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Targeted Mutagenesis Using RNA-guided Endonucleases in Mosses
在苔藓中使用RNA引导的内切核酸酶进行定向诱变   

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Abstract

RNA-guided endonucleases (RGENs) have been used for genome editing in various organisms. Here, we demonstrate a simple method for performing targeted mutagenesis and genotyping in a model moss species, Physcomitrella patens, using RGENs. We also performed targeted mutagenesis in a non-model moss, Scopelophilla cataractae, using a similar method (Nomura et al., 2016), indicating that this experimental system could be applied to a wide range of mosses species.

Keywords: Genome editing(基因组编辑), RNA-guided endonucleases(RNA引导的内切核酸酶), CRISPR/Cas system(CRISPR/Cas系统), Targeted mutagenesis(定向诱变), Mosses(苔藓)

Background

Targeted mutagenesis using RNA-guided endonucleases (RGENs) derived from the adaptive immune system, using the bacterial CRISPR (clusters of regularly interspaced palindromic repeats)/Cas (CRISPR-associated) systems, has dramatically advanced in recent years. In this method, the Cas9 endonuclease, derived from Streptococcus pyogenes, and an artificially designed single-chain guide RNA (sgRNA) are used. The Cas9-sgRNA complex recognizes the protospacer-adjacent motif (5’-NGG-3’) and cleaves 3 bp upstream of the target site (Jinek et al., 2012). Subsequently, random insertion and/or deletion mutations occur during the repair process for double-strand breaks (DSBs) in the DNA. As targeted mutagenesis using these RGENs is efficient as well as cost- and time-effective, it has been used for genome editing in various organisms, including many plant species. Here, we established a protocol for targeted mutagenesis using RGENs in mosses, and demonstrated it in a model and a non-model species (Nomura et al., 2016).

Materials and Reagents

  1. Washed and autoclaved cellophane (FUTAMURA CHEMICAL, catalog number: PS-1 )
  2. 9 cm plastic Petri dish (SANSEI MEDICAL, catalog number: 01-013 )
  3. 1.5 ml plastic tubes (FUKAEKASEI and WATSON, catalog number: 131-415C )
  4. 15 ml plastic tubes (FUKAEKASEI and WATSON, catalog number: 1332-015S )
  5. PCR tubes (NIPPON Genetics, FastGene, catalog number: FG-028DC )
  6. 10 µl plastic pipette tips (FUKAEKASEI and WATSON, catalog number: 123R-254CS )
  7. 200 µl plastic pipette tips (FUKAEKASEI and WATSON, catalog number: 123R-755CS )
  8. 1,000 µl plastic pipette tips (FUKAEKASEI and WATSON, catalog number: 122-804B )
  9. Protonemata of Physcomitrella patens (or other mosses)
  10. pSCgRNA, pSCOE1-fcoCas9 (Nomura et al., 2016, Figure 1A)
  11. DH5α-competent cells (Home-made)
  12. NEBuffer 2.1 (New England Biolabs, catalog number: R0539S )
  13. BbsI (New England Biolabs, catalog number: R0539S )
  14. Sterile Milli-Q water
  15. GEL/PCR Purification Mini Kit (Favorgen Biotech, catalog number: FAGCK 001-1 )
  16. Ligation high ver.2 (TOYOBO, catalog number: LGK-201 )
  17. ScU6p Ins. check F primer 5’-GAGGATCACGGTGTCACATGTCC-3’
  18. Quick Taq® HS DyeMix (TOYOBO, catalog number: DTM-101 )
  19. ScU6p Seq. check F primer 5’-ATGTCAAACATAACCTGG-3’
  20. Ampicillin (NACALAI TESQUE, catalog number: 02739-74 )
  21. Plasmid DNA Extraction Mini Kit (Favorgen Biotech, catalog number: FAPDE 001-1 )
  22. Agarose S (NIPPON GENE, catalog number: 312-01193 )
  23. DNA ladder markers (SMOBIO Technology, catalog number: DM3100 )
  24. NucleoBond® Xtra Midi (MACHEREY-NAGEL, catalog number: 740410.50 )
  25. G418 Disulfate (NACALAI TESQUE, catalog number: 16512-81 )
  26. BCDAT medium (Nishiyama et al., 2000)
  27. Glucose (Wako Pure Chemical Industries, catalog number: 049-31165 )
  28. Tks Gflex DNA polymerase (Takara Bio, catalog number: R060A )
  29. Zero Blunt PCR Cloning Kit (Thermo Fisher Scientific, Invitrogen, catalog number: K275020 )
  30. CloneJET PCR Cloning Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: K1231 )
  31. LB agar, Miller (BD, DifcoTM, catalog number: 244520 )
  32. LB broth, Miller (BD, DifcoTM, catalog number: 244620 )
  33. Tris (hydroxymethyl) aminomethane (NACALAI TESQUE, catalog number: 35434-21 )
  34. Potassium chloride (Wako Pure Chemical Industries, catalog number: 163-03545 )
  35. Ethylenediaminetetraacetic acid (EDTA) (DOJINDO, catalog number: 345-01865 )
  36. Acetic acid (Wako Pure Chemical Industries, catalog number: 017-00256 )
  37. Ethidium bromide solution (NACALAI TESQUE, catalog number: 14631-94 )
  38. LB agar plate with 50 µg/ml ampicillin (see Recipes)
  39. Lysis buffer (see Recipes)
  40. 50 x TAE buffer (see Recipes)
  41. 1x TAE buffer (see Recipes)

Equipment

  1. Plant growth chamber (SANYO, model: MLR-350HT )
  2. Micropipettes (Eppendorf, model: Reference® 2 )
  3. Sterile tweezers (Electron Microscopy Sciences, DUMONT, catalog number: 0108-5-PO )
  4. Thermal cycler (PCR Thermal Cycler Dice® Gradient, Takara Bio, model: TP600 )
  5. High-speed centrifuge (TOMY DIGITAL BIOLOGY, model: MX-300 )
  6. Block incubator (ASTEC, model: Bl-515A )
  7. Spectrophotometer (GE Healthcare, model: NanoVue )
  8. Autoclave (TOMY DIGITAL BIOLOGY, model: SX300 )
  9. Agarose gel electrophoresis equipment (Cosmo Bio, model: i-MyRun.N )
  10. Ultraviolet transilluminator (ATTO, models: DTB-20MP , TYPE-CX )
  11. Heated incubator (SANYO, model: MIR-262 )

Procedure

  1. Design and construction of sgRNA expression vector
    1. Selection of target sequence
      1. Choose the 20 bp target sequence in the gene or genomic region of interest (Figure 1B).
        1. Check the protospacer-adjacent motif (PAM) sequence (5’-NGG-3’) at the 3’-end of the target sequence.
        2. To induce a dysfunctional mutation in the gene, the target sequence should be designed near the 5’-end or in the functional domain.
        3. Avoid poly-T sequences, which work as transcriptional terminators for sgRNA; ensure that the G + C content is 40-80%.
        4. If the target site includes the recognition site of a restriction enzyme, it will be possible to perform restriction fragment length polymorphism (RFLP) analysis for genotyping.
          Note: It has been reported that truncated sgRNA could decrease off-target effects (Fu et al., 2014). We confirmed that truncated sgRNA (18 or 17 bp target sequences) works in our system; however, we have not yet evaluated their targeting efficiency.
      2. Add the following adapter sequence for cloning (Figure 1B): 5’-TCTG-3’ for sense oligonucleotides and 5’-AAAC-3’ for anti-sense oligonucleotides.
    2. Digestion of pSCgRNA vector (Figure 1C)
      1. Prepare the digestion mixture, as given below:


      2. Incubate the mixture at 37 °C for 3 h.
      3. Purify the BbsI-digested pSCgRNA vector using the DNA purification kit (Favorgen GEL/PCR Purification Mini Kit).
    3. Annealing of oligonucleotides (Figure 1C)
      1. Prepare the annealing mixture, as given below:


      2. Incubate at 95 °C for 5 min in a thermal cycler.
      3. Remove the tube from the thermal cycler and allow the mixture to cool down on the bench at 20-25 °C for 30 min.
    4. Cloning of annealed oligonucleotides into pSCgRNA vector (Figure 1C)
      1. Prepare the ligation mixture, as given below:


      2. Incubate the mixture at 16 °C for 3 h or more.
      3. Transform DH5α-competent cells using 2 µl ligation mixture and spread on an LB plate with 50 µg/ml ampicillin.
      4. Incubate the LB plate at 37 °C for 14-16 h.
      5. Mix a PCR reaction mixture, using the ScU6p Ins. check F primer (as the forward primer) and the antisense target sequence (as the reverse primer).


      6. Verify the insertion by performing a colony PCR with the following cycle:


        Note: If the cloning is successful, a 950 bp band will be amplified and detected (Figure 1D). We recommend testing at least 8-16 colonies.
      7. Grow the positive colonies in liquid LB medium with 50 µg/ml ampicillin.
      8. Purify the plasmids using a plasmid DNA mini prep kit (Favorgen Plasmid DNA Extraction Mini Kit).
      9. Confirm the inserted target sequence in the pSCgRNA vector using ScU6p Seq. check F primer using Sanger sequencing.


        Figure 1. Vector construction for targeted mutagenesis in mosses using RNA-guided endonucleases. A. Schematic illustration of the Cas9 and sgRNA expression vectors; B. Example of target sequence design; C. Procedure for constructing sgRNA expression vector; D. Representative results of colony-PCR for checking the insert target sequence. M: DNA ladder markers.

  2. PEG-mediated protoplast transformation (Figure 2A)
    1. Purify the pSCgRNA containing the target sequence and pSCOE1-fcoCas9, which backbone is pTN182 vector (Sakakibara et al., 2008), using the midi prep kit (NucleoBond® Xtra Midi) and dilute it with sterile TE buffer at 1 µg/µl. Use 15 µg of each plasmid for transformation.
    2. Co-transform both the vectors using the PEG-mediated protoplast transformation method, as described in a previous report (Nishiyama et al., 2000; PHYSCOmanual ver. 2.0 in NIBB PHYSCObase website: http://moss.nibb.ac.jp/protocol.html).


      Figure 2. Overview of protocol for acquiring mutant strains using RGENs

  3. Regeneration of protonemal cells and transient screening using antibiotic G418 (Geneticin). 
    1. After inoculating the transformed protoplasts in PRM/T on cellophane and on a PRM/B plate, incubate the mixture at 25 °C for 7 d in a plant growth chamber (Figure 2B).
    2. For transient screening, transfer the regenerated protonemata from the protoplasts in PRM/T on cellophane to new BCDAT plates with 20 µg/ml G418 (Figure 2C).
    3. Culture the plates in a plant growth chamber for 5 d (Figures 2C and 3).


      Figure 3. Example of protonemata after transient screening (PpFtsZ2-1 targeted). A. Representative example of dead protonemata; B. Representative example of surviving protonemata. Strain also shows the phenotype of PpFtsZ2-1 mutation. Scale bars = 50 µm.

    4. Transfer the surviving protonemata in PRM/T on cellophane to new BCDAT plates and culture again until small colonies have formed (Figure 2D).
    5. Using tweezers, choose the protonemata at the edge of the colonies and transfer them to new, numbered BCDAT or BCDAT + 0.5% glucose plates (Figure 2D).
    6. For genotyping, culture the protonemata until the colonies become 5-10 mm in diameter.
      Note: There is a possibility that the acquired strain is a mosaic plant, which is a mixture of strains with different mutations or wild type. If so, we can isolate the monogenic strain from the protoplast, single protonemal branch, or leaf of the gametophore.

  4. Genotyping
    1. Green PCR
      1. Transfer a pinch of fresh protonemata to PCR tubes (Figure 4A) and grind using the tip of the 200 µl pipette chip into 50 µl lysis buffer (see Recipes) (Figure 4B).


        Figure 4. Preparation of the template for Green PCR. A. A pinch of fresh protonemata in the lysis buffer; B. Protonemata crushed by the tip of the 200 µl chip in the lysis buffer.

      2. Incubate at 95 °C for 10 min.
      3. Spin down the debris at 3,500 x g for 10 sec and use the supernatant as the PCR template.
      4. Design the primer set using free online tool Primer 3 (http://primer3.ut.ee/) for amplifying the genomic DNA, including the target site for sequencing or for other genotyping such as RFLP.
      5. PreparePCR reaction mixture, as given below:


      6. Amplify the PCR product, including the target site of the genome, with the cycle given below:


    2. Restriction fragment length polymorphism (RFLP) analysis
      1. If the target site includes the recognition site of a restriction enzyme, it will be possible to observe the targeted mutagenesis by RFLP.
      2. After confirming the PCR products by agarose gel electrophoresis, digest the PCR products using the corresponding restriction enzymes.
      3. After sufficient digestion, analyze the enzyme-treated and untreated PCR products by agarose gel electrophoresis. It is possible that restriction enzyme-tolerant PCR products have mutations.
        Note: If a mixture of PCR products derived from wild type and mutant strains is used, the targeted mutagenesis can be checked using the T7 endonuclease I or cel1 assay, which can recognize and cleave non-perfectly matched DNA. As high throughput and comprehensive genotyping method, high resolution melting analysis or next-generation sequencing is also applicable.
    3. Sequencing of target sites in the genome
      1. After confirming the PCR product by agarose gel electrophoresis, clone the PCR products into the sequencing vector using the Zero Blunt PCR Cloning Kit or the CloneJET PCR Cloning Kit.
      2. After colony PCR and mini prep, check the DNA sequence of the inserted PCR product of several clones by Sanger sequencing.
        Note: If the PCR product is present as a single band in monogenic plant, it will be possible to check by direct sequencing.

Data analysis

According to our evaluation based on the phenotypic change caused by the mutation of a target gene, the target mutagenesis efficiency in P. patens was approximately 45% to 68% (Nomura et al., 2016). Since efficiency is dependent on the target sequence, we recommend using the ‘focas’ website (http://focas.ayanel.com/; Doench et al., 2014; Xiao et al., 2014; Osakabe et al., 2016) for designing and predicting the on- and off-target efficiency of the target genome. In this case, the target sequence with an on-target score of 0.6 or higher and a low possibility of off-target effect is desirable.

Notes

  1. It is possible to introduce long deletion mutations (~3 kb) through this method by using two gRNAs designed for distant sites on the genome (Nomura et al., 2016).
  2. In this method, the vector DNA is usually not integrated into the moss genome (Nomura et al., 2016).
  3. There is the possibility of the formation of polyploidy during the PEG-mediated protoplast transformation. DNA content in acquired strains should be assessed using a flow cytometer.
  4. If this protocol is applied to other species, it may be necessary to modify some experimental conditions such as the transformation (e.g., PEG treatment time) and screening method (e.g., antibiotic concentration and duration of selection).

Recipes

  1. LB agar plate with 50 µg/ml ampicillin
    LB agar, Miller 40 g
    Fill up to 1 L with Milli-Q water and autoclave (121 °C, 15 min)
    Add ampicillin to a final concentration of 50 µg/ml
  2. Lysis buffer
    100 mM Tris-HCl (pH 9.5)
    1 M KCl
    10 mM EDTA
  3. 50 x TAE buffer
    2 M Tris
    1 M acetic acid
    0.5 M EDTA (pH = 8.0)
  4. 1 x TAE buffer
    20 ml 50 x TAE buffer
    980 ml Milli-Q water

Acknowledgments

This protocol was adapted from a published paper (Nomura et al., 2016). This work was supported by the Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (B) (grant number 15K18824), and Grants-in-Aid for Scientific Research (C) (grant number 15K06905).

References

  1. Doench, J. G., Hartenian, E., Graham, D. B., Tothova, Z., Hegde, M., Smith, I., Sullender, M., Ebert, B. L., Xavier, R. J. and Root, D. E. (2014). Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol 32(12): 1262-1267.
  2. Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M. and Joung, J. K. (2014). Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol 32(3): 279-284.
  3. 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.
  4. Nishiyama, T., Hiwatashi, Y., Sakakibara, I., Kato, M. and Hasebe, M. (2000). Tagged mutagenesis and gene-trap in the moss, Physcomitrella patens by shuttle mutagenesis. DNA Res 7(1): 9-17.
  5. Nomura, T., Sakurai, T., Osakabe, Y., Osakabe, K. and Sakakibara, H. (2016). Efficient and heritable targeted mutagenesis in mosses using the CRISPR/Cas9 system. Plant Cell Physiol 57(12): 2600-2610.
  6. Osakabe, Y., Watanabe, T., Sugano, S. S., Ueta, R., Ishihara, R., Shinozaki, K. and Osakabe, K. (2016). Optimization of CRISPR/Cas9 genome editing to modify abiotic stress responses in plants. Sci Rep 6: 26685.
  7. Sakakibara, K., Nishiyama, T., Deguchi, H. and Hasebe, M. (2008). Class 1 KNOX genes are not involved in shoot development in the moss Physcomitrella patens but do function in sporophyte development. Evol Dev 10:555-566.
  8. Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B. C., Remm, M. and Rozen, S. G. (2012). Primer3—new capabilities and interfaces. Nucleic Acids Res 40(15): e115-e115.
  9. Xiao, A., Cheng, Z., Kong, L., Zhu, Z., Lin, S., Gao, G. and Zhang, B. (2014). CasOT: a genome-wide Cas9/gRNA off-target searching tool. Bioinformatics 30:1180-1182.

简介

RNA引导的核酸内切酶(RGENs)已被用于各种生物体的基因组编辑。 在这里,我们展示了使用RGENs在模型苔藓种类小立碗藓中进行定向诱变和基因分型的简单方法。 我们还使用类似的方法(Nomura等,2016)在非模型苔藓,Scopelophilla白内障中进行了定向诱变,表明该实验系统可以应用于广泛的苔藓物种。
【背景】使用来自适应性免疫系统的RNA引导内切核酸酶(RGEN)的靶向诱变,使用细菌CRISPR(定期间隔的回文重复序列)/ Cas(CRISPR相关)系统近年来已经急剧发展。在该方法中,使用源自化脓性链球菌的Cas9核酸内切酶和人工设计的单链导向RNA(sgRNA)。 Cas9-sgRNA复合物识别原始相邻基序(5'-NGG-3'),并在目标位点上游3 bp切割(Jinek等,2012)。随后,在DNA的双链断裂(DSB)修复过程中发生随机插入和/或缺失突变。使用这些RGEN的定向诱变是有效的以及成本和时间有效的,它已被用于各种生物体(包括许多植物物种)的基因组编辑。在这里,我们在青苔中建立了使用RGEN进行靶向诱变的方案,并在模型和非模型物种中进行了证明(Nomura等,2016)。

关键字:基因组编辑, RNA引导的内切核酸酶, CRISPR/Cas系统, 定向诱变, 苔藓

材料和试剂

  1. 洗涤和高压灭菌的玻璃纸(FUTAMURA CHEMICAL,目录号:PS-1)
  2. 9厘米塑料培养皿(SANSEI MEDICAL,目录号:01-013)
  3. 1.5ml塑料管(FUKAEKASEI和WATSON,目录号:131-415C)
  4. 15 ml塑料管(FUKAEKASEI和WATSON,目录号:1332-015S)
  5. PCR管(NIPPON Genetics,FastGene,catalog number:FG-028DC)
  6. 10μl塑料移液器吸头(FUKAEKASEI和WATSON,目录号:123R-254CS)
  7. 200μl塑料移液器吸头(FUKAEKASEI和WATSON,目录号:123R-755CS)
  8. 1,000μl塑料移液器吸头(FUKAEKASEI和WATSON,目录号:122-804B)
  9. 小立碗藓的质子(或其他苔藓)
  10. pSCgRNA,pSCOE1-fcoCas9(野村等,2016,图1A)
  11. DH5α感受态细胞(自制)
  12. NEBuffer 2.1(New England Biolabs,目录号:R0539S)
  13. I(New England Biolabs,目录号:R0539S)
  14. 无菌MilIi-Q水
  15. GEL/PCR纯化小套件(Favorgen Biotech,目录号:FAGCK 001-1)
  16. 连接高ver.2(TOYOBO,目录号:LGK-201)
  17. ScU6p插件检查F引物5'-GAGGATCACGGTGTCACATGTCC-3'
  18. Quick Taq ® HS DyeMix(TOYOBO,目录号:DTM-101)
  19. ScU6p Seq检查F引物5'-ATGTCAAACATAACCTGG-3'
  20. 氨苄青霉素(NACALAI TESQUE,目录号:02739-74)
  21. 质粒DNA提取微型试剂盒(Favorgen Biotech,目录号:FAPDE 001-1)
  22. 琼脂糖S(NIPPON GENE,目录号:312-01193)
  23. DNA梯子标记(SMOBIO Technology,目录号:DM3100)
  24. NucleoBond ® Xtra Midi(MACHEREY-NAGEL,目录号:740410.50)
  25. G418硫酸氢盐(NACALAI TESQUE,目录号:16512-81)
  26. BCDAT培养基(Nishiyama等人,2000)
  27. 葡萄糖(和光纯药,目录号:049-31165)
  28. Tks Gflex DNA聚合酶(Takara Bio,目录号:R060A)
  29. Zero Blunt PCR Cloning Kit(Thermo Fisher Scientific,Invitrogen,目录号:K275020)
  30. CloneJET PCR克隆试剂盒(Thermo Fisher Scientific,Thermo Scientific TM,目录号:K1231)
  31. LB琼脂,Miller(BD,Difco TM ,目录号:244520)
  32. LB肉汤,Miller(BD,Difco TM ,目录号:244620)
  33. 三羟甲基氨基甲烷(NACALAI TESQUE,目录号:35434-21)
  34. 氯化钾(Wako Pure Chemical Industries,目录号:163-03545)
  35. 乙二胺四乙酸(EDTA)(DOJINDO,目录号:345-01865)
  36. 乙酸(和光纯药,目录号:017-00256)
  37. 溴化乙锭溶液(NACALAI TESQUE,目录号:14631-94)
  38. 含有50μg/ml氨苄青霉素的LB琼脂平板(参见食谱)
  39. 裂解缓冲液(见配方)
  40. 50 x TAE缓冲液(见配方)
  41. 1x TAE缓冲区(见配方)

设备

  1. 植物生长室(SANYO,型号:MLR-350HT)
  2. 微量移液器(Eppendorf,型号:Reference ® 2)
  3. 无菌镊子(电子显微镜科学,DUMONT,目录号:0108-5-PO)
  4. 热循环仪(PCR Thermal Cycler Dice Gradient,Takara Bio,型号:TP600)
  5. 高速离心机(TOMY DIGITAL BIOLOGY,型号:MX-300)
  6. 块孵化器(ASTEC,型号:B1-515A)
  7. 分光光度计(GE Healthcare,型号:NanoVue)
  8. 高压灭菌器(TOMY DIGITAL BIOLOGY,型号:SX300)
  9. 琼脂糖凝胶电泳设备(Cosmo Bio,型号:i-MyRun.N)
  10. 紫外透射仪(ATTO,型号:DTB-20MP,TYPE-CX)
  11. 加热培养箱(SANYO,型号:MIR-262)

程序

  1. sgRNA表达载体的设计与构建
    1. 选择目标序列
      1. 选择目标基因或基因组区域中的20 bp靶序列(图1B)。
        1. 检查目标序列3'末端的原始相邻基序(PAM)序列(5'-NGG-3')。
        2. 为了诱导基因功能异常突变,靶序列应设计在5'末端或功能域附近。
        3. 避免poly-T序列作为sgRNA的转录终止子;确保G + C含量为40-80%
        4. 如果靶位点包含限制酶的识别位点,则可以对基因分型进行限制性片段长度多态性(RFLP)分析。
          注意:已经报道截短的sgRNA可以减少脱靶效应(Fu等,2014)。我们确认截断的sgRNA(18或17bp靶序列)在我们的系统中起作用;不过,我们尚未评估其目标效率
      2. 添加以下用于克隆的衔接子序列(图1B):正义寡核苷酸的5'-TCTG-3'和反义寡核苷酸的5'-AAAC-3'。
    2. 消化pSCgRNA载体(图1C)
      1. 准备消化混合物,如下所示:


      2. 将混合物在37℃下孵育3小时。
      3. 使用DNA纯化试剂盒(Favorgen GEL/PCR Purification Mini Kit)纯化I消化的pSCgRNA载体 Bbs。
    3. 寡核苷酸退火(图1C)
      1. 准备退火混合物,如下所示:


      2. 在热循环仪中在95℃孵育5分钟。
      3. 从热循环仪中取出管,并使混合物在工作台上在20-25℃冷却30分钟。
    4. 将退火的寡核苷酸克隆到pSCgRNA载体中(图1C)
      1. 准备连接混合物,如下所示:


      2. 将混合物在16℃下孵育3小时以上。
      3. 使用2μl连接混合物转化DH5α感受态细胞,并在含有50μg/ml氨苄青霉素的LB平板上铺展。
      4. 将LB板在37℃下孵育14-16小时
      5. 使用ScU6p Ins混合PCR反应混合物。检查F引物(作为正向引物)和反义靶序列(作为反向引物)

      6. 通过执行以下循环的菌落PCR验证插入:


        注意:如果克隆成功,则将扩增和检测到一个950bp的条带(图1D)。我们建议至少测试8-16个殖民地。
      7. 在50μg/ml氨苄青霉素的液体LB培养基中培养阳性菌落。
      8. 使用质粒DNA迷你准备试剂盒(Favorgen Plasmid DNA Extraction Mini Kit)纯化质粒
      9. 使用ScU6p Seq确认pSCgRNA载体中插入的靶序列。使用Sanger测序检查F引物。


        图1.使用RNA引导的内切核酸酶在苔藓中进行靶向诱变的载体构建。 A. Cas9和sgRNA表达载体的示意图; B.目标序列设计实例构建sgRNA表达载体的方法D.用于检查插入靶序列的菌落PCR的代表性结果。 M:DNA梯子标记。
  2. PEG介导的原生质体转化(图2A)
    1. 纯化含有靶序列的pSCgRNA和pSCOE1-fcoCas9,其中骨架是pTN182载体(Sakakibara等人,2008),使用midi制备试剂盒(NucleoBond,Xtra Midi ),并以1μg/μl的无菌TE缓冲液稀释。使用15μg每种质粒进行转化
    2. 使用PEG介导的原生质体转化方法共同转化两个载体,如先前的报告(Nishiyama等人,2000; PHIBCOmanual ver.2.0 in NIBB PHYSCObase website: http://moss.nibb.ac.jp/protocol.html )。


      图2.使用RGENs获取突变株的方案概述

  3. 使用抗生素G418(遗传霉素)进行质子细胞的再生和瞬时筛选。 
    1. 将转化的原生质体接种在玻璃纸上的PRM/T和PRM/B板上,在植物生长室中将混合物在25℃下孵育7天(图2B)。
    2. 对于瞬时筛选,将再生的质子转移到玻璃纸上的PRM/T中的原生质体到具有20μg/ml G418的新BCDAT平板(图2C)。
    3. 在植物生长室培养板5 d(图2C和3)

      图3.瞬时筛选后的质子的实例(ppFtsZ2-1靶向)。 A.死质子的代表性例子B.生存的代表性例子。菌株也显示了PpFtsZ2-1突变的表型。刻度棒=50μm。

    4. 将PRM/T玻璃纸中存活的质子转移到新的BCDAT平板上,再次培养直到形成小菌落(图2D)。
    5. 使用镊子,选择殖民地边缘的质子,并将其转移到新的BCDAT或BCDAT + 0.5%葡萄糖板(图2D)。
    6. 对于基因分型,培养质子直到菌落直径为5-10mm。
      注意:获得的菌株有可能是马赛克植物,其是具有不同突变或野生型的菌株的混合物。如果是这样,我们可以从原生质体,单个质子分枝或游戏头条的叶子中分离单基因株。

  4. 基因分型
    1. 绿色PCR
      1. 将一些新鲜的质子转移到PCR管(图4A),并使用200μl移液管芯片的尖端研磨成50μl裂解缓冲液(参见食谱)(图4B)。


        图4.绿色PCR的模板的制备 A.裂解缓冲液中新鲜的质子的一小部分; B.在裂解缓冲液中用200μl芯片的尖端压碎的质子
      2. 在95°C孵育10分钟。
      3. 将3,500 x g的碎屑旋转10秒,并使用上清液作为PCR模板。
      4. 使用免费在线工具Primer 3设计引物套件( http://primer3.ut.ee/),用于扩增基因组DNA,包括用于测序或其他基因分型的靶位点,如RFLP。
      5. PreparePCR反应混合物,如下所示:


      6. 扩增PCR产物,包括基因组的靶位点,其周期如下:


    2. 限制片段长度多态性(RFLP)分析
      1. 如果靶位点包含限制酶的识别位点,则可以通过RFLP观察到靶向的诱变。
      2. 通过琼脂糖凝胶电泳确认PCR产物后,使用相应的限制酶消化PCR产物
      3. 经过充分消化后,通过琼脂糖凝胶电泳分析酶处理和未处理的PCR产物。限制酶耐受性PCR产物可能有突变。
        注意:如果使用衍生自野生型和突变型菌株的PCR产物的混合物,则可以使用可以识别和切割非完全匹配的DNA的T7内切核酸酶I或cel1测定来检查靶向诱变。作为高通量和全面的基因分型方法,也可应用高分辨率熔解分析或下一代测序
    3. 基因组靶序列的测序
      1. 通过琼脂糖凝胶电泳确认PCR产物后,使用Zero Blunt PCR Cloning Kit或CloneJET PCR Cloning Kit将PCR产物克隆到测序载体中。
      2. 在菌落PCR和微型制备后,通过Sanger测序检查几个克隆的插入PCR产物的DNA序列 注意:如果PCR产物作为单基因植物中的单一条带存在,则可以通过直接测序来检查。

数据分析

根据我们基于目标基因突变引起的表型变化的评估,目标诱变效率在P < patens 约为45%至68%(Nomura等人,2016)。由于效率取决于目标序列,因此建议您使用"focas"网站( http://focas.ayanel.com/; Doench等人,2014; Xiao等人,2014; Osakabe等人 ,2016),用于设计和预测目标基因组的目标和离靶目标效率。在这种情况下,目标评分为0.6以上的靶序列和脱靶效果的可能性较低是理想的。

笔记

  1. 通过使用设计用于基因组上的远端位点的两种gRNA,可以通过该方法引入长缺失突变(〜3kb)(野村等,2016)。
  2. 在该方法中,载体DNA通常不被整合到苔藓基因组(Nomura等人,2016)中。
  3. 在PEG介导的原生质体转化过程中存在形成多倍体的可能性。获得性菌株中的DNA含量应使用流式细胞仪进行评估
  4. 如果该协议适用于其他物种,可能需要修改一些实验条件,例如转化(例如,PEG治疗时间)和筛选方法(例如)。 ,抗生素浓度和选择持续时间)

食谱

  1. LB琼脂平板与50μg/ml氨苄青霉素 LB琼脂,米勒40克
    用MilIi-Q水和高压釜(121℃,15分钟)填充1升 加氨苄青霉素至终浓度为50μg/ml
  2. 裂解缓冲液
    100mM Tris-HCl(pH9.5)
    1 M KCl
    10 mM EDTA
  3. 50 x TAE缓冲区
    2 M Tris
    1 M乙酸
    0.5 M EDTA(pH = 8.0)
  4. 1 x TAE缓冲区
    20 ml 50 x TAE缓冲液
    980 ml MilIi-Q水

致谢

该协议是从已发表的论文(Nomura等人,2016)改编而来的。这项工作得到了日本科学促进会,青年科学家赠款(B)(授权号15K18824)和科学研究助学金(C)(授权号15K06905)的支持。

参考

  1. Doench,JG,Hartenian,E.,Graham,DB,Tothova,Z.,Hegde,M.,Smith,I.,Sullender,M.,Ebert,BL,Xavier,RJ and Root,DE(2014) 用于CRISPR-Cas9介导的基因失活的高活性sgRNA的合理设计。 Nat Biotechnol 32(12):1262-1267。
  2. Fu,Y.,Sander,JD,Reyon,D.,Cascio,VM and Joung,JK(2014)。  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。
  3. Nishiyama,T.,Hiwatashi,Y.,Sakakibara,I.,Kato,M。和Hasebe,M。(2000)。< a class ="ke-insertfile"href ="http://www.ncbi。 nlm.nih.gov/pubmed/10718194"target ="_ blank">通过穿梭诱变在青苔,小立委乳头菌中标记的诱变和基因捕获。 DNA Res 7(1):9-17。
  4. Nomura,T.,Sakurai,T.,Osakabe,Y.,Osakabe,K.and Sakakibara,H。(2016)。< a class ="ke-insertfile"href ="http://www.ncbi。 nlm.nih.gov/pubmed/27986915"target ="_ blank">使用CRISPR/Cas9系统在苔藓中有效和可遗传的靶向诱变。植物细胞生理学57(12):2600 -2610。
  5. Osakabe,Y.,Watanabe,T.,Sugano,SS,Ueta,R.,Ishihara,R.,Shinozaki,K.and Osakabe,K。(2016)。< a class ="ke-insertfile"href = "http://www.ncbi.nlm.nih.gov/pubmed/27226176"target ="_ blank">优化CRISPR/Cas9基因组编辑以修改植物中的非生物胁迫反应 Sci Rep 6:26685.
  6. Sakakibara,K.,Nishiyama,T.,Deguchi,H。和Hasebe,M。(2008)。< a class ="ke-insertfile"href ="https://www.ncbi.nlm.nih.gov /pubmed/?term=1.%09Sakakibara%2C+K.%2C+Nishiyama%2C+T.%2C+Deguchi%2C+H.+and+Hasebe%2C+M.+%282008%29.+Class + 1 + KNOX +基因+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +不参与苔藓幼苗的发育,而是在孢子体发育中起作用。 Evol Dev 10:555-566。
  7. Untergasser,A.,Cutcutache,I.,Koressaar,T.,Ye,J.,Faircloth,BC,Remm,M.and Rozen,SG(2012)。< a class ="ke-insertfile"href = https://academic.oup.com/nar/article/40/15/e115/1223759/Primer3-new-capabilities-and-interfaces"target ="_ blank"> Primer3新功能和界面。 <核酸Res 40(15):e115-e115。
  8. Xiao,A.,Cheng,Z.,Kong,L.,Zhu,Z.,Lin,S.,Gao,G. and Zhang,B.(2014)。  CasOT:一个全基因组的Cas9/gRNA离靶搜索工具生物信息学 30:1180-1182。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Nomura, T. and Sakakibara, H. (2017). Targeted Mutagenesis Using RNA-guided Endonucleases in Mosses. Bio-protocol 7(12): e2359. DOI: 10.21769/BioProtoc.2359.
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