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Creating a RAW264.7 CRISPR-Cas9 Genome Wide Library
创建RAW264.7 CRISPR-Cas9全基因组文库   

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Abstract

The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing tools are used in mammalian cells to knock-out specific genes of interest to elucidate gene function. The CRISPR-Cas9 system requires that the mammalian cell expresses Cas9 endonuclease, guide RNA (gRNA) to lead the endonuclease to the gene of interest, and the PAM sequence that links the Cas9 to the gRNA. CRISPR-Cas9 genome wide libraries are used to screen the effect of each gene in the genome on the cellular phenotype of interest, in an unbiased high-throughput manner. In this protocol, we describe our method of creating a CRISPR-Cas9 genome wide library in a transformed murine macrophage cell-line (RAW264.7). We have employed this library to identify novel mediators in the caspase-11 cell death pathway (Napier et al., 2016); however, this library can then be used to screen the importance of specific genes in multiple murine macrophage cellular pathways.

Keywords: CRISPR(CRISPR), Screen(筛查), Macrophages(巨噬细胞), Library(文库), RAW264.7(RAW264.7)

Background

Historically, understanding the contribution of specific genes to phenotypes of interest in eukaryotic cells was possible using RNA interference (RNAi) or cells derived from knockout mice. However, within the last few years the new genome editing technique CRISPR-Cas9 has allowed for easy and efficient generation of knockout cell lines and genome-wide screens within eukaryotic cells. CRISPR-Cas9 genome-wide screens have expanded the toolbox for mammalian genetics and for the identification of novel proteins and their contributions to specific phenotype. Using this method, researchers have been able to identify novel genes involved in tumor growth (Chen et al., 2015; Kiessling et al., 2016; Steinhart et al., 2017), microbial entry and replication (Popov et al., 2015; Marceau et al., 2016), cell death pathways (Shi et al., 2015; Napier et al., 2106), and much more. Here we harness the CRISPR-Cas9 system, to create a genome-wide knockout library in a murine macrophage cell line. Macrophages are the crux of many innate immune responses to invading pathogens or danger signals. By creating a genome-wide knockout library in macrophages we can now begin to identify novel mediators of these innate immune responses to identify novel diagnostic and therapeutic targets for acute and chronic inflammation.

Materials and Reagents

  1. Pipette tips
  2. 10 cm bacteriological tissue culture (TC) treated Petri dish (Corning, Falcon®, catalog number: 353003 )
  3. 6-well plates TC treated (Corning, Costar®, catalog number: 353502 )
  4. 15 ml Falcon tube (E&K Scientific Products, catalog number: EK-4020 )
  5. 50 ml Falcon tube (E&K Scientific Products, catalog number: EK-4023 )
  6. Cell scraper (SARSTEDT, catalog number: 83.1830 )
  7. 0.45 μm syringe filters (Corning, catalog number: 431225 )
  8. T-75 flask (Corning, Falcon®, catalog number: 353136 )
  9. T-125 flask (Corning, Falcon®, catalog number: 353112 )
  10. Cryovials (Corning, catalog number: 430659 )
  11. RAW264.7 cells (ATCC, catalog number: TIB-71 )
  12. 293T cells (ATCC, catalog number: CRL-3216 )
  13. hCas9 plasmid (Addgene, catalog number: 52962 )
  14. FuGene (Promega, catalog number: E2311 )
  15. VSV-G (Addgene, catalog number: 8454 )
  16. pAdVAntage (Promega, catalog number: E1711 )
  17. △VPR (Addgene; catalog number: 8455 )
  18. Genome-wide Mouse lentiviral CRISPR gRNA library v1 (Addgene, catalog number: 50947 )
  19. Filtered DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 11995073 )
  20. Filtered (0.2 μm filter) heat-inactivated (30 min at 37 °C) FBS (Thermo Fisher Scientific, GibcoTM; stock specific)
  21. Protamine sulphate (Sigma-Aldrich, catalog number: P3369-10G ), keep stock at 8 mg/ml at 4 °C
  22. Blasticidin hydrochloride (MP Biomedicals, catalog number: 02150477-25 mg ), keep aliquoted stock at 10 mg/ml at -20 °C
  23. DMSO (Fisher Scientific, catalog number: BP231-100 )
  24. Puromycin dihydrochloride from Streptomyces alboniger (Sigma-Aldrich, catalog number: P8833 )
  25. RAW264.7 and 293T tissue culture media (see Recipes)
  26. Blasticidin selection media (see Recipes)
  27. Puromycin selection media (see Recipes)
  28. Tissue culture freezing media (see Recipes)

Equipment

  1. Pipettes
  2. Fluorescence microscope that can visualize BFP
  3. Incubator at 37 °C with 5% CO2
  4. Centrifuge

Procedure

  1. Making hCas9-expressing RAW264.7 cells
    1. D0: Plate 5 x 106 293T cells in a tissue culture (TC) treated 10 cm plate in 10 ml DMEM + 10% FBS, and incubate overnight at 37 °C with 5% CO2. We aim for 30-40% confluency.
    2. D1: Create hCas9-expressing retrovirus in 293T cells.
      1. Premix plasmids in 150 μl of serum-free DMEM
        For a 10 cm dish (retrovirus PMX-hCas9-blasti):
        1.3 μg △VPR
        0.87 μg VSV-G
        0.55 μg p-Advant
        0.73 μg PMX-hCas9-blasti (For the plasmid map see http://www.addgene.org/52962/)
      2. Add 15 μl of Fugene and let sit at 25 °C for 20 min.
      3. During incubation replace 293T media (see Recipes) with 10 ml of fresh 37 °C DMEM + 10% FBS.
      4. Add mixture drop by drop on 293T cells and mix by gently rocking plate back and forth.
      5. Incubate at 37 °C + 5% CO2 overnight.
    3. D2:
      1. Gently aspirate media off transfected 293T cells and replace with 6-8 ml of 37 °C DMEM + 10% FBS. The monolayer will be delicate (see Note 1).
      2. Plate RAW264.7 cells for transduction with Cas9-expressing virus on D3, at 3 x 105 cells/well in a 6-well plate, 1 well per sample. Additionally, plate 3x RAW264.7 cells at 3 x 105 cells/well in a 6-well plate for assessing viral titer.
    4. D3: Harvest virus and transduce RAW cells
      1. Harvest virus in the morning and the evening, from the same well (see Note 2).
      2. At each time point, gently remove supernatant from 293T cells that contains virus. For the morning time point, after removing supernatant add 6 ml of 37 °C DMEM + 10% FBS media to the 293T virus-producing cells. Again, be very careful with this monolayer.
      3. Filter virus-containing media with 0.45 μm syringe filter to ensure removal of cellular debris.
      4. Add protamine sulphate at 8 μg/ml to the filtered virus-containing media.
      5. Aspirate media off seeded RAW264.7 cells and add 2-5 ml of supernatant containing viruses (see Note 3). The control will just be DMEM only (see Note 4).
      6. For viral titer, prepare 3 10-fold dilutions of virus in serum-free DMEM and continue with assessing viral titer by plaque assay as previously published (Watanabe et al., 2016).
      7. Incubate at 37 °C + 5% CO2 overnight.
    5. D4: Aspirate media off RAW264.7 cells and add new 37 °C DMEM + 10% FBS media to transduced RAW cells.
    6. D5: Transfer transduced RAW264.7 cells from 6-well plate to TC treated 10 cm dish. This split should yield an ideal 30% confluence for antibiotic selection the next day. At this time, plate non-transduced RAW264.7 cells in 10 cm at a similar confluency as a negative control for your antibiotic selection (~1 x 106 cells per 10 cm dish).
      1. Remove media from transduced RAW264.7 cells, replace with 3 ml of fresh 37 °C DMEM + 10% FBS media.
      2. Using cell scrapers, scrape adherent cells off of bottom of wells and resuspend in media by pipetting up and down.
      3. Replate cells in a TC treated 10 cm dish in 10 ml of fresh 37 °C DMEM + 10% FBS media, aiming for 30% confluency. No cell count is necessary; this split should yield an ideal 30% confluency.
      4. Incubate at 37 °C + 5% CO2 overnight.
    7. D6: Antibiotic selection
      1. Removed media from transduced RAW264.7 cells.
      2. Add 4 ml of blasticidin selection media (see Recipes) (37 °C DMEM + 10% FBS + 10 μg/ml blasticidin) to transfected RAW264.7 cells and non-transfected RAW264.7 cells for a negative control.
      3. Incubate at 37 °C + 5% CO2 for 24-48 h.
    8. D7-8:
      1. Aspirate media off selected RAW264.7-hCas9 expressing cells and replace with 4 ml of fresh 37 °C DMEM + 10% FBS + 10 μg/ml blasticidin.
      2. Incubate at 37 °C + 5% CO2 for 24-48 h to expand cell line. Incubation period will vary, however you are aiming for > 90% cell death in your non-transfected RAW264.7 cell negative control plate.
    9. D8-9:
      1. Aspirate media off selected RAW264.7-hCas9 expressing cells and add 10 ml of 37 °C DMEM + 10% FBS. Incubate these cells for 2-4 days to allow RAW264.7 cell recovery from the selection process before replating for experiments or freezing back.
      2. When freezing back: Use the cell scrapers, scrape off selected RAW264.7-hCas9 expressing cells and either freeze back cells in tissue culture freezing media (see Recipes) (50% DMEM + 40% FBS + 10% DMSO) or re-plate for future experiments.

  2. Amplifying CRISPR gRNA library
    1. Follow protocol previously published (Koike-Yusa et al., 2014; https://media.addgene.org/data/67/85/9641c46c-8523-11e3-b6c5-000c298a5150.pdf). Use the ‘on-plate culture’ method of amplifying the library, as the ‘liquid culture’ method can lead to exclusion of some of the plasmids in the library (Figure 1).
    2. Freeze back amplified CRISPR gRNA plasmid library.


      Figure 1. Graphical abstract of Koike-Yusa et al. (2014) amplification protocol of the gRNA library

  3. Creating CRISPR gRNA lentiviral library
    1. Before starting, see Note 5.
    2. D0: Plate 1 x 106 293T cells per T-175 in 25 ml of DMEM + 10% FBS.
    3. D1: Creating CRISPR gRNA lentiviral library.
      1. Premix plasmids in 150 μl of serum-free DMEM.
        For a T-175 flask:
        4 μg △VPR
        2.6 μg VSV-G
        1.66 μg p-Advant
        6.6 μg CRISPR library
      2. Add 45 μl of Fugene and let sit at 25 °C for 20 min.
      3. During incubation replace 293T media with 25 ml of fresh, 37 °C DMEM + 10% FBS.
      4. Add mixture drop by drop on 293T cells and mix with soft horizontal movements.
        Incubate at 37 °C + 5% CO2 overnight.
    4. D2:
      1. Gently replace media on transfected 293T cells with 20 ml 37 °C DMEM + 10% FBS (see Note 3).
      2. Plate RAW264.7-hCas9 expressing cells for transfection on D3. To calculate needed RAW264.7 cells, see Procedure C. Additionally, plate 3x RAW264.7 cells at 3 x 105 cells/well in a 6-well plate for assessing viral titer.
    5. D3: Harvest virus and transduce RAW264.7 cells.
      1. Harvest virus in the AM and PM.
      2. Removed supernatant from 293T cells that contains virus.
      3. Filter with 0.45 μm syringe filter.
      4. Add protamine sulphate at 8 μg/ml.
      5. Aspirate media off seeded cells and add X ml of supernatant containing viruses, so that you have 30% transfection efficiency, and add 37 °C DMEM + 10% FBS up to 20 ml per T-175 flask (see Note 5).
      6. At this point, you can freeze back your virus to check for viral titer if you haven’t checked the transfection efficiency previously.
      7. Incubate at 37 °C + 5% CO2.
    6. D4: Plate 5 x 106 RAW264.7-hCas9 expressing cells in a T-175 flask in 25 ml of 37 °C DMEM + 10% FBS for antibiotic selection negative control.
    7. D6: Check transfection efficiency using fluorescence microscope and antibiotic selection.
      1. BFP is constitutively expressed on the lentiviral CRISPR library plasmid (Koike-Yusa et al., 2014), and you can visualize transfection efficiency by plating an aliquot of transfected cells and calculating the % fluorescence. This will not be a quantitative measure of transfection, but will allow you to be confident that your transfection worked before moving on to the antibiotic selection. Use only the cells that have been selected using antibiotics for future work.
      2. For puromycin selection for RAW264.7 transfected cells:
        1. Aspirate media off transfected RAW264.7 transfected cells and negative control, and replace with 20 ml of 37 °C DMEM + 10% FBS + 5 μg/ml puromycin.
        2. Incubate for 72 h at 37 °C + 5% CO2.
    8. D9: Split and expand RAW264.7 cells transfected with the CRISPR gRNA lentiviral library.
      1. Aspirate media off puromycin-selected RAW264.7 cells transfected with the CRISPR lentiviral library.
      2. Add 10 ml of 37 °C DMEM + 10% FBS + 5 μg/ml puromycin.
      3. Scrape adherent RAW264.7 cells off of the T-175 flask into the 10 ml of media.
      4. Pool all of selected RAW264.7 cells transfected with the CRISPR lentiviral library and pellet at 1,000 x g for 5 min.
      5. Resuspend the pellet of selected RAW264.7 cells transfected with the CRISPR lentiviral library in 37 °C DMEM + 10% FBS + 5 μg/ml puromycin and plate 1 x 106 cells per T-175 flask. There should be many T-175 flasks.
      6. Incubate for 72 h at 37 °C + 5% CO2.
    9. D13: Pool and freeze RAW264.7 cells transfected with the CRISPR gRNA lentiviral library.
      1. Each T-175 flask should contain many millions of selected RAW264.7 cells transfected with the CRISPR lentiviral library (on average 3-5 x 107).
      2. Aspirate media off selected RAW264.7 cells transfected with the CRISPR lentiviral library.
      3. Add 10 ml of 37 °C DMEM + 10% FBS.
      4. Scrape adherent RAW264.7 cells off of the T-175 flask into the 10 ml of media.
      5. Pool all of selected RAW264.7 cells transfected with the CRISPR lentiviral library and pellet at 1,000 x g for 5 min.
      6. Resuspend pellet in 50 ml of 37 °C DMEM + 10% FBS and count cells.
      7. At this point, freeze back the library at 1.5 x 107 cells per cryovial in 50% DMEM + 40% FBS + 10% DMSO media or use these cells for screening.

Data analysis

For sequencing your library and analyzing the coverage of your library or your library results, please refer to our original publication (Napier et al., 2016).

Notes

  1. Late in the day, you may start to see syncytia. Syncytia are a sign that the viruses are being produced.
  2. Retroviruses more easily infect mitotic cells, thus by harvesting twice from the same well in the AM and PM, you will double your chances of catching the RAW264.7 cells dividing.
  3. You can store the rest in 4 °C for re-infection later for up to 7 days. However, freezing decreases titers significantly.
  4. In our hands, transduction of RAW264.7 cells do not change the immunological profile of the cell line, however, if another cell line is being used in this protocol, it is imperative to use a retroviral control with a non-coding sequence to ensure that the phenotype of interest is not altered after transduction.
  5. This protocol has been standardized for cell transfection efficiency of 30% by lentivirus produced by 293T cells. This lower transfection efficiency is used to decrease the frequency of double or triple transfection events happening the same cell. You will be transfecting 1,000-fold the gRNA library in cells. For this protocol, there are 87,897 gRNAs (Koike-Yusa et al., 2014); therefore, we transfect (88 million RAW246.7 cells) x (30%) = 264 million RAW246.7 cells to maintain 1,000-fold coverage with 30% transfection efficiency.

Recipes

  1. RAW264.7 and 293T tissue culture media
    37 °C DMEM
    10% FBS
  2. Blasticidin selection media
    37 °C DMEM
    10% FBS
    10 μg/ml blasticidin
  3. Puromycin selection media
    37 °C DMEM
    10% FBS
    5 μg/ml puromycin
  4. Tissue culture freezing media
    50% DMEM
    40% FBS
    10% DMSO

Acknowledgments

This research was supported by the National Institute of Allergy and Infectious Diseases grants 1F32AI115959-01 (to B.A. Napier) and AI095396-05 (to D.M. Monack), and Defense Advanced Research Projects Agency (DARPA) grant DARPA-15-21-ThoR-FP-006 (to D.M. Monack).

References

  1. Chen, S., Sanjana, N. E., Zheng, K., Shalem, O., Lee, K., Shi, X., Scott, D. A., Song, J., Pan, J. Q., Weissleder, R., Lee, H., Zhang, F. and Sharp, P. A. (2015). Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell 160(6): 1246-1260.
  2. Kiessling, M. K., Schuierer, Sven., Stertz, Silke., Beibel, Martin., Bergling, Sebastian., Knehr, Judith., Carbone, Walter., de Vallière, Cheryl., Tchinda, Joelle., Bouwmeester, Tewis., Seuwen, Klaus., Rogler, Gerhard. and Roma, Guglielmo. (2016). Identification of oncogenic driver mutations by genome-wide CRISPR-Cas9 dropout screening. BMC genomics 17, 723.
  3. Koike-Yusa, H., Li, Y., Tan, E. P., Velasco-Herrera Mdel, C. and Yusa, K. (2014). Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat Biotechnol 32(3): 267-273.
  4. Marceau, C. D., Puschnik, A. S., Majzoub, K., Ooi, Y. S., Brewer, S. M., Fuchs, G., Swaminathan, K., Mata, M. A., Elias, J. E., Sarnow, P. and Carette, J. E. (2016). Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens. Nature 535(7610): 159-163.
  5. Napier, B. A., Brubaker, S. W., Sweeney, T. E., Monette, P., Rothmeier, G. H., Gertsvolf, N. A., Puschnik, A., Carette, J. E., Khatri, P. and Monack, D. M. (2016). Complement pathway amplifies caspase-11-dependent cell death and endotoxin-induced sepsis severity. J Exp Med 213(11): 2365-2382.
  6. Popov, L. M., Marceau, C. D., Starkl, P. M., Lumb, J. H., Shah, J., Guerrera, D., Cooper, R. L., Merakou, C., Bouley, D. M., Meng, W., Kiyonari, H., Takeichi, M., Galli, S. J., Bagnoli, F., Citi, S., Carette, J. E. and Amieva, M. R. (2015). The adherens junctions control susceptibility to Staphylococcus aureus α-toxin. Proc Natl Acad Sci U S A 112(46): 14337-14342.
  7. Shi, J., Zhao, Y., Wang, K., Shi, X., Wang, Y., Huang, H., Zhuang, Y., Cai, T., Wang, F. and Shao, F. (2015). Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526(7575): 660-665.
  8. Steinhart, Z., Pavlovic, Z., Chandrashekhar, M., Hart, T., Wang, X., Zhang, X., Robitaille, M., Brown, K. R., Jaksani, S., Overmeer, R., Boj, S. F., Adams, J., Pan, J., Clevers, H., Sidhu, S., Moffat, J. and Angers, S. (2017). Genome-wide CRISPR screens reveal a Wnt-FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant pancreatic tumors. Nat Med 23(1): 60-68.
  9. Watanabe, S., Chan, K. K. and Vasudevan, S. G. (2016). Mouse Model of Dengue Virus Infection with Serotypes 1 and 2 Clinical Isolates. Bio-protocol 6(23): e2040.

简介

细菌聚集的定期交织的短回文重复(CRISPR)-Cas9基因组编辑工具用于哺乳动物细胞敲除感兴趣的特定基因以阐明基因功能。 CRISPR-Cas9系统要求哺乳动物细胞表达Cas9核酸内切酶,引导RNA(gRNA)引导内切核酸酶到目的基因,以及连接Cas9与gRNA的PAM序列。使用CRISPR-Cas9基因组宽的文库以无偏倚的高通量方式筛选基因组中每个基因对感兴趣的细胞表型的影响。在本协议中,我们描述了我们在转化的鼠巨噬细胞细胞系(RAW264.7)中创建CRISPR-Cas9基因组文库的方法。我们已经使用该文库来鉴定胱天蛋白酶-11细胞死亡途径中的新型介质(Napier等人,2016);然而,该文库可用于筛选特定基因在多种鼠巨噬细胞通路中的重要性。

背景 历史上,使用RNA干扰(RNAi)或源自敲除小鼠的细胞,了解特定基因对真核细胞中感兴趣的表型的贡献是可能的。然而,在过去几年中,新的基因组编辑技术CRISPR-Cas9已经允许在真核细胞内容易且有效地产生敲除细胞系和全基因组筛选。 CRISPR-Cas9基因组范围的筛选扩大了哺乳动物遗传学的工具箱和新型蛋白质的鉴定及其对特定表型的贡献。使用这种方法,研究人员已经能够鉴定参与肿瘤生长的新基因(Chen等人,2015; Kiessling等人,2016; Steinhart et al。细胞死亡途径(Shi ,2017),微生物进入和复制(Popov等人,2015; Marceau等人,2016) em等人,2015; Napier等人,2106)等等。在这里,我们利用CRISPR-Cas9系统,在鼠巨噬细胞系中创建一个全基因组的敲除文库。巨噬细胞是入侵病原体或危险信号的许多先天免疫应答的关键。通过在巨噬细胞中创建全基因组敲除文库,我们现在可以开始鉴定这些先天免疫应答的新型介质,以鉴定急性和慢性炎症的新型诊断和治疗靶点。

关键字:CRISPR, 筛查, 巨噬细胞, 文库, RAW264.7

材料和试剂

  1. 移液器提示
  2. 10厘米细菌组织培养(TC)处理的培养皿(Corning,Falcon ®,目录号:353003)
  3. TC处理的6孔板(Corning,Costar ®,目录号:353502)
  4. 15ml Falcon管(E& K Scientific Products,目录号:EK-4020)
  5. 50ml Falcon管(E& K Scientific Products,目录号:EK-4023)
  6. 细胞刮刀(SARSTEDT,目录号:83.1830)
  7. 0.45μm注射器过滤器(Corning,目录号:431225)
  8. T-75烧瓶(Corning,Falcon ®,目录号:353136)
  9. T-125烧瓶(Corning,Falcon ®,目录号:353112)
  10. 冷冻保养瓶(Corning,目录号:430659)
  11. RAW264.7细胞(ATCC,目录号:TIB-71)
  12. 293T细胞(ATCC,目录号:CRL-3216)
  13. hCas9质粒(Addgene,目录号:52962)
  14. FuGene(Promega,目录号:E2311)
  15. VSV-G(Addgene,目录号:8454)
  16. pAdVAntage(Promega,目录号:E1711)
  17. △VPR(Addgene;目录号:8455)
  18. Genome-wide Mouse慢病毒CRISPR gRNA文库v1(Addgene,目录号:50947)
  19. 过滤的DMEM(Thermo Fisher Scientific,Gibco TM ,目录号:11995073)
  20. 过滤(0.2μm过滤器)热灭活(37℃30分钟)FBS(Thermo Fisher Scientific,Gibco TM,特异性)
  21. 硫酸鱼精蛋白(Sigma-Aldrich,目录号:P3369-10G),在4℃下保持8mg/ml的存储量
  22. 杀虫素氢氯化物(MP Biomedicals,目录号:02150477-25mg)在-20℃下保持10毫克/毫升的等分试样。
  23. DMSO(Fisher Scientific,目录号:BP231-100)
  24. 来自阿尔伯格链霉菌的嘌呤霉素二氢胆碱(Sigma-Aldrich,目录号:P8833)
  25. RAW264.7和293T组织培养基(参见食谱)
  26. 杀稻瘟素选择媒体(见配方)
  27. 嘌呤霉素选择培养基(见食谱)
  28. 组织培养冷冻介质(见食谱)

设备

  1. 移液器
  2. 荧光显微镜可以显示BFP
  3. 37℃的培养箱,5%CO 2
  4. 离心机

程序

  1. 制作表达hCas9的RAW264.7细胞
    1. D0:在10ml DMEM + 10%FBS中处理10cm板的组织培养物(TC)中的板5×10 6个细胞,并在37℃下用5%CO 2孵育过夜, 2 。我们的目标是达到30-40%的汇合。
    2. D1:在293T细胞中形成表达hCas9的逆转录病毒。
      1. 预混物质粒在150μl无血清DMEM中 对于10厘米盘(逆转录病毒PMX-hCas9-blasti):
        1.3μg△VPR
        0.87μgVSV-G
        0.55μgp-Advant
        0.73μgPMX-hCas9-blasti(质粒图见 http://www。 addgene.org/52962/
      2. 加入15微升Fugene,并在25℃下放置20分钟
      3. 在孵育期间用10ml新鲜的37℃DMEM + 10%FBS代替293T培养基(参见食谱)。
      4. 在293T细胞上逐滴加入混合物,并轻轻摇摆摇匀板混合
      5. 在37℃+ 5%CO 2孵育过夜。
    3. D2:
      1. 轻轻吸出转染293T细胞的培养基,并用6-8毫升37℃的DMEM + 10%FBS代替。单层会很细腻(见注1)
      2. 在6孔板中以3×10 5个细胞/孔在D3上以Cas9表达病毒转染的RAW264.7细胞用于转导,每个样品1孔。另外,在6孔板中以3×10 5个细胞/孔板3x RAW264.7细胞板,用于评估病毒滴度。
    4. D3:收获病毒并转染RAW细胞
      1. 早晨和晚上收获病毒,从同一口井(见注2)。
      2. 在每个时间点,轻轻地从含有病毒的293T细胞中去除上清液。对于早晨时间点,除去上清液后,向293T病毒产生细胞中加入6ml 37℃的DMEM + 10%FBS培养基。再次,要非常小心这个单层。
      3. 过滤含有0.45μm注射器过滤器的含病毒介质,以确保清除细胞碎片
      4. 将8μg/ml的硫酸精蛋白加到过滤的含病毒的培养基上
      5. 吸出培养基接种RAW264.7细胞,并加入2-5ml含有病毒的上清液(见注3)。控制只是DMEM(见注4)。
      6. 对于病毒滴度,在无血清的DMEM中制备3×10倍稀释的病毒,并继续通过以前公布的斑块测定来评估病毒滴度(Watanabe等人,2016)。
      7. 在37℃+ 5%CO 2孵育过夜。
    5. D4:将RAW264.7细胞吸出培养基,并加入新的37℃DMEM + 10%FBS培养基转导RAW细胞。
    6. D5:将转导的RAW264.7细胞从6孔板转移到TC处理的10cm培养皿中。这种分裂应该在第二天产生理想的30%汇合抗生素选择。此时,以相同的汇合为10cm的非转导的RAW264.7细胞作为抗生素选择的阴性对照(每10cm皿约1×10 6个细胞)。
      1. 从转染的RAW264.7细胞中取出培养基,用3 ml新鲜的37℃DMEM + 10%FBS培养基代替。
      2. 使用细胞刮刀,将粘附的细胞从井的底部刮掉,并通过上下移动将其悬浮在培养基中。
      3. 在TC新鲜的37℃DMEM + 10%FBS培养基中的TC处理的10cm培养皿中替换细胞,以达到30%融合。没有细胞计数是必要的,这种分裂应该产生理想的30%融合。
      4. 在37℃+ 5%CO 2孵育过夜。
    7. D6:抗生素选择
      1. 从转染的RAW264.7细胞中去除培养基
      2. 加入4毫升杀草丁素选择培养基(见食谱)(37℃DMEM + 10%FBS +10μg/ml杀稻瘟菌素)转染RAW264.7细胞和未转染的RAW264.7细胞用于阴性对照。
      3. 在37℃+ 5%CO 2孵育24-48小时。
    8. D7-8:
      1. 将所选RAW264.7-hCas9表达细胞吸出培养基,并用4ml新鲜37℃的DMEM + 10%FBS +10μg/ml杀稻瘟素代替。
      2. 在37℃+ 5%CO 2孵育24-48小时以扩增细胞系。孵化期非常非常,但是您正在瞄准>未转染的RAW264.7细胞阴性对照板中有90%的细胞死亡
    9. D8-9:
      1. 将所选的RAW264.7-hCas9表达细胞吸出培养基,并加入10ml 37℃的DMEM + 10%FBS。孵育这些细胞2-4天,以允许RAW264.7细胞从选择过程中恢复,然后再进行实验或冻结。
      2. 冷冻时:使用细胞刮刀,刮掉所选择的RAW264.7-hCas9细胞,并将组织培养冷冻培养基中的细胞冷冻(参见食谱)(50%DMEM + 40%FBS + 10%DMSO)或重新平板用于未来的实验。

  2. 扩增CRISPR gRNA文库
    1. 按照先前公布的协议(Koike-Yusa等人,2014年; https://media.addgene.org/data/67/85/9641c46c-8523-11e3-b6c5-000c298a5150.pdf )。使用扩增文库的"板上培养"方法,因为"液体培养"方法可导致排除图书馆中的一些质粒(图1)。
    2. 冷冻回收扩增的CRISPR gRNA质粒文库

      图1. Koike-Yusa等人的图形摘要(2014)gRNA文库的扩增方案

  3. 创建CRISPR gRNA慢病毒文库
    1. 开始之前,请参阅注释5.
    2. D0:在25ml DMEM + 10%FBS中每T-175的板1×10 6个/ml 293T细胞。
    3. D1:创建CRISPR gRNA慢病毒文库。
      1. 预混合质粒在150μl无血清DMEM中 对于T-175烧瓶:
        4μg△VPR
        2.6μgVSV-G
        1.66μgp-Advant
        6.6μgCRISPR库
      2. 加入45μlFugene,在25℃下静置20分钟
      3. 在孵育期间用25ml新鲜的37℃DMEM + 10%FBS代替293T培养基
      4. 在293T细胞上逐滴加入混合物,并用柔软的水平运动混合。
        在37℃+ 5%CO 2孵育过夜。
    4. D2:
      1. 用20ml 37℃的DMEM + 10%FBS轻轻取代转染的293T细胞上的培养基(参见附注3)。
      2. 在RAW上转染RAW264.7-hCas9表达细胞。为了计算所需的RAW264.7细胞,参见方法C.另外,在6孔板中以3×10 5个细胞/孔将3×RAW264.7细胞平板测定用于评估病毒滴度。
    5. D3:收获病毒并转染RAW264.7细胞
      1. 在AM和PM中收获病毒。
      2. 从含有病毒的293T细胞中除去上清液
      3. 用0.45μm注射器过滤器过滤。
      4. 加入8μg/ml的硫酸鱼精蛋白
      5. 将吸出的培养基吸除种子细胞,并加入含有病毒的Xml上清液,使您具有30%的转染效率,并将37℃的DMEM + 10%FBS加至每个T-175烧瓶的20ml(参见附注5)。 />
      6. 此时,如果您以前没有检查转染效率,您可以冻结病毒以检查病毒滴度。
      7. 在37℃+ 5%CO 2孵育。
    6. D4:板5×10 6在T-175烧瓶中的25ml 37℃DMEM + 10%FBS中的RAW264.7-hCas9表达细胞用于抗生素选择阴性对照。
    7. D6:使用荧光显微镜和抗生素选择检查转染效率
      1. BFP在慢病毒CRISPR文库质粒(Koike-Yusa等人,2014)上组成型表达,您可以通过电镀转染细胞的等分试样并计算%荧光来显现转染效率。这不会是转染的定量测量,而是让您确信您的转染有效,然后再进行抗生素选择。只能使用抗生素选择的细胞,以备将来工作。
      2. 对于RAW264.7转染细胞的嘌呤霉素选择:
        1. 吸出培养基转染RAW264.7转染的细胞和阴性对照,并用20ml 37℃的DMEM + 10%FBS +5μg/ml嘌呤霉素代替。
        2. 在37℃孵育72小时+ 5%CO 2
    8. D9:分离并扩增用CRISPR gRNA慢病毒文库转染的RAW264.7细胞
      1. 用嘌呤霉素选择的用CRISPR慢病毒文库转染的RAW264.7细胞的吸出培养基。
      2. 加入10ml 37℃的DMEM + 10%FBS +5μg/ml嘌呤霉素。
      3. 将粘附的RAW264.7细胞从T-175烧瓶中吸收到10ml的培养基中
      4. 将所有选择的用CRISPR慢病毒文库转染的RAW264.7细胞和1,000×g g的沉淀物沉淀5分钟。
      5. 重新悬浮在37℃的DMEM + 10%FBS +5μg/ml嘌呤霉素和每个T-175烧瓶的1×10 6个细胞中用CRISPR慢病毒文库转染的所选RAW264.7细胞沉淀。应该有很多T-175烧瓶。
      6. 在37℃孵育72小时+ 5%CO 2
    9. D13:用CRISPR gRNA慢病毒文库转染RAW264.7细胞并进行冷冻
      1. 每个T-175烧瓶应包含数百万种用CRISPR慢病毒文库转染的RAW264.7细胞(平均3-5×10 7)。
      2. 将所选的RAW264.7细胞用CRISPR慢病毒文库转染的吸出培养基
      3. 加入10ml 37℃的DMEM + 10%FBS。
      4. 将粘附的RAW264.7细胞从T-175烧瓶中吸收到10ml的培养基中
      5. 将所有选择的用CRISPR慢病毒文库转染的RAW264.7细胞和1,000×g g的沉淀物沉淀5分钟。
      6. 将沉淀重悬于50ml 37℃的DMEM + 10%FBS中并计数细胞
      7. 此时,在50%DMEM + 40%FBS + 10%DMSO培养基中,每个冷冻箱将1.5×10 7个细胞冷冻库,或者使用这些细胞进行筛选。

数据分析

对于您的图书馆排序和分析图书馆或图书馆结果的覆盖范围,请参阅我们的原始出版物(Napier 等人,2016年)。

笔记

  1. 晚上,您可能会开始看到合胞体。合胞体是正在产生病毒的迹象。
  2. 逆转录病毒更容易感染有丝分裂细胞,因此通过在AM和PM中从同一个孔中收获两次,您将使捕获RAW264.7细胞分裂的机会增加一倍。
  3. 您可以将其余部分储存在4°C,以便再次感染多达7天。然而,冻结显着降低滴度
  4. 在我们手中,RAW264.7细胞的转导不会改变细胞系的免疫学特征,但是如果在该方案中使用另一种细胞系,则必须使用具有非编码序列的逆转录病毒控制来确保感兴趣的表型在转导后不改变
  5. 该方案已被293T细胞产生的慢病毒细胞转染效率标准化为30%。这种较低的转染效率用于降低发生在同一细胞的双重或三重转染事件的频率。您将在细胞中转染1,000倍的gRNA文库。对于该协议,有87,897个gRNA(Koike-Yusa等人,2014);因此,我们转染(8800万RAW246.7细胞)x(30%)= 2.64亿RAW246.7细胞以保持1,000倍的覆盖率,转染效率为30%。

食谱

  1. RAW264.7和293T组织培养基
    37°C DMEM
    10%FBS
  2. 杀稻瘟菌素选择媒体
    37°C DMEM
    10%FBS
    10μg/ml杀稻瘟素
  3. 嘌呤霉素选择培养基
    37°C DMEM
    10%FBS
    5μg/ml嘌呤霉素
  4. 组织培养冻结媒体
    50%DMEM
    40%FBS
    10%DMSO

致谢

该研究得到了国家过敏和传染病研究所1F32AI115959-01(对BA Napier)和AI095396-05(DM Monack)和国防高级研究计划署(DARPA)的授权,授予DARPA-15-21-ThoR- FP-006(到DM Monack)。

参考

  1. Chen,S.,Sanjana,NE,Zheng,K.,Shalem,O.,Lee,K.,Shi,X.,Scott,DA,Song,J.,Pan,JQ,Weissleder,R.,Lee,H 。,Zhang,F.和Sharp,PA(2015)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/25748654"target ="_ blank "> Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis。 细胞 160(6):1246-1260。
  2. Kiessling,MK,Schuierer,Sven。,Stertz,Silke。,Beibel,Martin。,Bergling,Sebastian。,Knehr,Judith。,Carbone,Walter。,deVallière,Cheryl。,Tchinda,Joelle。,Bouwmeester,Tewis。 Seuwen,Klaus,Rogler,Gerhard。和罗马,古列利莫。 (2016)。识别通过全基因组CRISPR-Cas9缺失筛选进行致癌驱动突变。 BMC基因组学 17,723。
  3. Koike-Yusa,H.,Li,Y.,Tan,EP,Velasco-Herrera Mdel,C.and Yusa,K。(2014)。  使用慢病毒CRISPR引导RNA文库的哺乳动物细胞中的全基因组隐性遗传筛选 Nat Biotechnol 32(3):267-273。
  4. Marceau,CD,Puschnik,AS,Majzoub,K.,Ooi,YS,Brewer,SM,Fuchs,G.,Swaminathan,K.,Mata,MA,Elias,JE,Sarnow,P。和Carette,JE(2016) 。黄病毒科的遗传解剖主要因素通过基因组规模的CRISPR屏幕。自然 535(7610):159-163。
  5. Napier,BA,Brubaker,SW,Sweeney,TE,Monette,P.,Rothmeier,GH,Gertsvolf,NA,Puschnik,A.,Carette,JE,Khatri,P。和Monack,DM(2016) class ="ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pubmed/27697835"target ="_ blank">补体途径放大caspase-11依赖性细胞死亡和内毒素诱导的败血症严重程度。 J Exp Med 213(11):2365-2382。
  6. Popov,LM,Marceau,CD,Starkl,PM,Lumb,JH,Shah,J.,Guerrera,D.,Cooper,RL,Merakou,C.,Bouley,DM,Meng,W.,Kiyonari,H.,Takeichi ,M.,Galli,SJ,Bagnoli,F.,Citi,S.,Carette,JE和Amieva,MR(2015)。< a class ="ke-insertfile"href ="http://www.ncbi .nlm.nih.gov/pubmed/26489655"target ="_ blank">粘附连接处理对金黄色葡萄球菌α-毒素的敏感性。 Proc Natl Acad Sci USA em> 112(46):14337-14342。
  7. Shi,J.,Zhao,Y.,Wang,K.,Shi,X.,Wang,Y.,Huang,H.,Zhuang,Y.,Cai,T.,Wang,F. and Shao,F( 2015)。通过炎性胱天蛋白酶切割GSDMD决定了致热细胞死亡。 自然 526(7575):660-665。
  8. Steinhart,Z.,Pavlovic,Z.,Chandrashekhar,M.,Hart,T.,Wang,X.,Zhang,X.,Robitaille,M.,Brown,KR,Jaksani,S.,Overmeer,R.,Boj ,SF,Adams,J.,Pan,J.,Clevers,H.,Sidhu,S.,Moffat,J.and Angers,S。(2017)。< a class ="ke-insertfile"href = http://www.ncbi.nlm.nih.gov/pubmed/27869803"target ="_ blank">全基因组CRISPR屏幕显示Wnt-FZD5信号电路作为RNF43突变型胰腺肿瘤的药物易感性。 Nat Med 23(1):60-68。
  9. 渡边,S.,Chan,KK和Vasudevan,SG(2016)。登革热病毒感染与小鼠血清型1和2临床分离物的小鼠模型。 6(23):e2040。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Napier, B. A. and Monack, D. M. (2017). Creating a RAW264.7 CRISPR-Cas9 Genome Wide Library. Bio-protocol 7(10): e2320. DOI: 10.21769/BioProtoc.2320.
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