摘要
【目的】猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS),俗称"蓝耳病",是由猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)引起的一种高致死性传染病,对世界养猪业造成了巨大的经济损失。由于PRRSV遗传变异性较大,因此国内外并未有理想疫苗能够对此病进行有效防制。Cluster of differentiation 163(CD163)是PRRSV感染猪肺泡巨噬细胞(porcine alveolar macrophage,PAM)的受体之一,研究旨在利用CRISPR/Cas9技术结合体细胞核移植技术制备CD163基因编辑的大白猪。【方法】针对猪CD163基因的第7外显子设计构建CRISPR/Cas9基因编辑载体;转染大白猪胎儿成纤维细胞,获得基因编辑阳性细胞克隆;以基因编辑细胞为核供体、体外成熟的猪卵母细胞为核受体构建克隆胚胎;胚胎移植到受体母猪生产CD163基因编辑猪,并进行后续的扩繁试验。【结果】设计的g RNA能够高效的识别靶位点。对获得的127个细胞单克隆进行PCR和测序显示,共有21个克隆发生突变,其中14个克隆为单等位基因突变或双等位基因杂合突变,7个克隆为双等位基因纯合突变。通过体细胞核移植技术,成功获得了CD163双等位基因编辑的纯合大白猪;并获得首批F1代CD163基因编辑仔猪,目前健康状态良好。随后将有更多的F1代CD163基因编辑猪陆续出生。【结论】制备的无抗性筛选标记的CD163双等位基因编辑猪,能够安全并快速地为培育抗PRRSV新品种猪提供育种材料。
【Objective】 Porcine reproductive and respiratory syndrome(PRRS), commonly known as "blue ear disease", is a highly fatal infectious disease with porcine reproductive and respiratory syndrome virus(PRRSV) being the causative pathogen. PRRSV causes major economic losses in the pork industry world-wide. The genetic variability of PRRSV is high and an ideal vaccine to prevent the occurrence of this disease is not available. Cluster of differentiation 163(CD163) is the important receptor for the entry of PRRSV into the porcine alveolar macrophage(PAM) cells. The aim of this study was to generate CD163 gene edited Large White pigs by using the CRISPR/Cas9 and somatic cell nuclear transfer(SCNT) techniques.【Method】 CRISPR/Cas9 vector was constructed for editing the exon 7 of the porcine CD163 gene; The constructed vectors were transfected into pig fetal fibroblasts to obtain gene edited positive cell colonies; CD163 gene edited fibroblasts and in vitro matured porcine oocytes were employed as nuclear donors and nuclear receptors respectively to obtain reconstructed embryos; For obtaining CD163 gene edited pigs the reconstructed embryos ere transferred into recipient sows and performing the subsequent propagation experiment. 【Result】 The designed g RNAcould effectively recognize the intended site. Genotyping analysis of cloned cell showed that 21 colonies had mutations in the CD163 gene, of which 14 colonies had either a monoallelic mutation or a biallelic heterozygous mutation, and 7 colonies had a biallelic homozygous mutation. Through SCNT, we successfully obtained CD163 biallelic edited Large White pigs. Successful breeding allowed us to obtain F1 generation CD163 gene edited piglets, and they are all in good health. It is anticipated that more F1 piglets will be produced soon. 【Conclusion】The CD163 biallelic edited Large White pigs that do not harbor a drug resistant gene in their genome were produced and they can thus safely and quickly serve as a gene donor for breeding of PRRSV resistant pigs.
引文
[1]NIEDERWERDER M C,BAWA B,SERAO N V,TRIBLE B R,KERRIGAN M A,LUNNEY J K,DEKKERS J C,ROWLAND R R.Vaccination with a porcine reproductive and respiratory syndrome(PRRS)modified live virus vaccine followed by challenge with PRRS virus and porcine circovirus type 2(PCV2)protects against PRRS but enhances PCV2 replication and pathogenesis compared to results for nonvaccinated cochallenged controls.Clinical and Vaccine Immunology:CVI,2015,22(12):1244-1254.
[2]WENSVOORT G,TERPSTRA C,POL J M,TER LAAK E A,BLOEMRAAD M,DE KLUYVER E P,KRAGTEN C,VAN BUITEN L,DEN BESTEN A,WAGENAAR F,ET AL.Mystery swine disease in The Netherlands:The isolation of Lelystad virus.Veterinary Quarterly,1991,13(3):121-130.
[3]NELSEN C J,MURTAUGH M P,FAABERG K S.Porcine reproductive and respiratory syndrome virus comparison:divergent evolution on two continents.Journal of Virology,1999,73(1):270-280.
[4]ALLENDE R,LEWIS T L,LU Z,ROCK D L,KUTISH G F,ALI A,DOSTER A R,OSORIO F A.North American and European porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions.Journal of General Virology,1999,80(2):307-315.
[5]WELLS K D,BARDOT R,WHITWORTH K M,TRIBLE B R,FANG Y,MILEHAM A,KERRIGAN M A,SAMUEL M S,PRATHER R S,ROWLAND R R R.Replacement of porcine CD163scavenger receptor cysteine-rich domain 5 with a CD163-Like homolog confers resistance of pigs to genotype 1 but not genotype 2porcine reproductive and respiratory syndrome virus.Journal of Virology,2017,91(2):e01521-01516.
[6]NEUMANN E J,KLIEBENSTEIN J B,JOHNSON C D,MABRY J W,BUSH E J,SEITZINGER A H,GREEN A L,ZIMMERMAN J J.Assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the United States.Journal of the American Veterinary Medical Association,2005,227(3):385-392.
[7]ZHOU L,YANG H.Porcine reproductive and respiratory syndrome in China.Virus Research,2010,154(1-2):31-37.
[8]JUSA E R,INABA Y,KOUNO M,HIROSE O.Effect of heparin on infection of cells by porcine reproductive and respiratory syndrome virus.American Journal of Veterinary Research,1997,58(5):488-491.
[9]CALVERT J G,SLADE D E,SHIELDS S L,JOLIE R,MANNAN R M,ANKENBAUER R G,WELCH S K.CD163 expression confers susceptibility to porcine reproductive and respiratory syndrome viruses.Journal of Virology,2007,81(14):7371-7379.
[10]VAN GORP H,DELPUTTE P L,NAUWYNCK H J.Scavenger receptor CD163,a Jack-of-all-trades and potential target for celldirected therapy.Molecular Immunology,2010,47(7-8):1650-1660.
[11]PATTON J B,ROWLAND R R,YOO D,CHANG K O.Modulation of CD163 receptor expression and replication of porcine reproductive and respiratory syndrome virus in porcine macrophages.Virus Research,2009,140(1-2):161-171.
[12]VAN GORP H,VAN BREEDAM W,VAN DOORSSELAERE J,DELPUTTE P L,NAUWYNCK H J.Identification of the CD163protein domains involved in infection of the porcine reproductive and respiratory syndrome virus.Journal of Virology,2010,84(6):3101-3105.
[13]WHITWORTH K M,ROWLAND R R,EWEN C L,TRIBLE B R,KERRIGAN M A,CINO-OZUNA A G,SAMUEL M S,LIGHTNER J E,MCLAREN D G,MILEHAM A J,WELLS K D,PRATHER R S.Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus.Nature Biotechnology,2016,34(1):20-22.
[14]WHITWORTH K M,LEE K,BENNE J A,BEATON B P,SPATE L D,MURPHY S L,SAMUEL M S,MAO J,O'GORMAN C,WALTERS E M,MURPHY C N,DRIVER J,MILEHAM A,MCLAREN D,WELLS K D,PRATHER R S.Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos.Biology of Reproduction,2014,91(3):78.
[15]BURKARD C,LILLICO S G,REID E,JACKSON B,MILEHAM A J,AIT-ALI T,WHITELAW C B,ARCHIBALD A L.Precision engineering for PRRSV resistance in pigs:Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function.PLo S Pathogens,2017,13(2):e1006206.
[16]LI G,JIANG P,LI Y,WANG X,HUANG J,BAI J,CAO J,WU B,CHEN N,ZESHAN B.Inhibition of porcine reproductive and respiratory syndrome virus replication by adenovirus-mediated RNA interference both in porcine alveolar macrophages and swine.Antiviral Research,2009,82(3):157-165.
[17]LU T,SONG Z,LI Q,LI Z,WANG M,LIU L,TIAN K,LI N.Overexpression of Histone deacetylase 6 enhances resistance to porcine reproductive and respiratory syndrome virus in pigs.PLo S One,2017,12(1):e0169317.
[18]CONG L,RAN F A,COX D,LIN S,BARRETTO R,HABIB N,HSU P D,WU X,JIANG W,MARRAFFINI L A,ZHANG F.Multiplex genome engineering using CRISPR/Cas systems.Science,2013,339(6121):819-823.
[19]MIZUNO S,DINH T T,KATO K,MIZUNO-IIJIMA S,TANIMOTO Y,DAITOKU Y,HOSHINO Y,IKAWA M,TAKAHASHI S,SUGIYAMA F,YAGAMI K.Simple generation of albino C57BL/6J mice with G291T mutation in the tyrosinase gene by the CRISPR/Cas9 system.Mammalian Genome,2014,25(7-8):327-334.
[20]WANG H,YANG H,SHIVALILA C S,DAWLATY M M,CHENG A W,ZHANG F,JAENISCH R.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.Cell,2013,153(4):910-918.
[21]FUJIHARA Y,IKAWA M.CRISPR/Cas9-based genome editing in mice by single plasmid injection.Methods in Enzymology,2014,546:319-336.
[22]GUAN Y,SHAO Y,LI D,LIU M.Generation of site-specific mutations in the rat genome via CRISPR/Cas9.Methods in Enzymology,2014,546:297-317.
[23]HAI T,TENG F,GUO R,LI W,ZHOU Q.One-step generation of knockout pigs by zygote injection of CRISPR/Cas system.Cell Rresearch,2014,24(3):372-375.
[24]RUAN J,LI H,XU K,WU T,WEI J,ZHOU R,LIU Z,MU Y,YANG S,OUYANG H,CHEN-TSAI R Y,LI K.Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs.Scientific Reports,2015,5(14253).
[25]HUANG L,HUA Z,XIAO H,CHENG Y,XU K,GAO Q,XIA Y,LIU Y,ZHANG X,ZHENG X,MU Y,LI K.CRISPR-Cas9-mediated Apo E-/-and LDLR-/-double gene knockout in pigs elevates serum LDL-C and TC levels.Oncotarget,2017,8(23):37751-37760.
[26]WEHNES C A,REHBERGER T G,BARRANGOU R,SMITH A H.Short communication:determination of salmonella clustered regularly interspaced short palindromic repeats(CRISPR)diversity on dairy farms in Wisconsin and Minnesota.Journal of Dairy Science,2014,97(10):6370-6377.
[27]GAO Y,WU H,WANG Y,LIU X,CHEN L,LI Q,CUI C,LIU X,ZHANG J,ZHANG Y.Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects.Genome Biology,2017,18(1):13.
[28]CRISPO M,MULET A P,TESSON L,BARRERA N,CUADRO F,DOS SANTOS-NETO P C,NGUYEN T H,CRENEGUY A,BRUSSELLE L,ANEGON I,MENCHACA A.Efficient generation of myostatin knock-out sheep using CRISPR/Cas9 technology and microinjection into zygotes.PLo S One,2015,10(8):e0136695.
[29]KANG Y,ZHENG B,SHEN B,CHEN Y,WANG L,WANG J,NIU Y,CUI Y,ZHOU J,WANG H,GUO X,HU B,ZHOU Q,SHA J,JI W,HUANG X.CRISPR/Cas9-mediated Dax1 knockout in the monkey recapitulates human AHC-HH.Human Molecular Genetics,2015,24(25):7255-7264.
[30]VARSHNEY G K,PEI W,LAFAVE M C,IDOL J,XU L,GALLARDO V,CARRINGTON B,BISHOP K,JONES M,LI M,HARPER U,HUANG S C,PRAKASH A,CHEN W,SOOD R,LEDIN J,BURGESS S M.High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9.Genome Research,2015,25(7):1030-1042.
[31]GRATZ S J,UKKEN F P,RUBINSTEIN C D,THIEDE G,DONOHUE L K,CUMMINGS A M,O'CONNOR-GILES K M.Highly specific and efficient CRISPR/Cas9-catalyzed homologydirected repair in Drosophila.Genetics,2014,196(4):961-971.
[32]ZHENG J,JIA H,ZHENG Y.Knockout of leucine aminopeptidase in Toxoplasma gondii using CRISPR/Cas9.International Journal for Parasitology,2015,45(2-3):141-148.
[33]BARRANGOU R,DOUDNA J A.Applications of CRISPR technologies in research and beyond.Nature Biotechnology,2016,34(9):933-941.
