ShCAST技術可不造成DNA雙股斷裂地，將大片段DNA嵌入細菌的目標基因體，可應用於過去難以編輯的菌株。然而ShCAST具有特定位點嵌入效率低、脫靶、共嵌入等三種問題，嚴重影響其應用性。在本研究第一部分，我們透過增加Cas12k基因表達、降低培養溫度、增加Re-streak次數，開發出SHOT基因編輯技術，SHOT解決了ShCAST固有的三項問題，能高效率的將多達14.5 kb的DNA片段，嵌入細菌基因體中。本研究利用SHOT將pyc基因嵌入於大腸桿菌adhE基因增加琥珀酸產量，驗證了SHOT在基因體工程、代謝工程上的應用潛力。桿狀病毒是良好的基因遞送載體，能遞送DNA至多種哺乳動物細胞，現有編輯桿狀病毒的工具極少，因此在第二部份的研究中，我們嘗試以SHOT編輯DH10Bac菌株中的桿狀病毒人工染色體 (Bacmid)，然而最初的SHOT只能編輯28%的Bacmid，為了增強SHOT編輯能力，我們調整了質體的套數以及sgRNA表達量，發展了SHOT2.0技術。SHOT2.0可以嵌入高達14 kb的DNA至Bacmid，且編輯單一菌落內所有的Bacmid。SHOT2.0還可以跟商業化的Bac-to-Bac基因編輯技術共同協作，將兩種DNA片段嵌於單病毒載體中，我們利用SHOT2.0與Bac-to-Bac系統，建構出攜帶完整Prime editing系統的單病毒載體，並以此單病毒高效率的編輯人類細胞。
12-羥基十二烷酸 (HDA) 是合成長碳鏈尼龍的原料之一，目前台灣尚未有合成HDA的生物製程，因此第三部分中，我們選擇維斯假絲酵母菌株做為生物轉化平台，並利用CRISPR/Cas9基因編輯技術破壞消耗12-羥基十二烷酸 (HDA) 的關鍵基因，促使HDA產量增加，為生物轉化製程奠定基礎。

ShCAST can insert large DNA into the target genome of bacteria without causing double-strand DNA breaks. It can be applied to difficult-to-edit strains. However, ShCAST has three issues: low insertion efficiency in some target site, off-target effects, and co-integration. These issues seriously affect its applicability. In the first part of this study, we developed the SHOT method by increasing Cas12k gene expression, reducing editing temperature, and combining Re-streak. SHOT overcomes the inherent problems of ShCAST and efficiently inserts DNA fragments up to 14.5 kb into bacterial genomes. Using SHOT, we successfully inserted the pyc gene into the E. coli adhE loci to increase succinate production, demonstrating the potential of SHOT in genome engineering and metabolic engineering. Baculovirus are promising gene delivery vectors capable of delivering large DNA into various mammalian cells. However, there are limited tools available for editing baculovirus. Therefore, in the second part of this study, we attempted to edit the baculovirus artificial chromosome (Bacmid) in the DH10Bac strain using SHOT. Initially, SHOT could only edit 28% of the Bacmid. To enhance the editing efficiency of SHOT, we adjusted the plasmid copy numbers and sgRNA expression levels, resulting in SHOT2.0 system. SHOT2.0 can insert DNA up to 14 kb into Bacmid and edit all the Bacmids within a single colony. SHOT2.0 can also work orthogonally with the commercial Bac-to-Bac system to insert two kind of DNA fragments into a single Bacmid. Using SHOT2.0 and the Bac-to-Bac system, we constructed a single baculovirus carrying the whole Prime editing system and used it to efficiently edit human genome. 12-hydroxydodecanoic acid (HDA) is one of the materials for producing long-chain nylon. Currently, there is no bioprocess for synthesizing HDA in Taiwan. Therefore, in the third part of our study, we chose the Candida viswanathii ATCC20962 strain as the biotransformation platform and used CRISPR/Cas9 to disrupt key genes involved in HDA consumption, thereby enhancing HDA production and building the foundation for a HDA biotransformation process.

摘要--------------------------- I
Abstract----------------------- II
致謝--------------------------- III
目錄--------------------------- V
圖表目錄----------------------- VIII
第一章 文獻回顧----------------- 1
1-1大腸桿菌應用與基因工程-------- 1
1-2 CAST基因編輯技術------------ 2
1-3重組桿狀病毒之應用與製備------ 4
1-4 Prime editing基因編輯技術--- 6
1-5 12-羥基十二酸簡介----------- 8
1-6生產羥基脂肪酸之生物轉化法---- 8
1-7轉化羥基脂肪酸相關代謝途徑---- 9
1-8假絲酵母基因工程------------- 10
1-9研究動機--------------------- 11
第二章 材料與方法--------------- 19
2-1 實驗用大腸桿菌菌種培養------- 19
2-2 試劑配置-------------------- 20
2-3 質體建構-------------------- 21
2-4 以SHOT進行基因編輯---------- 24
2-5 重組桿狀病毒的製備與轉導----- 25
2-6 qRT-PCR、qPCR與dPCR定量分析- 26
2-7 以Prime editing編輯人類細胞- 26
2-8 製作維斯假絲酵母勝任細胞----- 27
2-9 以電穿孔將DNA轉型至酵母中---- 28
2-10 去除維斯假絲酵母中的質體---- 28
2-11 搖瓶生產HDA---------------- 29
2-12 分析產物HDA與DDA產量------- 29
2-13 數據處理與統計分析---------- 30
第三章 實驗結果與討論(1)-開發SHOT應用於代謝工程----37
3-1 評估ShCAST在各種菌株中的效能與缺點---- 37
3-1-1 ShCAST可在多種菌株中作用----------- 37
3-1-2 ShCAST有嚴重共嵌入及脫靶效應------- 38
3-2 建立優化的 ShCAST (SHOT)------------ 39
3-2-1 提升Cas12k表達與降低溫度增加靶向效率 39
3-2-2 全基因體測序分析編輯成果----------- 41
3-3 SHOT系統可以高效的嵌入大於14.5kb的DNA 41
3-4 用SHOT 改良菌株以提高琥珀酸產量------ 42
3-5 討論-開發SHOT應用於代謝工程--------- 43
第四章 實驗結果與討論(2)-開發SHOT2.0編輯桿狀病毒-- 47
4-1 以SHOT 編輯重組桿狀病毒------------- 47
4-2 建立SHOT2.0系統增加Bacmid編輯效率---- 48
4-2-1 篩選出最佳SHOT編輯系統------------- 49
4-2-2 分析各種SHOT質體套數與sgRNA表達---- 50
4-2-3 利用Re-streak完全編輯菌落中的所有Bacmid---- 51
4-2-4 利用SHOT2.0嵌入14kb大片段DNA------ 52
4-3 結合SHOT2.0與Bac-to-Bac製備重組桿狀病毒------ 52
4-4 利用SHOT2.0建立更穩定的病毒--------- 53
4-5 建立與測試all-in-one Prime editing病毒------ 54
4-6討論-開發SHOT2.0編輯桿狀病毒--------- 55
第五章 實驗結果與討論(3)-編輯維斯假斯酵母菌生產HDA- 59
5-1 建立CRISPR破壞目標基因技術---------- 59
5-2利用CRISPR破壞基因使HDA累積---------- 59
5-3討論-編輯維斯假斯酵母菌促進HDA生產---- 60
第六章 結語與未來展望-------------------- 62
第七章 參考資料------------------------- 82
附錄1 經費來源與研究分工----------------- 89

Airenne KJ, Hu YC, Kost TA, Smith RH, Kotin RM, Ono C, Matsuura Y, Wang S, Yla-Herttuala S. 2013. Baculovirus: an insect-derived vector for diverse gene transfer applications. Mol Ther 21: 739-749.
Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, Chen PJ, Wilson C, Newby GA, Raguram A, Liu DR. 2019. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576: 149-157.
Brinkman EK, Chen T, Amendola M, van Steensel B. 2014. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res 42: e168.
Bulaklak K, Gersbach CA. 2020. The once and future gene therapy. Nat Commun 11: 5820.
Chang CW, Huang JW, Lu YH, Pham NN, Tu J, Tung YT, Yen CY, Tu Y, Shen CC, Chien MC, Lin YH, Yang SW, Nguyen MTT, Pham DH, Hu YC. 2023. Metabolic engineering of Escherichia coli to enhance protein production by coupling ShCAST-based optimized transposon system and CRISPR interference. Journal of the Taiwan Institute of Chemical Engineers 144.
Chen CL, Wu JC, Chen GY, Yuan PH, Tseng YW, Li KC, Hwang SM, Hu YC. 2015. Baculovirus-mediated miRNA regulation to suppress hepatocellular carcinoma tumorigenicity and metastasis. Mol Ther 23: 79-88.
Chen PJ, Hussmann JA, Yan J, Knipping F, Ravisankar P, Chen PF, Chen C, Nelson JW, Newby GA, Sahin M, Osborn MJ, Weissman JS, Adamson B, Liu DR. 2021. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184: 5635-5652 e5629.
Chen W, Ren ZH, Tang N, Chai G, Zhang H, Zhang Y, Ma J, Wu Z, Shen X, Huang X, Luo GZ, Ji Q. 2021. Targeted genetic screening in bacteria with a Cas12k-guided transposase. Cell Rep 36: 109635.
Chen X, Zhou L, Tian K, Kumar A, Singh S, Prior BA, Wang Z. 2013. Metabolic engineering of Escherichia coli: a sustainable industrial platform for bio-based chemical production. Biotechnol Adv 31: 1200-1223.
Cheng ZH, Wu J, Liu JQ, Min D, Liu DF, Li WW, Yu HQ. 2022. Repurposing CRISPR RNA-guided integrases system for one-step, efficient genomic integration of ultra-long DNA sequences. Nucleic Acids Res 50: 7739-7750.
Chung ME, Yeh IH, Sung LY, Wu MY, Chao YP, Ng IS, Hu YC. 2017. Enhanced integration of large DNA into E. coli chromosome by CRISPR/Cas9. Biotechnol Bioeng 114: 172-183.
Clark DP. 1989. The fermentation pathways of Escherichia coli. FEMS Microbiol Rev 5: 223-234.
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. 2013. Multiplex genome engineering using CRISPR/Cas systems. Science 339: 819-823.
Eirich LD, Craft DL, Steinberg L, Asif A, Eschenfeldt WH, Stols L, Donnelly MI, Wilson CR. 2004. Cloning and characterization of three fatty alcohol oxidase genes from Candida tropicalis strain ATCC 20336. Appl Environ Microbiol 70: 4872-4879.
Ennis CL, Hernday AD, Nobile CJ. 2021. A Markerless CRISPR-Mediated System for Genome Editing in Candida auris Reveals a Conserved Role for Cas5 in the Caspofungin Response. Microbiol Spectr 9: e0182021.
Felberbaum RS. 2015. The baculovirus expression vector system: A commercial manufacturing platform for viral vaccines and gene therapy vectors. Biotechnol J 10: 702-714.
Friehs K. 2004. Plasmid copy number and plasmid stability. Adv Biochem Eng Biotechnol 86: 47-82.
Gatter M, Forster A, Bar K, Winter M, Otto C, Petzsch P, Jezkova M, Bahr K, Pfeiffer M, Matthaus F, Barth G. 2014. A newly identified fatty alcohol oxidase gene is mainly responsible for the oxidation of long-chain omega-hydroxy fatty acids in Yarrowia lipolytica. Fems Yeast Research 14: 858-872.
Ghosh S, Parvez MK, Banerjee K, Sarin SK, Hasnain SE. 2002. Baculovirus as mammalian cell expression vector for gene therapy: an emerging strategy. Mol Ther 6: 5-11.
He Q, Bennett GN, San KY, Wu H. 2019. Biosynthesis of Medium-Chain omega-Hydroxy Fatty Acids by AlkBGT of Pseudomonas putida GPo1 With Native FadL in Engineered Escherichia coli. Front Bioeng Biotechnol 7: 273.
Ho YC, Lee HP, Hwang SM, Lo WH, Chen HC, Chung CK, Hu YC. 2006. Baculovirus transduction of human mesenchymal stem cell-derived progenitor cells: variation of transgene expression with cellular differentiation states. Gene Ther 13: 1471-1479.
Hsu MN, Liao HT, Li KC, Chen HH, Yen TC, Makarevich P, Parfyonova Y, Hu YC. 2017. Adipose-derived stem cell sheets functionalized by hybrid baculovirus for prolonged GDNF expression and improved nerve regeneration. Biomaterials 140: 189-200.
Ibraheim R, Song CQ, Mir A, Amrani N, Xue W, Sontheimer EJ. 2018. All-in-one adeno-associated virus delivery and genome editing by Neisseria meningitidis Cas9 in vivo. Genome Biol 19: 137.
Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. 2013. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31: 233-239.
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. 2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337: 816-821.
Kim SK, Park YC. 2019. Biosynthesis of omega-hydroxy fatty acids and related chemicals from natural fatty acids by recombinant Escherichia coli. Appl Microbiol Biotechnol 103: 191-199.
Klompe SE, Vo PLH, Halpin-Healy TS, Sternberg SH. 2019. Transposon-encoded CRISPR-Cas systems direct RNA-guided DNA integration. Nature 571: 219-225.
Kluesner MG, Nedveck DA, Lahr WS, Garbe JR, Abrahante JE, Webber BR, Moriarity BS. 2018. EditR: A Method to Quantify Base Editing from Sanger Sequencing. CRISPR J 1: 239-250.
Lee H, Sugiharto YEC, Lee H, Jeon W, Ahn J, Lee H. 2019. Biotransformation of dicarboxylic acids from vegetable oil-derived sources: current methods and suggestions for improvement. Appl Microbiol Biotechnol 103: 1545-1555.
Lin MW, Tseng YW, Shen CC, Hsu MN, Hwu JR, Chang CW, Yeh CJ, Chou MY, Wu JC, Hu YC. 2018. Synthetic switch-based baculovirus for transgene expression control and selective killing of hepatocellular carcinoma cells. Nucleic Acids Res 46: e93.
Liu C, Liu F, Cai J, Xie W, Long TE, Turner SR, Lyons A, Gross RA. 2011. Polymers from fatty acids: poly(omega-hydroxyl tetradecanoic acid) synthesis and physico-mechanical studies. Biomacromolecules 12: 3291-3298.
Lu WH, Ness JE, Xie WC, Zhang XY, Minshull J, Gross RA. 2010. Biosynthesis of Monomers for Plastics from Renewable Oils. Journal of the American Chemical Society 132: 15451-15455.
Luckow VA, Lee SC, Barry GF, Olins PO. 1993. Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J Virol 67: 4566-4579.
Ma W, Xu YS, Sun XM, Huang H. 2021. Transposon-Associated CRISPR-Cas System: A Powerful DNA Insertion Tool. Trends Microbiol 29: 565-568.
Makkonen KE, Airenne K, Yla-Herttulala S. 2015. Baculovirus-mediated gene delivery and RNAi applications. Viruses 7: 2099-2125.
Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. 2013. RNA-guided human genome engineering via Cas9. Science 339: 823-826.
Nguyen N, Quail MMF, Hernday AD. 2017. An Efficient, Rapid, and Recyclable System for CRISPR-Mediated Genome Editing in Candida albicans. Msphere 2.
Park JU, Tsai AW, Mehrotra E, Petassi MT, Hsieh SC, Ke A, Peters JE, Kellogg EH. 2021. Structural basis for target site selection in RNA-guided DNA transposition systems. Science 373: 768-774.
Pham NN, Chang CW, Chang YH, Tu Y, Chou JY, Wang HY, Hu YC. 2023. Rational genome and metabolic engineering of Candida viswanathii by split CRISPR to produce hundred grams of dodecanedioic acid. Metab Eng 77: 76-88.
Picataggio S, Rohrer T, Deanda K, Lanning D, Reynolds R, Mielenz J, Eirich LD. 1992. Metabolic engineering of Candida tropicalis for the production of long-chain dicarboxylic acids. Biotechnology (N Y) 10: 894-898.
Pijlman GP, Grose C, Hick TAH, Breukink HE, van den Braak R, Abbo SR, Geertsema C, van Oers MM, Martens DE, Esposito D. 2020. Relocation of the attTn7 Transgene Insertion Site in Bacmid DNA Enhances Baculovirus Genome Stability and Recombinant Protein Expression in Insect Cells. Viruses 12.
Porto EM, Komor AC, Slaymaker IM, Yeo GW. 2020. Base editing: advances and therapeutic opportunities. Nat Rev Drug Discov 19: 839-859.
Querques I, Schmitz M, Oberli S, Chanez C, Jinek M. 2021. Target site selection and remodelling by type V CRISPR-transposon systems. Nature 599: 497-502.
Rakowski SA, Filutowicz M. 2013. Plasmid R6K replication control. Plasmid 69: 231-242.
Regev A, Rivkin H, Gurevitz M, Chejanovsky N. 2006. New measures of insecticidal efficacy and safety obtained with the 39K promoter of a recombinant baculovirus. FEBS Lett 580: 6777-6782.
Saleski TE, Chung MT, Carruthers DN, Khasbaatar A, Kurabayashi K, Lin XN. 2021. Optimized gene expression from bacterial chromosome by high-throughput integration and screening. Sci Adv 7.
Selas Castineiras T, Williams SG, Hitchcock AG, Smith DC. 2018. E. coli strain engineering for the production of advanced biopharmaceutical products. FEMS Microbiol Lett 365.
Shapiro RS, Chavez A, Porter CBM, Hamblin M, Kaas CS, DiCarlo JE, Zeng G, Xu X, Revtovich AV, Kirienko NV, Wang Y, Church GM, Collins JJ. 2018. A CRISPR-Cas9-based gene drive platform for genetic interaction analysis in Candida albicans. Nat Microbiol 3: 73-82.
Snoeckx RL, Huygen PL, Feldmann D, Marlin S, Denoyelle F, Waligora J, Mueller-Malesinska M, Pollak A, Ploski R, Murgia A, Orzan E, Castorina P, Ambrosetti U, Nowakowska-Szyrwinska E, Bal J, Wiszniewski W, Janecke AR, Nekahm-Heis D, Seeman P, Bendova O, Kenna MA, Frangulov A, Rehm HL, Tekin M, Incesulu A, Dahl HH, du Sart D, Jenkins L, Lucas D, Bitner-Glindzicz M, Avraham KB, Brownstein Z, del Castillo I, Moreno F, Blin N, Pfister M, Sziklai I, Toth T, Kelley PM, Cohn ES, Van Maldergem L, Hilbert P, Roux AF, Mondain M, Hoefsloot LH, Cremers CW, Lopponen T, Lopponen H, Parving A, Gronskov K, Schrijver I, Roberson J, Gualandi F, Martini A, Lina-Granade G, Pallares-Ruiz N, Correia C, Fialho G, Cryns K, Hilgert N, Van de Heyning P, Nishimura CJ, Smith RJ, Van Camp G. 2005. GJB2 mutations and degree of hearing loss: a multicenter study. Am J Hum Genet 77: 945-957.
Song CW, Lee J, Lee SY. 2015. Genome engineering and gene expression control for bacterial strain development. Biotechnol J 10: 56-68.
Stemmer WP, Morris SK. 1992. Enzymatic inverse PCR: a restriction site independent, single-fragment method for high-efficiency, site-directed mutagenesis. Biotechniques 13: 214-220.
Strecker J, Ladha A, Gardner Z, Schmid-Burgk JL, Makarova KS, Koonin EV, Zhang F. 2019. RNA-guided DNA insertion with CRISPR-associated transposases. Science 365: 48-53.
Sung LY, Chen CL, Lin SY, Li KC, Yeh CL, Chen GY, Lin CY, Hu YC. 2014. Efficient gene delivery into cell lines and stem cells using baculovirus. Nat Protoc 9: 1882-1899.
Sung LY, Wu MY, Lin MW, Hsu MN, Truong VA, Shen CC, Tu Y, Hwang KY, Tu AP, Chang YH, Hu YC. 2019. Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli. Biotechnol Bioeng 116: 1066-1079.
Thakker C, Zhu J, San KY, Bennett G. 2011. Heterologous pyc gene expression under various natural and engineered promoters in Escherichia coli for improved succinate production. J Biotechnol 155: 236-243.
Thompson MG, Sedaghatian N, Barajas JF, Wehrs M, Bailey CB, Kaplan N, Hillson NJ, Mukhopadhyay A, Keasling JD. 2018. Isolation and characterization of novel mutations in the pSC101 origin that increase copy number. Sci Rep 8: 1590.
Truong VA, Hsu MN, Kieu Nguyen NT, Lin MW, Shen CC, Lin CY, Hu YC. 2019. CRISPRai for simultaneous gene activation and inhibition to promote stem cell chondrogenesis and calvarial bone regeneration. Nucleic Acids Res 47: e74.
Truong VA, Hsu MN, Nguyen NTK, Lin MW, Shen CC, Lin CY, Hu YC. 2019. CRISPRai for simultaneous gene activation and inhibition to promote stem cell chondrogenesis and calvarial bone regeneration. Nucleic Acids Research 47.
Tsutsui H, Matsubara K. 1981. Replication control and switch-off function as observed with a mini-F factor plasmid. J Bacteriol 147: 509-516.
Uthayakumar D, Sharma J, Wensing L, Shapiro RS. 2020. CRISPR-Based Genetic Manipulation of Candida Species: Historical Perspectives and Current Approaches. Front Genome Ed 2: 606281.
van Loo ND, Fortunati E, Ehlert E, Rabelink M, Grosveld F, Scholte BJ. 2001. Baculovirus infection of nondividing mammalian cells: mechanisms of entry and nuclear transport of capsids. J Virol 75: 961-970.
van Oers MM. 2011. Opportunities and challenges for the baculovirus expression system. J Invertebr Pathol 107 Suppl: S3-15.
Vo PLH, Acree C, Smith ML, Sternberg SH. 2021. Unbiased profiling of CRISPR RNA-guided transposition products by long-read sequencing. Mob DNA 12: 13.
Vo PLH, Ronda C, Klompe SE, Chen EE, Acree C, Wang HH, Sternberg SH. 2021. CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering. Nat Biotechnol 39: 480-489.
Vyas VK, Bushkin GG, Bernstein DA, Getz MA, Sewastianik M, Barrasa MI, Bartel DP, Fink GR. 2018. New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi. mSphere 3.
Wan LL, Wang ZR, Tang M, Hong DF, Sun YH, Ren J, Zhang N, Zeng HX. 2021. CRISPR-Cas9 Gene Editing for Fruit and Vegetable Crops: Strategies and Prospects. Horticulturae 7.
Wang J, Peng J, Fan H, Xiu X, Xue L, Wang L, Su J, Yang X, Wang R. 2018. Development of mazF-based markerless genome editing system and metabolic pathway engineering in Candida tropicalis for producing long-chain dicarboxylic acids. J Ind Microbiol Biotechnol 45: 971-981.
Wessels HH, Mendez-Mancilla A, Guo X, Legut M, Daniloski Z, Sanjana NE. 2020. Massively parallel Cas13 screens reveal principles for guide RNA design. Nat Biotechnol 38: 722-727.
Wu MY, Sung LY, Li H, Huang CH, Hu YC. 2017. Combining CRISPR and CRISPRi Systems for Metabolic Engineering of E. coli and 1,4-BDO Biosynthesis. ACS Synth Biol 6: 2350-2361.
Xiao K, Yue XH, Chen WC, Zhou XR, Wang L, Xu L, Huang FH, Wan X. 2018. Metabolic Engineering for Enhanced Medium Chain Omega Hydroxy Fatty Acid Production in Escherichia coli. Front Microbiol 9: 139.
Yang C, Aslan H, Zhang P, Zhu S, Xiao Y, Chen L, Khan N, Boesen T, Wang Y, Liu Y, Wang L, Sun Y, Feng Y, Besenbacher F, Zhao F, Yu M. 2020. Carbon dots-fed Shewanella oneidensis MR-1 for bioelectricity enhancement. Nat Commun 11: 1379.
Yarnall MTN, Ioannidi EI, Schmitt-Ulms C, Krajeski RN, Lim J, Villiger L, Zhou W, Jiang K, Garushyants SK, Roberts N, Zhang L, Vakulskas CA, Walker JA, 2nd, Kadina AP, Zepeda AE, Holden K, Ma H, Xie J, Gao G, Foquet L, Bial G, Donnelly SK, Miyata Y, Radiloff DR, Henderson JM, Ujita A, Abudayyeh OO, Gootenberg JS. 2023. Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases. Nat Biotechnol 41: 500-512.
Zhang L, Zhang H, Liu Y, Zhou J, Shen W, Liu L, Li Q, Chen X. 2020. A CRISPR-Cas9 system for multiple genome editing and pathway assembly in Candida tropicalis. Biotechnol Bioeng 117: 531-542.
Zhang X, Zhao K, Lan L, Shi N, Nan H, Shi Y, Xu X, Chen H. 2021. Improvement of protein production by engineering a novel antiapoptotic baculovirus vector to suppress the expression of Sf-caspase-1 and Tn-caspase-1. Biotechnol Bioeng 118: 2977-2989.
呂祐萱. 2021. 在大腸桿菌中利用CRISPR基因編輯系統建立低產酸發酵平台. 國立清華大學, 新竹市.
李奎璋. 2015. 桿狀病毒改質間葉幹細胞的臨床前安全性評估及利用桿狀病毒調控MicroRNA Sponges修復骨質疏鬆骨缺陷模型. 國立清華大學, 新竹市.
范玉南. 2021. 以CRISPR基因編輯技術改質微生物用於高值化二元酸生產. 國立清華大學, 新竹市.
黃靖雯. 2021. 在大腸桿菌BL21(DE3)菌株中優化ShCAST基因編輯系統與其應用. 國立清華大學, 新竹市.
董彥孜. 2023. 利用CRISPR/Cas13d抑制關鍵基因促進ω-羥基十二烷酸產量. 國立清華大學, 新竹市.
顏佳怡. 2022. 以基因改質微生物生物合成ω-羥基十二烷酸. 國立清華大學, 新竹市.