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Use of Geminivirus for Delivery of CRISPR/Cas9 Components to Tobacco by Agro-infiltration
通过农杆菌浸润法使用双生病毒将CRISPR/Cas9组分转入烟草中   

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Abstract

CRISPR/Cas9 system is a recently developed genome editing tool, and its power has been demonstrated in many organisms, including some plant species (Wang et al., 2016). In eukaryotes, the Cas9/gRNA complexes target genome sites specifically and cleave them to produce double-strand breaks (DSBs), which can be repaired by non-homologous end joining (NHEJ) pathway (Wang et al., 2016). Since NHEJ is error prone, mutations are thus generated. In plants, delivery of genome editing reagents is still challenging. In this protocol, we detail the procedure of a virus-based gRNA delivery system for CRISPR/Cas9 mediated plant genome editing (VIGE). This method offers a rapid and efficient way to deliver gRNA into plant cells, especially for those that are recalcitrant to transformation with Agrobacterium.

Keywords: CRISPR(CRISPR), Cas9(Cas9), Geminivirus(双生病毒), VIGE(VIGE), Genome editing(基因组编辑), Tobacco(烟草)

Background

Genome editing technologies based on viruses have been reported using deconstructed DNA viruses and an RNA virus (Baltes et al., 2014; Ali et al., 2015). Recently, we used a full geminivirus - Cabbage Leaf Curl virus (CaLCuV) (a bipartite begomovirus which infects a wide range of members of the Brassicaceae, including cauliflower) for highly efficient genome editing in one of its hosts, Nicotiana benthamiana, for the first time (Yin et al., 2015).

Materials and Reagents

  1. 1-ml syringe
  2. 0.22-μm filter
  3. Wild type N. benthamiana plants; six to eight-leaf stage transgenic N. benthamiana plants stably expressing oCas9 (Arabidopsis codon optimized Cas9) - KQ334 plant
  4. Agrobacterium tumefaciens strain GV3101, E. coli DH5α, E. coli DB3.1 (for propagating vectors pJG081)
  5. The following plasmids are required: pJG081, pKQ334 (Yin et al., 2015), pT-U6p-scaffold-U6t (Yin et al., 2015), pCAMBIA1301, pCAMBIA2300 (www.cambia.org), pCVA (Tang et al., 2010), pCVB (Tang et al., 2010), and pMD18-T vector (Takara Bio, catalog number: D101A) 
    Notes:
    1. To generate a construct for constitutive expression of oCas9, oCas9 is PCR amplified and subsequently cloned into pJG081 by ligation independent cloning (LIC) technique (Aslanidis and de Jong, 1990) to generate pKQ334.
    2. pKQ334 is then used to transform N. benthamiana to generate Cas9 transgenic plant - KQ334 plant. pKQ334 can also be used in transient expression of Cas9 in N. benthamiana.
    3. The pT-U6p-scaffold-U6t is used to generate p-T-U6p-gRNA-scaffold containing gRNA of the target gene by PCR mutagenesis.
    4. pCVA and pCVB are CaLCuV-based T-DNA vectors. pCVA is used to generate pCVA-gRNA or pCVA-scaffold. pCVB is used in combination with pCVA or its derivatives to generate CaLCuV. pMD18-T vector is used to clone PCR amplicons.
    5. These vectors can be obtained by contacting Dr. Yule Liu.
  6. Primers (Table 1)

    Table 1. Primers used in this protocol


  7. TransStart FastPfu DNA polymerase (Beijing TransGen Biotech, catalog number: AP221 )
  8. Restriction enzymes (New England Biolabs, stored at -20 °C)
    1. DpnI (New England Biolabs, catalog number: R0176 )
    2. ApaI (New England Biolabs, catalog number: R0114 )
    3. SacI (New England Biolabs, catalog number: R0156 )
    4. PstI (New England Biolabs, catalog number: R3140S )
    5. KpnI (New England Biolabs, catalog number: R3142S )
    6. XbaI (New England Biolabs, catalog number: R0145S )
    7. MlyI (New England Biolabs, catalog number: R0610 )
  9. 10x Cutsmart buffer
  10. Antibiotics
    Ampicillin (AMRESCO, catalog number: 0339 )
    Kanamycin (AMRESCO, catalog number: 0408 )
    Rifampicin (Sigma-Aldrich, catalog number: R3501 )
  11. TIANprep Mini Plasmid Kit (TIANGEN Biotech, catalog number: DP103 ) for plasmid DNA extraction
  12. Ethanol (Beijing Chemical Factory, catalog number: B0301002 )
  13. T4 DNA polymerase (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EP0062 )
  14. dATP (Takara Bio, catalog number: 4026 )
  15. Chloroform (Sigma-Aldrich, catalog number: 1601383 )
  16. Phenol (Sigma-Aldrich, catalog number: P4557 )
  17. 3 M NaAc (Biovision, catalog number: 2118 )
  18. dTTP (Takara Bio, catalog number: 4029 )
  19. DNA Purification Kit (BioMED, catalog number: DH103-01 )
  20. T4 DNA ligase (Takara Bio, catalog number: 2011A )
  21. Tryptone (BD, BactoTM, catalog number: 211705 )
  22. Yeast extract (BD, BactoTM, catalog number: 212750 )
  23. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 31434 )
  24. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M7304 )
  25. MES (Sigma-Aldrich, catalog number: M3671 )
  26. Potassium hydroxide (KOH)
  27. Acetosyringone (Solarbio, catalog number: A8110-1 )
  28. DMSO
  29. Sodium phosphate monobasic (NaH2PO4) (Sigma-Aldrich, catalog number: S3139 )
  30. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S3264 )
  31. Sodium phosphate monobasic (NaH2PO4) (AMRESCO, catalog number: 0571 )
  32. Sodium phosphate dibasic (Na2HPO4) (AMRESCO, catalog number: 0404 )
  33. Sodium phosphate monobasic monohydrate (NaH2PO4·H2O)
  34. Sodium phosphate dibasic heptahydrate (Na2HPO4·7H2O)
  35. X-gluc (AMRESCO, catalog number: 0919 )
  36. Methanol (Beijing Chemical Factory, catalog number: B0301005 )
  37. Potassium ferricyanide (AMRESCO, catalog number: 0713 )
  38. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
  39. PEG 8000 (Sigma-Aldrich, catalog number: 89510 )
  40. Magnesium chloride hexahydrate (MgCl2·6H2O) (AMRESCO, catalog number: 0288 )
  41. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: 63068 )
  42. MES (Sigma-Aldrich, catalog number: M3671 )
  43. Acetosyringone (Sigma-Aldrich, catalog number: D134406 )
  44. EasyTaq DNA polymerase (Beijing TransGen Biotech, catalog number: AP111 )
  45. DNAsecure Plant Kit (TIANGEN Biotech, catalog number: DP320 ) for genomic DNA extraction
  46. Luria-Bertani medium (see Recipes)
  47. MgCl2 (1 M stock) (see Recipes)
  48. MES (0.5 M stock) (see Recipes)
  49. Acetosyringone (1 M stock) (see Recipes)
  50. Infiltration buffer (see Recipes)
  51. 2x phosphate buffer (pH 7) (see Recipes)
  52. X-gluc substrate solution (see Recipes)
  53. PEG-MgCl2 solution (see Recipes)

Equipment

  1. Microcentrifuge (Eppendorf)
  2. PCR cycler (Bio-Rad Laboratories)
  3. 37 °C and 28 °C incubator; 37 °C and 28 °C shaker
  4. NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, model: NanoDrop 1000 ) (for measuring the concentration of the DNA)
  5. Electrophoresis apparatus (Junyi, Beijing)
  6. Vacuum apparatus (WILMAD-LABGLASS)
  7. Glass/plastic beaker
  8. Autoclave

Software

  1. ImageJ (https://imagej.nih.gov/ij)

Procedure

  1. gRNA design for specific gene/site of interest
    For knocking out a gene of interest, choose gRNAs located in the exons. For editing the non CDS region in the genome, choose a region where disruption would have this desired effect. We strongly recommend using CRISPR-P (cbi.hzau.edu.cn) to design gRNAs (Lei et al., 2014). It provides a specificity score for each possible gRNA and lists the potential off-targets. Additionally, it provides a list of useful restriction enzymes that would recognize the gRNA spacer for RFLP analysis of this specific site.

  2. Construct gRNA plasmid
    To drive gRNA expression in plants, we constructed a gRNA plasmid targeting NbPDS. Any other genes-of-interest can also be targeted. To clone the gRNA easily, we first used a small vector (T-U6p-scaffold-U6t). Then, the promoter-gRNA-terminator construct is transferred to a larger binary vector, such as pCAMBIA2300 and pCVA.
    1. Perform inverse PCR (Stemmer and Morris, 1992) to add gRNA spacer between the AtU6 promoter and gRNA scaffold necessary for Cas9 binding (Figure 1). Setup reactions as follows:



      Figure 1. Illustration of pT-U6p-scaffold-U6t (Yin et al., 2015) and the use of inverse PCR for inserting gRNA spacer into pT-U6p-scaffold-U6t to generate pT-U6p-gRNA-scaffold-U6t. A. AtU6 promoter, scaffold, and AtU6 terminator are shown in orange boxes on the plasmid map. Two primers used for inserting gRNA spacer (targeting NbPDS) are indicated on the plasmid map with their respective directions. Parallel blacklines on the primers are complementary sequences. SacI and PstI are used to cut the gRNA expression cassette which is then cloned into a binary vector. The small arrow here is to indicate scaffold. B. Sequence information for inverse PCR. Here, gRNA spacer targeting NbPDS is used as an example. Partial sequence of the NbPDS locus is shown in black. PAM and MlyI site are shown. gRNA spacer is also shown in black above the NbPDS locus. Two primers (oYK1056 and oYK1057) for PCR are shown, the sequence from AtU6 is shown in red, and the sequence from scaffold is shown in blue. The two primers that bind to the template (vector) during the PCR process are also shown with primers and vector indicated. The change after inverse PCR is shown below the arrow.

    2. Perform PCR amplification using the following parameters (Tm represents the annealing temperature of the primer pair):


    3. Setup a restriction enzyme digest to remove the plasmid template in the above PCR product as follows:
      Note: DpnI is a methylation sensitive enzyme, and plasmid purified from E. coli DH5α is generally methylated. Thus, DpnI is often used to remove plasmid template. Here, a ligation or a denaturation/reannealing step is not necessary to connect the ends of the vector, because the PCR product with overlapping ends will re-circularize to form a doubly-nicked plasmid  and will be repaired further in bacteria to form an intact plasmid.


      Mix well and incubate at 37 °C for 2 h.
    4. Transform E.coli DH5α using 1-2 μl product of the above reaction with heat shock at 42 °C for 90 sec, recover at 37 °C for 1 h, plate on LB plate with 100 mg/L ampicillin, and then incubate overnight at 37 °C (Im, 2011).
    5. Select one to five transformants and culture overnight in LB with 100 mg/L ampicillin at 37 °C, 220 rpm. Extract plasmids from the transformants respectively with TIANprep Mini Plasmid Kit, and Sanger sequence the plasmid pT-U6p-gRNA:NbPDS-scaffold-U6t with sequencing primer M13-47 to confirm the presence of the gRNA spacer.

  3. Check Cas9 activity and gRNA efficiency using fsGUS (frameshift GUS) reporter system
    It’s necessary to confirm if KQ334 plants retain Cas9 activity and that Cas9 cleaves DNA under the guidance of gRNA. In this system, a 23 bp sequence with a PAM (AGG) at the end is inserted downstream of the start codon of the GUS gene, which causes a frame shift of the GUS gene (fsGUS). We co-expressed two vectors: one harboring the fsGUS gene driven by a 35S promoter termed pJG081-fsGUS and the other p2300-gRNA:fsGUS containing an Arabidopsis U6-26 gene promoter (AtU6 promoter)-driven gRNA containing a 20 bp sequence that matches the first 20 bp of the sequence inserted into the fsGUS gene.
    There is another highly efficient method to check gRNA efficiency based on protoplast transfection. Please refer to Li and co-workers’ protocol (Li et al., 2015)  
    1. Setup a PCR reaction to amplify an fsGUS fragment in which a short sequence containing the gRNA spacer sequence and a MlyI recognition site is inserted after the GUS start codon (Figure 2). Primers used at this step should be designed specifically to flank the cut sites of different gRNAs.



      Figure 2. Illustration of the fsGUS reporter system used to check gRNA efficiency. Illustration of the fsGUS reporter system. A 23 bp sequence containing a MlyI site is inserted after the start codon of the GUS gene, and the resulting sequence is designated as fsGUS. Due to the inserted sequence, fsGUS cannot be translated into a functional enzyme. fsGUS is driven by a CaMV35S promoter and placed in a binary vector. Another binary vector contains AtU6 promoter-driven gRNA that targets the first 20 bp of the inserted sequence in fsGUS. Two A. tumefaciens lines transformed with each binary vector are mixed together and infiltrated into KQ334 plant leaves. After two T-DNAs enter into the same cell, the first 20 bp of the inserted sequence in fsGUS is targeted by the Cas9/gRNA complex for cleavage. Repair via NHEJ will create in-dels at the cleavage site and, in some cases, recover the normal coding frame for fsGUS, thus creating a functional GUS which can be detected by GUS staining. (This figure is adapted and modified from Yin et al., 2015.)

      Perform PCR amplification using the following parameters (Tm represents the annealing temperature of the primer pair):


    2. Purify the PCR product using PEG/MgCl2 method (Paithankar and Prasad, 1991)
      1. Add 50 μl water to 50 μl PCR product and then add 50 μl 30% PEG8000/30 mM MgCl2.
      2. Mix well and centrifuge immediately at 12,000 x g for 15 min.
      3. Next, remove the supernatant and add 200 μl 70% ethanol.
      4. Centrifuge at 12,000 x g for 5 min and remove the supernatant. Finally, dry the pellet and dissolve the pellet in 10 μl sterile deionized water.
    3. Construct pJG081-fsGUS using LIC method.
      Note: LIC is a fast, simple, and very cheap cloning technique. It utilizes annealing of complementary overhangs of a vector and a PCR fragment consisting of 9-15 bases.
      1. Single-stranded overhangs are generated by using T4 DNA polymerase and the addition of only one dNTP in the reaction buffer, leading to an equilibrium of activity of polymerase and exonuclease at the first appearance of this nucleotide (Aslanidis and de Jong, 1990). For the generation of the single stranded tails, the purified DNA preparations are treated with T4 DNA polymerase. Setup T4 DNA polymerase treatment for the above PCR product:


      2. Mix gently and well by pipetting. Incubate at 37 °C for 20 min then at 75 °C for 20 min to inactivate T4 DNA polymerase using a PCR cycler. The resulting product is designated as fsGUS-LIC.
    4. Digest pJG081 for LIC reaction:


      1. Incubate at 37 °C in incubator for 3 h.
      2. Then, purify the product using phenol/chloroform method: add an equal volume of phenol/chloroform (1:1) and vortex vigorously for 20 sec; then centrifuge at 12,000 x g for 10 min at room temperature; retain the supernatant and add an equal volume of chloroform, vortex vigorously for 20 sec, and centrifuge at 12,000 x g for 10 min at room temperature; precipitate the DNA from the aqueous phase of the above extractions by adding 1/10 volume of 3 M NaAc and 2 volume of ethanol.
      3. Finally, pellet the DNA by centrifugation, and dissolve it with sterile deionized water.
    5. Setup T4 DNA polymerase treatment for this linear pJG081:


      The resulting product is designated as pJG081-LIC.
    6. Construct pJG081-fsGUS:
      1. Mix 5 μl fsGUS-LIC and 5 μl pJG081-LIC well, incubate at 70 °C for 10 min and then at 22 °C for 10 min.
      2. Next, transform this product into E. coli DH5α, which is then selected on medium with kanamycin.
    7. Construct p2300-gRNA:fsGUS.
      1. Setup a restriction enzyme reaction as follows:


      2. Incubate at 37 °C for 3 h. Then, purify the product with the DNA Purification Kit according to manufacturer recommendations.
      3. To clone a complete gRNA expression cassette into pCambia 2300, U6p-gRNA:NbPDS-scaffold-U6t is cut from pT-U6p-gRNA:NbPDS-scaffold-U6t. Setup a restriction enzyme reaction as follows:


      4. Incubate at 37 °C for 3 h. Then purify the product with the DNA Purification Kit according to manufacturer recommendations.
      5. Setup a ligation reaction as follows:


      6. Incubate at 16 °C for 8 h and transform it into E. coli DH5α, which is then selected on medium with kanamycin.
    8. Agrobacterium transformation
      1. Transform pKQ334, pJG081-fsGUS and p2300-gRNA:fsGUS respectively into Agrobacterium tumefaciens strain GV3101 by heat shock method (Höfgen and Willmitzer, 1988).
      2. Select the transformants on LB medium with 50 mg L-1 kanamycin and 50 mg L-1 rifampicin at 28 °C for two days.
    9. Transient expression
      Note: Either Cas9 transgenic plants (KQ334 plants) or another Agrobacterium culture containing pKQ334 plasmid (constitutive expression of Cas9) can be used for this experiment.
      1. The Agrobacterium cultures are inoculated in a 5 ml Luria-Bertani medium containing appropriate antibiotics (50 mg L-1 rifampicin, and 50 mg L-1 kanamycin) and grown overnight in a 28 °C shaker.
      2. Agrobacterium cells are harvested by centrifugation at 5,000 x g for 5 min and resuspended in infiltration buffer, adjusted to an optical density at 600 nm of 1.0 and incubated at room temperature for 3 to 4 h before infiltration.
      3. For transient expression, equal amounts of Agrobacterium strain GV3101 cultures containing either pKQ334 or pJG081-fsGUS or p2300-gRNA:fsGUS are mixed as experimental group and spot-infiltrated into leaves of six-leaf stage plants using a 1-ml syringe. As control, Agrobacterium cultures containing either pJG081-fsGUS or p2300-gRNA:fsGUS are spot-infiltrated separately into leaves of six-leaf stage plants using a 1-ml syringe.
      4. The first three leaves counting from the top of each plant are infiltrated; each leaf is spot-infiltrated with experimental group and control group. Midrib and main veins are avoided when infiltrating.
      5. Press the nozzle of a 1 ml syringe without needle against the abaxial surface of the leaf, and hold a gloved finger on the other side and inject slowly. The infiltration area is often seen as ‘wet’ (Video 1). 

        Video 1. Transient expression using agro-infiltration in N. benthamiana

      6. Alternatively, equal amounts of Agrobacterium cultures containing either pJG081-fsGUS or p2300-gRNA:fsGUS can be mixed together and infiltrated into leaves of six-leaf stage KQ334 plants using a 1-ml syringe (Figure 2). As control, Agrobacterium cultures containing either pJG081-fsGUS or p2300-gRNA:fsGUS are infiltrated separately into leaves of six-leaf stage KQ334 plants using a 1-ml syringe.
    10. GUS staining
      The GUS staining assay is based on Kabbage et al. (2011) and performed as follows:
      1. Two to three days after infiltration, infiltrated leaves are collected. Vacuum infiltrate the X-gluc substrate into the collected leaves. A glass/plastic beaker is used to put leaves in for vacuum infiltration and a vacuum level of 600 mm Hg is applied (for 3 min) and released for several times until the leaves become translucent.
      2. After infiltration, leaves are incubated in darkness at room temperature overnight.
      3. Rinse the leaves by changing the solution with deionized water and remove the chlorophyll by immersing the leaves in 70% ethanol for at least one day.

  4. Construct pCVA-gRNA and pCVA-scaffold
    To perform VIGE of the target gene, pCVA-gRNA is first generated by inserting a cassette containing an AtU6 promoter and gRNA::NbPDS into pCVA. The construct pCVA-scaffold is taken as a negative control.
    1. Setup PCR reactions as follows:


    2. Perform PCR amplification using the following parameters (Tm represents the annealing temperature of the primer pair):


    3. Then purify the product with the DNA Purification Kit according to manufacturer recommendations.
    4. Setup a restriction enzyme reaction as follows:


    5. Incubate at 37 °C for 3 h. Then purify the product with the DNA Purification Kit. The final products are U6p-gRNA-scaffold_KpnI/XbaI and U6p-scaffold_KpnI/XbaI.
    6. At the same time, setup a restriction enzyme reaction as follows:


    7. Incubate at 37 °C for 3 h. Then purify the product with the DNA Purification Kit according to manufacturer recommendations.
    8. Next, setup a ligation reaction as follows to generate pCVA-gRNA and pCVA-scaffold


    9. Incubate at 16 °C for 8 h and transform it into E. coli DH5α, which is then selected on medium with kanamycin.

  5. VIGE of a gene/site of interest
    1. First, transform pCVA-gRNA, pCVA-scaffold and pCVB respectively into Agrobacterium tumefaciens strain GV3101 by heat shock method. Select the transformants on medium with kanamycin and rifampicin.
    2. Next, the Agrobacterium cultures are inoculated to 5 ml Luria-Bertani medium containing appropriate antibiotics (50 mg L-1 rifampicin, and 50 mg L-1 kanamycin) and grown overnight in a 28 °C shaker. 
    3. Agrobacterium cells are harvested by centrifugation at 5,000 x g for 5 min and resuspended in infiltration buffer, adjusted to an optical density at 600 nm of 1.0 and incubated at room temperature for 3 to 4 h before infiltration.
    4. Agrobacterium cultures containing pCVB and pCVA or their derivatives are mixed at a 1:1 ratio and infiltrated into petioles of six to eight-leaf stage KQ334 plants using a 1-ml syringe (Tang et al., 2010) (Figure 3 and Video 2). As a control, they are also infiltrated into petioles of six to eight-leaf stage wild-type plants using the same method.


      Figure 3. Illustration of VIGE. The Agrobacterium containing either pCVA-gRNA or pCVB are mixed with each other and infiltrated into petiole of KQ334 plants (Cas9 transgenic plants). Systemic leaves are used to check the genome editing events. AtU6 promoter is used to drive the expression of gRNA. The gRNA spacer is shown in red. CR, common region; AL1, replication-associated protein; AL2, transcription activator; AL3, replication enhancer; AL4, putative pathogenesis-related protein; BL1 and BR1, movement proteins; LB, left border; RB, right border. (This figure is adapted and modified from Yin et al., 2015.)

      Video 2. Agro-infiltration into petiole of N. benthamiana

  6. Detection of VIGE mediated mutations in plant genomic DNA
    1. For the detection of VIGE results, genomic DNA extracted from systemic leaves according to the manufacturer’s instructions is used as template for PCR amplification (see below) of a targeted locus with primers (PDS_MlyIF and PDS_MlyIR) flanking the target site using EasyTaq DNA polymerase. PCR product is purified and digested with selected restriction enzyme (i.e., MlyI for NbPDS) and run on a 2% agarose gel.
    2. Mutation rate is estimated by comparing band intensities using ImageJ (https://imagej.nih.gov/ij). The uncut band is then cut from the gel and the DNA is recovered by using DNA Purification Kit according to the manufacturer’s instructions. The undigested purified DNA is subsequently cloned into pMD18-T vector (TAKARA) and transformed into E. coli DH5α. Clones are sequenced with M13-47 primer to check for mutations (Figure 3). An example of VIGE for NbPDS gene is shown in Figure 4, in which the PCR product of a specific primer pair targeting the NbPDS gene is digested with MlyI.
      PCR amplification is performed using the following parameters (Tm represents the annealing temperature of the primer pair):



      Figure 4. VIGE of NbPDS. A. illustration of VIGE targeted gene NbPDS. Cut site of Cas9/gRNA is under the red scissor. MlyI is used for discriminating the edited and wild-type sequence. B. schematic of the gel assay used to estimate the editing efficiency of VIGE of NbPDS gene.

Data analysis

To investigate whether endogenous NbPDS gene is edited, ImageJ is used to evaluate the editing efficiency by VIGE.

  1. Select Lanes for analysis and add frames to include bands with ImageJ (https://imagej.nih.gov/ij/).
  2. Measure the intensity of each band in each lane with ImageJ.
  3. The relative editing efficiency is calculated by dividing the intensity of uncut band by the total intensities of all the bands.

Recipes

  1. Luria-Bertani medium
    10 g/L tryptone
    5 g/L yeast extract
    10 g/L NaCl
    Adjust pH to 7, autoclave at 121 °C for 20 min and store at room temperature
  2. MgCl2 (1 M stock)
    Dissolve 203.3 g of MgCl2·6H2O in 800 ml of deionized H2O
    Adjust the volume to 1 L with deionized water
  3. MES (0.5 M stock)
    Dissolve 19.52 g MES in 200 ml deionized water
    Adjust to pH 5.6 with 1 N KOH
  4. Acetosyringone (1 M stock)
    Dissolve 1.962 g acetosyringone in 10 ml of DMSO
  5. Infiltration buffer
    Note: The stock buffers above (Recipes 2-4) are filter sterilized with a 0.22-μm filter and stored at room temperature. The infiltration buffer is prepared freshly for each time.
    10 mM MgCl2
    10 mM MES
    200 μM acetosyringone
  6. 2x phosphate buffer (pH 7)
    0.2 M NaH2PO4
    0.2 M Na2HPO4
    1. First, make two solutions:
      Solution A: dissolve 55.2 g NaH2PO4·H2O in 1 L deionized water
      Solution B: dissolve 107.3 g Na2HPO4·7H2O in 1 L deionized water
    2. Autoclave at 121 °C for 20 min and store them at room temperature
    3. To make 200 ml 2x phosphate buffer (pH 7), add 39.0 ml Solution A and 61.0 ml Solution B and then dilute with deionized water to 200 ml
  7. X-gluc substrate solution (Kabbage et al., 2011)
    1. Add 1 mg X-gluc in 0.1 ml methanol
    2. Then add 1 ml 2x phosphate buffer, 20 μl 0.1 M potassium ferricyanide, 10 μl 10% Triton X-100 and 850 μl deionized water
  8. PEG-MgCl2 solution
    1. Dissolve 30 g of PEG 8000 in 100 ml of 30 mM MgCl2
    2. Filter sterilize the solution with a 0.22-μm filter and store it at room temperature

Acknowledgments

The VIGE method is based on the report by Yin et al. (2015). The method of inoculating CaLCuV is based on Tang et al. (2010). The GUS staining assay is based on Kabbage et al. (2011). This work is supported by the National Basic Research Program of China (2014CB138400), the National Transgenic Program of China (2014ZX0800104B, 2014ZX08010-002 and 2014ZX08005-001) and the National Natural Science Foundation of China (31421001, 3142100007). We thank Alice L. Yu from University of North Carolina at Chapel Hill for proofreading the manuscript.

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  9. Nekrasov, V., Staskawicz, B., Weigel, D., Jones, J. D. and Kamoun, S. (2013). Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol 31(8): 691-693.
  10. Paithankar, K. R. and Prasad, K. S. (1991). Precipitation of DNA by polyethylene glycol and ethanol. Nucleic Acids Res 19(6): 1346.
  11. Stemmer, W. P. and Morris, S. K. (1992). Enzymatic inverse PCR: a restriction site independent, single-fragment method for high-efficiency, site-directed mutagenesis. Biotechniques 13(2): 214-220.
  12. Tang, Y., Wang, F., Zhao, J., Xie, K., Hong, Y. and Liu, Y. (2010). Virus-based microRNA expression for gene functional analysis in plants. Plant Physiol 153(2): 632-641.
  13. Wang, H., La Russa, M. and Qi, L. S. (2016). CRISPR/Cas9 in genome editing and beyond. Annu Rev Biochem 85: 227-264.
  14. Yin, K., Han, T., Liu, G., Chen, T., Wang, Y., Yu, A. Y. and Liu, Y. (2015). A geminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing. Sci Rep 5: 14926.

简介

CRISPR / Cas9系统是最近开发的基因组编辑工具,其功能已被证实在许多生物体中,包括一些植物物种(Wang等人,2016)。 在真核生物中,Cas9 / gRNA复合物特异性地靶向基因组位点并切割它们以产生双链断裂(DSB),其可以通过非同源末端连接(NHEJ)途径修复(Wang等人, 。,2016)。 由于NHEJ易出错,因此产生突变。 在植物中,基因组编辑试剂的递送仍然是挑战性的。 在本协议中,我们详细介绍了CRISPR / Cas9介导的植物基因组编辑(VIGE)的基于病毒的gRNA传递系统的过程。 该方法提供了将gRNA递送到植物细胞中的快速且有效的方式,特别是对于那些难以转基因农杆菌的方法。

已经报道了基于病毒的基因组编辑技术使用解构DNA病毒和RNA病毒(Baltes等人,2014; Ali等人,2015)。 最近,我们使用了一种完整的双因素病毒 - 卷心菜叶卷曲病毒(CaLCuV)(一种感染广西芥菜科的成员,包括花椰菜的二分酵母病毒),用于高效率 第一次(Yin等人,2015年),其主机之一的基因组编辑(Nicotiana benhamiana)首次进行基因组编辑。

关键字:CRISPR, Cas9, 双生病毒, VIGE, 基因组编辑, 烟草

材料和试剂

  1. 1-ml注射器
  2. 0.22-μm过滤器
  3. 野生型 N。本哈本植物;六至八叶期转基因N。本发明植物稳定表达oCas9(拟南芥密码子优化的Cas9) - KQ334植物
  4. 根癌土壤杆菌菌株GV3101,E。大肠杆菌DH5α,E。大肠杆菌DB3.1(用于繁殖载体pJG081)
  5. 需要以下质粒:pJG081,pKQ334(Yin等人,2015),pT-U6p-scaffold-U6t(Yin等人,2015),pCAMBIA1301, pCAMBIA2300( www.cambia.org ),pCVA(Tang等)。 ,2010),pCVB(Tang等人,2010)和pMD18-T载体(Takara Bio,目录号:D101A) 
    注意:
    1. 为了产生用于组成型表达oCas9的构建体,将oCas9进行PCR扩增,随后通过连接独立克隆(LIC)技术(Aslanidis和de Jong,1990)克隆到pJG081中以产生pKQ334。
    2. 然后使用pKQ334转化本氏烟草以产生Cas9转基因植物-KQ334植物。 pKQ334也可用于本氏烟草中Cas9的瞬时表达。
    3. pT-U6p-scaffold-U6t用于通过PCR诱变产生含有靶基因的gRNA的p-T-U6p-gRNA-支架。
    4. pCVA和pCVB是基于CaLCuV的T-DNA载体。 pCVA用于产生pCVA-gRNA或pCVA-支架。 pCVB与pCVA或其衍生物组合使用以产生CaLCuV。 pMD18-T载体用于克隆PCR扩增子。
    5. 这些载体可以通过联系Dr. Yule Liu获得。
  6. 引物(表1)

    表1.本协议中使用的引物


  7. TransStart FastPfu DNA聚合酶(北京TransGen Biotech,目录号:AP221)
  8. 限制酶(New England Biolabs,储存于-20℃)
    1. I(New England Biolabs,目录号:R0176)
    2. Apa I(New England Biolabs,目录号:R0114)
    3. 我(新英格兰Biolabs,目录号:R0156)

    4. I(New England Biolabs,目录号:R3140S)
    5. Kpn I(New England Biolabs,catalog number:R3142S)
    6. Xba I(New England Biolabs,目录号:R0145S)
    7. Mly I(New England Biolabs,目录号:R0610)
  9. 10x Cutsmart缓冲区
  10. 抗生素
    氨苄青霉素(AMRESCO,目录号:0339)
    卡那霉素(AMRESCO,目录号:0408)
    利福平(Sigma-Aldrich,目录号:R3501)
  11. TIANprep Mini Plasmid Kit(TIANGEN Biotech,目录号:DP103),用于质粒DNA纯化
  12. 乙醇(北京化工厂,目录号:B0301002)
  13. T4 DNA聚合酶(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EP0062)
  14. dATP(Takara Bio,目录号:4026)
  15. 氯仿(Sigma-Aldrich,目录号:1601383)
  16. 苯酚(Sigma-Aldrich,目录号:P4557)
  17. 3 M NaAc(Biovision,目录号:2118)
  18. dTTP(Takara Bio,目录号:4029)
  19. DNA纯化试剂盒(BioMED,目录号:DH103-01)
  20. T4 DNA连接酶(Takara Bio,目录号:2011A)
  21. Tryptone(BD,Bacto TM ,目录号:211705)
  22. 酵母提取物(BD,Bacto TM ,目录号:212750)
  23. 氯化钠(NaCl)(Sigma-Aldrich,目录号:31434)
  24. 氯化镁六水合物(MgCl 2·6H 2 O)(Sigma-Aldrich,目录号:M7304)
  25. MES(Sigma-Aldrich,目录号:M3671)
  26. 氢氧化钾(KOH)
  27. Acetosyringone(Solarbio,目录号:A8110-1)
  28. DMSO
  29. 磷酸二氢钠(NaH 2 PO 4)(Sigma-Aldrich,目录号:S3139)
  30. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S3264)
  31. 磷酸二氢钠(NaH 2 PO 4)(AMRESCO,目录号:0571)
  32. 磷酸氢二钠(Na 2 HPO 4)(AMRESCO,目录号:0404)
  33. 磷酸二氢钠一水合物(NaH 2 PO 4·2H 2 O)
  34. 磷酸氢二钠七水合物(Na 2 HPO 4·7H 2 O)
  35. X-gluc(AMRESCO,目录号:0919)
  36. 甲醇(北京化工厂,目录号:B0301005)
  37. 铁氰化钾(AMRESCO,目录号:0713)
  38. Triton X-100(Sigma-Aldrich,目录号:T9284)
  39. PEG 8000(Sigma-Aldrich,目录号:89510)
  40. 氯化镁六水合物(MgCl 2·6H 2 O)(AMRESCO,目录号:0288)
  41. 氯化镁六水合物(MgCl 2·6H 2 O)(Sigma-Aldrich,目录号:63068)
  42. MES(Sigma-Aldrich,目录号:M3671)
  43. Acetosyringone(Sigma-Aldrich,目录号:D134406)
  44. EasyTaq DNA聚合酶(北京TransGen Biotech,目录号:AP111)
  45. DNA安全植物试剂盒(TIANGEN Biotech,目录号:DP320),用于基因组DNA提取
  46. Luria-Bertani培养基(见食谱)
  47. MgCl 2(1M储备)(参见食谱)
  48. MES(0.5M库存)(见配方)
  49. Acetosyringone(1 M库存)(见配方)
  50. 渗透缓冲液(见配方)
  51. 2x磷酸盐缓冲液(pH 7)(参见食谱)
  52. X-gluc底物溶液(参见食谱)
  53. PEG-MgCl 2溶液(参见食谱)

设备

  1. 微量离心机(Eppendorf)
  2. PCR循环仪(Bio-Rad Laboratories)
  3. 37℃和28℃培养箱; 37°C和28°C振动筛
  4. NanoDrop 1000分光光度计(Thermo Fisher Scientific,型号:NanoDrop 1000)(用于测量DNA的浓度)
  5. 电泳仪(Junyi,北京)
  6. 真空装置(WILMAD-LABGLASS)
  7. 玻璃/塑料烧杯
  8. 高压灭菌器

软件

  1. ImageJ( https://imagej.nih.gov/ij/index/html

程序

  1. 特定基因/感兴趣位点的gRNA设计
    为了敲除感兴趣的基因,选择位于外显子中的gRNA。为了编辑基因组中的非CDS区域,请选择一个区域,其中中断将产生这种所需的效果。我们强烈建议您使用CRISPR-P( cbi.hzau.edu.cn )设计gRNA(Lei等人,2014)。它为每种可能的gRNA提供特异性评分,并列出潜在的脱靶。此外,它还提供了一些有用的限制性酶,其将识别用于该特异性位点的RFLP分析的gRNA间隔区。

  2. 构建gRNA质粒
    为了驱动植物中的gRNA表达,我们构建了靶向铌磷酸酶的gRNA质粒。任何其他感兴趣的基因也可以被靶向。为了容易地克隆gRNA,我们首先使用了一个小载体(T-U6p-scaffold-U6t)。然后,将启动子-GRNA终止子构建体转移到较大的二进制载体,例如pCAMBIA2300和pCVA。
    1. 进行反向PCR(Stemmer和Morris,1992),以在Cas9结合所需的AtU6启动子和gRNA支架之间添加gRNA间隔(图1)。设置反应如下:



      图1. pT-U6p-scaffold-U6t (Yin等人,2015)的示意图,以及使用反向PCR将gRNA间隔区插入pT- U6p-scaffold-U6t产生pT-U6p-gRNA-scaffold-U6t。在AtU6启动子,支架和AtU6终止子上显示橙色盒子在质粒图上。用于插入gRNA间隔区(靶向NbPDS )的两个引物在质粒图上用它们各自的方向表示。引物上的平行黑线是互补序列。 我和Pst 我用于切割gRNA表达盒,然后将其克隆到二元载体中。这里的小箭头是指示脚手架。 B.反向PCR的序列信息。这里,以gPN间隔物靶向为NbPDS 为例。 NbPDS 轨迹的部分序列显示为黑色。 PAM和Mly I网站已显示。 gRNA间隔区也显示在NbPDS基因座上方的黑色上。显示用于PCR的两个引物(oYK1056和oYK1057),来自AtU6的序列以红色显示,并且来自支架的序列显示为蓝色。在PCR过程中结合模板(载体)的两个引物也用引物和载体表示。反向PCR后的变化如箭头所示。

    2. 使用以下参数进行PCR扩增(T 表示引物对的退火温度):


    3. 设置限制酶消化以除去上述PCR产物中的质粒模板,如下所示:
      注意:DpnI是甲基化敏感酶,从大肠杆菌DH5α纯化的质粒通常被甲基化。因此,DpnI通常用于去除质粒模板。在这里,连接或变性/再退火步骤不需要连接载体的末端,因为具有重叠末端的PCR产物将重新循环以形成双重切割的质粒并将在细菌中进一步修复以形成完整质粒。


      混匀,37℃孵育2小时。
    4. 转化大肠杆菌DH5α,使用上述反应的1-2μl产物,在42℃热休克90秒,在37℃回收1小时,在LB板上用100mg/L氨苄青霉素,然后在37℃下孵育过夜(Im,2011)。
    5. 选择一至五个转化体,并在含有100mg/L氨苄青霉素的LB中在37℃,220rpm下培养过夜。分别用TIANprep Mini Plasmid Kit提取质粒,并用测序引物M13-47 Sanger序列质粒pT-U6p-gRNA:NbPDS -scaffold-U6t,证实gRNA间隔物的存在。

  3. 使用fsGUS(移动GUS)记者系统检查Cas9活性和gRNA效率 有必要确认KQ334植物是否保留Cas9活性,Cas9在gRNA的指导下切割DNA。在该系统中,末端具有PAM(AGG)的23bp序列被插入到GUS基因的起始密码子的下游,这导致GUS 基因的帧位移>基因( fsGUS )。我们共同表达了两个载体:一个携带被称为pJG081-fsGUS的35S启动子的驱动的 GUS 基因,另一个含有拟南芥U6-26基因的p2300-gRNA:fsGUS启动子(AtU6启动子) - 包含与插入到基因中的序列的前20bp匹配的20bp序列的驱动的gRNA。
    基于原生质体转染检测gRNA效率的另一种高效方法。请参阅李和同事协议(Li等等,2015年) 
    1. 设置PCR反应以扩增其中在GUS起始密码子(图2)之后插入含有gRNA间隔区序列和MlyI识别位点的短序列的fsGUS 片段。在此步骤中使用的引物应特别设计用于侧接不同gRNA的切割位点。



      图2.用于检查gRNA效率的fsGUS记者系统的图示。 fsGUS记者系统的插图。含有Mly I位点的23bp序列被插入到GUS 基因的起始密码子之后,并将得到的序列指定为fsGUS 。由于插入的序列,fsGUS 不能翻译成功能酶。 fsGUS 由CaMV35S启动子驱动并置于二进制载体中。另一个二进制载体包含启动子驱动的gRNA,其靶向插入序列在fsGUS 中的前20bp。两个。将每种二元载体转化的根瘤农杆菌线混合在一起并渗透到KQ334植物叶中。两个T-DNA进入相同的细胞后,fsGUS 中插入的序列的前20bp被Cas9/gRNA复合物靶向用于切割。通过NHEJ修复将在切割位点产生内含子,并且在某些情况下恢复fsGUS 的正常编码框架,从而产生可通过GUS染色检测的功能性GUS。 (这个数字是从尹等人,2015年改编和修改的),

      使用以下参数进行PCR扩增(T 表示引物对的退火温度):


    2. 使用PEG/MgCl 2方法纯化PCR产物(Paithankar和Prasad,1991)
      1. 向50μlPCR产物中加入50μl水,然后加入50μl30%PEG8000/30mM MgCl 2。
      2. 混匀并立即以12,000 x g离心15分钟。
      3. 接下来,去除上清液并加入200μl70%乙醇。
      4. 以12,000 x g离心5分钟,取出上清液。最后,将颗粒干燥并将沉淀溶解于10μl无菌去离子水中
    3. 使用LIC方法构建pJG081-fsGUS。
      注意:LIC是一种快速,简单,非常便宜的克隆技术。它利用载体的互补突出端的退火和由9-15个碱基组成的PCR片段。
      1. 通过使用T4 DNA聚合酶和在反应缓冲液中加入仅一种dNTP产生单链突出端,导致在该核苷酸的第一次出现时聚合酶和外切核酸酶的活性平衡(Aslanidis和de Jong,1990)。为了产生单链尾,用T4 DNA聚合酶处理纯化的DNA制剂。对上述PCR产物进行T4 DNA聚合酶处理:


      2. 通过移液轻轻混匀。在37℃孵育20分钟,然后在75℃孵育20分钟,使用PCR循环仪使T4DNA聚合酶失活。得到的产品被指定为fsGUS-LIC。
    4. 摘要pJG081用于LIC反应:


      1. 在37℃孵育3小时后孵育。
      2. 然后,使用苯酚/氯仿法纯化产物:加入等体积的苯酚/氯仿(1:1),剧烈旋涡20秒;然后在室温下以12,000 x g离心10分钟;保留上清液并加入等体积的氯仿,剧烈旋转20秒,并在室温下以12,000×g离心10分钟;通过加入1/10体积的3M NaAc和2体积乙醇从上述提取物的水相中沉淀DNA。
      3. 最后,通过离心沉淀DNA,并用无菌去离子水溶解
    5. 为此线性pJG081设置T4 DNA聚合酶处理:


      得到的产品被指定为pJG081-LIC。
    6. 构造pJG081-fsGUS:
      1. 混合5μlfsGUS-LIC和5μlpJG081-LIC,在70℃下孵育10分钟,然后在22℃下孵育10分钟。
      2. 接下来,将该产品转换为E。大肠杆菌DH5α,然后在含有卡那霉素的培养基上选择
    7. 构建p2300-gRNA:fsGUS。
      1. 设置限制酶反应如下:


      2. 在37℃孵育3小时。然后,根据制造商的建议,用DNA纯化试剂盒纯化产物。
      3. 为了将完整的gRNA表达盒克隆到pCambia 2300中,从pT-U6p-gRNA:NbPDS -scaffold-U6t切割U6p-gRNA:NbPDS - 支架-U6t。设置限制酶反应如下:


      4. 在37℃孵育3小时。然后根据制造商的建议,用DNA纯化试剂盒纯化产物。
      5. 设置结扎反应如下:


      6. 在16℃下孵育8小时,并将其转化为E.大肠杆菌DH5α,然后在含有卡那霉素的培养基上选择
    8. 转基因农杆菌
      1. 通过热休克法(Höfgenand Willmitzer,1988)将pKQ334,pJG081-fsGUS和p2300-gRNA:fsGUS分别转化到根癌土壤杆菌菌株GV3101中。
      2. 在28℃的LB培养基上选择具有50mg L 卡那霉素和50mg L 利福平两天的转化体。
    9. 瞬态表达式
      注意:可以使用Cas9转基因植物(KQ334植物)或含有pKQ334质粒(Cas9的组成型表达)的另一种农杆菌培养物进行该实验。
      1. 将土壤杆菌培养物接种在含有适当抗生素(50mg L -1)利福平和50mg L -1 sup suprampilin的5ml Luria-Bertani培养基中>卡那霉素),并在28℃振荡器中生长过夜。
      2. 通过在5,000xgg离心5分钟收获土壤杆菌细胞,并重新悬浮于渗透缓冲液中,调节至600nm的光密度为1.0,并在室温下温育3至渗透前4 h。
      3. 对于瞬时表达,将等量的含有pKQ334或pJG081-fsGUS或p2300-gRNA:fsGUS的土壤杆菌菌株GV3101培养物作为实验组混合,并用六叶期植物的叶片点渗入1 ml注射器。作为对照,使用1-ml注射器将含有pJG081-fsGUS或p2300-gRNA:fsGUS的土壤杆菌培养物分别点入六叶期植物的叶中。
      4. 从每个植物顶部计数的前三个叶子被渗透;每个叶片用实验组和对照组进行点渗。浸润时避免中脉和主脉。
      5. 将1毫升注射器的针头按压到叶片的背面,将另一侧的戴手套的手指按压,并缓慢注射。渗透区域通常被视为"湿"(视频1)。 

        Video 1. Transient expression using agro-infiltration in N. benthamiana

        To play the video, you need to install a newer version of Adobe Flash Player.

        Get Adobe Flash Player


      6. 或者,将含有pJG081-fsGUS或p2300-gRNA:fsGUS的等量的土壤杆菌培养物可以混合在一起,并使用1ml注射器将其浸入六叶期KQ334植物的叶中(图2) 。作为对照,使用1-ml注射器将含有pJG081-fsGUS或p2300-gRNA:fsGUS的土壤杆菌培养物分别渗入六叶期KQ334植物的叶中。
    10. GUS染色
      GUS染色测定基于Kabbage等人。 (2011),执行情况如下:
      1. 渗透两到三天后,收集渗透叶。真空将X-gluc底物浸入收集的叶中。使用玻璃/塑料烧杯将叶子置于真空浸渗中,并施加600mm Hg的真空度(3分钟)并释放数次直到叶片变得透明。
      2. 渗透后,将叶子在黑暗中室温孵育过夜。
      3. 通过用去离子水改变溶液冲洗叶子并通过将叶子浸入70%乙醇中至少一天来除去叶绿素。

  4. 构建pCVA-gRNA和pCVA支架
    为了进行靶基因的VIGE,首先通过将含有AtU6启动子和gRNA :: NbPDS的盒插入pCVA来产生pCVA-gRNA。将构建体pCVA-支架作为阴性对照。
    1. 设置PCR反应如下:


    2. 使用以下参数进行PCR扩增(T 表示引物对的退火温度):


    3. 然后根据制造商的建议,用DNA纯化试剂盒纯化产品。
    4. 设置限制酶反应如下:


    5. 在37℃孵育3小时。然后用DNA纯化试剂盒纯化产物。最终产品是U6p-gRNA-scaffold_Kpn I/Xba I和U6p-scaffold_Kpn I/Xba I.
    6. 同时设置限制酶反应如下:


    7. 在37℃孵育3小时。然后根据制造商的建议,用DNA纯化试剂盒纯化产物。
    8. 接下来,如下设置连接反应产生pCVA-gRNA和pCVA-支架


    9. 在16℃下孵育8小时,并将其转化为E.大肠杆菌DH5α,然后在含有卡那霉素的培养基上选择
  5. 感兴趣的基因/位点的VIGE
    1. 首先,通过热休克法将pCVA-gRNA,pCVA-支架和pCVB分别转化到根癌土壤杆菌菌株GV3101中。用卡那霉素和利福平选择中等转化体
    2. 接下来,将农杆菌培养物接种到含有合适抗生素(50mg L -1,利福平,50mg L -1)的5ml Luria-Bertani培养基中, sup>卡那霉素),并在28℃振荡器中生长过夜。 
    3. 通过在5,000xgg离心5分钟收获土壤杆菌细胞,并重新悬浮于渗透缓冲液中,调节至600nm的光密度为1.0,并在室温下温育3至渗透前4 h
    4. 将含有pCVB和pCVA或其衍生物的农杆菌培养物以1:1的比例混合,并使用1ml注射器渗透入6至8叶期KQ334植物的叶柄(Tang等人,2010 )(图3和视频2)。作为对照,它们也使用相同的方法渗透入6至8叶期野生型植物的叶柄。


      图3. VIGE的图示含有pCVA-gRNA或pCVB的土壤杆菌彼此混合并渗透到KQ334植物(Cas9转基因植物)的叶柄中。 系统叶用于检查基因组编辑事件。 使用AtU6启动子来驱动gRNA的表达。 gRNA间隔区显示为红色。 CR,公共区域; AL1,复制相关蛋白; AL2,转录激活因子; AL3,复制增强子; AL4,推定发病相关蛋白; BL1和BR1,运动蛋白; LB,左边界; RB,右边界。 (这个数字是从尹等人,2015年改编和修改的),

      Video 2. Agro-infiltration into petiole of N. benthamiana

      To play the video, you need to install a newer version of Adobe Flash Player.

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  6. 检测植物基因组DNA中VIGE介导的突变
    1. 为了检测VIGE结果,根据制造商的说明书从系统叶提取的基因组DNA用作使用EasyTaq DNA聚合酶在靶位点侧翼的引物(PDS_MlyIF和PDS_MlyIR)的靶向位点的PCR扩增(见下文)的模板。将纯化的产物纯化并用选择性的限制性内切酶(即,用于NbPDS的Mly I)消化,并在2%琼脂糖凝胶上运行。
    2. 通过使用ImageJ比较带强度估计突变率( https://imagej .nih.gov/ij/index/html )。然后从凝胶切割未切割的条带,并根据制造商的说明书使用DNA纯化试剂盒回收DNA。未消化的纯化DNA随后克隆到pMD18-T载体(TAKARA)中并转化到E中。大肠杆菌DH5α。用M13-47引物对克隆进行测序以检测突变(图3)。 NbPDS基因的VIGE的一个例子显示于图4中,其中靶向于NbPDS基因的特异性引物对的PCR产物用MlyI /我>我
      使用以下参数进行PCR扩增(T 表示引物对的退火温度):



      图4. NbPDS的VIGE 。 A. VIGE靶向基因的说明NbPDS 。 Cas9/gRNA的切割位点处于红色剪刀下。 Mly 我用于区分编辑和野生型序列。 B.用于估计NbPDS基因VIGE的编辑效率的凝胶测定示意图。

数据分析

为了研究是否编辑了内源性的NbPDS基因,ImageJ用于评估VIGE的编辑效率。

  1. 选择用于分析的泳道,并添加框架以包含ImageJ的乐队( https://imagej.nih.gov/ij/index/html )。
  2. 用ImageJ测量每个泳道中每个乐队的强度。
  3. 相对编辑效率通过将未切割频带的强度除以所有频带的总强度来计算

食谱

  1. Luria-Bertani培养基
    10g/L胰蛋白胨
    5克/升酵母提取物
    10g/L NaCl
    将pH调节至7,在121℃高压灭菌20分钟,并在室温下储存
  2. MgCl 2(1M储备)
    将203.3g MgCl 2·6H 2 O溶解在800ml去离子H 2 O 2 / 用去离子水调节体积至1升
  3. MES(0.5M库存)
    将19.52 g MES溶解于200 ml去离子水中 用1N KOH调节至pH 5.6
  4. Acetosyringone(1 M库存)
    将1.962 g乙酰丁香酮溶于10ml DMSO中
  5. 渗透缓冲液
    注意:上面的库存缓冲液(配方2-4)用0.22μm过滤器过滤灭菌,并在室温下储存。每次新鲜地制备渗透缓冲液。
    10mM MgCl 2
    10 mM MES
    200μM乙酰丁香酮
  6. 2x磷酸盐缓冲液(pH 7)
    0.2M NaH 2 PO 4
    0.2M Na 2 HPO 4
    1. 首先,做出两个解决方案:
      溶液A:在1L去离子水中溶解55.2g NaH 2 PO 4·2H 2 O / 溶液B:在1L去离子水中溶解107.3g Na 2 HPO 4·7H 2 O·
    2. 在121℃高压灭菌20分钟,并将其储存在室温下
    3. 为了制备200ml 2x磷酸盐缓冲液(pH7),加入39.0ml溶液A和61.0ml溶液B,然后用去离子水稀释至200ml
  7. X-gluc底物溶液(Kabbage等人,2011)
    1. 在0.1ml甲醇中加入1mg X-gluc
    2. 然后加入1ml 2x磷酸盐缓冲液,20μl0.1M铁氰化钾,10μl10%Triton X-100和850μl去离子水
  8. PEG-MgCl 2 溶液
    1. 将30g PEG 8000溶于100ml 30mM MgCl 2
    2. 用0.22μm过滤器过滤消毒溶液,并将其储存在室温

致谢

VIGE方法是基于尹等人的报告。 (2015)。接种CaLCuV的方法是基于Tang等人的。 (2010)。 GUS染色测定基于Kabbage等人。 (2011)。这项工作得到了中国国家基础研究计划(2014CB138400),中国全国转基因项目(2014ZX0800104B,2014ZX08010-002,2014ZX08005-001)和国家自然科学基金(31421001,3142100007)的支持。我们感谢北卡罗莱纳大学教堂山大学的爱丽丝·俞校对校对手稿。

参考文献

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引用:Yin, K., Han, T. and Liu, Y. (2017). Use of Geminivirus for Delivery of CRISPR/Cas9 Components to Tobacco by Agro-infiltration. Bio-protocol 7(7): e2209. DOI: 10.21769/BioProtoc.2209.
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