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Jan 2020
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A Novel Method to Construct Binary CRISPR Vectors for Plant Transformation by Single Round of PCR Amplification
一种通过单轮PCR扩增构建用于植物转化的二元CRISPR载体的新方法   

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Abstract

CRISPR/Cas9 is an established and flexible tool for genome editing. However, most methods used to generate expression clones for the CRISPR/Cas9 are time-consuming. Hence, we have developed a one-step protocol to introduce sgRNA expression cassette(s) directly into binary vectors (Liu et al., 2020). In this approach, we have optimized the multiplex PCR to produce an overlapping PCR product in a single reaction to generate the sgRNA expression cassette. We also amplified two sgRNA expression cassettes through a single round of PCR. Then, the sgRNA expression cassette(s) is cloned into the binary vectors in a Gateway LR or Golden gate reaction. The system reported here provides a much more efficient and simpler procedure to construct expression clones for CRISPR/Cas9-mediated genome editing. In this protocol, we describe the detailed step-by-step instructions for using this system.

Keywords: Cloning system (克隆系统), Multiplex PCR (多重PCR), CRISPR/Cas9 (CRISPR/Cas9), Genome editing (基因组编辑), Rice (水稻)

Background

Bacteria defend against viruses through a protein system, consisting of the clustered regularly interspaced short palindromic repeat (CRISPR), the CRISPR-associated (Cas) protein, CRISPR RNAs (crRNAs) and trans-encoded crRNA (tracrRNA). Researchers have now developed this system into a key tool for targeted genome editing. CRISPR – binary vectors express two elements – the sgRNAs with a target sequence (target-sgRNAs) and Cas9 protein – to cleave target genomic regions. Feng et al. (2013) have constructed gateway vectors to co-express Cas9 and sgRNAs in plants through Agrobacterium sp.-mediated transformation. In a restriction-ligation reaction, a gene-specific sgRNA spacer substitutes the target region in the entry clone, which encodes attL recombination sites. Then, an “LR Clonase” reaction transfers the target-sgRNA cassette into a destination clone, which contains a Cas9 expression cassette (Feng et al., 2013). Ma et al. (2016) have developed this basic system into a CRISPR/Cas9 system for multiplex genome editing in rice. In this approach, a restriction-ligation reaction inserts a spacer into intermediate vectors to produce an sgRNA expression cassette, which fuses with adaptors for Golden Gate cloning or Gibson Assembly (Ma et al., 2016). In an alternative approach, an overlapping PCR, with two rounds of reactions, can also establish a sgRNA expression cassette with adaptors. The sgRNA expression cassette can then be introduced into a binary vector via Golden Gate cloning or Gibson Assembly (Ma et al., 2016). However, traditional cloning, based on restriction-ligation reactions or two-round overlapping PCRs, is time-consuming.


Herein, we report a novel method to construct the binary vectors with one or two targets by a single round of PCR and a single LR reaction or Golden Gate cloning (Liu et al., 2020). Using this system, an expression clone can be constructed within 36 hours, which significantly improves efficiency and reduces costs.


Materials and Reagents

  1. 200 µl PCR tubes (Biosharp, catalog number: BS-02-P )

  2. 1.5 ml microcentrifuge 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 T1 cells (TransGen, catalog number: CD501-02 )

  5. LR clonase (GatewayTM LR ClonaseTM Enzyme Mix, catalog number: 11791-043 )

  6. NEB Cutsmart buffer (New England BioLabs, catalog number: B7204S )

  7. NEBuffer 3.1 (New England BioLabs, catalog number: B7203S )

  8. EcoRV (New England BioLabs, catalog number: R0195L )

  9. BsaI-HF (New England BioLabs, catalog number: R3733L )

  10. T4 DNA ligase (New England BioLabs, catalog number: M0202L )

  11. PCR SuperMix (TransGen, catalog number: AS111-11 )

  12. KOD FX (Toyobo, catalog number: KFX-101 )

  13. dNTPs Mixture (2mM) (Toyobo, catalog number: NTP-201 )

  14. Sterilized double distilled H2O (Phygene, catalog number: PH0727 )

  15. DreamTaq or other equivalent DNA polymerase (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: K1072 )

  16. Agarose (Biowest, catalog number: 111860 )

  17. Plasmid prep mini kit (OMEGA, catalog number: D3350-01 )

  18. Gel Extraction Kit (OMEGA, catalog number: D2500-01 )

  19. Spectinomycin (Sigma-Aldrich, catalog number: PHR1441 )

  20. Tris (Solarbio Life Scientific, catalog number: T8060 )

  21. Acetic acid (MERCK, catalog number: M10006307 )

  22. 0.5 M EDTA (Solarbio Life Scientific, catalog number: E1170 )

  23. Tryptone (Oxoid, catalog number: LP0042 )

  24. Yeast extract (Oxoid, catalog number: LP0021 )

  25. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S5886 )

  26. Ethidium bromide (EB) (Sigma-Aldrich, catalog number: E8751 )

  27. Universal primers for PCR screening (see Table 1)


    Table 1. Universal primers

    Primers Sequences (5′ → 3′)
    OJP001 TCGCGTTAACGCTAGCATGGATCTC
    OJP002 GTAACATCAGAGATTTTGAGACAC
    OJP008 ACCACCTCGGCTATCCACA
    OJP026 ATAGCCTTATGCAGTTGCTCT
    OJP065 CGACTCGGTGCCACTTTTTC


  28. PJF997 (the donor vector containing OsU3-sgRNA expression cassette), PJF999 (the donor vector containing OsU6-sgRNA expression cassette), PJG090 (the donor vector for amplification of two spacers), PJG097 (the destination vector for one target), PJG112 (the destination vector for two targets). All the vectors were developed in our previous work (Liu et al., 2020)

  29. 50× TAE electrophoresis buffer (see Recipes)

  30. LB medium (see Recipes)

  31. LB agar medium (see Recipes)

Equipment

  1. Pipettes (Eppendorf)

  2. Microcentrifuge (Eppendorf, model: Centrifuge 5424 )

  3. Heating block (Hangzhou Allsheng Instruments, model: MK-20 )

  4. Thermal cycler (Thermo Fisher Scientific, Applied BiosystemsTM, model: Veriti® 96 well thermal cycler )

  5. Water bath (Shanghai Binglin, model: BLHH-6N )

  6. NanoDrop (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 )

  7. Gel Imaging System (The ChemiDoc XRS+ System, BIO-RAD, model: 1708265 )

Software

  1. SnapGene (GSL Biotech LLC, https://www.snapgene.com/)

  2. WPS Excel (Kingsoft Office, https://www.wps.cn/)

Procedure

  1. Design sgRNA primers

    1. Design sgRNA spacers using any online tools, such as CRISPR-P (http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR).

      Note: The transcription of sgRNAs is derived by OsU3 or OsU6 promoters, which have different transcription initiation sites (the first nucleotide of spacer). The transcription initiation site of OsU3 promoter is nucleotide A, while that of OsU6 promoter is nucleotide G.

    2. To construct binary vectors harboring one spacer starting with an ‘A’, add the following nucleotides to 3′ downstream of the sense (SS) and antisense (AS) sgRNA spacers to obtain sgRNA primers:

      SS: 5′-gttttagagctatgctgaaa-3′

      AS: 5′-tgccacggatcatctgcac-3′

    3. To construct binary vectors harboring one target starting with a ‘G’, add the following nucleotides to 3′ downstream of the sense (SS) and antisense (AS) sgRNA spacers to obtain sgRNA primers:

      SS: 5′-gttttagagctagaaatag-3′

      AS: 5′-ggcagccaagccagcaccc-3′

    4. To construct binary vectors harboring two targets, starting with an ‘A’ and a ‘G’, respectively:

      1. Add the following nucleotides to 5′ upstream of the sense of ‘A’-started sgRNAs (A-sgRNA-SS) and antisense of ‘G’-started sgRNAs (G-sgRNA-AS) to obtain sgRNA primers:

        A-sgRNA-SS: 5′-agGGTCTCAggca-3′

        G-sgRNA-AS: 5′-agGGTCTCAaaac-3′

      2. Add the following nucleotides to 3′ downstream of the sense of ‘A’-started sgRNAs (A-sgRNA-SS) and antisense of ‘G’-started sgRNAs (G-sgRNA-AS) oligonucleotides:

        A-sgRNA-SS: 5′-gttttagagctatgc-3′

        G-sgRNA-AS: 5′-ggcagccaagccagc-3′

    5. Order the primers from any qualified company.

      1. To construct binary vectors harboring one target, dilute the primers to a work concentration of 1 μM with sterilized double distilled H2O.

      2. To construct binary vectors harboring two targets, dilute the primers to a work concentration of 10 μM with sterilized double distilled H2O.

      Notes:

      1. When you are designing sgRNAs to binary vectors harboring two targets, it is important but unnecessary to exclude sgRNA sequences containing BsaI restriction sites.

      2. The concatenate function in WPS Excel is very useful in obtaining the correct sequences of sgRNA primers. For example, in order to obtain sgRNA forward primer in Excel, add spacer sequence in cell A1 and SS sequence in cell B1. Set cell C1=CONCATENATE(A1, B1), and C1 is sgRNA forward primer.


  2. Prepare PCR templates

    1. Linearize the donor vectors with EcoRV.

      PJF997 or PJF999         2-3 μg

      10× Cutsmart buffer   5 μl

      EcoRV                            1 μl

      ddH2O                           to 50 μl

      Incubate at 37 °C for 3 h

    2. Recycle the two fragments produced by digestion reaction into the same EP tube and purify them together using a Gel Extraction Kit according to the manufacturer’s instructions.

    3. Measure the DNA concentration using the NanoDrop then dilute the DNA product to a work concentration of 3 ng/μl.


  3. Construct a binary vector harboring one target (Figure 1)

    1. Set up an optimized multiplex PCR reaction:

      2× KOD-FX buffer                                5 μl

      dNTPs                                                   2 μl

      KOD-FX                                                0.2 μl

      Linearized donor vectors (3 ng/μl) 0.2 μl

      OJP001 (10 μM)                                  0.3 μl

      OJP002 (10 μM)                                  0.3 μl

      sgRNA-primer-F (1 μM)                     0.2 μl

      sgRNA-primer-R (1 μM)                     0.2 μl

      ddH2O                                                  to 10 μl

    2. Run PCR in a thermal cycle with the following program:

    3. Load 2 μl PCR products onto 1% agarose gel for electrophoresis.

      Notes:

      1. The size of the PCR product is 774bp (linearized PJF997 as template) or 872bp (linearized PJF999 as template).

      2. The rest of the PCR product does not need to be purified.

    4. Set up an LR reaction to clone one sgRNA into the destination vector.
      PCR product                     0.4 μl

      LR clonase                        0.4 μl

      PJG097                              0.2 μl

      ddH2O                              1 μl

      Incubate at 25 °C for 3 h

      Note: For the method to prepare PJG097 for the LR reaction, refer to Liu et al. (2020).

    5. Transform 1 μl of the reaction products into 20 μl competent T1 cells. Spread the transformed cells on LB agar plate containing 50 μg/ml spectinomycin. Then, incubate the plates at 37 °C overnight.

      The steps of transformation:

      1. T1 chemically competent cells were taken out from -80 °C and quickly inserted into the ice. After about 5 min, the target reaction products were added into the melted competent cells and gently mixed. The competent cells were left for 25 min in the ice.

      2. The competent cells were heated in a water bath at 42 °C for 45 s and then quickly placed on ice for 2 min.

      3. 700 μl sterilized LB medium without antibiotics was added into the centrifuge tube, and shaken for 60 min at 200 rpm at 37 °C.

      Note: This step needs to be carried out in aseptic conditions.

    6. Set up a colony PCR reaction to verify the positive clones. Pick two to four colonies from each plate. A portion of each clone is directly diluted into the PCR mix and the remainder of the each clone is retained.

      PCR Supermix             5 μl

      OJP008                         0.3 μl

      OJP026                         0.3 μl

      ddH2O                         to 10 μl

      Note: If the linearized PJF999 was used as the template of the multiplex PCR, use OJP065 instead of OJP008.

    7. Colony PCR Conditions:

    8. Load 5 μl PCR products onto 1% agarose gel for electrophoresis.

    9. Select one to three positive colony from each transformation and transfer them into 6 ml LB medium containing 50 μg/ml spectinomycin. Then, incubate overnight at 37 °C in an orbital shaker at 220 rpm.

    10. Isolate the plasmid DNA from the overnight cultures using a commercial miniprep kit according to the manufacturer’s instructions.

    11. Validate the plasmids by Sanger sequencing using OJP026.



      Figure 1. Schematic illustration of the cloning procedure described in the protocol


  4. Construct a binary vector harboring two targets (Figure 1)

    1. Set up an optimized multiplex PCR reaction.

      2× KOD-FX buffer                         5 μl

      dNTPs                                            2 μl

      KOD-FX                                         0.2 μl

      PJG090 (3 ng/μl)                          0.1 μl

      sgRNA-primer-F (10 μM)           0.2 μl

      sgRNA-primer-R (10 μM)          0.2 μl

      ddH2O to 10 μl

    2. Run PCR in a thermocycling with the following program:

    3. Load 2 μl PCR products onto 1% agarose gel for electrophoresis.

    4. Set up a Golden Gate reaction to clone two sgRNAs into the destination vector.

      Non-purified PCR product                          1 μl

      PJG112                                                            50 ng

      Cutsmart Buffer (NEB)                                 1 μl

      T4 ligase buffer (NEB)                                  0.4 μl

      BsaI (NEB)                                                      5 U

      T4 DNA ligase (NEB)                                     20 U

      ddH2O                                                            to 15 μl

    5. Golden Gate reaction Conditions:

    6. Transform 4 μl of the reaction products into 20 μl competent T1 cells. Spread the transformed cells on LB agar plate containing 50 μg/ml spectinomycin. Then, incubate the plates at 37 °C overnight.

      Note: This step needs to be carried out under aseptic conditions.

    7. Set up a colony PCR reaction to verify the positive clones. Pick two to four colonies from each plate.

      PCR mix               5 μl

      OJP008                 0.3 μl

      OJP026                 0.3 μl

      ddH2O                  4.4 μl

    8. Colony PCR Conditions:

    9. Load PCR products onto 1% agarose gel for electrophoresis.

    10. Select one to three positive colony from each transformation and transfer them into 6 ml LB medium containing 50 μg/ml spectinomycin under aseptic conditions. Then, incubate overnight at 37 °C in an orbital shaker.

    11. Isolate the plasmid DNA from the overnight cultures using a commercial miniprep kit according to the manufacturer’s instructions.

    12. Validate the plasmids by Sanger sequencing using OJP026.

Recipes

  1. 50× TAE electrophoresis buffer

    Tris 242 g/L

    Acetic acid 57.1 ml/L

    0.5 M EDTA (pH 8.0) 100 ml/L

  2. LB medium

    Tryptone 10 g/L

    NaCl 10 g/L

    Yeast extract 5 g/L

    Autoclave sterilization for 15 min at 121 °C

  3. LB agar medium

    Tryptone 10 g/L

    NaCl 10 g/L

    Yeast extract 5 g/L

    Agar 12 g/L

    Atoclave sterilization for 15 min at 121 °C

Acknowledgments

We thank Luis Alejandro José Mur (Aberystwyth University) for his critical reading and editing on the manuscript. C.F. was supported by grants from the National Science Foundation of China (31800250 and 31960063). This protocol is developed based on our previous study published in PeerJ (Liu et al., 2020).

Competing interests

The authors declare no conflict of interest.

References

  1. Feng, Z. Y., Zhang, B. T., Ding, W. N., Liu, X. D., Yang, D. L., Wei, P. L., Cao, F. Q., Zhu, S. H., Zhang, F., Mao, Y. F. and Zhu, J. K. (2013). Efficient genome editing in plants using a CRISPR/Cas system. Cell Res 23(10): 1229-1232.
  2. Liu, X., Zhou, X., Li, K., Wang, D., Ding, Y., Liu, X., Luo, J. and Fang, C. (2020). A simple and efficient cloning system for CRISPR/Cas9-mediated genome editing in rice. PeerJ 8: e8491.
  3. Ma, X. L., Zhu, Q. L., Chen, Y. L. and Liu, Y. G. (2016). CRISPR/Cas9 Platforms for Genome Editing in Plants: Developments and Applications.Molecular Plant 9(7): 961-974.

简介

[摘要] CRISPR / Cas9是一种成熟且灵活的基因组编辑工具。但是,大多数用于生成CRISPR / Cas9表达克隆的方法都很耗时。因此,我们开发了一种将sgRNA表达盒直接引入二元载体的一步协议(Liu等人,2020年)。在这种方法中,我们优化了多重PCR,以在单个反应中产生重叠的PCR产物,从而生成sgRNA表达盒。我们还通过单轮PCR扩增了两个sgRNA表达盒。然后,在Gateway LR或Golden gate反应中将sgRNA表达盒克隆到二元载体中。本文报道的系统为构建用于CRISPR / Cas9介导的基因组编辑的表达克隆提供了更有效,更简单的程序。在此协议中,我们描述了使用此系统的详细分步说明。


[背景]乙acteria保卫针对病毒通过蛋白系统,由群集规则间隔开的短回文重复序列(CRISPR)中,CRISPR相关(CAS)蛋白质,CRISPR的RNA(crRNAs)和反式编码crRNA(tracrRNA)。现在,研究人员已经将其系统开发为用于靶向基因组编辑的关键工具。CRISPR –二元载体表达两个元素–具有靶序列的sgRNA(target-sgRNA)和Cas9蛋白–切割靶基因组区域。冯等人。(2013年)已经构建了网关载体,通过农杆菌介导的转化在植物中共表达Cas9和sgRNA 。在限制性连接反应中,基因特异性sgRNA间隔子替代了进入克隆中的靶区域,该区域编码atL重组位点。然后,“ LR Clonase”反应将靶标-sgRNA盒转移至目的克隆,该克隆包含Cas9表达盒(Feng等,2013)。Ma等。(2016)已将该基本系统开发为用于水稻多基因组编辑的CRISPR / Cas9系统。在这种方法中,限制性连接反应将间隔子插入中间载体以产生sgRNA表达盒,该sgRNA表达盒与用于金门克隆或吉布森装配的衔接子融合(Ma等人,2016)。在另一种方法中,具有两轮反应的重叠PCR也可以建立带有衔接子的sgRNA表达盒。然后可以通过金门克隆或Gibson Assembly将sgRNA表达盒引入二元载体(Ma等,2016)。然而,基于限制性-连接反应或两轮重叠PCR的传统克隆是费时的。

本文中,我们报道了一种通过单轮PCR和单次LR反应或金门克隆构建具有一个或两个靶标的二元载体的新方法(Liu等人,2020)。使用该系统,一个表达克隆可以被构建在36小时内,这显著提高效率并减少小号成本小号。

关键字:克隆系统, 多重PCR, CRISPR/Cas9, 基因组编辑, 水稻



材料和试剂


1. 200 µl PCR管(Biosharp,目录号:BS-02-P)     

2. 1.5 ml微量离心管(Biosha rp,目录号:BS-15-M)     

3.移液器吸头(Biosharp,目录号s :BS-10-T,BS-200-T,BS-1000-T)     

4.感受态大肠杆菌T1细胞(TransGen,目录号:CD501-02 )     

5. LR克隆酶(网关TM LR克隆酶TM酶混合物,目录号:11791-043)     

6. NEB Cutsmart缓冲液(新英格兰生物实验室,目录号:B7204S)     

7. NEBuffer 3.1(新英格兰生物实验室,目录号:B7203S)     

8. EcoR V(新英格兰生物实验室,目录号:R0195L)     

9. Bsa I-HF(新英格兰生物实验室,目录号:R3733L)     

10. T4 DNA连接酶(新英格兰生物实验室,目录号:M0202L) 

11. PCR SuperMix(TransGen,目录号:AS111-11) 

12. KOD FX(Toyobo,目录号:KFX-101) 

13. dNTPs混合物(2mM)(Toyobo,    目录号: NTP-201)


14.灭菌的双蒸H 2 O(Phygene,目录号:PH0727) 

15. DreamTaq或其他等效的DNA聚合酶(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:K1072) 

16.琼脂糖(Biowest,目录号:111860) 

17.质粒制备微型试剂盒(OMEGA,目录号:D3350-01) 

18.凝胶提取试剂盒(OMEGA,目录号:D2500-01) 

19.大观霉素(Sigma-Aldrich,目录号:PHR1441) 

20. Tris(Solarb io Life Scientific,目录号:T8060) 

21.乙酸(MERCK,目录号:M10006307 ) 

22. 0.5 M EDTA(Solarbio Life Scientific,目录号:E1170) 

23.胰蛋白Try(类毒素,目录号:LP0042) 

24.酵母菌提取物(类毒素,目录号:LP0021) 

25.氯化钠(NaCl)(    Sigma-Aldrich,目录号:S5886)


26.溴化乙锭(EB)(Sigma-Aldrich,目录号:E8751) 

27.用于PCR筛选的通用引物(见表1) 





表1.通用引物


28. PJF997(含有OsU3-sgRNA表达盒的供体载体),PJF999(含有OsU6-sgRNA表达盒的供体载体),PJG090(用于扩增两个间隔子的供体载体),PJG097(一个靶标的目的载体), PJG112(两个目标的目标向量)。所有的载体都在我们之前的工作中得到了发展(Liu等,2020)。 

29. 50 × TAE电泳缓冲液(请参阅“配方” ) 

30. LB培养基(请参阅食谱) 

31. LB琼脂培养基(请参见食谱) 



设备


Pi pettes(Eppendorf)
微量离心机(Eppendorf,型号:Centrifuge 5424)
加热块(杭州奥盛仪器有限公司,型号:MK-20)
热循环仪(赛默飞世尔科技,Applied Biosystems公司TM ,型号:Veriti ® 96孔热循环仪)
水浴锅(上海冰林,型号:BLHH-6N)
纳米滴(赛默飞世尔科技,赛科技TM ,型号:NanoDropTM 2000)
凝胶成像系统(ChemiDoc XRS +系统,BIO-RAD,型号:1708265)


软件


SnapGene(GSL Biotech LLC,https: //www.snapgene.com/ )
WPS Excel (金山办事处,https://www.wps.cn/)


程序


设计sgRNA引物
使用任何在线工具(例如CRISPR-P (http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR))设计sgRN A间隔子。
注意:sgRNA的转录是由OsU3或OsU6启动子衍生的,它们具有不同的转录起始位点(间隔子的第一个核苷酸)。OsU3启动子的转录起始位点是 核苷酸A,而Os U6启动子的是核苷酸G.


要构建带有一个以'A'开头的间隔子的二元载体,请将以下核苷酸添加到有义(SS)和反义(AS)sgRNA间隔子下游的3'处,以获得sgRNA引物:
SS:5 ' -gttttagagctatgctgaaa-3 '


AS:5 ' -tgccacggatcatctgcac-3 '


要构建带有一个以'G'开头的靶标的二元载体,请将以下核苷酸添加到有义(SS)和反义(AS)sgRNA间隔子下游的3'处,以获得sgRNA引物:
SS:5 ' -gttttagagctagaaatag-3 '


AS:5 ' -ggcagccaagccagcaccc-3 '


构建包含两个目标的二进制向量,分别以“ A”和“ G”开头:
甲DD以下个核苷酸至5 A'-开始sgRNAs(A-因组-SS)和'G'-开始sgRNAs(G-因组-AS),以获得因组的引物的反义“的意义上的上游':
A-因组-SS:5 ' - agGGTCTCAggca -3 '


G-因组-AS:5 ' - agGGTCTCAaaac -3 '


甲DD以下个核苷酸至3 A'-开始sgRNAs(A-因组-SS)和'G'-开始sgRNAs(G-因组-AS)寡核苷酸的反义“的意义上的下游':
A-因组-SS:5 ' - gttttagagctatgc -3 '


G-因组-AS:5 ' - ggcagccaagccagc -3 '


从任何合格的公司订购底漆。
要构建包含一个靶标的二元载体,请将引物稀释至1μM的工作浓度用无菌双蒸馏水2 ö。
要构建包含两个靶标的二元载体,请使用灭菌的双蒸馏水H 2 O将引物稀释至10μM的工作浓度。
笔记:


当您将sgRNA设计为带有两个靶标的二元载体时,重要但不必要的是排除包含BsaI限制性酶切位点的sgR NA序列。
WPS Excel中的连接功能对于获取正确的sgRNA引物序列非常有用。例如,为了在Excel中获得sgRNA正向引物,请在单元格A1中添加间隔序列,在单元格B1中添加SS序列。设置单元格C1 = CONCATENATE(A1,B1),并且C1是sgRNA正向引物。
 
准备PCR模板
使用Eco RV线性化供体向量。
PJF997或PJF999 2-3 μ克           

10 × Cutsmart缓冲器5微升           

EcoR V 1微升           

ddH 2 O至50μl           

在37°C下孵育3小时


将消化反应产生的两个片段回收到同一EP管中,并根据制造商的说明使用凝胶提取试剂盒一起纯化。
使用NanoDrop测量DNA浓度,然后将DNA产物稀释至3 ng / μl的工作浓度。


构造一个包含一个目标的二进制向量(图1)
建立优化的多重PCR反应:
2 × KOD-FX缓冲器5微升           

dNTPs 2微升           

KOD-FX 0.2微升           

线性化供体载体(3纳克/微升)0.2微升           

OJP001(10μM)0.3μL           

OJP002(10μM)0.3μL           

sgRNA引物F(1μM)0.2μl           

sgRNA引物R(1μM)0.2μl           

ddH 2 O至10μl           

使用以下程序在热循环中运行PCR:



温度


时间


初始变性


94°摄氏度


2分钟


98°摄氏度


10秒


40个循环


55°摄氏度


20秒


68°摄氏度


30秒


最终扩展


68°摄氏度


5分钟


抓住


25°摄氏度


负载2 μ升的PCR产物在1%琼脂糖凝胶电泳。
笔记:


PCR产物的大小为774bp(线性化的PJF997作为模板)或872bp(线性化的PJF999作为模板)。
其余的PCR产物无需纯化。
                                                                                                                                            设置LR反应,以将一个sgRNA克隆到目的载体中。
PCR产物0.4微升             
LR克隆酶0.4微升           

PJG097 0.2微升           

ddH 2 O 1微升           

在25°C下孵育3小时


注意:有关制备用于LR反应的PJG097的方法,请参见Liu等。(2020)。


将1μl反应产物转化为20μl感受态T1细胞。将转化的细胞铺在含有50μg/ ml大观霉素的LB琼脂平板上。然后,将板在37°C下孵育过夜。
转换步骤:


从-80取出T1化学感受态细胞 °C并迅速插入冰中。约5分钟后,将目标反应产物添加到融化的感受态细胞中并轻轻混合。将感受态细胞在冰中放置25分钟。
将感受态细胞在42 °C的水浴中加热45 s,然后迅速置于冰上2 min。
700微升无抗生素无菌LB培养基加入到离心管中,并摇动以200rpm在37 60分钟℃下。
注意:此步骤需要在无菌条件下进行。


设置菌落PCR反应以验证阳性克隆。从每个板中选择2-4个菌落。每个克隆的一部分直接稀释到PCR混合物中,其余的每个克隆都保留下来。
PCR Supermix    5微升


OJP008 0.3微升


OJP026 0.3微升


ddH 2 O           至10μl


注意:如果将一分子化的PJF999用作多重PCR的模板,请使用OJP065代替OJP008。


菌落PCR条件:



温度


时间


初始变性


94°摄氏度


5分钟


94°摄氏度


30秒


35个循环


55°摄氏度


30秒


72°摄氏度


1分钟


最终扩展


72°摄氏度


5分钟


抓住


25°摄氏度


将5μlPCR产物上样至1%琼脂糖凝胶上进行电泳。
从每个转化中选择一到三个阳性菌落,并将其转移到含有50μg/ ml大观霉素的6 ml LB培养基中。然后,在定轨摇床上于37℃在220 rpm下孵育过夜。
根据生产商的说明,使用市售的miniprep试剂盒从过夜培养物中分离质粒DNA。
使用OJP026通过Sanger测序验证质粒。




图1.协议中描述的克隆过程的示意图


构造一个包含两个目标的二进制向量(图1)
设置优化的多重PCR反应。
2 × KOD-FX缓冲液5μl


dNTPs 2微升


KOD-FX 0.2微升


PJG090(3 ng /μl)0.1微升


sgRNA引物F(10μM)0.2μl


sgRNA引物R(10μM)0.2μl


ddH 2 O至10μl


使用以下程序在热循环中运行PCR:



温度


时间


初步d enaturation


94°摄氏度


2分钟


98°摄氏度


10秒


40个循环


55°摄氏度


20秒


68°摄氏度


30秒


最终扩展


68°摄氏度


5分钟


抓住


25°摄氏度


将2μlPCR产物加到1%琼脂糖凝胶上进行电泳。
设置金门反应,将两个sgRNA克隆到目的载体中。
非纯化的PCR产物1μl


PJG112 50 ng


Cutsmart缓冲液(NEB)1μl


T4连接酶缓冲液(NEB)0.4μl


BsaI(NEB)5 U


T4 DNA连接酶(NEB)20 U


ddH 2 O至15μl


金门反应条件:


温度


时间


37°摄氏度


10分钟


20个循环


37°摄氏度


2分钟


20°摄氏度


5分钟


37°摄氏度


5分钟


将4μl反应产物转化为20μl感受态T1细胞。将转化细胞铺在含50μg/ ml大观霉素的LB琼脂平板上。然后,将板在37°C下孵育过夜。
              注意:此步骤需要在无菌条件下进行。


设置菌落PCR反应以验证阳性克隆。从每个板中选择2-4个菌落。
PCR混合液5μl


OJP008 0.3微升


OJP026 0.3微升


ddH 2 O 4.4微升






菌落PCR条件:



温度


时间


初始变性


94°摄氏度


5分钟


94°摄氏度


30秒


35个循环


55°摄氏度


30秒


72°摄氏度


1分钟


最终扩展


72°摄氏度


5分钟


抓住


25°摄氏度


将PCR产物加载到1%琼脂糖凝胶上进行电泳。
从每个转化中选择1-3个阳性菌落,并在无菌条件下将其转移至6 ml含50μg/ ml大观霉素的LB培养基中。然后,在定轨振荡器中于37°C孵育过夜。
根据生产商的说明,使用市售的miniprep试剂盒从过夜培养物中分离质粒DNA。
使用OJP026通过Sanger测序验证质粒。


菜谱


50 × TAE电泳缓冲液
特里斯242克/升


甲cetic酸57.1毫升/ L


0.5 M EDTA(pH 8.0)100毫升/升


LB培养基
Ť ryptone 10g / L的


氯化钠10克/升


Y东提取物5 g / L


在121 °C下高压灭菌15分钟


LB琼脂培养基
Ť ryptone 10g / L的


氯化钠10克/升


Y东提取物5 g / L


琼脂12克/升


在121 °C下进行15分钟的灭菌器灭菌


致谢


感谢阿伯斯威斯大学的路易斯·亚历杭德罗·何塞·穆尔(Luis AlejandroJoséMur)对手稿的批判性阅读和编辑。CF获得了中国国家科学基金(31800250和31960063)的资助。该协议是基于我们先前在PeerJ上发表的研究(Liu等人,2020年)开发的。


利益争夺


作者宣称没有利益冲突。


参考


冯ZY,张,BT,丁,文,刘,XD,杨,DL,魏,PL,曹,FQ,朱,SH,张,F.,毛,YF和朱,JK(2013)。使用CRISPR / Cas系统在植物中进行有效的基因组编辑。Cell Res 23(10):1229-1232。              
Liu X.,Zhou,X.,Li,K.,Wang,D.,Ding,Y.,Liu X.,Luo,J. and Fang,C.(2020年)。一个简单,高效的克隆系统,用于水稻中CRISPR / Cas9介导的基因组编辑。PeerJ 8:e8491。              
马XL,朱QL,陈YL和刘YG(2016)。用于植物基因组编辑的CRISPR / Cas9平台:开发和应用。分子植物9(7):961-974。
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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用:Li, K., Wang, Y. and Fang, C. (2021). A Novel Method to Construct Binary CRISPR Vectors for Plant Transformation by Single Round of PCR Amplification. Bio-protocol 11(7): e3971. DOI: 10.21769/BioProtoc.3971.
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