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Knock-in Blunt Ligation Utilizing CRISPR/Cas9
利用CRISPR/Cas9敲入平端连接   

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Abstract

The incorporation of the CRISPR/Cas9 bacterial immune system into the genetic engineering toolbox has led to the development of several new methods for genome manipulation (Auer et al., 2014; Byrne et al., 2015). We took advantage of the ability of Cas9 to generate blunt-ended double-strand breaks (Jinek et al., 2012) to introduce exogenous DNA in a highly precise manner through the exploitation of non-homologous end-joining DNA repair machinery (Geisinger et al., 2016). This protocol has been successfully applied to traditional immortalized cell lines and human induced pluripotent stem cells. Here we present a generalized protocol for knock-in blunt ligation, using HEK293 cells as an example.

Keywords: CRISPR/Cas9(CRISPR/Cas9), Genome engineering(基因组工程), NHEJ(NHEJ), Double-strand breaks(双链断裂), Cell culture(细胞培养)

Background

At the time we conceptualized knock-in blunt ligation (Geisinger et al., 2016), the vast majority of methods developed for use with CRISPR/Cas9 were focused on enhancing the efficiency of homologous recombination. However, there was one exception: a homology-independent, plasmid-based knock-in method developed in zebrafish (Auer et al., 2014). This method, like knock-in blunt ligation, relies on the machinery of canonical non-homologous end-joining to insert a linearized, blunt-ended, double-stranded DNA fragment into a genomic double-strand break with a high degree of precision and minimal loss of nucleotides. Both methods are similar to a method developed for zinc-finger nucleases and TALENs known as obligate ligation-gated recombination (ObLiGaRe; Maresca et al., 2013), which relied on the generation of compatible overhangs to facilitate insertion of target DNA into the genome. Both the Auer method and ObLiGaRe rely on delivery of a vector bearing the desired transgene construct, which could lead to incorporation of undesirable exogenous sequences. Because of the propensity of Staphylococcus pyogenes Cas9 to make blunt-ended double-strand breaks, we reasoned that exogenous sequence delivery in genome engineering experiments could be limited to solely the CRISPR/Cas9 expression vector and a PCR-generated amplicon of solely the sequence of interest. Thus, knock-in blunt cloning possesses the dual advantages of minimizing the introduction of exogenous sequences and its reliance on canonical non-homologous end-joining rather than homologous recombination. While the following protocol is specifically for human HEK293 cells, we note that this method is likely to be broadly applicable to eukaryotic cells.

Materials and Reagents

  1. ChromaSpin+TE-1,000 chromatography columns (Takara Bio, Clontech, catalog number: 636079 )
  2. Falcon® polystyrene 24-well tissue culture microplates (Corning, Falcon®, catalog number: 353226 )
  3. Thin-walled PCR tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: AB1182 )
  4. Falcon® test tube with cell strainer snap cap (Corning, Falcon®, catalog number: 352235 )
  5. Falcon® polystyrene 96-well tissue culture microplates, flat bottom (Corning, Falcon®, catalog number: 353916 )
  6. 1.75 ml microcentrifuge tubes
    Note: Manufacturer does not matter.
  7. Optional: 10-cm tissue culture dishes (Corning, catalog number: 353003 )
  8. A human cell line, such as HEK293
  9. Avector that expresses the guide RNA or RNAs of interest and Cas9 (e.g., Addgene, catalog number: 42230 )
  10. Double-stranded DNA template, preferably plasmid
  11. DNA oligos containing three phosphorothioate bonds at the 5’ end of each oligo for PCR amplification of a template
  12. A high-fidelity, blunt-end generating DNA polymerase: Phusion (New England Biolabs, catalog number: E0553S ) or Q5 (New England Biolabs, catalog number: M0491S ), with associated buffer, are the only acceptable polymerases for this protocol
  13. 10 mM dNTPs (New England Biolabs, catalog number: N0446S )
  14. MinElute PCR Purification Kit (QIAGEN, catalog number: 28004 )
  15. FastDigest DpnI (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD1703 )
  16. Nuclease-free water
  17. A transfection reagent such as FuGENE® HD (Promega, catalog number: E2311 )
  18. Opti-MEM I media (Thermo Fisher Scientific, GibcoTM, catalog number: 31985062 )
  19. NucBlue® Fixed Cell Stain ReadyProbes® reagent (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: R37606 )
  20. Phosphate buffered saline (PBS)
  21. 0.05% trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
  22. DMEM, high glucose (Thermo Fisher Scientific, GibcoTM, catalog number: 11965092 )
  23. Fetal bovine serum (FBS)
  24. 0.5 M EDTA, pH 8.0 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15575020 )
  25. Growth media (see Recipes)
  26. FACS media (see Recipes)

Equipment

  1. PCR thermocycler (Bio-Rad Laboratories, catalog number: 1861096 )
  2. Tissue culture hood, Type B2 biological safety cabinet (e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: 1300 Series Class II )
  3. Incubator (37 °C and 5% CO2) (e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: Series 8000 )
  4. Microcentrifuge (e.g., Eppendorf, model: 5424 )
  5. FACSAria II cell sorter (BD)
  6. Hemocytometer (Neubauer chamber) or equivalent method

Procedure

  1. Generation of the knock-in blunt ligation cassette
    1. Set up a minimum of 8 PCR reactions according to the manufacturer’s instructions using 1 ng of double-stranded DNA template and a minimum reaction volume of 50 µl. Carry out the PCR on a thermocycler using appropriate parameters.
    2. Following PCR, pool the reactions and purify the product using the MinElute PCR Purification Kit according to the manufacturer’s instructions. Elute in 20 µl of the provided EB buffer.
    3. To remove carried-over plasmid, perform a restriction digest using FastDigest DpnI. Use a reaction volume of 30 µl and the following thermocycler protocol:
      37 °C for 1 h
      80 °C for 10 min
      12 °C for infinity
    4. Bring the reaction to 100 µl with nuclease-free water and purify the cassette product from the digest using a CHROMASpin+TE-1000 chromatography column according to the manufacturer’s instructions (see Note 1).

  2. Knock-in blunt ligation
    1. One day prior to transfection, plate 150,000 to 500,000 HEK293 cells per well in a 24-well plate in 500 µl of growth media and place in a tissue culture incubator.
    2. On the day of transfection, assemble the transfection reactions in microcentrifuge tubes in a tissue culture hood using 100-200 ng of purified cassette, 0.5-1.5 µg of guide RNA + Cas9 vector, FuGENE® HD reagent at a 3:1 reagent-to-DNA ratio, and Opti-MEM I to 50 µl. Perform each reaction in triplicate, making sure to include a negative (transfection reagent alone) control. Vortex the reactions briefly and incubate at room temperature for 30 min.
    3. After 30 min, aspirate media from cells and replace with 500 µl of fresh growth media. Remove 50 µl of media from each well. Then, add the transfection reaction dropwise to each well, followed by gently swirling the plate to mix. Place the plate in a tissue culture incubator.
    4. Two days post-transfection, trypsinize cells (see Note 2), harvest in microcentrifuge tubes, spin down at 300 x g for 5 min in a microcentrifuge, and aspirate the supernatant. Then, resuspend the pellets in 200 µl of FACS media supplemented with NucBlue® Fixed Cell Stain ReadyProbes® reagent and filter the cell suspension through the strainer of a Falcon test tube. Place tubes on ice, protected from light.
    5. Using a FACSAria II flow cytometer, clone individual cells into individual wells of a 96-well plate containing 200 µl growth media. Briefly centrifuge (300 x g) the plate after cloning and place in a tissue culture incubator for 5 days undisturbed (see Note 3). Note that this cloning process is greatly facilitated by a fluorescent reporter, either in the cassette or on the Cas9 expression vector (see Note 4).
    6. After 5 days, replace old media with 200 µl of fresh media regularly until colonies are large enough to passage.
    7. When passaging colonies for the first time, reserve half of the cells in each colony for diagnostic PCR to identify which colonies contain the desired knock-in event.

Data analysis

Examples of flow plots and average percentages of transfected cells along with sequencing data from successfully knocked-in alleles for HEK293 and human induced pluripotent stem cells can be found in the original paper (Geisinger et al., 2016; Link to paper). Additionally, diagrams of the procedure, as well as examples of knock-in cassettes, can be found in the original paper.

Notes

  1. Alternatively, steps A2-A4 can be replaced with gel electrophoresis followed by gel extraction.
  2. To trypsinsize cells, first aspirate media. Then, wash with 1 ml of PBS followed by 200 µl of 0.05% trypsin-EDTA. Place cells in tissue culture incubator at 37 °C for 10 min. Remove cells from incubator and add 800 µl growth media before harvesting.
  3. As an alternative to cloning individual cells, serial dilution plating may be used, with the caveat that more time and more plates may be required to obtain a desired clone.
  4. If using a fluorescent reporter or drug resistance-based selection, cells from the transfection alternatively can be sorted into one pool and plated sparsely on 10-cm tissue culture dishes in growth media to generate colonies.

Recipes

  1. Growth media
    450 ml high-glucose DMEM
    50 ml FBS
  2. FACS media
    488 ml PBS
    10 ml FBS
    2 ml 0.5 M EDTA

Acknowledgments

This protocol was originally published as part of Geisinger et al. (2016). The authors wish to thank past members of the Calos lab for helpful discussions. Special thanks to the Stanford Shared FACS Facility and NIH S10 Shared Instrument Grant S10RR025518-01 and the California Institute for Regenerative Medicine TR4-06711 for funding.

References

  1. Auer, T. O., Duroure, K., De Cian, A., Concordet, J. P. and Del Bene, F. (2014). Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. Genome Res 24(1): 142-153.
  2. Byrne, S. M., Ortiz, L., Mali, P., Aach, J. and Church, G. M. (2015). Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells. Nucleic Acids Res 43(3): e21.
  3. Geisinger, J. M., Turan, S., Hernandez, S., Spector, L. P. and Calos, M. P. (2016). In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining. Nucleic Acids Res 44(8): e76.
  4. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A. and Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096): 816-821.
  5. Maresca, M., Lin, V. G., Guo, N. and Yang, Y. (2013). Obligate ligation-gated recombination (ObLiGaRe): custom-designed nuclease-mediated targeted integration through nonhomologous end joining. Genome Res 23(3): 539-546.

简介

将CRISPR / Cas9细菌免疫系统并入基因工程工具箱已经导致了几种用于基因组操作的新方法的开发(Auer等人,2014; Byrne等人,2015)。我们利用Cas9产生平端双链断裂的能力(Jinek等人,2012),以高度精确的方式通过开发非同源末端引物来引入外源DNA,加入DNA修复机械(Geisinger等人,2016)。该方案已成功应用于传统的永生化细胞系和人诱导多能干细胞。在这里,我们提出了使用HEK293细胞作为例子的敲入钝性连接的一般化方案。

背景 当我们概念化敲门钝性结扎(Geisinger等人,2016)时,开发用于CRISPR / Cas9的绝大多数方法都集中在提高同源重组的效率。然而,有一个例外:在斑马鱼中开发的同源性独立的基于质粒的敲入方法(Auer等人,2014)。这种方法,如敲入钝性连接,依赖于典型的非同源末端连接的机制,以线性化的,平端的双链DNA片段以高精度插入到基因组双链断裂中,核苷酸损失最小。这两种方法类似于为锌指核酸酶和被称为专性连接门控重组的TALEN开发的方法(ObLiGaRe; Maresca等人,2013),其依赖于产生相容的突出端以促进将靶DNA插入基因组。 Auer方法和ObLiGaRe都依赖于携带所需转基因构建体的载体的递送,这可能导致不期望的外源序列的引入。由于化脓性葡萄球菌Cas9进行平端双链断裂的倾向,我们推断基因组工程实验中的外源序列传递可能仅限于CRISPR / Cas9表达载体和PCR-仅产生感兴趣的序列的扩增子。因此,敲入钝性克隆具有最小化引入外源序列和依赖于典型非同源末端连接而不是同源重组的双重优点。虽然以下方案专门针对人类HEK293细胞,但我们注意到,该方法可能广泛适用于真核细胞。

关键字:CRISPR/Cas9, 基因组工程, NHEJ, 双链断裂, 细胞培养

材料和试剂

  1. ChromaSpin + TE-1000色谱柱(Takara Bio,Clontech,目录号:636079)
  2. Falcon ®聚苯乙烯24孔组织培养微孔板(Corning,Falcon ®,目录号:353226)
  3. 薄壁PCR管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:AB1182)
  4. Falcon ®试管与细胞过滤器卡帽(Corning,Falcon ®,目录号:352235)
  5. 聚苯乙烯96孔组织培养微孔板,平底(Corning,Falcon ®,目录号:353916)
  6. 1.75 ml微量离心管
    注意:制造商无所谓。
  7. 可选:10厘米组织培养皿(康宁,目录号:353003)
  8. 人类细胞系,如HEK293
  9. 表达感兴趣的引导RNA或RNA的载体和Cas9(例如,Addgene,目录号:42230)
  10. 双链DNA模板,优选质粒
  11. 在每个寡核苷酸的5'末端含有三个硫代磷酸酯键用于PCR扩增模板的DNA寡核苷酸
  12. 具有相关缓冲液的高保真,平端产生DNA聚合酶:Phusion(New England Biolabs,目录号:E0553S)或Q5(New England Biolabs,目录号:M0491S)是该方案唯一可接受的聚合酶
  13. 10mM dNTPs(New England Biolabs,目录号:N0446S)
  14. MinElute PCR Purification Kit(QIAGEN,目录号:28004)
  15. FastDigest I(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:FD1703)
  16. 无核酸酶的水
  17. 转染试剂如FuGENE HD(Promega,目录号:E2311)
  18. Opti-MEM I介质(Thermo Fisher Scientific,Gibco TM ,目录号:31985062)
  19. NucBlue ®固定细胞染色ReadyProbes ®试剂(Thermo Fisher Scientific,Molecular Probes TM,目录号:R37606)
  20. 磷酸盐缓冲盐水(PBS)
  21. 0.05%胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM,目录号:25300054)
  22. DMEM,高葡萄糖(Thermo Fisher Scientific,Gibco TM,目录号:11965092)
  23. 胎牛血清(FBS)
  24. 0.5M EDTA,pH8.0(Thermo Fisher Scientific,Invitrogen TM,目录号:15575020)
  25. 成长媒介(见食谱)
  26. FACS媒体(见配方)

设备

  1. PCR热循环仪(Bio-Rad Laboratories,目录号:1861096)
  2. 组织培养罩,B2型生物安全柜(Thermo Fisher Scientific,Thermo Scientific TM,型号:1300 Series Class II)
  3. 孵育器(37℃和5%CO 2)(Thermo Fisher Scientific,Thermo Scientific,Sup。TM,型号:Series 8000)(例如, br />
  4. 微量离心机(例如,Eppendorf,型号:5424)
  5. FACSAria II细胞分选机(BD)
  6. 血细胞计数器(Neubauer chamber)或等效方法

程序

  1. 敲入钝性结扎盒的产生
    1. 根据制造商的说明书使用1ng双链DNA模板和50μl的最小反应体积设置至少8个PCR反应。使用适当的参数在热循环仪上进行PCR
    2. PCR后,使用MinElute PCR纯化试剂盒根据制造商的说明进行反应并纯化产物。在20μl提供的EB缓冲液中洗脱。
    3. 为了除去携带的质粒,使用FastDigestIp进行限制性消化。使用30μl的反应体积和以下热循环仪方案:
      37°C 1小时
      80°C 10分钟
      12°C无限远
    4. 使用无核酸酶的水将反应物加入到100μl中,并使用CHROMASpin + TE-1000色谱柱根据制造商的说明书从消化物中纯化盒产品(见注1)。

  2. 敲入钝性结扎
    1. 转染前一天,在500μl生长培养基中的24孔板中每孔铺板150,000至500,000个HEK293细胞,并置于组织培养箱中。
    2. 在转染当天,使用100-200ng纯化的盒,0.5-1.5μg的引导RNA + Cas9载体,FuGENE HD试剂在组织培养罩的微量离心管中组装转染反应3:1试剂与DNA的比例,Opti-MEM I至50μl。一式三份进行一次反应,确保包括阴性(单独转染试剂)对照。短暂旋转反应,室温孵育30分钟
    3. 30分钟后,从细胞吸出培养基,并用500μl新鲜生长培养基代替。从每个孔中取出50μl的培养基。然后,将转染反应滴加到各孔中,然后轻轻旋转平板混匀。将板放在组织培养箱中
    4. 转染后两天,胰蛋白酶消化细胞(参见注释2),在微量离心管中收获,在微量离心机中以300×g离心5分钟,并吸出上清液。然后,将颗粒重新悬浮在200μl补充有NucBlue固定细胞染色ReadyProbes 试剂的FACS培养基中,并通过Falcon试管的过滤器过滤细胞悬浮液。将管放在冰上,防止光照。
    5. 使用FACSAria II流式细胞仪,将单个细胞克隆到含有200μl生长培养基的96孔板的各个孔中。克隆后将板简单离心(300×g),并将其置于组织培养箱中5天而不受干扰(见注3)。请注意,这种克隆过程可以通过荧光报告员在盒式磁带或Cas9表达载体中大大方便(见注4)。
    6. 5天后,定期用200μl新鲜培养基更换旧培养基,直到菌落足够大才能通过。
    7. 当第一次传代菌落时,将每个菌落中的一半细胞保留用于诊断PCR,以鉴定哪个菌落含有所需的敲入事件。

数据分析

原始文献(Geisinger等人,2016年)中可以找到流式图和转染细胞的平均百分比以及来自成功敲入等位基因HEK293和人诱导多能干细胞的测序数据的实例。 链接到纸)。另外,在原始的论文中可以找到程序的图表,以及敲入盒的例子。

笔记

  1. 或者,步骤A2-A4可以用凝胶电泳代替,随后进行凝胶提取
  2. 为了细胞培养,首先吸出培养基。然后用1ml PBS洗涤,然后用200μl的0.05%胰蛋白酶-EDTA洗涤。将组织培养箱中的细胞在37℃下放置10分钟。从培养箱中取出细胞,然后在收获前加入800μl生长培养基
  3. 作为克隆单个细胞的替代方案,可以使用连续稀释电镀,注意可能需要更多的时间和更多的平板来获得所需的克隆。
  4. 如果使用荧光报告基因或耐药基选择,则可将转染细胞分选成一个池,稀释地铺在生长培养基上的10cm组织培养皿上以产生菌落。

食谱

  1. 成长媒体
    450毫升高糖DMEM
    50ml FBS
  2. FACS媒体
    488毫升PBS
    10 ml FBS
    2 ml 0.5 M EDTA

致谢

该协议最初作为Geisinger等人的一部分发布。 (2016)。作者希望感谢Calos实验室的过去成员进行有益的讨论。特别感谢斯坦福共享FACS设备和NIH S10共享仪器授权S10RR025518-01和加利福尼亚再生医学研究所TR4-06711资助。

参考文献

  1. Auer,TO,Duroure,K.,De Cian,A.,Concordet,JP and Del Bene,F.(2014)。  高效CRISPR / Cas9介导的通过同源性DNA修复的斑马鱼敲入。 Genome Res 24( 1):142-153。
  2. Byrne,SM,Ortiz,L.,Mali,P.,Aach,J.和Church,GM(2015)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm。 nih.gov/pubmed/25414332"target ="_ blank">在人诱导的多能干细胞中的多千碱基型纯合靶向基因替换。(c)核酸Res 43(3):e21。 br />
  3. Geisinger,JM,Turan,S.,Hernandez,S.,Spector,LP和Calos,MP(2016)。&lt; a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih .gov / pubmed / 26762978"target ="_ blank"> 体内通过CRISPR / Cas9促进的非同源末端连接进行平端克隆。核酸Res < / em> 44(8):e76。
  4. Jinek,M.,Chylinski,K.,Fonfara,I.,Hauer,M.,Doudna,JA和Charpentier,E.(2012)。&nbsp; 可编程双RNA引导的DNA内切核酸酶在适应性细菌免疫中的应用。 科学 337(6096) :816-821。
  5. Maresca,M.,Lin,VG,Guo,N。和Yang,Y。(2013)。&nbsp; 有义连接门控重组(ObLiGaRe):通过非同源末端连接定制设计的核酸酶介导的靶向整合。 Genome Res 23(3): 539-546。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Geisinger, J. M. and Calos, M. P. (2017). Knock-in Blunt Ligation Utilizing CRISPR/Cas9. Bio-protocol 7(5): e2163. DOI: 10.21769/BioProtoc.2163.
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