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May 2020
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A New Method for Studying RNA-binding Proteins on Specific RNAs
一种研究特异性RNA结合蛋白的新方法   

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Abstract

Proximity-based protein labeling has been developed to identify protein-nucleic acid interactions. We have reported a novel method termed CRUIS (CRISPR-based RNA-United Interacting System), which captures RNA-protein interactions in living cells by combining the RNA-binding capacity of CRISPR/Cas13 and the proximity-tagging activity of PUP-IT. Enzymatically deactivated Cas13a (dCas13a) is fused to the proximity labeling enzyme PafA. In the presence of a guide RNA, dCas13a binds specific target RNA region, while the fused PafA mediates the labeling of biotin-tagged Pup on proximal proteins. The labeled proteins can be enriched by streptavidin pull-down and identified by mass spectrometry. Here we describe the general procedure for capturing RNA-protein interactions using this method.

Keywords: Proximity-based protein labeling (临近蛋白标记), CRUIS (基于CRISPR的RNA联合相互作用系统), RNA-binding protein (RNA结合蛋白), PUP-IT (PUP-IT), CRISPR (CRISPR)

Background

RNA-binding proteins (RBPs) play important roles in regulating cellular processes and function. Mapping RNA-protein interactions is a key endeavor to address how proteins regulate the location and function of target RNAs. The traditional methods are mostly protein-centric methods, including RNA immunoprecipitation (RIP) and crosslinking and immunoprecipitation (CLIP). They rely on specific antibodies to immunoprecipitate RNA-protein complexes and identify the RNAs by sequencing. Although some RNA-centric methods have been developed, they focused on systematic labeling of all RNAs, rather than one specific RNA, for the purification of RNA-protein complexes (Chu et al., 2015; McHugh et al., 2015). Therefore, there is an urgent need to develop a method that can be widely used to study RNA-protein interactions of the specific RNA in living cells. Since the CRISPR-based RNA-targeting Cas nucleases have been reported, it provides us a new powerful tool to bind and cleave specific RNAs to regulate RNA function (Abudayyeh et al., 2016; Cox et al., 2017). In addition, the enzymatically deactivated Cas13a provides a new strategy for tracking the specific RNA. The proximity labeling system PUP-IT has been widely used to identify protein-protein interactions. It employs the proximity ligase PafA to mediate the ligation of a small protein (PupE) to lysines on the surrounding proteins (Liu et al., 2018). Combining PUP-IT with the RNA-targeting Cas nucleases (Figure 1), we have developed a new class of RNA-centric methods for studying RNA-protein interactions in living cells (Zhang et al., 2020).



Figure 1. The workflow of capturing RNA-protein interactions. A. The workflow for isolating HEK293T cell line stably expressing CRUIS. B. The workflow of mass spectrometry sample preparation.


Materials and Reagents

  1. Pipette tips (Filtered) (Thermo, catalog numbers: TF112-1000-Q, T104RS-Q, TF140-200-Q)

  2. 0.2 ml PCR tubes (Thermo, catalog number: 431-MIXED-Q)

  3. 1.5 ml Microtubes (AXYGEN, catalog number: MCT-150-L-C)

  4. 50 ml Tubes (Falcon, catalog number: 352098)

  5. 96-well plates (Corning, catalog number: 3599)

  6. 24-well plates (Corning, catalog number: 3524-ND)

  7. 6-well plates (Corning, catalog number: 3516)

  8. 10 cm dishes (Corning, catalog number: 430167)

  9. pC0040-LwaCas13a crRNA backbone (Addgene, catalog number: 103851)

  10. pB-CAGGS-dCas9 (Addgene, catalog number: 110823)

  11. Super PiggyBac Transposase (SBI, catalog number: PB210PA-1)

  12. DMEM (Life, catalog number: C11995500CP)

  13. FBS (Gemini, catalog number: 100-307)

  14. Penicillin-Streptomycin (Life, catalog number: 15140122)

  15. Opti-MEM (Gibco, catalog number: 31985062)

  16. PEI (Polysciences, catalog number: 23966)

  17. PBS (Life, catalog number: C20012500CP)

  18. 0.25% Trypsin-EDTA (Life, catalog number: 25200072)

  19. Biotin (SCRC, catalog number: 67000260)

  20. Triton® X-100 (Sangon Biotech, catalog number: T0694-100 ml)

  21. 1 M Tris pH 7.5, sterile (Sangon Biotech, catalog number: B548127-0500)

  22. 100× protease inhibitor cocktail (Biotool, catalog number: B14001)

  23. Urea (Sangon Biotech, catalog number: UT0907)

  24. DTT (MDBio, catalog number: D023-5G)

  25. Iodoacetamide (SIGMA, catalog number: I1149-5G)

  26. Streptavidin magnetic beads (PIERCE, catalog number: 88816)

  27. 1 M Tris pH 8.0, sterile (Sangon Biotech, catalog number: B548127-0500)

  28. NaCl (Sangon Biotech, catalog number: A501218-0001)

  29. SDS (MDBio, catalog number: S001-100)

  30. EDTA (Sangon Biotech, catalog number: E0105-500g)

  31. Ammonium bicarbonate reagent plus(R) (Sigma, catalog number: A6141-500G)

  32. RapiGest SF (Waters, catalog number: 186008090)

  33. Sequencing Grade Modified Trypsin (Promega, catalog number: V5113)

  34. Formic acid (Sigma, catalog number: 94318-250ML)

  35. Acetonitrile (Merck Chemicals, catalog number: 1.00030.4008)

  36. Trifluoroacetic acid LC-MC Ultra (Sigma, catalog number: 14264-50ML)

  37. Amino acid sequence information of CRUIS and Bio-PupE are available at https://doi.org/10.1093/nar/gkaa143

  38. ZIPTIP C18 (Millipore,catalog number: ZTC18S960)

  39. Anti-myc antibody (Cell Signaling, catalog number: 2276s)

  40. Goat anti-mouse IgG Antibody (H&L) [HRP] (GenScript, catalog number: A00160)

  41. Streptavidin-HRP (Cell Signaling, catalog number: 3999s)

  42. BSA (AMRESCO, catalog number: 0903-5G)

  43. SurePAGE (GenScript, catalog number: M00657)

  44. PVDF Western blotting membranes (Roche, catalog number: 30100400)

  45. BbsI-HF (NEB, catalog number: R3539)

  46. Lysis buffer (see Recipes)

  47. Buffer 1 (see Recipes)

  48. Buffer 2 (see Recipes)

  49. Buffer 3 (see Recipes)

  50. Buffer 4 (see Recipes)

Equipment

  1. Standard molecular biology lab equipment

  2. Eppendorf mixer (Eppendorf, catalog number: 5382000074)

  3. Magnetic separator(Bimake, catalog number: B23803)

  4. -80°C freezer (Therom, catalog number: 905)

  5. FACSAriaTM III (BD)

  6. Trans-blot turbo (Biorad, catalog number: 1704150)

  7. Chemiluminescence imaging system (GE, Amersham Imager 600)

  8. Vacuum concentrator (Thermo, SpeedVac)

  9. Spectrometer (Thermo, Orbitrap Fusion)

Procedure

  1. Molecular cloning: design and assembly of sgRNA expression vector for target gene, subclone CRUIS into the plasmid for generating the stable cell line


    For the sgRNA expression vector
    1. Design sgRNAs for the target gene. We recommend design with CRISPR-RT. The parameters can be set as the length of the target complementarity region of crRNA at 28 nt, the length of the seed region at 10 nt (default parameters of CRISPR-RT). The system will give a series of crRNAs, if you are interested in a specific region of the target RNA, you can choose the crRNA targeted to the corresponding position. For more information and guidelines, please follow the link to CRISPR-RT (http://bioinfolab.miamioh.edu/CRISPR-RT/interface/C2c2.php) (Zhu et al., 2018).

    2. The sgRNA expression plasmids cloned by inserting annealed oligos into the U6 promoter-based expression vector between LwaCas13a-DR and poly-T that digested by BbsI (For the forward oligo should add AAAC in the 5’, reverse oligo should add AAAA at 5’ as an overhand to insert the LwaCas13a crRNA backbone digested by BbsI). For cloning the gRNAs into the plasmid by annealed oligo cloning, we recommend the protocol provided by Addgene as a reference (http://www.addgene.org/protocols/annealed-oligo-cloning).

      Note: Any plasmid with LwaCas13a crRNA is suitable, here we take pC0040-LwaCas13a crRNA backbone (Addgene #103851) as an example.


    For the plasmid used to construct stable cell line

    1. Clone CRUIS-P2A-EGFP into the backbone of pB-CAGGS-dCas9 vector between the restriction enzyme sites NheI and HpaI.

      Notes:

      1. Any PiggyBac expression vector is suitable, here we take pB-CAGGS-dCas9 (Addgene #110823) as an example, the sequence information of CRUIS-P2A-EGFP is available at https://doi.org/10.1093/nar/gkaa143.

      2. In order to facilitate the following description, we use CRUIS, which is the method’s name to represent the dCas13-PafA fusion gene; and use 293T-CRUIS to represent the 293T cells stably expressing dCas13-PafA.

    2. Preparation of endotoxin-free plasmids for subsequent transfection with PEI.


  2. Generation of 293T-CRUIS stable cell line

    Note: Any method that can be used to construct a stable cell line is suitable. We prefer to recommend the lentivirus transfection system. The size of the CRUIS is about 5k bp, which is close to the upper limit of lentiviral vectors. If you need to introduce some selection marker, it is recommended to apply the PiggyBac system.

    1. Culture 293T cells in DMEM with 10% FBS at 37°C in 5% CO2.

    2. The day before transfection, seed 5 × 105 cells/well in one well of a 6-well dish with 2.5 ml medium.

    3. Co-transfect 293T cells with PB-CRUIS-P2A-EGFP and Super PiggyBac Transposase (SBI: PB210PA-1) by PEI follow Steps B4-B6 when cells grow to 70% cell confluency.

    4. Dilute 1.5 μg PB-CRUIS-P2A-EGFP plasmids and 1.5 μg Super PiggyBac Transposase plasmids in 125 μl Opti-MEM, dilute 7.5 μl of 1 mg/ml PEI in 125 μl Opti-MEM.

    5. Mix the diluted plasmids and PEI in a 1.5 ml tube, incubate the mixture at room temperature for 20 min.

    6. Add PEI-DNA complex to the cells dropwise.

    7. Three days after transfection, EGFP positive cells were sorted by flow cytometry for continuous culture.

      Note: This step is to sort out the successfully transfected cells; For the sorting, wild-type 293T cells were used as control.

    8. Ten days after transfection, EGFP positive cells were sorted into 96-well plates with 1 cell/well for single clone selection.

      Note: The purpose of this step is to sort out the cells with CRUIS-P2A-EGFP stable expression.

    9. Three weeks after sorting, 10 EGFP positive clones were selected for further amplification.

    10. Western Blot is used to test the expression of CRUIS in each clone. 1 million cells were harvested, washed with cold PBS, and lysed in 200 μl lysis buffer supplemented with 1× protease inhibitor for 1 h on ice. After centrifugation at 13,000 rcf, 150 μl supernatant was mixed with 30 μl 6× protein loading buffer and denatured at 100°C for 10 min. 20 μl sample was loaded on 4-20% SDS-PAGE gels, followed by immune-blotting with anti-myc antibody (1:3,000 dilute) to detect the expression of CRUIS.

    11. To test the proximity labeling activity of CRUIS in those clones stably expressing CRUIS. Cells were transfected with pcDNA3.1-Bio-PupE in a 6-well plate by PEI.

    12. Twelve hours after transfection, supplement fresh media with biotin at the final concentration of 20 μM (For convenience, biotin is prepared as 100× concentrated stock in DMEM, and stored at 4°C).

    13. After adding biotin for 24-48 h, cells are harvested and further examined for PafA activity, indicated by biotin signals on western blot. The cell clone with high enzymatic activity (with strong biotin signals) will be selected for future experiments.

      Note: The blocking reagent used for detecting biotin by western is 5% BSA, since most commonly used milk based blocking reagents contain biotin and result in background.


  3. CRUIS labeling in cells

    1. Prepare 20 million cells in a 150 mm dish as the experimental group and another one as the control group.

      Note: The number of required cells depends on the abundance of target RNA. More cells generally produce better results.

    2. When the CRUIS cells reach about 70% confluence, co-transfect the cells with sgRNA and pcDNA3.1-Bio-PupE plasmids by PEI. A co-transfection of non-targeting sgRNA and pcDNA3.1-Bio-PupE are used as a control (the ratio of sgRNA and pcDNA3.1-Bio-PupE is 1:1, totally 15 μg).

    3. Twelve hours after transfection, replace with fresh medium containing biotin at the final concentration of 20 μM.

    4. After adding biotin for 24-48 h, cells are harvested, washed with cold PBS 3 times, stored or lysed for the mass spectrometry sample preparation.


  4. Mass spectrometry preparation

    Day 1
    1. Lyse about 30 million cells by 2 ml lysis buffer with 1× protease inhibitor at 4°C with shaking for one hour.

    2. Clarify the lysates by centrifuging at 13,000 rcf for 10 min at 4°C.

    3. Transfer 900 μl supernatant into 1.5 ml microtubes, with the addition of 576 mg urea to a final concentration of about 8 M (The final volume is about 1.2 ml).

    4. Add 12 μl 1 M DTT to a final concentration of 10 mM, then incubate lysate for 1 h at 56°C.

    5. Treat lysate with 30 μl 1 M iodoacetamide to a final concentration of 25 mM, incubate in dark at room temperature for 45 min to aminocarbonyl modify the Cys site of proteins.

    6. Add 30 μl 1 M DTT to a final concentration of 25 mM, and incubate at room temperature for 30 min to terminate the modification.

    7. Take 50 μl streptavidin magnetic beads (stock in PBS containing 0.1% BSA, 0.05% NaN3 and 0.05% Tween 20) into 1.5 ml microtubes, put microtubes on a magnetic stand for 3 min at room temperature, then remove the supernatant with pipette and wash beads three times with 500 μl PBS, then resuspend beads with 50 μl lysis buffer.

    8. Transfer beads into the lysate and incubated overnight on a rotator at 4°C.


    Day 2
    1. After incubation, the microtubes were placed into magnetic stand for 3 min, then aspirate out lysate and wash beads on Eppendorf mixer with the following four buffers step by step.

    2. Add 1 ml buffer 1 to each microtube and shake on Eppendorf mixer at 600 rpm for 5 min at room temperature.

    3. Place the microtube into magnetic band for 3 min and aspirate out buffer 1.

    4. Repeat the Steps D10 and D11 for once.

    5. Add 1 ml buffer 2 to each microtube and shake on Eppendorf mixer at 600 rpm for 5 min at room temperature.

    6. Place the microtube into magnetic band for 3 min and aspirate out buffer 2.

    7. Add 1 ml buffer 3 to each microtube and shake on Eppendorf mixer at 600 rpm for 5 min at room temperature.

    8. Place the microtube into magnetic band for 3 min and aspirate out buffer 3.

    9. Repeat Steps D15 and D16 for once.

    10. Add 1 ml buffer 4 to each microtube and shake at room temperature for 5 min on Eppendorf mixer at 600 rpm.

    11. Place the microtube into magnetic band for 3 min and aspirate out buffer 4.

    12. Repeat Steps D18 and D19 for twice.

    13. Resuspend beads by 100 μl buffer 5 with addition of 6 μg trypsin, transfer the mixture into new PCR tubes and shake samples at 37°C for overnight.


    Day 3
    1. After incubation, place the tubes into magnetic stand and transfer the supernatant to a new PCR tube, adjust sample pH to a lower than 3 with addition of 8 μl 10% formic acid (Under normal circumstances, the pH of the sample will be below 3, which can pass the PH paper test).

    2. Wash Ziptip with 100 μl 100% ACN (Acetonitrile) with pipette.

    3. Aspirate out ACN slowly with pipette.

    4. Balance Ziptip with 100 μl 0.1% TFA (Trifluoroacetic acid) with pipette.

    5. Repeat Step D25 for once.

    6. Bind peptides to Ziptip carefully for three times with pipette.

    7. Desalt peptides with 100 μl 0.1% TFA.

    8. Repeat Step D28 for once.

    9. Elute peptides into new 1.5 ml microtubes by addition of 50 μl 70% ACN-0.1% TFA.

    10. Repeat step D30 for twice.

    11. Dry peptides in the SpeedVac concentrator. The samples can be stored in -80°C or analyzed on an Orbitrap Fusion.

Recipes

  1. Lysis buffer

    1% TritonX-100

    50 mM Tris pH 7.5, sterile

    150 mM NaCl

    Note: Prepared in advance and store at 4°C.

  2. Buffer 1

    50 mM Tris pH 8.0, sterile

    8 M urea

    200 mM NaCl

    0.2% SDS

    Note: Prepared in advance and stored at room temperature).

  3. Buffer 2

    50 mM Tris 8.0

    200 mM NaCl

    8 M urea

    Note: Prepared in advance and store at room temperature.

  4. Buffer 3

    50 mM Tris 8.0

    0.5 mM EDTA

    1 mM DTT(store at -80°C)

    Note: Prepared in advance and store at room temperature,add DTT to a final concentration of 1 mM before used.

  5. Buffer 4

    50 mM Ammonium bicarbonate

    Note: Prepared in advance and store at room temperature.

  6. Buffer 5

    100 mM Ammonium bicarbonate with 1 mg/ml RapiGest

    Note: Prepared 100 mM Ammonium bicarbonate in H2O in advance and store at room temperature,add RapiGest to a final concentration of 1 mg/ml before used.

Acknowledgments

We thank the Molecular Imaging Core Facility (MICF), the Molecular and Cell Biology Core Facility (MCBCF) and the Multi-Omics Core Facility (MOCF) at the School of Life Science and Technology, ShanghaiTech University for providing technical support. This work has been supported by Shanghai Municipal Science and Technology Commission (19JC1413700 to M.Z.) and National Natural Science Foundation of China (31922038 to M.Z.; 31771490 to J.L.L).

Competing interests

None declared.

References

  1. Abudayyeh, O. O., Gootenberg, J. S., Konermann, S., Joung, J., Slaymaker, I. M., Cox, D. B., Shmakov, S., Makarova, K. S., Semenova, E., Minakhin, L., Severinov, K., Regev, A., Lander, E. S., Koonin, E. V. and Zhang, F. (2016). C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353(6299): aaf5573.
  2. Chu, C., Zhang, Q. C., da Rocha, S. T., Flynn, R. A., Bharadwaj, M., Calabrese, J. M., Magnuson, T., Heard, E. and Chang, H. Y. (2015). Systematic discovery of Xist RNA binding proteins. Cell 161(2): 404-416.
  3. Cox, D. B. T., Gootenberg, J. S., Abudayyeh, O. O., Franklin, B., Kellner, M. J., Joung, J. and Zhang, F. (2017). RNA editing with CRISPR-Cas13. Science 358(6366): 1019-1027.
  4. Liu, Q., Zheng, J., Sun, W., Huo, Y., Zhang, L., Hao, P., Wang, H. and Zhuang, M. (2018). A proximity-tagging system to identify membrane protein-protein interactions. Nat Methods 15(9): 715-722.
  5. McHugh, C. A., Chen, C. K., Chow, A., Surka, C. F., Tran, C., McDonel, P., Pandya-Jones, A., Blanco, M., Burghard, C., Moradian, A., et al. (2015). The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3. Nature 521: 232-236.
  6. Zhang, Z., Sun, W., Shi, T., Lu, P., Zhuang, M. and Liu, J. L. (2020). Capturing RNA-protein interaction via CRUIS. Nucleic Acids Res 48(9): e52.
  7. Zhu, H., Richmond, E. and Liang, C. (2018). CRISPR-RT: a web application for designing CRISPR-C2c2 crRNA with improved target specificity. Bioinformatics 34(1): 117-119.

简介

[摘要]已经开发了基于邻近的蛋白质标记来鉴定蛋白质-核酸相互作用。W¯¯ Ë已经报道了一种新方法被称为CRUIS(CRISPR-基于RNA的联合相互作用系统),其捕获在通过结合活细胞RNA-蛋白质相互作用的RNA结合能力的CRISPR / Cas13和所述接近标记活性的PUP-IT 。酶促去ACTIV ated Cas13a(dCas13a )被融合至所述接近标记酶PAFA 。在存在指导RNA的情况下,dCas13a 结合特异性靶RNA区域,而融合的PafA介导近端蛋白质上生物素标记的Pup的标记。可以通过抗生蛋白链菌素下拉来富集标记的蛋白质,并通过质谱鉴定。在这里,我们ð escribe的一般程序,用于捕获使用这种方法RNA-蛋白质相互作用。

[背景技术] RNA结合蛋白(限制性商业惯例)中发挥重要的作用调节的细胞过程和功能。绘制RNA-蛋白质相互作用的图谱是解决蛋白质如何调节靶RNA的位置和功能的一项关键工作。传统的方法大多蛋白为中心的方法,包括- [R NA我mmuno p recipitation(RIP)和Ç罗斯升我nking和我mmuno p recipitation(CLIP)。他们依靠特异性抗体免疫沉淀RNA-蛋白质复合物,并通过测序鉴定RNA。尽管一些RNA为中心的方法已经被开发,他们专注于所有RNA的系统标签,而不是一个特定的RNA,为的净化RNA-蛋白复合体ES (楚等人。2015年,麦克休等人,2015)。因此,迫切需要开发可广泛用于研究RNA-蛋白质相互作用的方法将特定的RNA在活细胞中。由于已经报道了基于CRISPR的靶向RNA的Cas核酸酶,它为我们提供了一种强大的工具来结合和切割特定的RNA以调节RNA功能(Abudayyeh等人,2016; Cox等人,2017 )。此外,酶失活Cas13a提供用于跟踪新战略的具体RNA。在P roximity标签系统PUP-IT公顷š被广泛用于识别蛋白质-蛋白质相互作用。它采用邻近连接酶PafA介导小蛋白(PupE )与周围蛋白上的赖氨酸的连接(Liu等人,2018 )。将PUP-IT与靶向RNA的Cas核酸酶结合起来(图1),我们已经开发出了一类以RNA为中心的新方法,用于研究活细胞中RNA-蛋白质的相互作用(Zhang等人,2020年)。



图1中。T他工作流捕获RNA-蛋白的相互作用。A.分离稳定表达CRUIS的HEK293T细胞的工作流。B.质谱样品制备的工作流程。

关键字:临近蛋白标记, 基于CRISPR的RNA联合相互作用系统, RNA结合蛋白, PUP-IT, CRISPR



材料和试剂


移液器吸头(已过滤)(Thermo,目录号:TF112-1000-Q,T104RS-Q,TF140-200-Q)
0.2 ml PCR管(Thermo,货号:431-MIXED-Q)
1.5 ml微型管(AXYGEN,目录号:MCT-150-LC)
50毫升试管(猎鹰,货号:352098)
96孔板(Corning,目录号:3599)
24孔板(Corning,目录号:3524-ND)
6孔板(Corning,目录号:3516)
10厘米盘子(Corning,货号:430167)
pC0040-LwaCas13a crRNA主链(Addgene ,目录号:103851)
pB-CAGGS-dCas9(Addgene ,目录号:110823)
超级PiggyBac转座酶(SBI,目录号:PB210PA-1)
DMEM(生命,目录号:C11995500CP)
FBS(Gemini,目录号:100-307)
青霉素-链霉素(生命,目录号:15140122)
Opti-MEM(Gibco,目录号:31985062)
PEI(Polysciences ,目录号:23966)
PBS(生活,货号:C20012500CP)
0.25%胰蛋白酶-EDTA(生命,目录号:25200072)
生物素(SCRC,目录号:67000260)
的Triton ® X-100(生工生物,目录号:T0694-100毫升)
1的1M Tris pH值7.5,无菌(生工生物,目录号:B548127-0500)
100×蛋白酶抑制剂混合物(Biotool ,目录号:B14001)
脲(生工生物,目录号:UT0907)
DTT(MDBio ,目录号:D023-5G)
碘乙酰胺(SIGMA,货号:I1149-5G)
链霉亲和素磁珠(PIERCE,目录号:88816)
的1M Tris pH 8.0中,无菌(生工生物,目录号:B548127-0500)
的NaCl(生工生物,目录号:A501218-0001)
SDS(MDBio ,目录号:S001-100)
EDTA(生工生物,目录号:E0105-500g)
碳酸氢铵试剂plus(R)(Sigma,目录号:A6141-500G)
RapiGest SF(Waters,货号:186008090)
测序级修饰胰蛋白酶(Promega,目录号:V5113)
甲酸(Sigma,目录号:94318-250ML)
乙腈(Merck Chemicals,目录号:1.00030.4008)
三氟乙酸LC-MC Ultra(Sigma,目录号:14264-50ML)
CRUIS和BioPupE的氨基酸序列信息可在https://doi.org/10.1093/nar/gkaa143获得
ZIPTIP C18(密理博,目录号:ZTC18S960 )
抗MYC抗体(Cell š ignaling,目录号:2276s)
山羊一个nti-米乌斯IgG抗体(H&L)[HRP(金斯瑞,目录号:A00160)
链霉亲和素-HRP(小区š ignaling,目录号:3999s)
BSA (AMRESCO ,目录号0903-5G )
SurePAGE (金斯瑞,目录号:M00657 )
PVDF西方b印迹膜(罗氏,目录号:30100400 )
Bbs I -HF(NEB,货号:R3539 )
裂解缓冲液(请参见食谱)
缓冲区1(请参阅食谱)
缓冲区2(请参阅食谱)
缓冲区3(请参阅食谱)
缓冲区4(请参阅食谱)


设备


标准分子生物学实验室设备
Eppendorf搅拌机(Eppendorf,目录号:5382000074)
              磁性小号eparator(Bimake ,目录号:B23803)
-80°C冰箱(Therom ,目录号:905)
FACSAria TM III(BD)
反式- b很多吨urbo(Biorad公司,目录号:1704150)
化学发光成像系统(GE,Amersham Imager 600)
真空浓缩器(Thermo,SpeedVac )
光谱仪(Thermo,Orbitrap Fusion)


程序


分子克隆:D ESIGN和组装为靶基因因组表达载体,亚克隆CRUIS成的质粒用于产生稳定细胞系


对于sgRNA表达载体


设计sgRNAs的靶基因。w ^ ê建议设计与CRISPR-RT。可以将参数设置为crRNA的靶互补区的长度为28 nt ,种子区的长度为10 nt (CRISPR-RT的默认参数)。系统将提供一系列crRNA,如果您对目标RNA的特定区域感兴趣,则可以选择针对相应位置的crRNA。欲了解更多信息和指南,请按照链接到CRISPR-RT(http://bioinfolab.miamioh.edu/CRISPR-RT/interface/C2c2.php)(朱等人,2018 )。
通过将退火的寡核苷酸插入由BbsI消化的LwaCas13a -DR和poly-T之间基于U 6启动子的表达载体中克隆的sgRNA表达质粒(对于正向寡核苷酸应在5'中添加AAAC,反向寡核苷酸应在5'处添加AAAA作为插入被BbsI消化的LwaCas13a crRNA主链的开销。为了通过退火寡核苷酸克隆将gRNA克隆到质粒中,我们建议使用Addgene提供的方案作为参考(http://www.addgene.org/protocols/annealed-oligo-cloning)。
注意:任何带有LwaCas13a crRNA的质粒都适用,这里我们以pC0040-LwaCas13a crRNA骨架(Addgene #103851)为例。


F或用于构建稳定细胞系的质粒


将CRUIS-P2A-EGFP克隆到限制性酶切位点NheI和HpaI之间的pB-CAGGS-dCas9载体的骨架中。
注意小号:


任何PiggyBac表达载体都是合适的,这里以pB-CAGGS-dCas9(Addgene #110823)为例,CRUIS-P2A-EGFP的序列信息可在https://doi.org/10.1093/nar/gkaa143获得。
在邻刻申到便于下面的描述中,我们使用CRUIS,其是方法“的名称来表示dCas13-PAFA融合基因; 并使用293T-CRUIS代表稳定表达dCas13-PafA的293T细胞。
制备无内毒素质粒,用于随后的PEI转染。


293T - CRUIS稳定细胞系的产生
注意:可被用于构建任何方法一个稳定的细胞系是合适的。我们更愿意推荐的慢病毒转染系统。CRUIS的大小约为5k bp,接近慢病毒载体的上限。如果需要引入一些选择标记,建议使用PiggyBac系统。


在37 %10%FBS的DMEM中培养293T细胞 在5%CO 2中为°C。
转染前一天,种子5 × 10 5细胞/孔,一个孔的一个6孔培养皿用2.5ml培养基。
当细胞生长至70%细胞融合时,通过PEI与PB-CRUIS-P2A-EGFP和Super PiggyBac转座酶(SBI:PB210PA-1)共转染293 T细胞,遵循步骤B4-B6 。
稀1.5 μ克PB-CRUIS-P2A-EGFP质粒和1.5 μ克超级piggyBac转转座的质粒在125微升的Opti-MEM ,稀7.5微升1毫克/毫升的PEI在125微升的Opti-MEM。
将稀释的质粒和PEI在1.5 ml试管中混合,在室温下孵育20分钟。
滴加PEI-DNA复合物到细胞中。
转染三天后,通过流式细胞仪分选EGFP阳性细胞以进行连续培养。
注意:此步骤是为了筛选成功转染的细胞;对于分选,野生型的293T细胞瓦特ERE用作对照。


十转染后的天数,EGFP阳性细胞分选入96 -孔板1个细胞/孔的单克隆选择。
注意:此步骤的目的是筛选出具有CRUIS-P2A-EGFP稳定表达的细胞。


分选三周后,选择了10个EGFP阳性克隆进行进一步扩增。
Western Blot用于测试每个克隆中CRUIS的表达。1个是百万细胞收获,用冷PBS洗涤,并在裂解2 00 μ升裂解缓冲液补充有1 ×蛋白酶抑制剂在冰上1个小时。甲压脚提升离心13 ,000 RCF ,150 μ升上清液中混合与30 μ升6 ×蛋白加载缓冲液和变性d在100 ℃下进行10分钟。20 μ升样品被加载到4-20%SDS-PAGE凝胶上,然后免疫-B很多婷用抗MYC抗体(1:3 ,000稀释的)来检测的表达CRUIS 。
到t EST的接近贴标活性CRUIS的在那些克隆稳定表达荷兰国际集团CRUIS 。细胞是在与的pcDNA3.1-生物PupE转染一个由PE 6孔板I.
Ť welve ħ我们的转染后,补充新鲜的培养基在20的最终浓度的生物素μ中号(为了方便起见,生物素被制备成100×DMEM中浓缩储备,并保存在4 ℃)
加入生物素24-48小时后,细胞被收获,并进一步检查PAFA活性,通过生物素信号指示对蛋白印迹分析。Ť与他细胞克隆高酶活性(具有强生物素信号小号)将被选择用于将来的实验。
注意:西方人用于检测生物素的封闭剂为5%BSA,因为最常用的牛奶基封闭剂含有生物素并导致本底。


细胞中的CRUIS标记
制备20个百万个细胞在一150毫米盘作为所述实验组,另一个作为所述对照组。
注意:所需细胞的数量取决于靶RNA的丰度。更多的细胞通常会产生更好的结果。


当CRUIS细胞达到约70%融合时,可通过PEI用sgRNA和pcDNA3.1-Bio-PupE质粒进行c -o转染。的共转染非靶荷兰国际集团因组和pCDNA3.1-生物PupE用来作为对照(因组的比率和pCDNA3.1-生物PupE为1:1,共15 μ克)。
Ť welve小时转染后,在20的最终浓度含有生物素的新鲜培养基替换μM 。
加入生物素24-48小时后,细胞被收获,洗涤用冷PBS 3次,贮存或裂解的质谱分析样品制备。


质谱制备
第一天


1.裂解约30亿个细胞通过2毫升裂解缓冲液以1×蛋白酶抑制剂在4℃下振荡一小时。     

2.通过在13离心澄清裂解物,000 RCF 10分钟在4℃下。     

3.转移9 00微升上清液至1.5mL微管,在添加576毫克尿素至约8 M的最终浓度(终体积为约1.2毫升)。     

4.添加1 2微升的1M DTT至10mM的终浓度,然后裂解物孵育1个小时在56℃。     

5.款待裂解物与30微升的1M碘乙酰胺至25mM的终浓度,孵化在黑暗在室温下45分钟到氨基羰修改的Cys蛋白质的位点。     

6.添加30微升的1M DTT至25mM的终浓度,在室温下孵育30分钟以终止改性。     

7.取50个微升链霉亲和磁珠(股票在含有PBS 0.1%BSA,0.05%的NaN 3和0.05%吐温20)至1.5mL微管中,把微管上的磁性支架为3分钟,在室温下,然后删除与该上清液移液管和洗涤珠三次用500微升PBS,然后重悬珠子用50 μ升裂解缓冲液。     

8.将珠子转移到裂解物中,并在4°C的旋转器上孵育过夜。     



第二天


9.温育后,将微管置于磁力架中3分钟,然后吸出裂解物,并在Eppendorf混合器上逐步用以下四个缓冲液洗涤珠子。     

10.向每个微管中添加1 ml缓冲液1,并在Eppendorf混合器上于室温以600 rpm摇动5分钟。 

11.将微管放入磁力带中3分钟,然后吸出缓冲液1。 

12.重复小号TEPS D10和D11为一次。 

13.加入1ml缓冲液2至每个微管,并在600rpm下摇动的Eppendorf上混合器,在室温5分钟温度。 

14.将微管放入磁力带中3分钟,然后吸出缓冲液2。 

15.向每个微管中添加1 ml缓冲液3,并在Eppendorf混合器上于室温以600 rpm摇动5分钟。 

16.将微管放入磁力带中3分钟,然后吸出缓冲液3。 

17.重复小号TEPS D15 D16和一次。 

18.向每个微管中加入1 ml缓冲液4,并在室温下在Eppendorf混合器上以600 rpm摇动5分钟。 

19.将微管放入磁力带中3分钟,然后吸出缓冲液4。 

20.重复小号TEPS D18 D19及两次。 

21.重悬珠子由100微升缓冲液5并加入6微克胰蛋白酶,将混合物转移到新的PCR管中,并在37℃下摇动样品过夜。 



第三天


22.孵育后,将试管置于磁力架中,并将上清液转移至新的PCR试管中,并通过添加8μl10 %甲酸将样品的pH调节至低于3 (在正常情况下,样品的p H低于3,可以通过PH纸测试)。 

23.洗涤Ziptip用100微升100%ACN (乙腈)用移液管。 

24.用移液管缓慢吸出ACN 。 

25.余额Ziptip用100微升0.1%TFA (三氟乙酸)与移液管。 

26.重复步骤D25一次。 

27.用移液管小心地将肽与Ziptip结合3次。 

28.脱盐肽用100微升0.1%TFA。 

29.重复步骤D28一次。 

30.通过添加50μl70 %ACN -0.1%TFA将肽洗脱到新的1.5 ml微管中。 

31.重复步骤D30两次。 

32. SpeedVac浓缩器中的干肽。样品可以存储在-80°C或在Orbitrap Fusion上进行分析。 



菜谱


裂解缓冲液
1%的Triton X-100


50 mM Tris pH 7.5,无菌


150毫米氯化钠


注意:P事先准备好并储存在4 °C下。


缓冲区1
50 mM Tris pH 8.0,无菌


8 M尿素


200毫米氯化钠


0.2%SDS


注:P事先准备好并在室温下保存)。


缓冲区2
50毫米Tris 8.0


200毫米氯化钠


8 M尿素


注意:P事先准备好并在室温下保存。


缓冲区3
50毫米Tris 8.0


0.5毫米EDTA


1 mM DTT (储存在-80°C )


注意:P预先制备并在室温下保存,使用前将DTT加入终浓度为1 mM 。


缓冲区4
50 mM碳酸氢铵


注意:P事先准备好并在室温下保存。


缓冲区5
100 mM碳酸氢铵和1 mg / ml RapiGest


注:P repared 100mM碳酸氢铵H中2 ö提前,并储存在室温下,加入RapiGest至终浓度为1mg / ml使用之前。


参考


Abudayyeh,OO,Gootenberg ,JS,Konermann ,S.,Joung,J.,Slaymaker,IM,Cox,DB,Shmakov,S.,Makarova,KS,Semenova,E.,Minakhin ,L.,Severinov,K., Regev,A.,Lander,ES,Koonin ,EV和Zhang F.(2016)。C2c2是单组分可编程RNA引导的RNA靶向CRISPR效应子。科学353(6299):aaf5573。
Chu,C.,Zhang,QC,da Rocha,ST,Flynn,RA,Bharadwaj,M.,Calabrese,JM,Magnuson,T.,Heard,E.和Chang,HY(2015)。Xist RNA结合蛋白的系统发现。单元格161(2):404-416。
Cox,DBT,Gootenberg ,JS,Abudayyeh,OO,Franklin,B.,Kellner,MJ,Joung,J.和Zhang,F.(2017年)。使用CRISPR-Cas13进行RNA编辑。科学358(6366):1019-1027。
刘强,郑洁,孙伟,霍勇,张丽,郝平,王洪,庄明(2018)。邻近标签系统,用于识别膜蛋白之间的相互作用。Nat Methods 15(9):715-722。
McHugh,CA ,Chen,CK,Chow,A.,Surka,CF,Tran,C.,McDonel,P.,Pandya-Jones,A.,Blanco,M.,Burghard,C.,Moradian,A。,等。人。(2015)。Xist lncRNA直接与SHARP相互作用,通过HDAC3沉默转录。自然521 :232-236。
Zhang,Z.,Sun,W.,Shi,T.,Lu,P.,Zhuang,M. and Liu,JL(2020)。通过CRUIS捕获RNA-蛋白质相互作用。Nucleic Acids Res 48(9):e52。
Zhu H.,Richmond E.和Liang C.(2018年)。CRISPR-RT:用于设计具有改善的靶标特异性的CRISPR-C2c2 crRNA的Web应用程序。生物信息学34(1):117-119。
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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sun, W., Zhang, Z., Liu, J. and Zhuang, M. (2021). A New Method for Studying RNA-binding Proteins on Specific RNAs. Bio-protocol 11(10): e4022. DOI: 10.21769/BioProtoc.4022.
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