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Jul 2016

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Construction of a Single Transcriptional Unit for Expression of Cas9 and Single-guide RNAs for Genome Editing in Plants
构建共表达Cas9和sgRNAs的单一独立转录单元用于植物基因组编辑   

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Abstract

The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein9 (Cas9) is a simple and efficient tool for genome editing in many organisms including plant and crop species. The sgRNAs of the CRISPR/Cas9 system are typically expressed from RNA polymerase III promoters, such as U6 and U3. In many transformation events, more nucleotides will increase the difficulties in plasmid construction and the risk of wrong integration in genome such as base-pair or fragment missing (Gheysen et al., 1990). And also, in many organisms, Pol III promoters have not been well characterized, and heterologous Pol III promoters often perform poorly (Sun et al., 2015). Thus, we have developed a method using single transcriptional unit (STU) CRISPR-Cas9 system to drive the expression of both Cas9 and sgRNAs from a single RNA polymerase II promoter to achieve effective genome editing in plants.

Keywords: CRISPR (CRISPR), Cas9 (Cas9), Genome editing (基因组编辑), Single transcriptional unit (STU) (单一独立转录单元), Ribozyme (核酶)

Background

The sgRNA of the CRISPR-Cas9 system is mainly promoted by the small nuclear RNA promoters such as U6 and U3. Although it has been tested with prospered efficiency in many cases, it also has some limitations: (1) it is hard to achieve coordinated and/or inducible expression of Cas9 and the sgRNAs; (2) manipulating multiple sgRNAs for multiplexed gene editing can be tedious, requiring multiple Pol III promoters. The traditional RNA polymerase II promoter can’t be used in driving sgRNA expression, extra nucleotides will be added to the 5’- and 3’-ends of gRNA by RNA polymerase II and may interrupt the normal gRNA function. Additionally, RNAs transcribed by RNA polymerase II are exported rapidly into the cytoplasm while nuclear localization is required for the CRISPR-Cas9/gRNA duplex to access the genome editing (Lei et al., 2001). To overcome these obstacles, we use the ribozyme’s self-catalyzed cleavage to release the precise processing mature sgRNA under a RNA polymerase II promoter which drives expression of both Cas9 and sgRNA (named STU CRISPR-Cas9 system, Figure 1). Compared to the traditional small nuclear RNA promoters used in sgRNA expression, our STU CRISPR-Cas9 system has some advantages: (1) it’s shorter and easier in vector construction, and it will increase the transformation efficiency under some circumstances; (2) it only needs extra ribozyme flanking sequence (shorter than any RNA polymerase III promoter we are currently using) for multiple sgRNAs expression;(3) it has shown higher deletion efficiency induced by double sgRNAs. Thus, the STU CRISPR-Cas9 system driven by a single RNA polymerase II promoter can replace the traditional CRISPR-Cas9 system now we are using whether in vivo or in vitro if appropriate promoters are chosen.


Figure 1. Schematic illustration of the single transcription unit (STU) CRISPR-Cas9 system. Once transcribed by a Pol II promoter, the STU CRISPR-Cas9 primary transcripts will undergo self-cleavage by hammerhead ribozyme (RZ) to release the mature Cas9 mRNA and sgRNA. The Cas9 mRNA is terminated with a synthetic polyA (pA) sequence to facilitate translation, The RZ sequence (in blue) and its target sequence (in black) are illustrated.

Materials and Reagents

  1. 0.2 ml PCR tubes (Biosharp, catalog number: BS-02-P )
  2. 1.5 ml Eppendorf tubes (Biosharp, catalog number: BS-15-M )
  3. Pipette tips (Biosharp, catalog numbers: BS-10-T , BS-200-T , BS-1000-T )
  4. Competent E. coli DH5α cells (Homemade)
  5. pTX171 plasmids (Addgene, catalog number: 89258 )
  6. pTX172 plasmids (Addgene, catalog number: 89259 )
  7. BsaI (New England Biolabs, catalog number: R0535L )
  8. Deionized water (sterile)
  9. Agarose (Biowest, catalog number: 111860 )
  10. Ethidium bromide (Solarbio Life Scientific, catalog number: E1020 )
  11. AxyPrepTM DNA Gel Extraction Kit (Corning, Axygen®, catalog number: AP-GX-250 )
  12. T4 DNA ligase (New England Biolabs, catalog number: M0202L )
  13. dNTPs mixture (Tiangen Biotech, catalog number: CD117-11 )
  14. Taq DNA polymerase (Tiangen Biotech, catalog number: ET101-01-02 )
  15. Q5® High-Fidelity DNA polymerase (New England Biolabs, catalog number: M0491L )
  16. AxyPrepTM Plasmid Miniprep Kit (Corning, Axygen®, catalog number: AP-MN-P-250 )
  17. Kanamycin (Solarbio Life Scientific, catalog number: K8020 )
  18. TAE electrophoresis buffer (see Recipes)
    Tris (Solarbio Life Scientific, catalog number: T8060 )
    Acetic acid (Kelong)
    0.5 M EDTA (Solarbio Life Scientific, catalog number: E1170 )
  19. LB medium (see Recipes)
    Tryptone (Oxoid, catalog number: LP0042 )
    Yeast extract (Oxoid, catalog number: LP0021 )
    Sodium chloride (NaCl) (Kelong)

Equipment

  1. Pipettes (Dragon-Lab)
  2. Heating block (Hangzhou Allsheng Instruments, model: MK-20 )
  3. Thermal cycler (Thermo Fisher Scientific, Thermo ScientificTM, model: ArktikTM Thermal Cycler )
  4. Water bath (Yongguangming, model: DZKW-S-4 )
  5. Microcentrifuge (Eppendorf, model: 5424 )
  6. DNA electrophoresis apparatus (Bio-Rad Laboratories, model: Mini-Sub® Cell GT Systems )
  7. NanoDrop (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 )

Procedure

  1. Design sgRNAs to target the genes of interest
    1. Select appropriate sgRNA targets for the genes of interest using the online sgRNA design tools such as CRIPSR-P v2.0 (http://cbi.hzau.edu.cn/CRISPR2/); CRISPR RGEN tools (http://www.rgenome.net/cas-offinder/); E-CRISP (http://www.e-crisp.org/E-CRISP/designcrispr.html). These are several web-based tools available for sgRNA design. They have mainly the same functions in sgRNA design and Off-target prediction. The main difference is the algorithm of the scoring system.
      Notes: sgRNA targets containing a restriction enzyme site at the Cas9 cleavage site would contribute to identify mutant using polymerase chain reaction-restriction endonuclease digestion assay.
    2. Design and order forward and reverse oligonucleotides for cloning sgRNA into the STU CRISPR-Cas9 expression vector. (1) the forward sgRNA oligonucleotide contains a ‘CGGA’ sequence at the 5’ end followed by 20 bases of sgRNA targets without PAM sites (N20); (2) the reverse sgRNA oligonucleotide contains an ‘AAAC’ at the 5’ end followed by the reverse complement of N20.
      For example, if the target site is GTTGGTCTTTGCTCCTGCAGAGG (AGG is PAM), the forward and reverse oligonucleotides should be:
      Forward oligonucleotide: 5’-CGGAGTTGGTCTTTGCTCCTGCAG-3’
      Reverse oligonucleotide: 5’-AAACCTGCAGGAGCAAAGACCAAC-3’

  2. Annealing of sgRNA oligos
    1. Mix 10 µl of the forward and reverse oligos (100 µM) of each sgRNA in separate microtubes.
    2. Incubate the microtubes at 95 °C for 5 min in a heating block or thermal cycler.
    3. Allow the microtubes to slowly cool down to room temperature.
    4. Make a 1:200 dilution of the annealed mixture with deionized water.

  3. Vector cloning
    Two methods could be used to clone sgRNAs into the STU CRISPR-Cas9 expression vector: Cut and ligation or Golden Gate method (Figure 2). These two methods are the same in procedure including BsaI digestion and T4 DNA ligase ligation. The Golden Gate reaction is much easier because the digestion and ligation will process in one PCR tube, and it may save some time.


    Figure 2. Schematic illustration of the cloning procedure described in the protocol

    1. Cut and ligation
      1. Linearize the STU CRISPR-Cas9 plasmid pTX171 or pTX172 with BsaI. Incubate at 37 °C for 2-4 h.


      2. Load digestion products onto a 1% agarose gel for electrophoresis. Purify the digested vector using the AxyPrepTM DNA Gel Extraction Kit, and quantify DNA concentration using NanoDrop.
      3. Ligate the diluted annealed oligos into linearized STU CRISPR-Cas9 expression vector, incubate at 16 °C overnight or room temperature for 1-2 h.


      4. Transform 5 μl of the reaction into 50 μl competent DH5ɑ cells, spread the transformed cells on LB plates supplemented with 50 mg/L kanamycin, and then incubate overnight at 37 °C.
      5. Verify the positive clones by colony PCR and Sanger sequencing.
    2. Golden Gate method (Make sure annealed sgRNA oligos don’t contain BsaI site)
      1. Set up a Golden Gate reaction for cloning sgRNAs into the STU Cas9 expression vector.


      2. Incubate Golden Gate reactions in a thermal cycler using the following program: 10 cycles of 5 min at 37 °C and 10 min at 16 °C, then heat to 37 °C for another 5 min and 80 °C for 10 min.
      3. Transform 5 μl of the reaction into 50 μl competent DH5α cells, spread the transformed cells on LB plates supplemented with 50 mg/L kanamycin, and then incubate overnight at 37 °C.
      4. Verify the positive clones by colony PCR and Sanger sequencing. Colony PCR can be performed with the forward sgRNA oligonucleotide (e.g., see step A2) and ZY065-RB: (5’-ttctaataaacgctcttttctct-3’). The expected product size is approximately 230 bp. ZY065-RB can be used for sequencing.

  4. Two sgRNAs can be cloned into the STU CRISPR-Cas9 expression vector to target two sites simultaneously (Figure 2).
    1. Design two primers as follows:
      BsaI-sgRNA01-F: 5’-CAGGTCTCACGGA-N20-gttttagagctagaaatagcaagttaa-3’
      BsaI-sgRNA02-R: 5’-TCGGTCTCCAAAC-N20-tccggtgacaaaagcaccga-3’
      GGTCTC is the BsaI recognition sequence;
      N20’ is same as the sgRNA01 target-specific sequence;
      N20’ is the reverse complement of the sgRNA02 target-specific sequence;
      The lowercase letters are complementary with the STU CRISPR-Cas9 expression vector.
      Note: PAGE purified oligos are highly recommended, desalted is also OK.
    2. Set up a 50 μl PCR reaction to amplify DNA for STU CRISPR-Cas9 expression vector construction.


    3. Run PCR in a thermal cycler with the following program:


    4. Load PCR products onto a 1% agarose gel for electrophoresis. Purify the PCR products using the AxyPrepTM DNA Gel Extraction Kit, and quantify DNA concentration using NanoDrop.
    5. Set up a Golden Gate reaction for cloning two sgRNAs into the STU CRISPR-Cas9 expression vector.


    6. Incubate Golden Gate reactions in a thermal cycler using the following program: 10 cycles of 5 min at 37 °C and 10 min at 16 °C, then heat to 37 °C for other 5 min and 80 °C for 10 min.
    7. Transform 5 μl of the reaction products into 50 μl competent DH5α cells, spread the transformed cells on LB plates supplemented with 50 mg/L kanamycin, and then incubate overnight at 37 °C.
    8. Verify the positive clones by colony PCR and Sanger sequencing.

  5. Our STU CRISPR-Cas9 system has the potential for multiplex sites genome editing. For more than two sites within one STU CRISPR-Cas9 vector, two-round PCR could be performed to clone different sgRNAs into the expression vector (Figure 2).
    1. Design primers as follows:
      BsaI-sgRNA01-F: 5’-CAGGTCTCACGGA-N20-3’
      sgRNA01-F: 5’-N20-gttttagagctagaaatagcaagttaa-3’
      sgRNA02-F: 5’-N20-gttttagagctagaaatagcaagttaa-3’
      sgRNA02-R: 5’-N20-tccggtgacaaaagcaccga-3’

      sgRNA(n-1)-F: 5’-N20-gttttagagctagaaatagcaagttaa-3’
      sgRNA(n-1)-R: 5’-N20-tccggtgacaaaagcaccga-3’
      sgRNA(n)-R: 5’-N20-tccggtgacaaaagcaccga-3’
      BsaI-sgRNA(n)-R: 5’-TCGGTCTCCAAAC-N20-3’
      GGTCTC is the BsaI recognition sequence;
      ‘N20’ is same as the target-specific sequence;
      N20’ is the reverse complement of the target-specific sequence;
      The different colors represent different target-specific sequences (e.g., N20, N20, N20, N20).
      The lowercase letters are complementary with the STU CRISPR-Cas9 expression vector.
    2. Set up 1st round PCR reactions to amplify sgRNAs fragments with ribozyme cleavage site flanked using primer pairs (sgRNA01-F/sgRNA02-R, sgRNA02-F/sgRNA03-R…sgRNA(n-1)-F/sgRNA(n)-R).


    3. Run PCR in a thermal cycler with the following program:


    4. Load PCR products onto a 1% agarose gel for electrophoresis. Purify the PCR products using the AxyPrepTM DNA Gel Extraction Kit, and quantify DNA concentration using NanoDrop.
    5. Set up 2nd round PCR reaction to link the different sgRNAs with ribozyme cleavage site into one fragment.


    6. Run PCR in a thermal cycler with the following program:


    7. Load PCR products onto a 1% agarose gel for electrophoresis. Purify the PCR products using the AxyPrepTM DNA Gel Extraction Kit, and quantify DNA concentration using NanoDrop.
    8. Set up a Golden Gate reaction for cloning multiplex sgRNA Fragment into the STU CRISPR-Cas9 expression vector.


    9. Incubate Golden Gate reactions in a thermal cycler using the following program: 10 cycles of 5 min at 37 °C and 10 min at 16 °C, then heat to 37 °C for other 5 min and 80 °C for 10 min.
    10. Transform 5 μl of the reaction products into 50 μl competent DH5α cells, spread the transformed cells on LB plates supplemented with 50 mg/L kanamycin, and then incubate overnight at 37 °C.
    11. Verify the positive clones by colony PCR and Sanger sequencing.

Data analysis

Examples of STU CRISPR-Cas9 system application including gene editing and gene deletion with sequencing data in rice, tobacco and Arabidopsis can be found in the original paper (Tang et al., 2016; Link to paper). Additionally, diagrams of the procedure, as well as examples of genome editing and sgRNA multiplex construction, can also be found in the original research paper (Tang et al., 2016).

Notes

  1. If failed to get colony using Golden Gate method, increase the number of cycles to 15-20 times.
  2. We tested the STU CRISPR-Cas9 system in several organisms (including rice, tobacco and Arabidopsis) and successfully achieved efficient genome editing.

Recipes

  1. 50x TAE electrophoresis buffer
    242 g/L Tris
    57.1 ml/L acetic acid
    100 ml/L 0.5 M EDTA (pH 8.0)
  2. LB medium
    10 g/L tryptone
    10 g/L NaCl
    5 g/L yeast extract

Acknowledgments

Y.Z. was supported by grants from the National Science Foundation of China (31330017 and 31371682), the Sichuan Youth Science and Technology Foundation (2017JQ0005) and the Fundamental Research Funds for the Central Universities (ZYGX2016J119 and ZYGX2016J122). This protocol is developed based on our previous study published in Molecular Plant (Tang et al., 2016).

References

  1. Gheysen, G., Herman, L., Breyne, P., Gielen, J., Montagu, M. V. and Depicker, A. (1990). Cloning and sequence analysis of truncated T-DNA inserts from nicotiana tabacum. Gene 94(2): 155-63.
  2. Lei, E. P., Krebber, H. and Silver, P. A. (2001). Messenger RNAs are recruited for nuclear export during transcription. Genes Dev 15(14): 1771-1782.
  3. Sun, X., Hu, Z., Chen, R., Jiang, Q., Song, G., Zhang, H. and Xi, Y. (2015). Targeted mutagenesis in soybean using the CRISPR-Cas9 system. Sci Rep 5: 10342.
  4. Tang, X., Zheng, X., Qi, Y., Zhang, D., Cheng, Y., Tang, A., Voytas, D. F. and Zhang, Y. (2016). A single transcript CRISPR-Cas9 system for efficient genome editing in plants. Mol Plant 9(7): 1088-1091.

简介

相关蛋白9(Cas9)的CRISPR(聚集的定期交织的短回文重复序列)是许多生物体(包括植物和作物物种)中基因组编辑的简单有效的工具。 CRISPR / Cas9系统的sgRNA通常由RNA聚合酶III启动子(如U6和U3)表达。 在许多转化事件中,更多的核苷酸将增加质粒构建中的困难和基因组错误整合的风险,如碱基对或片段缺失(Gheysen等,1990)。 而且,在许多生物体中,Pol III启动子没有得到很好的表征,异源Pol III启动子通常表现不佳(Sun等,2015)。 因此,我们开发了使用单转录单位(STU)CRISPR-Cas9系统来驱动来自单个RNA聚合酶II启动子的Cas9和sgRNA的表达以在植物中实现有效的基因组编辑的方法。
【背景】CRISPR-Cas9系统的sgRNA主要由小核RNA启动子如U6和U3促进。虽然在许多情况下已经进行了繁殖效率的测试,但也有一些局限性:(1)难以实现Cas9和sgRNA的协调和/或诱导表达; (2)操作多个sgRNA用于多重基因编辑可能是乏味的,需要多个Pol III启动子。传统的RNA聚合酶II启动子不能用于驱动sgRNA表达,通过RNA聚合酶II将多余的核苷酸加入到gRNA的5'和3'末端,并可能中断正常的gRNA功能。另外,由RNA聚合酶II转录的RNA被快速输出到细胞质中,而CRISPR-Cas9 / gRNA双链体需要核定位才能进入基因组编辑(Lei et al。,2001)。为了克服这些障碍,我们使用核酶的自催化裂解来释放驱动Cas9和sgRNA表达的RNA聚合酶II启动子(称为STU CRISPR-Cas9系统,图1)的精确加工成熟sgRNA。与sgRNA表达中使用的传统小核RNA启动子相比,我们的STU CRISPR-Cas9系统有一些优点:(1)矢量构建更简便,在某些情况下会提高转化效率; (2)多个sgRNA表达只需要额外的核酶侧翼序列(比目前使用的任何RNA聚合酶III启动子短);(3)它显示出由双sgRNA诱导的更高的缺失效率。因此,由单个RNA聚合酶II启动子驱动的STU CRISPR-Cas9系统可以取代传统的CRISPR-Cas9系统,现在如果选择适当的启动子,我们正在使用体内或体外。

关键字:CRISPR, Cas9, 基因组编辑, 单一独立转录单元, 核酶

材料和试剂

  1. 0.2ml PCR管(Biosharp,目录号:BS-02-P)
  2. 1.5ml Eppendorf管(Biosharp,目录号:BS-15-M)
  3. 移液器吸头(Biosharp,目录号:BS-10-T,BS-200-T,BS-1000-T)
  4. 主管人员大肠杆菌DH5α细胞(自制)
  5. pTX171质粒(Addgene,目录号:89258)
  6. pTX172质粒(Addgene,目录号:89259)
  7. Bsa I(New England Biolabs,目录号:R0535L)
  8. 去离子水(无菌)
  9. 琼脂糖(Biowest,目录号:111860)
  10. 溴化乙锭(Solarbio Life Scientific,目录号:E1020)
  11. AxyPrep TM DNA凝胶提取试剂盒(Corning,Axygen ,目录号:AP-GX-250)
  12. T4 DNA连接酶(New England Biolabs,目录号:M0202L)
  13. dNTPs混合物(Tiangen Biotech,目录号:CD117-11)
  14. Taq DNA聚合酶(Tiangen Biotech,目录号:ET101-01-02)
  15. Q5 ®高保真DNA聚合酶(New England Biolabs,目录号:M0491L)
  16. AxyPrep TM质粒Miniprep试剂盒(Corning,Axygen,目录号:AP-MN-P-250)
  17. 卡那霉素(Solarbio Life Scientific,目录号:K8020)
  18. TAE电泳缓冲液(参见食谱)
    Tris(Solarbio Life Scientific,目录号:T8060)
    乙酸(Kelong)
    0.5M EDTA(Solarbio Life Scientific,目录号:E1170)
  19. LB培养基(见食谱)
    胰蛋白胨(Oxoid,目录号:LP0042)
    酵母提取物(Oxoid,目录号:LP0021)
    氯化钠(NaCl)(Kelong)

设备

  1. 移液器(龙实验室)
  2. 加热块(杭州Allsheng Instruments,型号:MK-20)
  3. 热循环仪(Thermo Fisher Scientific,Thermo Scientific TM,型号:Arktik TM 热循环仪)
  4. 水浴(永光明,型号:DZKW-S-4)
  5. 微量离心机(Eppendorf,型号:5424)
  6. DNA电泳装置(Bio-Rad Laboratories,型号:Mini-Sub Cell GT Systems)
  7. NanoDrop(Thermo Fisher Scientific,Thermo Scientific TM ,型号:NanoDrop TM 2000)

程序

  1. 设计sgRNA以靶向感兴趣的基因
    1. 使用在线sgRNA设计工具(如CRIPSR-P v2.0)( http://cbi.hzau.edu.cn/CRISPR2/ ); CRISPR RGEN工具( http://www.rgenome.net/cas-offinder / ); E-CRISP( http://www.e- crisp.org/E-CRISP/designcrispr.html )。这些是可用于sgRNA设计的几种基于Web的工具。它们在sgRNA设计和非目标预测中主要具有相同的功能。主要区别是评分系统的算法。
      注意:在Cas9切割位点含有限制酶位点的sgRNA靶标将有助于使用聚合酶链反应限制性内切核酸酶消化测定鉴定突变体。
    2. 设计和命令将sgRNA克隆入STU CRISPR-Cas9表达载体的正向和反向寡核苷酸。 (1)前向sgRNA寡核苷酸在5'端含有“'序列,其后是没有PAM位点的sgRNA靶标的20个碱基(N <子> 20 ); (2)反向sgRNA寡核苷酸在5'端含有“ AAAC ',后跟N 20的反向互补 例如,如果目标站点是GTTGGTCTTTGCTCCTGCAG AGG AGG 是PAM),正向和反向寡核苷酸应该是:
      正向寡核苷酸:5' - CGGA GTTGGTCTTTGCTCCTGCAG-3'
      反向寡核苷酸:5' - AAAC CTGCAGGAGCAAAGACCAAC-3'

  2. sgRNA寡核苷酸退火
    1. 将10μl的每个sgRNA的正向和反向寡核苷酸(100μM)混合在分开的微管中
    2. 在加热块或热循环仪中将微管在95℃孵育5分钟。
    3. 让微管缓慢冷却至室温。
    4. 用去离子水稀释退火的混合物1:200
  3. 矢量克隆
    可以使用两种方法将sgRNA克隆入STU CRISPR-Cas9表达载体:切割和连接或金门法(图2)。这两种方法在包括Bsa I消化和T4DNA连接酶连接在内的方法中是相同的。金门反应更容易,因为消化和连接将在一个PCR管中处理,可能会节省一些时间。


    图2.在协议
    中描述的克隆程序的示意图
    1. 切割和结扎
      1. 使用Bsa I将STU CRISPR-Cas9质粒pTX171或pTX172线性化。在37℃孵育2-4小时。


      2. 将消化产物负载到1%琼脂糖凝胶上进行电泳。使用AxyPrep DNA凝胶提取试剂盒纯化消化的载体,并使用NanoDrop定量DNA浓度。
      3. 将稀释退火的寡核苷酸连接成线性化的STU CRISPR-Cas9表达载体,在16℃过夜或室温孵育1-2小时。


      4. 将5μl反应转化为50μl感受态DH5ɑ细胞,将转化的细胞扩增到补充有50 mg / L卡那霉素的LB平板上,然后在37°C孵育过夜。
      5. 通过菌落PCR和Sanger测序验证阳性克隆。
    2. 金门法(确保退火的sgRNA寡核苷酸不包含Bsa I位点)
      1. 建立一个金门反应,将sgRNAs克隆入STU Cas9表达载体。


      2. 使用以下程序在热循环仪中孵育金门反应:在37℃下5分钟和16℃10分钟的10个循环,然后加热至37℃另外5分钟和80℃10分钟。 />
      3. 将5μl的反应转化成50μl感受态的DH5α细胞,将转化的细胞在补充有50mg / L卡那霉素的LB平板上扩增,然后在37℃下孵育过夜。
      4. 通过菌落PCR和Sanger测序验证阳性克隆。可以使用正向sgRNA寡核苷酸(例如,参见步骤A2)和ZY065-RB:(5'-ttctaataaacgctcttctct-3')进行集落PCR。预期的产品大小约为230bp。 ZY065-RB可用于排序。

  4. 两个sgRNA可以克隆到STU CRISPR-Cas9表达载体中,同时靶向两个位点(图2)。
    1. 设计两个底漆如下:
      Bsa I-sgRNA01-F:5'-CA GGTCTC ACGGA- N20 -gttttagagctagaaatagcaagttaa-3'
      Bsa I-sgRNA02-R:5'-TC GGTCTC CAAAC- N20 -tccggtgacaaaagcaccga-3'
      GGTCTC 是Bsa I识别序列;
      ' N20 '与sgRNA01目标特异性序列相同;
      ' N20 '是sgRNA02目标特异序列的相反补体;
      小写字母与STU CRISPR-Cas9表达载体互补。
      注意:强烈建议使用PAGE纯化的寡核苷酸,脱盐也可以。
    2. 设置50μlPCR反应以扩增STU CRISPR-Cas9表达载体构建的DNA

    3. 使用以下程序在热循环仪中运行PCR:


    4. 将PCR产物加载到1%琼脂糖凝胶上进行电泳。使用AxyPrep TM凝胶提取试剂盒纯化PCR产物,并使用NanoDrop定量DNA浓度。
    5. 建立一个金门反应,将两条sgRNA克隆入STU CRISPR-Cas9表达载体

    6. 使用以下程序在热循环仪中孵育金门反应:在37℃下5分钟和16℃10分钟的10个循环,然后加热至37℃另外5分钟和80℃10分钟。 />
    7. 将5μl反应产物转化成50μl感受态DH5α细胞,将转化的细胞在补充有50mg / L卡那霉素的LB平板上扩增,然后在37℃下孵育过夜。
    8. 通过菌落PCR和Sanger测序验证阳性克隆。

  5. 我们的STU CRISPR-Cas9系统具有多重位点基因组编辑的潜力。对于一个STU CRISPR-Cas9载体中的两个以上的位点,可以进行双向PCR以将不同的sgRNA克隆到表达载体中(图2)。
    1. 设计底稿如下:
      Bsa I-sgRNA01-F:5'-CA GGTCTC ACGGA- N20 -3'
      sgRNA01-F:5' - N20 - gttttagagctagaaatagcaagttaa -3'
      sgRNA02-F:5' - N20 - gttttagagctagaaatagcaagttaa -3'
      sgRNA02-R:5' - N20 -tccggtgacaaaagc accga -3'
      ...
      sgRNA(n-1)-F:5' - N20 - gttttagagctagaaatagcaagttaa -3'
      sgRNA(n-1)-R:5' - N20 -tccggtgacaaaagc accga -3'
      sgRNA(n)-R:5' - N20 -tccggtgacaaaagc accga -3'
      Bsa I-sgRNA(n)-R:5'-TC GGTCTC CAAAC- N20 -3'
      GGTCTC 是Bsa I识别序列;
      “N20”与目标特定序列相同;
      'N20 是目标特异性序列的反向互补;
      不同的颜色表示不同的目标特定序列(例如, N20 N20 N20 N20 )。
      小写字母与STU CRISPR-Cas9表达载体互补。
    2. 设置1个 st 循环PCR反应以使用引物对(sgRNA01-F / sgRNA02-R,sgRNA02-F / sgRNA03-R ... sgRNA(n-1) - 侧翼的核酶切割位点扩增sgRNA片段F / sgRNA(n)-R)

    3. 使用以下程序在热循环仪中运行PCR:


    4. 将PCR产物加载到1%琼脂糖凝胶上进行电泳。使用AxyPrep TM凝胶提取试剂盒纯化PCR产物,并使用NanoDrop定量DNA浓度。
    5. 设置2个 nd 圆形PCR反应,将不同的sgRNA与核酶切割位点连接成一个片段。


    6. 使用以下程序在热循环仪中运行PCR:


    7. 将PCR产物加载到1%琼脂糖凝胶上进行电泳。使用AxyPrep TM凝胶提取试剂盒纯化PCR产物,并使用NanoDrop定量DNA浓度。
    8. 建立一个金门反应,将多重sgRNA片段克隆到STU CRISPR-Cas9表达载体中。


    9. 使用以下程序在热循环仪中孵育金门反应:在37℃下5分钟和16℃10分钟的10个循环,然后加热至37℃另外5分钟和80℃10分钟。 />
    10. 将5μl反应产物转化成50μl感受态DH5α细胞,将转化的细胞在补充有50mg / L卡那霉素的LB平板上扩增,然后在37℃下孵育过夜。
    11. 通过菌落PCR和Sanger测序验证阳性克隆。

数据分析

在原始论文(Tang等人)中可以找到STU CRISPR-Cas9系统应用的实例,包括基因编辑和基因缺失以及水稻,烟草和拟南芥中的测序数据。 2016; 链接到论文)。此外,程序图以及基因组编辑和sgRNA多重构建的实例也可以在原始研究论文(Tang等人,2016)中找到。

笔记

  1. 如果使用Golden Gate方法无法获得殖民地,则将周期数增加到15-20次。
  2. 我们在几种生物(包括水稻,烟草和拟南芥)中测试了STU CRISPR-Cas9系统,并成功实现了有效的基因组编辑。

食谱

  1. 50倍TAE电泳缓冲液
    242g / L Tris
    57.1ml / L乙酸
    100 ml / L 0.5 M EDTA(pH 8.0)
  2. LB培养基
    10g / L胰蛋白胨
    10g / L NaCl
    5克/升酵母提取物

致谢

Y.Z.获得中国国家科学基金(31330017和31371682),四川青年科技基金(2017JQ0005)和中央大学基础研究基金(ZYGX2016J119和ZYGX2016J122)的资助。该协议是基于我们以前在Molecular Plant(Tang等人,2016)发表的研究开发的。

参考

  1. Gheysen,G.,Herman,L.,Breyne,P.,Gielen,J.,Montagu,MV and Depicker,A.(1990)。&lt; a class =“ke-insertfile”href =“https:// www.ncbi.nlm.nih.gov/pubmed/1701747“target =”_ blank“>来自烟草的截短的T-DNA插入片段的克隆和序列分析。(基因)94(2) :155-63。
  2. Lei,EP,Krebber,H.和Silver,PA(2001)。信使RNA在转录过程中被招募用于核出口。 Genes Dev 15(14):1771-1782。
  3. Sun,X.,Hu,Z.,Chen,R.,Jiang,Q.,Song,G.,Zhang,H. and Xi,Y.(2015)。&nbsp; 使用CRISPR-Cas9系统的大豆中的靶向诱变。 5:10342。
  4. Tang,X.,Zheng,X.,Qi,Y.,Zhang,D.,Cheng,Y.,Tang,A.,Voytas,DF and Zhang,Y。(2016)。&lt; a class = -insertfile“href =”http://www.ncbi.nlm.nih.gov/pubmed/27212389“target =”_ blank“>用于在植物中有效进行基因组编辑的单一记录CRISPR-Cas9系统。 > Mol Plant 9(7):1088-1091。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tang, X., Zhong, Z., Zheng, X. and Zhang, Y. (2017). Construction of a Single Transcriptional Unit for Expression of Cas9 and Single-guide RNAs for Genome Editing in Plants. Bio-protocol 7(17): e2546. DOI: 10.21769/BioProtoc.2546.
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