CRISPR/Cas9 Gene Editing in the Marine Diatom Phaeodactylum tricornutum
海洋硅藻三角褐指藻中CRISPR / Cas9基因编辑技术   

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Scientific Reports
May 2016



The establishment of the CRISPR/Cas9 technology in diatoms (Hopes et al., 2016; Nymark et al., 2016) enables a simple, inexpensive and effective way of introducing targeted alterations in the genomic DNA of this highly important group of eukaryotic phytoplankton. Diatoms are of interest as model microorganisms in a variety of areas ranging from oceanography to materials science, in nano- and environmental biotechnology, and are presently being investigated as a source of renewable carbon-neutral fuel and chemicals. Here we present a detailed protocol of how to perform CRISPR/Cas9 gene editing of the marine diatom Phaeodactylum tricornutum, including: 1) insertion of guide RNA target site in the diatom optimized CRISPR/Cas9 vector (pKS diaCas9-sgRNA), 2) biolistic transformation for introduction of the pKS diaCas9-sgRNA plasmid to P. tricornutum cells and 3) a high resolution melting based PCR assay to screen for CRISPR/Cas9 induced mutations.

Keywords: CRISPR/Cas9 technology (CRISPR/Cas9技术), Diatoms (硅藻), Phaeodactylum tricornutum (三角褐指藻), Biolistic transformation (基因枪转化), HRM analyses (HRM 分析)


The CRISPR/Cas9 system has proven to be a very efficient and successful genome editing system in a number of eukaryotic organisms, now also including microalgae (Hopes et al., 2016; Nymark et al., 2016; Shin et al., 2016). The CRISPR/Cas9 system includes a guide RNA (gRNA) and a nuclease called Cas9 (Sander and Joung, 2014). These two molecules form a complex where the gRNA directs the complex to the target of interest. The Cas9 nuclease induces double strand brakes at the target site that can be repaired by nonhomologous end joining (NHEJ) which can result in indel mutations, or via the homology-directed repair (HDR) pathway that can be exploited to create defined alterations of the DNA. The presented protocol is the first to describe a step-by-step procedure for applying the CRISPR/Cas9 system to create gene-targeted mutations (indels ranging from one to hundreds of nucleotides) in one of the main diatom model species, P. tricornutum.

Materials and Reagents

  1. Pipette tips, 1,000 µl (SARSTEDT, catalog number: 70.762.100 )
  2. Pipette tips, 200 µl (SARSTEDT, catalog number: 70.760.502 )
  3. Pipette tips, 20 µl (Biosphere® Tip 20 µl neutral) (SARSTEDT, catalog number: 70.1116.200 )
  4. Filter tips, 20 µl (Biosphere® Fil. Tip 20 µl neutral) (SARSTEDT, catalog number: 70.1116.210 )
  5. 24 or 48-cell multiwell cell culture plates, flat bottom, TC treated (VWR, catalog number: 734-2325 or 734-2326 )
  6. 1.5 ml tube
  7. Parafilm
  8. 50 ml centrifuge tube (SARSTEDT, catalog number: 62.547.254 )
  9. 0.2 µm sterile filter
  10. Macrocarriers (Bio-Rad Laboratories, catalog number: 1652335 )
  11. 1,550 psi rupture discs (Bio-Rad Laboratories, catalog number: 1652331 )
  12. Stopping screens (Bio-Rad Laboratories, catalog number: 1652336 )
  13. Phaeodactylum tricornutum cells (NCMA Bigelow Laboratory for Ocean Sciences, Bigelow, catalog number: CCMP2561 starter culture)
  14. Competent DH5α E.coli cells (‘home-made’ RbCl competent cells [efficiency ≥ 1.0 x 106 cfu/µg])
  15. pKS diaCas9-sgRNA plasmid (Addgene, catalog number: 74923 )
  16. pAF6 plasmid (Falciatore et al., 1999) containing the ShBle gene conferring resistance to zeocin
  17. BsaI-HF restriction endonuclease (New England Biolabs, catalog number: R3535S )
  18. Complementary oligos (24 nt) with 5’ TCGA and AAAC overhangs (for creation of the adapter for targeting the gene of interest). Custom DNA oligos can be ordered from Sigma-Aldrich
  19. Wizard® SV Gel and PCR Clean-Up Kit (Promega, catalog number: A9282 )
  20. T4 DNA ligase buffer (New England Biolabs, catalog number: M0202S )
  21. ExTaq DNA polymerase and buffer system (AH Diagnostics) (Takara Bio, catalog number: RR001A )
  22. QIAprep Spin Miniprep Kit (QIAGEN, catalog number: 27106 )
  23. 50% (v/v) seawater plates supplemented with f/2-Si, 1% (w/v) agar plates
  24. Tungsten M10 or M17 microcarriers (Bio-Rad Laboratories, catalog number: 1652266 or 1652267 )
  25. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C7902 )
  26. Spermidine (Sigma-Aldrich, catalog number: S2626 )
  27. 70% (v/v) and 100% EtOH
  28. 70% (v/v) isopropanol
  29. 50% (v/v) seawater plates supplemented with f/2-Si, 1% (w/v) agar plates, 100 µg/ml zeocin
  30. Zeocin (InvivoGen, catalog number: ant-zn-5p )
  31. TOPO® TA Cloning® Kit for Sequencing (Thermo Fisher Scientific, InvitrogenTM, catalog number: 450030 )
  32. LightCycler 480 High Resolution Melting Master Kit (Roche Molecular Systems, catalog number: 04909631001 )
  33. TritonTM X-100 (Sigma-Aldrich, catalog number: X100 )
  34. Trizma® base (Sigma-Aldrich, catalog number: T6066 )
  35. Ethylenediaminetetraacetate acid disodium salt (EDTA) (Sigma-Aldrich, catalog number: E5134 )
  36. Pancreatic peptone (VWR, catalog number: 26208.297 )
  37. Bacto yeast extract (BD, BactoTM, catalog number: 212750 )
  38. Bacteriological agar (VWR, catalog number: 84609.5000 )
  39. Sodium chloride (NaCl) (Merck, catalog number: 106404 )
  40. Lysis buffer (Buffer for lysis of P. tricornutum cells) (see Recipes)
  41. LB medium (see Recipes)
  42. LB agar plates (see Recipes) containing 100 µg/ml ampicillin
  43. f/2 growth medium with natural (or artificial) seawater (see Recipes) (Guillard, 1975)


  1. NanoDrop ND-1000 or ND-2000 spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 1000 or NanoDropTM 2000 )
  2. Heating block (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88870005 )
  3. Water bath (Grant Instruments)
  4. Micropipettes (Eppendorf, model: Eppendorf Research® , variable volume)
  5. Table top centrifuges (Fisher Scientific, model: accuspinTM Micro 17R , catalog number: 13-100-676)
  6. Incubator shaker (Eppendorf, New BrunswickTM, model: Innova® 44 , catalog number: M1282-0002)
  7. Sterile bench (Thermo Fisher Scientific, Thermo ScientificTM, model: Holten Horizontal Laminar Airflow )
  8. PCR thermal cycler (Bio-Rad Laboratories, model: T100TM, catalog number: 1861096 )
  9. LightCycler® 96 Real-Time PCR system (Roche Molecular Systems, catalog number: 05815916001 )
  10. Biolistic PDS-1000/He Particle Delivery System (Bio-Rad Laboratories, catalog numbers: 165-2257 and 165-2250LEASE to 165-2255LEASE )
  11. Vortex
  12. Autoclave
  13. Refrigerator


  1. LightCycler® 96 Software Version 1.1 (Roche Molecular Systems)


  1. Selection of Cas9-target sites
    1. Use a custom made or publicly available CRISPR/Cas9 target finding tool (e.g., PhytoCRISP-Ex [Rastogi et al., 2016]) to identify Cas9 target sites (N20-NGG) with low or no homology to other genomic loci. The gene of interest should be sequenced before choosing the target site, or checked for polymorphisms using RNAseq data, because unpublished single nucleotide polymorphism is a frequent finding in the P. tricornutum genome.
    2. Order 24 nt long oligos with 20 complementary nt and 5’ TCGA and AAAC overhangs for creation of the adapter for targeting the gene of interest (Figure 1).
      Optional: Length of the target site can be ± 2 bp.

      Figure 1. Example of adapter with 5’ TCGA and AAAC overhangs

  2. Ligation of adapter for target of interest into the pKS diaCas9-sgRNA plasmid
    1. Exploit the two BsaI restriction sites that are placed immediately 5’- to the sgRNA in the vector (Figure 2) to ligate the adapter with 5’-TCGA and 5’-AAAC overhangs into the pKS diaCas9-sgRNA plasmid (Nymark et al., 2016). Digest 2 µg of pKS diaCas9-sgRNA plasmid with 1 µl BsaI-HF (20,000 U/ml, NEB) in a reaction with 5 µl 10x CutSmart buffer (NEB) and DNase-free water to a total reaction volume of 50 µl. Incubate for ≥ 2 h or overnight at 37 °C to ensure complete digestion of the plasmid DNA at both BsaI restriction sites producing incompatible ends. Heat inactivate the BsaI-HF enzyme at 65 °C for 20 min. Clean up the digestion reaction with Wizard® SV Gel and PCR Clean-Up Kit (Promega). Estimate the linearized plasmid concentration by measuring the absorbance at 260 nm using a NanoDrop spectrophotometer.

      Figure 2. Schematic illustration of the pKS diaCas9-sgRNA plasmid. A diatom codon optimized Cas9 is placed under control of a promoter for a fucoxanthin chlorophyll a/c binding gene (LHCF2), and the expression of the sgRNA is driven by the P. tricornutum U6 promoter. Two BsaI cutting sites are located within the sgRNA cassette to enable insertion of a small adapter for targeting the gene of interest (Nymark et al., 2016).

    2. Anneal the oligos by setting up an annealing reaction containing 1 µg of each oligo, 5 µl 10x T4 ligase buffer (NEB), and DNase-free water to a total reaction volume of 50 µl. Incubate the annealing reaction at 85 °C for 10 min in a heating block for disruption of any secondary structures within each oligo. Slowly cool down the reaction to room temperature (~60-90 min) by turning off the heating block while leaving the tube with the annealing reaction in the heating block. Slow cooling facilitates hybridization as new hydrogen bonds form between the complementary oligos. (Alternatively the annealing reaction can be performed in a thermocycler with a 1 °C/min temperature decrease to room temperature following the 10 min incubation at 85 °C).
    3. Ligate the adapter into the linearized pKS diaCas9-sgRNA plasmid using a molar vector to insert ratio of 1:20. Set up a ligation reaction using 1 µl T4 DNA ligase (400,000 U/ml; NEB) and 2 µl 10x T4 DNA ligase buffer (NEB) in a total reaction volume of 20 µl for 1 h at 16 °C. Heat inactivate the reaction for 10 min at 65 °C.
    4. Chill on ice, and transform 5 µl of the ligation reaction into competent cells (e.g., competent DH5α E.coli cells).
    5. Perform PCR colony screening of the transformed E. coli cells. To identify a pKS diaCas9-sgRNA plasmid containing the insert of interest, use one of the oligos used for creation of the adapter as forward primer in combination with a vector specific primer as reverse primer (e.g., the universal M13 reverse [-29] primer [5’-CAG GAA ACA GCT ATG ACC-3’]) in the PCR reaction.
      Note: Screening of 4-8 bacterial colonies is usually sufficient to identify a pKS diaCas9-sgRNA plasmid containing the adapter.
    6. Isolate pKS diaCas9-sgRNA plasmids (e.g., QIAprep Spin Miniprep Kit [QIAGEN]) containing the correct insert from the transformed E.coli cells. Relatively high concentrations (> 400-500 ng/µl) is preferable for coating of the tungsten particles during the preparations for biolistic transformation of the diatom cells.
      Note: To achieve such concentrations it might be necessary to pellet 10-15 ml of bacterial culture, since the yield of the pKS diaCas9-sgRNA plasmid is often low.

  3. Biolistic transformation for introduction of the pKS diaCas9-sgRNA plasmid to P. tricornutum cells
    Note: P. tricornutum cells can be grown under a wide range of light and temperature conditions. Suggested growth condition: 16 h photoperiod, 22 °C, 65 µmol m-2 sec-1.
    1. DAY 1
      1. Harvest P. tricornutum cells that are in the exponential growth phase by centrifugation (4,500 x g, 10 min). Remove the supernatant and resuspend the cells in f/2 medium to obtain a concentration of 1 x 108 cells/ml. Spread 500 µl of the high-density cell suspension on 50% (v/v) seawater 1% (w/v) agar plates supplemented with f/2-Si. Allow the plate with the cell suspension to dry in a sterile hood (~15-30 min).
        Note: The P. tricornutum cells should be kept in exponential growth phase (cell density ~0.2-2 x 106 cells/ml) for approx. 1-2 weeks before performing the biolistic transformation. Cell numbers can be checked by manual counting using a counting chamber or a flow cytometer. As long as the cells have been well maintained during the period before performing the biolistic transformation, the number of cells on the day of harvesting does not seem to be of importance for the transformation efficiency. For practical reasons cell numbers of approx. 0.5-1.5 x 106 cells/ml are preferable before up-concentration of the cell density as described above.
      2. Transfer the agar plates with P. tricornutum cells to the growth room (16 h photoperiod, 22 °C, 65 µmol m-2 sec-1) for overnight storage.
    2. DAY 2
      Note: Preparation and coating of microcarriers, and washing/sterilization of all materials and equipment necessary for the biolistic bombardment procedure, were performed exactly as described in the Biolistic PSD-1000/He Particle Delivery System manual (Bio-Rad Laboratories).
      1. Vortex a 50 µl aliquot containing 3 mg washed microcarriers (Tungsten M10 or M17 [Bio-Rad Laboratories]) for 5 min to disrupt agglomerated particles.
      2. Continue vortexing while adding in the following order 2.5 µg pKS diaCas9_sgRNA and 2.5 µg pAF6 plasmid DNA (maximum total volume of plasmid DNA ≤ 10 µl), 50 µl 2.5 M CaCl2 and 20 µl 0.1 M spermidine (free base, tissue culture grade). Continue vortexing for another 3 min.
      3. Allow the microcarriers to settle for 1 min.
      4. Pellet the tungsten particles by briefly (~2 sec) spinning the tube in a microfuge.
      5. Remove supernatant.
      6. Add 140 µl of 70% (v/v) EtOH and remove it again by pipetting (no mixing or centrifugation).
      7. Add 140 µl of 100% EtOH and remove it again by pipetting (no mixing or centrifugation).
      8. Add 50 µl of 100% EtOH.
      9. Resuspend by carefully tapping the tube several times.
      10. While vortexing at a low speed, remove 10 µl of the coated tungsten particles and spread the particles at the center of a macrocarrier using a pipette with a 10-200 µl tip. Repeat this step four times (loading in total five macrocarriers). After the ethanol has evaporated from the macrocarriers assemble all parts necessary for performing the bombardment according to the manufacturer’s instructions (Biolistic PSD-1000/He Particle Delivery System manual, Bio-Rad Laboratories). Use 1,550 psi rupture discs, and sterilize the rupture discs by dipping them briefly (~1-2 sec) in 70% (v/v) isopropanol just prior to insertion in the Retaining Cap.
      11. Place the target shelf with the agar plate containing P. tricornumtum cells 6 cm below the stopping screen (level 2) in the bombardment chamber.
      12. Set the vacuum level in the bombardment chamber to 26.5 psi.
      13. Bombard the same agar plate five times. After each shot, mark the impact area, and change the position of the plate to ensure that the subsequent shots are delivered on another part of the plate.
      14. Return the plate to the growth room for a minimum time of 24 h.
    3. DAY 3
      1. After a minimum incubation time of 24 h, transfer the algae to selection plates (50% [v/v] seawater plates supplemented with f/2-Si, 1% [w/v] agar plates, 100 µg/ml zeocin). Add 1 ml f/2 to the plate used for microparticle bombardment. Wash off the cells and transfer cells suspension (using a 1 ml pipette) to two selection plates.
      2. Wash the plate used for microparticle bombardment with an additional 0.5 ml f/2 medium. Transfer the cell suspension to a third selection plate.
        Note: Spreading of the cell suspension to three selection plates is performed to lower the cell density on the selection plates and to increase the sensitivity to zeocin, thereby avoiding growth of non-transformed cells.
      3. Dry the plates in the sterile bench (10-15 min)
      4. Seal the plates with Parafilm and incubate them in the growth room (growth conditions as above) for 2-4 weeks. (Expected number of transformed colonies: ~10-100)

  4. Screening for CRISPR/Cas9 induced mutations (see Figure 3 for overview of screening procedure)
    Note: To avoid PCR product contamination, filter pipette tips should be used during preparation for and performance of High Resolution Melting (HRM) Analyses.

    Figure 3. Overview of the screening procedure for detecting CRISPR/Cas9 induced mutations. A. Wait 2-4 weeks after biolistic transformation of P. tricornutum cells with the pKS diaCas9-sgRNA plasmid for colonies to appear on selection plates; B. Transfer the desired number of putative mutant colonies to a 24 or 48-well plate. Grow the cells for 1-2 weeks. C. Remove a small amount of cells from each well and lysate the cells; D. Use the lysate as template and amplify a 500-1,000 bp region around the target site by PCR; E. Check the PCR products by gel electrophoresis to identify large indels; F. Dilute the PCR product 1:4 x 106; G. Perform HRM analyses to detect smaller indels. Use the diluted PCR product from (F) as template in a PCR reaction that amplifies a region of around 100 bp surrounding the target site. H. Analyze the HRM data. Identify samples with CRISPR/Cas9 induced mutations by studying the difference in melting temperature between mutants and WT samples. I. Confirm mutations detected by HRM analyses by sequencing.

    1. Preparations for High Resolution Melting (HRM) Analyses
      1. Pick transformed colonies and transfer them to 24 or 48-cell multiwell cell culture plates (VWR) containing f/2 medium with 50 µg/ml zeocin.
      2. After approx. 1-2 weeks of growth, transfer a small amount of cells (100-200 µl) to a 1.5 ml tube.
        Note: Include a P. tricornutum wild type (WT) sample in addition to the transformed cells to be used as a control during the HRM analyses.
      3. Centrifuge for 1 min at 17,000 x g, and remove the supernatant.
      4. Add 20 µl lysis buffer (see Recipes) and vortex for 30 sec.
      5. Place the tubes on ice for 15 min.
      6. Transfer the tubes to a heating block, and incubate for 10 min at 85 °C.
      7. Dilute the lysate 1:5 with DNase-free water, and centrifuge briefly to pellet cell debris. The diluted lysate can be used directly as template for PCR or stored at -20 °C.
      8. Amplify by PCR an amplicon with a size of approx. 500-1,000 bp surrounding the target of interest using the lysate from step D1g as template.
        Note: A DNA polymerase that adds single deoxyadenosine (A) to the 3’ ends of the PCR products should be used to enable subsequent TOPO® TA Cloning (Invitrogen) of PCR products.
      9. Check the PCR products by agarose gel electrophoresis. Transformants with large indels can be detected directly. HRM based PCR assays can be used to detect smaller indels (including single nucleotide indels).
    2. HRM analyses
      1. Design HRM primers enabling amplification of a PCR product of approx. 100 bp surrounding the target site. The HRM primers should have annealing temperatures around 60 °C. Order HPLC purified primers.
      2. Make dilution series of the PCR products from step D1h from 1:40 to 1:4,000,000.
      3. Use 5 µl of the 1:4,000,000 diluted PCR product as template in a 20 µl PCR reaction using the LightCycler® 480 High Resolution Melting Master kit (Roche), with 1x LightCycler® 480 High Resolution Melting Master Mix, 200 nM forward and reverse primers and 3.0 mM MgCl2, final concentrations. Amplify the ~100 bp PCR product surrounding the target site in a LightCycler® 96 instrument (Roche), using (for most target sequences) the following cycling conditions: pre-incubation 10 min 95 °C, 45 cycles 3 step amplification (95 °C for 10 sec, 55 °C for 10 sec, 72 °C for 20 sec) followed by one cycle high resolution melting (95 °C for 60 sec, 40 °C for 60 sec, 65 °C for 1 sec, and finally using ramp rate 0.07 °C/sec and acquisition mode setting 15 readings/sec to reach target temperature 95 °C [1 sec]). Include 2-3 technical replicates for each sample.
      4. Analyze the raw data with the LightCycler® 96 software 1.1 (Roche) that sort the samples into groups based on normalized melting curves (Figure 4). Samples that show melting peaks that differ from WT are likely to contain PCR products with indels as a result of targeted genome editing induced by the CRISPR/Cas9 system.
      5. Confirm mutations detected by the HRM analysis by Sanger sequencing.

        Figure 4. HRM data presented as normalized melting peaks. Purple line: profile of a WT PCR product. Red line: profile of a PCR product containing a single biallelic G-insert. Yellow line: profile of a PCR product containing a single biallelic A-insert. Brown line: profile of a mix of PCR products containing a variety of deletions of different sizes.

  5. Identification of mutant lines originating from single cells with biallelic mutations
    Note: The primary P. tricornutum colonies/cultures identified by HRM to contain targeted mutations are often a mix of cells with different indels. Some colonies also contain a low percentage of WT cells. Single cells must therefore be isolated from the primary cultures to obtain ‘clean’ mutant lines with biallelic mutations.
    1. Spread 500 µl of a highly diluted cell suspension (~200 cells/ml) containing mutated P. tricornutum cells on f/2-Si, 1% (w/v) agar plates, 100 µg/ml Zeocin.
    2. Repeat the screening process (described in step D1) on the resulting colonies.
    3. Perform TOPO® TA Cloning (Invitrogen) to insert the 500-1,000 bp PCR product into a TOPO® TA-vector (e.g., pCRTM4-TOPO® TA vector [Invitrogen]) to identify the mutagenic event in the two different alleles. Direct Sanger sequencing of the PCR product will produce double peaks after the Cas9 target site if the two alleles contain different indels.

Data analysis

HRM analysis is a post-PCR method that can be performed using the LightCycler® 96 software 1.1 (Roche) enabling easy screening of genetic variations in the PCR amplicons based on differences in the melting points of the amplicons. When performing HRM analysis, at least two technical replicates should be included per sample. Before the HRM analysis, the amplification curves and the melting curves from qPCR raw data should be evaluated. Amplification curves with high crossing point (Cp) values may contain unspecific PCR products, and curves giving > 30 Cp units should be excluded. The cycling conditions need to be optimized if the melting curves indicate unspecific PCR products in the WT samples or in the Non-template controls (NTCs). The crossing point between samples should not vary more than 5 Cp units. Mutations indicated by the HRM analyses must be confirmed by sequencing.


  1. Lysis buffer
    1% (v/v) Triton X-100
    20 mM Tris-HCl pH 8
    2 mM EDTA
  2. LB medium/agar (per litre)
    10 g pancreatic peptone
    5 g Bacto yeast extract
    5 g NaCl
    15 g bacteriological agar (only for LB agar plates)
  3. f/2 growth medium with natural (or artificial) seawater (Guillard’s medium for diatoms)
    Filter seawater through a 0.2 µm sterile filter and autoclave at 120 °C for 20 min. Add f/2 trace elements, Vitamins and inorganic nutrients (see below) aseptically after autoclaving
    Note: Filtering is necessary for the removal of inorganic and organic suspended particles from natural seawater, whereas autoclaving inactivates organisms small enough to pass through the pore size of the filter.
    1. Trace elements (per litre)
      4.36 g NA2EDTA
      3.15 g FeCl2·6H2O
      0.01 g CuSO4·5H2O
      0.022 g ZnSO4·7H2O
      0.01 g CoCl2·6H2O
      0.18 g MnCl2·4H2O
      0.006 g Na2Mo4·2H2O
      Sterilize by filtering through a 0.2 µm sterile filter. Store in refrigerator (or freezer for long-term storage)
    2. Vitamin mix (per litre)
      0.0005 g cyanocobalamin (Vitamin B12)
      0.1 g thiamine HCl (Vitamin B1)
      0.0005 g biotin
      Sterilize by filtering through a 0.2 µm sterile filter. Store in refrigerator (or freezer for long-term storage)
    3. 30.0 g/L Na2SiO3·9H2O
      Sterilize by autoclaving. Store in refrigerator
    4. 75 g/L NaNO3
      Sterilize by autoclaving. Store in refrigerator
    5. 5.65 g/L NaH2PO4·2H2O
      Sterilize by autoclaving. Store in refrigerator.
    Medium (per litre):
    1. 1 ml trace elements stock solution (1)
    2. 1 ml Vitamin mix stock solution (2)
    3. 1 ml Na2SiO3·9H2O stock solution (3)
    4. 1 ml NaNO3 stock solution (4)
    5. 1 ml NaH2PO4·2H2O stock solution (5)


This work was funded by a grant from the Gordon and Betty Moore Foundation GBMF 4966 to Atle Bones and the NTNU enabling technologies program.


  1. Falciatore, A., Casotti, R., Leblanc, C., Abrescia, C. and Bowler, C. (1999). Transformation of nonselectable reporter genes in marine diatoms. Mar Biotechnol (NY) 1(3): 239-251.
  2. Guillard, R. R. L. (1975). Culture of phytoplankton for feeding marine invertebrates. In: Smith, W. L. and Chanley, M. H. (Eds.). Culture of marine invertebrate animals: Proceedings–1st conference on culture of marine invertebrate animals Greenport. Springer 29-60.
  3. Hopes, A., Nekrasov, V., Kamoun, S. and Mock, T. (2016). Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana. Plant Methods 12: 49.
  4. Nymark, M., Sharma, A. K., Sparstad, T., Bones, A. M. and Winge, P. (2016). A CRISPR/Cas9 system adapted for gene editing in marine algae. Sci Rep 6: 24951.
  5. Rastogi, A., Murik, O., Bowler, C. and Tirichine, L. (2016). PhytoCRISP-Ex: a web-based and stand-alone application to find specific target sequences for CRISPR/CAS editing. BMC Bioinformatics 17(1): 261.
  6. Sander, J. D. and Joung, J. K. (2014). CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32(4): 347-355.
  7. Shin, S. E., Lim, J. M., Koh, H. G., Kim, E. K., Kang, N. K., Jeon, S., Kwon, S., Shin, W. S., Lee, B., Hwangbo, K., Kim, J., Ye, S. H., Yun, J. Y., Seo, H., Oh, H. M., Kim, K. J., Kim, J. S., Jeong, W. J., Chang, Y. K. and Jeong, B. R. (2016). CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii. Sci Rep 6: 27810.


在硅藻(Hopes ,2016; Nymark等人,2016)中建立了CRISPR / Cas9技术,使得能够简单,廉价和有效地引入目标 这个非常重要的真核浮游植物群的基因组DNA的改变。 硅藻在纳米和环境生物技术领域从海洋学到材料科学,各种领域的示范性微生物都是有意义的,目前正在作为可再生碳中和燃料和化学品的来源进行调查。 在这里,我们提出了如何进行海洋硅藻三角褐指藻CRISPR / Cas9基因编辑的详细方案,包括:1)在硅藻优化的CRISPR / Cas9载体(pKS diaCas9)中插入引导RNA靶位点 -sgRNA),2)用于将pKS diaCas9-sgRNA质粒导入P的生物弹道转化。 三分支毛细胞和3)基于高分辨率熔融的PCR测定以筛选CRISPR / Cas9诱导的突变。
【背景】CRISPR / Cas9系统已被证明是许多真核生物中非常有效和成功的基因组编辑系统,现在也包括微藻(Hopes等人,2016; Nymark等人)。 ,2016; Shin 等人,2016)。 CRISPR / Cas9系统包括引导RNA(gRNA)和称为Cas9的核酸酶(Sander and Joung,2014)。 这两个分子形成复合物,其中gRNA将复合物引导至感兴趣的靶标。 Cas9核酸酶在靶位点诱导双链制动,可以通过非同源末端连接(NHEJ)修复,可导致indel突变,或通过同源性定向修复(HDR)途径修复,可用于创建定义的改变 脱氧核糖核酸。 所提出的方案是首先描述应用CRISPR / Cas9系统以在主要硅藻模型物种之一中创建基因靶向突变(从1到数百个核苷酸的indel)的分步骤方法,P.藻。

关键字:CRISPR/Cas9技术, 硅藻, 三角褐指藻, 基因枪转化, HRM 分析


  1. 移液器提示,1,000μl(SARSTEDT,目录号:70.762.100)
  2. 移液器提示,200μl(SARSTEDT,目录号:70.760.502)
  3. 移液头,20μl(生物圈提示20μl中性)(SARSTEDT,目录号:70.1116.200)
  4. 过滤嘴,20微升(生物圈 Fil。Tip 20微升中性)(SARSTEDT,目录号:70.1116.210)
  5. 24或48细胞多孔细胞培养板,平底,TC处理(VWR,目录号:734-2325或734-2326)
  6. 1.5 ml管子
  7. 石蜡膜
  8. 50ml离心管(SARSTEDT,目录号:62.547.254)
  9. 0.2μm无菌过滤器
  10. 大载体(Bio-Rad Laboratories,目录号:1652335)
  11. 1,550psi断裂盘(Bio-Rad Laboratories,目录号:1652331)
  12. 停止筛选(Bio-Rad Laboratories,目录号:1652336)
  13. 三角褐指藻细胞(NCMA Bigelow海洋科学实验室,Bigelow,目录号:CCMP2561启动子培养物)
  14. 有效的DH5α大肠杆菌细胞(“自制的”RbCl感受态细胞[效率≥1.0×10 6] / cfu /μg])
  15. pKS diaCas9-sgRNA质粒(Addgene,目录号:74923)
  16. 含有赋予zeocin抗性的ShBle 基因的pAF6质粒(Falciatore等人,1999),
  17. Bsa I-HF限制性内切核酸酶(New England Biolabs,目录号:R3535S)
  18. 具有5'TCGA和AAAC突出端的互补寡核苷酸(24nt)(用于创建用于靶向感兴趣的基因的衔接子)。定制DNA寡核苷酸可以从Sigma-Aldrich
  19. 向导® SV凝胶和PCR清洁试剂盒(Promega,目录号:A9282)
  20. T4 DNA连接酶缓冲液(New England Biolabs,目录号:M0202S)
  21. ExTaq DNA聚合酶和缓冲液系统(AH Diagnostics)(Takara Bio,目录号:RR001A)
  22. QIAprep Spin Miniprep Kit(QIAGEN,目录号:27106)
  23. 补充有f / 2-Si,1%(w / v)琼脂板的50%(v / v)海水板
  24. 钨M10或M17微载体(Bio-Rad Laboratories,目录号:1652266或1652267)
  25. 氯化钙二水合物(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:C7902)
  26. 亚精胺(Sigma-Aldrich,目录号:S2626)
  27. 70%(v / v)和100%EtOH
  28. 70%(v / v)异丙醇
  29. 补充有f / 2-Si,1%(w / v)琼脂平板,100μg/ ml zeocin的50%(v / v)海水板
  30. Zeocin(InvivoGen,目录号:ant-zn-5p)
  31. TOPO ® TA克隆®测序试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:450030)
  32. LightCycler 480高分辨率熔融主试剂盒(Roche Molecular Systems,目录号:04909631001)
  33. Triton TM X-100(Sigma-Aldrich,目录号:X100)
  34. Trizma ®碱(Sigma-Aldrich,目录号:T6066)
  35. 乙二胺四乙酸酸二钠盐(EDTA)(Sigma-Aldrich,目录号:E5134)
  36. 胰蛋白胨(VWR,目录号:26208.297)
  37. 细菌酵母提取物(BD,Bacto TM,目录号:212750)
  38. 细菌琼脂(VWR,目录号:84609.5000)
  39. 氯化钠(NaCl)(Merck,目录号:106404)
  40. 裂解缓冲液(用于裂解三角褐指杆细胞的缓冲液)(参见食谱)
  41. LB培养基(参见食谱)
  42. 含有100μg/ ml氨苄青霉素的LB琼脂平板(参见食谱)
  43. f / 2具有天然(或人工)海水的生长培养基(参见食谱)(Guillard,1975)


  1. NanoDrop ND-1000或ND-2000分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM 1000或NanoDrop TM 2000) br />
  2. 加热块(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:88870005)
  3. 水浴(Grant Instruments)
  4. 微量移液器(Eppendorf,型号:Eppendorf Research ®,变量)
  5. 台式离心机(Fisher Scientific,型号:accuspin TM Micro 17R,目录号:13-100-676)
  6. 孵化器(Eppendorf,New Brunswick TM ,型号:Innova ® 44,目录号:M1282-0002)
  7. 无菌台(Thermo Fisher Scientific,Thermo Scientific TM,型号:Holten Horizontal Laminar Airflow)
  8. PCR热循环仪(Bio-Rad Laboratories,型号:T100 TM,目录号:1861096)
  9. LightCycler ®96实时PCR系统(Roche Molecular Systems,目录号:05815916001)
  10. 生物学PDS-1000 / He颗粒递送系统(Bio-Rad Laboratories,目录号:165-2257和165-2250LEASE至165-2255LEASE)
  11. 涡流
  12. 高压灭菌器
  13. 冰箱


  1. LightCycler ® 96软件版本1.1(Roche Molecular Systems)


  1. 选择Cas9靶位点
    1. 使用定制或公开提供的CRISPR / Cas9目标发现工具(例如,PhytoCRISP-Ex [Rastogi等人,2016])来鉴定Cas9靶位点(N < sub-20-NGG),与其他基因组基因座具有低或不同源性。感兴趣的基因应该在选择靶位点之前进行测序,或使用RNAseq数据检查多态性,因为未发表的单核苷酸多态性是“P”中的常见发现。三角褐斑基因组。
    2. 订购具有20个互补nt和5'TCGA和AAAC突出端的24个长的寡核苷酸,用于创建用于靶向感兴趣的基因的适配器(图1)。
      可选:目标站点的长度可以为±2 bp。


  2. 将感兴趣靶标的适配器连接到pKS diaCas9-sgRNA质粒中
    1. 利用载体(图2)中立即5'-位置的两个Bsa I限制性位点将衔接子与5'-TCGA和5'-AAAC突出端连接到pKS diaCas9中-sgRNA质粒(Nymark等人,2016)。在与5μl10×CutSmart缓冲液(NEB)和不含DNA酶的水反应中将2μg具有1μlBsa I-HF(20,000U / ml,NEB)的pKS diaCas9-sgRNA质粒消化至总反应体积为50μl。在37℃下孵育≥2小时或过夜,以确保在产生不相容的末端的Bsa I限制性位点处完全消化质粒DNA。在65℃下热灭活I-HF酶20分钟。使用Wizard ® SV Gel和PCR Clean-Up Kit(Promega)清洁消化反应。通过使用NanoDrop分光光度计测量260nm处的吸光度来估计线性化质粒浓度。

      图2. pKS diaCas9-sgRNA质粒的示意图。 将硅藻土密码子优化Cas9 置于岩藻黄质叶绿素a / c结合基因(LHCF2)的启动子的控制下,并且sgRNA的表达是由

      P驱动。三角褐质 U6启动子。两个Bsa 我切割位点位于sgRNA盒内,以使得能够插入用于靶向感兴趣的基因的小适配器(Nymark等人,2016)。 >

    2. 通过设置含有1μg每种寡核苷酸,5μl10×T4连接酶缓冲液(NEB)和不含DNA酶的水,使总反应体积为50μl的退火反应来退火寡核苷酸。在加热块中将85℃的退火反应温育10分钟,以破坏每个寡核苷酸内的任何二级结构。通过关闭加热块,同时将加热块中的退火反应离开管,将反应缓慢冷却至室温(〜60-90分钟)。由于在互补寡核苷酸之间形成新的氢键,缓慢冷却便于杂交。 (或者,退火反应可以在温度为1℃/分钟的温度下在85℃温育10分钟后降至室温的热循环仪中进行)。
    3. 使用摩尔向量插入比例为1:20将适配器连接到线性化的pKS diaCas9-sgRNA质粒中。使用1μlT4 DNA连接酶(400,000 U / ml; NEB)和2μl10x T4 DNA连接酶缓冲液(NEB),在总反应体积为20μl的条件下,于16℃设置连接反应1 h。加热使反应在65℃灭活10分钟。
    4. 在冰上冷却,并将5μl连接反应转化成感受态细胞(例如,有感受力的DH5α大肠杆菌细胞)。
    5. 对转化的E进行PCR集落筛选。大肠杆菌细胞。为了鉴定包含感兴趣的插入片段的pKS diaCas9-sgRNA质粒,使用用于产生衔接子的寡核苷酸作为正向引物与作为反向引物的载体特异性引物(例如,通用的) M13反向[-29]引物[5'-CAG GAA ACA GCT ATG ACC-3'])。 注意:筛选4-8个细菌菌落通常足以鉴定含有适配体的pKS diaCas9-sgRNA质粒。
    6. 从转化的大肠杆菌细胞中分离含有正确插入片段的pKS diaCas9-sgRNA质粒(例如,QIAprep Spin Miniprep Kit [QIAGEN])。在硅藻细胞的生物弹性转化制备过程中,相对高的浓度(> 400-500ng /μl)优选用于涂覆钨颗粒。
      注意:为了达到这样的浓度,可能需要沉淀10-15ml的细菌培养物,因为pKS diaCas9-sgRNA质粒的产量通常很低。

  3. 将pKS diaCas9-sgRNA质粒导入到P的生物学转化。三角褐斑细胞
    注意:三角肌细胞可以在宽和轻的条件下生长。建议的生长条件:16小时光周期,22℃,65μmol/秒 秒 1 。
    1. 第一天
      1. 收获。通过离心(4,500xg,10分钟)处于指数生长阶段的三角褐质细胞。去除上清液并将细胞重悬于f / 2培养基中,得到1×10 8细胞/ ml的浓度。将500μl高密度细胞悬浮液在补充有f / 2-Si的50%(v / v)海水1%(w / v)琼脂平板上扩散。让细胞悬浮液在无菌罩中干燥(约15-30分钟)注意:三角褐斑细胞应保持指数生长期(细胞密度〜0.2-2×10 6细胞/细胞/ ml) 1-2周前进行生物弹性变换。可以使用计数室或流式细胞仪通过手动计数检查细胞编号。只要细胞在进行生物弹性变换之前的时期内保持良好,收获当天的细胞数量对转化效率似乎并不重要。细胞数约为如上所述,在细胞密度上升浓度之前优选0.5-1.5×10 6细胞/ ml。
      2. 转移琼脂平板与P。三角褐斑细胞向生长室(16小时光周期,22℃,65μmol/平方米)至保存时间过夜。
    2. 第2天
      注意:精确地按照Biolistic PSD-1000 / He Particle Delivery System手册(Bio-Rad Laboratories)中描述的方式精确地进行微生物体的制备和包被以及对于生物射弹轰击过程所需的所有材料和设备的洗涤/灭菌。 。
      1. 旋转含有3毫克洗涤的微载体(钨M10或M17 [Bio-Rad Laboratories])的50μl等分试样5分钟,以破坏附聚颗粒。
      2. 继续涡旋,同时按以下顺序加入2.5μgpKS diaCas9_sgRNA和2.5μgpAF6质粒DNA(质粒DNA的最大总体积≤10μl),50μl2.5M CaCl 2和20μl0.1M亚精胺(游离碱,组织培养级)。继续涡旋3分钟。
      3. 让微载体沉降1分钟
      4. 短时间(〜2秒)在微量离心机中旋转管子,使钨颗粒成粒。
      5. 去除上清液
      6. 加入140μl70%(v / v)乙醇,并通过移液(无混合或离心)再次取出
      7. 加入140μl100%乙醇,并通过移液(无混合或离心)再次除去
      8. 加入50μl100%EtOH。
      9. 通过仔细点击管子几次重悬。
      10. 当以低速涡旋时,除去10微升的涂覆的钨颗粒,并使用10-200微升的移液管在大载流子的中心扩散颗粒。重复此步骤四次(载入总共五个宏载波)。乙醇从大载体蒸发后,根据制造商的说明书(Biolistic PSD-1000 / He Particle Delivery System manual,Bio-Rad Laboratories)组装进行轰击所需的所有部件。使用1550psi的破裂片,并在插入保持盖之前,将它们在70%(v / v)异丙醇中短暂浸泡(〜1-2秒),对破裂片进行灭菌。
      11. 将目标架放在含有P的琼脂板上。在轰炸室内的停止屏幕(2级)以下6厘米处的三角锥细胞。
      12. 将轰炸室中的真空度设置为26.5 psi。
      13. 轰炸相同的琼脂板五次。每次拍摄后,标记冲击区域,并更改板的位置,以确保随后的拍摄在纸张的另一部分投放。
      14. 将板返回生长室至少24小时。
    3. 第三天
      1. 在24小时的最小孵育时间后,将藻类转移到选择板(补充有f / 2-Si,1%[w / v]琼脂平板,100μg/ ml zeocin的50%[v / v]海水板)上。向用于微粒轰击的板上加入1ml f / 2。洗涤细胞并将细胞悬浮液(使用1毫升移液管)转移到两个选择板上。
      2. 用额外的0.5ml f / 2培养基洗涤用于微粒轰击的板。将细胞悬浮液转移到第三个选择板。
      3. 干燥无菌台(10-15分钟)
      4. 用Parafilm密封板,并在生长室(如上所述的生长条件)孵育2-4周。 (预期转化菌落数:〜10-100)

  4. 筛选CRISPR / Cas9诱导突变(见图3,筛选程序概述)

    图3.检测CRISPR / Cas9诱导突变的筛选程序概述。 A.等待2-4周后,用pKS diaCas9- sgRNA质粒用于菌落出现在选择板上; B.将所需数量的推定的突变菌落转移到24孔或48孔板中。生长细胞1-2周。 C.从每个孔中取出少量细胞并裂解细胞; D.使用裂解物作为模板,通过PCR扩增靶位点周围的500-1,000bp区域; E.通过凝胶电泳检查PCR产物,鉴定大型indels; F.稀释PCR产物1:4×10 6。 G.进行人力资源管理分析以检测较小的indel。在PCR反应中使用来自(F)的稀释的PCR产物作为模板,其扩增靶位点周围约100bp的区域。 H.分析人力资源管理数据。通过研究突变体和WT样品之间的融合温度差异,识别CRISPR / Cas9诱导突变的样品。 I.通过测序确认HRM分析检测到的突变
    1. 高分辨率熔融(HRM)分析的准备工作
      1. 挑取转化的菌落并将其转移到含有50μg/ ml zeocin的含有f / 2培养基的24或48细胞多孔细胞培养板(VWR)中。
      2. 大约1-2周的生长,将少量细胞(100-200μl)转移到1.5ml管中。
      3. 以17,000 x g离心1分钟,并除去上清液
      4. 加入20μl裂解缓冲液(参见食谱)并旋涡30秒
      5. 将管放在冰上15分钟
      6. 将管转移到加热块,并在85℃下孵育10分钟。
      7. 用无DNA酶的水稀释裂解物1:5,并短暂离心以沉淀细胞碎片。稀释的裂解液可以直接用作PCR的模板或-20℃储存
      8. 通过PCR扩增大小约为使用步骤D1g的裂解物作为模板,围绕感兴趣的靶标500-1,000bp 注意:应将PCR产物的3'末端添加单一脱氧腺苷(A)的DNA聚合酶用于启用后续TOPO PCR产物的TA克隆(Invitrogen)。
      9. 通过琼脂糖凝胶电泳检查PCR产物。可以直接检测到具有大型indel的转化体。基于HRM的PCR检测可用于检测较小的indel(包括单核苷酸indels)
    2. 人力资源管理分析
      1. 设计HRM引物,使PCR扩增产物约为围绕目标地点的100 bp。 HRM引物应具有约60℃的退火温度。订购HPLC纯化引物
      2. 将步骤D1h的PCR产物的稀释系列从1:40稀释至1:4,000,000。
      3. 使用LightCycler 480高分辨率熔融主试剂盒(Roche),用1x LightCycler 在20μlPCR反应中使用5μl的1:4,000,000稀释的PCR产物作为模板> 480高分辨率熔融主混合物,200nM正向和反向引物和3.0mM MgCl 2终浓度。在以下循环条件下使用(对于大多数靶序列),在LightCycler 96仪器(Roche)中扩增靶标位点周围约100bp的PCR产物:预孵育10分钟95℃,45循环3步扩增(95℃10秒,55℃10秒,72℃20秒),然后循环高分辨率熔融(95℃,60秒,40℃,60秒,65度) C,持续1秒,最后使用斜率0.07°C /秒,采集模式设置15个读数/秒,达到目标温度95°C [1秒])。每个样品包括2-3个技术重复。
      4. 使用LightCycler 96软件1.1(Roche)分析原始数据,根据归一化的熔解曲线将样品分组成组(图4)。显示与WT不同的融合峰的样品可能含有由CRISPR / Cas9系统诱导的靶向基因组编辑的结果的indels的PCR产物。
      5. 通过Sanger测序确定HRM分析检测到的突变

        图4.以归一化熔融峰表示的HRM数据。紫色线:WT PCR产物的简图。红线:含有单个双重G-插入物的PCR产物的谱图。黄线:含有单一双液A型插入物的PCR产物的谱图。棕色线:含有各种不同大小缺失的PCR产物的混合物的概况
  5. 鉴定来源于具有双重突变的单细胞的突变体系 注意:由HRM鉴定的含有靶向突变的主要三角褐指菌菌落/培养物通常是具有不同indel的细胞混合物。一些菌落也含有低百分比的WT细胞。因此,单细胞必须与原代培养物分离,以获得具有双重突变的“干净”突变株
    1. 将含有突变的三角褐指瘤细胞的500μl高度稀释的细胞悬浮液(〜200个细胞/ ml)扩散到f / 2-Si,1%(w / v)琼脂平板上,100μg/ ml zeocin。
    2. 对产生的菌落重复筛选过程(在步骤D1中描述)。
    3. 执行TOPO ® TA克隆(Invitrogen)将500-1,000bp PCR产物插入TOPO TA载体(例如,pCR < sup> TM 4-TOPO TA载体[Invitrogen])以鉴定两种不同等位基因中的诱变事件。如果两个等位基因含有不同的indel,PCR产物的直接Sanger测序将在Cas9靶位点后产生双峰。


HRM分析是可以使用LightCycler 96软件1.1(Roche)进行的PCR后方法,其能够基于扩增子的熔点的差异容易地筛选PCR扩增子中的遗传变异。在进行人力资源管理分析时,每个样本至少应包含两项技术重复。在HRM分析之前,应对qPCR原始数据的扩增曲线和熔解曲线进行评估。具有高交叉点(Cp)值的扩增曲线可能含有非特异性PCR产物,应排除30个Cp单位。如果熔解曲线表示WT样品或非模板对照(NTC)中的非特异性PCR产物,则需要优化循环条件。样品之间的交叉点不应超过5个Cp单位。 HRM分析表明的突变必须通过测序确认。


  1. 裂解缓冲液
    1%(v / v)Triton X-100
    20mM Tris-HCl pH 8
    2 mM EDTA
  2. LB培养基/琼脂(每升)
  3. f / 2天然(或人工)海水生长培养基(Guillard的硅藻培养基)
    过滤海水通过0.2μm无菌过滤器,并在120°C高压灭菌20分钟。高压灭菌后无菌添加f / 2微量元素,维生素和无机营养素(见下文) 注意:过滤对于从天然海水中去除无机和有机悬浮颗粒是必要的,而高压灭菌使得足够小的生物通过过滤器的孔径。
    1. 微量元素(每升)
      4.36g NA 2 EDTA
      3.15g FeCl 2·6H 2 O - /
      0.01g CuSO 4·5H 2 O
      0.022g ZnSO 4·7H 2 O
      0.01克CoCl 2·6H 2 O
      0.18g MnCl 2·4H 2 O
      0.006g Na 2 Mo 4 O 2·2H 2 O
    2. 维生素混合(每升)
      0.0005g氰钴胺素(维生素B 12
      0.1g硫胺素HCl(维生素B 1
    3. 30.0g / L Na 2 SiO 3·9H 2 O 通过高压灭菌消毒。存放在冰箱里
    4. 75g / L NaNO 3
    5. 5.65g / L NaH 2 PO 4·2H 2 O
    1. 1ml微量元素储备溶液(1)
    2. 1 ml维生素混合原液(2)
    3. 1ml Na 2 SiO 3·9H 2 O储备溶液(3)
    4. 1ml NaNO 3储备溶液(4)
    5. 1ml NaH 2 PO 4·2H 2 O储备溶液(5)


这项工作由Gordon和Betty Moore基金会GBMF 4966授予Atle Bones以及NTNU授权技术计划的资助。


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引用:Nymark, M., Sharma, A. K., Hafskjold, M. C., Sparstad, T., Bones, A. M. and Winge, P. (2017). CRISPR/Cas9 Gene Editing in the Marine Diatom Phaeodactylum tricornutum. Bio-protocol 7(15): e2442. DOI: 10.21769/BioProtoc.2442.