参见作者原研究论文

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Feb 2018

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CRISPR/Cas Gene Editing of a Large DNA Virus: African Swine Fever Virus
一种大DNA病毒(非洲猪瘟病毒)的CRISPR/Cas基因编辑   

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Abstract

Gene editing of large DNA viruses, such as African swine fever virus (ASFV), has traditionally relied on homologous recombination of a donor plasmid consisting of a reporter cassette with surrounding homologous viral DNA. However, this homologous recombination resulting in the desired modified virus is a rare event. We recently reported the use of CRISPR/Cas9 to edit ASFV. The use of CRISPR/Cas9 to modify the African swine fever virus genome resulted in a fast and relatively easy way to introduce genetic changes. To accomplish this goal we first infect primary swine macrophages with a field isolate, ASFV-G, and transfect with the CRISPR/Cas9 donor plasmid along with a plasmid that will express a specific gRNA that targets our gene to be deleted. By inserting a reporter cassette, we are then able to purify our recombinant virus from the parental by limiting dilution and plaque purification. We previously reported comparing the traditional homologous recombination methodology with CRISPR/Cas9, which resulted in over a 4 log increase in recombination.

Keywords: ASFV (ASFV), African swine fever (非洲猪瘟), ASF (ASF), CRISPR (CRISPR), CRISPR/Cas9 (CRISPR/Cas9)

Background

African Swine Fever (ASF) is a highly lethal contagious viral disease of swine caused by ASF virus (ASFV). The genome of ASFV consists of a double-stranded DNA genome of approximately 180-190 kilobase pairs. ASFV causes a spectrum of disease, from highly lethal to sub-clinical, depending on host characteristics and the virus strain (Tulman et al., 2009). There is no commercial vaccine for ASFV; experimentally, the only vaccines that have shown to protect against the current circulating strain from the outbreak in Georgia in 2007 (ASFV-G) are live attenuated vaccines that contain one or more deletions to the viral genome, for example: (O' Donnell et al., 2015). Traditionally, gene deletions for ASFV have been performed by homologous recombination where a donor plasmid containing homologous genomic sequences is used for gene deletion (O' Donnell et al., 2015), however this only occurs at a very low rate, making production of recombinant ASFV difficult (Borca et al., 2018).

Recently we reported the use of CRISPR/Cas9 as an alternative approach to introduce gene deletions in ASFV, with a 4 log increase over traditional methods (Borca et al., 2018). This increase, in recombination using CRISPR/Cas9, allows for easier production and purification of recombinant viruses from the parental wild-type. It is possible that using CRISPR/Cas9 will allow for viral protein mutations, expanding our abilities to dissect critical domains for virulence, possibly even in genes that have been previously determined to be essential. This approach has successfully been reported with other large DNA viruses including Orthopox (Okoli et al., 2018), Vaccinia virus (Yuan et al., 2015 and 2016), Herpes Simplex virus (Suenaga et al., 2014) and Pseudorabies (Tang et al., 2016).

Materials and Reagents

  1. Pipette tips 10 μl, 200 μl, 1,000 μl (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 2140-05 , 2160P , 2079 )
  2. PrimariaTM 6-well plates (Corning, catalog number: 353846 )
  3. PrimariaTM Tissue culture flasks T-75 filtered flasks (Corning, Falcon®, catalog number: 353824 )
  4. 50 ml Conical tube (Corning, Falcon®, catalog number: 352098 )
  5. Polypropylene microcentrifuge tubes
  6. T-25 flask (Corning, catalog number: 3056 )
  7. T-150 flask (Corning, catalog number: 3151 )
  8. 0.22 μm filter (Corning, catalog number: 430769 )
  9. African swine fever virus field isolates such as ASFV-G (isolated from infected swine serum and adding serum to swine macrophages to propagate the virus. Titrations were done as described by Enjuanes et al., 1976). 
  10. Yorkshire pigs aged 3-12 months as blood donors
  11. Cas9 donor plasmid (custom designed for target of interest by blue heron bio. See attached insertion cassette sequence (Supplemental file)
  12. Fugene HD (Active Motif, catalog number: 32042 )
  13. gRNA plasmids (custom designed for target of interest by blue heron bio.)
  14. OptiMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 31985062 )
  15. Phosphate Buffered Saline (PBS) 1x, pH 7.0-7.3 (Thermo Fisher Scientific, GibcoTM, catalog number: 14190144 )
  16. 0.5 M EDTA pH 8.0 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15575020 )
  17. Ficoll-Paque PLUS density gradient of 1.077 g/ml (GE Healthcare, catalog number: 17144002 )
  18. RPMI 1640 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 11875093 )
  19. Gamma Irradiated Fetal bovine serum (GE Healthcare, HycloneTM, catalog number: SH30071.03 )
  20. Antibiotic-Antimycotic (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15240096 )
  21. Gentamycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15750060 )
  22. HEPES 1 M (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
  23. L-Glutamine, 200 nM (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  24. L929 media (L cell, L929, derivative of Strain L) (ATCC, catalog number: CCL-1 )
  25. Heparin (Sagent Pharmaceuticals, catalog number: 400-10 )
  26. L929 conditioned media (see Recipes)
  27. Macrophage Wash Media (see Recipes)
  28. Macrophage complete media (see Recipes)
  29. PBS + EDTA (see Recipes)

Equipment

  1. P1,000, P200, P20, P10 Pipettes (Gilson)
  2. 2 Liter roller bottles (Corning, catalog number: 431329 )
  3. 250 ml centrifuge bottles (Thermo Fisher Scientific, Thermo ScientificTM, NalgeneTM, catalog number: 3141-0250 )
  4. Inverted Fluorescent Microscope (Carl-Zeiss)
  5. Hematocytometer
  6. Pipet aid (Drummond Scientific, catalog number: 4-000-101 )
  7. Tabletop Centrifuge (Eppendorf, model: 5810 )
  8. Floor Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: Sorvall® RC-4 )
  9. Sorval SLA-1500 fixed angle rotor (Thermo Fisher Scientific, model: SLA-1500 )
  10. Vortex mixer (Fisher Scientific, catalog number: 12-812 )
  11. -70 °C freezer (Thermo Scientific, model: ULT2586-9-A40 )

Procedure

  1. Macrophage isolation
    1. Obtain 500 ml of swine blood by venipuncture and treat with heparin.
    2. Incubate the blood at 37 °C for 1-2 h until you observe erythrocyte sedimentation and blood leukocyte enrichment into a supernatant plasma.
    3. Collect plasma and float over Ficoll-Paque (35 ml plasma, 10 ml Ficoll-Paque) in a 50 ml Falcon tube.
    4. Centrifuge in an Eppendorf 5810 at 367 x g for 30 min at room temperature.
    5. Collect top layers and pool into a 250 ml centrifuge bottle.
    6. Centrifuge at 7,500 x g in a Sorval SLA-1500 for 20 min at room temperature.
    7. Collect mononuclear leukocytes and wash with macrophage media by adding an equal volume of media as collected cells, inverting several times to mix.
    8. Centrifuge in an Eppendorf 5810 at 367 x g for 10 min at room temperature.
    9. Remove supernatant, and wash cells by adding 10 ml macrophage media and centrifuging as in Step 8 again for a total of three washes.
    10. Resuspend cells in 10 ml of Macrophage complete media.
    11. Plate cells into five PrimariaTM T-75 flasks (each with 1 ml of cells and 10 ml of macrophage media) and incubate overnight.
    12. Adherent cells are now considered macrophages.
    13. Detach cells using PBS EDTA, washing with 10 ml and incubating with 10 ml at 37 °C until cells detach (typically 15-20 min).
    14. Count macrophages using 10 μl of cell suspension in a hematocytometer.
    15. Plate macrophages at a density of 5 x 106 per well in PrimariaTM 6-well plates and allow to attach overnight.

  2. Infection/Transfection with CRISPR/Cas9
    1. Inoculate each well with a field isolate of ASFV, such as ASFV-G or other field isolates of ASFV, at a multiplicity of infection (MOI) of 1.0. Inoculate by adding virus directly to the media of each well containing 2 ml of media, gently shake plates side to side to mix and incubate at 37 °C under 5% CO2 for 1 h.
    2. Add 1.6 µg of donor plasmid and 1.6 µg of gRNA plasmid suspended in ddH2O to 150 µl of optiMEM in polypropylene microcentrifuge tubes. 
    3. Carefully mix by pipetting or vortexing briefly.
    4. Add 10 µl of Fugene HD mix by pipetting and incubate for 10 min.
    5. Add dropwise 150 µl of complex to macrophages containing 2 ml of media.
    6. Incubate at 37 °C under 5% CO2 for 24 h.
    7. Observe under a fluorescent microscope 24 h after transfection for your reporter gene to determine the rate of transfection. Calculate the percentage by observing both attached and unattached cells that are expressing the fluorescent marker. 
    8. Freeze plate at -70 °C, and store until ready to use.
    9. Take 1 ml of frozen media from transfection to make serial 1:10 dilutions (1:10, 1:100, 1:1,000; etc.) in macrophage media.
    10. Add 100 µl of dilutions to 6 well plates of macrophages prepared as in Step B7, in a final volume of 2 ml of media. Incubate at 37 °C under 5% CO2.
    11. Observe every 24 h for complete CPE (cytopathic effect) (typically 1-7 days), mark wells that are positive for your fluorescent marker. If complete CPE occurs quickly (< 48 h) without seeing your fluorescent marker in any of the dilutions, additional dilutions may be required–further diluting the transfection allows for expression of your reporter gene.
    12. Freeze all plates as CPE occurs, and take the lowest dilution that has your recombinant gene expressed to purify further.
    13. Continue diluting and passing recombinant virus until you can observe fluorescence in all cells showing CPE. At least 5 passages are recommended. 
    14. Confirm purity by the absence of the deleted gene by conducting PCR of an internal portion of the deleted gene or by Whole Genome Sequencing.

Data analysis

In our previous work (Borca et al., 2018), we used CRISPR/Cas9 as an alternative approach to introduce gene deletions in ASFV in primary swine macrophage cell lines and compared the homologous recombination rate with traditional homologous recombination methods. It showed that using the approach as described in the above procedure, has a 4 log increase in the homologous recombination rate. This method will allow for manipulation of outbreak strains without tissue culture adaption (a timely process, that often results in genome instability), and customized production using new emerging ASFV isolates as the backbone for a live-attenuated vaccine.

Notes

Troubleshooting

  1. Low levels of recombinant virus
    A high-transfection efficiency is required for recombination to occur with the viral genome. Observing a high transfection efficiency doesn’t mean recombinant virus has been produced. Passage 1 of the virus determines recombination efficiency and the amount of recombinant virus in the sample. The amount of recombinant virus should be increasing with increasing passages. If this is not the case, go back to the previous passage. 
  2. Essential genes
    If the recombination efficiency is low, it is possible that the virus gene being targeted could be essential. If this is the case, try partial gene deletions or gene mutations. It is possible that you can observe a small amount of what appears to be recombinant virus in Passage 1 even if the gene is essential, if the gene can be complemented by the non-recombinant virus. It is unlikely that this will be observed in Passage 2 of the recombinant virus. 
  3. Poor infection of macrophages
    Improper handling of macrophages and possible animal variation can lead to isolation of cells that are not capable of supporting ASFV infection. Although this is rare, use of a different animal and careful handling of macrophages can improve infection.

Recipes

  1. L929 conditioned media
    500 ml RPMI 1640 medium
    55 ml Fetal Bovine Serum-Gamma Irradiated
    12.5 ml HEPES 1 M
    5.5 ml L-Glutamine 200 nM
    5.5 ml Antibiotic-Antimycotic
    0.55 ml Gentamicin
    Condition media as follows: 
    1. Seed a T-25 flask between 5 x 106 and 1 x 107 cells in total 15 ml of media 
    2. Transfer to a T150 flask in total 35 ml volume when confluent
    3. When confluent, detach and resuspend cells in 12 ml media in T150
    4. Add 3 ml of cells to each of (d) 2 L Roller Bottles, and add 250 ml L929 media to each
    5. Allow growing for 11 days
    6. Pour contents/media into 250 ml centrifuge bottles
    7. Centrifuge at 5,500 x g in a Sorval SLA-1500 for 20 min
    8. Collect supernatant and filter through a 0.22 μm filter
    9. Freeze at -20 °C until use
  2. Macrophage Wash Media
    250 ml RPMI 1640
    100 ml FBS
    5 ml Anti-Anti
    0.5 ml Gentamycin 50 mg/ml
  3. Macrophage complete media
    250 ml RPMI 1640
    150 ml conditioned L929 media
    100 ml FBS (16.5% final concentration)
    5 ml Anti-Anti
    0.5 ml Gentamycin 50 mg/ml
    150 ml donor swine plasma
  4. PBS + EDTA
    500 ml PBS
    10 ml 0.5 M EDTA

Acknowledgments

This project was funded under Award Numbers HSHQDC-11-X-00077 and HSHQPM-12-X-00005 through an interagency agreement with the Science and Technology Directorate of the U.S. Department of Homeland Security. The macrophage isolation protocol was adapted from Zsak et al. (1996).

Competing interests

The authors declare no conflicts of interest or competing interests.

References

  1. Borca, M. V., Holinka, L. G., Berggren, K. A. and Gladue, D. P. (2018). CRISPR-Cas9, a tool to efficiently increase the development of recombinant African swine fever viruses. Sci Rep 8(1): 3154.
  2. Enjuanes, L., Carrascosa, A. L., Moreno, M. A. and Vinuela, E. (1976). Titration of African swine fever (ASF) virus. J Gen Virol 32(3): 471-477.
  3. O'Donnell, V., Holinka, L. G., Krug, P. W., Gladue, D. P., Carlson, J., Sanford, B., Alfano, M., Kramer, E., Lu, Z., Arzt, J., Reese, B., Carrillo, C., Risatti, G. R. and Borca, M. V. (2015). African swine fever virus Georgia 2007 with a deletion of virulence-associated gene 9GL (b119L), when administered at low doses, leads to virus attenuation in swine and induces an effective protection against homologous challenge. J Virol 89(16): 8556-8566.
  4. Okoli, A., Okeke, M. I., Tryland, M. and Moens, U. (2018). CRISPR/Cas9-advancing orthopoxvirus genome editing for vaccine and vector development. Viruses 10(1).
  5. Suenaga, T., Kohyama, M., Hirayasu, K. and Arase, H. (2014). Engineering large viral DNA genomes using the CRISPR-Cas9 system. Microbiol Immunol 58(9): 513-522.
  6. Tang, Y. D., Liu, J. T., Wang, T. Y., An T. Q., Sun, M. X., Wang, S. J., Fang, Q. Q., Hou, L. L., Tian, Z. J. and Cai, X. H. (2016). Live attenuated pseudorabies virus developed using the CRISPR/Cas9 system. Virus Res 225(2): 33-39.
  7. Tulman, E. R., Delhon, G. A., Ku, B. K., Rock, D. L. (2009). African swine fever virus. In: Lesser Known Large dsDNA Viruses. Vol. 328 43-87. Current Topics in Microbiology and Immunology. Springer.
  8. Yuan, M., Gao, X., Chard, L. S., Ali, Z., Ahmed, J., Li, Y., Liu, P., Lemoine, N. R. and Wang, Y. (2015). A marker-free system for highly efficient construction of vaccinia virus vectors using CRISPR Cas9. Mol Ther Methods Clin Dev 2: 15035.
  9. Yuan, M., Wang, P., Chard, L. S., Lemoine, N. R. and Wang, Y. (2016). A simple and efficient approach to construct mutant vaccinia virus vectors. J Vis Exp (116).
  10. Zsak, L., Lu, Z., Kutish, G. F., Neilan, J. G. and Rock, D. L. (1996). An African swine fever virus virulence-associated gene NL-S with similarity to the herpes simplex virus ICP34.5 gene. J Virol 70(12): 8865-8871.

简介

大型DNA病毒(例如非洲猪瘟病毒(ASFV))的基因编辑传统上依赖于由报道盒组成的供体质粒与周围同源病毒DNA的同源重组。然而,这种导致所需修饰病毒的同源重组是罕见的事件。我们最近报道了使用CRISPR / Cas9编辑ASFV。使用CRISPR / Cas9修饰非洲猪瘟病毒基因组导致了引入遗传变化的快速且相对简单的方法。为了实现这一目标,我们首先用田间分离株ASFV-G感染原代猪巨噬细胞,并用CRISPR / Cas9供体质粒转染质粒,该质粒将表达靶向我们基因的特异性gRNA被删除。通过插入报告盒,我们能够通过有限稀释和噬菌斑纯化从亲本中纯化我们的重组病毒。我们以前曾报道将传统的同源重组方法与CRISPR / Cas9进行比较,结果导致重组增加超过4个对数。

【背景】 非洲猪瘟(ASF)是一种由ASF病毒(ASFV)引起的高度致命的猪传染性病毒性疾病。 ASFV的基因组由大约180-190千碱基对的双链DNA基因组组成。 ASFV引起一系列疾病,从高度致命到亚临床,取决于宿主特征和病毒株(Tulman et al。,2009)。 ASFV没有商业疫苗;实验上,2007年格鲁吉亚爆发的唯一能够抵御目前流行病毒株的疫苗(ASFV-G)是含有一个或多个病毒基因组缺失的减毒活疫苗,例如:( O'Donnell et al。,2015)。传统上,ASFV的基因缺失已通过同源重组进行,其中含有同源基因组序列的供体质粒用于基因缺失(O'Donnell 等人,2015),但这仅发生在非常低速率,使重组ASFV的生产变得困难(Borca et al。,2018)。

最近,我们报道了使用CRISPR / Cas9作为在ASFV中引入基因缺失的替代方法,与传统方法相比增加了4个对数(Borca et al。,2018)。在使用CRISPR / Cas9的重组中,这种增加允许从亲本野生型更容易地生产和纯化重组病毒。有可能使用CRISPR / Cas9将允许病毒蛋白突变,扩展我们解剖关键域的毒力的能力,甚至可能在以前被确定为必需的基因中。这种方法已经成功地报道了其他大型DNA病毒,包括Orthopox(Okoli et al。,2018),痘苗病毒(Yuan et al。,2015和2016),疱疹单纯疱疹病毒(Suenaga et al。,2014)和伪狂犬病(Tang et al。,2016)。

关键字:ASFV, 非洲猪瘟, ASF, CRISPR, CRISPR/Cas9

材料和试剂

  1. 移液器吸头10μl,200μl,1,000μl(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:2140-05,2160P,2079)
  2. Primaria TM 6孔板(Corning,目录号:353846)
  3. Primaria TM 组织培养瓶T-75过滤瓶(Corning,Falcon®,目录号:353824)
  4. 50毫升锥形管(Corning,Falcon ®,目录号:352098)
  5. 聚丙烯微量离心管
  6. T-25烧瓶(康宁,目录号:3056)
  7. T-150烧瓶(康宁,目录号:3151)
  8. 0.22μm过滤器(Corning,目录号:430769)
  9. 非洲猪瘟病毒野外分离株如ASFV-G(从受感染的猪血清中分离并向猪巨噬细胞中添加血清以繁殖病毒。按照Enjuanes 等人的描述进行滴定,1976)。&nbsp ;
  10. 约克郡猪3-12个月作为献血者
  11. Cas9供体质粒(通过蓝鹭生物定制设计用于感兴趣的目标。参见附带的插入盒序列(补充文件)
  12. Fugene HD(Active Motif,目录号:32042)
  13. gRNA质粒(由蓝鹭生物定制设计用于感兴趣的目标。)
  14. OptiMEM(Thermo Fisher Scientific,Gibco TM ,目录号:31985062)
  15. 磷酸盐缓冲盐水(PBS)1x,pH 7.0-7.3(Thermo Fisher Scientific,Gibco TM ,目录号:14190144)
  16. 0.5 M EDTA pH 8.0(Thermo Fisher Scientific,Invitrogen TM ,目录号:15575020)
  17. Ficoll-Paque PLUS密度梯度为1.077 g / ml(GE Healthcare,目录号:17144002)
  18. RPMI 1640培养基(Thermo Fisher Scientific,Gibco TM ,目录号:11875093)
  19. γ辐照胎牛血清(GE Healthcare,Hyclone TM ,目录号:SH30071.03)
  20. 抗生素 - 抗真菌药(100x)(Thermo Fisher Scientific,Gibco TM ,目录号:15240096)
  21. 庆大霉素(Thermo Fisher Scientific,Gibco TM ,目录号:15750060)
  22. HEPES 1 M(Thermo Fisher Scientific,Gibco TM ,目录号:15630080)
  23. L-谷氨酰胺,200 nM(Thermo Fisher Scientific,Gibco TM ,目录号:25030081)
  24. L929培养基(L细胞,L929,菌株L的衍生物)(ATCC,目录号:CCL-1)
  25. 肝素(Sagent Pharmaceuticals,目录号:400-10)
  26. L929条件培养基(见食谱)
  27. 巨噬细胞洗涤培养基(见食谱)
  28. 巨噬细胞完全培养基(见食谱)
  29. PBS + EDTA(见食谱)

设备

  1. P1,000,P200,P20,P10移液器(Gilson)
  2. 2升滚瓶(康宁,目录号:431329)
  3. 250毫升离心瓶(Thermo Fisher Scientific,Thermo Scientific TM ,Nalgene TM ,目录号:3141-0250)
  4. 倒置荧光显微镜(Carl-Zeiss)
  5. 血细胞计数
  6. 移液器(Drummond Scientific,目录号:4-000-101)
  7. 台式离心机(Eppendorf,型号:5810)
  8. 落地式离心机(Thermo Fisher Scientific,Thermo Scientific TM ,型号:Sorvall ® RC-4)
  9. Sorval SLA-1500固定角转子(Thermo Fisher Scientific,型号:SLA-1500)
  10. 涡旋混合器(Fisher Scientific,目录号:12-812)
  11. -70°C冰箱(Thermo Scientific,型号:ULT2586-9-A40)

程序

  1. 巨噬细胞分离
    1. 通过静脉穿刺获得500ml猪血并用肝素治疗。
    2. 将血液在37°C孵育1-2小时,直到观察到红细胞沉降和血液白细胞富集到上清血浆中。
    3. 收集血浆并漂浮在50ml Falcon管中的Ficoll-Paque(35ml血浆,10ml Ficoll-Paque)上。
    4. 在Eppendorf 5810中以367 x g 在室温下离心30分钟。
    5. 收集顶层并将池装入250ml离心瓶中。
    6. 在Sorval SLA-1500中在7,500 x g 下在室温下离心20分钟。
    7. 收集单核白细胞并通过添加等体积的培养基作为收集的细胞用巨噬细胞培养基洗涤,倒置数次以混合。
    8. 在Eppendorf 5810中以367 x g 在室温下离心10分钟。
    9. 除去上清液,加入10ml巨噬细胞培养基洗涤细胞,再按步骤8离心,共洗涤3次。
    10. 将细胞重悬于10ml巨噬细胞完全培养基中。
    11. 将细胞培养成5个Primaria TM T-75烧瓶(每个含有1ml细胞和10ml巨噬细胞培养基)并孵育过夜。
    12. 粘附细胞现在被认为是巨噬细胞。
    13. 使用PBS EDTA分离细胞,用10ml洗涤并在37℃下用10ml孵育直至细胞脱离(通常15-20分钟)。
    14. 在血细胞计数器中使用10μl细胞悬浮液计数巨噬细胞。
    15. 在Primaria TM 6孔板中以每孔5×10 6 的密度平板巨噬细胞,并允许连接过夜。

  2. CRISPR / Cas9的感染/转染
    1. 用ASFV的野外分离物接种每个孔,例如ASFV-G或ASFV的其他野外分离株,感染复数(MOI)为1.0。通过将病毒直接添加到含有2ml培养基的每个孔的培养基中接种,轻轻摇动平板以混合并在37℃,5%CO 2 下孵育1小时。
    2. 将悬浮在ddH 2 O中的1.6μg供体质粒和1.6μggRNA质粒加入150μl在聚丙烯微量离心管中的optiMEM中。&nbsp;
    3. 通过移液或短暂涡旋小心混合。
    4. 通过移液加入10μlFugeneHD混合物并孵育10分钟。
    5. 向含有2ml培养基的巨噬细胞中滴加150μl复合物。
    6. 在37℃,5%CO 2 下孵育24小时。
    7. 转染后24小时在荧光显微镜下观察报告基因,以确定转染率。通过观察表达荧光标记物的附着细胞和未附着细胞来计算百分比。&nbsp;
    8. 在-70°C冷冻板,并储存直至准备使用。
    9. 从转染中取1 ml冷冻培养基,在巨噬细胞培养基中进行1:10稀释(1:10,1:100,1:1,000; 等)。
    10. 将100μl稀释液加入到如步骤B7中制备的6孔巨噬细胞平板中,最终体积为2ml培养基。在37℃,5%CO 2 下孵育。
    11. 每24小时观察完整的CPE(细胞病变效应)(通常为1-7天),标记对您的荧光标记呈阳性的孔。如果完全CPE快速发生(<48小时)而没有在任何稀释液中看到您的荧光标记物,则可能需要额外的稀释液 - 进一步稀释转染物可以表达您的报告基因。
    12. 当CPE发生时冻结所有平板,并采用表达重组基因的最低稀释液进一步纯化。
    13. 继续稀释并通过重组病毒,直到您可以在所有显示CPE的细胞中观察到荧光。建议至少传递5个段落。&nbsp;
    14. 通过对缺失基因的内部部分进行PCR或通过全基因组测序确认缺失基因的纯度。

数据分析

在我们之前的工作中(Borca et al。,2018),我们使用CRISPR / Cas9作为替代方法在原代猪巨噬细胞系中引入ASFV中的基因缺失,并将同源重组率与传统同源物进行比较。重组方法。它表明,使用上述方法中描述的方法,同源重组率增加4个对数。该方法将允许在没有组织培养适应的情况下操纵爆发菌株(及时的过程,通常导致基因组不稳定),并使用新出现的ASFV分离株作为减毒活疫苗的骨干进行定制生产。

笔记

故障排除

  1. 低水平的重组病毒
    与病毒基因组重组发生需要高转染效率。观察到高转染效率并不意味着已经产生了重组病毒。病毒的第1代确定了重组效率和样品中重组病毒的量。随着传代的增加,重组病毒的数量应该增加。如果不是这种情况,请返回上一段。&nbsp;
  2. 必需基因
    如果重组效率低,则靶向的病毒基因可能是必需的。如果是这种情况,尝试部分基因缺失或基因突变。如果基因可以被非重组病毒补充,即使基因是必需的,你也可以在第1代中观察到少量似乎是重组病毒的东西。在重组病毒的第2代中不太可能观察到这种情况。&nbsp;
  3. 巨噬细胞感染不良
    巨噬细胞的处理不当和可能的动物变异可导致分离不能支持ASFV感染的细胞。虽然这种情况很少见,但使用不同的动物和小心处理巨噬细胞可以改善感染。

食谱

  1. L929条件媒体
    500毫升RPMI 1640培养基
    55毫升胎牛血清-γ辐射
    12.5毫升HEPES 1 M
    5.5毫升L-谷氨酰胺200 nM
    5.5毫升抗生素 - 抗真菌药 0.55毫升庆大霉素
    条件媒体如下:&nbsp;
    1. 将T-25烧瓶置于总共15ml培养基中的5×10 6 和1×10 5个细胞之间。
    2. 当汇合时转移到总共35ml体积的T150烧瓶中
    3. 在T150中将细胞汇合,分离并重悬于12ml培养基中
    4. 向(d)2L滚瓶中的每一个中加入3ml细胞,并向每个滚瓶中加入250ml L929培养基
    5. 允许生长11天
    6. 将内容物/培养基倒入250ml离心瓶中
    7. 在Sorval SLA-1500中以5,500 x g 离心20分钟
    8. 收集上清液并通过0.22μm过滤器过滤
    9. 在-20°C冷冻直至使用
  2. 巨噬细胞清洗培养基
    250毫升RPMI 1640
    100毫升FBS
    5毫升抗 - 抗 0.5ml庆大霉素50mg / ml
  3. 巨噬细胞完整的媒体
    250毫升RPMI 1640
    150毫升条件L929培养基
    100ml FBS(终浓度16.5%)
    5毫升抗 - 抗 0.5毫升庆大霉素50毫克/毫升
    150毫升供体猪血浆
  4. PBS + EDTA
    500毫升PBS
    10毫升0.5毫升EDTA

致谢

该项目由奖项编号HSHQDC-11-X-00077和HSHQPM-12-X-00005通过与美国国土安全部科学技术局的机构间协议资助。巨噬细胞分离方案改编自Zsak 等人(1996)。作者声明没有利益冲突或竞争利益。

参考

  1. Borca,M.V.,Holinka,L.G.,Berggren,K.A。和Gladue,D.P。(2018)。 CRISPR-Cas9,一种有效增加重组非洲猪瘟病毒发展的工具。 Sci Rep 8(1):3154。
  2. Enjuanes,L.,Carrascosa,A.L.,Moreno,M。A.和Vinuela,E。(1976)。 滴定非洲猪瘟(ASF)病毒。 J Gen Virol < / em> 32(3):471-477。
  3. O'Donnell,V.,Holinka,LG,Krug,PW,Gladue,DP,Carlson,J.,Sanford,B.,Alfano,M.,Kramer,E.,Lu,Z.,Arzt,J.,Reese ,B.,Carrillo,C.,Risatti,GR和Borca,MV(2015)。 非洲猪瘟病毒格鲁吉亚2007年删除毒力相关基因 9GL (b119L),当以低剂量施用时,导致猪中的病毒减毒并诱导针对同源攻击的有效保护。 J Virol 89(16):8556-8566。
  4. Okoli,A.,Okeke,M。I.,Tryland,M。和Moens,U。(2018)。 用于疫苗和载体开发的CRISPR / Cas9-推进正痘病毒基因组编辑。 病毒 10(1)。
  5. Suenaga,T.,Kohyama,M.,Hirayasu,K。和Arase,H。(2014)。 使用CRISPR-Cas9系统设计大型病毒DNA基因组。 Microbiol Immunol 58(9):513-522。
  6. Tang,Y.D.,Liu,J.T。,Wang,T.Y.,An T.Q.,Sun,M.X.,Wang,S.J.Fang,Q.Q.,Hou,L.L.Tan,Z.J。和Cai,X.H。(2016)。 使用CRISPR / Cas9系统开发的减毒伪狂犬病病毒。 Virus Res 225(2):33-39。
  7. Tulman,E.R.,Delhon,G.A.,Ku,B.K.,Rock,D.L。(2009)。 非洲猪瘟病毒。在:鲜为人知的大型dsDNA病毒。卷。 328 43-87。 当前微生物学和免疫学专题。 Springer。
  8. Yuan,M.,Gao,X.,Chard,L。S.,Ali,Z.,Ahmed,J.,Li,Y.,Liu,P.,Lemoine,N.R。和Wang,Y。(2015)。 使用CRISPR Cas9高效构建痘苗病毒载体的无标记系统。 Mol Ther Methods Clin Dev 2:15035。
  9. Yuan,M.,Wang,P.,Chard,L。S.,Lemoine,N。R. and Wang,Y。(2016)。 构建突变痘苗病毒载体的简单有效方法。 J Vis Exp (116)。
  10. Zsak,L.,Lu,Z.,Kutish,G.F.,Neilan,J.G。和Rock,D.L。(1996)。 非洲猪瘟病毒毒力相关基因NL-S与单纯疱疹病毒相似 ICP34.5 基因。 J Virol 70(12):8865-8871。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Borca, M. V., Berggren, K. A., Ramirez-Medina, E., Vuono, E. A. and Gladue, D. P. (2018). CRISPR/Cas Gene Editing of a Large DNA Virus: African Swine Fever Virus. Bio-protocol 8(16): e2978. DOI: 10.21769/BioProtoc.2978.
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