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Cytosolic and Nuclear Delivery of CRISPR/Cas9-ribonucleoprotein for Gene Editing Using Arginine Functionalized Gold Nanoparticles
使用精氨酸功能化金纳米粒子进行CRISPR/Cas9-核糖核蛋白的细胞溶质和细胞核递送以用于基因编辑   

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Abstract

In this protocol, engineered Cas9-ribonucleoprotein (Cas9 protein and sgRNA, together called Cas9-RNP) and gold nanoparticles are used to make nanoassemblies that are employed to deliver Cas9-RNP into cell cytoplasm and nucleus. Cas9 protein is engineered with an N-terminus glutamic acid tag (E-tag or En, where n = the number of glutamic acid in an E-tag and usually n = 15 or 20), C-terminus nuclear localizing signal (NLS), and a C-terminus 6xHis-tag. [Cas9En hereafter]

To use this protocol, the first step is to generate the required materials (gold nanoparticles, recombinant Cas9En, and sgRNA). Laboratory-synthesis of gold nanoparticles can take up to a few weeks, but can be synthesized in large batches that can be used for many years without compromising the quality. Cas9En can be cloned from a regular SpCas9 gene (Addgene plasmid id = 47327), and expressed and purified using standard laboratory procedures which are not a part of this protocol. Similarly, sgRNA can be laboratory-synthesized using in vitro transcription from a template gene (Addgene plasmid id = 51765) or can be purchased from various sources.

Once these materials are ready, it takes about ~30 min to make the Cas9En-RNP complex and 10 min to make the Cas9En-RNP/nanoparticles nanoassemblies, which are immediately used for delivery (Figure 1). Complete delivery (90-95% cytoplasmic and nuclear delivery) is achieved in less than 3 h. Follow-up editing experiments require additional time based on users’ need.

Synthesis of arginine functionalized gold nanoparticles (ArgNPs) (Yang et al., 2011), expression of recombinant Cas9En, and in vitro synthesis of sgRNA is reported elsewhere (Mout et al., 2017). We report here only the generation of the delivery vehicle i.e., the fabrication of Cas9En-RNP/ArgNPs nanoassembly.

Keywords: Cas9-ribonucleoprotein delivery(Cas9-核糖核蛋白递送), Protocol for CRISPR/Cas9 delivery(CRISPR/Cas9递送方案), Cytosolic delivery(细胞溶质递送), Nanoparticles(纳米粒子), Gene editing(基因编辑)

Background

Delivery of Cas9-ribonucleoprotein provides an alternative strategy for CRISPR gene delivery, offering a transient way of editing genes. Although a few strategies for Cas9-RNP delivery have been reported, these strategies suffer from endosomal entrapment of both Cas9 protein and sgRNA (Liu et al., 2015). Mechanical methods including membrane deformation (Han et al., 2015), electroporation (Schumann et al., 2015), and the use of hypertonic agents (D’Astolfo et al., 2015) provide direct delivery, however, they require specialized instrumentations and are generally not practical for in vivo therapeutic applications. Our protocol provides an approach for direct cytoplasmic and nuclear delivery of Cas9-RNP that can find applications in both gene editing and genome imaging.

Materials and Reagents

  1. Round bottom 35 mm confocal dish (MATTEK, catalog number: P35G-0-14-C )
  2. 24-well plates (Corning, Costar®, catalog number: 3524 )
  3. Sterile 1.5 ml tubes (Fisher Scientific, catalog number: 05-408-129 )
  4. Sterile pipette tips
  5. Cell lines (i.e., HeLa)
  6. Stock solution of ArgNP gold nanoparticles (~50 μM in water), freshly purified Cas9En protein (~10-20 μM), and sgRNA (~150 μM)
  7. 1x phosphate-buffered saline (PBS) (GE Healthcare, HyCloneTM, catalog number: SH30028.02 )
  8. Plain DMEM media (No serum and antibiotics, appropriate media for cell culture, i.e., HeLa cells) (Thermo Fisher Scientific, GibcoTM, catalog number: 10567014 )
  9. Alexa Fluor 488 NHS Ester (Thermo Fisher Scientific, InvitrogenTM, catalog number: A20000 )
  10. AmpliScribe T7-Flash- Transcription Kit (Epicentre, catalog numbers: ASF3257 and ASF3507 )

Equipment

  1. Pipettes
  2. Cell culture incubator at 5% CO2 and 37 °C
  3. Fluorescence microscope (any confocal microscope)

Procedure


Figure 1. Schematic overview of the protocol. Step 1: formation of Cas9En-sgRNA complex (takes 30 min); Step 2: formation of Cas9En-RNP/ArgNPs nanoassembly (takes 10 min); and Step 3: Cas9En-RNP delivery (takes 3 h).

Final working nanoassembly concentration is 125 nM of ArgNPs and 62 nM of Cas9En-RNP complex, which is at a 2:1 molar ratio of ArgNPs/Cas9En-RNP. The total volume of the nanoassembly samples required for delivery depends on the kind of cell culture plate used. We generally use 1 ml for round bottom 35 mm confocal dishes, 500 μl for 24-well plates, and 200 μl for 96-well plates, per well. Therefore, the nanoassemblies should be made and scaled up according to users’ need. The following calculation is for one sample in a 24-well plate (i.e., 500 μl total volume). Additionally, the following protocol is for HeLa cells, however, we verified the Cas9En-RNP delivery in other cell lines including mouse macrophage RAW 264.7, human embryonic kidney HEK cells, and human primary mammary epithelial cells.

  1. First, 24 h before the delivery experiment, seed cells on a 24-well plate at a cell density of 8 x 104-10 x 105 cells/well.
  2. On the day of delivery, prepare the Cas9En-RNP complex by simply mixing Cas9En and sgRNA at a 1:1 molar ratio in 10-20 μl of 1x PBS buffer. Keep in mind that the final Cas9En-RNP concentration in 500 μl sample should be 62 nM (~5 μg Cas9En, and 1 μg sgRNA).
  3. Incubate the complex at room temperature for 30 min.
  4. In the meantime, add 100 μl of 1x PBS into a sterile 1.5 ml tube.
  5. Add required amount of ArgNPs into the tube containing 1x PBS to make the final concentration of ArgNPs 125 nM for 500 μl sample. Mix by pipetting up and down.
  6. Now add the premixed Cas9En-RNP complex to the ArgNPs solution in the tube.
  7. Mix well by pipetting up and down.
  8. Incubate the sample at room temperature for 10 min. The nanoassemblies will be ready in 10 min and therefore users should prepare the cells for delivery in the meantime.
    Note: Don’t incubate for more than 10 min, as this may cause aggregate formation instead of well-defined assemblies.
  9. Wash the cells with 1x PBS, twice.
  10. When the nanoassembly sample is ready after 10 min, add ~400 μl of DMEM plain media (or any media of interest) into the nanoassembly solution in the tube. Mix well.
    Note: Media with serum should be strictly avoided at this step; media with serum may require further optimization such as the ratio of nanoparticles to Cas9En:sgRNA, and incubation time.
  11. Add the whole (500 μl) sample into the washed-cells.
  12. Incubate the cells at an appropriate condition (usually 5% CO2 and 37 °C) for 3 h. Complete Cas9En-RNP delivery should be achieved after 3 h of incubation.
  13. Wash away the media after 3 h, and replace with fresh desired media (DMEM with 10% serum and 1% antibiotics, and any supplements if needed).

Data analysis

Cytoplasmic delivery efficiency of Cas9En or Cas9En-RNP was determined by confocal microscopy imaging. Even distribution of fluorophore labelled Cas9En in the cytosol and nucleus is considered as effective delivery, whereas any punctate distribution in the cytoplasm is considered as endosomal delivery (Figures 2A and 2B). Briefly, ~400 cells were counted manually to estimate effective cytosolic and nuclear delivery. Any punctate distribution (see Figure 2B) of labelled-Cas9En should be avoided from counting as direct cytoplasmic and nuclear delivery. Please see Mout et al., 2017 for more details.


Figure 2. Confocal microscopy images showing examples of efficient cytosolic and nuclear delivery of Cas9E20 (Alexa Fluor 488 labelled) in cultured HeLa cells. A. High efficient cytoplasmic delivery at a 2:1 molar ratio of ArgNP/Cas9E20; B. Low efficient delivery of Cas9E20 that occurs through endocytosis at a 1:1 molar ratio of ArgNP/Cas9E20. Note that, maximum cytoplasmic delivery efficiency can be achieved through screening different molar ratio of ArgNPs/Cas9En or Cas9En-RNP, as noted below.

Notes

  1. As a point of reference, gene editing verification is performed 48 h after nanoassembly treatment.
  2. The ratio ArgNPs/Cas9En is the most crucial factor for delivery efficiency. Every batch of ArgNPs can be slightly deferent in terms of surface ligand coverage and therefore users are suggested to test different ratios of ArgNPs/Cas9En to find out maximum delivery. Fluorophore labelled (FITC/Alexa Fluor 488) Cas9En should be used to evaluate delivery efficiency (see manufacturer’s protocol for labelling: Alexa Fluor 488 NHS Ester, Thermo Fisher Scientific).
  3. Freshly prepared (not more than a week old, and preserved at 4 °C) Cas9En should be used for better activity. Flash-frozen Cas9En at -80 °C with glycerol should be avoided as our preliminary investigation shows that glycerol hampers assembly formation.

Acknowledgments

This research was supported by the NIH (GM077173), NSF (CHE-1307021) and a UMass OTCV grant.

References

  1. D’Astolfo, D. S., Pagliero, R. J., Pras, A., Karthaus, W. R., Clevers, H., Prasad, V., Lebbink, R. J., Rehmann, H. and Geijsen, N. (2015). Efficient intracellular delivery of native proteins. Cell 161(3): 674-690.
  2. Han, X., Liu, Z., Jo, M. C., Zhang, K., Li, Y., Zeng, Z., Li, N., Zu, Y. and Qin, L. (2015). CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation. Sci Adv 1(7): e1500454.
  3. Liu, J., Gaj, T., Yang, Y., Wang, N., Shui, S., Kim, S., Kanchiswamy, C. N., Kim, J. S. and Barbas, C. F., 3rd (2015). Efficient delivery of nuclease proteins for genome editing in human stem cells and primary cells. Nat Protoc 10(11): 1842-1859.
  4. Mout, R., Ray, M., Tonga, G. Y., Lee, Y. W., Tray, T., Sasaki, K. and Rotello, V. M. (2017). Direct cytoplasmic delivery of CRISPR/Cas9-ribonucleoprotein for efficient gene editing. ACS Nano 11(3): 2452-2458.
  5. Schumann, K., Lin, S., Boyer, E., Simeonov, D. R., Subramaniam, M., Gate, R. E., Haliburton, G. E., Ye, C. J., Bluestone, J. A., Doudna, J. A. and Marson, A. (2015). Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc Natl Acad Sci U S A 112(33): 10437-10442.
  6. Yang, X. C., Samanta, B., Agasti, S. S., Jeong, Y., Zhu, Z. J., Rana, S., Miranda, O. R. and Rotello, V. M. (2011). Drug delivery using nanoparticle-stabilized nanocapsules. Angew Chem Int Ed Engl 50(2): 477-481.

简介

在该方案中,使用工程化的Cas9-核糖核蛋白(Cas9蛋白和sgRNA,一起称为Cas9-RNP)和金纳米颗粒制备用于将Cas9-RNP递送至细胞质和细胞核的纳米组件。 Cas9蛋白用N末端谷氨酸标签(E标签或En,其中n = E-标签中的谷氨酸数目,通常n = 15或20),C末端核定位信号(NLS) ,和C末端6xHis标签。 [Cas9En]

为了使用该方案,第一步是产生所需的材料(金纳米颗粒,重组Cas9En和sgRNA)。金纳米颗粒的实验室合成可能需要长达数周,但可以批量合成,可以使用多年,而不损害质量。可以从常规SpCas9基因(Addgene plasmid id = 47327)克隆Cas9En,并使用不属于该方案的标准实验室程序进行表达和纯化。类似地,可以使用来自模板基因(Addgene plasmid id = 51765)的体外转录实验室合成sgRNA,或者可以从各种来源购买。

一旦这些材料准备就绪,制作Cas9En-RNP复合物大约需要30分钟,并使得Cas9En-RNP /纳米纳米组件立即用于输送(图1)。完成交付(90-95%细胞质和核递送)在不到3小时内实现。后续编辑实验需要根据用户需要额外的时间。

报道了精氨酸功能化金纳米粒子(ArgNPs)(Yang等人,2011),重组Cas9En的表达和sgRNA的体外合成的合成(Mout > et al。,2017)。我们在这里仅报告传送车辆的生成,即制造Cas9En-RNP / ArgNP纳米组件。
【背景】Cas9-核糖核蛋白的递送为CRISPR基因递送提供了替代策略,提供了一种编辑基因的瞬时方式。 虽然已经报道了Cas9-RNP递送的几种策略,但是这些策略受到Cas9蛋白和sgRNA的内体捕获的困扰(Liu等人,2015)。 包括膜变形的机械方法(Han et al。,2015),电穿孔(Schumann等人,2015),以及使用高渗剂(D'Astolfo >等),2015)提供直接递送,但是,它们需要专门的仪器,并且对于体内治疗应用通常不实用。 我们的方案提供了Cas9-RNP的直接细胞质和核递送的方法,可以在基因编辑和基因组成像中找到应用。

关键字:Cas9-核糖核蛋白递送, CRISPR/Cas9递送方案, 细胞溶质递送, 纳米粒子, 基因编辑

材料和试剂

  1. 圆底35毫米共焦盘(MATTEK,目录号:P35G-0-14-C)
  2. 24孔板(Corning,Costar ®,目录号:3524)
  3. 无菌1.5ml管(Fisher Scientific,目录号:05-408-129)
  4. 无菌移液器吸头
  5. 细胞系(,即,HeLa)
  6. ArgNP金纳米粒子(水中约50μM),新鲜纯化的Cas9En蛋白(〜10-20μM)和sgRNA(〜150μM)的储备溶液
  7. 1x磷酸盐缓冲盐水(PBS)(GE Healthcare,HyClone TM,目录号:SH30028.02)
  8. 平板DMEM培养基(无血清和抗生素,适用于细胞培养的培养基,例如HeLa细胞)(Thermo Fisher Scientific,Gibco ,目录号:10567014) >
  9. Alexa Fluor 488 NHS酯(Thermo Fisher Scientific,Invitrogen TM,目录号:A20000)
  10. AmpliScribe T7-Flash-Transcription Kit(Epicentre,目录号:ASF3257和ASF3507)

设备

  1. 移液器
  2. 在5%CO 2和37℃下的细胞培养培养箱
  3. 荧光显微镜(任何共焦显微镜)

程序


图1.方案的示意图。步骤1:形成Cas9En-sgRNA复合物(需要30分钟);步骤2:形成Cas9En-RNP / ArgNPs纳米组装(需要10分钟);和步骤3:Cas9En-RNP递送(需要3小时)。

最终工作纳米组装浓度为125nM的ArgNP和62nM的Cas9En-RNP复合物,其浓度为ArgNPs / Cas9En-RNP的2:1摩尔比。所需的纳米组装样品的总体积取决于所用细胞培养板的种类。我们通常使用1毫升圆底35毫米共焦盘,500微升24孔板,每孔200微升96孔板。因此,纳米组件应根据用户的需要进行制作和扩大。以下计算是对于24孔板中的一个样品(即,,总体积为500μl)。此外,以下协议是用于HeLa细胞,然而,我们验证了其他细胞系中的Cas9En-RNP递送,包括小鼠巨噬细胞RAW 264.7,人胚胎肾HEK细胞和人类初级乳腺上皮细胞。

  1. 首先,在递送实验之前24小时,将细胞密度为8×10 4个细胞/孔的24孔板上的种子细胞置于细胞/孔中。 br />
  2. 在交货的当天,通过在10-20μl的1x PBS缓冲液中以1:1摩尔比混合Cas9En和sgRNA来制备Cas9En-RNP复合物。请记住,500μl样品中最终的Cas9En-RNP浓度应为62 nM(〜5μgCas9En和1μgsgRNA)。
  3. 在室温下孵育复合物30分钟
  4. 同时,将100μl1x PBS加入无菌的1.5 ml管中
  5. 将需要量的ArgNPs加入到含有1×PBS的管中,使500μl样品的ArgNP最终浓度为125nM。通过上下移动混合。
  6. 现在,将预混合的Cas9En-RNP复合物添加到管中的ArgNPs溶液中
  7. 通过上下移动进行混合。
  8. 在室温下孵育样品10分钟。纳米组件将在10分钟内准备就绪,因此用户应在此期间准备传送细胞。
    注意:不要孵育超过10分钟,因为这可能导致聚集体形成,而不是明确的组件。
  9. 用1x PBS洗涤细胞两次。
  10. 当纳米组装样品在10分钟后准备好后,加入〜400μl的DMEM平板培养基(或任何感兴趣的培养基)到管中的纳米组装溶液中。混合好
    注意:本步骤应严格避免含有血清的培养基;具有血清的培养基可能需要进一步优化,例如纳米颗粒与Cas9En:sgRNA的比例和孵育时间。
  11. 将全部(500μl)样品加入洗涤细胞中。
  12. 在适当的条件(通常为5%CO 2和37℃)下孵育细胞3小时。完成Cas9En-RNP交付应在孵化3小时后实现。
  13. 3小时后清洗培养基,更换新鲜所需的培养基(含有10%血清和1%抗生素的DMEM,如有需要可补充任何补充剂)。

数据分析

通过共焦显微镜成像测定Cas9En或Cas9En-RNP的细胞质传递效率。在细胞质和细胞核中标记的Cas9En的荧光团的均匀分布被认为是有效递送,而细胞质中的任何点状分布被认为是内体递送(图2A和2B)。简言之,手动计数〜400个细胞以估计有效的胞质和核递送。应该避免标记的Cas9En的任何点状分布(见图2B)计数为直接的细胞质和核递送。请参阅Mout 等。,2017年了解更多详情。


图2.共聚焦显微镜图像,显示培养的HeLa细胞中Cas9E20(Alexa Fluor 488标记)的有效胞质和核递送的实例。A.以2:1摩尔比的ArgNP / Cas9E20; B.通过ArgNP / Cas9E20 1:1摩尔比的内吞作用,Cas9E20的低效递送。请注意,如下所述,通过筛选不同摩尔比的ArgNPs / Cas9En或Cas9En-RNP可以实现最大的细胞质输送效率。

笔记

  1. 作为参考,在纳米组装处理48小时后进行基因编辑验证
  2. ArgNPs / Cas9En的比例是提供效率最关键的因素。每一批ArgNPs在表面配体覆盖方面可能略有不同,因此建议用户测试不同比例的ArgNPs / Cas9En,以找出最大的传递。应使用标记有荧光素(FITC / Alexa Fluor 488)的Cas9En来评估传递效率(参见制造商标签协议:Alexa Fluor 488 NHS Ester,Thermo Fisher Scientific)。
  3. 新鲜准备(不超过一周,并保存在4°C)Cas9En应用于更好的活动。应该避免在-80℃下用甘油冷冻的Cas9En,因为我们的初步调查显示甘油阻碍了组装形成。

致谢

这项研究得到NIH(GM077173),NSF(CHE-1307021)和UMass OTCV授权的支持。

参考

  1. D'Astolfo,D. S.,Pagliero,R.J.,Pras,A.,Karthaus,W.R.,Clevers,H.,Prasad,V.,Lebbink,R.J.,Rehmann,H.and Geijsen,N。(2015)。 天然蛋白质的高效细胞内转运细胞 161( 3):674-690。
  2. Han,X.,Liu,Z.,Jo,M. C.,Zhang,K.,Li,Y.,Zeng,Z.,Li,N.,Zu,Y.and Qin,L。(2015)。 CRISPR-Cas9通过膜变形传递到难以转染的细胞。 > Sci Adv 1(7):e1500454。
  3. Liu,J.,Gaj,T.,Yang,Y.,Wang,N.,Shui,S.,Kim,S.,Kanchiswamy,C.N.,Kim,J.S。和Barbas,C.F.,3rd(2015)。 在人类干细胞和原代细胞中有效提供用于基因组编辑的核酸酶蛋白质。 Nat Protoc 10(11):1842-1859。
  4. Mout,R.,Ray,M.,Tonga,G.Y.,Lee,Y.W.,Tray,T.,Sasaki,K.and Rotello,V.M。(2017)。 用于有效基因编辑的CRISPR / Cas9-ribonucleoprotein的直接细胞质递送。 ACS Nano 11(3):2452-2458。
  5. Schumann,K.,Lin,S.,Boyer,E.,Simeonov,DR,Subramaniam,M.,Gate,RE,Haliburton,GE,Ye,CJ,Bluestone,JA,Doudna,JA and Marson, )。 使用Cas9核糖核蛋白生成敲入的原代人类T细胞。 Natl Acad Sci USA 112(33):10437-10442。
  6. Yang,X.C.,Samanta,B.,Agasti,S.S.,Jeong,Y.,Zhu,Z.J.,Rana,S.,Miranda,O.R。和Rotello,V.M。(2011)。 使用纳米颗粒稳定纳米胶囊的药物递送 Angew Chem Int Ed Engl < 50(2):477-481。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Mout, R. and Rotello, V. M. (2017). Cytosolic and Nuclear Delivery of CRISPR/Cas9-ribonucleoprotein for Gene Editing Using Arginine Functionalized Gold Nanoparticles. Bio-protocol 7(20): e2586. DOI: 10.21769/BioProtoc.2586.
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