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Jun 2020
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In vitro Cleavage and Electrophoretic Mobility Shift Assays for Very Fast CRISPR
快速CRISPR的体外裂解和电泳迁移率位移测定    

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Abstract

CRISPR-Cas9 has transformed biomedical research and medicine through convenient and targeted manipulation of DNA. Time- and spatially-resolved control over Cas9 activity through the recently developed very fast CRISPR (vfCRISPR) system have facilitated comprehensive studies of DNA damage and repair. Understanding the fundamental principles of Cas9 binding and cleavage behavior is essential before the widespread use of these systems and can be readily accomplished in vitro through both cleavage and electrophoretic mobility shift assays (EMSA). The protocol for in vitro cleavage consists of Cas9 with guide RNA (gRNA) ribonucleoprotein (RNP) formation, followed by incubation with target DNA. For EMSA, this reaction is directly loaded onto an agarose gel for visualization of the target DNA band that is shifted due to binding by the Cas9 RNP. To assay for cleavage, Proteinase K is added to degrade the RNP, allowing target DNA (cleaved and/or uncleaved) to migrate consistently with its molecular weight. Heating at 95°C rapidly inactivates the RNP on demand, allowing time-resolved measurements of Cas9 cleavage kinetics. This protocol facilitates the characterization of the light-activation mechanism of photocaged vfCRISPR gRNA.

Keywords: CRISPR-Cas9 (CRISPR-Cas9), In vitro (活体外 ), Cleavage (分裂), Electrophoretic mobility shift assay (电泳迁移率实验), EMSA (电泳迁移率实验), Genome editing (基因组编辑)

Background

Endonucleases, especially Cas9, from clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CRISPR-Cas) adaptive immune systems (Marraffini et al., 2015) have found widespread use as highly versatile tools in the biomedical sciences from genome editing, functional screening, to imaging (Adli et al., 2018). Recently, highly resolved spatiotemporal control over Cas9 activity via very fast CRISPR (vfCRISPR) has enabled detailed studies of the DNA damage response through highly synchronized activation of Cas9 in space and time (Liu et al., 2020). For all uses, characterization of these nucleases is essential and can be most readily accomplished in vitro (Singh et al., 2016). Electrophoretic mobility shift assays (EMSA) are powerful tools to characterize protein-DNA binding that can be directly applied to determine the binding affinity of Cas9 to nucleic acids (Hellman et al., 2007). In contrast, in vitro cleavage assays allow characterization of the Cas9 DNA cleavage mechanism. In this manuscript, we outline detailed protocols for both strategies to characterize the properties of the vfCRISPR light-inducible Cas9 system. We demonstrate how both EMSA and in vitro cleavage assays characterize the binding, speed, and efficiency of vfCRISPR, which paves the way for its use in biological systems.

Materials and Reagents

  1. 10 mg/ml SpCas9 (purified in-house from BL21 CodonPlus (DE3)-RIL cells [Agilent 230245], but can also be purchased from Integrated DNA Technologies) (Alt-R®, catalog number: 1081058, storage temperature: -80°C)

  2. tracrRNA (Integrated DNA Technologies) (Alt-R®, catalog number: 1072533, storage temperature: -80°C)

  3. Photocaged crRNA (cgRNA) from vfCRISPR targeting PPP1R2 (BioSynthesis, sequence: GACUUCCUCUAUGGUGGCGUGUUUUAGAGCUAUGCUGUUUUG; U corresponds to replacements of uracil with NPOM-dT photocaged nucleotides, storage temperature: -80°C)

  4. HEK293T cells (ATCC, catalog number: CRL-3216, storage temperature: liquid nitrogen vapor)

  5. Nuclease-Free Duplex Buffer (Integrated DNA Technologies, catalog number: 11-01-03-01, storage temperature: 4°C)

  6. PPP1R2 Fwd PCR primer (Integrated DNA Technologies, storage temperature: -20°C, sequence: 5’-GTTTCCGAGGCAGCAGTTG-3’)

  7. PPP1R2 Rev PCR primer (Integrated DNA Technologies, storage temperature: -20°C, sequence: 5’-GCATGATAAACGTCATCGCCC-3’)

  8. DNeasy Blood & Tissue Kit (Qiagen, DNeasy, catalog number: 69504)

  9. QIAquick PCR purification kit (Qiagen, QIAquick, catalog number: 28104)

  10. Q5® High-Fidelity 2X Master Mix (New England BioLabs, Q5®, catalog number: M0492, storage temperature: -20°C)

  11. NEBufferTM 3.1 (New England BioLabs, catalog number: B7203S, storage temperature: -20°C)

  12. Proteinase K, Molecular Biology Grade (New England BioLabs, catalog number: P8107S, storage temperature: -20°C)

  13. E-GelTM Agarose Gels with SYBRTM Safe DNA Gel Stain, 4% (Thermo Fisher, E-GelTM, catalog number: A45206, storage temperature: room temperature)

  14. 100 bp DNA ladder (New England BioLabs, catalog number: N3231S)

  15. HEPES (Sigma-Aldrich, catalog number: H3375)

  16. KCl (Sigma-Aldrich, catalog number: P3911)

  17. Glycerol (Sigma-Aldrich, catalog number: G5516)

  18. NaOH (Sigma-Aldrich, catalog number: 221465)

  19. Aluminum foil (Reynolds Wrap)

  20. Nuclease-Free Water (not DEPC-treated) (InvitrogenTM, catalog number: AM9932)

Equipment

  1. C1000 TouchTM Thermo Cycler (Bio-Rad, model number: 1851148), or any alternative thermocycler that can perform polymerase chain reaction (PCR)

  2. E-Gel® iBaseTM Power System (Thermo Fisher, E-GelTM, catalog number: G6465), or alternative gel electrophoresis system

  3. JAXMAN 365 nm LED flashlight (Amazon, JAXMAN, https://www.amazon.com/JAXMAN-Ultraviolet-365nm-Detector-Flashlight/dp/B06XW7S1CS/)

  4. TyphoonTM FLA 9000 (GE Healthcare), or any alternative gel imager that can image ethidium bromide or SYBR Gold agarose gels

Procedure

  1. Generation of target DNA at PPP1R2 for in vitro cleavage or EMSA

    1. Purify genomic DNA (gDNA) from HEK293T cells using the DNeasy Blood & Tissue Kit following manufacturer’s instructions. Elute in 200 μl AE provided by the kit. Store gDNA in -20°C.

    2. Prepare PCR primer mixture by mixing 10 μM of Fwd primer with 10 μM of Rev primer, both diluted in water.

    3. Set up PCR reaction.

      Component Volume
      Nuclease-Free Water (NFW)       3 μl
      Genomic DNA (10-50 ng)       1 μl
      Fwd/Rev primer mixture (10 μM)      1 μl
      Q5® High-Fidelity 2× Master Mix      5 μl
      Total    10 μl

    4. Start thermocycling protocol on the thermocycler.

      Step   Temp Time
      Initial Denaturation    98°C   30 s
      35 cycles    98°C   10 s
         68°C (PPP1R2)   10 s
         72°C   20 s
      Final extension    72°C   2 min
      Hold     4°C    Inf

    5. Extract PCR-amplified target DNA with the QIAquick PCR Purification Kit following manufacturer instructions. Elute in 30 μl EB provided by the kit.


  2. Prepare 10 ml of Cas9 Dilution Buffer

    1. Make solution composed of 20 mM HEPES, 500 mM KCl, and 20% glycerol.

    2. Add sufficient 1 M NaOH to bring pH to 7.5.


  3. Prepare 10 μM Cas9

    Dilute 5 μl of 10 mg/ml Cas9 with 25 μl of Cas9 Dilution Buffer to make 30 μl of 10 μM SpCas9.


  4. Anneal light-activatable crRNA with tracrRNA to form cgRNA

    1. Resuspend photocaged crRNA and tracrRNA separately to 100 μM with Duplex Buffer.

    2. Mix 3 μl of 100 μM photocaged crRNA with 3 μl of 100 μM tracrRNA in PCR tube.

    3. Heat at 95°C for 3 min in the thermocycler with heated lid.

    4. Cool on benchtop for 5 min.

    5. Mix in 24 μl of Duplex Buffer to make 30 μl of 10 μM annealed cr/trRNA.


  5. In vitro cleavage to test light activation capability of cgRNA

    1. Mix the following components together in order. Thoroughly mix NFW and NEBufferTM 3.1 before adding cgRNA and Cas9 and mix again. Prepare 11 identical volumes, all in separate PCR tubes.

      Component Volume
      NFW     8.1 μl
      NEBufferTM 3.1        1 μl
      10 μM cgRNA targeting PPP1R2     0.5 μl
      10 μM Cas9     0.4 μl
      Total      10 μl

    2. Leave on benchtop (room temperature) for 30 min to form RNP complex. Cover with aluminum film because the photocaged gRNA are light-sensitive.

    3. Add 2 μl (~60 fmol) of PCR-amplified PPP1R2 target DNA to each RNP tube (11 in total). Mix well with pipette. For a highly efficient reaction, Cas9/cgRNA should be in at least 100-fold excess relative to target DNA. The target DNA should also be easily visible on an agarose gel with a gel imager.

    4. Set up two thermocyclers – one to 37°C and the other to 95°C.

    5. Move one tube to the 37°C thermocycler, open lid, and wait for 1 min. Turn on 365 nm LED flashlight 10 cm above tube for 1 s; then, immediately transfer tube to 95°C for 5 min and then to ice (which corresponds to ‘1 s’ sample) (Figure 1).



      Figure 1. In vitro cleavage setup. (a) Cas9, gRNA, and target DNA are added to tubes labeled with the appropriate incubation times. (b) Tubes are first placed in the 37°C incubator. After light exposure and 37°C incubation for the specified time, each tube is transferred to the 95°C incubator for heat-deactivation of Cas9. (c) Light exposure using a commercially available LED flashlight. The tube is first opened, the flashlight is held 10 cm above the tube, and the light is turned on for 30 s.


    6. Repeat using three other tubes for 5 s, 10 s, and 30 s of light illumination (which corresponds to ‘5 s’, ’10 s’, and ’30 s’ samples, respectively).

    7. Move another tube to the 37°C thermocycler, open lid, and wait for 1 min. Turn on 365 nm LED flashlight 10 cm above tube for 30 s, turn off flashlight and incubate tube with cap closed for another 30 s; transfer to 95°C for 5 min and then to ice (which corresponds to ‘1 min’ sample).

    8. Repeat using five other tubes with 30 s light illumination, followed by 1.5 min, 4.5 min, 9.5 min, 19.5 min, and 29.5 min incubations at 37°C; transfer to 95°C for 5 min, and then to ice (which corresponds to ‘2 min’, ‘5 min’, ’10 min’, ’20 min’, and ’30 min’ samples, respectively).

    9. The one remaining sample should not be exposed to light but directly moved to 95°C for 5 min and then to ice (which corresponds to ‘no light’ sample).

    10. Add 0.5 μl of Proteinase K to all 11 tubes and incubate at 55°C for 15 min.

    11. Load 5 μl of each tube (in ascending order of time-duration) mixed with 15 μl of water into each well of a 4% agarose E-Gel and run for 10 min. Add 100 bp ladder to the left-most lane. An alternative agarose gel electrophoresis system may also be used.

    12. Visualize with TyphoonTM FLA 9000 using the SYBR Gold setting. An alternative gel imager may also be used (Figure 2).



      Figure 2. In vitro cleavage results. The top band migrating at 444 bp corresponds to the uncleaved PCR product of PPP1R2. The bottom band is formed by two unresolved bands, both migrating around 220 bp, which correspond to the cleavage products of the 444 bp top band. Each lane corresponds to either no light exposure or a different light exposure/incubation condition.


  6. EMSA

    1. Mix the following components together in order. Thoroughly mix NFW and NEBufferTM 3.1 before adding cgRNA and Cas9 and mixing again. Prepare 2 identical volumes, both in PCR tubes as samples {B} and {C}.

      Component Volume
      NFW    8.1 μl
      NEBufferTM 3.1       1 μl
      10 μM cgRNA targeting PPP1R2     0.5 μl
      10 μM Cas9     0.4 μl
      Total      10 μl

    2. Prepare sample {A} by replacing Cas9 and cgRNA with water (negative control).

    3. Leave on benchtop (room temperature) for 30 min to form RNP complex. Cover with aluminum film because photocaged gRNAs are light-sensitive.

    4. Add 2 μl (~60 fmol) of PCR-amplified PPP1R2 target DNA to each of the three tubes. Mix well with pipette. For a highly efficient reaction, Cas9/cgRNA should be in at least 100-fold excess relative to target DNA. The target DNA should also be easily visible on an agarose gel with a gel imager.

    5. Add 0.5 μl of Proteinase K to {C}, incubate at 55°C for 15 min.

    6. Load 5 μl of each tube (in ascending order of time-duration) mixed with 15 μl water into each well of a 4% agarose E-Gel and run for 10 min. Add 100 bp ladder to the left-most lane. An alternative agarose gel electrophoresis system may also be used.

    7. Visualize with TyphoonTM FLA 9000 using SYBR Gold setting. An alternative gel imager may also be used (Figure 3).



      Figure 3. EMSA results. Lane 1 is the ladder; lane 2 is sample {A} (negative control without RNP); lane 3 is sample {B} (EMSA); lane 4 is sample {C} (Proteinase K digested sample). There is no cleavage product because the cgRNA was not exposed to any light, demonstrating that Cas9 with cgRNA binds to DNA even in the absence of light activation.

Acknowledgments

This work was supported by grants from the National Institutes of Health (R35 GM 122569 to T.H., and T32 GM 73009 and F30 CA 254160 to R. S. Z.) and the National Science Foundation (PHY 1430124 to T.H.). T.H. is an investigator of the Howard Hughes Medical Institute.

This protocol was derived from: Liu, Y., Zou, R. S., He, S., Nihongaki, Y., Li, X., Razavi, S., Wu, B. and Ha, T. (2020). Very fast CRISPR on demand. Science 368(6496): 1265-1269.

Competing interests

The authors and Johns Hopkins University have filed patent application PCT/US20/57256 on the method of spatiotemporal control of Cas9 activities via cgRNA.

References

  1. Adli, M. (2018). The CRISPR tool kit for genome editing and beyond. Nat Commun 9(1): 1911.
  2. Hellman, L. M. and Fried, M. G. (2007). Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nat Protoc 2(8): 1849-1861.
  3. Liu, Y., Zou, R. S., He, S., Nihongaki, Y., Li, X., Razavi, S., Wu, B. and Ha, T. (2020). Very fast CRISPR on demand. Science 368(6496): 1265-1269.
  4. Marraffini, L. A. (2015). CRISPR-Cas immunity in prokaryotes. Nature 526(7571): 55-61.
  5. Singh, D., Sternberg, S. H., Fei, J., Doudna, J. A. and Ha, T. (2016). Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9. Nat Commun 7: 12778.

简介

[摘要] CRISPR-Cas9 通过方便和有针对性的 DNA 操作改变了生物医学研究和医学。通过最近开发的非常快速的 CRISPR (vfCRISPR) 系统对 Cas9 活动的时间和空间分辨控制促进了 DNA 损伤和修复的综合研究。在广泛使用这些系统之前,了解 Cas9 结合和切割行为的基本原理是必不可少的,并且可以通过切割和电泳迁移率变化分析 (EMSA)在体外轻松完成。体外实验方案切割由 Cas9 与向导 RNA (gRNA) 核糖核蛋白 (RNP) 形成组成,然后与目标 DNA 孵育。对于 EMSA,该反应直接加载到琼脂糖凝胶上,用于可视化由于 Cas9 RNP 结合而移动的目标 DNA 条带。以测定裂解,加入蛋白酶K降解的RNP,从而允许靶DNA(切割的和/或未切割的)迁移一致LY与它的分子量。在 95 ° C加热可根据需要快速灭活 RNP,从而可以对 Cas9 裂解动力学进行时间分辨测量。该协议有利于所述光笼vfCRISPR gRNA的光活化机理的表征。


[背景]来自成簇的规则间隔短回文重复序列 (CRISPR) 和 CRISPR 相关 (CRISPR-Cas) 适应性免疫系统(Marraffini等,2015)的核酸内切酶,尤其是 Cas9已被广泛用作生物医学科学中的高度通用工具基因组编辑、功能筛选、成像(Adli等人,2018 年)。最近,通过非常快速的 CRISPR (vfCRISPR) 对 Cas9 活动的高度解析时空控制使得通过 Cas9 在空间和时间上的高度同步激活对 DNA 损伤反应的详细研究成为可能(Liu等,2020)。对于所有使用小号,这些核酸酶的表征是必不可少的,并且可以最容易地实现在体外(辛格等人。,2016)。电泳迁移率变化分析 (EMSA) 是表征蛋白质-DNA 结合的强大工具,可直接用于确定 Cas9 与核酸的结合亲和力(Hellman等,2007)。我Ñ相反,我Ñ体外切割分析允许Cas9 DNA切割机构的表征。在这份手稿中,我们概述了两种策略的详细协议,以表征 vfCRISPR 光诱导 Cas9 系统的特性。我们展示了 EMSA 和体外裂解测定如何表征 vfCRISPR 的结合、速度和效率,这为其在生物系统中的使用铺平了道路。

关键字:CRISPR-Cas9, 活体外 , 分裂, 电泳迁移率实验, 电泳迁移率实验, 基因组编辑


材料和试剂


1. 10毫克/毫升SpCas9纯化(在内部从BL21 CodonPlus(DE3)-RIL细胞[安捷伦230245],但可也可从集成DNA技术购买)(Alt键-R ® ,目录号:1081058,贮存温度: -80°C)      
2. tracrRNA(集成DNA技术)(Alt键-R ® ,目录号:1072533,贮存温度:-80℃)      
3.来自 vfCRISPR 的P hotocaged crRNA (cgRNA),靶向PPP1R2 (BioSynthesis,序列:GAC UU CC U CUAUGGUGGCGUGUUUUAGAGCUAUGCUGUUUUG;U对应于用 NPOM-dT photocaged 核苷酸替代尿嘧啶,储存温度:-80°C)      
4. HEK293T细胞(ATCC,目录号:CRL-3216,储存温度:液氮蒸气)      
5.无核酸酶双链体缓冲液(Integrated DNA Technologies,目录号:11-01-03-01,储存温度:4°C)      
6. PPP1R2 Fwd PCR 引物(Integrated DNA Technologies,储存温度:-20°C,序列:5' - GTTTCCGAGGCAGCAGTTG - 3')      
7. PPP1R2 Rev PCR 引物(Integrated DNA Technologies,储存温度:-20°C,序列:5' - GCATGATAAACGTCATCGCCC - 3')      
8. DNeasy Blood & Tissue Kit(Qiagen,DNeasy,目录号:69504)      
9. QIAquick PCR纯化试剂盒(Qiagen,QIAquick,目录号:28104)      
10. Q5 ®高保真2X预混(新英格兰生物实验室,Q5 ® ,目录号:M0492,储存温度:-20℃)   
11. NEBuffer TM 3.1(New England BioLabs,目录号:B7203S,储存温度:-20°C)   
12.蛋白酶K,分子生物学级(New England BioLabs,目录号:P8107S,储存温度:-20°C)   
13. E-凝胶TM用SYBR琼脂糖凝胶TM安全DNA凝胶染色,4%(赛默飞世,E-凝胶TM ,目录号:A45206,贮存温度:室温下)   
14. 100 bp DNA 阶梯(New England BioLabs,目录号:N3231S)   
15. HEPES(Sigma-Aldrich,目录号:H3375)   
16. KCl(Sigma-Aldrich,目录号:P3911)   
17.甘油(Sigma-Aldrich,目录号:G5516)   
18. NaOH(Sigma-Aldrich,目录号:221465)   
19.铝箔(雷诺包装)   
20.核酸酶-游离水(未DEPC-吨reated)(Invitrogen公司TM ,目录号:AM9932 )   
 
设备
 
C1000 Touch TM热循环仪(Bio-Rad,型号:1851148 ),或任何可进行聚合酶链反应 (PCR) 的替代热循环仪
E-Gel ® iBase TM Power System(Thermo Fisher,E-Gel TM ,目录号:G6465)或替代凝胶电泳系统
              JAXMAN 365 nm LED 手电筒(亚马逊、JAXMAN、https: //www.amazon.com/JAXMAN-Ultraviolet-365nm-Detector-Flashlight/dp/B06XW7S1CS/ )
Typhoon TM FLA 9000(GE Healthcare),或任何可对溴化乙锭或 SYBR Gold 琼脂糖凝胶成像的替代凝胶成像仪
 
程序
 
在PPP1R2处生成用于体外切割或 EMSA的靶 DNA
从HEK293T细胞用净化的基因组DNA(gDNA中)的的DNeasy血液和组织试剂盒按照生产商的说明进行操作。在试剂盒提供的 200 μl AE 中洗脱。将 gDNA 储存在 -20 °C。
将 10 μ M 的 Fwd 引物与 10 μ M 的 Rev 引物混合,制备 PCR 引物混合物,两者均在水中稀释。
设置 PCR 反应。
在热循环仪上启动热循环协议。
按照制造商的说明,使用QIAquick PCR 纯化试剂盒提取 PCR 扩增的目标 DNA 。用试剂盒提供的30 μl EB洗脱。
 
准备 10 ml Cas9 稀释缓冲液
1.配制由 20 mM HEPES、500 mM KCl和20% 甘油组成的溶液。      
2.加入足量的 1 M NaOH 使 pH 值达到 7.5。      
 
准备 10 μ M Cas9
稀5微升10毫克/毫升Cas9与25微升Cas9稀释缓冲液,使30微升的10 μ中号SpCas9。
 
将可光激活的 crRNA 与 tracrRNA 退火形成 cgRNA
1.重悬光笼crRNA和tracrRNA分别至100 μ与双工缓冲微米。      
2.混合3微升的100 μ中号光笼与crRNA 3微升的100 μ中号tracrRNA在PCR管中。      
3.在带有加热盖的热循环仪中在 95°C 下加热3 分钟。      
4.在台式上冷却 5 分钟。      
5.混合在24微升双工缓冲液使30微升10的μ中号退火CR / trRNA。      
 
体外切割以测试 cgRNA 的光激活能力
1.将以下成分按顺序混合在一起。在加入 cgRNA 和 Cas9 之前彻底混合 NFW 和 NEBuffer TM 3.1,然后再次混合。准备 11 个相同的体积,全部在单独的 PCR 管中。      
2.在台式(室温)上放置 30 分钟以形成 RNP 复合物。用铝膜覆盖,因为光笼式 gRNA 对光敏感。      
3.添加2 PCR扩增的微升(〜60飞摩尔)PPP1R2靶DNA的每个管RNP(11中总计)。用移液器充分混合。˚F或高效率的反应,Cas9 / cgRNA应该在至少相对于靶DNA过量100倍。靶DNA也应与琼脂糖凝胶容易看见一个凝胶成像仪。      
4.设置两个热循环仪       –一个到 37°C,另一个到 95°C。
5.移动一个管到所述37℃热循环仪,打开盖子,并等待1分钟。在管子上方 10 厘米处打开 365 nm LED 手电筒 1 秒;然后,立即转移至管95℃5分钟和然后到冰(其对应于“1秒”样品)(图1)。      
 
 
图1.我Ñ体外裂解设置。(a) 将 Cas9、gRNA 和目标 DNA 添加到标有适当孵育时间的试管中。(b) 试管首先放入 37°C 培养箱中。在光照和 37°C 孵育指定时间后,将每个管转移到 95°C 培养箱中以热灭活 Cas9。(c) 使用市售 LED 手电筒进行曝光。首先打开管子,将手电筒置于管子上方 10 cm 处,打开灯 30 s。
 
6.使用其他三个管重复进行 5 秒、10 秒和 30 秒的光照(分别对应于“5 秒”、“10 秒”和“30 秒”样本)。      
7.移动另一个管的37℃热循环仪,开盖,并等待1分钟。在管子上方 10 厘米处打开 365 nm LED 手电筒 30 秒,然后关闭手电筒并将管子盖上盖子再孵育 30 秒;转移到95℃持续5分钟,并然后到冰(其对应于“1分钟”样品)。      
8.用 30 秒光照的其他五个管重复,然后在 37°C 下孵育1.5 分钟、4.5 分钟、9.5 分钟、19.5 分钟和 29.5分钟;转移至95℃5分钟,并然后以冰(其分别对应于“2分钟”,“5分钟”,'10分钟,'20分钟和'30分钟的样品,)。      
9.所述一个剩余的样品不应当被暴露于光而直接移动到95℃5分钟和然后到冰(其对应于“没有光”样品)。      
10.向所有 11 个管中加入 0.5 μl蛋白酶 K,并在 55°C 下孵育 15 分钟。   
11.负载5微升每个管的(在时间持续时间的升序)用15混合微升的琼脂糖4%的水到每个孔ë - G ^ e1和10分钟运行。将 100 bp 阶梯添加到最左侧的车道。也可以使用替代的琼脂糖凝胶电泳系统。   
12.可视化台风与TM FLA 9000使用的SYBR金设置。也可以使用替代的凝胶成像仪(图 2)。   
 
 
图2 。我ñ体外裂解结果。在 444 bp 处迁移的顶部条带对应于PPP1R2的未切割 PCR 产物。底部条带由两个未解析的条带形成,均迁移约 220 bp,对应于 444 bp 顶部条带的裂解产物。每个泳道对应于无光照或不同的光照/孵育条件。
 
EMSA
1.将以下成分按顺序混合在一起。在加入 cgRNA 和 Cas9 并再次混合之前,彻底混合 NFW 和 NEBuffer TM 3.1。准备 2 个相同的体积,均在 PCR 管中作为样品 {B} 和 {C}。      
2.通过用水代替 Cas9 和 cgRNA(阴性对照)来制备样品 {A}。      
3.在台式(室温)上放置 30 分钟以形成 RNP 复合物。盖与铝膜因为光笼gRNA小号是光敏感的。      
4.将 2 μl (~60 fmol) PCR 扩增的PPP1R2目标 DNA 加入三个管中。用移液器充分混合。˚F或高效率的反应,Cas9 / cgRNA应该在至少相对于靶DNA过量100倍。靶DNA也应与琼脂糖凝胶容易看见一个凝胶成像仪。      
5.加入 0.5 μl蛋白酶 K 至 {C},55°C 孵育 15 分钟。      
6.负载5微升各管(以上升时间持续时间的顺序)15混合微升的琼脂糖4%的水到每个孔ë - G ^ e1和10分钟运行。将 100 bp 阶梯添加到最左侧的车道。也可以使用替代的琼脂糖凝胶电泳系统。      
7.使用 SYBR Gold 设置,使用Typhoon TM FLA 9000 进行可视化。也可以使用替代的凝胶成像仪(图 3)。      
 
 
图3 。EMSA 结果。泳道1是该阶梯; 泳道2是样品{A}(没有RNP的阴性对照);泳道3是样品{B}(EMSA);泳道 4 是样品 {C}(蛋白酶 K 消化的样品)。没有切割产物,因为 cgRNA 没有暴露在任何光线下,这表明即使在没有光激活的情况下,带有 cgRNA 的 Cas9 也能与 DNA 结合。
 
致谢
 
这项工作是由美国国立卫生研究院(R35 GM 122569到TH,资金支持和T32 GM 73009和F30 CA 254160到RSZ)和美国国家科学基金会(PHY 1430124至TH)。TH 是霍华德休斯医学研究所的研究员。
  该协议源自:Liu, Y.、Zou, RS、He, S.、Nihongaki, Y.、Li, X.、Razavi, S.、Wu, B. 和 Ha, T. (2020)。按需提供非常快的 CRISPR。科学368(6496):1265-1269。
 
利益争夺
 
作者和约翰霍普金斯大学已经提交了关于通过 cgRNA 对 Cas9 活动进行时空控制的方法的专利申请 PCT/US20/57256。
 
参考
 
Adli, M. (2018)。用于基因组编辑及其他方面的 CRISPR 工具包。国家通讯社9(1): 1911。
Hellman, LM 和 Fried, MG (2007)。用于检测蛋白质-核酸相互作用的电泳迁移率变化测定 (EMSA)。国家议定书2(8):1849-1861。
Liu, Y.、Zou, RS、He, S.、Nihongaki, Y.、Li, X.、Razavi, S.、Wu, B. 和 Ha, T. (2020)。按需提供非常快的 CRISPR。科学368(6496):1265-1269。
马拉菲尼,洛杉矶(2015 年)。原核生物中的 CRISPR-Cas 免疫。自然526(7571):55-61。              
Singh, D.、Sternberg, SH、Fei, J.、Doudna, JA 和 Ha, T.(2016 年)。通过 RNA 引导的核酸内切酶 Cas9 实时观察 DNA 识别和排斥。国家通讯社 7:12778。              
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Zou, R. S., Liu, Y. and Ha, T. (2021). In vitro Cleavage and Electrophoretic Mobility Shift Assays for Very Fast CRISPR. Bio-protocol 11(17): e4138. DOI: 10.21769/BioProtoc.4138.
  2. Liu, Y., Zou, R. S., He, S., Nihongaki, Y., Li, X., Razavi, S., Wu, B. and Ha, T. (2020). Very fast CRISPR on demand. Science 368(6496): 1265-1269.
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