1 user has reported that he/she has successfully carried out the experiment using this protocol.
Targeted Genome Editing of Virulent Phages Using CRISPR-Cas9
使用CRISPR-Cas9对烈性噬菌体进行靶向基因组编辑   

引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
ACS Synthetic Biology
Mar 2017

 

Abstract

This protocol describes a straightforward method to generate specific mutations in the genome of strictly lytic phages. Briefly, a targeting CRISPR-Cas9 system and a repair template suited for homologous recombination are provided inside a bacterial host, here the Gram-positive model Lactococcus lactis MG1363. The CRISPR-Cas9 system is programmed to cleave a specific region present on the genome of the invading phage, but absent from the recombination template. The system either triggers the recombination event or exerts the selective pressure required to isolate recombinant phages. With this methodology, we generated multiple gene knockouts, a point mutation and an insertion in the genome of the virulent lactococcal phage p2. Considering the broad host range of the plasmids used in this protocol, the latter can be extrapolated to other phage-host pairs.

Keywords: Phage (噬菌体), Lactococcus lactis (乳酸乳球菌), Genome editing (基因组编辑), CRISPR-Cas9 (CRISPR-Cas9), Homologous recombination (同源重组)

Background

Phages are bacterial viruses found in abundance in every ecosystem (Suttle, 2005; Breitbart and Rohwer, 2005) and unsurprisingly, they are natural inhabitants of milk. Phage p2 is a model for the most prevalent group (Sk1virus) of virulent lactococcal phages found in the dairy industry (Deveau et al., 2006; Mahony et al., 2012) and it infects the Gram-positive bacterium Lactococcus lactis MG1363, also a model strain for basic research. Despite the status of p2 as a reference phage, almost half of its genes encode uncharacterized proteins. Likewise, a clear majority of phage genes identified by metagenomics have no functional assignment and no homolog in public databases (Hurwitz et al., 2016; Paez-Espino et al., 2016).

One of the ways to study genes is through their modification and subsequent observation of the resulting phenotypes. Phage genomes can only be modified inside a host, in their biologically active form. Virulent phages are strictly lytic; thus, their genome never integrates into the bacterial chromosome. This adds a time constraint for the in vivo modification of their DNA, which can be manipulated only during their short infection cycle. The emergence of CRISPR-Cas research in the last decade lead to the adaptation of this natural prokaryotic defense mechanism into a powerful tool to edit the genome of a plethora of organisms and viruses, including virulent phages (Kiro et al., 2014; Martel and Moineau, 2014; Box et al., 2015; Pires et al., 2016; Bari et al., 2017; Lemay et al., 2017; Manor and Qimron, 2017; Tao et al., 2017).

Here, we detail a simple and reproducible protocol to edit the genome of phage p2 using the well-known Streptococcus pyogenes Cas9 (SpCas9) cloned into the lactic acid bacterium L. lactis MG1363 (Lemay et al., 2017). Within our laboratory, this protocol has also been successfully applied to edit the genome of a virulent phage infecting the Gram-negative E. coli (unpublished), illustrating its broad applicability.

Materials and Reagents

  1. Materials
    1. Disposable Pasteur pipettes (VWR, catalog number: 14672-200 )
    2. 3 mm glass beads (VWR, catalog number: 26396-508 )
      Manufacturer: Walter Stern, catalog number: 100C .
    3. 100 mm glass tubes with caps (Fisher Scientific, catalog numbers: 14-961-27 and 05-888-1A )
      Manufacturer: Bal Supply, catalog number: 13144UL .
    4. 150 mm glass tubes with caps (Fisher Scientific, catalog numbers: 14-961-32 and 05-888C )
      Manufacturer: Bal Supply, catalog number: 18144CL .
    5. 0.5 ml micro-tubes (SARSTEDT, catalog number: 72.699 )
    6. 1.5 ml micro-tubes (SARSTEDT, catalog number: 72.690 )
    7. Sterile 100 x 15 mm plastic Petri dishes (‘plates’) (VWR, catalog number: 25384-302 )
    8. 10 ml sterile BD Luer-LokTM Tip syringe (BD, catalog number: 309604 )
    9. 0.2 µm sterile PES syringe filter (SARSTEDT, catalog number: 83.1826.001 )
    10. 0.45 µm sterile PES syringe filter (SARSTEDT, catalog number: 83.1826 )

  2. Phage and bacterial strain
    1. Lactococcus lactis MG1363 (Félix d’Hérelle Reference Center for Bacterial Viruses, catalog number: HER1439 )
      Note: L. lactis is generally recognized as a safe bacterium and all the experiments can be performed in a biosafety level 1 laboratory.
    2. Phage p2 (Félix d’Hérelle Reference Center for Bacterial Viruses, catalog number: HER457 )

  3. Plasmids
    1. pL2Cas9 (Lemay et al., 2017) (Addgene, catalog number: 98841 )
    2. pNZ123 (de Vos, 1987) (L. lactis MG1363 (pNZ123)) (Félix d’Hérelle Reference Center for Bacterial Viruses, catalog number: HER1532 )

  4. Enzymes
    1. Antarctic phosphatase (5,000 U per ml) (New England Biolabs, catalog number: M0289S )
    2. BsaI (10,000 U per ml) (New England Biolabs, catalog number: R0535S )
    3. Lysozyme (20,000 U per mg dry weight) (Thermo Fisher Scientific, catalog number: 89833 )
    4. Q5 DNA polymerase (2,000 U per ml) (New England Biolabs, catalog number: M0491S )
    5. T4 DNA ligase (1,000 U per ml) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15224017 )
    6. T4 polynucleotide kinase (10,000 U per ml) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EK0031 )
    7. Taq DNA polymerase (5,000 U per ml) (NBS Biologicals, catalog number: 9K-001-0034 )
    8. XbaI (20,000 U per ml) (New England Biolabs, catalog number: R0145S )

  5. Reagents
    1. AccuGENETM molecular biology water (Lonza, catalog number: 51200 )
    2. Agarose LE (Roche Diagnostics, catalog number: 11685678001 )
    3. BDTM BactoTM Brain Heart Infusion (BHI) (Fisher Scientific, catalog number: DF0037-17-8 )
      Manufacturer: BD, catalog number: 237500 .
    4. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C5080 )
    5. Chloramphenicol (Sigma-Aldrich, catalog number: C0378 )
    6. EDTA-Na2 (Sigma-Aldrich, catalog number: E5134 )
    7. Erythromycin (Fisher Scientific, catalog number: 10583315 )
    8. 95% ethanol (Commercial Alcohols, catalog number: P016EA95 )
    9. 100% ethanol (Commercial Alcohols, catalog number: P016EAAN )
    10. EZ-Vision® Three (VWR, catalog number: 97063-166 )
    11. Glacial acetic acid (Caledon Laboratories, catalog number: 1000-1-29 )
    12. Glucose monohydrate (Sigma-Aldrich, catalog number: 49159 )
    13. Glycerol (Merck, catalog number: GX0185-2 )
    14. Glycine (Merck, catalog number: 4810-OP )
    15. Granulated agar (Fisher Scientific, catalog number: BP1423-500 )
    16. Hydrochloric acid ( HCl) (Fisher Scientific, catalog number: 351285-212 )
    17. High DNA Mass Ladder (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10496016 )
    18. 1 Kb Plus DNA Ladder (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10787018 )
    19. Low DNA Mass Ladder (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10068013 )
    20. OxoidTM M17 broth (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: CM0817 )
    21. Magnesium sulfate heptahydrate ( MgSO4·7H2O) (Caledon Laboratories, catalog number: 4860-1-70 )
    22. Magnesium chloride hexahydrate (MgCl2·6H2O) (VWR, catalog number: BDH9244 )
    23. Sodium chloride (NaCl) (Anachemia, catalog number: 81708-380 )
    24. Primers (Table 1) (customized by Thermo Fisher Scientific, www.thermofisher.com)

      Table 1. Primers used in this protocol

      aRestriction sites for ligation and overhangs for Gibson Assembly are underlined. ‘N’ stands for any nucleotides and ‘x’ for any number of nucleotides required for the amplification of a fragment of interest.

    25. Sodium acetate (NaAc) (Sigma-Aldrich, catalog number: S2889 )
    26. Sucrose (Sigma-Aldrich, catalog number: S0389 )
    27. Tris (Base), Ultrapure (Avantor Performance Materials, catalog number: 4109-6 )
    28. 0.8% and 2% agarose gel (see Recipes)
    29. BHI agar medium supplemented with erythromycin (see Recipes)
    30. BHI medium supplemented with erythromycin (see Recipes)
    31. 2 M CaCl2 (see Recipes)
    32. 10 mg/ml chloramphenicol stock solution (see Recipes)
    33. 10 mg/ml and 75 mg/ml erythromycin stock solution (see Recipes)
    34. 70% ethanol (see Recipes)
    35. GM17 agar medium supplemented with CaCl2 or antibiotics (see Recipes)
    36. GM17 medium (see Recipes)
    37. GM17 soft agar medium supplemented with CaCl2 (see Recipes)
    38. Glycine shock solution (see Recipes)
    39. 1 M MgCl2 (see Recipes)
    40. 10x Phage buffer (see Recipes)
    41. Recovery solution (see Recipes)
    42. 50x TAE buffer (see Recipes)
    43. Wash solution (see Recipes)

  6. Kits
    1. Gibson Assembly® Master Mix (New England Biolabs, catalog number: E2611S )
    2. QIAGEN Plasmid Maxi Kit (QIAGEN, catalog number: 12162 )
    3. QIAquick PCR Purification Kit (QIAGEN, catalog number: 28104 )

Equipment

  1. Autoclave
  2. Benchtop microcentrifuge (Eppendorf, model: 5415 D )
  3. Bunsen burner
  4. Centrifuge bottles (SS-34)
  5. Centrifuge Sorvall RC5C and rotor SS-34 (Thermo Fisher Scientific, catalog number: 28020 )
  6. Dry bath with heating block (VWR, catalog numbers: 13259-034 and 13259-130 )
  7. Electroporation cuvettes, 0.2 cm electrode gap (Bio-Rad Laboratories, catalog number: 1652086 )
  8. Gene Pulser II electroporator (Bio-Rad Laboratories, catalog number: 165-2109 )
  9. Incubator-shaker set to 37 °C
    Note: Incubator that can be set to 30 °C (shaking not necessary) and 37 °C.
  10. Micropipettes (Nichiryo, catalog numbers: 00-NPX2-10 , 00-NPX2-100 , 00-NPX2-1000 )
    Note: Sterile filtered pipette tips to reduce contamination of the micropipettes by bioaerosols.
  11. Microwave
  12. MultiDoc-ItTM Imaging System (UVP, catalog number: 97-0200-01 )
  13. PCR Thermal Cycler (MJ Research, model: PTC-200 )
  14. Power supply (Bio-Rad Laboratories, model: PowerPac 300 )
  15. Spectra/Por® 1 dialysis membranes, MWCO 6-8000 (VWR, catalog number: 28170-138 )
    Manufacturer: Spectrum, catalog number: S632650 .
  16. Spectrophotometer Spectronic 20D
  17. Tweezers
  18. VWR® Midi Plus 15 Horizontal Electrophoresis Systems (VWR, catalog number: 89032-296 )

Procedure

  1. Spacer cloning in pL2Cas9
    Plasmid pL2Cas9 encodes the CRISPR-Cas9 components derived from the strain Streptococcus pyogenes SF370 as well as an erythromycin resistance gene. To avoid plasmid loss in E. coli or L. lactis, erythromycin is added to media to a final concentration of 150 µg/ml (Em 150) or 5 µg/ml (Em 5), respectively. When purchased from Addgene, pL2Cas9 is sent as a stab culture of transformed E. coli NEB5α strain. Steps A2 to A4 are adapted from Jiang et al. (2013).
    1. Purification pL2Cas9
      1. Streak the bacterial strain from the stab to a BHI Em 150 plate (see Recipes).
      2. Incubate overnight at 37 °C.
      3. Pick a single colony with a sterile pipette tip using sterile tweezers and drop the tip into 500 ml of BHI Em 150 (see Recipes).
      4. Incubate overnight at 37 °C in a shaking incubator at 200 rpm.
      5. Follow QIAGEN plasmid maxi kit protocol for isolating low copy plasmid.
        Note: We transfer the isopropanol precipitate in 16 x 1.5 ml micro-tubes to minimize loss of DNA during the washing step. Each DNA pellet is then redissolved in 20 µl of the provided plasmid resuspension buffer and pooled in a 1.5 ml micro-tube.
      6. Run a sample of the purified plasmid DNA preparation on a 0.8% agarose gel (see Recipes) with the High DNA Mass Ladder to estimate DNA concentration.
        Note: To visualize DNA, we use EZ-Vision® Three, a 6x loading buffer containing a fluorescent DNA dye.
      7. Keep the purified plasmid DNA at -20 °C until needed.
    2. Spacer design and annealing
      1. Search for a 5’-NGG-3’ sequence in the locus to modify (Figure 1). This signature sequence constitutes the protospacer adjacent motif (PAM) recognized by SpCas9.
      2. To clone the desired spacer in pL2Cas9, design and synthesize two ssDNA oligos of 35 nucleotides corresponding to both strands of the selected protospacer with BsaI restriction sites (Figure 1, Table 1).
        Note: The GC content of the spacer sequences should not influence the efficiency of SpCas9 to cleave its target DNA (Tao et al., 2017). To avoid off-target activity, make sure that the protospacer is not found elsewhere in the phage or the cloning host genome next to a functional PAM.
      3. Dilute the oligos to 50 μM in molecular biology water.
      4. For 5’-phosphorylation of the oligos, mix 2 μl of each diluted oligo with 10 µl of 5x T4 ligase buffer, 1 µl of T4 polynucleotide kinase (PNK) and 32 µl of distilled water in a PCR tube to a final volume of 50 µl.
        Note: 5x T4 ligase buffer is supplied with T4 DNA ligase. Divide the ligase buffer into single-use aliquots as ATP in the buffer can be degraded by repeated freeze-thaw cycles.
      5. Perform the phosphorylation for 30 min at 37 °C and heat inactivate the PNK for 20 min at 65 °C (in a PCR machine or a water bath).
      6. Add 2.5 µl of 1 M NaCl to the phosphorylated oligos.
      7. Place the tube in a PCR thermal cycler and heat the sample for 5 min at 95 °C before cooling down to 25 °C at a rate of 0.1 °C per sec.
      8. Keep at -20 °C for up to 2 months.


        Figure 1. Graphical representation of a spacer design. In this example, a PAM is identified in the phage genome. The 30 bp sequence upstream the PAM is called the ‘protospacer’, and must be incorporated into the CRISPR array (crRNA) in the form of a ‘spacer’ to dictate target specificity. BsaI restriction sites are added to the ends of the spacer for ligation into pL2Cas9.

    3. Digestion of pL2Cas9 with BsaI
      1. Digest 1 µg of purified pL2Cas9 with BsaI by following the manufacturer’s instructions with an overnight incubation at 37 °C (final volume of 50 µl).
      2. Dephosphorylate the 5’-ends of the digested pL2Cas9 with the Antarctic phosphatase by following the manufacturer’s instructions.
      3. Purify the digested and dephosphorylated vector by precipitation with salts and ethanol.
        Note: Purification by gel extraction removes uncut vector, but the yield is poor.
        1. Add 1:10 volume of NaAc 3 M pH 5.2 to DNA.
        2. Add 2 volumes of 100% ethanol.
        3. Mix thoroughly and incubate on ice for 15 min.
        4. Centrifuge in a benchtop microcentrifuge at 16,000 x g for 15 min.
        5. Carefully remove the supernatant and wash the pellet with 70% ethanol (see Recipes).
        6. Air dry the pellet for 5-10 min.
        7. Resuspend the pellet in 30 µl 10 mM Tris-HCl pH 8.5.
      4. Run the purified vector on a 0.8% agarose gel with the annealed oligos as well as both the High DNA Mass and 1 Kb Plus DNA Ladders in separate wells to estimate concentrations.
      5. Store the digested pL2Cas9 at -20 °C until needed.
        Note: Aliquot into smaller volumes to prevent DNA degradation that may occur during repeated freeze-thaw cycles.
    4. Ligation of the targeting plasmid
      1. Set up ligation reactions with the T4 DNA ligase following the manufacturer’s instructions. Use a molar ratio of approximately 3:1 of insert to vector.
      2. Prepare another ligation reaction without the insert (negative ligation control) to evaluate vector re-circularization.
      3. Perform the ligations overnight at 16 °C (in a PCR machine or a water bath).
      4. Heat inactivate the ligase at 65 °C for 10 min, put on ice, and proceed immediately to the transformation (Steps A5 and A6).
        Note: Ligation products can be kept a few days at -20 °C, but transformation efficiency will decrease.
    5. Preparation of L. lactis MG1363 competent cells
      Note: If working with other phage-host, transformation of DNA constructs can be done directly into the bacterial host of interest or first into L. lactis MG1363. In the latter case, DNA constructs have to be extracted from the cloning host and then transferred into the host of interest. Cells should be transformed with one plasmid, selected with the appropriate antibiotics, made competent, transformed with the second plasmid, and selected with both antibiotics. We do not recommend double transformation.
      1. Inoculate 10 ml of GM17 broth (see Recipes) with L. lactis MG1363 and incubate overnight at 30 °C.
      2. Inoculate 5 tubes containing 9.7 ml glycine shock solution (see Recipes) with 300 µl of the overnight culture from Step A5a.
        Note: A 10-ml bacterial culture is sufficient to prepare 50 µl of competent cells. Typically, 5 x 50 µl competent cells are prepared for the transformation of: (1) the ligation product, (2) uncut vector (positive transformation control), (3) dephosphorylated cut vector (negative digestion control) (4) negative ligation control, and (5) no DNA (negative transformation control). If needed, additional cultures can be prepared concomitantly.
      3. Incubate at 30 °C until the bacterial cultures reach an OD600 of 0.2.
      4. Transfer the cultures into sterile centrifuge bottles (SS-34) and centrifuge for 5 min at 4 °C and 12,000 x g.
        Note: From here onwards, cultures and solutions should be kept on ice whenever possible.
      5. Discard supernatant and resuspend each pellet in 1 ml of sterile cold wash solution (see Recipes).
      6. Transfer cells into 1.5 ml micro-tubes.
      7. Centrifuge the resuspensions in a benchtop microcentrifuge at 16,000 x g for 1 min.
        Note: We perform this step at room temperature (RT), but a refrigerated microcentrifuge may be used to keep the cultures cold (~4 °C).
      8. Repeat the last three steps twice.
      9. Resuspend each pellet of competent cells in 50 μl of sterile cold wash solution before pooling them in a 1.5 ml micro-tube and proceed immediately to Step A6.
    6. Electroporation of L. lactis MG1363
      1. Dialyse assembled DNA using a membrane (Figure 2).
        Note: Ligation and Gibson Assembly products should be desalted prior to electroporation.


        Figure 2. Quick and easy DNA dialysis. A. Heat the wide end of a Pasteur pipette with a Bunsen burner flame until the glass turns red. B. Quickly pierce the lid of a 1.5 ml micro-tube. C. Add 2 ml of distilled water in the micro-tube. Due to surface tension, a convex meniscus should be above the edge of the micro-tube. D. Place a 4-cm square of dialysis membrane (MWCO 6-8000) in distilled water. Once it is hydrated, cut the dialysis membrane tubing to the length to have two separate layers of membrane. Put a single layer of membrane onto the water and close the lid of the micro-tube. Make sure there is no air bubble between the water and the dome-shaped membrane. E. Remove excess water on top of the membrane. F. Put 5 µl of DNA sample on top of the membrane. G. Wait approximately 30 sec. H. Collect the desalted DNA. Repeat the whole process for every sample to dialyse.

      2. Add 5 µl of DNA (or water for negative transformation control) and 45 µl of competent cells to cold electroporation cuvettes.
        Note: Keep the cuvettes and the recovery solution on ice.
      3. Set up the Gene Pulser to 25 µF, 200 Ω and 2.5 KV.
      4. Place a cuvette in the track and simultaneously push both red buttons until the tone.
      5. Quickly add 500 µl of cold recovery solution (see Recipes) to cells and leave on ice for 10 min.
      6. Transfer the content of the cuvettes into 1.5 ml micro-tubes.
      7. Repeat the last three steps for every transformation.
      8. Incubate for 2 h at 30 °C.
      9. Plate the cells on GM17 supplemented with the appropriate antibiotic (see Recipes). Use 5-10 glass beads per plate to spread the transformed cells evenly.
      10. Incubate for 24-48 h at 30 °C.
    7. Colony PCR
      1. To confirm the presence of the DNA insert in the plasmid constructs, pick several single colonies with sterile pipette tips and place each of them in 50 µl molecular biology water.
        Note: The number of colonies to screen will depend on the number of background colonies on the digested vector control plate.
      2. Using the same pipette tips, streak the colonies on a GM17 plate supplemented with the appropriate antibiotic and incubate overnight at 30 °C.
        Note: This plate will later serve to start liquid cultures of positive clones.
      3. Use 5 µl of DNA template (water-bacteria suspension) per 50 µl PCR reactions with the Taq DNA Polymerase. Always follow the manufacturer’s instructions.
        Note: We use the backbone-specific primers Cas9_S.pyo_F6 and crRNA_S.pyo_R (Table 1) to screen for new spacers in the CRISPR array of pL2Cas9. Amplification of the uncut vector or desired ligation products will both generate an 815 bp fragment. To screen colonies for the right repair template, we use the backbone-specific primers pNZins_F and pNZins_R (Table 1). Amplification of uncut pNZ123 will generate a 145 bp fragment, while the assembled repair template will generate a bigger fragment of variable length.
      4. Run the PCR products on a 2% agarose gel (see Recipes) with the 1 Kb Plus DNA Ladder to determine their size.
      5. To confirm the sequence of the inserts, submit PCR products for Sanger sequencing.
        Note: To reduce the number of PCR products sent for sequencing, positive clones can also be identified with insert-specific primers. To screen for new spacers in pL2Cas9, we use the oligo I from Step A2 (Table 1) and crRNA_S.pyo_R. While a positive clone results in the amplification of a 391 bp fragment, a negative clone results in no product.
      6. Inoculate 10 ml of GM17 broth medium supplemented with the appropriate antibiotics with positive clones and incubate overnight at 30 °C.
      7. Store cells at -80 °C by adding 850 µl of overnight culture to 150 µl of sterile glycerol.

  2. Construction of homologous repair template
    We use the broad-host-range and high-copy-number plasmid pNZ123 to construct our recombination templates. It confers chloramphenicol resistance to the bacterial cells carrying it. To avoid plasmid loss in L. lactis, chloramphenicol is supplied at a final concentration of 5 µg/ml (Cm 5). Other plasmids compatible with pL2Cas9 could be used. Most importantly, the repair template must be designed to lack the Cas9 target sequence so that recombinant phages, and the template itself, can avoid cleavage. The most effective approach is to create a deletion, which removes the whole target sequence (PAM and protospacer) (Figure 3). Otherwise, a single mutation can be introduced in the PAM and/or multiple mutations in the protospacer to prevent DNA cleavage. If the target sequence is in a coding region, mutations should be designed based on codon usage patterns.


    Figure 3. Construction of homologous repair template with a desired deletion. Insert A (left) contains the 5’-end of a gene of interest (yellow) and insert B (right) contains the 3’-end of the same gene. The two fragments, amplified from the phage genome, are the homologous arms of the repair template. The external primers have overlaps (green) for inserting the amplicons into linearized pNZ123 using Gibson Assembly (pNZ_insertA and pNZ_insertB, Table 1). The inner primers have complementary overhangs (yellow) for annealing together. Assembly of the two inserts removes part of the yellow gene, and the target sequence (blue box) is absent from the repair template. A similar strategy can be used to generate deletions, point mutations and insertions.

    1. Purification pNZ123
      1. Streak L. lactis MG1363 (pNZ123) to a GM17 Cm 5 plate (see Recipes).
      2. Incubate overnight at 30 °C.
      3. Pick a single colony with a sterile pipette tip using sterile tweezers and drop the tip into 500 ml of GM17 Cm 5 (see Recipes).
      4. Incubate statically overnight at 30 °C.
      5. Harvest the overnight culture by centrifugation at 6,000 x g for 15 min.
      6. Resuspend the bacterial pellet in 20 ml buffer P1 (from QIAGEN plasmid maxi kit) supplemented with lysozyme 30 mg/ml.
      7. Incubate for 30 min at 37 °C.
      8. Follow QIAGEN plasmid maxi kit protocol.
      9. Run a sample of the purified plasmid DNA preparation on a 0.8% agarose gel (see Recipes) with the High DNA Mass Ladder to estimate DNA concentration.
      10. Keep the purified plasmid DNA at -20 °C until needed.
    2. Linearization of pNZ123
      1. Digest 1 µg of purified pNZ123 with XbaI following the manufacturer’s instructions with an overnight incubation at 37 °C.
      2. Amplify the digested vector with primers pNZ_XbaI_F and pNZ_XbaI_R (Table 1) and the Q5 DNA polymerase following the manufacturer’s instructions. We use an annealing temperature of 58 °C and an elongation time of 75 sec.
        Note: Amplification of the digested vector significantly reduces background from uncut or re-circularized vector.
      3. Clean the resulting PCR products with the QIAquick PCR Purification kit following the manufacturer’s instructions. Elute in molecular biology water.
      4. Store the linearized and PCR-amplified pNZ123 at -20 °C until needed.
        Note: Aliquot into smaller volumes to prevent DNA degradation that may occur during repeated freeze-thaw cycles.
    3. Amplification of inserts (Figure 3)
      1. Design primers with overlaps suited for Gibson Assembly into linearized pNZ123.
        Note: Even though the manufacturer recommends a 20 bp overlap between the fragments to be assembled, we found that > 30 bp overlaps significantly increased the efficiency of assembly. While 250-500 bp homologous arms are preferred, shorter arms are generally sufficient for recombination with the phage genome.
      2. PCR amplify the inserts with the Q5 DNA polymerase using the phage genome as a template and following the manufacturer’s instructions.
      3. Run on a 2% agarose gel with the linearized pNZ123 and both the Low DNA Mass and 1 Kb Plus Ladders in separate wells to confirm and determine the concentration of fragments.
    4. Assembly and transformation of the repair template
      1. Assemble the fragments from Steps B1 and B2 using Gibson Assembly Master Mix according to the manufacturer’s instructions.
        Note: For best results, assemble and transform the repair template on the same day.
      2. Incubate for 1 h at 50 °C.
      3. Keep on ice (or -20 °C) until needed.
      4. Prepare competent cells of L. lactis MG1363 (Step A5).
        Note: The repair template can also be electroporated into L. lactis MG1363 already harboring the targeting plasmid pL2Cas9.
      5. Proceed immediately to the electroporation (Steps A6) of the repair template and plate the cells on GM17 supplemented with the appropriate antibiotics.
      6. Incubate 24-48 h at 30 °C.
        Note: Selection with two antibiotics often slows down bacterial growth.
      7. Analyze the transformants by colony PCR (Step A7).

  3. Phage engineering (Figure 4)
    We perform double layer plaque assays to obtain isolated phage plaques and purify recombinant phages. This step could easily be adapted to other phage-host pairs, provided that the bacterial host harbors the two DNA constructs obtained in Steps A and B. We do not use antibiotic selection during phage infection since the plasmids are stable in L. lactis MG1363. Plasmid stability may vary in different hosts, and antibiotic selection should be considered in some cases.


    Figure 4. Targeted genome editing of phage p2 using CRISPR-Cas9. Phage p2 infects L. lactis MG1363 harboring a targeting plasmid (pL2Cas9) and a repair template. The CRISPR array is depicted as black diamonds (repeats) and a blue box (spacer). Shortly after the viral DNA enters the bacterium, the CRISPR-Cas9 complex recognizes and cleaves its target. The genomic lesion can then be repaired with a template suited for homologous recombination and harboring a desired mutation (here a deletion). Recombinant phages avoid cleavage by the CRISPR-Cas9 system as they lack the target sequence.

    1. Phage infection
      1. Inoculate 10 ml of GM17 supplemented with Em 5 and Cm 5 with L. lactis MG1363 harboring the targeting plasmid and the repair template.
      2. Incubate overnight at 30 °C.
      3. Perform a ten-fold serial dilution of a phage p2 lysate. Prepare six sterile 1.5 ml micro-tubes containing 900 µl of 1x sterile phage buffer (see Recipes). Add 100 µl of undiluted phage lysate to the first micro-tube and mix by gently pipetting up and down. This is dilution 10-1. Using a new pipette tip, transfer 100 µl of the 10-1 dilution to the second micro tube (dilution 10-2), gently mix, and repeat up to dilution 10-6.
      4. Add 300 µl of overnight bacterial culture to 3 ml GM17 soft agar medium supplemented with CaCl2 (see Recipes) kept at 50 °C.
      5. Add 100 µl of undiluted phage lysate.
      6. Pour rapidly on top of a GM17 agar medium supplemented with CaCl2 and swirl the plate to spread the soft agar evenly.
      7. Repeat the last three steps for dilutions 10-2, 10-4 and 10-6.
      8. Incubate 24 h at 30 °C.
      9. Pick 3 phage plaques with a truncated sterile pipette tip and put each phage-containing agar plug into a separate sterile 1.5 ml micro-tube containing 500 µl of 1x phage buffer.
        Note: Phage plaques can be kept in buffer at 4 °C for a few weeks.
      10. Let the phages diffuse in the buffer for > 20 min at RT.
      11. Repeat two more rounds of infection on the same bacterial strain (Steps C1a to C1j) to purify the recombinant phages.
    2. Mutant phage analysis
      1. Design pairs of primers amplifying the mutated region of the phage genome and absent from the repair template.
      2. Use 5 µl of the phage suspension per 50 µl PCR reactions with the Taq DNA Polymerase and follow the manufacturer’s instructions. Use wild-type phage p2 as a template for positive control.
      3. Run the PCR products on a 2% agarose gel with the 1 Kb Plus DNA Ladder to determine their size.
        Note: Gene deletions can be observed on gel by shorter PCR products obtained with recombinant phages compared to those obtained with the wild-type phage p2.
      4. Submit PCR products for Sanger sequencing and align them with the phage genome to confirm the desired mutation in the recombinant phages.
        Note: Knockout of genes that are nonessential for phage multiplication under laboratory conditions always resulted in a homogeneous population of recombinant phages.
      5. Sequence the whole genome of the engineered phages to confirm the absence of additional mutations.
        Note: To date, we have sequenced the full genome of several mutant phages and no off-target mutations were detected following CRISPR-Cas9-mediated genome engineering. Unexpected mutations could occur if genetic compensation is needed to buffer against deleterious mutations.

Data analysis

Multiple mutants of phage p2 were generated with this methodology. We refer the readers to the original paper (Lemay et al., 2017).

Notes

  1. In interest of time, some of the steps from the protocol can be done concomitantly (Figure 5).


    Figure 5. Flowchart illustrating the protocol to generate a mutant phage with CRISPR-Cas9

  2. Gene deletions are not suitable for in vivo investigation of genes essential for phage multiplication. When knockouts are not possible, non-disruptive mutations, such as point mutations or insertions, can be generated.

Recipes

  1. 0.8% and 2% agarose gel
    1. Add 0.8 g or 2 g of agarose LE to 100 ml of 1x TAE
    2. Heat in microwave until melted
    3. Cool down to ~50 °C
    4. Pour the appropriate volume into the electrophoresis apparatus and add the desired comb. Allow agarose to solidify, remove the comb and cover the gel with 1x TAE buffer
  2. BHI agar medium supplemented with erythromycin
    Note: Important to use BHI as E. coli is more resistant to erythromycin in lysogeny broth (LB).
    1. Dissolve 3.7 g of BHI and 1 g of agar in 75 ml of distilled water
    2. Complete to 100 ml with distilled water
    3. Sterilize by autoclaving and cool down to 50 °C
    4. Add 200 µl of 75 mg/ml erythromycin (150 µg/ml)
    5. Pour approximately 20 ml of medium per plate
    6. Store at 4 °C in the dark
  3. BHI medium supplemented with erythromycin
    1. Dissolve 18.5 g of BHI in 500 ml of distilled water
    2. Sterilize by autoclaving and cool down
    3. Store at room temperature (RT)
    4. When ready to grow the bacterial culture, add 1 ml of 75 mg/ml erythromycin (150 µg/ml)
  4. 2 M CaCl2
    Note: CaCl2 is required for phage p2 infection.
    1. Dissolve 29.4 g of CaCl2·2H2O in 100 ml of distilled water
    2. Sterilize by autoclaving and cool down
    3. Aliquot in smaller volumes to limit contamination of the solution
    4. Store at RT
  5. 10 mg/ml chloramphenicol stock solution
    Note: Chloramphenicol is required for selection of bacteria harboring pNZ123 and derivatives.
    1. Dissolve 0.1 g chloramphenicol in 10 ml 95% ethanol
    2. Store at -20 °C
  6. 10 mg/ml and 75 mg/ml erythromycin stock solution
    Note: Erythromycin is required for selection of bacteria harboring pL2Cas9 and its derivatives.
    1. Dissolve 0.1 g or 0.75 g erythromycin in 10 ml 95% ethanol
    2. Store at -20 °C
  7. 70% ethanol
    1. Mix 2.8 L 100 % ethanol with 1.2 L of distilled water
    2. Store at RT
  8. GM17 agar medium supplemented with CaCl2 or antibiotics
    1. Dissolve 37.25 g of M17, 5 g glucose monohydrate and 10 g agar in 850 ml distilled water
    2. Complete to 1 L with distilled water
    3. Sterilize by autoclaving and cool down to 50 °C
    4. Add 5 ml of sterile 2 M CaCl2 (final concentration of 10 mM) or 0.5 ml of the appropriate 10 mg/ml antibiotic stock (final concentration of 5 µg/ml)
    5. Pour approximately 20 ml of medium per plate
    6. Store at RT or at 4 °C in the dark if supplemented with antibiotics
  9. GM17 medium
    1. Dissolve 37.25 g of M17 and 5 g glucose monohydrate in 1 L of distilled water
    2. Sterilize by autoclaving and cool down
    3. Store at RT
  10. GM17 soft agar medium supplemented with CaCl2
    1. Dissolve 3.73 g M17, 0.5 g glucose monohydrate and 0.75 g agar in 85 ml distilled water
    2. Complete to 100 ml with distilled water
    3. Sterilize by autoclaving and cool down to 50 °C
    4. Add 0.5 ml of sterile 2 M CaCl2 (final concentration of 10 mM)
    5. Pour 3 ml of medium in as many 13 x 100 mm sterile glass tubes as needed
    6. Keep the aliquots at 50 °C in a dry bath with heating block until ready to use
    7. Store remaining of the media at RT and melt in the microwave whenever needed
  11. Glycine shock solution
    1. Dissolve 1 g of glycine and 17.1 g of sucrose in GM17 broth medium for a final volume of 100 ml
    2. Filter sterilize through 0.2 µm sterile PES syringe filter
    3. Store the solution at RT
  12. 1 M MgCl2
    1. Dissolve 203.3 g of MgCl2·6H2O in 1 L of distilled water
    2. Sterilize by autoclaving
    3. Store at RT
  13. 10x Phage buffer
    1. Dissolve 58 g of NaCl and 20 g of MgSO4·7H2O in 500 ml of 1 M Tris-HCl pH 7.5
    2. Complete to 1 L with distilled water
    3. Sterilize by autoclaving
    4. Store at RT
    5. For 1x phage buffer, dilute 100 ml of 10x phage buffer with 900 ml distilled water, sterilize it by autoclaving and aliquot in smaller volumes to prevent contamination
  14. Recovery solution
    1. For a final volume of 100 ml, dissolve 17.1 g of sucrose in GM17 broth medium supplemented with 2 ml of 1 M MgCl2 and 100 µl of 2 M CaCl2
    2. Filter sterilize through a 0.2 µm sterile PES syringe filter
    3. Store the solution at 4 °C
  15. 50x TAE buffer
    1. Mix 242 g Tris Base, 57.1 ml glacial acetic acid, 100 ml 0.5 M EDTA pH 8.0
    2. Complete to 1 L with distilled water
    3. Store at RT
    4. For 1x TAE buffer, dilute 20 ml of TAE 50x with 980 ml of distilled water
  16. Wash solution
    1. Mix 100 ml of glycerol and 171 g of sucrose (0.5 M) in 800 ml of distilled water
    2. Complete to 1 L with distilled water
    3. Sterilize by autoclaving
    4. Store the solution at 4 °C

Acknowledgments

This protocol details the methodology used in the original paper (Lemay et al., 2017). We thank Witold Kot and Hanne Hendrix for insightful discussions. M.-L.L. is supported by a scholarship from the Natural Sciences and Engineering Research Council of Canada (NSERC). S.M. acknowledges funding from the NSERC Discovery program. S.M. holds a Tier 1 Canada Research Chair in Bacteriophages. The authors declare no competing financial interest.

References

  1. Bari, S. M. N., Walker, F. C., Cater, K., Aslan, B. and Hatoum-Aslan, A. (2017). Strategies for editing virulent staphylococcal phages using CRISPR-Cas10. ACS Synth Biol 6(12): 2316-2325.
  2. Box, A. M., McGuffie, M. J., O'Hara, B. J. and Seed, K. D. (2015). Functional analysis of bacteriophage immunity through a type I-E CRISPR-Cas system in vibrio cholerae and its application in bacteriophage genome engineering. J Bacteriol 198(3): 578-590.
  3. Breitbart, M. and Rohwer, F. (2005). Here a virus, there a virus, everywhere the same virus? Trends Microbiol 13:278-284.
  4. Deveau, H., Labrie, S. J., Chopin, M. C. and Moineau, S. (2006). Biodiversity and classification of lactococcal phages. Appl Environ Microbiol 72(6): 4338-4346.
  5. De Vos, W. M. (1987). Gene cloning and expression in lactic streptococci. FEMS Microbiol Lett 46: 281-295.
  6. Hurwitz, B. L., U'Ren, J. M. and Youens-Clark, K. (2016). Computational prospecting the great viral unknown. FEMS Microbiol Lett 363(10).
  7. Jiang, W., Bikard, D., Cox, D., Zhang, F. and Marraffini, L. A. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31(3): 233-239.
  8. Kiro, R., Shitrit, D. and Qimron, U. (2014). Efficient engineering of a bacteriophage genome using the type I-E CRISPR-Cas system. RNA Biol 11(1): 42-44.
  9. Lemay, M. L., Tremblay, D. M. and Moineau, S. (2017). Genome engineering of virulent lactococcal phages using CRISPR-Cas9. ACS Synth Biol 6(7): 1351-1358.
  10. Mahony, J., Murphy, J. and van Sinderen, D. (2012). Lactococcal 936-type phages and dairy fermentation problems: from detection to evolution and prevention. Front Microbiol 3: 335.
  11. Manor, M. and Qimron, U. (2017). Selection of genetically modified bacteriophages using the CRISPR-Cas system. Bio Protoc 7(15) e2431.
  12. Martel, B. and Moineau, S. (2014). CRISPR-Cas: an efficient tool for genome engineering of virulent bacteriophages. Nucleic Acids Res 42(14): 9504-9513.
  13. Paez-Espino, D., Eloe-Fadrosh, E. A., Pavlopoulos, G. A., Thomas, A. D., Huntemann, M., Mikhailova, N., Rubin, E., Ivanova, N. N. and Kyrpides, N. C. (2016). Uncovering Earth’s virome. Nature 536: 425-430.
  14. Pires, D. P., Cleto, S., Sillankorva, S., Azeredo, J. and Lu, T. K. (2016). Genetically engineered phages: a review of advances over the last decade. Microbiol Mol Biol Rev 80(3): 523-543.
  15. Suttle, C. A. (2005). Viruses in the sea. Nature 437(7057): 356-361.
  16. Tao, P., Wu, X., Tang, W. C., Zhu, J. and Rao, V. (2017). Engineering of bacteriophage T4 genome using CRISPR-Cas9. ACS Synth Biol 6(10): 1952-1961.

简介

该协议描述了一个直接的方法来产生严格裂解噬菌体的基因组中的特定突变。 简而言之,在细菌宿主(此处为革兰氏阳性模型乳酸乳球菌MG1363)内提供靶向CRISPR-Cas9系统和适合于同源重组的修复模板。 CRISPR-Cas9系统被编程为切割入侵噬菌体的基因组上存在的特定区域,但是缺少重组模板。 该系统触发重组事件或施加分离重组噬菌体所需的选择性压力。 利用这种方法,我们在毒性乳酸球菌噬菌体p2的基因组中产生了多个基因敲除,点突变和插入。 考虑到本协议中使用的质粒的广泛宿主范围,后者可以外推到其他噬菌体 - 宿主对。

【背景】噬菌体是在每个生态系统中发现丰富的细菌病毒(Suttle,2005; Breitbart and Rohwer,2005),毫不奇怪,它们是牛奶的天然居民。噬菌体p2是乳品工业中发现的强毒乳球菌噬菌体的最普遍组( Sk1virus )的模型(Deveau等人,2006; Mahony等人。,2012),它感染革兰氏阳性细菌乳酸乳球菌MG1363,也是基础研究的模式菌株。尽管p2作为参照噬菌体的地位,但几乎一半的基因编码未表征的蛋白质。同样,由宏基因组学确定的绝大多数噬菌体基因在公共数据库中没有功能分配和同系物(Hurwitz等人,2016; Paez-Espino等人, 2016)。

研究基因的方法之一是通过修饰和随后观察所得到的表型。噬菌体基因组只能在宿主内以其生物活性形式进行修饰。强毒噬菌体严格裂解;因此,它们的基因组从未整合到细菌染色体中。这为DNA的体内修饰增加了一个时间限制,只能在短的感染周期内对其进行操作。 CRISPR-Cas研究在过去十年的出现导致将这种天然的原核生物防御机制适应于编辑包括毒性噬菌体在内的多种生物体和病毒的基因组的有力工具(Kiro等, 2014; Martel和Moineau,2014; Box等人,2015; Pires等人,2016; Bari等人, ,2017; Lemay等人,2017; Manor和Qimron,2017; Tao等人,2017)。

在这里,我们详细描述了使用克隆到乳酸细菌L中的熟知的酿脓链球菌Cas9(SpCas9)编辑噬菌体p2基因组的简单和可重复的方案。乳酸杆菌MG1363(Lemay等人,2017)。在我们的实验室中,该方案也被成功地用于编辑感染革兰氏阴性E的毒性噬菌体的基因组。 (未发表),说明其广泛的适用性。

关键字:噬菌体, 乳酸乳球菌, 基因组编辑, CRISPR-Cas9, 同源重组

材料和试剂

  1. 物料
    1. 一次性巴斯德吸管(VWR,目录号:14672-200)
    2. 3毫米玻璃珠(VWR,目录号:26396-508)
      制造商:Walter Stern,目录号:100C。
    3. 带盖的100毫米玻璃管(Fisher Scientific,产品目录号:14-961-27和05-888-1A)
      制造商:Bal Supply,目录号:13144UL。
    4. 带盖的150毫米玻璃管(Fisher Scientific,目录号:14-961-32和05-888C)
      制造商:Bal Supply,目录号:18144CL。
    5. 0.5毫升微型管(SARSTEDT,目录号:72.699)
    6. 1.5毫升微型管(SARSTEDT,目录号:72.690)
    7. 无菌100 x 15毫米塑料培养皿('盘')(VWR,目录号:25384-302)
    8. 10ml无菌BD Luer-Lok TM Tip注射器(BD,目录号:309604)

    9. 0.2μm无菌PES注射器过滤器(SARSTEDT,目录号:83.1826.001)
    10. 0.45μm无菌PES注射器过滤器(SARSTEDT,目录号:83.1826)

  2. 噬菌体和细菌菌株
    1. 乳酸乳球菌MG1363(Félixd'Hérelle细菌病毒参考中心,目录号:HER1439)
      注意:乳酸乳球菌一般被认为是一种安全的细菌,所有的实验都可以在一级生物安全实验室进行。
    2. 噬菌体p2(Félixd'Hérelle细菌病毒参考中心,目录号:HER457)

  3. 质粒
    1. pL2Cas9(Lemay等人,2017)(Addgene,目录号:98841)
    2. pNZ123(de Vos,1987)(乳酸乳球菌MG1363(pNZ123))(Félixd'Hérelle细菌病毒参考中心,目录号:HER1532)

    1. 南极磷酸酶(5,000 U / ml)(New England Biolabs,目录号:M0289S)
    2. BsaI(10,000 U / ml)(New England Biolabs,目录号:R0535S)
    3. 溶菌酶(20,000 U / mg干重)(Thermo Fisher Scientific,目录号:89833)
    4. Q5 DNA聚合酶(2,000U / ml)(New England Biolabs,目录号:M0491S)
    5. T4 DNA连接酶(1,000U / ml)(Thermo Fisher Scientific,Invitrogen TM,目录号:15224017)。
    6. T4多核苷酸激酶(10,000U / ml)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EK0031)
    7. Taq DNA聚合酶(5,000 U / ml)(NBS Biologicals,目录号:9K-001-0034)
    8. XbaI(20,000U / ml)(New England Biolabs,目录号:R0145S)

  4. 试剂
    1. AccuGENE TM分子生物学水(Lonza,目录号:51200)
    2. 琼脂糖LE(Roche Diagnostics,目录号:11685678001)
    3. BD TM TM Bacto TM脑心浸液(BHI)(Fisher Scientific,目录号:DF0037-17-8)
      制造商:BD,目录号:237500。
    4. 氯化钙二水合物(CaCl 2•2H 2 O)(Sigma-Aldrich,目录号:C5080)
    5. 氯霉素(Sigma-Aldrich,目录号:C0378)
    6. EDTA-Na 2(Sigma-Aldrich,目录号:E5134)
    7. 红霉素(Fisher Scientific,目录号:10583315)
    8. 95%乙醇(商业醇,目录号:P016EA95)
    9. 100%乙醇(商业醇,目录号:P016EAAN)
    10. EZ-Vision ®三(VWR,目录号:97063-166)
    11. 冰醋酸(Caledon Laboratories,目录号:1000-1-29)
    12. 葡萄糖一水合物(Sigma-Aldrich,目录号:49159)
    13. 甘油(Merck,目录号:GX0185-2)
    14. 甘氨酸(Merck,目录号:4810-OP)
    15. 颗粒琼脂(Fisher Scientific,目录号:BP1423-500)
    16. 盐酸(HCl)(Fisher Scientific,目录号:351285-212)
    17. 高DNA质量梯(Thermo Fisher Scientific,Invitrogen TM,目录号:10496016)
    18. 1 Kb Plus DNA Ladder(Thermo Fisher Scientific,Invitrogen TM,产品目录号:10787018)
    19. 低DNA质量梯(Thermo Fisher Scientific,Invitrogen TM,目录号:10068013)
    20. Oxoid TM M17肉汤(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:CM0817)
    21. 硫酸镁七水合物(MgSO 4•7H 2 O)(Caledon Laboratories,目录号:4860-1-70)
    22. 氯化镁六水合物(MgCl 2•6H 2 O)(VWR,目录号:BDH9244)
    23. 氯化钠(NaCl)(Anachemia,目录号:81708-380)
    24. 引物(表1)(由Thermo Fisher Scientific定制, www.thermofisher.com

      表1.本协议中使用的引物

      a 下划线标出了Gibson Assembly的连接和突出的限制性位点。 'N'代表任何核苷酸,'x'代表扩增感兴趣片段所需的任何数量的核苷酸。

    25. 乙酸钠(NaAc)(Sigma-Aldrich,目录号:S2889)
    26. 蔗糖(Sigma-Aldrich,目录号:S0389)
    27. Tris(Base),Ultrapure(Avantor Performance Materials,目录号:4109-6)
    28. 0.8%和2%琼脂糖凝胶(见食谱)
    29. BHI琼脂培养基补充红霉素(见食谱)
    30. BHI培养基补充红霉素(见食谱)
    31. 2 M CaCl 2(见食谱)
    32. 10毫克/毫升氯霉素原液(见食谱)
    33. 10毫克/毫升和75毫克/毫升红霉素原液(见食谱)
    34. 70%乙醇(见食谱)
    35. GM17琼脂培养基补充有CaCl 2或抗生素(见食谱)
    36. GM17中等(见食谱)
    37. GM17软琼脂培养基补充CaCl 2(见食谱)
    38. 甘氨酸休克溶液(见食谱)
    39. 1 M MgCl 2(见食谱)
    40. 10倍噬菌体缓冲液(见食谱)
    41. 恢复解决方案(请参阅食谱)
    42. 50倍TAE缓冲液(见食谱)
    43. 洗液(见食谱)

  5. 套件
    1. Gibson Assembly Master Mix(New England Biolabs,目录号:E2611S)
    2. QIAGEN Plasmid Maxi Kit(QIAGEN,目录号:12162)
    3. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28104)

设备

  1. 高压灭菌器
  2. 台式微量离心机(Eppendorf,型号:5415 D)
  3. 本生燃烧器
  4. 离心瓶(SS-34)
  5. 离心机Sorvall RC5C和转子SS-34(Thermo Fisher Scientific,目录号:28020)
  6. 加热块干燥浴(VWR,目录号:13259-034和13259-130)
  7. 电穿孔小杯,0.2cm电极间隙(Bio-Rad Laboratories,产品目录号:1652086)
  8. Gene Pulser II电穿孔仪(Bio-Rad Laboratories,目录号:165-2109)
  9. 孵化器摇床设置为37°C
    注意:培养箱可以设置为30°C(无需摇动)和37°C。
  10. Micropipettes(Nichiryo,产品目录号:00-NPX2-10,00-NPX2-100,00-NPX2-1000)
    注意:无菌过滤枪头可减少生物气溶胶污染微量移液管。
  11. 微波炉
  12. MultiDoc-It TM成像系统(UVP,目录号:97-0200-01)
  13. PCR热循环仪(MJ Research,型号:PTC-200)
  14. 电源(Bio-Rad Laboratories,型号:PowerPac 300)
  15. Spectra / Por透析膜,MWCO 6-8000(VWR,目录号:28170-138)
    制造商:Spectrum,产品目录号:S632650。
  16. 分光光度计Spectronic 20D
  17. 镊子
  18. VWR Midi Plus 15水平电泳系统(VWR,目录号:89032-296)

程序

  1. Spacer克隆在pL2Cas9中
    质粒pL2Cas9编码源自酿脓链球菌SF370的CRISPR-Cas9组分以及红霉素抗性基因。为了避免E中质粒的丢失。大肠杆菌或 L。向培养基中加入红霉素至终浓度分别为150μg/ ml(Em 150)或5μg/ ml(Em 5)。当从Addgene购买时,pL2Cas9作为转化的大肠杆菌NEB5α菌株的刺状培养物发送。步骤A2至A4由Jiang等人改编。 (2013年)。
    1. 纯化pL2Cas9
      1. 将刺中的细菌菌株划到BHI Em 150平板(参见食谱)。
      2. 在37°C孵育过夜。
      3. 用无菌镊子用无菌吸头挑一个菌落,将其滴入500毫升BHI Em 150中(见食谱)。

      4. 在37℃摇床培养过夜
      5. 按照QIAGEN质粒Maxi试剂盒说明书分离低拷贝质粒。
        注意:我们将异丙醇沉淀物转移到16×1.5ml的微管中,以减少洗涤过程中DNA的损失。然后将每个DNA沉淀重新溶解于20μl提供的质粒再悬浮缓冲液中,并收集在1.5ml微量管中。
      6. 在0.8%琼脂糖凝胶(见食谱)上用高DNA质量梯(Ladder)运行纯化的质粒DNA制备样品,以估计DNA浓度。
        注意:为了使DNA可视化,我们使用EZ-Vision三个6x上样缓冲液,其中含有荧光DNA染料。 em>
      7. 将纯化的质粒DNA保存在-20°C直到需要。
    2. 垫片设计和退火
      1. 在基因座中搜索5'-NGG-3'序列进行修饰(图1)。这个特征序列构成了SpCas9识别的protospacer毗邻基序(PAM)。
      2. 为了在pL2Cas9中克隆所需的间隔物,设计并合成对应于具有BsaI限制性位点的所选原始空间的两条链的35个核苷酸的两个ssDNA寡核苷酸(图1,表1)。
        注意:间隔序列的GC含量不应该影响SpCas9切割其靶DNA的效率(Tao et al。,2017)。为了避免脱靶活动,确保在功能性PAM旁边的噬菌体或克隆宿主基因组的其他位置没有发现原型间隔区。
      3. 将分子生物学水中的寡核苷酸稀释至50μM。
      4. 为了寡核苷酸的5'-磷酸化,在PCR管中将2μl各稀释的寡核苷酸与10μl5x T4连接酶缓冲液,1μlT4多核苷酸激酶(PNK)和32μl蒸馏水混合至最终体积为50 μl。
        注:5×T4连接酶缓冲液提供T4 DNA连接酶。将连接酶缓冲液分成一次性等分试样,因为缓冲液中的ATP可以通过反复冻融循环而降解。
      5. 在37°C下进行磷酸化30分钟,然后在65°C(在PCR仪器或水浴中)加热使PNK失活20分钟。
      6. 添加2.5微升的1M氯化钠的磷酸化寡核苷酸。
      7. 将管置于PCR热循环仪中,在95°C下加热样品5分钟,然后以0.1°C /秒的速率冷却至25°C。
      8. 保持在-20°C最长2个月。


        图1.隔垫设计的图形表示。在这个例子中,在噬菌体基因组中鉴定出PAM。 PAM上游的30bp序列称为“原型间隔区”,并且必须以“间隔区”的形式并入CRISPR阵列(crRNA)以指定目标特异性。将BsaI限制性位点添加到间隔物的末端用于连接到pL2Cas9中。

    3. 用BsaI消化pL2Cas9
      1. 按照制造商的说明,在37℃孵育过夜(最终体积为50μl),用BsaI消化1μg纯化的pL2Cas9。
      2. 按照生产商的说明,用南极磷酸酶去磷酸化消化的pL2Cas9的5'端。
      3. 通过用盐和乙醇沉淀纯化消化的和去磷酸化的载体。
        注意:通过凝胶提取纯化除去未切割的载体,但收率差。

        1. 加入1:10体积的NaAc 3 M pH 5.2至DNA
        2. 添加2卷100%乙醇。
        3. 充分混合,并在冰上孵育15分钟。
        4. 在台式微量离心机中以16,000×gg离心15分钟。
        5. 小心地取出上清液,用70%乙醇清洗沉淀(见食谱)。
        6. 将颗粒风干5-10分钟。

        7. 在30μl10mM Tris-HCl pH 8.5中重悬沉淀
      4. 在0.8%的琼脂糖凝胶上使用退火的寡核苷酸,以及高DNA质量和1 Kb Plus DNA Ladders在分开的孔中运行纯化的载体,以估计浓度。
      5. 将消化的pL2Cas9保存在-20°C直到需要。
        注意:分装成较小的体积以防止在反复冻融循环过程中可能发生的DNA降解。
    4. 靶向质粒的连接
      1. 按照生产商的说明,用T4 DNA连接酶建立连接反应。
        使用插入物与载体的摩尔比约为3:1
      2. 准备另一个连接反应没有插入(阴性连接控制)来评估载体重新环化。

      3. 在16°C(在PCR仪器或水浴)中过夜
      4. 在65°C加热灭活连接酶10分钟,置于冰上,立即进行转化(步骤A5和A6)。
        注意:连接产品可以在-20°C保存几天,但转化效率会降低。
    5. 准备 L。 lactis MG1363感受态细胞
      注意:如果与其他噬菌体宿主一起工作,DNA构建体的转化可以直接进入感兴趣的细菌宿主,或者首先进入乳酸乳球菌MG1363。在后一种情况下,DNA构建体必须从克隆宿主中提取,然后转移到感兴趣的宿主中。应该用一个质粒转化细胞,用合适的抗生素选择,制成感受态,用第二个质粒转化,并用两种抗生素选择。我们不建议进行双重转换。
      1. 用乳酸乳球菌MG1363接种10毫升GM17肉汤(见食谱),并在30℃孵育过夜。

      2. 用300μl来自步骤A5a的过夜培养物接种含有9.7ml甘氨酸休克溶液(参见食谱)的5个管。 注:10毫升的细菌培养物足以制备50微升的感受态细胞。 (1)连接产物,(2)未切割载体(阳性转化对照),(3)去磷酸化切割载体(阴性消化对照),(4)阴性连接对照,(5)没有DNA(阴性转化对照)。如果需要,可以同时准备更多的培养物。
      3. 在30°C孵育,直到细菌培养物达到0.2的OD 600。
      4. 将培养物转移到无菌离心瓶(SS-34)中,并在4℃和12,000×g g离心5分钟。
        注意:从此之后,文化和解决方案应尽可能地保存在冰上。
      5. 丢弃上清,并重悬在1毫升无菌冷洗液(见食谱)每个颗粒。
      6. 将细胞转移到1.5毫升的微管中。
      7. 在台式微量离心机中以16,000×g g离心1分钟使再悬浮液离心。
        注意:我们在室温下(RT)执行这一步骤,但可以使用冷冻微量离心机来保持培养物冷(〜4°C)。
      8. 重复最后三个步骤两次。
      9. 将每一粒感受态细胞重悬于50μl无菌冷洗液中,然后将其置于1.5ml微量管中,立即进入步骤A6。
    6. 电击 L。乳糖 MG1363
      1. 透析器使用膜组装DNA(图2)。
        注意:Ligation和Gibson组装产品应在电穿孔之前进行脱盐。


        图2.快速简单的DNA透析A.用本生灯火焰加热巴斯德移液管的宽端,直到玻璃变红。 B.迅速刺穿1.5毫升微管的盖子。 C.在微管中加入2ml蒸馏水。由于表面张力,凸形弯月面应位于微管边缘的上方。 D.将4厘米见方的透析膜(MWCO 6-8000)置于蒸馏水中。一旦水合,将透析膜管切成一定长度以具有两个单独的膜层。把单层膜放在水上,关闭微管的盖子。确保水和圆顶膜之间没有气泡。 E.除去膜上多余的水分。 F.将5μl的DNA样品放在膜上。 G.等待约30秒。 H.收集脱盐的DNA。对每个样品重复整个过程进行透析。

      2. 加入5μlDNA(或用于阴性转化控制的水)和45μl感受态细胞到冷电击杯中。
        注意:将比色杯和回收溶液放在冰上。
      3. 将基因脉冲发生器设置为25μF,200Ω和2.5 KV。
      4. 将一个比色皿放在轨道上,同时按下两个红色按钮,直到声音。
      5. 向细胞中迅速加入500μl冷回收溶液(参见食谱),并在冰上放置10分钟。
      6. 将比色杯的内容转移到1.5毫升的微管中。

      7. 重复最后三步

      8. 在30°C孵育2小时
      9. 在补充有适当抗生素的GM17上铺板细胞(见食谱)。
        每个平板使用5-10个玻璃珠将转化的细胞均匀分散

      10. 在30°C孵育24-48小时
    7. 菌落PCR
      1. 为了确认质粒构建物中DNA插入物的存在,用无菌吸头挑出几个单菌落,并将它们放入50μl分子生物学水中。
        注:筛选的菌落数取决于消化的载体对照板上的背景菌落数。
      2. 使用相同的移液器吸头,将菌落划线在补充有适当抗生素的GM17平板上,并在30℃孵育过夜。
        注意:此板块稍后将启动阳性克隆的液体培养。
      3. 每50μlPCR反应用Taq DNA聚合酶使用5μl的DNA模板(水细菌悬浮液)。始终遵守制造商的说明。
        注意:我们使用骨架特异性引物Cas9_S.pyo_F6和crRNA_S.pyo_R(表1)来筛选pL2Cas9的CRISPR阵列中的新的间隔区。未切割的载体或所需的连接产物的扩增都将产生815bp的片段。为了筛选正确修复模板的克隆,我们使用骨架特异性引物pNZins_F和pNZins_R(表1)。未切割的pNZ123的扩增将产生145bp的片段,而组装的修复模板将产生更大的可变长度的片段。
      4. 使用1 Kb Plus DNA Ladder,在2%琼脂糖凝胶(参见食谱)上运行PCR产物,以确定它们的大小。
      5. 为了确认插入序列,提交PCR产物用于Sanger测序。
        注:为减少PCR产物的测序数量,也可用插入特异性引物鉴定阳性克隆。为了在pL2Cas9中筛选新的间隔区,我们使用来自步骤A2(表1)和crRNA_S.pyo_R的寡核苷酸I.当一个阳性克隆导致扩增一个391bp片段时,一个阴性克隆没有产物。
      6. 接种10毫升的GM17肉汤培养基,补充适当的抗生素与阳性克隆,并在30°C孵育过夜。

      7. 在-80°C储存细胞,加入850μl过夜培养物到150μl无菌甘油。

  2. 同源修复模板的构建
    我们使用广泛的主机范围和高拷贝数质粒pNZ123构建我们的重组模板。它赋予氯霉素抗性的细菌细胞携带它。为了避免质量损失。以最终浓度5μg/ ml(Cm 5)提供氯霉素。可以使用其他与pL2Cas9相容的质粒。最重要的是,修复模板必须设计为缺乏Cas9靶序列,以便重组噬菌体和模板本身可以避免切割。最有效的方法是创建删除整个目标序列(PAM和protospacer)的删除(图3)。否则,可以在PAM中引入单个突变和/或在原型间隔中的多个突变以防止DNA切割。如果目标序列位于编码区域,则应根据密码子使用模式设计突变。


    插入A(左侧)含有感兴趣基因的5'末端(黄色),插入B(右侧)含有3'末端同一个基因的末端。从噬菌体基因组扩增的两个片段是修复模板的同源臂。使用吉布森组装(pNZ_insertA和pNZ_insertB,表1),外部引物具有重叠(绿色)以将扩增子插入到线性化的pNZ123中。内部引物具有互补的突出端(黄色)用于一起退火。两个插入物的组装去除部分黄色基因,并且修复模板中没有靶序列(蓝色框)。类似的策略可以用来产生缺失,点突变和插入。

    1. 纯化pNZ123
      1. Streak L。乳酸MG1363(pNZ123)转化为GM17Cm5板(参见食谱)。
      2. 在30°C孵育过夜。
      3. 用无菌镊子用无菌吸头挑一个菌落,将其尖端放入500ml GM17 Cm 5中(见食谱)。

      4. 在30°C静夜培养

      5. 在6,000×g×15分钟的条件下离心收获过夜培养物
      6. 在20毫升缓冲液P1(从QIAGEN质粒Maxi试剂盒)补充溶菌酶30毫克/毫升的重悬细菌颗粒。

      7. 在37°C孵育30分钟
      8. 遵循QIAGEN质粒Maxi试剂盒方案。
      9. 在0.8%琼脂糖凝胶(见食谱)上用高DNA质量梯(Ladder)运行纯化的质粒DNA制备样品,以估计DNA浓度。
      10. 将纯化的质粒DNA保存在-20°C直到需要。
    2. pNZ123的线性化
      1. 按照制造商的说明,用XbaI消化1μg纯化的pNZ123并在37℃过夜温育。
      2. 用引物pNZ_XbaI_F和pNZ_XbaI_R(表1)和Q5 DNA聚合酶按照生产商的说明扩增消化的载体。我们使用58℃的退火温度和75秒的延伸时间。
        注意:消化的载体的扩增显着减少了未切割或重新环化的载体的背景。
      3. 用QIAquick PCR纯化试剂盒按照生产商的说明清洁产生的PCR产物。在分子生物学水中洗脱。
      4. 将线性化和PCR扩增的pNZ123保存在-20°C直到需要。
        注意:分装成较小的体积以防止在反复冻融循环过程中可能发生的DNA降解。
    3. 插入物的扩增(图3)
      1. 将具有适合吉布森组装的重叠的引物设计成线性化的pNZ123。
        注意:即使制造商推荐在要装配的片段之间有20bp的重叠,我们发现> 30 bp的重叠显着提高了装配效率。尽管250-500bp的同源臂是优选的,但较短的臂通常足以与噬菌体基因组重新组合。
      2. PCR使用噬菌体基因组作为模板并按照生产商的说明用Q5 DNA聚合酶扩增插入物。
      3. 在线性化的pNZ123上进行2%琼脂糖凝胶电泳,分别在不同的孔中加入Low DNA Mass和1 Kb Plus Ladders,以确定和确定片段的浓度。
    4. 装配和转换修复模板
      1. 根据制造商的说明,使用Gibson Assembly Master Mix组装步骤B1和B2中的片段。
        注意:为获得最佳效果,请在同一天组装并转换修复模板。

      2. 在50°C孵育1小时
      3. 保持冰(或-20°C),直到需要。
      4. 准备 L的感受态细胞。乳酸MG1363(步骤A5)。
        注:还可以将修复模板电穿孔到已经含有靶向质粒pL2Cas9的乳酸乳球菌MG1363中。
      5. 立即进行修复模板的电穿孔(步骤A6),并在补充适当抗生素的GM17上铺板细胞。

      6. 在30°C孵育24-48小时 注意:用两种抗生素进行选择通常会减慢细菌的生长。
      7. 通过菌落PCR分析转化体(步骤A7)。

  3. 噬菌体工程(图4)
    我们进行双层噬斑试验以获得分离的噬菌斑并纯化重组噬菌体。如果细菌宿主含有步骤A和B中获得的两种DNA构建体,则该步骤可容易地适用于其他噬菌体 - 宿主对。我们在噬菌体感染期间不使用抗生素选择,因为质粒在λ中是稳定的。乳糖 MG1363。
    不同宿主的质粒稳定性可能有所不同,在某些情况下应考虑抗生素的选择

    图4.使用CRISPR-Cas9靶向基因组编辑噬菌体p2。噬菌体p2感染L。含有靶向质粒(pL2Cas9)的MG1363和修复模板。 CRISPR阵列被描绘成黑色菱形(重复)和蓝色框(间隔)。病毒DNA进入细菌后不久,CRISPR-Cas9复合物识别并裂解其靶标。然后可以用适于同源重组并且具有期望的突变(这里是缺失)的模板修复基因组损伤。
    重组噬菌体避免CRISPR-Cas9系统切割,因为它们缺少靶序列
    1. 噬菌体感染
      1. 用含有靶向质粒和修复模板的乳酸乳球菌MG1363接种10ml的补充有Em5和Cm5的GM17。

      2. 在30°C孵育过夜
      3. 进行噬菌体p2裂解物的十倍连续稀释。准备六个无菌1.5毫升微管含有900微升1x无菌噬菌体缓冲液(见食谱)。添加100微升未稀释的噬菌体裂解物到第一个微管和上下轻轻吹打混合。这是稀释10 -1 -1。使用新的移液管吸头,将100μl10-1稀释液转移至第二个微量管(稀释度10 -2 -2),轻轻混匀,重复至稀释10 -6 。
      4. 添加300μL的过夜细菌培养到补充有CaCl 2(参见食谱)的3ml GM17软琼脂培养基中,保持在50℃。
      5. 加入100μl未稀释的噬菌体裂解液。
      6. 快速倒入补充有CaCl 2的GM17琼脂培养基的顶部,并且将板旋转以均匀地铺展软琼脂。
      7. 重复最后三个步骤进行稀释10 - 2 - , - 10 - 4和10 - 6 - 。

      8. 在30°C孵育24小时
      9. 用截短的无菌吸头挑取3个噬菌斑,并将每个含有噬菌体的琼脂塞放入含有500μl1x噬菌体缓冲液的单独的无菌1.5ml微管中。
        注意:噬菌斑可以保存在4℃的缓冲液中数周。
      10. 让噬菌体在缓冲液中扩散>在室温20分钟。
      11. 在相同的细菌菌株上重复两轮感染(步骤C1a至C1j)以纯化重组噬菌体。
    2. 突变噬菌体分析
      1. 设计扩增噬菌体基因组的突变区域和缺失修复模板的引物对。
      2. 使用Taq DNA聚合酶每50μlPCR反应5μL噬菌体悬浮液,并按照制造商的说明。使用野生型噬菌体p2作为阳性对照的模板。
      3. 使用1 Kb Plus DNA Ladder在2%琼脂糖凝胶上运行PCR产物,以确定它们的大小。
        注意:与用野生型噬菌体p2获得的那些相比,用重组噬菌体获得的较短的PCR产物可在凝胶上观察到基因缺失。
      4. 提交用于Sanger测序的PCR产物,并将它们与噬菌体基因组进行比对以确认重组噬菌体中所需的突变。
        注意:在实验室条件下敲除对于噬菌体增殖不重要的基因总是会导致重组噬菌体的同质群体。
      5. 测序工程噬菌体的全基因组,以确认没有额外的突变。
        注:迄今为止,我们测序了几个突变噬菌体的全基因组,并且在CRISPR-Cas9介导的基因组工程之后没有检测到脱靶突变。

        如果需要遗传补偿来抵御有害的突变,可能会出现意想不到的突变。

数据分析

用这种方法生成噬菌体p2的多个突变体。我们引用读者的原始文件(Lemay 等,2017年)。

笔记

  1. 为了节省时间,协议中的一些步骤可以同时完成(图5)。


    图5.说明使用CRISPR-Cas9生成突变噬菌体的方案的流程图

  2. 基因缺失不适合体内研究噬菌体增殖所必需的基因。当敲除不可能时,可以产生非破坏性突变,如点突变或插入。

食谱

  1. 0.8%和2%琼脂糖凝胶
    1. 加入0.8克或2克琼脂糖LE到100毫升的1x TAE
    2. 加热到微波融化
    3. 冷却到〜50°C
    4. 将合适的体积倒入电泳装置中并添加所需的梳子。让琼脂糖固化,删除梳子,并用1倍TAE缓冲液覆盖凝胶
  2. BHI琼脂培养基补充红霉素
    注意:重要的是使用BHI,因为大肠杆菌对溶原培养液(LB)中的红霉素更具抗性。
    1. 将3.7克BHI和1克琼脂溶于75毫升蒸馏水中
    2. 用蒸馏水完成100毫升

    3. 高压灭菌消毒并冷却至50°C
    4. 加入200微升75毫克/毫升红霉素(150微克/毫升)
    5. 每盘倒入约20毫升的培养基
    6. 在4°C的黑暗中保存
  3. BHI培养基补充红霉素
    1. 将18.5克BHI溶于500毫升蒸馏水中
    2. 通过高压灭菌和冷却来消毒
    3. 在室温(RT)
      存放
    4. 准备好培养细菌时,加入1毫升75毫克/毫升的红霉素(150微克/毫升)
  4. 2 M CaCl 2 2
    注意:噬菌体p2感染需要CaCl 2 。
    1. 将29.4克CaCl 2•2H 2 O溶于100毫升蒸馏水中
    2. 通过高压灭菌和冷却来消毒
    3. 少量分装以限制溶液污染
    4. 在RT
      存储
  5. 10毫克/毫升氯霉素原液
    注:氯霉素是选择含有pNZ123和衍生物的细菌所必需的。
    1. 将0.1克氯霉素溶于10毫升95%乙醇中
    2. 在-20°C储存
  6. 10毫克/毫升和75毫克/毫升红霉素原液
    注:红霉素是选择含有pL2Cas9及其衍生物的细菌所必需的。
    1. 将0.1克或0.75克红霉素溶于10毫升95%乙醇中
    2. 在-20°C储存
  7. 70%乙醇
    1. 将2.8升100%乙醇与1.2升蒸馏水混合
    2. 在RT
      存储
  8. GM17琼脂培养基中补充有CaCl 2或抗生素

    1. 溶解37.25克M17,5克葡萄糖一水合物和10克琼脂在850毫升蒸馏水中
    2. 完成1升蒸馏水

    3. 高压灭菌消毒并冷却至50°C
    4. 加入5ml无菌2M CaCl 2(终浓度10mM)或0.5ml适当的10mg / ml抗生素原液(最终浓度为5μg/ ml)。
    5. 每盘倒入约20毫升的培养基

    6. 如果补充了抗生素,应在室温或4°C避光保存
  9. GM17中等
    1. 将37.25克M17和5克葡萄糖一水合物溶于1升蒸馏水中
    2. 通过高压灭菌和冷却来消毒
    3. 在RT
      存储
  10. GM17软琼脂培养基补充CaCl 2 2
    1. 将3.73克M17,0.5克葡萄糖一水合物和0.75克琼脂溶于85毫升蒸馏水中
    2. 用蒸馏水完成100毫升

    3. 高压灭菌消毒并冷却至50°C
    4. 加入0.5ml无菌2M CaCl 2(终浓度10mM)
    5. 根据需要将3毫升培养基倒入多达13 x 100毫米的无菌玻璃管中
    6. 保持在50°C在加热块干燥浴等分直到准备使用
    7. 将剩余的介质存放在室温下,并在需要时融化在微波炉中
  11. 甘氨酸休克溶液
    1. 在GM17肉汤培养基中溶解1克甘氨酸和17.1克蔗糖,最终体积为100毫升。
    2. 过滤通过0.2微米无菌PES注射器过滤器消毒
    3. 将解决方案存储在RT
  12. 1M MgCl 2 2
    1. 将203.3克MgCl 2•6H 2 O溶于1升蒸馏水中
    2. 通过高压灭菌来消毒
    3. 在RT
      存储
  13. 10倍噬菌体缓冲液
    1. 将58克NaCl和20克MgSO 4•7H 2 O溶于500毫升1M Tris-HCl pH7.5中
    2. 完成1升蒸馏水
    3. 通过高压灭菌来消毒
    4. 在RT
      存储
    5. 对于1x噬菌体缓冲液,用900ml蒸馏水稀释100ml 10x噬菌体缓冲液,通过高压灭菌将其灭菌,并分装成小份以防止污染。
  14. 恢复解决方案
    1. 对于100ml的最终体积,将17.1g蔗糖溶解在补充有2ml 1M MgCl 2和100μl2M CaCl 2 2的GM17肉汤培养基中/>
    2. 过滤通过一个0.2微米无菌PES注射器过滤器消毒
    3. 将溶液储存在4°C
  15. 50倍TAE缓冲液
    1. 混合242克Tris碱,57.1毫升冰醋酸,100毫升0.5M EDTA pH8.0
    2. 完成1升蒸馏水
    3. 在RT
      存储
    4. 对于1x TAE缓冲液,用980 ml蒸馏水稀释20 ml TAE 50x
  16. 洗涤溶液

    1. 在800毫升蒸馏水中混合100毫升甘油和171克蔗糖(0.5M)
    2. 完成1升蒸馏水
    3. 通过高压灭菌来消毒
    4. 将溶液储存在4°C

致谢

该协议详细介绍了原始论文中使用的方法(Lemay等人,2017)。我们感谢Witold Kot和Hanne Hendrix的深入讨论。 M.-L.L.由加拿大自然科学和工程研究委员会(NSERC)的一个奖学金支持。 S.M.承认NSERC发现计划的资助。 S.M.在Bacteriophages拥有一级加拿大研究主席。作者声明没有竞争的经济利益。

参考

  1. Bari,S. M. N.,Walker,F. C.,Cater,K.,Aslan,B.和Hatoum-Aslan,A.(2017)。 使用CRISPR-Cas10编辑毒性葡萄球菌噬菌体的策略 ACS Synth Biol 6(12):2316-2325。
  2. Box,A.M.,McGuffie,M.J。,O'Hara,B.J。和Seed,K.D。(2015)。 IE型CRISPR-Cas系统在霍乱弧菌中对噬菌体免疫的功能分析及其在噬菌体中的应用基因组工程。 J Bacteriol 198(3):578-590。
  3. Breitbart,M.和Rohwer,F。(2005)。 这里有病毒,有病毒,到处都是同一种病毒吗?趋势Microbiol 13:278-284。
  4. Deveau,H.,Labrie,S.J。,Chopin,M.C。和Moineau,S。(2006)。 乳酸菌噬菌体的生物多样性和分类 Appl Environ Microbiol 72(6):4338-4346。
  5. De Vos,W.M。(1987)。 乳酸链球菌的基因克隆和表达 FEMS Microbiol Lett 46:281-295。
  6. Hurwitz,B.L.,U'Ren,J.M。和Youens-Clark,K。(2016)。 计算探索伟大的病毒未知数 FEMS Microbiol Lett 363(10)。
  7. Jiang,W.,Bikard,D.,Cox,D.,Zhang,F。和Marraffini,L.A。(2013)。 使用CRISPR-Cas系统对RNA进行细菌基因组编辑。 Nat Biotechnol 31(3):233-239。
  8. Kiro,R.,Shitrit,D.和Qimron,U。(2014)。 使用IE CRISPR-Cas系统高效工程化噬菌体基因组。 > RNA Biol 11(1):42-44。
  9. Lemay,M.L。,Tremblay,D.M。和Moineau,S。(2017)。 使用CRISPR-Cas9进行强毒乳球菌噬菌体的基因组工程 ACS Synth Biol 6(7):1351-1358。
  10. Mahony,J.,Murphy,J.和van Sinderen,D.(2012)。 乳酸乳球菌936型噬菌体和乳品发酵问题:从检测到进化和预防。前微生物 3:335。
  11. Manor,M.和Qimron,U.(2017)。 使用CRISPR-Cas系统选择基因修饰的噬菌体 Bio Protoc 7(15)e2431。
  12. Martel,B。和Moineau,S。(2014)。 CRISPR-Cas:一种有效的毒力噬菌体基因组工程工具 Nucleic Acids Res 42(14):9504-9513。
  13. Paez-Espino,D.,Eloe-Fadrosh,E.A.,Pavlopoulos,G.A.,Thomas,A.D.,Huntemann,M.,Mikhailova,N.,Rubin,E.,Ivanova,N.N.和Kyrpides,N.C。(2016)。 揭开地球的漩涡 Nature 536:425-430 。
  14. Pires,D.P.,Cleto,S.,Sillankorva,S.,Azeredo,J。和Lu,T.K。(2016)。 基因工程噬菌体:回顾过去十年的进展。 Microbiol Mol Biol Rev 80(3):523-543。
  15. Suttle,C.A。(2005)。 海中的病毒 Nature 437(7057) :356-361。
  16. Tao,P.,Wu,X.,Tang,W.C.,Zhu,J。和Rao,V.(2017)。 使用CRISPR-Cas9设计噬菌体T4基因组 ACS Synth Biol 6(10):1952-1961。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lemay, M., Renaud, A., Rousseau, G. M. and Moineau, S. (2018). Targeted Genome Editing of Virulent Phages Using CRISPR-Cas9. Bio-protocol 8(1): e2674. DOI: 10.21769/BioProtoc.2674.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。