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Multiplexed GuideRNA-expression to Efficiently Mutagenize Multiple Loci in Arabidopsis by CRISPR-Cas9
多重向导RNA表达通过CRISPR Cas9对拟南芥中的多个基因座进行高效诱变   

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Abstract

Since the discovery of the CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein (Cas) as an efficient tool for genome editing in plants (Li et al., 2013; Shan et al., 2013; Nekrasov et al., 2013), a large variety of applications, such as gene knock-out, knock-in or transcriptional regulation, has been published. So far, the generation of multiple mutants in plants involved tedious crossing or mutagenesis followed by time-consuming screening of huge populations and the use of the Cas9-system appeared a promising method to overcome these issues. We designed a binary vector that combines both the coding sequence of the codon optimized Streptococcus pyogenes Cas9 nuclease under the control of the Arabidopsis thaliana UBIQUITIN10 (UBQ10)-promoter and guide RNA (gRNA) expression cassettes driven by the A. thaliana U6-promoter for efficient multiplex editing in Arabidopsis (Yan et al., 2016). Here, we describe a step-by-step protocol to cost-efficiently generate the binary vector containing multiple gRNAs and the Cas9 nuclease based on classic cloning procedure.

Keywords: CRISPR-Cas9(CRISPR-Cas9), Multiplexing(多重), gRNA(gRNA), Gene editing(基因编辑), Arabidopsis(拟南芥)

Background

The RNA-guided Cas9-system is derived from the bacterial defense system against foreign DNA (Sorek et al., 2013). It has been recognized as a method of choice for genome editing because of its high efficiency, easy handling and possibility of multiplex editing. In general, the Cas9-gene editing system involves a single synthetic RNA molecule, the gRNA that directs the Cas9 protein to target the desired DNA site for genome modification or transcriptional control. The gRNA-Cas9 complex recognizes the targeted DNA by gRNA-DNA pairing and requires the presence of a protospacer-adjacent motif (PAM). The PAM is represented by the nucleotides NGG or less specific NAG (with N for any nucleotide) in the target site following the gRNA-DNA pairing region. Thus, the approximate 20 nucleotides long gRNA spacer sequence, i.e., the part of the gRNA sequence complementary to the DNA target site, determines the specificity of the complex. In this protocol, we describe the details to generate a binary vector that contains both the gRNA and the Cas9 coding sequence by classic cloning (Figure 1). As our vector system allows for subsequent addition of further gRNAs, it can be used for multiplex editing of the Arabidopsis genome and to obtain multiple, stably inherited alleles. Strong expression of the Cas9 protein and the gRNAs especially in proliferating tissues is achieved by the use of the A. thaliana UBQ10- and the U6-promoter, respectively. First mutations can be detected in the T1 generation, and T-DNA- and Cas9-free mutant plants may already be selected in the T2 generation. Besides the selection of the gRNA and the construction of the plasmid, we give an overview of efficient genotyping methods required for detection of small or large deletions.


Figure 1. Scheme of the cloning procedure described in the protocol. Only a series of restriction and ligation steps is required to obtain a plant transformation vector equipped with a set of AtU6-driven gRNAs and the Cas9 enzyme under the control of the UBQ10 promoter. Restriction enzyme cutting sites are displayed in italics. Arrows on vectors indicate primer binding sites. TDNA-R and TDNA-L point out T-DNA right and left borders, respectively. (B) and (P) indicate selection markers for selection in bacteria or plants, respectively.

Materials and Reagents

Note: The protocol described here is based on the RNA-guided Cas9 system which was published recently (Yan et al., 2016). Only classical cloning methods such as restriction enzyme digestion and cohesive end ligation are required to construct plasmids ready for plant transformation.

  1. Consumables
    1. 200 µl PCR tubes (Kisker Biotech, catalog number: G003-SF )
      or 96-well PCR plates (SARSTEDT, catalog number: 72.1978.202 )
      StarSeal sealing tape (STARLAB, catalog number: E2796-9793 )
    2. 1.5 ml reaction tubes (SARSTEDT, catalog number: 72.690.001 )
    3. Pipette tips  
    4. Scalpel
    5. Petri dishes, round, 9.2 x 1.6 cm (SARSTEDT, catalog number: 82.1472.001 )
    6. Petri dishes, square, 10 x 10 x 2 cm (SARSTEDT, catalog number: 82.9923.422 )
    7. Cuvettes for electroporation, e.g., Gene Pulser® cuvette 0.1 cm (Bio-Rad Laboratories, catalog number: 1652089 )
    8. 5 ml glass pipette
    9. MicroporeTM tape (VWR, catalog number: 115-8172 )
      Note: Any appropriate consumable can be used.
  2. Competent cells
    1. One Shot® TOP10 chemically competent Escherichia coli (Thermo Fisher Scientific, InvitrogenTM, catalog number: C4040 )
    2. Electro-competent Agrobacterium tumefaciens, strain pGV3101 (for preparation of electro-competent Agrobacteria, please see Mersereau et al., 1990)
  3. Plant material
    1. Arabidopsis thaliana Col-0
  4. Plasmids
    1. AtU6-26-V4 (3.5 kb, ampicillin resistance marker [AmpR], available on request)
    2. UBQ10::pcoCas9p1300 (14.3 kb, kanamycin resistance marker [KanR] in bacteria, hygromycin or kanamycin resistance marker [HygR or KanR] in plants, available on request)
    3. Optional: pGEM®-T easy (Promega, catalog number: A3600 )
  5. Enzymes and buffers
    Restriction
    1. BpiI (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: ER1011 )
    2. KpnI-HF (New England Biolabs, catalog number: R3142 )
    3. XbaI (New England Biolabs, catalog number: R0145 )
    4. SpeI-HF (New England Biolabs, catalog number: R3133 )
    5. SbfI-HF (New England Biolabs, catalog number: R3642 )
    6. Cutsmart® buffer (New England Biolabs, catalog number: B7204S , supplied with the enzyme)
    Ligation
    1. T4 DNA ligase (New England Biolabs, catalog number: M0202 )
    2. T4 DNA ligase reaction buffer (New England Biolabs, catalog number: B0202S , supplied with the enzyme)
    Genotyping (bacteria)
    1. Green Taq DNA polymerase (GenScript, catalog number: E00043 )
    2. 10x Taq buffer (GenScript, catalog number: B0005 , supplied with the enzyme)
    3. 10 mM dNTPs (Carl Roth, catalog number: K039 )
    4. Any DNA-loading dye, e.g., 6x gel loading dye, purple (New England Biolabs, catalog number: B7024 )
    5. Any DNA-size standard, e.g., GeneRulerTM 1 kb Plus DNA ladder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: SM1331 )
    6. Agarose (Carl Roth, catalog number: 3810 )
    7. 10x TBE electrophoresis buffer (see Recipes)
      Tris (Applichem, catalog number: A1379 )
      Boric acid (Applichem, catalog number: 131015 )
      EDTA (Applichem, catalog number: 131669 )
  6. Deionized water (sterile)
  7. Antibiotics (store stocks at -20 °C)
    1. Ampicillin (Carl Roth, catalog number: K029 , stock 50 mg/ml in deionized water)
    2. Kanamycin (Carl Roth, catalog number: T832 , stock 30 mg/ml in deionized water)
    3. Rifampicin (Applichem, catalog number: A2220 , stock 25 mg/ml in DMSO)
    4. Gentamycin (Carl Roth, catalog number: 0233 , stock 10 mg/ml in deionized water)
    5. Hygromycin (Carl Roth, catalog number: CP13 , stock 10 mg/ml in deionized water)
  8. Media (see Recipes)
    1. YEB medium for Agrobacterium
      Meat extract (Carl Roth, catalog number: 5770 )
      Yeast extract (Carl Roth, catalog number: 2904 )
      Peptone (Sigma-Aldrich, catalog number: 82303 )
      Sucrose (Carl Roth, catalog number: 4621 )
      Magnesium sulfate (MgSO4) (Carl Roth, catalog number: P027 )
      Bacto-agar (Th. Geyer, CHEMSOLUTE®, catalog number: 9914-500G )
      Sodium hydroxide (NaOH) (Merck, catalog number: 28245 )
    2. YT medium for E. coli
      Sodium chloride (NaCl) (Carl Roth, catalog number: 9265 )
      Yeast extract (Carl Roth, catalog number: 2904)
      Peptone (Sigma-Aldrich, catalog number: 82303)
      Bacto-agar (Th. Geyer, CHEMSOLUTE®, catalog number: 9914-500G)
      Sodium hydroxide (NaOH) (Merck, catalog number: 28245)
    3. ½ MS for Arabidopsis
      MS + B5 Vitamins (Duchefa Biochemie, catalog number: M0231 )
      Potassium hydroxide (KOH) (Merck Millipore, catalog number: 105033 )
  9. DNA purification
    1. Gel-purification, e.g., NucleoSpin® Gel and PCR clean-up (MACHEREY-NAGEL, catalog number: 740609 )
    2. Plasmid isolation, e.g., NucleoSpin® Plasmid EasyPure (MACHEREY-NAGEL, catalog number: 740727 )
  10. Silwet L-77 (Lehle seeds, catalog number: VIS-30 ) for plant transformation
  11. Bleach (Carl Roth, catalog number: 9062 )
  12. 37% HCl (Merck Millipore, catalog number: 100317 )
  13. Genotyping (plants)
    1. Optional: Phire Plant Direct PCR Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: F-130WH )
    2. Optional: T7 endonuclease I (New England Biolabs, catalog number: M0302 ) plus NEBuffer 2 (New England Biolabs, catalog number: B7002 ), 250 mM EDTA
    3. Optional: pGEM®-T easy (Promega, catalog number: A3600)
  14. Oligonucleotides (5’-3’), 10 pmol/µl
    1. M13F_TGTAAAACGACGGCCAGT
    2. M13R_CAGGAAACAGCTATGACC
    3. K197 _CTGTTAATCAGAAAAACTCAG

Equipment

Note: No specific equipment is required. Any appropriate device can be used.

  1. Computer with internet access
  2. Thermocycler (e.g., Eppendorf, model: Mastercycler® nexus )
  3. Thermoblock (e.g., Eppendorf, model: Thermomixer® comfort )
  4. Plate incubators (28 °C, 37 °C)
  5. Shakers (28 °C, 37 °C)
  6. Horizontal gel-electrophoresis system (e.g., Bio-Rad Laboratories, model: Mini-Sub® Cell GT System )
  7. UV transilluminator (e.g., Bio-Rad Laboratories, model: GelDocTM XR+ System )
  8. Electroporator, (e.g., Bio-Rad Laboratories, model: MicroPulserTM Electroporator )
  9. 10 L desiccator
  10. Fume hood
  11. 200 ml beaker

Procedure

  1. Preparation of gRNA-spacers with CRISPR-PLANT© (Xie et al., 2014) and primer design (Figure 1I)
    1. Go to http://www.genome.arizona.edu/crispr/index.html.
    2. Select ‘Search’.
    3. Select ‘Select Species’ and choose ‘Arabidopsis thaliana’.
    4. Select ‘Chromosome’ and enter the coordinates of the target region in ‘From’ and ‘To’.
    5. Confirm with ‘Search by region’.
      You will be returned Class 0.0 and Class 1.0 gRNAs. Class 0.0 gRNAs are supposed to be more specific. The SeqID is the coordinate of the gRNA in your predefined region. The minMM_GG (and minMM_AG) value refers to the minimum amount of mismatches the 12 bp sequence adjacent to the PAM motif has to any other sequence in the A. thaliana genome. The higher the value the smaller is the risk for off-targeting. Therefore, sequences with a minMM_GG higher than three should be considered.
    6. Copy the optimal 20 bp long spacer sequence. Here, X represents any nucleotide.
      Forward sequence: 5’-XXXXXXXXXXXXXXXXXXXX-3’
      Reverse sequence: 5’-XXXXXXXXXXXXXXXXXXXX-3’
      As the U6-promoter drives the expression of the gRNA, the initiation of transcription requires a guanine (G) as the transcription start site. If the chosen spacer sequence does not start with a ‘G’, change the first base. This will have no considerable effects on the specificity of Cas9 as the specificity largely depends on the 12 bp adjacent to the PAM motif.
      Forward sequence: 5’-GXXXXXXXXXXXXXXXXXXX-3’
      Reverse sequence: 5’-XXXXXXXXXXXXXXXXXXXC-3’
    7. Add ‘GATT’ and ‘AAAC’ to the 5’-ends of the forward and reverse sequences, respectively, to generate BpiI-compatible overhangs.
      Forward primer: 5’-GATTGXXXXXXXXXXXXXXXXXXX-3’
      Reverse primer: 5’-AAACXXXXXXXXXXXXXXXXXXXC-3’
    8. Now you have a complementary primer pair ready to order.
  2. Anneal the gRNA-primers (Figure 1II)
    1. Mix 10 µl of each primer (10 pmol/µl). Denature primers for 3 min at 98 °C. Hybridize primers in a thermocycler by decreasing the temperature from 98 °C to 22 °C. Pause at each degree for 30 sec.
  3. Digest the AtU6-26-V4-vector with BpiI according to manufacturer’s instructions (Figure 1III)
    1. Mix the digest with DNA-loading dye and load it on a 1% agarose gel. Separate digested from undigested bands by gel electrophoresis.
    2. Purify the linear vector backbone (3,517 bp) from gel.
  4. Ligate the annealed gRNA-primers and the BpiI-digested AtU6-26-V4-vector in a 10 µl ligation reaction (Figure 1IV).
    1. Mix 25 ng to 50 ng vector, 1 µl of 10x T4 DNA ligase reaction buffer and 0.5 µl T4 DNA ligase (200 U). Fill the rest of the volume with the hybridized primer.
    2. Incubate the reaction for 10 min at room temperature.
      Note: Use 4 °C overnight incubation if you encounter problems getting positive transformants in step 6.
  5. Use 3 µl of the ligation reaction to transform chemically competent TOP10 E. coli cells.
    1. Thaw 50 µl competent cells on ice.
    2. Add 3 µl of the ligation reaction to the cells.
    3. Incubate on ice for 15 min.
    4. Heat shock the cells for 45 sec at 42 °C in a thermoblock and place back on ice for 2 min.
    5. Add 400 µl of YT medium and incubate the cells in a thermoblock for 60 min at 37 °C and 750 rpm.
    6. Plate 60 to 80 µl of the transformation on solid YT medium with 50 µg/ml ampicillin (YT-amp).
  6. Do a colony-PCR to genotype transformants. Usually testing 5 to 10 colonies is sufficient to obtain positive transformants.
    1. Set up a 20 µl PCR reaction per colony as follows. Final concentrations are indicated in brackets:
      2 µl 10x Taq buffer (1x)
      0.6 µl M13F (0.5 µM)
      0.6 µl gRNA-specific reverse primer (0.5 µM)
      0.4 µl dNTPs (0.2 mM)
      0.1 µl Green Taq DNA polymerase (0.5 U)
      16.3 µl deionized water
    2. Use a pipette tip to pick a colony and restreak the cells on an YT-amp plate. Directly afterwards dip the tip into the PCR reaction to transfer a small amount of cells. Incubate the plate at 37 °C for approximately 5 h and run the PCR in the thermocycler using the following conditions:

      Note: The annealing temperature depends on the gRNA-specific reverse primer. M13F works well within the range of 54 °C to 60 °C during annealing. Instead of M13F and the gRNA-specific reverse primer you can also use M13R and the gRNA-specific forward primer with an elongation time of 20 sec.
    3. Add 4 µl of 6x DNA loading dye to the reaction and load it on a 1% agarose gel. The expected amplicon size is 581 bp for the PCR reaction using M13F and the gRNA-specific reverse primer or 257 bp for the reaction with M13R and the gRNA-specific forward primer.
    4. Prepare 3 ml of YT-amp and pick the PCR-positive clones from the restreak plate. Incubate over night at 37 °C with 190 rpm.
    5. Isolate the plasmid from positive clones and check for the correct insert by Sanger sequencing using M13R as sequencing primer.
  7. Optional (for multiplexing):
    If more than one gRNA is needed, prepare each gRNA in a separate AtU6-26-V4-vector first (gRNA1-AtU6-26-V4, gRNA2-AtU6-26-V4, etc.) and combine them into one single gRNA2-gRNA1-AtU6-26-V4-vector.
    1. Cut gRNA1-AtU6-26-V4 open with KpnI-HF and SpeI-HF (3,519 bp, Figure 1V).
    2. Cut gRNA2 out of gRNA2-AtU6-26-V4 using KpnI-HF and XbaI (643 bp, Figure 1VI).
    3. Load reactions on gel, gel purify positive bands.
    4. Ligate KpnI-gRNA2-XbaI and SpeI-gRNA1-AtU6-26-V4-KpnI (XbaI and SpeI produce compatible ends, Figure 1VII).
    5. Transform TOP10 E. coli cells, select transformants on YT-amp plates.
    6. Genotype clones with M13F and a specific reverse primer for the last added gRNA (amplicon size = 581 bp).
    7. Isolate plasmids and sequence with M13R.
      Repeat this step to add more gRNAs.
  8. Digest gRNA2-gRNA1-AtU6-26-V4 with KpnI-HF and SbfI-HF according to manufacturer’s instructions. With this step, you will cut out the gRNA2-gRNA1 unit including U6-promoters (Figure 1VIII).
    1. Mix the digest with DNA-loading dye and load it on a 1% agarose gel. Separate digested from undigested bands by gel electrophoresis.
    2. Purify the gRNA-fragment (660 bp for a single gRNA-fragment, 1,287 bp for a double gRNA-fragment) from gel.
  9. Digest UBQ10::pcoCas9p1300 with KpnI-HF and SbfI-HF according to manufacturer’s instructions to open the vector (Figure 1IX).
    1. Mix the digest with DNA-loading dye and load it onto a 1% agarose gel. Separate digested from undigested bands by gel electrophoresis.
    2. Purify the linearized vector backbone (14.3 kb) from gel.
  10. Ligate the gRNA2-gRNA1 unit and UBQ10::pcoCas9p1300 in a 20 µl reaction (Figure 1X).
    1. Mix 25 ng to 50 ng vector, 10 ng to 20 ng gRNA2-gRNA1, respectively, 2 µl of 10x T4 DNA ligase reaction buffer, deionized water ad 19 µl, and 1 µl T4 DNA ligase (400 U).
    2. Incubate the reaction at 4 °C overnight.
  11. Use 3 µl of the ligation reaction to transform chemically competent TOP10 E. coli cells. Select transformants on YT plates with 30 µg/ml kanamycin.
  12. Do a colony-PCR to genotype transformants as described in step 6.
    1. Genotype clones with K197 and a specific forward primer for the first added gRNA (here gRNA1, product size 801 bp).
    2. Isolate and sequence plasmids with M13F (optional with K197).
  13. Use 50 ng AtU6::gRNA2-AtU6::gRNA1-UBQ10::pcoCas9p1300 to transform A. tumefaciens via electroporation.
    1. Thaw 50 µl competent A. tumefaciens on ice.
    2. Pre-cool the cuvette on ice.
    3. Add 50 ng final vector to the A. tumefaciens cells, transfer to the cuvette and place the cuvette in the electroporator.
    4. Choose the ‘Agr’ settings at the MicroPulserTM electroporator (Bio-Rad, 1 pulse at 2.2 kV) or equivalent settings to transform the cells.
    5. Add 1 ml of YEB medium and transfer into a fresh 1.5 ml reaction tube. Incubate at 28 °C and 750 rpm for 2 to 3 h.
    6. Plate 60 µl of the transformation and select on YEB medium with 30 µg/ml kanamycin, 100 µg/ml rifampicin and 25 µg/ml gentamycin at 28 °C for 2 days.
  14. Use the transformed A. tumefaciens to transform A. thaliana by floral dip with infiltration (Clough and Bent, 1998; Chen, 2011)
  15. Sterilize seeds, e.g., by vapor-phase sterilization.
    1. Transfer seeds to 1.5 ml reaction tubes.
    2. Place open tubes in a 10 L desiccator under the fume hood.
    3. Place a 200 ml beaker filled with 100 ml bleach (6%) into the desiccator.
    4. Use a glass pipette to add 3 ml HCl (37%) to the bleach and close desiccator lid quickly.
      Note: Careful: The vapor can chemically burn skin and eyes!
    5. Sterilize for 3 to 4 h.
  16. Sow seeds on ½ MS with 20 µg/ml hygromycin or kanamycin (depending on the resistance marker from the UBQ10::pcoCas9p1300 of your choice) to select for positive transformants. Seal plates with MicroporeTM tape. Stratify seeds in darkness at 4 °C for 3 days. Germinate seeds in long day conditions (22 °C, 16:8 h light:dark photoperiod) until transformants reach 4 true-leaf stage. Transplant seedlings to soil and continue growth in long day conditions.
  17. Genotype the transformants.
    As in the T1 generation the activity and efficiency of the Cas9 enzyme but also of the DNA repair mechanisms might differ from cell to cell, each plant most likely will be a mosaic of different genotypes. Therefore we suggest to pooling genotyping samples of one plant, such as pieces of cauline leaves from different branches. If a plant with the expected mutation was identified in T1, label and genotype the single branches as they can have different genotypes (Figures 2C and 2D). The method of genotyping depends on the anticipated results.
    1. Big fragment deletions which had been generated by multiple gRNAs, can be identified by PCR followed by gel electrophoresis of the PCR products (Figures 2A and 2B).
      Note: Design primers flanking the expected deletion in a distance of approximately 200 bp to 400 bp to each gRNA target site. For high throughput genotyping we suggest to prepare 10 µl PCR reactions using the Phire Plant Direct PCR Kit.


      Figure 2. Detection of big fragment deletions in the AP1 locus. A. The AP1 locus was targeted using two different gRNAs (scissors). Target sites are sequences in the third and sixth intron of AP1. B. Genotyping of a phenotypically wild type (WT) and an ap1-like (m) branch in the T1 generation revealed the presence of a 680 bp deletion in the AP1 locus in the ap1-like branch (m). C and D. Plants in the T1 generation can be a mosaic of different genotypes. Two different flower phenotypes, wild type (C, left; D, blue arrow) and ap1 (C, right; D, yellow arrow) can be found on two different branches of the same plant.

    2. Small deletions or polymorphisms that produce a minimum mismatch of 2 bp to the wild type allele can be detected by T7 endonuclease digestion (Vouillot et al., 2015).
      1. Perform the genotyping PCR as mentioned for big fragment deletions.
      2. Set up the hybridization reaction by adding 1.12 µl NEBuffer 2 to the PCR product without purification of the DNA. Prepare WT control and sample separately. When pooling different branches in T1, it is not necessary to add wild type PCR product to the sample as we usually do not expect Cas9 efficiencies to be higher than the T7 endonuclease detection limit (Vouillot et al., 2015). But if you already see a clear phenotype when genotyping single branches, you can replace 20% of the sample volume by wild type to not miss homozygous mutant branches.
      3. Hybridize PCR products using the following conditions:
      4. Add 0.1 µl of T7 endonuclease (1 U) and incubate for 30 min at 37 °C.
      5. Stop the reaction with 0.8 µl 250 mM EDTA.
      6. Separate cleavage products on a 2% agarose gel.
    3. Point mutations are not detected by T7 endonuclease digest efficiently (Vouillot et al., 2015) and the IDT® Surveyor® nuclease (Integrated DNA Technologies), which is supposed to cut at point mutations, appeared to be incompatible with the Phire Plant Direct PCR Mix. Considering the economic factors of high throughput mutant screening, we did not increase the amount of Surveyor® enzyme beyond manufacturer’s recommendations. Here, we suggest to using the T7 endonuclease assay to exclude plants with mutations of 2 bp or more. Then PCR products from the rest of the samples can be purified and sent for Sanger sequencing.
  18. Confirm the mutation by sequencing.
  19. Harvest the seeds from the branch that has a mutation.
  20. Sow the seeds to generate a small T2 population of approximately 100 individuals. Select the plant that carries the mutation but lost the T-DNA.
    1. To identify plants that still carry the transgene, use M13F and the gRNA-specific reverse primer (product size 557 bp). Exclude PCR-positive plants.
    2. Genotype for the expected mutation as described in step 17.
      Note: To verify small fragment deletions in T2 (Figure 3), prepare WT, sample and a 1:1 mix of WT and sample prior to hybridization. The WT-sample-mix is necessary as plants can be homozygous for the mutant allele. In that case, they do not produce heteroduplex DNA when hybridized and will not be digested by T7 endonuclease.


      Figure 3. Detection of small deletions or polymorphisms by T7 digest. PCR samples of T2 wild type (WT), mutant (m) and a 1:1 mix of wild type and mutant (WT+m) were hybridized. Half of the reaction volume was incubated without T7 endonuclease (undigested) and the other half was incubated with 1 U of the enzyme (T7 digest) and separated by gel electrophoresis on 2% agarose. The presence of cleavage products in the mutant sample is the result of at least two different alleles, which can be wild type and mutant or different mutant alleles or a mix of both options. As the addition of wild type to the mutant sample (WT+m) does not dilute the signal of the cleavage products, there are most likely different mutant alleles, which later was confirmed by sequencing (not shown). Asterisk indicates undigested PCR products, black arrows point out cleavage products. GR indicates the GeneRulerTM 1 kb Plus DNA ladder.
      Note: Once the protocol is set up, the ‘undigested’ controls can be omitted.

Data analysis

Mutations obtained in the T1 generation can be confirmed by Sanger sequencing as mentioned in step 17. But, as neighboring cells might harbor different mutations and as the mutation might be biallelic, we recommend to gel-purify the mutant PCR product and subclone it to pGEM®-T or any other cloning vectors. Sequence five to ten positive colonies to get an overview of gained mutations. After the selection of a T-DNA-free mutant plant in the T2 generation (step 19) confirm the presence and type of the mutation again by sequencing.
Note: PCR products generated by the Phire Plant Direct PCR Kit are blunt ended and require A-tailing before ligation with pGEM®-T.

Notes

This protocol is simple and straightforward. So far, more than 50 binary vectors containing different sets of gRNAs have been generated by our group and used for genome editing of Arabidopsis.

Recipes

  1. 10x TBE electrophoresis buffer (1 L)
    1 M Tris
    1 M boric acid
    0.02 M EDTA
    Deionized water ad 1,000 ml
    Dilute 10x TBE buffer with deionized water to 1:10 to use as gel running buffer
  2. YEB medium for Agrobacterium (1 L)
    5 g beef extract
    1 g yeast extract
    5 g peptone
    5 g sucrose
    500 mg MgSO4
    10 g Bacto-agar
    Deionized water ad 1,000 ml
    Adjust pH to 7.0 using NaOH
    Autoclave YEB medium at 121 °C and 2 bar for 15 min
  3. YT medium for E. coli (1 L)
    5 g NaCl
    5 g yeast extract
    8 g peptone
    (15 g Bacto-agar)
    Deionized water ad 1,000 ml
    Adjust pH to 7.0 using NaOH
    Autoclave YT medium at 121 °C and 2 bar for 15 min
  4. ½ MS for Arabidopsis (1 L)
    2.3 g MS + B5 Vitamins
    8 g Phyto-agar
    Deionized water ad 1,000 ml
    Adjust pH to 5.7 using KOH
    Autoclave ½ MS at 121 °C and 2 bar for 15 min
    Prepare sterile 50% sucrose (filter sterilize or autoclave separately)
    Add 1% sucrose to ½ MS prior to pouring the plates
    Note: The addition of sucrose is not required. The advantage is that seedlings recover better on selective media. The disadvantage is that it promotes fungal growth on the medium.

Acknowledgments

This protocol is based on our work previously published in Plant Methods (Yan et al., 2016). Work of J.S. was supported by the IMPRS-PMPG fellowship. K.K. wishes to thank the Alexander-von-Humboldt foundation and the BMBF for support. The authors declare that they have no competing interests.

References

  1. Chen, L. (2011). Simplified Agrobacterium-mediated transformation of Arabidopsis thaliana. Bio-protocol Bio101: e41.
  2. Clough, S. J. and Bent, A. F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16(6): 735-743.
  3. Li, J. F., Norville, J. E., Aach, J., McCormack, M., Zhang, D., Bush, J., Church, G. M. and Sheen, J. (2013). Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol 31(8): 688-691.
  4. Mersereau, M., Pazour, G. J. and Das, A. (1990). Efficient transformation of Agrobacterium tumefaciens by electroporation. Gene 90(1):149-51.
  5. 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.
  6. Shan, Q., Wang, Y., Li, J., Zhang, Y., Chen, K., Liang, Z., Zhang, K., Liu, J., Xi, J. J., Qiu, J. L. and Gao, C. (2013). Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol 31(8): 686-688.
  7. Sorek, R., Lawrence, C. M. and Wiedenheft, B. (2013). CRISPR-mediated adaptive immune systems in bacteria and archaea. Annu Rev Biochem 82: 237-266.
  8. Vouillot, L., Thelie, A. and Pollet, N. (2015). Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3 (Bethesda) 5(3): 407-415.
  9. Xie, K., Zhang, J. and Yang, Y. (2014). Genome-wide prediction of highly specific guide RNA spacers for CRISPR-Cas9-mediated genome editing in model plants and major crops. Mol Plant 7(5): 923-926.
  10. Yan, W., Chen, D. and Kaufmann, K. (2016). Efficient multiplex mutagenesis by RNA-guided Cas9 and its use in the characterization of regulatory elements in the AGAMOUS gene. Plant Methods 12: 23.

简介

自从发现CRISPR(聚集的定期交织的短回文重复) - 相关蛋白(Cas)作为植物基因组编辑的有效工具(Li等人,2013; Shan等人已经出版了诸如基因敲除,敲入或转录调控等各种各样的应用,例如,2013; Nekrasov等人,2013)。到目前为止,植物中多种突变体的产生涉及繁琐的杂交或诱变,随后大量人群的耗时筛选,Cas9系统的使用似乎是有希望的方法来克服这些问题。我们设计了一种二元载体,其结合了在拟南芥UBIQUITIN10(UBQ10)启动子和引导RNA(gRNA)控制下的优化的化脓性链球菌(Caspase)密码子的编码序列)由 A驱动的表现盒。拟南芥U6 - 启动子,用于在拟南芥中进行有效的多重编辑(阎等人,2016年)。在这里,我们描述了一个逐步的方案,以经济有效的方式生成含有多个gRNA的二元载体和基于经典克隆方法的Cas9核酸酶。

背景 RNA引导的Cas9系统源于针对外源DNA的细菌防御系统(Sorek等人,2013)。由于其高效率,易于处理和多重编辑的可能性,已经被认为是基因组编辑的选择方法。通常,Cas9基因编辑系统涉及单个合成RNA分子,其指导Cas9蛋白质靶向所需DNA位点以进行基因组修饰或转录控制的gRNA。 gRNA-Cas9复合物通过gRNA-DNA配对识别靶向的DNA,并需要存在原始相邻基序(PAM)。 PAM由在gRNA-DNA配对区后面的靶位点中的核苷酸NGG或更少的特异性NAG(任何核苷酸为N)表示。因此,大约20个核苷酸长的gRNA间隔区序列,即与DNA靶位点互补的gRNA序列的部分决定了复合物的特异性。在该协议中,我们描述了通过经典克隆生成包含gRNA和Cas9编码序列的二元载体的细节(图1)。由于我们的载体系统允许随后添加进一步的gRNA,因此可用于多重编码拟南芥基因组并获得多个稳定遗传的等位基因。 Cas9蛋白和gRNA特别是增殖组织中的强表达可以通过使用A实现。 thaliana UBQ10 - 和 U6 - 启动器。可以在T1代中检测到第一个突变,而在T2代可能已经选择了T-DNA和Cas9的突变植物。除了选择gRNA和构建质粒外,我们还概述了检测小或大缺失所需的有效基因分型方法。


图1.在方案中描述的克隆程序方案 仅需要一系列限制和连接步骤来获得装备有一组AtU6的植物转化载体 - 驱动的gRNA和在UBQ10启动子控制下的Cas9酶。限制酶切位点用斜体显示。载体上的箭头表示引物结合位点。 TDNA-R和TDNA-L分别指出了T-DNA的左右边界。 (B)和(P)分别表示在细菌或植物中选择的选择标记。

关键字:CRISPR-Cas9, 多重, gRNA, 基因编辑, 拟南芥

材料和试剂

注意:这里描述的协议基于最近出版的RNA引导的Cas9系统( Yan et al。,2016 )。只有经典的克隆方法,如限制性内切酶消化和内聚端连接才能构建准备进行植物转化的质粒。

  1. 消耗品
    1. 200μlPCR管(Kisker Biotech,目录号:G003-SF)
      或96孔PCR板(SARSTEDT,目录号:72.1978.202)
      StarSeal密封带(STARLAB,目录号:E2796-9793)
    2. 1.5ml反应管(SARSTEDT,目录号:72.690.001)
    3. 移液器提示
    4. Scalpel
    5. 圆形9.21厘米(SARSTEDT,目录号:82.1472.001)
    6. 培养皿,方形,10 x 10 x 2厘米(SARSTEDT,目录号:82.9923.422)
    7. 用于电穿孔的比色皿,例如,Gene Pulser 比色杯0.1cm(Bio-Rad Laboratories,目录号:1652089)
    8. 5毫升玻璃移液器
    9. Micropore TM 磁带(VWR,目录号:115-8172)
      注意:可以使用任何适当的耗材。
  2. 主管单元格
    1. (Thermo Fisher Scientific,Invitrogen TM,目录号:C4040)
    2. 电生物根癌土壤杆菌,菌株pGV3101(用于制备电抗病毒农杆菌,请参见Mersereau等人,1990)
  3. 植物材料
    1. 拟南芥 Col-0
  4. 质粒
    1. AtU6-26-V4(3.5 kb,氨苄青霉素抗性标记[AmpR],可根据要求提供)
    2. UBQ10 :: pcoCas9p1300(14.3 kb,卡那霉素抗性标记[KanR]在植物中的细菌,潮霉素或卡那霉素抗性标记[HygR或KanR]可根据要求提供)
    3. 可选:pGEM ® -T easy(Promega,目录号:A3600)
  5. 酶和缓冲液
    限制
    1. Bpi I(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:ER1011)
    2. I-HF(New England Biolabs,目录号:R3142)
    3. Xba I(New England Biolabs,目录号:R0145)
    4. I-HF(New England Biolabs,目录号:R3133)
    5. I-HF(New England Biolabs,目录号:R3642)
    6. Cutsmart ®缓冲液(New England Biolabs,目录号:B7204S,随酶提供)
    连接
    1. T4 DNA连接酶(New England Biolabs,目录号:M0202)
    2. T4 DNA连接酶反应缓冲液(New England Biolabs,目录号:B0202S,用酶提供)
    基因分型(细菌)
    1. 绿色Taq DNA聚合酶(GenScript,目录号:E00043)
    2. 缓冲区(GenScript,目录号:B0005,随酶提供)
    3. 10mM dNTP(Carl Roth,目录号:K039)
    4. 任何DNA加载染料,例如6倍凝胶加载染料,紫色(New England Biolabs,目录号:B7024)
    5. 任何DNA大小的标准,例如,GeneRuler TM 1 kb Plus DNA梯(Thermo Fisher Scientific,Thermo Scientific TM,目录号:SM1331 )
    6. 琼脂糖(Carl Roth,目录号:3810)
    7. 10x TBE电泳缓冲液(见配方)
      Tris(Applichem,目录号:A1379)
      硼酸(Applichem,目录号:131015)
      EDTA(Applichem,目录号:131669)
  6. 去离子水(无菌)
  7. 抗生素(-20°C储存库存)
    1. 氨苄青霉素(Carl Roth,目录号:K029,在去离子水中储存50mg/ml)
    2. 卡那霉素(Carl Roth,目录号:T832,去离子水中30mg/ml)
    3. 利福平(申请,目录号:A2220,在DMSO中25mg/ml)
    4. 庆大霉素(Carl Roth,目录号:0233,去离子水中为10毫克/毫升)
    5. 潮霉素(Carl Roth,目录号:CP13,在去离子水中储存10毫克/毫升)
  8. 媒体(见食谱)

    1. 的YEB培养基 肉提取物(Carl Roth,目录号:5770)
      酵母提取物(Carl Roth,目录号:2904)
      胨(Sigma-Aldrich,目录号:82303)
      蔗糖(Carl Roth,目录号:4621)
      硫酸镁(MgSO 4)(Carl Roth,目录号:P027)
      Bacto-agar(Th。Geyer,CHEMSOLUTE ®,目录号:9914-500G)
      氢氧化钠(NaOH)(Merck,目录号:28245)
    2. YT培养基。大肠杆菌
      氯化钠(NaCl)(Carl Roth,目录号:9265)
      酵母提取物(Carl Roth,目录号:2904)
      胨(Sigma-Aldrich,目录号:82303)
      Bacto-agar(Th。Geyer,CHEMSOLUTE ®,目录号:9914-500G)
      氢氧化钠(NaOH)(Merck,目录号:28245)

    3. MS + B5维生素(Duchefa Biochemie,目录号:M0231)
      氢氧化钾(KOH)(Merck Millipore,目录号:105033)
  9. DNA纯化
    1. 凝胶纯化,例如,NucleoSpin凝胶和PCR清除(MACHEREY-NAGEL,目录号:740609)
    2. 质粒分离,例如NucleoSpin质粒EasyPure(MACHEREY-NAGEL,目录号:740727)
  10. Silwet L-77(Lehle种子,目录号:VIS-30)用于植物转化
  11. 漂白剂(Carl Roth,目录号:9062)
  12. 37%HCl(Merck Millipore,目录号:100317)
  13. 基因分型(植物)
    1. 可选:Phire Plant Direct PCR Kit(Thermo Fisher Scientific,Thermo Scientific TM,目录号:F-130WH)
    2. 可选:T7内切核酸酶I(New England Biolabs,目录号:M0302)加上NEBuffer 2(New England Biolabs,目录号:B7002),250mM EDTA
    3. 可选:pGEM ® -T easy(Promega,目录号:A3600)
  14. 寡核苷酸(5'-3'),10pmol/μl
    1. M13F_TGTAAAACGACGGCCAGT
    2. M13R_CAGGAAACAGCTATGACC
    3. K197 _CTGTTAATCAGAAAAACTCAG

设备

注意:不需要特定的设备。可以使用任何适当的设备。

  1. 带互联网接入的电脑
  2. 热循环仪(例如,Eppendorf,型号:Mastercycler nexus)
  3. Thermoblock(例如,Eppendorf,型号:Thermomixer ®舒适)
  4. 板培养箱(28℃,37℃)
  5. 振荡器(28°C,37°C)
  6. 水平凝胶电泳系统(例如,Bio-Rad Laboratories,型号:Mini-Sub Cell GT System)
  7. 紫外线透照仪(例如,Bio-Rad Laboratories,型号:GelDoc TM) XR +系统)
  8. 电生理器(例如,Bio-Rad Laboratories,型号:MicroPulser TM Electroporator)
  9. 10 L干燥器
  10. 通风柜
  11. 200毫升烧杯

程序

  1. 使用CRISPR-PLANT制备gRNA-间隔物(Xie等人,2014)和引物设计(图1I)
    1. 转到 http://www.genome.arizona.edu/crispr/index.html
    2. 选择"搜索"。
    3. 选择"选择物种",然后选择"拟南芥拟南芥"
    4. 选择'染色体',并在'From'和'To'中输入目标区域的坐标。
    5. 用"按地区搜索"确认。
      您将返回Class 0.0和Class 1.0 gRNAs。 0.0类gRNA应该更具体。 SeqID是您预定义区域中gRNA的坐标。 minMM_GG(和minMM_AG)值是指与PAM基序相邻的12bp序列具有与A中任何其它序列的最小错配量。 thaliana 基因组。价值越高,违规定位风险就越小。因此,应考虑minMM_GG高于3的序列。
    6. 复制最佳的20 bp长间隔序列。这里,X表示任何核苷酸 转发顺序:5'-XXXXXXXXXXXXXXXXXXXX-3'
      反向顺序:5'-XXXXXXXXXXXXXXXXXXXX-3'
      当U6 启动子驱动gRNA的表达时,转录起始需要鸟嘌呤(G)作为转录起始位点。如果所选择的间隔序列不以"G"开头,则更改第一个基座。这对Cas9的特异性没有太大的影响,因为特异性主要取决于邻近PAM基序的12 bp。
      转发顺序:5'-GXXXXXXXXXXXXXXXXXXX-3'
      反向顺序:5'-XXXXXXXXXXXXXXXXXXXC-3'
    7. 将"GATT"和"AAAC"分别添加到正向和反向序列的5'端,以产生与I/O兼容的突出端。 正向引物:5'-GATTGXXXXXXXXXXXXXXXXXXX-3'
      反向引物:5'-AAACXXXXXXXXXXXXXXXXXXXC-3'
    8. 现在你有一个互补的引物对可以订购。
  2. gRNA引物退火(图1II)
    1. 混合10μl每个引物(10pmol/μl)。变性引物在98℃下3分钟。通过将温度从98℃降低到22℃来将引物杂交到热循环仪中。暂停每个学位30秒。
  3. 根据制造商的说明书(图1III),将AU6-26-V4-矢量与
    1. 将消化物与DNA负载染料混合,并将其装载在1%琼脂糖凝胶上。通过凝胶电泳分离消化未消化的条带
    2. 从凝胶中纯化线性载体骨架(3,517bp)
  4. 在10μl连接反应(图1IV)中,对已退火的gRNA引物和Ipi6消化的AtU6-26-V4载体进行退火(图1IV)。
    1. 混合25ng至50ng载体,1μl10×T4 DNA连接酶反应缓冲液和0.5μlT4 DNA连接酶(200U)。用混合的底漆填充剩余的体积。
    2. 在室温下孵育反应10分钟。
      注意:如果在步骤6中遇到阳性转化体遇到问题,请使用4°C过夜孵化。
  5. 使用3μl连接反应来转化化学感受态的TOP10E。大肠杆菌细胞。
    1. 在冰上解冻50μl感受态细胞。
    2. 向细胞中加入3μl连接反应。
    3. 在冰上孵育15分钟。
    4. 在42℃,热电冲击细胞45秒,并放置在冰上2分钟
    5. 加入400μlYT培养基,并在37℃和750 rpm下将细胞孵化60分钟。
    6. 在含有50μg/ml氨苄青霉素(YT-amp)的固体YT培养基上将板60至80μl转化。
  6. 做一个菌落PCR来转基因。通常测试5至10个菌落足以获得阳性转化体
    1. 如下设置每个菌落20μlPCR反应。最终浓度用括号表示:
      2μl10×Taq缓冲液(1x)
      0.6μlM13F(0.5μM)
      0.6μlgRNA特异性反向引物(0.5μM)
      0.4μldNTPs(0.2 mM)
      0.1μl绿色Taq DNA聚合酶(0.5U)
      16.3微升去离子水
    2. 使用移液器吸头挑取菌落并将细胞重新排列在YT放大器板上。直接将尖端浸入PCR反应中以转移少量细胞。在37℃下孵育板约5小时,并使用以下条件在热循环仪中运行PCR:

      注意:退火温度取决于gRNA特异性反向引物。 M13F在退火过程中在54°C至60°C的范围内工作良好。代替M13F和gRNA特异性反向引物,您还可以使用M13R和gRNA特异性正向引物,延伸时间为20秒。
    3. 向反应中加入4μl6x DNA负载染料,并将其加载到1%琼脂糖凝胶上。使用M13F和gRNA特异性反向引物的PCR反应的预期扩增子大小为581bp,与M13R和gRNA特异性正向引物的反应为257bp。
    4. 准备3毫升YT-amp,并从修复板中挑选PCR阳性克隆。在37°C下以190 rpm的速度孵育过夜。
    5. 从阳性克隆中分离质粒,并使用M13R作为测序引物,通过Sanger测序检查正确的插入。
  7. 可选(用于复用):
    如果需要多于一种的gRNA,首先在单独的AtU6-26-V4载体(gRNA1-AtU6-26-V4,gRNA2-AtU6-26-V4,等等)中制备每个gRNA,并且将它们组合成单个gRNA2-gRNA1-AtU6-26-V4载体。
    1. 用gp1-AtU6-26-V4切割用Kpn I-HF和I-HF(3,519bp,图1V)打开。
    2. 使用Kpn I-HF和Xba I(643bp,图1VI)从gRNA2-AtU6-26-V4切割gRNA2。
    3. 凝胶上加载反应,凝胶纯化阳性条带
    4. I-gRNA1-AtU6-26-V4-Kpn I-gRNA2-Xb I I gRNA2-I-gRNA1-AtU6-26-V4-Kpn I ( Xba I和 Spe 我产生兼容的目的,图1VII)。
    5. 转换TOP10 E。大肠杆菌细胞,在YT-amp板上选择转化体
    6. 具有M13F的基因型克隆和最后添加的gRNA的特异性反向引物(扩增子大小= 581bp)。
    7. 分离质粒和序列与M13R。
      重复此步骤以添加更多gRNA。
  8. 根据制造商的说明书,将具有kpn I-HF和Sbf I-HF的gRNA2-gRNA1-AtU6-26-V4消化。通过此步骤,您将切除包含U6启动子的gRNA2-gRNA1单位(图1VIII)。
    1. 将消化物与DNA负载染料混合,并将其装载在1%琼脂糖凝胶上。通过凝胶电泳分离消化未消化的条带
    2. 从凝胶中纯化gRNA片段(单个gRNA片段的660bp,双gRNA片段的1,287bp)。
  9. 根据制造商的说明书,将UBQ10 :: pcoCas9p1300与 Kpn I-HF和Sbf I-HF进行消化以打开载体(图1IX)。
    1. 将消化物与DNA负载染料混合并将其装载到1%琼脂糖凝胶上。通过凝胶电泳分离消化未消化的条带
    2. 从凝胶纯化线性化载体骨架(14.3 kb)
  10. 在20μl反应中调节gRNA2-gRNA1单位和UBQ10 :: pcoCas9p1300(图1X)。
    1. 分别混合25ng至50ng载体,10ng至20ng gRNA2-gRNA1,2μl10×T4 DNA连接酶反应缓冲液,去离子水,19μl和1μlT4 DNA连接酶(400U)。
    2. 在4℃下反应过夜,
  11. 使用3μl连接反应来转化化学感受态的TOP10E。大肠杆菌细胞。用30μg/ml卡那霉素选择YT平板上的转化体
  12. 按照步骤6所述进行菌落PCR以转基因转基因
    1. 具有K197的基因型克隆和第一次加入的gRNA的特异性正向引物(这里是gRNA1,产品大小为801bp)。
    2. 分离和序列质粒与M13F(可选与K197)
  13. 使用50ng AtU6 :: gRNA2-AtU6 :: gRNA1-UBQ10 :: pcoCas9p1300转化A。根癌土壤杆菌通过电穿孔
    1. 解冻50μl有效的。冰川上的根癌土壤杆菌
    2. 在冰上预先冷却比色皿。
    3. 将50ng最终向量添加到 A。根癌土壤杆菌细胞,转移到比色杯并将比色皿置于电穿孔器中。
    4. 选择MicroPulser TM 电穿孔仪(Bio-Rad,2.2 kV的1个脉冲)或等效设置的"Agr"设置,以转换细胞。
    5. 加入1ml YEB培养基并转移到新鲜的1.5ml反应管中。在28℃和750rpm下孵育2至3小时。
    6. 将板60μl进行转化,并在28℃下,在30℃/30μg/ml卡那霉素,100μg/ml利福平和25μg/ml庆大霉素的YEB培养基上选择2天。
  14. 使用转换的 A。 tumefaciens 转换 A。 thaliana 通过浸润浸润(Clough and Bent,1998; Chen,2011)
  15. 通过气相灭菌灭菌种子,例如。
    1. 将种子转移到1.5ml反应管中
    2. 将通风管上的10L干燥器放开管。
    3. 将装有100ml漂白剂(6%)的200ml烧杯放入干燥器中
    4. 使用玻璃移液管向漂白剂中加入3ml HCl(37%),并快速关闭干燥器盖。
      注意:仔细:蒸汽可以化学地烧伤皮肤和眼睛!
    5. 具有M13F的基因型克隆和最后添加的gRNA的特异性反向引物(扩增子大小= 581bp)。
    6. 分离质粒和序列与M13R。
      重复此步骤以添加更多gRNA。
  16. 根据制造商的说明书,将具有kpn I-HF和Sbf I-HF的gRNA2-gRNA1-AtU6-26-V4消化。通过此步骤,您将切除包含U6启动子的gRNA2-gRNA1单位(图1VIII)。
    1. 将消化物与DNA负载染料混合,并将其装载在1%琼脂糖凝胶上。通过凝胶电泳分离消化未消化的条带
    2. 从凝胶中纯化gRNA片段(单个gRNA片段的660bp,双gRNA片段的1,287bp)。
  17. 根据制造商的说明书,将UBQ10 :: pcoCas9p1300与 Kpn I-HF和Sbf I-HF进行消化以打开载体(图1IX)。
    1. 将消化物与DNA负载染料混合并将其装载到1%琼脂糖凝胶上。通过凝胶电泳分离消化未消化的条带
    2. 从凝胶纯化线性化载体骨架(14.3 kb)
  18. 在20μl反应中调节gRNA2-gRNA1单位和UBQ10 :: pcoCas9p1300(图1X)。
    1. 分别混合25ng至50ng载体,10ng至20ng gRNA2-gRNA1,2μl10×T4 DNA连接酶反应缓冲液,去离子水,19μl和1μlT4 DNA连接酶(400U)。
    2. 在4℃下反应过夜,
  19. 使用3μl连接反应来转化化学感受态的TOP10E。大肠杆菌细胞。用30μg/ml卡那霉素选择YT平板上的转化体
  20. 按照步骤6所述进行菌落PCR以转基因转基因
    1. 具有K197的基因型克隆和第一次加入的gRNA的特异性正向引物(这里是gRNA1,产品大小为801bp)。
    2. 分离和序列质粒与M13F(可选与K197)
  21. 使用50ng AtU6 :: gRNA2-AtU6 :: gRNA1-UBQ10 :: pcoCas9p1300转化A。根癌土壤杆菌通过电穿孔
    1. 解冻50μl有效的。冰川上的根癌土壤杆菌
    2. 在冰上预先冷却比色皿。
    3. 将50ng最终向量添加到 A。根癌土壤杆菌细胞,转移到比色杯并将比色皿置于电穿孔器中。
    4. 选择MicroPulser TM 电穿孔仪(Bio-Rad,2.2 kV的1个脉冲)或等效设置的"Agr"设置,以转换细胞。
    5. 加入1ml YEB培养基并转移到新鲜的1.5ml反应管中。在28℃和750rpm下孵育2至3小时。
    6. 将板60μl进行转化,并在28℃下,在30℃/30μg/ml卡那霉素,100μg/ml利福平和25μg/ml庆大霉素的YEB培养基上选择2天。
  22. 使用转换的 A。 tumefaciens 转换 A。 thaliana 通过浸润浸润(Clough and Bent,1998; Chen,2011)
  23. 通过气相灭菌灭菌种子,例如。
    1. 将种子转移到1.5ml反应管中
    2. 将通风管上的10L干燥器放开管。
    3. 将装有100ml漂白剂(6%)的200ml烧杯放入干燥器中
    4. 使用玻璃移液管向漂白剂中加入3ml HCl(37%),并快速关闭干燥器盖。
      注意:仔细:蒸汽可以化学地烧伤皮肤和眼睛!
    5. 灭菌3至4小时。
  24. 用20微克/毫升潮霉素或卡那霉素(取决于您选择的UBQ10 :: pcoCas9p1300的抗性标记),在1/2 MS上播种种子以选择阳性转化体。使用Micropore TM 胶带的密封板。在黑暗中将种子在4℃下分层3天。在长的天气条件下(22°C,16:8 h光:暗光周期)发芽种子,直到转化株达到4个真叶期。移植幼苗到土壤,并在长的日子条件下继续生长
  25. 基因型转化体。
    如在T1代,Cas9酶的活性和效率以及DNA修复机制的活性可能因细胞而异,每种植物最有可能是不同基因型的镶嵌。因此,我们建议汇集一个植物的基因分型样品,如来自不同分支的花生叶片。如果在T1中鉴定出具有预期突变的植物,则可以对单个分枝标记和基因型,因为它们可以具有不同的基因型(图2C和2D)。基因分型的方法取决于预期的结果
    1. 由多个gRNA产生的大片段缺失可以通过PCR鉴定,然后通过PCR产物的凝胶电泳鉴定(图2A和2B)。
      注意:在每个gRNA靶位点约200bp到400bp的距离内设计预期缺失侧翼的引物。对于高通量基因分型,我们建议使用Phire Plant Direct PCR Kit制备10μlPCR反应。


      图2.检测AP1 基因座中的大片段缺失。 A.使用两种不同的gRNA(剪刀)对AP1 基因座进行靶向。目标网站是AP1 的第三和第六内含子中的序列。 B.在T1代中表型野生型(WT)和类似于(a)的分支的基因分型显示在AP1基因座中存在680bp缺失在 ap1 样分支(m)中。 C和D. T1代植物可以是不同基因型的马赛克。在同一植物的两个不同分支上可以发现两种不同的花表型,即野生型(C,左; D,蓝色箭头)和 ap1 (C,右; D,黄色箭头)。 />
    2. 通过T7核酸内切酶消化(Vouillot et al。,2015)可以检测到产生与野生型等位基因2 bp的最小错配的小缺失或多态性。
      1. 执行基因分型PCR,如提及大片段缺失。
      2. 通过向PCR产物中加入1.12μlNEBuffer 2而不纯化DNA来设置杂交反应。分别准备WT控制和样品。当在T1中合并不同的分支时,不需要向样品中添加野生型PCR产物,因为我们通常不会期望Cas9效率高于T7内切核酸酶检测限(Vouillot等人, 2015)。但是,如果您在单分支基因分型时已经看到了明确的表型,您可以用野生型取代20%的样本量,以免遗漏纯合突变体分支。
      3. 使用以下条件杂交PCR产物:
      4. 加入0.1μlT7内切核酸酶(1U),37℃孵育30分钟
      5. 用0.8μl250 mM EDTA停止反应
      6. 在2%琼脂糖凝胶上分离切割产物
    3. T7内切核酸酶消化不能有效地检测到点突变(Vouillot等,2015)和IDT Surveyor 核酸酶(Integrated DNA Technologies )被认为是切点突变,似乎与Phire Plant Direct PCR Mix不相容。考虑到高产量突变体筛选的经济因素,超出制造商的建议,我们没有增加Surveyor ®酶的量。在这里,我们建议使用T7内切核酸酶测定法排除具有2 bp或更高突变的植物。然后将其余样品的PCR产物纯化并送至Sanger测序
  26. 通过测序确认突变。
  27. 从具有突变的分支中收获种子。
  28. 播种种子以产生约100人的小T2群体。选择携带突变但丢失T-DNA的植物。
    1. 为了鉴定仍携带转基因的植物,使用M13F和gRNA特异性反向引物(产品大小557bp)。排除PCR阳性植物
    2. 针对预期突变的基因型,如步骤17所述 注意:为了验证T2中的小片段缺失(图3),在杂交前准备WT,样品和WT和样品的1:1混合物。 WT样品混合物是必需的,因为植物对于突变体等位基因可以是纯合的。在这种情况下,它们在杂交时不产生异源双链DNA,不会被T7内切核酸酶消化。


      图3.通过T7消化检测小缺失或多态性
      T2野生型(WT),突变体(m)和野生型和突变体(WT + m)的1:1混合物的PCR样品杂交。反应体积的一半在没有T7内切核酸酶(未消化)的情况下孵育,另一半与1U酶(T7消化物)一起孵育,并通过在2%琼脂糖上的凝胶电泳分离。突变样品中切割产物的存在是至少两种不同等位基因的结果,其可以是野生型和突变体或不同突变体等位基因或两种选择的混合物。由于向突变体样品(WT + m)添加野生型不会稀释切割产物的信号,所以很可能是不同的突变体等位基因,其后来通过测序证实(未显示)。星号表示未消化的PCR产物,黑色箭头指出裂解产物。 GR表示GeneRuler TM 1 kb Plus DNA梯。
      注意:协议设置完成后,可以省略"未消息"控件。

数据分析

在T1代中获得的突变可以通过步骤17所述的Sanger测序来确认。但是,由于相邻细胞可能具有不同的突变,并且由于突变可能是双重的,因此我们建议将突变型PCR产物凝胶纯化并将其亚克隆到pGEM或其他任何克隆载体。序列5至10个阳性菌落,以获得获得突变的概述。在T2代中选择无T-DNA的突变植物(步骤19),通过测序再次确认突变的存在和类型。
注意:由Phire Plant Direct PCR Kit产生的PCR产物是平端的,并且在连接pGEM -T之前需要A-tailing。

笔记

这个协议很简单直接。到目前为止,我们的组已经产生了含有不同组gRNA的50多个二元载体,并用于拟南芥的基因组编辑。

食谱

  1. 10倍TBE电泳缓冲液(1升)
    1 M Tris
    1 M硼酸
    0.02 M EDTA
    去离子水广告1,000 ml
    用去离子水稀释10倍TBE缓冲液至1:10,用作凝胶运行缓冲液
  2. (1L)
    的YEB培养基 5克牛肉提取物
    1克酵母提取物
    5克蛋白胨
    5克蔗糖
    500mg MgSO 4
    10克Bacto-agar
    去离子水广告1,000 ml
    使用NaOH调节pH至7.0 高压灭菌器在121°C和2 bar 15分钟的YEB培养基
  3. YT培养基。大肠杆菌(1L)
    5克NaCl
    5克酵母提取物
    8克蛋白胨
    (15克Bacto-agar)
    去离子水广告1,000 ml
    使用NaOH调节pH至7.0 高压灭菌YT培养基在121°C和2 bar 15分钟
  4. (1 L)
    2.3克MS + B5维生素
    8 g Phyto-agar
    去离子水广告1,000 ml
    使用KOH调节pH至5.7 高压灭菌½MS在121°C和2 bar 15分钟
    准备无菌50%蔗糖(单独过滤灭菌或高压釜)
    在浇注板之前,加入1%蔗糖至1/2 MS 注意:不需要加入蔗糖。优势在于选择性培养基上的幼苗恢复得更好。缺点是它可以促进培养基上的真菌生长。

致谢

这个协议是基于我们以前在Plant Methods中发表的工作( Yan 等。,2016 )。 J.S.的工作得到IMPRS-PMPG奖学金的支持。公司感谢Alexander-von-Humboldt基金会和BMBF的支持。作者宣称他们没有竞争的利益。

参考文献

  1. Chen,L.(2011)。简化的农杆菌介导的拟南芥转化。 Bio-protocol Bio101:e41。
  2. Clough,SJ和Bent,AF(1998)。  花卉浸渍:拟南芥农杆菌介导的转化的简化方法。植物J 16(6):735-743。
  3. Li,JF,Norville,JE,Aach,J.,McCormack,M.,Zhang,D.,Bush,J.,Church,GM and Sheen,J.(2013)。  拟南芥中的多重和同源重组介导的基因组编辑和Nicotiana本指南使用指南RNA和Cas9。生物技术 31(8):688-691。
  4. Mersereau,M.,Pazour,GJ和Das,A.(1990)。< a class ="ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pubmed/?term= Mersereau%201990"target ="_ blank">通过电穿孔有效转化根癌土壤杆菌。 90(1):149-51。
  5. Nekrasov,V.,Staskawicz,B.,Weigel,D.,Jones,JD和Kamoun,S。(2013)。  使用Cas9 RNA引导的内切核酸酶在模型植物中本发明的烟草Nicamina本发明的靶向诱变。 Nat Biotechnol 31(8):691-693。
  6. Shan,Q.,Wang,Y.,Li,J.,Zhang,Y.,Chen,K.,Liang,Z.,Zhang,K.,Liu,J.,Xi,JJ,Qiu,JL and Gao, C.(2013)。作物的目标基因组修饰使用CRISPR-Cas系统。 Nat Biotechnol 31(8):686-688。
  7. Sorek,R.,Lawrence,CM和Wiedenheft,B。(2013)。  CRISPR介导的细菌和古细菌中的适应性免疫系统。 Annu Rev Biochem 82:237-266。
  8. Vouillot,L.,Thelie,A.and Pollet,N。(2015)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/25566793"目标="_ blank"> T7E1和测量师错配切割测定的比较,以检测由工程化核酸酶触发的突变。(Bethesda)5(3):407-415。
  9. Xie,K.,Zhang,J.and Yang,Y。(2014)。  在模式植物和主要作物中用于CRISPR-Cas9介导的基因组编辑的高度特异性指导RNA间隔区的基因组预测 Mol Plant 7(5):923 -926。
  10. Yan,W.,Chen,D. and Kaufmann,K.(2016)。  通过RNA引导的Cas9进行有效的多重诱变及其在AGAMOUS 基因的表征调控元件中的应用植物方法 12:
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引用:Schumacher, J., Kaufmann, K. and Yan, W. (2017). Multiplexed GuideRNA-expression to Efficiently Mutagenize Multiple Loci in Arabidopsis by CRISPR-Cas9. Bio-protocol 7(5): e2166. DOI: 10.21769/BioProtoc.2166.
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