Genomic Edition of Ashbya gossypii Using One-vector CRISPR/Cas9

Materials and Reagents

Note: Materials and reagents can be purchased from different suppliers. Here, we show one list of all reagents and materials that we used to develop the protocol.

Materials

1. Pipette tips (e.g., VWR, catalog numbers: 613-0340 , 613-0360 , 613-1068 )
2. PCR microtubes (e.g., Sarstedt, catalog number: 72.737 )
3. 1.5 ml microtube (Deltalab, catalog number: 200400P )
4. 50 ml centrifuge tube (Corning, catalog number: 352070 )
5. Graduated pipettes 10 ml (Brand, catalog number: 27011 )
6. Petri dishes (Thermo Scientific, catalog number: 11309283 )
7. Microscope slides (e.g., VWR, catalog number: 630-2099 )
8. Cover slides (e.g., VWR, catalog number: 631-0125 )
9. 0.4 cm electroporation cuvettes (Bio-Rad, catalog number: 1652088 )
10. Syringe 50 ml (Omnifix, catalog number: 4616502F )
11. Sterile syringe filter w/0.45 µm cellulose acetate membrane (VWR, catalog number: 28145-481 )
12. Filter paper

Reagents

1. PCR primers (Invitrogen), desalted, standard 25-50 nmol scale
2. Escherichia coli DH5α (Thermo Fisher Scientific, catalog number: 18265017 )
3. D(+)-Glucose monohydrate (Acros Organics, catalog number: 450740050 )
4. D(+)-Sucrose (PanReac AppliChem, catalog number: 131621.1211 )
5. Peptone (Condalab, catalog number: 1616.05 )
6. Tryptone (Condalab, catalog number: 1612.05 )
7. Yeast extract (Condalab, catalog number: 1702.05 )
8. Malt extract (VWR, catalog number: J873-500G )
9. Myo-inositol 99% (Sigma-Aldrich, CAS number: 87-89-8 )
10. Soybean oil (Santa Cruz Biotechnology, catalog number: 8001-22-7 )
    
11. Corn steep liquor (Sigma-Aldrich, CAS number: 66071-94-1 )
12. European bacteriological agar (Condalab, catalog number: 1800.05 )
13. Tris Base (Fisher BioReagents, catalog number: BP152-500 )
14. Boric acid (PanReac AppliChem, catalog number: 131015 )
15. EDTA (Ethylenediaminetetraacetic acid disodium salt 2-hydrate) (PanReac AppliChem, catalog number: 131669 )
16. Magnesium chloride hexahydrate (MgCl₂·6H₂O) (Sigma-Aldrich, CAS number: 7791-18-6 )
17. Sodium chloride (NaCl) (PanReac AppliChem, catalog number: 131659.1214 )
18. Potassium phosphate monobasic (KH₂PO₄) (Fisher BioReagents, CAS number: 7778-77-0 )
19. Potassium phosphate dibasic (K₂HPO₄) (Fisher BioReagents, CAS number: 7758-11-4)
20. Sodium carbonate anhydrous (Na₂CO₃) (PanReac AppliChem, catalog number: 131648.1211 )
21. Sodium hydrogen carbonate (NaHCO₃) (PanReac AppliChem, catalog number: 141638.121 0)
22. Triton X-100 (Acros Organics, catalog number: 215680010 )
23. DTT (dithiothreitol) (Thermo Scientific, catalog number: R0861 )
24. PVP 40 (Polyvinylpyrrolidone) (Sigma-Aldrich, CAS number: 9003-39-8 )
25. Tween 20 (PanReac AppliChem, catalog number: 142312.1611 )
26. Bovine Serum Albumin (Sigma-Aldrich, CAS number: 9048-46-8 )
27. G-418 Disulphate (Sigma-Aldrich, CAS number: 108321-42-2 )
28. Kanamycin sulfate (Sigma-Aldrich, CAS number: 25389-94-0 )
29. Ampicillin sodium salt (Sigma-Aldrich, CAS number: 69-52-3 )
30. Zymolyase^®, 20T (Nacalai Tesque, catalog number: 07663-91 )
31. FavorPrep Plasmid Extraction Mini Kit (Favorgen, catalog number: FADPE 300 )
32. DreamTaq Green PCR Master Mix (2x) (Thermo Fisher Scientific, catalog number: K1082 )
33. RedSafe Nucleic Acid Staining Solution (iNtRON Biotechnology, catalog number: 21141 )
34. Agarose D1 Low EEO (Condalab, catalog number: 8010.01 )
35. LB medium (see Recipes)
36. MA2 medium (see Recipes)
37. SPA medium (see Recipes)
38. STM buffer (see Recipes)
39. TBE 20x (see Recipes)
40. DNA extraction buffer (see Recipes)
41. Transformation buffer (see Recipes)
42. Potassium phosphate buffer (see Recipes)

Equipment

1. Pipettes P2, P20, P200 and P1000 (e.g., Gilson, PIPETMAN Classic^TM, catalog numbers: F144801 , F144056M , F144058M , F144059M )
2. Motorized pipette filler (e.g., Eppendorf, catalog number: 4430000018 )
3. Sentino Magnetic Filter Funnels (Pall, catalog number: 4273 )
4. Sterile glass handle or glass beads
5. Erlenmeyer flasks 500 ml (VWR, catalog number: 10536-926 )
6. Filtering flask 250 ml (Simax, catalog number: PJH006 )
7. Gene-Pulser (Bio-Rad, model: MicroPulser )
8. Heat block (e.g., Eppendorf, catalog number: 5355 000.011 )
9. Thermal Cycler (e.g., Bio-Rad, catalog number: 170-6700 )
10. Microcentrifuge (e.g., Thermo Scientific, catalog number: 10236249 )
11. Centrifuge (e.g., Eppendorf, catalog number: 5703000010 , model: 5702R )
12. Power Source (VWR, catalog number: 700-0115 , model: 250 V )
13. Gel Doc EZ System (Bio-Rad, catalog number: 1708270 )
14. Optical microscope
15. Vacuum pump
16. Microflow vertical laminar flow workstation
17. 28 °C incubator
18. Shaker incubator 28 °C
19. Autoclave
    

Software

1. Image lab^TM software 6.0.1. (Bio-Rad, catalog number: 1709690)

Procedure

The following protocol can be divided into three different sections (see Figure 2 for a schematic workflow):
•Isolation of the CRISPR/Cas9 plasmid. The design and assembly of CRISPR/Cas9 plasmids has been previously described in Jiménez et al., 2019. See Figure 3A for a schematic design of the synthetic gRNA-dDNA.
• A. gossypii transformation. In this section, a detailed method for A. gossypii transformation is provided.
•Isolation and screening of engineered homokaryotic strains. The methodology for the screening of positive clones depends on the phenotype produced by the corresponding genomic edition. Phenotypes such as colored mycelia can be followed directly by visual observation (Jiménez et al., 2019). Alternatively, the genomic editions can be confirmed by analytical PCR (see Figure 3B for a typical three primer PCR strategy) using the corresponding primer pair for each modification (Jiménez et al., 2019).

Figure 2. Workflow of the CRISPR/Cas9 genomic edition in A. gossypii


Figure 3. CRISPR/Cas9 design and analysis. A. Schematic design of the synthetic gRNA-dDNA. B. Analytical three primer PCR strategy.

Note: Perform all steps involving microbial culture under sterile environment in laminar airflow.

1. Isolate the CRISPR/Cas9 plasmid from the corresponding E. coli strain
   1. Inoculate the corresponding E. coli strain harboring the CRISPR/Cas9 plasmid in 4 ml of LB plus 50 µg/ml kanamycin and 100 µg/ml ampicillin.
   2. Incubate at 37 °C in a shaker incubator overnight.
   3. Collect the cells and extract the plasmid DNA using the FavorPrep Plasmid Extraction Mini Kit.
      
      
      
2. A. gossypii transformation
   Note: Perform Steps B8 to B20 strictly on ice.
   1. Inoculate 750 µl of spores (approx. 10⁸ spores) of the target strain of A. gossypii in a flask with 100 ml of liquid MA2 medium.
      Note: A counting chamber such as Neubauer/Thoma can be used for calculation of spore number.
   2. Incubate at 28 °C for 10-12 h at 180 rpm.
   3. After this time, check the status of the spores under a microscope. The spores must be in a germination state called “germlings” for the optimal transformation efficiency (Figure 4A) (Jiménez et al., 2005).
   4. Collect the spores by vacuum filtration using a sterile paper filter.
      Note: Use an autoclave sterilized sentino magnetic filter funnel.
   5. Prepare the transformation buffer (see Recipes).
   6. Introduce the paper filter with the spores in a tube with transformation buffer and resuspend the spores by inversion.
      Note: Remove the paper filter after resuspending the spores.
   7. Incubate the tube with the spore suspension at 28 °C for 30 min with agitation at 180 rpm.
      Note: Pre-chill liquid MA2 medium, STM buffer, electroporation cuvettes and Eppendorf tubes on ice.
   8. Centrifuge for 10 min at 3,000 x g at 4 °C.
   9. Carefully discard the supernatant without resuspending the spores.
   10. Gently wash the spores with 20 ml of ice-cold STM buffer.
   11. Repeat Steps B8-B10.
   12. Centrifuge for 10 min at 3,000 x g at 4 °C.
   13. Resuspend the spores in 2 ml of ice-cold STM buffer and aliquot the spores in Eppendorf tubes.
   14. Centrifuge for 1 min at 17,000 x g at 4 °C.
   15. Discard the supernatant and resuspend the spores to a final volume of 400 µl with ice-cold STM buffer.
   16. Mix 190 µl of the spore suspension and 5-10 µg of DNA (15-20 µl approx.) in an electroporation cuvette.
       Notes:
       a.Use cut pipette tips to avoid clogging of the spore suspension.
       b.For the negative control use the same volume of water instead of DNA.
   17. Use the following settings in the Gene-Pulser: 1,500 V, 200 W and 25 mF.
   18. Pulse the mixture and immediately add 1 ml of ice-cold MA2 medium to resuspend the spores. Transfer the spores to a new Eppendorf.
   19. Centrifuge for 1 min at 17,000 x g at 4 °C.
   20. Discard 1 ml of the supernatant and plate 200 µl of the spores onto an MA2 agar plate.
       Note: Use a sterile glass handle to plate the spores.
   21. Incubate at 28 °C for 6 h for the regeneration of spores.
   22. After this time, the transformation plates must be covered with an upper agarose layer containing G418. Add 6 ml of 0.4% agarose, previously melted at 95 °C and cooled to 42 °C, with 200 µl of G418 at 50 µg/ml (stock concentration).
   23. Incubate the plates at 28 °C for 2 days.
       
       
       
3. Isolation and screening of primary heterokaryotic transformants
   1. Isolate G418-resistant heterokaryotic clones from the transformation plates using fresh MA2-G418 plates (Figures 4B and 4C). Incubate the heterokaryotic transformants at 28 °C for 2 days.
      
      
      Figure 4. A. gossypii germlings and transformation isolates. A. Micrograph of A. gossypii germling. B. Transformation plate. C. Isolated heterokaryotic transformants.
      
      
   2. Extraction of genomic DNA for analytical PCR of the heterokaryotic clones:
      1. Prepare DNA extraction buffer (Recipe 6).
      2. Pick approximately 2 mm² of mycelia from the isolated G418-resistant heterokaryotic clones and put into 20 µl of DNA extraction buffer.
      3. Incubate at 100 °C for 10 min.
      4. Centrifuge for 2 min at 17,000 x g.
      5. Keep the supernatant at 4 °C for analytical PCR. The pellet can be discarded.
         
      Notes:
      
      1. Perform a reaction for each primary heterokaryotic transformant.
      2. If possible, a three primer PCR (TP-PCR) strategy should be followed (Figure 3B) (Jiménez et al., 2019): design a pair of primers for the amplification of the same region in both edited and non-edited nuclei/genomes/DNAs. Additionally, design a third primer that exclusively aligns in edited genome templates for the amplification of the edited amplicon.
   3. Analytical PCR.
      
      1. Set up the following reaction using PCR microtubes:
         15 µl of DreamTaq Green PCR Master Mix (2x)
         1 µl of each PCR primer (10 µM)
         1 µl of the supernatant from DNA extraction (50-100 ng/µl)
         dH₂O up to 30 µl
      2. Set up the following conditions in the thermal cycler:
         A denaturation step at 95 °C for 3 min
         35 cycles of:
           Denaturation: 95 °C for 30 s.
           Annealing: 50-65 °C (adjust to the primers Tm) for 30 s.
           Elongation: 72 °C for 1 minute (adjust to the amplicon length by 1 kb/min).
         Final elongation step at 72 °C for 10 min.
         Store at 4 °C.
   4. Run a 0.8% agarose gel electrophoresis with RedSafe (0.5 µg/ml).
      
      1. Use 5 µl of the PCR product.
      2. Prepare TBE 1x Buffer from TBE 20x buffer (Recipe 5).
      3. Expose the agarose gel under a UV transilluminator (e.g., Gel Doc EZ System, Bio-Rad).
      
      
4. Sporulation of the primary heterokaryotic positives transformants
   Note: The mycelium of A. gossypii is organized as multinucleated cells that are separated by septa along the hyphae. Only a limited number of nuclei within each syncytium are recipients of the transforming DNA. Sporulation of the primary heterokaryotic transformants is a required step for the isolation of homokaryotic clones, which are derived from uninucleated spores.
   1. Spread mycelia from the positive G418-resistant clones onto SPA plates lacking G418 and incubate at 28 °C for 2 days. This step promotes the loss of the CRISPR/Cas9 plasmid, since episomic plasmids are not fully stable in A. gossypii.
   2. After two days on SPA medium, confirm the presence of spores under a microscope.
   3. Prepare a 1 mg/ml zymolyase-20T buffer to isolate the spores. Sterilize by filtration through a 0.45 µm filter.
   4. Collect 1 cm² of mycelia and transfer into an Eppendorf tube with 1 ml of 1mg/ml of zymolyase buffer.
   5. Incubate at 37 °C for 50 min with gentle agitation at 850 rpm.
   6. After this time, check the mycelial lysis under a microscope.
   7. Centrifuge for 2 min at 17,000 x g.
   8. Discard the supernatant carefully by pipetting and wash the spores with 1 ml of 0.03% Triton X-100.
   9. Repeat Steps D7 and D8.
   10. Centrifuge for 2 min at 17,000 x g.
   11. Discard the supernatant and resuspend the spores with 1 ml of MA2.
   12. Finally, make appropriate dilutions of the spore suspension and plate approx. 10² spores onto MA2 solid medium lacking G418.
       Note: Use a sterile glass handle to plate.
   13. Incubate at 28 °C for 2 days.
       
       
       
5. Isolation and screening of homokaryotic transformants:
   1. Isolate homokaryotic clones using fresh MA2 plates lacking G418. Incubate the homokaryotic transformants 28 °C for 2 days. Use MA2 plates plus G418 to confirm the loss of the CRISPR/Cas9 plasmid.
   2. Repeat Steps C2, C3 and C4 to verify the presence of the designed mutation in the genome.
   3. Sequence the amplicons with appropriate primers to further confirm the presence of the desired genomic edition.

Data analysis

The editing efficiency is estimated as the frequency of primary heterokaryotic transformants containing edited nuclei. For calculation of the editing efficiency, 10-30 heterokaryotic transformants are usually analyzed. On average, an efficiency of 60% is obtained using different sgRNA-dDNA combinations.

Recipes

1. LB medium
   1% Tryptone
   0.5% Yeast extract
   1% NaCl
   1.5% Agar (for solid media)
   Dissolve completely in distilled water and sterilize by autoclaving.
   Notes:
   
   1. Adjust pH to 7.2 before autoclaving.
   2. Add 50 µg/ml of kanamycin and 100 µg/ml ampicillin for selective medium after autoclaving.
2. MA2 medium
   2% Glucose
   2% Peptone
   0.2% Yeast extract
   0.06% Myo-inositol
   2% Agar (for solid media)
   Dissolve completely in distilled water and sterilize by autoclaving
   Notes:
   
   1. Adjust pH to 6.8 for solid media.
   2. Add 200 µg/ml of G418 for selective media after autoclaving.
3. SPA medium
   2% Corn steep liquor
   1% Glucose
   0.3% Soybean oil
   0.3% Yeast extract
   0.3% Malt extract
   4% Agar (for solid media)
   Dissolve completely in distilled water and sterilize by autoclaving
   Notes:
   
   1. Adjust pH to 6.8 for solid media.
   2. Add 200 µg/ml G418 for selective media after autoclaving.
4. STM buffer
   270 mM sucrose
   10 mM Tris base pH 7.5
   1 mM MgCl₂
   Dissolve completely in distilled water and sterilize by autoclaving
5. TBE buffer (20x final concentration)
   90 mM Tris base
   90 mM Boric acid
   2 mM EDTA
   Dissolve completely and sterilize by autoclaving
6. DNA extraction buffer
   2% PVP 40
   0.2% BSA
   0.05 M Tween 20
   0.05 M sodium carbonate buffer pH 9.6
   Note: For the sodium carbonate buffer pH 9.6, add 0.45 ml solution A and 0.182 ml solution B in 9.36 ml of distilled water.
   Solution A = Sodium hydrogen carbonate (NaHCO₃) 0.1 M (Add 0.84 g in 10 ml of dH₂O).
   Solution B = Sodium carbonate anhydrous (Na₂CO₃) 0.1 M (Add 1.06 g in 10 ml of dH₂O).
   Store the DNA extraction buffer at -20 °C.
7. Transformation buffer
   0.5 ml of DTT 1M
   1 ml of potassium phosphate buffer (see below)
   18.5 ml of distilled water
   Mix and sterilize by filtration through a 0.45 µm filter
   Note: Prepare 20 ml of transformation buffer for each strain of transformation.
8. Potassium phosphate buffer
   
   1. Mix potassium phosphate monobasic (KH₂PO₄) 1 M with potassium phosphate dibasic (K₂HPO₄) 1 M in equal volumes
   2. Adjust the pH to 7.0
   3. Sterilize by autoclaving

Acknowledgments

This work was financed by grants from the Spanish Ministerio de Economía y Competitividad (BIO2017-88435-R) and Junta de Castilla y León (SA016P17) to JLR and AJ. GM-F was recipient of an FPI predoctoral contract from the Spanish Ministerio de Economía y Competitividad. We thank María Dolores Sánchez and Silvia Domínguez for excellent technical help.
  This protocol was derived from the following original research paper (Jiménez et al., 2019).

Competing interests

The authors declare no financial or commercial conflict of interest.

References

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