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Feb 2018

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Delivery of the Cas9 or TevCas9 System into Phaeodactylum tricornutum via Conjugation of Plasmids from a Bacterial Donor
通过细菌供体质粒接合转移的方法将Cas9或 TevCas9系统导入三角褐指藻   

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Abstract

Diatoms are an ecologically important group of eukaryotic microalgae with properties that make them attractive for biotechnological applications such as biofuels, foods, cosmetics and pharmaceuticals. Phaeodactylum tricornutum is a model diatom with defined culture conditions, but routine genetic manipulations are hindered by a lack of simple and robust genetic tools. One obstacle to efficient engineering of P. tricornutum is that the current selection methods for P. tricornutum transformants depend on the use of a limited number of antibiotic resistance genes. An alternative and more cost-effective selection method would be to generate auxotrophic strains of P. tricornutum by knocking out key genes involved in amino acid biosynthesis, and using plasmid-based copies of the biosynthetic genes as selective markers. Previous work on gene knockouts in P. tricornutum used biolistic transformation to deliver CRISPR-Cas9 system into P. tricornutum. Biolistic transformation of non-replicating plasmids can cause undesired damage to P. tricornutum due to random integration of the transformed DNA into the genome. Subsequent curing of edited cells to prevent long-term overexpression of Cas9 is very difficult as there is currently no method to excise integrated plasmids. This protocol adapts a new method to deliver the Cas9 or TevCas9 system into P. tricornutum via conjugation of plasmids from a bacterial donor cell. The process involves: 1) design and insertion of a guideRNA targeting the P. tricornutum urease gene into a TevCas9 expression plasmid that also encodes a conjugative origin of transfer, 2) installation of this plasmid in Escherichia coli containing a plasmid (pTA-Mob) containing the conjugative machinery, 3) transfer of the TevCas9 expression plasmid into P. tricornutum by conjugation, 4) screening of ex-conjugants for urease knockouts using T7 Endonuclease I and phenotypic screening, and 5) curing of the plasmid from edited cells.

Keywords: CRISPR-Cas9 (CRISPR-Cas9), Conjugation (结合), Phaeodactylum tricornutum (三角褐指藻), Auxotroph (营养缺陷型), Genome editing (基因组编辑), Diatoms (硅藻)

Background

The CRISPR system is a bacterial immune system that recognizes foreign DNA and leads to the activation of a targeted endonuclease, such as Cas9, which will generate a double strand break (DSB) in the invading DNA (Jinek et al., 2012; Wright et al., 2016). This system has been co-opted for genome editing applications whereby a single chimeric guide RNA (gRNA) can program Cas9 to target genes in model organisms, including in P. tricornutum (Daboussi et al., 2014). However, Cas9 generates blunt end DSB and the length of indels generated upon DNA repair is difficult to predict. Thus, in this study we used a dual endonuclease, TevCas9 to target the urease gene. TevCas9 is a dual endonuclease that is generated through the fusion of the I-TevI nuclease domain to Cas9 via a linker region (Wolfs et al., 2016). TevCas9 creates 33-36 bp deletions with non-compatible DNA ends (Wolfs et al., 2016). A previous study used transcription activator-like effector nucleases (TALENs) delivered by biolistic transformation (Weyman et al., 2015) to generate knockouts of the urease gene in P. tricornutum. To create an efficient system for gene editing in P. tricornutum, we adapted and optimized a plasmid-based system to deliver Cas9 or TevCas9 into P. tricornutum via conjugation from a bacterial donor cell (Karas et al., 2015). The new conjugation-based method to deliver Cas9 or TevCas9 described here is simpler, more efficient, and does not require specialized equipment for biolistic transformation (Slattery et al., 2018).

Materials and Reagents

  1. Pipette tips, 100-1,250 μl (VWR, catalog number: 89079-470 )
  2. Pipette tips, 1-200 μl (VWR, catalog number: 89079-478 )
  3. Pipette tips, 0.1-10 μl (VWR, catalog number: 89079-464 )
  4. 1.5 ml Eppendorf tubes (Corning, Axygen®, catalog number: MCT-150-C )
  5. 0.2 ml PCR tubes (VWR, catalog number: 20170-012 )
  6. Parafilm (Bemis, catalog number: PM996 )
  7. 50 ml centrifuge tubes (Greiner Bio One, catalog number: 227261 )
  8. NalgeneTM filters (Thermo Fisher Scientific, catalog number: 595-4520 )
  9. Phaeodactylum tricornutum cells [Culture Collection of Algae and Protozoa (CCAP, UK), catalog number: 1055/1 ] grown in 50 ml Falcon tubes
  10. TransforMaxTM EPI300TM Chemically competent E. coli cells (Lucigen, catalog number: C300C105 )
  11. pKS diaCas9_sgRNA plasmid (Addgene, catalog number: 74923
  12. BsaI-HF restriction endonuclease (New England Biolabs, catalog number: R0535S )
  13. P1 buffer (QIAGEN, catalog number: 19051 )
  14. β-mercaptoethanol (VWR, catalog number: 97064-588 )
  15. Alkaline lysis (P2) buffer (QIAGEN, catalog number: 19052 )
  16. Neutralization (P3) buffer (QIAGEN, catalog number: 19053 )
  17. Isopropanol (Sigma-Aldrich, catalog number: 190764 )
  18. 95% (v/v) EtOH (Commercial Alcohols, catalog number: P016EA95 )
  19. T4 DNA ligase with buffer (New England Biolabs, catalog number: M0202S )
  20. 2-Propanol-205 (Caledon Laboratories, catalog number: 8601-7-40 )
  21. BSA (Sigma-Aldrich, catalog number: A2058 )
  22. BsaI (New England Biolabs, catalog number: R0535S )
  23. EZ-10 spin column Miniprep Kit (Bio Basic, catalog number: BS614 )
  24. EZ-10 spin column PCR product purification kit (Bio Basic, catalog number: BS363 )
  25. Bacto agar (BD, BactoTM, catalog number: 214030 )
  26. Agar A (Bio Basic, catalog number: FB0010 )
  27. Yeast extract (BioShop, catalog number: YEX401 )
  28. Bacteriological tryptone (BioShop, catalog number: TRP402 )
  29. Sodium chloride (NaCl) (Fisher Scientific, Fisher Chemical, catalog number: S271-500 )
  30. AmpliTaq 360 DNA polymerase, 10x buffer, and 25 mM Magnesium Chloride (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4398828 )
  31. ZeocinTM (InvivoGen, catalog number: ant-zn-5p )
  32. Ampicillin (BioShop, catalog number: AMP201.100 )
  33. Gentamicin sulfate (Bio Basic, catalog number: GB0217 )
  34. D-Glucose (BioShop, catalog number: GLU501.1 )
  35. Zymolyase 20 T (BioShop, catalog number: ZYM001.1 )
  36. Tris (Fisher Scientific, Fisher BioReagents, catalog number: BP152-5 )
  37. Glycerol (Fisher Scientific, Fisher Chemical, catalog number: G33-1 )
  38. Sodium sulfate (Na2SO4) (Fisher Scientific, Fisher Chemical, catalog number: S421-300LB )
  39. Potassium chloride (KCl) (Merck, EMD Millipore, catalog number: PX1405-1 )
  40. Sodium bicarbonate (NaHCO3) (Merck, EMD Millipore, catalog number: SX0320-1 )
  41. Potassium bromide (KBr) (The Science Company, catalog number: NC-2136 )
  42. Boric acid (H3BO3) (BioShop, catalog number: BOR001.1 )
  43. Sodium fluoride (NaF) (Sigma-Aldrich, catalog number: S7920 )
  44. Magnesium chloride hexahydrate (MgCl2•6H2O) (BioShop, catalog number: MAG510.1 )
  45. Calcium chloride dihydrate (CaCl2•2H2O) (Fisher Scientific, catalog number: C79-3 )
  46. Strontium chloride hexahydrate (SrCl2•6H2O) (Sigma-Aldrich, catalog number: 255521 )
  47. Sodium nitrate (NaNO3) (Fisher Scientific, catalog number: S343-500 )
  48. Sodium phosphate monobasic monohydrate (NaH2PO4•H2O) (Sigma-Aldrich, catalog number: S9638 )
  49. Iron(III) chloride hexahydrate (FeCl3•6H2O) (Sigma-Aldrich, catalog number: 236489 )
  50. Na2EDTA•2H2O (Bio Basic, catalog number: EB0185 )
  51. Copper (II) sulfate pentahydrate (CuSO4•5H2O) (Bio Basic, catalog number: CDB0063 )
  52. Sodium molybdate dihydrate (Na2MoO4•2H2O) (Avantor Performance Materials, catalog number: 3764-01 )
  53. Zinc sulfate heptahydrate (ZnSO4•7H2O) (Alfa Aesar, catalog number: A12915-36
  54. Cobalt (II) chloride hexahydrate (CoCl2•6H2O) (Sigma-Aldrich, catalog number: C3169 )
  55. Manganese(II) chloride tetrahydrate (MnCl2•4H2O) (BioShop, catalog number: MAN222 )
  56. Selenous acid (H2SeO3) (Sigma-Aldrich, catalog number: 229857 )
  57. Nickel(II) sulfate (NiSO4) (Sigma-Aldrich, catalog number: 656895 )
  58. Sodium orthovanadate (Na3VO4) (BioShop, catalog number: SPP310 )
  59. Potassium chromate (K2CrO4) (Sigma-Aldrich, catalog number: 216615 )
  60. Thiamine-HCl (Sigma-Aldrich, catalog number: 47858 )
  61. Biotin (Sigma-Aldrich, catalog number: B4639 )
  62. Cyanocobalamin (Sigma-Aldrich, catalog number: C3607 )
  63. Zymogen solution (see Recipes) 
  64. Lysis buffer for P. tricornutum cells (see Recipes)
  65. LB medium (see Recipes)
  66. SOC media (see Recipes)
  67. LB agar plates (see Recipes) containing 100 μg ml-1 ampicillin or 100 μg ml-1 ampicillin with 40 μg ml-1 gentamicin
  68. 50% L1, 5% LB, 1% agar plate (1 L) (see Recipes)
  69. L1 media (see Recipes)
  70. L1 agar plates (see Recipes) containing 50 μg ml-1 ZeocinTM 
  71. 2x Aquil salt (see Recipes)
  72. NP stock (see Recipes)
  73. 1,000x L1 trace metals (see Recipes)
  74. Vitamin solution (see Recipes)

Equipment

  1. Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, InvitrogenTM, model: Qubit® 2.0 , catalog number: Q32866)
  2. Heat block (VWR, catalog number: 13259-030 )
  3. Water bath (Fisher Scientific, model: IC 2100 )
  4. Micropipettes (PIPETMAN, variable volume)
  5. Binocular Microscope (Leitz Wetzlar, model: Labolux12 )
  6. Hemocytometer 
  7. Table top centrifuge (Hettich Lab Technology, catalog number: 2004-01 )
  8. Centrifuge (Beckman Coulter, model: Avanti® J-26 XPI , catalog number: 393127)
  9. PCR thermal cycler (Bio-Rad Laboratories, model: T100TM, catalog number: 1861096 )
  10. UV-visible spectrophotometer (Biochrom, model: ULTROSPEC 2100® , catalog number: 80-2112-21)
  11. Incubator shaker (Thermo Fisher Scientific, model: Large Incubated and Refrigerated, catalog number: SHKE5000-7 )
  12. Vortex
  13. Autoclave
  14. Refrigerator

Procedure

  1. Designing Cas9-sgRNA targeting the urease gene
    1. Obtain the pKSconj diaCas9 or diaTevCas9 (pPtGE34 or pPtGE35) plasmid (Figure 1) from Addgene (107932 or 107999).


      Figure 1. Plasmid map for pKSconj. diaCas9/diaTevCas9 (pPtGE34/pPtGE35)

    2. Download the P. tricornutum urease gene sequence. NCBI Gene ID: 7194881 (PHATRDRAFT_29702).
    3. Format the sequence in FASTA format with a sequence identifier on line 1 preceded by a > followed by the nucleotide sequence. 
    4. Use a custom Perl script to search for Cas9 and TevCas9 gRNA target sites in the urease gene, ~15 bp downstream of a 5’-CNNNG-3’ I-TevI cleavage site (see Supplemental files). A typical TevCas9 target site is illustrated in Figure 2.


      Figure 2. Illustration of TevCas9 target site with predicted deletion size

    5. Select four target sites from the list of predicted sites for the P. trincornutum urease gene. Order an oligonucleotide corresponding to the predicted gRNA binding site (underlined sequence in Table 1), and the complementary oligonucleotide. Design the oligonucleotides with top strand 5’-TCGA-3’ and bottom strand 3’-CAAA-5’ BsaI restriction cut site overhangs to facilitate cloning into the Cas9 and TevCas9 vectors (Table 1).

      Table 1. Sequences of gRNAs targeting the urease gene in P. tricornutum


  2. Cloning gRNAs into pKSconj diaCas9 and pKSconj diaTevCas9 plasmids
    1. Phosphorylate and anneal the ordered oligos to generate gRNA cloning inserts. Prepare a 50 μl gRNA phosphorylation reaction (Table 2) in a 0.2 ml PCR tube. Place the reaction in a thermocycler and incubate at 37 °C for 30 min and then denature at 95 °C for 5 min. Next, slowly cool to 50 °C at a rate of -1 °C min-1 to allow annealing of gRNA top and bottom strands.

      Table 2. Reagents for gRNA phosphorylation


    2. Clone gRNAs into the pKSconj diaCas9 and pKSconj diaTevCas9 plasmids using Golden Gate assembly (Table 3). Place the reaction in a thermocycler and run the protocol as described in Table 4.

      Table 3. Reagents for Golden Gate assembly



      Table 4. Thermocycler setting for Golden Gate assembly


    3. Transform the product from the Golden Gate reaction into Epi300 E. coli cells using heat shock as follows: For one reaction, mix 3 μl of Golden Gate assembly product with 30 μl of Epi300 E. coli cells. Incubate the reaction on ice for 30 min and heat shock at 42 °C for 45 sec, then incubate on ice for 2 min. Add 400 μl of SOC to the reaction and recover cells at 37 °C with shaking at 225 RPM for 90 min. Plate 150 μl of recovered cells onto LB + Ampicillin media and incubate at 37 °C for 16 h/overnight.
    4. The following day, inoculate 1 to 5 colonies into separate glass test tubes containing 3 ml of LB + Ampicillin and incubate at 37 °C with shaking at 225 RPM for 16 h/overnight.
    5. After overnight growth, take 3 ml of overnight culture to isolate plasmid DNA (Bio Basic Inc. plasmid miniprep kit). Send isolated plasmid DNA for Sanger sequencing at the London Regional Genomics Centre using a sequencing primer upstream of the gRNA insertion site to identify a plasmid containing a properly cloned gRNA. 
    6. Transform the properly cloned gRNA plasmid into Epi300 E. coli containing the pTA-Mob (Strand et al., 2014) plasmid, obtained from Dr. Rahmi Lale, using the heat shock protocol described in Step 3 with 1 μl of plasmid and 30 μl of E. coli cells.

  3. Transfer of plasmid into P. tricornutum via conjugation from E. coli
    Note: This protocol was adapted from a published protocol (Karas et al., 2015).
    1. P. tricornutum growth conditions: 18 °C under cool white fluorescent light (75 μE m-2 sec-1) with a photoperiod of 16 h light and 8 h dark in 50 ml Falcon tubes at the Biotron Experimental Climate Change Research Centre at Western University. 
    2. Briefly, adjust 250 μl of P. tricornutum liquid culture to 1.0 x 108 cells ml-1, plate onto a 50% L1, 1% agar plate, and grow for 4 days.
    3. Add 2 ml of L1 media to the plate, scrape and collect cells into a 1.5 ml Eppendorf tube.
    4. Count the cells using a hemocytometer and adjust the concentration to 5 x 108 cells ml-1.
    5. Grow 3 ml E. coli cultures containing the diaCas9 or diaTevCas9 plasmids with gRNAs at 37 °C overnight. Dilute 1 ml of overnight culture to 50 ml of LB with 100 μg ml-1 ampicillin and 40 μg ml-1 gentamicin and grow the culture to an OD600 of 0.8-1.0.
    6. Centrifuge the culture for 10 min at 3,000 x g in a 50 ml Falcon tube.
    7. Discard the supernatant and resuspend the pellet in 500 μl SOC media.
    8. Mix 200 μl of P. tricornutum from Step 4 with 200 μl of E. coli from Step 7 and plate them onto a 50% L1, 5% LB, 1% agar plate.
    9. Incubate the plate for 90 min at 30 °C, then for 2 days at 18 °C with light.
    10. After 2 days, add 1.5 ml of L1 media to the plate, scrape and collect cells in a 1.5 ml Eppendorf tube.
    11. Plate 500 μl of the scraped cells onto a 50% L1, 1% agar plate containing 50 μg ml-1 ZeocinTM.
    12. Parafilm the plate and incubate the plate for 14 days at 18 °C with light. A typical conjugation experiment should yield 1.0 x 102 - 1.3 x 104 total exconjugants.

  4. Screening for urease knockouts in P. tricornutum induced by Cas9 and TevCas9 (see Figure 3 for an overview of screening procedure) - T7 Endonuclease I assay


    Figure 3. Overview of screening procedure for P. tricornutum urease knockouts. pKSconj diaCas9 or pKSconj diaTevCas9 plasmid is delivered to P. tricornutum by E. coli. Screen the exconjugants with T7EI assay. Isolate sub-clones from exconjugants and perform phenotypic screen by plating sub-clones onto urea and nitrate plates. Verify the deletion length by Sanger Sequencing.

    1. After 14 days, randomly select ten P. tricornutum exconjugants and streak them onto an L1 + ZeocinTM plate and grow for 7 days.
    2. Scrape 50% of the cells from one streak and place it into 3 ml of L1 media with 50 μg ml-1 ZeocinTM. Suspend the other 50% of cells in 100 μl TE (pH 8.0) buffer, flash freeze at -80 °C for 15 min, and heat lyse at 95 °C for 5 min. 
    3. Use the lysed P. tricornutum exconjugant cells as the template for PCR amplification of the gRNA target site using AmpliTaq DNA polymerase. Design the PCR so the product will be ~500 bp using primers located ~250 bp upstream and downstream of the gRNA target site. Analyze the PCR products on a 1% agarose gel to confirm the size and presence of the PCR product. 
    4. Denature the PCR product at 95 °C for 5 min and slowly cool to 50 °C and then freeze the sample at -20 °C for 2 min to generate heteroduplexes of edited and unedited target sites.
    5. Use the PCR product as the substrate for a T7EI assay (Table 5). Incubate the reaction at 37 °C for 15 min and analyze the digested product on a 1% agarose gel. PCR products from exconjugant that yield two bands on the agarose gel (500 bp and 250 bp) contains editing at the target site by Cas9 or TevCas9. An example of the T7EI assay gel is shown in Figure 4.

      Table 5. Reagents for T7EI assay



      Figure 4. Example analysis of Cas9 activity by T7EI assay. P. tricornutum colonies were resuspended in TE (pH 8.0) buffer followed by flash freezing at -80 °C for 15 min followed by cell lysis at 95 °C for 12 min. The urease gene target site was PCR amplified. PCR product was denatured at 95 °C and cooled down to 50 °C to allow random reannealing. T7E1 assay was performed using the randomly annealed PCR product as template and incubated at 37 °C for 15 min with T7EI and NEB 2 buffer. Products were loaded onto a 1% agarose gel at 100 V for 40 min. Asterisks (*) denote colonies positive for editing. The numbers (11.12, 11.13….) stand for different colonies.

    6. Dilute the liquid cultures from Step D2 of P. tricornutum exconjugants that were identified to have editing to 10-4 and plate onto L1+ ZeocinTM plates and grow for 10-14 days to obtain sub-clones.

  5. Phenotypic screening of urease knockouts (see Figure 5 for an example of the phenotypic screen)
    1. Streak sub-clones first onto L1 + ZeocinTM plates containing 2.1 mM urea with a pipette tip, use the same tip to streak the sub-clone onto an L1 + ZeocinTM plate containing 4.2 mM nitrate and grow for 5 days. Sub-clones that grow on L1 + ZeocinTM with nitrate but not on L1 + ZeocinTM with urea are urease knockouts.


      Figure 5. Example phenotypic screen of P. tricornutum sub-clones on urea and nitrate plates. Dilutions of Cas9 and TevCas9 sub-clones were spot plated onto L1 with nitrate and L1 with urea plates.

    2. PCR amplify the target site, as in Steps D2-D3, from sub-clones identified as urease knockouts and send the PCR product for Sanger sequencing.

  6. Curing of the pKSconj diaCas9 and diaTevCas9 plasmids from urease knockout sub-clones
    1. Inoculate identified urease knockout sub-clones in 3 ml of L1 media without ZeocinTM and grow for 7 days.
    2. After 7 days, dilute (10-4) and plate the liquid cultures onto L1 plates and grow for 7 days to obtain single colonies.
    3. Randomly select 10 colonies and streak them onto L1 and L1 + ZeocinTM plates and grow for 5 days. Growth on L1 plates but not on L1 + ZeocinTM plates indicates colonies that are successfully cured of the plasmid.

Data analysis

  1. Ten exconjugants of P. tricornutum containing the pKSconj diaCas9 or diaTevCas9 plasmid with different gRNAs were randomly selected for T7EI analysis. 
  2. From those, 5 sub-clones were randomly selected for phenotypic screening. 
  3. To determine the removal of the plasmid, 10 colonies were randomly selected for Zeocin sensitivity screening.

Notes

  1. Multiple gRNAs may need to be designed as the activity of Cas9 and TevCas9 varies when using different gRNAs.
  2. The number of edited colonies may vary, more exconjugants may have to be selected to identify edited clones and obtain more accurate editing efficiencies.

Recipes

  1. Zymolyase solution
    200 mg zymolyase 20 T (USB)
    9 ml dH2O
    1 ml 1 M Tris pH 7.5
    10 ml 50% glycerol
  2. Lysis buffer for P. tricornutum cells (1 L)
    10 ml 1 M Tris pH 8.0
    2 ml 0.5 M EDTA
    988 ml dH2O
  3. LB medium (1 L)
    10 g tryptone
    5 g yeast extract
    10 g NaCl
    1 L dH2O
    Sterilize by autoclave at 121 °C with 1 atmosphere pressure for 15 min
  4. SOC media (1 L)
    20 g tryptone
    5 g yeast extract
    0.5 g NaCl
    10 ml 250 mM KCl
    5 ml 2 M MgCl
    20 ml 1 M glucose
    900 ml dH2O
  5. LB agar plates (containing 100 μg ml-1 ampicillin or 100 μg ml-1 ampicillin with 40 μg ml-1 gentamicin) (1 L)
    10 g tryptone
    5 g yeast extract
    10 g NaCl
    15 g Agar
    0.1 g ampicillin or 0.1 g ampicillin with 0.4 g gentamicin
    1 L dH2O
  6. 50% L1, 5% LB, 1% agar plate (1 L)
    500 ml L1
    50 ml LB
    10 g bacto agar
  7. L1 media (1 L)
    500 ml 2x Aquil salt
    2 ml NP stock
    500 μl vitamin solution
    1 ml 1,000x L1 trace metals
    497.5 ml dH2O
    Sterilize by vacuum filtration with NalgeneTM filters
  8. L1 agar containing 50 μg ml-1 ZeocinTM (1 L)
    500 ml L1 media
    10 g bacto agar
    0.5 g ZeocinTM
    500 ml dH2O
  9. 2x Aquil salt (1 L)
    24.5 g NaCl
    4.09 g Na2SO4
    0.7 g KCl
    0.2 g NaHCO3
    0.1 KBr
    0.03 g H3BO3
    0.003 g NaF
    11.1 g MgCl2•6H2O
    1.54 g CaCl2•2H2O
    0.017 g SrCl2•6H2O
  10. NP stock (100 ml)
    37.5 g NaNO3
    2.5 g NaH2PO4•H2O
  11. 1,000x L1 trace metals (1 L)
    3.15 g FeCl3•6H2O
    4.36 g Na2EDTA•2H2O
    0.25 ml CuSO4•5H2O (9.8 g L-1 dH2O)
    3.0 ml Na2MoO4•2H2O (6.3 g L-1 dH2O)
    1.0 ml ZnSO4•7H2O (22.0 g L-1 dH2O)
    1.0 ml CoCl2•6H2O (10.0 g L-1 dH2O)
    1.0 ml MnCl2•4H2O (180.0 g L-1 dH2O)
    1.0 ml H2SeO3 (1.3 g L-1 dH2O)
    1.0 ml NiSO4•6H2O (2.7 g L-1 dH2O)
    1.0 ml Na3VO4 (1.84 g L-1 dH2O)
    1.0 ml K2CrO4 (1.94 g L-1 dH2O)
  12. Vitamin solution (1 L)
    200 mg Thiamine-HCl
    10 ml of 0.1 g L-1 biotin
    1 ml of 1 g L-1 cyanocobalamin

Acknowledgments

This work was supported by Designer Microbes Inc., an NSERC ENGAGE Grant to D.R.E. and Designer Microbes Inc. (EGP/486420-2015), an Ontario Centres of Excellence VIP-1 Grant to Designer Microbes Inc. (OCE-VIP-1/23879), and NSERC Discovery Grant to D.R.E. (RGPIN-2015-04800).
This protocol was developed from the following published paper: Slattery et al. (2018).

Competing interests

The authors declare no conflicts of interests.

References

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  11. Wright, A. V., Nuñez, J. K., Doudna, J. A. (2016). Biology and applications of CRISPR systems: Harnessing nature's toolbox for genome engineering. Cell 164(1-2): 29-44.

简介

硅藻是一种具有重要生态意义的真核微藻类,其特性使其对生物燃料,食品,化妆品和药品等生物技术应用具有吸引力。 Phaeodactylum tricornutum 是具有确定培养条件的模型硅藻,但缺乏简单而强大的遗传工具阻碍了常规遗传操作。有效设计 P的一个障碍。 tricornutum 是 P的当前选择方法。 tricornutum 转化体依赖于使用有限数量的抗生素抗性基因。另一种更具成本效益的选择方法是产生 P的营养缺陷型菌株。通过敲除参与氨基酸生物合成的关键基因,并使用基于质粒的生物合成基因拷贝作为选择标记,使三角酵母。以前关于 P基因敲除的研究。 tricornutum 使用biolistic转换将CRISPR-Cas9系统传递到 P.藻。非复制质粒的生物射弹转化可导致对 P的不期望的损伤。由于转化的DNA随机整合到基因组中,tricornutum 。随后固化编辑的细胞以防止Cas9的长期过表达是非常困难的,因为目前没有方法来切除整合的质粒。该协议采用新方法将Cas9或TevCas9系统传送到 P. tricornutum 通过来自细菌供体细胞的质粒的缀合。该过程涉及:1)设计和插入靶向 P的guideRNA。将tricornutum 尿素酶基因导入TevCas9表达质粒,该质粒也编码转移的接合起点,2)将该质粒安装在含有含有接合机制的质粒(pTA-Mob)的大肠杆菌中, 3)将TevCas9表达质粒转移到 P中。通过缀合,tricornutum ,4)使用T7核酸内切酶I筛选前缀合物的脲酶敲除和表型筛选,和5)从编辑的细胞中固化质粒。

【背景】 CRISPR系统是一种细菌免疫系统,可识别外源DNA并导致靶向核酸内切酶(如Cas9)的激活,这将在入侵的DNA中产生双链断裂(DSB)(Jinek 等。,2012; Wright et al。,2016)。该系统已被选择用于基因组编辑应用,其中单个嵌合指导RNA(gRNA)可以编程Cas9以靶向模型生物中的基因,包括 P. tricornutum (Daboussi et al。,2014)。然而,Cas9产生钝端DSB并且难以预测DNA修复时产生的indel长度。因此,在这项研究中,我们使用双核酸内切酶TevCas9靶向脲酶基因。 TevCas9是双核酸内切酶,其通过I-TevI核酸酶结构域经由接头区域与Cas9融合而产生(Wolfs 等人,2016)。 TevCas9通过不相容的DNA末端产生33-36bp的缺失(Wolfs et al。,2016)。先前的研究使用通过生物射弹转化(Weyman 等人,,2015)递送的转录激活因子样效应核酸酶(TALEN)来产生 P中尿素酶基因的敲除。藻。在 P中创建一个有效的基因编辑系统。 tricornutum ,我们调整并优化了基于质粒的系统,将Cas9或TevCas9递送到 P中。 tricornutum 通过细菌供体细胞的结合(Karas et al。,2015)。这里描述的用于递送Cas9或TevCas9的新的基于缀合的方法更简单,更有效,并且不需要用于生物射弹转化的专用设备(Slattery et al。,2018)。

关键字:CRISPR-Cas9, 结合, 三角褐指藻, 营养缺陷型, 基因组编辑, 硅藻

材料和试剂

  1. 移液器吸头,100-1,250μl(VWR,目录号:89079-470)
  2. 移液器吸头,1-200μl(VWR,目录号:89079-478)
  3. 移液器吸头,0.1-10μl(VWR,目录号:89079-464)
  4. 1.5毫升Eppendorf管(Corning,Axygen ®,目录号:MCT-150-C)
  5. 0.2 ml PCR管(VWR,目录号:20170-012)
  6. Parafilm(Bemis,目录号:PM996)
  7. 50毫升离心管(Greiner Bio One,目录号:227261)
  8. Nalgene TM 过滤器(Thermo Fisher Scientific,目录号:595-4520)
  9. Phaeodactylum tricornutum 细胞[藻类和原生动物的培养物收集(CCAP,UK),目录号:1055/1]在50 ml Falcon管中生长
  10. TransforMax TM EPI300 TM 化学上有能力的 E.大肠杆菌细胞(Lucigen,目录号:C300C105)
  11. pKS diaCas9_sgRNA质粒(Addgene,目录号:74923) 
  12. BsaI-HF限制性内切酶(New England Biolabs,目录号:R0535S)
  13. P1缓冲液(QIAGEN,目录号:19051)
  14. β-巯基乙醇(VWR,目录号:97064-588)
  15. 碱性裂解(P2)缓冲液(QIAGEN,目录号:19052)
  16. 中和(P3)缓冲液(QIAGEN,目录号:19053)
  17. 异丙醇(Sigma-Aldrich,目录号:190764)
  18. 95%(v / v)EtOH(商品醇,目录号:P016EA95)
  19. 带缓冲液的T4 DNA连接酶(New England Biolabs,目录号:M0202S)
  20. 2-丙醇-205(Caledon Laboratories,目录号:8601-7-40)
  21. BSA(Sigma-Aldrich,目录号:A2058)
  22. BsaI(New England Biolabs,目录号:R0535S)
  23. EZ-10旋转柱Miniprep Kit(Bio Basic,目录号:BS614)
  24. EZ-10离心柱PCR产物纯化试剂盒(Bio Basic,目录号:BS363)
  25. Bacto琼脂(BD,Bacto TM ,目录号:214030)
  26. Agar A(Bio Basic,目录号:FB0010)
  27. 酵母提取物(BioShop,目录号:YEX401)
  28. 细菌学胰蛋白胨(BioShop,目录号:TRP402)
  29. 氯化钠(NaCl)(Fisher Scientific,Fisher Chemical,目录号:S271-500)
  30. AmpliTaq 360 DNA聚合酶,10x缓冲液和25 mM氯化镁(Thermo Fisher Scientific,Applied Biosystems TM ,目录号:4398828)
  31. Zeocin TM (InvivoGen,目录号:ant-zn-5p)
  32. 氨苄青霉素(BioShop,目录号:AMP201.100)
  33. 硫酸庆大霉素(Bio Basic,目录号:GB0217)
  34. D-葡萄糖(BioShop,目录号:GLU501.1)
  35. Zymolyase 20 T(BioShop,目录号:ZYM001.1)
  36. Tris(Fisher Scientific,Fisher BioReagents,目录号:BP152-5)
  37. 甘油(Fisher Scientific,Fisher Chemical,目录号:G33-1)
  38. 硫酸钠(Na 2 SO 4 )(Fisher Scientific,Fisher Chemical,目录号:S421-300LB)
  39. 氯化钾(KCl)(Merck,默克密理博中国,目录号:PX1405-1)
  40. 碳酸氢钠(NaHCO 3 )(Merck,默克密理博中国,目录号:SX0320-1)
  41. 溴化钾(KBr)(科学公司,目录号:NC-2136)
  42. 硼酸(H 3 BO 3 )(BioShop,目录号:BOR001.1)
  43. 氟化钠(NaF)(Sigma-Aldrich,目录号:S7920)
  44. 氯化镁六水合物(MgCl 2 •6H 2 O)(BioShop,目录号:MAG510.1)
  45. 氯化钙二水合物(CaCl 2 •2H 2 O)(Fisher Scientific,目录号:C79-3)
  46. 氯化锶六水合物(SrCl 2 •6H 2 O)(Sigma-Aldrich,目录号:255521)
  47. 硝酸钠(NaNO 3 )(Fisher Scientific,目录号:S343-500)
  48. 磷酸二氢钠一水合物(NaH 2 PO 4 •H 2 O)(Sigma-Aldrich,目录号:S9638)
  49. 氯化铁(III)六水合物(FeCl 3 •6H 2 O)(Sigma-Aldrich,目录号:236489)
  50. Na 2 EDTA•2H 2 O(Bio Basic,目录号:EB0185)
  51. 硫酸铜(II)五水合物(CuSO 4 •5H 2 O)(Bio Basic,目录号:CDB0063)
  52. 钼酸钠二水合物(Na 2 MoO 4 •2H 2 O)(Avantor Performance Materials,目录号:3764-01)
  53. 硫酸锌七水合物(ZnSO 4 •7H 2 O)(Alfa Aesar,目录号:A12915-36) 
  54. 氯化钴(II)六水合物(CoCl 2 •6H 2 O)(Sigma-Aldrich,目录号:C3169)
  55. 氯化锰(II)四水合物(MnCl 2 •4H 2 O)(BioShop,目录号:MAN222)
  56. 亚硒酸(H 2 SeO 3 )(Sigma-Aldrich,目录号:229857)
  57. 硫酸镍(II)(NiSO 4 )(Sigma-Aldrich,目录号:656895)
  58. 原钒酸钠(Na 3 VO 4 )(BioShop,目录号:SPP310)
  59. 铬酸钾(K 2 CrO 4 )(Sigma-Aldrich,目录号:216615)
  60. 硫胺素-HCl(Sigma-Aldrich,目录号:47858)
  61. 生物素(Sigma-Aldrich,目录号:B4639)
  62. Cyanocobalamin(Sigma-Aldrich,目录号:C3607)
  63. 酶原解决方案(见食谱) 
  64. P的裂解缓冲液。三角酵母细胞(见食谱)
  65. LB培养基(见食谱)
  66. SOC媒体(见食谱)
  67. LB琼脂平板(见食谱)含有100μg/ ml -1 氨苄青霉素或100μg/ ml -1 氨苄青霉素和40μg/ ml -1 庆大霉素
  68. 50%L1,5%LB,1%琼脂平板(1升)(见食谱)
  69. L1媒体(见食谱)
  70. 含有50μgml- 1 Zeocin TM的L1琼脂平板(见食谱)
  71. 2x含水盐(参见食谱)
  72. NP库存(见食谱)
  73. 1,000x L1痕量金属(参见配方)
  74. 维生素溶液(见食谱)

设备

  1. Qubit ® 2.0荧光计(Thermo Fisher Scientific,Invitrogen TM ,型号:Qubit ® 2.0,目录号:Q32866)
  2. 加热块(VWR,目录号:13259-030)
  3. 水浴(Fisher Scientific,型号:IC 2100)
  4. 微量移液管(PIPETMAN,可变容积)
  5. 双目显微镜(Leitz Wetzlar,型号:Labolux12)
  6. 血球 
  7. 台式离心机(Hettich Lab Technology,目录号:2004-01)
  8. 离心机(Beckman Coulter,型号:Avanti ® J-26 XPI,目录号:393127)
  9. PCR热循环仪(Bio-Rad Laboratories,型号:T100 TM ,目录号:1861096)
  10. 紫外可见分光光度计(Biochrom,型号:ULTROSPEC 2100 ®,目录号:80-2112-21)
  11. 培养箱摇床(Thermo Fisher Scientific,型号:Large Incubated and Refrigerated,目录号:SHKE5000-7)
  12. 涡流
  13. 高压灭菌器
  14. 冰箱

程序

  1. 设计靶向脲酶基因的Cas9-sgRNA
    1. 从Addgene(107932或107999)获得pKSconj diaCas9或diaTevCas9(pPtGE34或pPtGE35)质粒(图1)。


      图1. pKSconj的质粒图谱。 diaCas9 / diaTevCas9(pPtGE34 / pPtGE35)

    2. 下载 P. tricornutum 脲酶基因序列。 NCBI Gene ID:7194881(PHATRDRAFT_29702)。
    3. 使用序列标识符格式化FASTA格式的序列,第1行前面带有>然后是核苷酸序列。 
    4. 使用自定义Perl脚本搜索脲酶基因中的Cas9和TevCas9 gRNA靶位点,在5'-CNNNG-3'I-TevI切割位点下游约15bp处(参见补充文件)。典型的TevCas9目标站点如图2所示。


      图2.具有预测删除大小的TevCas9目标站点的插图

    5. 从 P的预测站点列表中选择四个目标站点。 trincornutum 脲酶基因。订购对应于预测的gRNA结合位点的寡核苷酸(表1中用下划线的序列)和互补的寡核苷酸。设计具有顶部链5'-TCGA-3'和底部链3'-CAAA-5'BsaI限制性切割位点突出端的寡核苷酸,以便于克隆到Cas9和TevCas9载体中(表1)。

      表1. P中靶向脲酶基因的gRNA序列。 tricornutum


  2. 将gRNA克隆到pKSconj diaCas9和pKSconj diaTevCas9质粒中
    1. 对有序寡核苷酸进行磷酸化和退火以产生gRNA克隆插入物。在0.2ml PCR管中制备50μlgRNA磷酸化反应(表2)。将反应物置于热循环仪中并在37℃下孵育30分钟,然后在95℃下变性5分钟。接下来,以-1℃min -1 的速率缓慢冷却至50℃,以允许gRNA顶部和底部链的退火。

      表2. gRNA磷酸化的试剂


    2. 使用Golden Gate组装将gRNA克隆到pKSconj diaCas9和pKSconj diaTevCas9质粒中(表3)。将反应物置于热循环仪中并按照表4中所述运行方案。

      表3.金门装配的试剂


      表4.金门组件的热循环仪设置


    3. 将产品从金门反应转换为Epi300 E。使用热休克的大肠杆菌细胞如下:对于一次反应,将3μl金门装配产品与30μlEpi300 E混合。大肠杆菌细胞。将反应在冰上孵育30分钟并在42℃热激45秒,然后在冰上孵育2分钟。向反应中加入400μlSOC并在37℃下以225RPM振荡回收细胞90分钟。将150μl回收的细胞置于LB +氨苄青霉素培养基上,并在37℃下孵育16小时/过夜。
    4. 第二天,将1至5个菌落接种到含有3ml LB +氨苄青霉素的单独玻璃试管中,并在37℃下以225RPM振荡孵育16小时/过夜。
    5. 过夜生长后,取3ml过夜培养物以分离质粒DNA(Bio Basic Inc.质粒小量制备试剂盒)。使用gRNA插入位点上游的测序引物,在伦敦地区基因组学中心发送用于Sanger测序的分离的质粒DNA,以鉴定含有正确克隆的gRNA的质粒。 
    6. 将正确克隆的gRNA质粒转化到Epi300 E中。含有pTA-Mob(Strand et al。,2014)质粒的大肠杆菌,来自Rahmi Lale博士,使用步骤3中描述的热休克方案,含有1μl质粒和30 μl的 E.大肠杆菌细胞。

  3. 将质粒转移到 P中。 tricornutum 通过 E的结合。大肠杆菌
    注意:该协议改编自已发布的协议(Karas等,2015)。
    1. P上。三角褐指藻生长条件:冷白色荧光灯下的温度为18°C(75μEm -2 sec -1 ),光周期为16 h,光照时间为8 h在西部大学Biotron实验气候变化研究中心的50毫升Falcon管中黑暗。 
    2. 简而言之,调整250μl P.将tricornutum 液体培养至1.0×10 8个细胞ml -1 ,平板接种到50%L1,1%琼脂平板上,并生长4天。
    3. 向板中加入2ml L1培养基,刮取细胞并收集到1.5ml Eppendorf管中。
    4. 使用血细胞计数器计数细胞,并将浓度调节至5×10 5 sup / ml细胞ml-1。
    5. 增长3毫升 E.含有带有gRNA的diaCas9或diaTevCas9质粒的大肠杆菌培养物在37℃过夜。将1 ml过夜培养物稀释至50 ml含有100μg/ ml -1 氨苄青霉素和40μg/ ml -1 庆大霉素的LB,并将培养物培养至OD 600 0.8-1.0。
    6. 将培养物在50ml Falcon管中以3,000μLg/ g离心培养物10分钟。
    7. 弃去上清液,将沉淀重悬于500μlSOC培养基中。
    8. 混合200μl P.步骤4中的tricornutum 与200μl E。来自步骤7的大肠杆菌,将它们铺在50%L1,5%LB,1%琼脂平板上。
    9. 将板在30℃孵育90分钟,然后在18℃光照下孵育2天。
    10. 2天后,向板中加入1.5ml L1培养基,刮擦并收集1.5ml Eppendorf管中的细胞。
    11. 将500μl刮下的细胞置于含有50μg/ ml -1 Zeocin TM 的50%L1,1%琼脂平板上。
    12. 将板封口并将板在18℃下用光孵育14天。典型的缀合实验应该产生1.0×10 2 - 1.3×10 4 总共结合物。

  4. 在 P中筛选脲酶敲除。由Cas9和TevCas9诱导的三角褐指藻(参见图3的筛选程序概述) - T7核酸内切酶I检测


    图3. P的筛选程序概述。 tricornutum 尿素酶敲除。 pKSconj diaCas9或pKSconj diaTevCas9质粒被递送至 P. tricornutum 由 E。大肠杆菌。用T7EI试验筛选出结合物。从excjugants中分离亚克隆,并通过将亚克隆接种到尿素和硝酸盐平板上进行表型筛选。通过Sanger Sequencing验证删除长度。

    1. 14天后,随机选择10个 P.将三角酵母与结合物偶联并将它们划线到L1 + Zeocin TM 平板上并生长7天。
    2. 从一条条纹上刮下50%的细胞,并将其置于3ml含有50μg/ ml 1 Zeocin TM 的L1培养基中。将其他50%的细胞悬浮在100μlTE(pH 8.0)缓冲液中,在-80℃下快速冷冻15分钟,并在95℃下加热裂解5分钟。 
    3. 使用裂解的 P.使用AmpliTaq DNA聚合酶将tricornutum excjugant细胞作为用于PCR扩增gRNA靶位点的模板。设计PCR,使用位于gRNA靶位点上游和下游约250bp的引物,产物约为500bp。在1%琼脂糖凝胶上分析PCR产物以确认PCR产物的大小和存在。 
    4. 使PCR产物在95℃下变性5分钟并缓慢冷却至50℃,然后在-20℃下冷冻样品2分钟以产生编辑和未编辑的靶位点的异源双链体。
    5. 使用PCR产物作为T7EI测定的底物(表5)。将反应在37℃孵育15分钟,并在1%琼脂糖凝胶上分析消化的产物。来自excjugant的PCR产物在琼脂糖凝胶(500bp和250bp)上产生两条带,包含Cas9或TevCas9在靶位点的编辑。 T7EI测定凝胶的一个例子如图4所示。

      表5.T7EI检测的试剂



      图4.通过T7EI测定分析Cas9活性的实例。 P上。将三角褐藻菌落重悬于TE(pH8.0)缓冲液中,然后在-80℃下快速冷冻15分钟,然后在95℃下细胞裂解12分钟。对脲酶基因靶位点进行PCR扩增。将PCR产物在95℃变性并冷却至50℃以允许随机再退火。使用随机退火的PCR产物作为模板进行T7E1测定,并在37℃下用T7EI和NEB 2缓冲液孵育15分钟。将产物在100V下加载到1%琼脂糖凝胶上40分钟。星号(*)表示对编辑有积极作用的菌落。数字(11.12,11.13 ......)代表不同的殖民地。

    6. 从 P的步骤D2稀释液体培养物。鉴定出具有编辑至10 -4 并且在L1 + Zeocin TM 平板上培养并且生长10-14天以获得亚克隆的三角酵母结合物。

  5. 脲酶敲除的表型筛选(参见图5的表型筛选实例)
    1. 首先使用移液管尖端将含有2.1 mM尿素的L1 + Zeocin TM 平板亚克隆,使用相同的尖端将亚克隆划线到L1 + Zeocin TM上含有4.2mM硝酸盐的平板并生长5天。使用硝酸盐在L1 + Zeocin TM 上生长但在L1 + Zeocin TM 上与尿素生长的亚克隆是脲酶敲除。


      图5. P的表型屏幕示例。尿酸盐和硝酸盐平板上的亚克隆 Cas9和TevCas9亚克隆的稀释液用硝酸盐和L1与尿素平板一起点在L1上。

    2. 如在步骤D2-D3中,PCR扩增靶位点,从鉴定为脲酶敲除的亚克隆中发送PCR产物用于Sanger测序。

  6. 从尿素酶敲除亚克隆中固化pKSconj diaCas9和diaTevCas9质粒
    1. 将鉴定的脲酶敲除亚克隆接种在3ml不含Zeocin TM 的L1培养基中并生长7天。
    2. 7天后,稀释(10 -4 )并将液体培养物平板接种到L1平板上并生长7天以获得单菌落。
    3. 随机选择10个菌落并将它们划线到L1和L1 + Zeocin TM 平板上并生长5天。在L1平板上但不在L1 + Zeocin TM 平板上生长表明质粒成功固化的菌落。

数据分析

  1. P的十个exconjugants。随机选择含有不同gRNA的pKSconj diaCas9或diaTevCas9质粒的tricornutum 进行T7EI分析。 
  2. 从那些中,随机选择5个亚克隆进行表型筛选。 
  3. 为了确定质粒的去除,随机选择10个菌落进行Zeocin灵敏度筛选。

笔记

  1. 当使用不同的gRNA时,可能需要设计多个gRNA,因为Cas9和TevCas9的活性变化。
  2. 编辑的菌落数可能会有所不同,可能必须选择更多的exconjugants来识别已编辑的克隆并获得更准确的编辑效率。

食谱

  1. Zymolyase解决方案
    200毫克酶解酶20 T(USB)
    9毫升dH 2 O
    1毫升1M Tris pH 7.5
    10毫升50%甘油
  2. P的裂解缓冲液。 tricornutum 细胞(1 L)
    10毫升1M Tris pH 8.0
    2毫升0.5毫升EDTA
    988ml dH 2 O.
  3. LB培养基(1升)
    10克胰蛋白胨
    5克酵母提取物
    10克NaCl
    1 L dH 2 O
    通过高压灭菌器在121℃,1大气压下灭菌15分钟
  4. SOC媒体(1升)
    20克胰蛋白胨
    5克酵母提取物
    0.5克NaCl
    10毫升250毫米KCl
    5毫升2M MgCl
    20毫升1M葡萄糖
    900毫升dH 2 O.
  5. LB琼脂平板(含有100μg/ ml -1 氨苄青霉素或100μg/ ml -1 氨苄青霉素含40μg/ ml -1 庆大霉素)(1 L )
    10克胰蛋白胨
    5克酵母提取物
    10克NaCl
    15克琼脂
    0.1克氨苄青霉素或0.1克氨苄青霉素和0.4克庆大霉素
    1 L dH 2 O.
  6. 50%L1,5%LB,1%琼脂平板(1L)
    500毫升L1
    50毫升LB
    10克bacto琼脂
  7. L1媒体(1 L)
    500毫升2x Aquil盐
    2毫升NP库存
    500μl维生素溶液
    1毫升1,000x L1微量金属
    497.5 ml dH 2 O
    用Nalgene TM 过滤器真空过滤灭菌
  8. L1琼脂含有50μg/ ml -1 Zeocin TM (1 L)
    500毫升L1媒体
    10克bacto琼脂
    0.5克ZeocinTM
    500毫升dH 2 O.
  9. 2x Aquil盐(1升)
    24.5克NaCl
    4.09g Na 2 SO 4
    0.7克KCl
    0.2克NaHCO 3
    0.1 KBr
    0.03g H 3 BO 3
    0.003克NaF
    11.1g MgCl 2 •6H 2 O
    1.54g CaCl 2 •2H 2 O
    0.017g SrCl 2 ·6H 2 O.
  10. NP库存(100毫升)
    37.5g NaNO 3
    2.5 g NaH 2 PO 4 •H 2 O
  11. 1,000x L1痕量金属(1 L)
    3.15 g FeCl 3 •6H 2 O
    4.36g Na 2 EDTA•2H 2 O
    0.25 ml CuSO 4 •5H 2 O(9.8 g L -1 dH 2 O)
    3.0 ml Na 2 MoO 4 •2H 2 O(6.3 g L -1 dH 2 < / sub> O)
    1.0 ml ZnSO 4 •7H 2 O(22.0 g L -1 dH 2 O)
    1.0 ml CoCl 2 •6H 2 O(10.0 g L -1 dH 2 O)
    1.0 ml MnCl 2• 4H 2 O(180.0 g L -1 dH 2 O)
    1.0ml H 2 SeO 3 (1.3 g L -1 dH 2 O)
    1.0 ml NiSO 4 •6H 2 O(2.7 g L -1 dH 2 O)
    1.0 ml Na 3 VO 4 (1.84 g L -1 dH 2 O)
    1.0 ml K 2 CrO 4 (1.94 g L -1 dH 2 O)
  12. 维生素溶液(1升)
    200毫克硫胺素-HCl
    10毫升0.1克L -1 生物素
    1毫升1克L -1 氰钴胺素

致谢

这项工作得到了设计师微生物公司的支持,这是NSERC ENGAGE Grant给D.R.E.和Designer Microbes Inc.(EGP / 486420-2015),安大略卓越中心VIP-1授予Designer Microbes Inc.(OCE-VIP-1/23879),以及NSERC Discovery Grant授予D.R.E. (RGPIN-2015-04800)。
该协议是从以下发表的论文开发的:Slattery et al。(2018)。作者声明没有利益冲突。

参考

  1. Daboussi,F.,Leduc,S.,Marechal,A.,Dubois,G.,Guyot,V.,Perez-Michaut,C.,Amato,A.,Falciatore,A.,Juillerat,A.,Beurdeley,M 。,Voytas,DF,Cavarec,L。和Duchateau,P。(2014)。 基因组工程授权硅藻 Phaeodactylum tricornutum 用于生物技术。 Nat Commun 5:3831。
  2. Gibson,D.G。(2009)。 通过重叠寡核苷酸的一步组装在酵母中合成DNA片段。 Nucleic Acids Res 37:6984-6990。&nbsp;
  3. 吉布森,D.G。 (2011年)。酵母中的基因和基因组构建。 Curr Protoc Mol Biol 章 3 ,单位3.22。
  4. Jinek,M.,Chylinski,K.,Fonfara,I.,Hauer,M.,Doudna,J。A.和Charpentier,E。(2012)。 自适应细菌免疫中可编程的双RNA指导DNA核酸内切酶。 科学 337(6096):816-821。
  5. Karas,BJ,Diner,RE,Lefebvre,SC,McQuaid,J.,Phillips,AP,Noddings,CM,Brunson,JK,Valas,RE,Deerinck,TJ,Jablanovic,J.,Gillard,JT,Beeri,K。 ,Ellisman,MH,Glass,JI,Hutchison,CA,3rd,Smith,HO,Venter,JC,Allen,AE,Dupont,CL and Weyman,PD(2015)。通过细菌结合传递的设计师硅藻附加物。 Nat Commun 6:6925。
  6. Noskov,VN,Karas,BJ,Young,L.,Chuang,RY,Gibson,DG,Lin,YC,Stam,J.,Yonemoto,IT,Suzuki,Y.,Andrews-Pfannkoch,C.,Glass,JI, Smith,HO,Hutchison,CA,3rd,Venter,JC和Weyman,PD(2012)。 在酵母中装配大的高G + C细菌DNA片段。 ACS Synth Biol 1(7):267-273。&nbsp;
  7. Slattery,SS,Diamond,A.,Wang,H.,Therrien,JA,Lant,JT,Jazey,T.,Lee,K.,Klassen,Z.,Desgagné-Penix,I.,Karas,BJ,等人(2018)。 扩展的基于质粒的遗传工具箱可以在中实现Cas9基因组编辑和合成途径的稳定维持phaeodactylum tricornutum 。 ACS Synth Biol 7:328-338。
  8. Strand,T.A.,Lale,R.,Degnes,K.F.,Lando,M。和Valla,S。(2014)。 用于基于RK2的结合转移的新的和改进的宿主非依赖性质粒系统。 PLoS One 9(3):e90372。
  9. Weyman,P.D.,Beeri,K.,Lefebvre,S.C.,Rivera,J.,McCarthy,J.K.,Heuberger,A.L.,Peers,G.,Allen,A.E。和Dupont,C.L。(2015)。 使用转录激活因子样效应核酸酶灭活 Phaeodactylum tricornutum 脲酶基因基于定向诱变。 Plant Biotechnol J 13(4):460-470。
  10. Wolfs,J.M.,Hamilton,T.A.,Lant,J.T。,Laforet,M.,Zhang,J.,Salemi,L.M.,Gloor,G.B.,Schild-Poulter,C。和Edgell,D.R。(2016)。 使用RNA引导的TevCas9双核酸酶偏向基因组编辑事件,以实现精确的长度缺失。 Proc Natl Acad Sci USA 113(52):14988-14993。&nbsp;
  11. Wright,A。V.,Nuñez,J。K.,Doudna,J。A.(2016)。 CRISPR系统的生物学和应用:利用大自然的基因组工程工具箱。 Cell 164(1-2):29-44。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wang, H., Slattery, S. S., Karas, B. J. and Edgell, D. R. (2018). Delivery of the Cas9 or TevCas9 System into Phaeodactylum tricornutum via Conjugation of Plasmids from a Bacterial Donor. Bio-protocol 8(16): e2974. DOI: 10.21769/BioProtoc.2974.
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xuehua liu
IOCAS
Dear Dr.Wang, When I transformed the TevCas9 vector into the Phaeodactylum tricornutum, I found there were multiple cuts of the target gene of the single clone grown on the zeocin plate . I only design the gRNA of Cas9 and ignore the TEV site. I used gene gunning to bombard the vector into the Phaeodactylum tricornutum. I would appreciate it if you could help me solve this problem. My Emai is Liuxuehua1213@163.com
9/18/2019 10:07:58 AM 回复
xuehua liu
IOCAS
Dear Dr.Wang, When I transformed the TevCas9 vector into the Phaeodactylum tricornutum, I found there were multiple cuts of the target gene of the single clone grown on the zeocin plate . I only design the gRNA of Cas9 and ignore the TEV site. I used gene gunning to bombard the vector into the Phaeodactylum tricornutum. I would appreciate it if you could help me solve this problem. My Emai is Liuxuehua1213@163.com
9/18/2019 10:07:52 AM 回复