Fonsecaea monophora聚酮合酶基因的CRISPR-Cas9敲除载体的构建

中国真菌学杂志　 2021, Vol. 16 　Issue (5): 303-307,313.

Fonsecaea monophora聚酮合酶基因的CRISPR-Cas9敲除载体的构建

李敏英¹, 黄欢¹, 李倩¹, 骆明芬¹, 王晓悦¹, 刘红芳¹, 曾维英¹, 席丽艳^1,2

1. 南方医科大学皮肤病医院, 实验研究中心, 广州 510091;
2. 中山大学孙逸仙纪念医院皮肤科, 广州 510120

收稿日期:2020-01-25 发布日期:2021-10-28
通讯作者: 席丽艳,E-mail:xiliyan@mail.sysu.edu.cn E-mail:xiliyan@mail.sysu.edu.cn
作者简介:李敏英,女(汉族),硕士,实习研究员.E-mail:1138800144@qq.com
基金资助:
国家自然科学基金（81873960）；国自然青年科学基金（81601746）；广东省医学科学技术研究基金（A2019345）

Construction of CRISPR-Cas9 knockout vector of Fonsecaea monophora polyketide synthase gene

LI Minying¹, HUANG Huan¹, LI Qian¹, LUO Mingfen¹, WANG Xiaoyue¹, LIU Hongfang¹, ZENG Weiying¹, XI Liyan^1,2

1. Dermatology Hospital, Southern Medical University, Experimental Research Center, Guangzhou 510091, China;
2. Department of Dermatology, SunYat-Sen Hospital, SunYat-Sen University, Guangzhou 510120, China

Received:2020-01-25 Published:2021-10-28

摘要/Abstract

摘要： 目的改造pFC332质粒，简化实验流程，构建Fonsecaea monophora PKS1基因的CRISPR-Cas9敲除载体。方法设计引物PCR扩增pFC334质粒上的gRNA骨架，获得已经插入AarⅠ酶切位点的gRNA骨架片段后，使用PstI和MerI酶线性化pFC334载体，构建pFC334-AarⅠ载体，使用MreⅠ和BglⅡ酶对其和pFC332质粒进行双酶切，连接后获得pFC332-gRNA-AarⅠ载体，设计gRNA，最后将各片段插入到载体中。结果测序得到16533bp大小的改造后的pFC332-gRNA-AarⅠ质粒，构建了PKS1基因的CRISPR-Cas9敲除载体，通过PCR，酶切以及测序的方法确定敲除载体构建成功。结论成功构建Fonsecaea monophora聚酮合酶基因的CRISPR-Cas9敲除载体，为研究PKS1基因及DHN-黑素在Fonsecaea monophora的生物学功能奠定了良好的基础。

关键词: Fonsecaea monophora, 聚酮合酶基因, CRISPR-Cas9, 载体构建

Abstract: Objective To construct a Fonsecaea monophora polyketide synthase 1 (PKS1) gene knock-out vector by CRISPR/Cas9 system with pFC332 plasmid. Methods Primers of the gRNA skeleton fragment from pFC334 plasmid were designed, and then the gRNA skeleton fragment inserted with the AarⅠ restriction site was amplified by PCR. After that, the pFC334 plasmid was linearized with PstⅠ and MerI enzymes and a pFC334-AarⅠ vector was constructed. Subsequently, both pFC334-AarⅠ vector and pFC332 plasmid were digested with MreI and BglII. Digested products were ligated together in order to produce a novel recombinant vector called pFC332-gRNA-AarⅠ, with gRNA skeleton fragment. Finally, multiple gRNAs targeting PKS1 were designed and integrated into the pFC332-gRNA-AarⅠ vector, respectively. Results The obtained pFC332-gRNA-AarⅠ plasmid was 16533bp long, identified by sequencing. PCR, enzyme digestion and sequencing were applied to indicate that the PKS1 gene knock-out vector by CRISPR/Cas9 system was successfully constructed. Conclusion The Fonsecaea monophora polyketide synthase 1 (PKS1) gene knock-out vector by CRISPR/Cas9 system was successfully constructed, which laid a good foundation for the research of PKS1 gene and the biological functions of DHN-melanin in Fonsecaea monophora.

Key words: Fonsecaea monophora, polyketide synthase gene, CRISPR/Cas9, vector construction

中图分类号:

R379.9

李敏英, 黄欢, 李倩, 骆明芬, 王晓悦, 刘红芳, 曾维英, 席丽艳. Fonsecaea monophora聚酮合酶基因的CRISPR-Cas9敲除载体的构建[J]. 中国真菌学杂志, 2021, 16(5): 303-307,313.

LI Minying, HUANG Huan, LI Qian, LUO Mingfen, WANG Xiaoyue, LIU Hongfang, ZENG Weiying, XI Liyan. Construction of CRISPR-Cas9 knockout vector of Fonsecaea monophora polyketide synthase gene[J]. Chinese Journal of Mycology, 2021, 16(5): 303-307,313.

参考文献

[1] Cunha MM, Franzen AJ, Alviano DS, et al. Inhibition of melanin synthesis pathway by tricyclazole increases susceptibility of Fonsecaea pedrosoi against mouse macrophages[J]. Microsc Res Tech, 2005, 68(6):377-384.
[2] Franzen AJ, Cunha MM, Batista EJ, et al. Effects of tricyclazole (5-methyl-1, 2, 4-triazol benzothiazole), a specific DHN-melanin inhibitor, on the morphology of Fonsecaea pedrosoi conidia and sclerotic cells[J]. Microsc Res Tech, 2006, 69(9):729-737.
[3] Santos AL, Palmeira VF, Rozental S, et al. Biology and pathogenesis of Fonsecaea pedrosoi, the major etiologic agent of chromoblastomycosis[J]. FEMS Microbiol Rev, 2007, 31(5):570-591.
[4] Farbiarz SR, de Carvalho TU, Alviano C, et al. Inhibitory effect of melanin on the interaction of Fonsecaea pedrosoi with mammalian cells in vitro[J]. J Med Vet Mycol, 1992(4):265-273.
[5] Nosanchuk JD, Rosas AL, Casadevall A. The antibody response to fungal melanin in mice[J]. J Immunol, 1998, 160(12):6026-6031.
[6] Polak A, Dixon DM. Loss of melanin in Wangiella dermatitidis does not result in greater susceptibility to antifungal agents.[J]. Antimicrob Agents Chemother, 1989, 33(9):1639-1640.
[7] van de Sande WW, de Kat J, Coppens J, et al. Melanin biosynthesis in Madurella mycetomatis and its effect on susceptibility to itraconazole and ketoconazole[J]. Microbes Infect, 2007, 9(9):1114-1123.
[8] Sun J, Zhang J, Najafzadeh MJ, et al. Melanization of a meristematic mutant of Fonsecaea monophora increases tolerance to stress factors while no effects on antifungal susceptibility[J]. Mycopathologia, 2011, 172(5):373-380.
[9] De Hoog GS, Attili-Angelis D, Vicente VA, et al. Molecular ecology and pathogenic potential of Fonsecaea species[J]. Med Mycol, 2004, 42(5):405-416.
[10] Queiroz-Telles F, Esterre P, Perez-Blanco M, et al. Chromoblastomycosis:an overview of clinical manifestations, diagnosis and treatment[J]. Med Mycol, 2009, 47(1):3-15.
[11] Xi L, Sun J, Lu C, et al. Molecular diversity of Fonsecaea (Chaetothyriales) causing chromoblastomycosis in southern China[J]. Med Mycol, 2009, 47(1):27-33.
[12] 蒋丽, 张军民, 孙九峰, 等. Fonsecaea monophora对巨噬细胞TLR2、TLR4、Dectin-1和TNF-α表达的影响[J]. 中国真菌学杂志, 2014, 9(3):134-138.
[13] Zhang J, Wang L, Xi L, et al. Melanin in a meristematic mutant of Fonsecaea monophora inhibits the production of nitric oxide and Th1 cytokines of murine macrophages[J]. Mycopathologia, 2013, 175(5-6):515-522.
[14] Fetzner R, Seither K, Wenderoth M, et al. Alternaria alternata transcription factor CmrA controls melanization and spore development[J]. Microbiology (Reading), 2014, 160(Pt 9):1845-1854.
[15] Akoumianaki T, Kyrmizi I, Valsecchi I, et al. Aspergillus cell wall melanin blocks LC3-associated phagocytosis to promote pathogenicity[J]. Cell Host Microbe, 2016, 19(1):79-90.
[16] Nóbrega YK, Lozano VF, de Araújo TS, et al. The cell wall fraction from Fonsecaea pedrosoi stimulates production of different profiles of cytokines and nitric oxide by murine peritoneal cells in vitro[J]. Mycopathologia, 2010, 170(2):89-98.
[17] Da SM, Marques AF, Nosanchuk JD, et al. Melanin in the dimorphic fungal pathogen Paracoccidioides brasiliensis:effects on phagocytosis, intracellular resistance and drug susceptibility[J]. Microbes Infect, 2006, 8(1):197-205.
[18] Nosanchuk JD, Casadevall A. Impact of melanin on microbial virulence and clinical resistance to antimicrobial compounds[J]. Antimicrob Agents Chemother, 2006, 50(11):3519-3528.
[19] Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J]. Science, 2012, 337(6096):816-821.
[20] Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering[J]. Cell, 2014, 157(6):1262-1278.
[21] Fuller KK, Chen S, Loros JJ, et al. Development of the CRISPR/Cas9 system for targeted gene disruption in Aspergillus fumigatus[J]. Eukaryot Cell, 2015, 14(11):1073-1080.
[22] Nødvig CS, Nielsen JB, Kogle ME, et al. A CRISPR-Cas9 system for genetic engineering of filamentous fungi[J]. PLoS One, 2015, 10(7):e133085.
[23] Wenderoth M, Pinecker C, Voß B, et al. Establishment of CRISPR/Cas9 in Alternaria alternata[J]. Fungal Genet Biol, 2017, 101:55-60.
[24] Liu R, Chen L, Jiang Y, et al. Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system[J]. Cell Discov, 2015, 1:15007.
[25] Arazoe T, Ogawa T, Miyoshi K, et al. Tailor-made TALEN system for highly efficient targeted gene replacement in the rice blast fungus[J]. Biotechnol Bioeng, 2015, 112(7):1335-1342.
[26] Schuster M, Trippel C, Happel P, et al. Single and multiplexed gene editing in Ustilago maydis using CRISPR-Cas9[J]. Bio-protocol, 2018, 8(14):e2928.