse model,colorectal cancer, | en |
| dc.relation.page | 73 | |
| dc.identifier.doi | 10.6342/NTU202200254 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2022-02-08 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 基因體暨蛋白體醫學研究所 | zh_TW |
| 顯示於系所單位： | 基因體暨蛋白體醫學研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| U0001-2801202216463700.pdf 授權僅限NTU校內IP使用（校園外請利用VPN校外連線服務） | 2.84 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。