基于CRISPR/Cas9系统的大豆疫霉转化

Jingting  Cao; Min  Qiu; Wenwu  Ye; Yuanchao Wang

doi:10.21769/BioProtoc.4352

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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 email

发布: 2022年03月20日第12卷第6期 DOI: 10.21769/BioProtoc.4352 浏览次数: 2455

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

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Oct 2020

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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)

Genome editing (基因组编辑)

Phytophthora sojae (大豆疫霉)

Knockout (敲除)

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

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
Competent cells
1. E. coli JM109 competent cells (homemade)
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.

Primers (see Table 1)

Table 1. List of oligonucleotides (see Supplementary Information)

Primer name	Sequence ( 5'→3' )
139767-sgRNA1-F	CTAGCTAGCCCGTCACTGATGAGTCCGTGAGGAC
139767-sgRNA1-R	CTAGGGTCTCGAAACCAATGGTGACTACACCGTCAGACGAGCTTACTCGTTTCG
139767-sgRNA2-F	CTAGCTAGCTAGTAGCTGATGAGTCCGTGAGGAC
139767-sgRNA2-R	CTAGGGTCTCGAAACCCACTAGGTTGCAGTAGTAGGACGAGCTTACTCGTTTCG
139767-up1kb-F	CTATAGGGCGAATTGGGTACCGCATTTCCGCAGTTCTCGTC
139767-up1kb-R	GTCGCGGTGAGTTCAGGCATCGTCTACCTCCACTACGCG
139767-down1kb-F	GTCCGAGGGCAAAGGAATAGACAAGCTACTCTTAGACTTTT
139767-down1kb-R	AGGGAACAAAAGCTGGAGCTCAACGCAAGCACTGTCAAAGC
HPH-F	ATGCCTGAACTCACCGCGAC
HPH-R	CTATTCCTTTGCCCTCGGAC
139767-genome-F	TTTGAAGACAAAAGCGGGCG
139767-genome-R	TACTCGACGATACAGCACGC
139767-overup-F1	GGCCCGTGAATAAAACCCCT
Hph-overup-R1	ACCATCGGCGCAGCTATTT
Hph-overdown-F2	CGGGGATTCCCAATACGAGG
139767-overdown-R2	TAGCGTGTGTCAGATCCACC
M13F	GTAAAACGACGGCCAGT
M13R	CAGGAAACAGCTATGAC

Strain
1. Phytophthora sojae strain P6497
2. E. coli JM109 (KangTi Life Technology, catalog number: KTSM107L)
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 Taq^TM,10× PCR Buffer(Mg²⁺ plus) and dNTP Mixture (Takara, catalog number: R001)
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)
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)
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)
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)
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

Pipettes (Eppendorf)
Rotating mixer (Select BioProducts, model: SBS550-2)
PCR machine (Bio-Rad, model: T100^TM)
Light microscope (Leica, model: DM500)
Incubator (25°C) (Ningbo Jiangnan, model: HWS-1000)
Water bath (42°C) (SHELLAB, model: SWB7-2)
Tabletop centrifuge(4°C) (Thermofisher, model: ST8)
Incubator-shaker (37°C) (Crystal, model: IS-RSD3)
DNA electrophoresis apparatus (Bio-Rad, model: 1704469)

Software

sgRNA design software: EuPaGDT (http://grna.ctegd.uga.edu/)
Sequence alignment: SeqHunter2 (https://sourceforge.net/projects/seqhunter2)

Procedure

English

中文翻译

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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.