家蚕β-呋喃果糖苷酶基因BmSuc1的CRISPR敲除载体的构建与导入
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摘要
【目的】CRISPR/Cas9是近些年报道较多的基因编辑新工具,具有简便高效、特异性强等优势。BmSuc1是从鳞翅目经济昆虫家蚕Bombyx mori体内发现的编码β-呋喃果糖苷酶(β-fructofuranosidase,β-FFase)的基因,也是首个被克隆和鉴定的动物型β-FFase编码基因。β-FFase是作用于果糖基的蔗糖水解酶,BmSUC1可能与家蚕防御桑叶生物碱的生理过程有关,但目前其发挥作用的分子机制尚不清晰。为了解析BmSuc1在蚕体的作用途径及其生理功能,本研究基于CRISPR/Cas9基因编辑系统构建表达双元sgRNA的CRISPR载体,用于敲除家蚕基因组中的BmSuc1基因。【方法】根据目的基因BmSuc1的ORF序列设计2条sgRNA,通过同源重组的方法分别插入CRISPR载体的Sal I和Nhe I酶切位点。进而利用转基因显微注射技术将该编辑载体注入G_0代蚕卵,经催青孵化饲养家蚕并自交制备G_1代蚕种。【结果】PCR验证及测序结果均表明CRISPR编辑载体构建成功。根据Ds Red2红色蛋白的荧光标记,从G_1代家蚕幼虫中成功筛选出阳性转基因个体。【结论】本文详细介绍了表达双元sgRNA的CRISPR载体的构建方法,所获得的阳性转基因家蚕对于下一步探讨BmSuc1在家蚕糖类营养的吸收与利用途径中的作用奠定了重要的实验基础,有助于阐明蚕-桑相互选择和适应的分子机制。
[Objectives] CRISPR/Cas9 is a novel genome-editing tool that has recently had broad application in many fields of research. CRISPR/Cas9 has the advantages of simplicity, efficiency and high specificity. BmSuc1 encodes β-fructofuranosidase(β-FFase, a fructosyl-hydrolytic sucrase) in the silkworm Bombyx mori(Lepidoptera). BmSuc1 is also the first β-FFase gene to have been cloned and identified in an animal. The physiological function of BmSUC1 in the silkworm is probably related to defense against mulberry alkaloids. However, further research is needed to confirm this. In order to clarify the role of BmSUC1 we used the CRISPR/Cas9 genome-editing system to construct a dual-sgRNA expressing vector. [Methods] We first designed two sgRNAs against BmSuc1 ORF and inserted these into the Sal I and Nhe I restriction enzyme sites of a CRISPER edition vector using the homologous recombination method. We then microinjected the recombinant CRISPR plasmid into G_0 silkworm eggs and created a G_1 generation by G_0 sib-crossing. [Results] Both sgRNA insertions were verified by polymerase chain reaction and sequencing. Ds Red2 fluorescence confirmed that we successfully obtained positive transgenic G_1 individuals. [Conclusion] This study provides a detailed description of the construction of a CRISPR edition vector to express dual sgRNAs. The resultant positive transgenic silkworms will help clarify the biological role of BmSuc1-coding β-FFase in the silkworm.
[Objectives] CRISPR/Cas9 is a novel genome-editing tool that has recently had broad application in many fields of research. CRISPR/Cas9 has the advantages of simplicity, efficiency and high specificity. BmSuc1 encodes β-fructofuranosidase(β-FFase, a fructosyl-hydrolytic sucrase) in the silkworm Bombyx mori(Lepidoptera). BmSuc1 is also the first β-FFase gene to have been cloned and identified in an animal. The physiological function of BmSUC1 in the silkworm is probably related to defense against mulberry alkaloids. However, further research is needed to confirm this. In order to clarify the role of BmSUC1 we used the CRISPR/Cas9 genome-editing system to construct a dual-sgRNA expressing vector. [Methods] We first designed two sgRNAs against BmSuc1 ORF and inserted these into the Sal I and Nhe I restriction enzyme sites of a CRISPER edition vector using the homologous recombination method. We then microinjected the recombinant CRISPR plasmid into G_0 silkworm eggs and created a G_1 generation by G_0 sib-crossing. [Results] Both sgRNA insertions were verified by polymerase chain reaction and sequencing. Ds Red2 fluorescence confirmed that we successfully obtained positive transgenic G_1 individuals. [Conclusion] This study provides a detailed description of the construction of a CRISPR edition vector to express dual sgRNAs. The resultant positive transgenic silkworms will help clarify the biological role of BmSuc1-coding β-FFase in the silkworm.
引文
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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(6121):819-823.
Daimon T,Kiuchi T,Takasu Y,2014.Recent progress in genome engineering techniques in the silkworm,Bombyx mori.Dev.Growth Differ,56(1):14-25.
Daimon T,Taguchi T,Meng Y,Katsuma S,Mita K,Shimada T,2008.Beta-fructofuranosidase genes of the silkworm,Bombyx mori:insights into enzymatic adaptation of B.mori to toxic alkaloids in mulberry latex.J.Biol.Chem.,283(22):15271-15279.
Jiang W,Bikard D,Cox D,Zhang F,Marraffini LA,2013.RNA-guided editing of bacterial genomes using CRISPR-Cas systems.Nature Biotechnology,31(3):233-239.
Ma SY,Xia QY,2015.Genome editing brings a new era of silkworm research.Science of Sericulture,2:195-203.[马三垣,夏庆友,2015.基因组编辑开启家蚕研究新纪元.蚕业科学,2:195-203.]
Ran FA,Hsu PD,Lin CY,Gootenberg JS,Konermann S,Trevino AE,Scott DA,Inoue A,Matoba S,Zhang Y,Zhang F,2013.Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.Cell,154(6):1380-1389.
Tamura T,Thibert C,Royer C,Kanda T,Abraham E,Kamba M,Komoto N,Thomas JL,Mauchamp B,Chavancy G,Shirk P,Fraser M,Prudhomme JC,Couble P,2000.Germline transformation of the silkworm Bombyx mori L.using a piggy Bac transposonderived vector.Nature Biotechnology,18(1):81-84.
Veres A,Gosis BS,Ding Q,Collins R,Ragavendran A,Brand H,Erdin S,Cowan CA,Talkowski ME,Musunuru K,2014.Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing.Cell Stem Cell,15(1):27-30.
Wang H,Yang H,Shivalila CS,Dawlaty MM,Cheng AW,Zhang F,Jaenisch R,2013.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.Cell,153(4):910-918.
Wang WY,Fu Y,Reyon D,Maeder ML,Tsai SQ,Sander JD,Peterson RT,Yeh JR,Joung JK,2013.Efficient genome editing in zebrafish using a CRISPR-Cas system.Nature Biotechnology,31(3):227-229.
Xia Q,Guo Y,Zhang Z,Li D,Xuan Z,Li Z,Dai F,Li Y,Cheng D,Li R,Cheng T,Jiang T,Becquet C,Xu X,Liu C,Zha X,Fan W,Lin Y,Shen Y,Jiang L,Jensen J,Hellmann I,Tang S,Zhao P,Xu H,Yu C,Zhang G,Li J,Cao J,Liu S,He N,Zhou Y,Liu H,Zhao J,Ye C,Du Z,Pan G,Zhao A,Shao H,Zeng W,Wu P,Li C,Pan M,Li J,Yin X,Li D,Wang J,Zheng H,Wang W,Zhang X,Li S,Yang H,Lu C,Nielsen R,Zhou Z,Wang J,Xiang Z,Wang J,2009.Complete resequencing of 40 genomes reveals domestication events and genes in silkworm(Bombyx).Science,326(5951):433-436.
Xia Q,Li S,Feng Q,2014.Advances in silkworm studies accelerated by the genome sequencing of Bombyx mori.Annual Review of Entomology,59:513-536.
Xia Q,Wang J,Zhou Z,Li R,Fan W,Cheng D,Cheng T,Qin J,Duana J,Xu H,Li Q,Li N,Wang M,Dai F,Liu C,Lin Y,Zhao P,Zhang H,Liu S,Zha X,Li C,Zhao A,Pan M,Pan G,Shen Y,Gao Z,Wang Z,Wang G,Wu Z,Hou Y,Chai C,Yu Q,He N,Zhang Z,Li S,Yang H,Lu C,Wang J,Xiang Z,Mita K,Kasahara M,Nakatani Y,Yamamoto K,Abe H,Ahsan B,Daimoni T,Doi K,Fujii T,Fujiwara H,Fujiyama A,Futahashi R,Hashimotol S,Ishibashi J,Iwami M,Kadono-Okuda K,Kanamori H,Kataoka H,Katsuma S,Kawaoka S,Kawasaki H,Kohara Y,Kozaki T,Kuroshu RM,Kuwazaki S,Matsushima K,Minami H,Nagayasu Y,Nakagawa T,Narukawa J,Nohata J,Ohishi K,Ono Y,Osanai-Futahashi M,Ozaki K,Qu W,Roller L,Sasaki S,Sasaki T,Seino A,Shimomura M,Shimomura M,Shin-I T,Shinoda T,Shiotsuki T,Suetsugu Y,Sugano S,Suwa M,Suzuki Y,Takiya S,Tamura T,Tanaka H,Tanaka Y,Touhara K,Yamada T,Yamakawa M,Yamanaka N,Yoshikawa H,Zhong YS,Shimada T,Morishita S,2008.The genome of a lepidopteran model insect,the silkworm Bombyx mori.Insect Biochemistry and Molecular Biology,38(12):1036-1045.
Xia Q,Zhou Z,Lu C,Cheng D,Dai F,Li B,Zhao P,Zha X,Cheng T,Chai C,Pan G,Xu J,Liu C,Lin Y,Qian J,Hou Y,Wu Z,Li G,Pan M,Li C,Shen Y,Lan X,Yuan L,Li T,Xu H,Yang G,Wan Y,Zhu Y,Yu M,Shen W,Wu D,Xiang Z,Yu J,Wang J,Li R,Shi J,Li H,Li G,Su J,Wang X,Li G,Zhang Z,Wu Q,Li J,Zhang Q,Wei N,Xu J,Sun H,Dong L,Liu D,Zhao S,Zhao X,Meng Q,Lan F,Huang X,Li Y,Fang L,Li C,Li D,Sun Y,Zhang Z,Yang Z,Huang Y,Xi Y,Qi Q,He D,Huang H,Zhang X,Wang Z,Li W,Cao Y,Yu Y,Yu H,Li J,Ye J,Chen H,Zhou Y,Liu B,Wang J,Ye J,Ji H,Li S,Ni P,Zhang J,Zhang Y,Zheng H,Mao B,Wang W,Ye C,Li S,Wang J,Wong GK,Yang H,2004,Biology Analysis Group,2004.A draft sequence for the genome of the domesticated silkworm(Bombyx mori).Science,306(5703):1937-1940.
Xiang H,Zhu J,Chen Q,Dai F,Li X,Li M,Zhang H,Zhang G,Li D,Dong Y,Zhao L,Lin Y,Cheng D,Yu J,Sun J,Zhou X,Ma K,He Y,Zhao Y,Guo S,Ye M,Guo G,Li Y,Li R,Zhang X,Ma L,Kristiansen K,Guo Q,Jiang J,Beck S,Xia Q,Wang W,Wang J,2010.Single base-resolution methylome of the silkworm reveals a sparse epigenomic map.Nat.Biotechnol.,28(5):516-520.
Xing Y,Yang Q,Ren J,2016.Application of CRISPR/Cas9mediated genome editing in farm animals.Hereditas,38(3):217-226.[幸宇云,杨强,任军,2016.CRISPR/Cas9基因组编辑技术在农业动物中的应用.遗传,38(3):217-226.]
Yang K,Tang F,Liu Z,Zhong J,Dong Z,2016.Transcriptional expression ofβ-fructofuranosidase gene in midgut of the 5th instar Bombyx mori larvae.Journal of Southern Agriculture,47(5):721-725.[杨伟克,唐芬芬,刘增虎,钟健,董占鹏,2016.5龄家蚕中肠β-呋喃果糖苷酶基因转录表达分析.南方农业学报,47(5):721-725.]
Zhang Z,Aslam AF,Liu X,Li M,Huang Y,Tan A,2015.Functional analysis of Bombyx Wnt1 during embryogenesis using’the CRISPR/Cas9 system.J.Insect Physiol.,79:73-79.
Barrangou R,2012.RNA-mediated programmable DNA cleavage.Nature Biotechnology 30(9):836-838.
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(6121):819-823.
Daimon T,Kiuchi T,Takasu Y,2014.Recent progress in genome engineering techniques in the silkworm,Bombyx mori.Dev.Growth Differ,56(1):14-25.
Daimon T,Taguchi T,Meng Y,Katsuma S,Mita K,Shimada T,2008.Beta-fructofuranosidase genes of the silkworm,Bombyx mori:insights into enzymatic adaptation of B.mori to toxic alkaloids in mulberry latex.J.Biol.Chem.,283(22):15271-15279.
Jiang W,Bikard D,Cox D,Zhang F,Marraffini LA,2013.RNA-guided editing of bacterial genomes using CRISPR-Cas systems.Nature Biotechnology,31(3):233-239.
Ma SY,Xia QY,2015.Genome editing brings a new era of silkworm research.Science of Sericulture,2:195-203.[马三垣,夏庆友,2015.基因组编辑开启家蚕研究新纪元.蚕业科学,2:195-203.]
Ran FA,Hsu PD,Lin CY,Gootenberg JS,Konermann S,Trevino AE,Scott DA,Inoue A,Matoba S,Zhang Y,Zhang F,2013.Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.Cell,154(6):1380-1389.
Tamura T,Thibert C,Royer C,Kanda T,Abraham E,Kamba M,Komoto N,Thomas JL,Mauchamp B,Chavancy G,Shirk P,Fraser M,Prudhomme JC,Couble P,2000.Germline transformation of the silkworm Bombyx mori L.using a piggy Bac transposonderived vector.Nature Biotechnology,18(1):81-84.
Veres A,Gosis BS,Ding Q,Collins R,Ragavendran A,Brand H,Erdin S,Cowan CA,Talkowski ME,Musunuru K,2014.Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing.Cell Stem Cell,15(1):27-30.
Wang H,Yang H,Shivalila CS,Dawlaty MM,Cheng AW,Zhang F,Jaenisch R,2013.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.Cell,153(4):910-918.
Wang WY,Fu Y,Reyon D,Maeder ML,Tsai SQ,Sander JD,Peterson RT,Yeh JR,Joung JK,2013.Efficient genome editing in zebrafish using a CRISPR-Cas system.Nature Biotechnology,31(3):227-229.
Xia Q,Guo Y,Zhang Z,Li D,Xuan Z,Li Z,Dai F,Li Y,Cheng D,Li R,Cheng T,Jiang T,Becquet C,Xu X,Liu C,Zha X,Fan W,Lin Y,Shen Y,Jiang L,Jensen J,Hellmann I,Tang S,Zhao P,Xu H,Yu C,Zhang G,Li J,Cao J,Liu S,He N,Zhou Y,Liu H,Zhao J,Ye C,Du Z,Pan G,Zhao A,Shao H,Zeng W,Wu P,Li C,Pan M,Li J,Yin X,Li D,Wang J,Zheng H,Wang W,Zhang X,Li S,Yang H,Lu C,Nielsen R,Zhou Z,Wang J,Xiang Z,Wang J,2009.Complete resequencing of 40 genomes reveals domestication events and genes in silkworm(Bombyx).Science,326(5951):433-436.
Xia Q,Li S,Feng Q,2014.Advances in silkworm studies accelerated by the genome sequencing of Bombyx mori.Annual Review of Entomology,59:513-536.
Xia Q,Wang J,Zhou Z,Li R,Fan W,Cheng D,Cheng T,Qin J,Duana J,Xu H,Li Q,Li N,Wang M,Dai F,Liu C,Lin Y,Zhao P,Zhang H,Liu S,Zha X,Li C,Zhao A,Pan M,Pan G,Shen Y,Gao Z,Wang Z,Wang G,Wu Z,Hou Y,Chai C,Yu Q,He N,Zhang Z,Li S,Yang H,Lu C,Wang J,Xiang Z,Mita K,Kasahara M,Nakatani Y,Yamamoto K,Abe H,Ahsan B,Daimoni T,Doi K,Fujii T,Fujiwara H,Fujiyama A,Futahashi R,Hashimotol S,Ishibashi J,Iwami M,Kadono-Okuda K,Kanamori H,Kataoka H,Katsuma S,Kawaoka S,Kawasaki H,Kohara Y,Kozaki T,Kuroshu RM,Kuwazaki S,Matsushima K,Minami H,Nagayasu Y,Nakagawa T,Narukawa J,Nohata J,Ohishi K,Ono Y,Osanai-Futahashi M,Ozaki K,Qu W,Roller L,Sasaki S,Sasaki T,Seino A,Shimomura M,Shimomura M,Shin-I T,Shinoda T,Shiotsuki T,Suetsugu Y,Sugano S,Suwa M,Suzuki Y,Takiya S,Tamura T,Tanaka H,Tanaka Y,Touhara K,Yamada T,Yamakawa M,Yamanaka N,Yoshikawa H,Zhong YS,Shimada T,Morishita S,2008.The genome of a lepidopteran model insect,the silkworm Bombyx mori.Insect Biochemistry and Molecular Biology,38(12):1036-1045.
Xia Q,Zhou Z,Lu C,Cheng D,Dai F,Li B,Zhao P,Zha X,Cheng T,Chai C,Pan G,Xu J,Liu C,Lin Y,Qian J,Hou Y,Wu Z,Li G,Pan M,Li C,Shen Y,Lan X,Yuan L,Li T,Xu H,Yang G,Wan Y,Zhu Y,Yu M,Shen W,Wu D,Xiang Z,Yu J,Wang J,Li R,Shi J,Li H,Li G,Su J,Wang X,Li G,Zhang Z,Wu Q,Li J,Zhang Q,Wei N,Xu J,Sun H,Dong L,Liu D,Zhao S,Zhao X,Meng Q,Lan F,Huang X,Li Y,Fang L,Li C,Li D,Sun Y,Zhang Z,Yang Z,Huang Y,Xi Y,Qi Q,He D,Huang H,Zhang X,Wang Z,Li W,Cao Y,Yu Y,Yu H,Li J,Ye J,Chen H,Zhou Y,Liu B,Wang J,Ye J,Ji H,Li S,Ni P,Zhang J,Zhang Y,Zheng H,Mao B,Wang W,Ye C,Li S,Wang J,Wong GK,Yang H,2004,Biology Analysis Group,2004.A draft sequence for the genome of the domesticated silkworm(Bombyx mori).Science,306(5703):1937-1940.
Xiang H,Zhu J,Chen Q,Dai F,Li X,Li M,Zhang H,Zhang G,Li D,Dong Y,Zhao L,Lin Y,Cheng D,Yu J,Sun J,Zhou X,Ma K,He Y,Zhao Y,Guo S,Ye M,Guo G,Li Y,Li R,Zhang X,Ma L,Kristiansen K,Guo Q,Jiang J,Beck S,Xia Q,Wang W,Wang J,2010.Single base-resolution methylome of the silkworm reveals a sparse epigenomic map.Nat.Biotechnol.,28(5):516-520.
Xing Y,Yang Q,Ren J,2016.Application of CRISPR/Cas9mediated genome editing in farm animals.Hereditas,38(3):217-226.[幸宇云,杨强,任军,2016.CRISPR/Cas9基因组编辑技术在农业动物中的应用.遗传,38(3):217-226.]
Yang K,Tang F,Liu Z,Zhong J,Dong Z,2016.Transcriptional expression ofβ-fructofuranosidase gene in midgut of the 5th instar Bombyx mori larvae.Journal of Southern Agriculture,47(5):721-725.[杨伟克,唐芬芬,刘增虎,钟健,董占鹏,2016.5龄家蚕中肠β-呋喃果糖苷酶基因转录表达分析.南方农业学报,47(5):721-725.]
Zhang Z,Aslam AF,Liu X,Li M,Huang Y,Tan A,2015.Functional analysis of Bombyx Wnt1 during embryogenesis using’the CRISPR/Cas9 system.J.Insect Physiol.,79:73-79.