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Phytophthora sojae Transformation Based on the CRISPR/Cas9 System

基于CRISPR/Cas9系统的大豆疫霉转化

JC Jingting Cao
MQ Min Qiu
WY Wenwu Ye
Yuanchao Wang Yuanchao Wang

发布: 2022年03月20日第12卷第6期 DOI: 10.21769/BioProtoc.4352 浏览次数: 3826

评审: Zhibing LaiFernando A Gonzales-ZubiateAnonymous reviewer(s)

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Abstract

Phytophthora sojae is a model species for the study of plant pathogenic oomycetes. The initial research on gene function using Phytophthora was mainly based on gene silencing technology. Recently, the CRISPR/Cas9-mediated genome editing technology was successfully established in P. sojae and widely used in oomycetes. In this protocol, we describe the operating procedures for the use of CRISPR/Cas9-based genome editing technology and PEG-mediated stable transformation of P. sojae protoplasts. Two plasmids were co-transformed into P. sojae: pYF515 expressing Cas9 and the single guide RNA, and the homologous replacement vector of the candidate gene. Finally, the ORF of candidate gene were replaced with the ORF of the entire hygromycin B phosphotransferase gene (HPH), to achieve precise knockout.

Keywords: CRISPR/Cas9 (CRISPR/Cas9) search Genome editing (基因组编辑) search Phytophthora sojae (大豆疫霉) search Knockout (敲除) search

Background

The probability of homologous recombination of Phytophthora itself is extremely low, and it is difficult to use methods based on homologous recombination to knock out genes. Therefore, a CRISPR/Cas9-based technology was developed to allow gene editing in P. sojae (Fang and Tyler, 2016). CRISPR/Cas9 is an adaptive immune response found in Bacteria and Archaea to prevent phage infection (Deveau et al., 2010; Garneau et al., 2010; Horvath and Barrangou, 2010). The CRISPR/Cas9 system consists of two components, namely Cas9 and single guide RNA (sgRNA) (Hsu et al., 2014). The single guide RNA (sgRNA) contains 20 nucleotides, which can mediate Cas9 to target the specific sequence region based on the principle of double-strand complementarity; Cas9 protein acts as a nuclease to cut specific DNA sites to produce double-strand break nicks (DSB).


In the presence of a homologous template (donor DNA), the cell uses the homologous template to repair, resulting in the homologous replacement of the target gene by the donor DNA to complete precise gene editing. Here, we provide detailed steps for the transformation of P. sojae based on the CRISPR/Cas9 system, including sgRNA design, Cas9-sgRNA plasmid construction, homologous replacement vector construction, P. sojae transformation, and detection of mutations, using Ps139767 as an example.

Materials and Reagents

  1. Consumables

    1. Sterile pipette tips (10 μL, 100 μL, 1,000 μL, 5 mL)

    2. Petri dishes (60 mm × 15 mm, 90 mm ×15 mm)

    3. 250 mL Erlenmeyer flasks

    4. Miracloth

    5. Stainless steel laboratory spatulas

    6. 50 mL Falcon tubes

    7. Microcentrifuge tubes (1.5 mL, 2.0 mL)

    8. 200 μL PCR tubes

    9. 20 mL Syringe

    10. Beaker (50 mL, 200 mL)

    11. Bacteria filter (0.45 nm)

    12. Parafilm


  2. Competent cells

    1. E. coli JM109 competent cells (homemade)


  3. Plasmids

    1. pBluescript SK II+(pBS-SK II+)

    2. pYF515 (constructed by Fang and Tyler, 2016) (Figure 1)



      Figure 1. The pYF515 vector expresses both the Cas9 gene and the sgRNA. The expression of Cas9 and the selection marker NPT II is driven by the Ham34 promoter, and the transcription of sgRNA [including the flanking ribozyme, hammerhead ribozyme (HH ribozyme) and HDV ribozyme] is driven by the RPL41 promoter.


  4. Primers (see Table 1)


    Table 1. List of oligonucleotides (see Supplementary Information)

    Primer nameSequence ( 5'→3' )
    139767-sgRNA1-FCTAGCTAGCCCGTCACTGATGAGTCCGTGAGGAC
    139767-sgRNA1-RCTAGGGTCTCGAAACCAATGGTGACTACACCGTCAGACGAGCTTACTCGTTTCG
    139767-sgRNA2-FCTAGCTAGCTAGTAGCTGATGAGTCCGTGAGGAC
    139767-sgRNA2-RCTAGGGTCTCGAAACCCACTAGGTTGCAGTAGTAGGACGAGCTTACTCGTTTCG
    139767-up1kb-FCTATAGGGCGAATTGGGTACCGCATTTCCGCAGTTCTCGTC
    139767-up1kb-RGTCGCGGTGAGTTCAGGCATCGTCTACCTCCACTACGCG
    139767-down1kb-FGTCCGAGGGCAAAGGAATAGACAAGCTACTCTTAGACTTTT
    139767-down1kb-RAGGGAACAAAAGCTGGAGCTCAACGCAAGCACTGTCAAAGC
    HPH-FATGCCTGAACTCACCGCGAC
    HPH-RCTATTCCTTTGCCCTCGGAC
    139767-genome-FTTTGAAGACAAAAGCGGGCG
    139767-genome-RTACTCGACGATACAGCACGC
    139767-overup-F1GGCCCGTGAATAAAACCCCT
    Hph-overup-R1ACCATCGGCGCAGCTATTT
    Hph-overdown-F2CGGGGATTCCCAATACGAGG
    139767-overdown-R2TAGCGTGTGTCAGATCCACC
    M13FGTAAAACGACGGCCAGT
    M13RCAGGAAACAGCTATGAC


  5. Strain

    1. Phytophthora sojae strain P6497

    2. E. coli JM109 (KangTi Life Technology, catalog number: KTSM107L)


  6. Enzymes and buffers

    1. BsaI and 10× Cutsmart Buffer (New England Biolabs, catalog number: R3733S)

    2. NheI (New England Biolabs, catalog number: R3131S)

    3. T4 DNA Ligase and 10× T4 DNA Ligase Buffer (Takara, catalog number: 2011A)

    4. 2× Phanta® Max Master Mix (Dye Plus) (Vazyme, catalog number: P525)

    5. TaKaRa TaqTM,10× PCR Buffer(Mg2+ plus) and dNTP Mixture (Takara, catalog number: R001)


  7. Kits

    1. FastPure® Gel DNA Extraction Mini Kit (Vazyme, catalog number: DC301)

    2. ClonExpress® ULtra One Step Cloning Kit (Vazyme, catalog number: C115)

    3. FastPure® Plasmid Mini Kit (Vazyme, catalog number: DC201-01)

    4. DNAsecure New Plant Genomic DNA Extraction Kit (TIANGEN BIOTECH, catalog number: DP320)

    5. TaKaRa MiniBEST DNA Fragment Purification Kit Ver.4.0 (Takara, catalog number: 9761)


  8. Antibiotics

    1. Ampicillin (to a final concentration of 50 mg/mL) (Aladdin, catalog number: 69-52-3)

    2. G418 (to a final concentration of 50 mg/mL) (Warbio, 108321-42-2)


  9. Media (see Recipes)

    1. Luria-Bertani agar plates + 50 mg/mL ampicillin

    2. Luria-Bertani liquid medium

    3. 10% V8 agar plates +50 mg/mL G418

    4. 10% V8 liquid medium

    5. Nutrient pea broth and agar medium (agar plates and liquid)

    6. Mannitol-pea broth (agar plates and liquid)


  10. Reagents

    1. V8 vegetable juice (Campbell Soup Company, catalog number: 051000153159)

    2. Frozen green peas

    3. Lysing Enzymes (Sigma-Aldrich, catalog number: L1412)

    4. CELLULYSIN Cellulase (Millipore, catalog number: 9012-54-8)

    5. Bacto tryptone (OXOID, catalog number: 73049-73-7)

    6. Yeast extract (Sigma-Aldrich, catalog number: 8013-01-2)

    7. NaCl (Aladdin, catalog number: 7647-14-5)

    8. Agar (Aladdin, catalog number: 9002-18-0)

    9. CaCO3 (Sangon, catalog number: 471-34-1)

    10. CaCl2 (Sangon, catalog number: 10043-52-4)

    11. KCl (Sangon,catalog number: 7447-40-7)

    12. PEG4000 (Aladdin, catalog number:25322-68-3)

    13. Biotin (Aladdin, catalog number:58-85-5)

    14. Folic acid (Aladdin, catalog number: 59-30-3)

    15. L-inositol (Sigma-Aldrich, catalog number: 551-72-4)

    16. Nicotinic acid (Aladdin, catalog number: 59-67-6)

    17. Pyridoxine-HCl (Aladdin, catalog number: 58-56-0)

    18. Riboflavin (Aladdin, catalog number: 83-88-5)

    19. Thiamine-HCl (Aladdin, catalog number: 59-43-8)

    20. FeC6H5O7·3H2O (Sangon, catalog number: 17217-76-4)

    21. ZnSO4·7H2O (Sangon, catalog number: 7446-20-0)

    22. CuSO4·5H2O (Sangon, catalog number: 7758-99-8)

    23. MgSO4·H2O (Sangon, catalog number: 14567-64-7)

    24. H3BO3 (Phytotech, catalog number: 10043-35-3)

    25. MoO3 (Phytotech, catalog number: 1313-27-5)

    26. KH2PO4 (Sangon, catalog number: 7778-77-0)

    27. K2HPO4 (Sangon, catalog number: 7758-11-4)

    28. KNO3 (Aladdin, catalog number: 7757-79-1)

    29. D-sorbitol (Aladdin, catalog number: 50-70-4)

    30. D-mannitol (Aladdin, catalog number: 69-65-8)

    31. Glucose (Aladdin, catalog number: 50-99-7)

    32. MgCl2·6H2O (Diamond, catalog number: 7791-18-6)

    33. MES (Aladdin, catalog number: 4432-31-9)

    34. Tris-HCl (Diamond, catalog number: 1185-53-1)

    35. EDTA (Phytotech, catalog number: 60-00-4)

    36. Hexadecyl trimethyl ammonium Bromide(CTAB) (Phytotech, catalog number: 57-09-0)


  11. Solutions

    1. 0.8 M Mannitol solution, autoclave at 121°C for 20 min

    2. 0.5 M CaCl2 solution, sterilize using a 0.45-mm filter

    3. 0.5 M KCl solution, sterilize using a 0.45-mm filter

    4. Luria-Bertani medium (see Recipes)

    5. 10% V8 agar (see Recipes)

    6. Pea broth (see Recipes)

    7. Nutrient pea broth and agar medium (see Recipes)

    8. 0.5 M MES-KOH solution (see Recipes)

    9. W5 solution (see Recipes)

    10. MMg solution (see Recipes)

    11. Enzyme solution (see Recipes)

    12. 40% PEG solution (see Recipes)

    13. Mannitol-pea broth (see Recipes)

    14. 2% CTAB solution (see Recipes)

    15. Vitamin stock (see Recipes)

    16. Trace elements (see Recipes)

Equipment

  1. Pipettes (Eppendorf)

  2. Rotating mixer (Select BioProducts, model: SBS550-2)

  3. PCR machine (Bio-Rad, model: T100TM)

  4. Light microscope (Leica, model: DM500)

  5. Incubator (25°C) (Ningbo Jiangnan, model: HWS-1000)

  6. Water bath (42°C) (SHELLAB, model: SWB7-2)

  7. Tabletop centrifuge(4°C) (Thermofisher, model: ST8)

  8. Incubator-shaker (37°C) (Crystal, model: IS-RSD3)

  9. DNA electrophoresis apparatus (Bio-Rad, model: 1704469)

Software

  1. sgRNA design software: EuPaGDT (http://grna.ctegd.uga.edu/)

  2. Sequence alignment: SeqHunter2 (https://sourceforge.net/projects/seqhunter2)

Procedure

English
中文翻译

文章信息

版权信息

© 2022 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Cao, J., Qiu, M., Ye, W. and Wang, Y. (2022). Phytophthora sojae Transformation Based on the CRISPR/Cas9 System. Bio-protocol 12(6): e4352. DOI: 10.21769/BioProtoc.4352.

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分类

微生物学 > 微生物遗传学 > CRISPR-Cas9

分子生物学 > DNA > 转化

分子生物学 > DNA > 诱/突变

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