利用CRISPR/Cas 9技术构建大肠杆菌aroA基因的敲除系统及其初步应用

doi:10.11843/j.issn.0366-6964.2016.04.016

Abstract

Abstract:

CRISPR/Cas 9 system is a novel gene editing technology.This study was aimed to construct CRISPR/Cas 9-based knockout system for E.coli aroA gene，and analyze knockout efficiencies in different E.colis. In the present study，aroA gene-targeting short guide RNA (sgRNA) was designed and constructed together with Donor sequence for homologous repair，then this resulting vector and pEwt-Cas 9 vector co-constituted the CRISPR/Cas 9 system.Preliminary application was performed on E.coli DH10B，DH5α and JM109，respectively，and then knockout efficiencies of aroA gene were analyzed by PCR amplification，TA cloning and subsequent DNA sequencing.The restriction enzyme digestion analysis and sequencing results showed that homologous repair vector was successfully constructed.PCR results indicated that the established CRISPR/Cas 9 system could be used for aroA gene knockout in multiple E.coli with efficiency ranging from 46% to 50%.Sequencing results further confirmed the successful and accurate knockout of aroA gene.In conclusion，CRISPR/Cas 9-based knockout system for E.coli aroA gene was successfully established，which would provide a novel and effective tool for functional studies on aroA genes of other pathogenic microorganisms and further development of attenuated vaccine.

CLC Number:

S852.612

YU Shen-yi，ZHAO Jin-rong，ZHENG Ling-hong，ZHU Er-peng，ZHOU Wu-duo，WU Bao-cheng. The Application of CRISPR/Cas 9 Technology for aroA Gene Knockout in Escherichia coli[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(4): 762-770.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.xmsyxb.com/EN/10.11843/j.issn.0366-6964.2016.04.016

https://www.xmsyxb.com/EN/Y2016/V47/I4/762

References

［1］BOOCOCK M R，COGGINS J R.Kinetics of 5-enolpyruvylshikimate-3-phosphate synthase inhibition by glyphosate［J］.FEBS Lett，1983，154(1)：127-133.
［2］STEINRÜCKEN H C，AMRHEIN N.5-Enolpyruvylshikimate-3-phosphate synthase of Klebsiella pneumonia 2.Inhibition by glyphosate［N-(phosphonomethyl)glycine］［J］.Eur J Biochem，1984，143(2)：351-357.
［3］HOISETH S K，STOCKER B A.Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines［J］.Nature，1981，291(5812)：238-239.
［4］GARSIDE L H，COLLINS M，LANGFORD P R，et al.Actinobacillus pleuropneumoniae serotype 1 carrying the defined aroA mutation is fully avirulent in the pig［J］.Res Vet Sci，2002，72(2)：163-167.
［5］KIM T，TOAN N T，SEO J，et al.Bordetella bronchiseptica aroA mutant as a live vaccine vehicle for heterologous porcine circovirus type 2 major capsid protein expression［J］.Vet Microbiol，2009，138(3-4)：318-324.
［6］ISHINO Y，SHINAGAWA H，MAKINO K，et al.Nucleotide sequence of the iap gene，responsible for alkaline phosphatase isozyme conversion in Escherichia coli，and identification of the gene product［J］. J Bacteriol，1987，169(12)：5429-5433.
［7］BARRANGOU R，FREMAUX C，DEVEAU H，et al.CRISPR provides acquired resistance against viruses in prokaryotes［J］.Science，2007，315(5819)：1709-1712.
［8］JANSEN R，EMBDEN J D，GAASTRA W，et al.Identification of genes that are associated with DNA repeats in prokaryotes［J］.Mol Microbiol，2002，43(6)：1565-1575.
［9］JANSEN R，VAN EMBDEN J D，GAASTRA W，et al.Identification of a novel family of sequence repeats among prokaryotes［J］.OMICS，2002，6(1)：23-33．
［10］KUNIN V，SOREK R，HUGENHOLTZ P.Evolutionary conservation of sequence and secondary structures in CRISPR repeats［J］.Genome Biol，2007，8(4)：R61.
［11］DEVEAU H，GARNEAU J E，MOINEAU S.CRISPR/Cas system and its role in phage-bacteria interactions［J］.Annu Rev Microbiol，2010，64：475-493.
［12］HWANG W Y，FU Y，REYON D，et al.Efficient genome editing in zebrafish using a CRISPR-Cas system［J］.Nat Biotechnol，2013，31(3)：227-229.
［13］GARNEAU J E，DUPUIS M È，VILLION M，et al.The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA［J］.Nature，2010，468(7320)：67-71.
［14］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.
［15］JIANG W，BIKARD D，COX D，et al.RNA-guided editing of bacterial genomes using CRISPR-Cas systems［J］.Nat Biotechnol，2013，31(3)：233-239.
［16］MUSSOLINO C，CATHOMEN T.RNA guides genome engineering［J］.Nat Biotechnol，2013，31(3)：208-209.
［17］GEURTS A M，COST G J，FREYVERT Y，et al.Knockout rats via embryo microinjection of zinc-finger nucleases［J］.Science，2009，325(5939)：433.
［18］MALI P，ESVELT K M，CHURCH G M.Cas9 as a versatile tool for engineering biology［J］.Nat Methods，2013，10(10)：957-963.
［19］魏泽辉，贾存灵，张智英.CRISPR/Cas9系统在基因表达调控中的应用［J］.畜牧兽医学报，2014，45(9)：1387-1392.
WEI Z H，JIA C L，ZHANG Z Y.Application of CRISPR/Cas9 system for gene regulation［J］.Acta Veterinaria et Zootechnica Sinica，2014，45(9)：1387-1392.（in Chinese）
［20］HARPER M，BOYCE J D，ADLER B.Pasteurella multocida pathogenesis：125 years after Pasteur［J］.FEMS Microbiol Lett，2006，265(1)：1-10.
［21］SCHUBERT S，RAKIN A，KARCH H，et al.Prevalence of the “high-pathogenicity island”of Yersinia species among Escherichia coli strains that are pathogenic to humans［J］.Infect Immun，1998，66(2)：480-485.
［22］COPELAND N G，JENKINS N A，COURT D L.Recombineering：A powerful new tool for mouse functional genomics［J］.Nat Rev Genet，2001，2(10)：769-779.
［23］SHARAN S K，THOMASON L C，KUZNETSOV S G，et al.Recombineering：a homologous recombination-based method of genetic engineering［J］. Nat Protoc，2009，4(2)：206-223.
［24］JIANG Y，CHEN B，DUAN C，et al.Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system［J］.Appl Environ Microbiol，2015，81(7)：2506-2514.

[1]	ZHI Yan, MEI Chen, LIU Zhenyi, WUYUN Gerile, WANG Hongjun, HU Ge. Research Progress on the Virulence Factors of Avibacterium paragallinarum [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 4934-4942.
[2]	WANG Jianing, ZHANG Ziqiang, KONG Dejing, FENG Caicai, ZHANG Feike, LIU Yumei. Isolation and Identification of Klebsiella pneumoniae in Rabbits [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5198-5206.
[3]	LI Lili, CHEN Kaifeng, CHEN Bing, ZHOU Zhouping, WANG Nanwei, QU Xiaoyun, XU Chenggang, LIAO Ming, ZHANG Jianmin. Regulatory Role of STM1827 in the Biofilm Formation and Environmental Stress of Salmonella Typhimurium [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5207-5217.
[4]	TONG Panpan, HUANG Shunmin, WANG Yudan, SHI Xuhui, CHEN Wenxia, SONG Xinlong, ZHANG Yi, SU Zhanqiang, XIE Jinxin. Phylogenetic Clustering, Serotype and Drug Resistance Analysis of Escherichia coli from Diarrhea with Piglets in Xinjiang [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 414-420.
[5]	WANG Fei, YANG Jie, Lü Qingjie, WANG Mixue, LIU Peng, ZHANG Ruoyu, SHI Congcong, WANG Xueying, LIN Lin, HUA Lin, SONG Wenbo, LIANG Wan, CHEN Huanchun, WU Bin, PENG Zhong. Isolation and Genomic Characterization of a Meningitis Causing Pasteurella multocida [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(12): 4346-4355.
[6]	MEN Kaikai, LIU Jialong, GUO Yage, HE Lei, JIA Yanyan, YU Zuhua. Effects of Chicken TGF-β1 on the Adhesion of Escherichia coli and Salmonella pullorum to DF1 Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 4000-4007.
[7]	YANG Yang, XIE Liqing, HU Pei, GAO Lixu, YUAN Xiang, LI Pan, PENG Yuanyi, LI Nengzhang. Construction of Bovine Pasteurella multocida Type A hyaD Mutant and Its Cross-protection Analysis in Mice [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3582-3597.
[8]	SHAO Ying, FU Dandan, WU Xiaoyan, GU Yi, SONG Xiangjun, TU Jian, QI Kezong. Effect of the ETT2 Structural Gene epaPQR on the Biological Properties and Pathogenicity of Avian Pathogenic Escherichia coli [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3631-3641.
[9]	XU Wenbo, WU Limei, LIU Xin, LI Sheng, HUANG Fushen, LI Runcheng. Analysis on Complete Genome Sequence and Pathogenic Genes of a Pasteurella multocida Strain [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(6): 1858-1869.
[10]	WANG Xi, LI Ke, LI Tingcui, YAN Hongya, ZHAO Rong, CHANG Zhishun, LIAO Ming, SUN Minhua, XIN Aiguo. MLST Typing and Drug Resistance Analysis of 75 Salmonella Strains Isolated from Laying Hens [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(5): 1626-1631.
[11]	WANG Jun, LI Jun, CUI Guolin. The Effect of R91S Mutation in FliC on the Flagellar Shape and Salmonella Enteritidis Colonization in BALB/c Mice [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(2): 607-617.
[12]	TONG Panpan, ZHANG Mengmeng, CHEN Wenxia, LIU Luyao, ZHANG Ling, TANG Xuelin, SU Zhanqiang, XIE Jinxin. Phylogenetic Clustering, Serotype, Virulence Genes and Drug Resistance Analysis of Shiga Toxin-producing Escherichia coli from Cattle, Sheep and Camel in Xinjiang [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(12): 3660-3668.
[13]	CHEN Chaoqun, CHEN Jiali, ZHOU Xuanyi, WU Xue, WANG Jincheng, HUANG Anxiong, HAO Haihong. Establishment of Epidemiological Cut-off Values and Determination of Drug Resistance of Haemophilus parasuis with β-lactam Drugs [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(11): 3234-3245.
[14]	HE Zhuolin, TANG Minjia, ZHANG Xuejing, HOU Xiao, PU Wanxia. ERIC-PCR Genotyping and Phylogenetic Grouping of Escherichia coli from a Dairy Farm Environment [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(7): 2059-2064.
[15]	YI Zhengfei, ZHANG Yaodong, WANG Yao, ZHU Hong, TAO Chenglin, AFAYIBO Dossêh Jean Apôtre, LI Tao, QI Jingjing, TIAN Mingxing, DING Chan, YU Shengqing, WANG Shaohui. Two Component System CpxR Contributes to the Antibiotic Resistance, Serum Bactericidal Resistance and Virulence of Avian Pathogenic Escherichia coli [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(4): 1053-1060.

The Application of CRISPR/Cas 9 Technology for aroA Gene Knockout in Escherichia coli

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments