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Selection of Genetically Modified Bacteriophages Using the CRISPR-Cas System
利用CRISPR-Cas系统筛选基因组编辑的噬菌体   

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Abstract

We present a CRISPR-Cas based technique for deleting genes from the T7 bacteriophage genome. A DNA fragment encoding homologous arms to the target gene to be deleted is first cloned into a plasmid. The T7 phage is then propagated in Escherichia coli harboring this plasmid. During this propagation, some phage genomes undergo homologous recombination with the plasmid, thus deleting the targeted gene. To select for these genomes, the CRISPR-Cas system is used to cleave non-edited genomes, enabling isolation of the desired recombinant phages. This protocol allows seamless deletion of desired genes in a T7 phage, and can be expanded to other phages and other types of genetic manipulations as well.

Keywords: Bacteriophage(噬菌体), Escherichia coli(大肠埃希杆菌), Homologous recombination(同源重组), Positive selection(正向选择)

Background

Bacteriophages (phages) are the most prevalent and widely distributed biological entity in the biosphere, highlighting their ecological importance (Suttle, 2007). Many studies also propose using phages for medical purposes (Weber-Dabrowska et al., 2001; Merril et al., 2003; Harper and Enright, 2011; Edgar et al., 2012; Bikard et al., 2014; Citorik et al., 2014; Yosef et al., 2014 and 2015). Unfortunately, only a few published methods detail genetic engineering of phage genomes (Selick et al., 1988; Marinelli et al., 2008; Pires et al., 2016) and in addition, some of these methods are tedious, and some cannot achieve desired results such as seamless deletions. A simple and efficient technique for seamless genetic engineering of phages is thus desired. In this protocol, we present a technique described in 2014 (Kiro et al., 2014) for deleting genes of the E. coli phage, T7. We first designed genetic constructs that facilitate desired homologous recombination events. We then used the CRISPR-Cas type I-E system to select desired engineered phages. Several other studies have also reported the use of CRISPR-Cas systems to engineer phages and the reader is referred to them for selecting the most appropriate and fitting protocols for the specific requirements (Martel and Moineau, 2014; Box et al., 2015; Lemay et al., 2017).

Materials and Reagents

  1. Materials
    1. 1.7 ml microfuge tube (Corning, Axygen®, catalog number: MCT-175-C )
    2. 15 ml tube (Corning, catalog number: 430052 )
    3. PCR tubes (Corning, Axygen®, catalog number: PCR-0208-C )
    4. Pipette tips
  2. Bacterial strains
    1. Electro-competent BL21-AI (Invitrogen, Genotype: F− ompT hsdSB(rB−, mB−) gal dcm araB::T7RNAP-tetA, tetr)
    2. Electro-competent NEB5α (New England Biolabs)
  3. Phage
    1. WT T7 phage (laboratory collection. Available at ATCC, catalog number: BAA-1025-B2 )
  4. Plasmids
    1. pUC19 (Yanisch-Perron et al., 1985)
    2. pWUR397 (Brouns et al., 2008. cas3 under T7 promoter, KanR)
    3. pWUR400 (Brouns et al., 2008. cascade genes under T7 promoter, StrR)
    4. pWUR477 (Brouns et al., 2008. pACYCDuet-1 (Novagen) cloned with control spacers under T7 promoter, camR)
  5. Enzymes
    1. DpnI restriction enzyme (New England Biolabs, catalog number: R0176S )
    2. T4 polynucleotide kinase (New England Biolabs, catalog number: M0201S ) used with T4 DNA ligase buffer (New England Biolabs, catalog number: M0202S )
  6. Kits
    1. Gel and PCR Clean-up Kit (MACHEREY-NAGEL, catalog number: 740609.50 )
    2. KAPA HiFi PCR Kit (Roche Diagnostics, catalog number: 07958935001 )
    3. Lamda Taq PCR Master mix (Lamda Biotech, catalog number: D123P-200 )
    4. Quick Ligation Kit (New England Biolabs, catalog number: M2200L )
  7. Reagents
    1. Agar (BD, DifcoTM, catalog number: 214010 )
    2. Ampicillin (Merck, catalog number: 171254 ; Stock 100 mg/ml in double-distilled water, filtered, -20 °C)
    3. Chloramphenicol (Merck, catalog number: 220551 ; Stock 35 mg/ml in ethanol, filtered, -20 °C)
    4. Isopropyl-β-D-thiogalactopyranoside (IPTG) (Bio-Lab, catalog number: 16242352 ; Stock 1 M in double-distilled water, filtered, -20 °C)
    5. Kanamycin (Merck, catalog number: 420311 ; Stock 50 mg/ml in 50% glycerol and double-distilled water, filtered, -20 °C)
    6. L-arabinose (Gold Bio, catalog number: A-300-1 ; Stock 20% in double-distilled water, filtered, RT)
    7. LB (Luria-Bertani) medium (10 g/L tryptone, 5 g/L yeast extract and 5 g/L NaCl) (Acumedia)
    8. Molecular biology water (Bio-Lab, catalog number: 232123 )
    9. PEG 8000 (polyethylene glycol) (Promega, catalog number: V3011 ; Stock 50% in double-distilled water, filtered, RT)
    10. Streptomycin (EMD Millipore, catalog number: 5711 ; Stock 50 mg/ml in double-distilled water, filtered, -20 °C)
    11. TAE buffer (Bio-Lab, catalog number: 20502323 )
  8. LB medium (see Recipes)
  9. LB agar plates (see Recipes)
  10. Soft agar (see Recipes)
  11. TAE buffer (see Recipes)

Equipment

  1. Cell density meter (OD600 reader) (Amersham Biosciences, model: Ultrospec 10 )
  2. Electroporation device (Bio-Rad Laboratories, model: MicroPulserTM )
  3. Gel electrophoresis apparatus (Cleaver Scientific, model: MultiSUB Choice )
  4. Micro-centrifuge (Eppendorf, model: MiniSpin® )
  5. NanoDrop spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000c )
  6. Pipettes
  7. Shaker (for 50 ml tubes, at 37 °C) (Thermo Fisher Scientific, Thermo ScientificTM, model: MaxQTM 2000 )
  8. Thermal Cycler (Bio-Rad Laboratories, model: C1000 TouchTM )
  9. Thermoblock (Labnet International, model: AccuBlockTM Digital Dry Baths )

Procedure

  1. Plasmid construction
    1. Construction of pGeneX
      This plasmid encodes DNA homologous to the upstream and downstream sequences of the target genetic locus. These DNA will recombine with the desired locus thus generating the desired recombinant phages (Figure 1).
      1. Perform a PCR amplification reaction with pUC19 plasmid as a template, using primers with 60 bp 5’ overhangs homologous to sequences flanking the target gene to be deleted (hereafter, geneX). The forward primer should carry a 60 bp overhang sequence identical to the 60 bp downstream to geneX. The reverse primer should carry a 60 bp overhang matching the upstream sequence. See design in Figure 1.





        Figure 1. Schematics of the pGeneX plasmid. A. An example of forward and reverse primers encoding sequences annealing to the pUC19 backbone (gray) with 60 bp 5’ overhang sequence identical to the upstream (red) and downstream (blue) flanking sequences of geneX. B. PCR amplification with these primers on the plasmid and self-ligation yield the pGeneX plasmid.

      2. Add 1 µl of restriction enzyme DpnI directly to the PCR mixture to eliminate residues of the pUC19.
      3. Incubate for 1 h at 37 °C.
      4. Isolate DNA product according to size using gel electrophoresis.
      5. Purify the product using Gel and PCR Clean-up kit according to the manufacturer’s instructions. Elute in 20 µl.
      6. Phosphorylate the purified DNA:
        1. Incubate the purified DNA at 70 °C for 5 min.
        2. Cool on ice for 5 min.
        3. Add to the 20 µl purified DNA:
          3 µl T4 DNA ligase buffer
          1 µl T4 polynucleotide kinase
          3 µl PEG-800 (50%)
          3 µl molecular biology water
        4. Incubate for 30 min at 37 °C.

          Note: DNA oligos could be also synthesized with a 5’-phosphate. In such cases, step A1f should be skipped.

      7. Purify the product using Gel and PCR Clean-up kit according to the manufacturer’s instructions. Elute in 20 µl.
      8. Self-ligate the phosphorylated DNA:
        1. Add to 10 µl phosphorylated DNA:
          10 µl Quick ligation buffer
          1 µl Quick ligase enzyme
        2. Incubate for 15 min at 25 °C.
      9. Purify the product using Gel and PCR Clean-up kit according to the manufacturer’s instructions. Elute in 20 µl.
    2. Construction of pAnti-GeneX
      This plasmid encodes specific spacers that will serve to target the unmodified phage, thus enabling selection of the modified phage.
      1. Perform a PCR amplification reaction with pWUR477 plasmid as a template using primers with 5’ overhangs that form a new spacer upon ligation. See Figure 2 below.



        Note: Minimum reaction volume of 30 µl.


        Figure 2. Schematics of the pAnti-GeneX construct. A. An example of a primer design yielding a spacer to target gene X. The targeted DNA is in pink. This protospacer is a 33 bp sequence in the gene of interest and is preceded by an AA, yielding a functional protospacer-adjacent motif (PAM). The forward and reverse primers encode annealing sequences to the pWUR477 template, and 5’ overhangs which together form the desired spacer sequence. B. PCR amplification with the forward and reverse primers on the pWUR477 plasmid and self-ligation to construct the pAnti-GeneX plasmid. Grey rectangles represent repeat sequences, pink represents the new spacer, orange represents spacer 2 and red represents spacer 3, and black represents the plasmid backbone.

      2. Add 1 µl of restriction enzyme DpnI directly to the PCR mixture to eliminate residues of the pWUR477.
      3. Incubate for 1 h at 37 °C.
      4. Isolate DNA product according to size using gel electrophoresis.
      5. Purify the product using Gel and PCR Clean-up kit according to the manufacturer’s instructions. Elute in 20 µl.
      6. Phosphorylate the purified DNA:
        1. Incubate the purified DNA at 70 °C for 5 min.
        2. Cool on ice for 5 min.
        3. Add to the 20 µl purified DNA:
          3 µl T4 DNA ligase buffer.
          1 µl T4 polynucleotide kinase.
          3 µl PEG-800 (50%).
          3 µl molecular biology water.
        4. Incubate for 30 min at 37 °C.

        Note: DNA oligos could also be synthesized with a 5’-phosphate. In such cases, step A2f should be skipped.

      7. Purify the product using Gel and PCR Clean-up kit the according to the manufacturer’s instructions. Elute in 20 µl.
      8. Self-ligate the phosphorylated DNA:
        1. Add to 10 µl phosphorylated DNA:
          10 µl Quick ligation buffer
          1 µl Quick ligase enzyme
        2. Incubate for 15 min at 25 °C.
      9. Purify the product using Gel and PCR Clean-up kit according to the manufacturer’s instructions. Elute in 20 µl.

  2. Preparation of recombinant T7 lysate
    1. Transform the pGeneX and the pUC19 plasmid (lacking the homologous sequences) as a control into NEB5α cells.
    2. Inoculate one colony of each transformant into 3 ml LB supplemented with 100 µg/ml ampicillin and shake at 37 °C, overnight.
    3. Dilute overnight cultures 1:100 into 6 ml fresh LB, supplemented with 100 µg/ml ampicillin.
    4. Shake at 37 °C to an OD600 ~1.
    5. Infect 5 ml of each culture with WT T7 phage at an MOI of ~0.1 (107-108 PFU). Leave the remaining 1 ml culture as an uninfected control.
    6. Shake the infected bacteria at 37 °C until lysis occurs (usually 2 h).
      Note: Lysis can be recognized by ‘clear’ culture, strings of cell debris or a translucent appearance.

  3. Select T7 engineered phages
    1. Transform pWUR397, pWUR400 and targeting plasmid (pAnti-GeneX) or a control plasmid (pWUR477), into BL21-AI cells.
    2. Inoculate one colony of each transformant into 3 ml LB supplemented with 50 µg/ml streptomycin, 50 µg/ml kanamycin and 35 µg/ml chloramphenicol and shake at 37 °C, overnight.
    3. Prepare at least 4 LB agar plates (see Recipes) supplemented with 50 µg/ml streptomycin, 50 µg/ml kanamycin and 35 µg/ml chloramphenicol.
    4. Dilute overnight cultures 1:100 into 5 ml fresh LB supplemented with the appropriate antibiotics.
    5. Shake at 37 °C (without inducers) until OD600 ~1.
    6. Add 0.2% L-arabinose and 0.1 mM IPTG (final concentrations) to the cultures.
    7. Shake at 37 °C for an additional hour.
    8. Pre-warm soft agar (0.7% agar, see Recipes) to a liquid phase. Add 0.2% L-arabinose and 0.1 mM IPTG to the agar and keep at 50 °C.
    9. Pre-warm LB agar plates with the antibiotics prepared in step C3 for at least 1 h at 37 °C.
    10. For every combination of phage and bacteria (see Table 1), mix in a 15 ml tube:
      100 µl bacteria (from step C7).
      10 µl phage lysates prepared in ‘Preparation of recombinant T7 lysate’.
      3.5 ml of the pre-warmed soft agar (0.7%) with inducers.

      Table 1. Combinations of phage with bacteria. All four different combinations: targeting/non-targeting bacteria with recombinant/non-recombinant lysates.


    11. Vortex briefly and IMMEDIATELY pour onto the pre-warmed plates. Gently tilt and rotate plate to spread top agar evenly.
    12. Allow the plates to cool for 5 min, invert, and incubate overnight at RT or for 4-6 h at 37 °C (until plaques are visible).

  4. Isolation and validation
    1. Inoculate one colony of targeting bacteria (BL21-AI/pWUR397, pWUR400, pAnti-GeneX) into 3 ml LB supplemented with 50 µg/ml streptomycin, 50 µg/ml kanamycin and 35 µg/ml chloramphenicol.
    2. Shake at 37 °C overnight.
    3. Dilute plaques larger than 0.4 cm into 100 µl LB.
      Note: Small plaques are expected even on the control plates. Desired plaques (i.e., ones that have the desired deletion mutation) should appear 3-5 times larger than the background plaques.
    4. Perform a PCR amplification reaction on the diluted plaque for validation: Use primers upstream and downstream to geneX. Determine gene deletion according to PCR product size. Use wild-type T7 as a template for positive control. See Figure 3.
      Note: Deletion of a fragment encoding the corresponding protospacer can result in isolated phages that do not encode the desired deletion (by escaping the CRISPR-Cas targeting).





      Figure 3. Illustration for PCR validation. Forward and reverse primers anneal upstream and downstream to geneX (green). PCR amplification on the edited phage results in a smaller DNA product. PCR amplification of the WT phage or phage with deletion of a fragment encoding the corresponding protospacer results in a full length DNA product. Black square represents gel electrophoresis, white bands represent DNA products.

    5. Pre-warm soft agar (0.7% agar) to a liquid phase. Add 0.2% L-arabinose and 0.1 mM IPTG (final concentrations) to the agar and keep at 50 °C.
    6. Pre-warm LB agar plates for at least 1 h at 37 °C. One plate for every modified phage.
    7. Streak positive deleted phages on the pre-warmed LB agar plate.
    8. For each modified phage, mix in a 15 ml tube:
      200 µl of the targeting bacteria
      3 ml of the pre-warmed soft agar with the inducers
    9. Gently pour the soft-agar with the bacteria on the streaked plates (from step D6).
      Note: Pour the soft-agar with the bacteria on the lower concentration area and let the fluid spread to the high concentration end.
    10. Allow the plates to cool for 5 min, invert, and incubate overnight at RT or for 4-6 h at 37 °C (until plaques are visible).
    11. Pick one plaque from each plate and repeat steps D3-D9, 3 times.
    12. Perform a PCR amplification on an isolated phage and validate desired sequence with DNA sequencing.
    13. Dilute 1:50 targeting bacteria into 5 ml LB supplemented with 50 µg/ml streptomycin, 50 µg/ml kanamycin and 35 µg/ml chloramphenicol, 0.2% L-arabinose and 0.1 mM IPTG.
    14. Shake at 37 °C to an OD600 ~0.5.
    15. Infect the culture with 10 µl of the validated engineered phage.
    16. Shake the infected bacteria at 37 °C until lysis occurs (usually 2 h).

Data analysis

A flow chart of the protocol is shown in Figure 4. An overview of the procedures for isolating desired recombinant bacteriophage and percentages of engineered cells can be found in the original paper (Kiro et al., 2014).


Figure 4. Flow chart of the protocol

Recipes

  1. LB medium
    1. Measure 20 g LB (Luria-Bertani) medium powder and fill up to 1 L with deionized water
    2. Autoclave solution and cool down
    3. Store at RT
  2. LB agar plates
    1. Measure 7.5 g agar and add sterile LB medium up to 500 ml
    2. Autoclave the solution, cool down and add antibiotics
    3. Pour into Petri dishes
    4. Wait until the agar solidifies
    5. Store at 4 °C
  3. Soft agar
    1. Measure 1.75 g agar and add sterile LB medium up to 250 ml
    2. Autoclave the solution and cool down
    3. Store at RT
  4. TAE buffer
    1. Pour 200 ml of TAE buffer (stock) and add deionized water up to 10 L
    2. Mix thoroughly
    3. Store at RT

Acknowledgments

The research leading to these results was funded by the European Research Council under the European Community’s Seventh Framework Programme (FP7/207-2013)/ERC grant agreement No. 336079 (EcCRISPR). The protocol described here is adapted from (Kiro et al., 2014).

References

  1. Bikard, D., Euler, C. W., Jiang, W., Nussenzweig, P. M., Goldberg, G. W., Duportet, X., Fischetti, V. A. and Marraffini, L. A. (2014). Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Nat Biotechnol 32(11): 1146-1150.
  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. Brouns, S. J., Jore, M. M., Lundgren, M., Westra, E. R., Slijkhuis, R. J., Snijders, A. P., Dickman, M. J., Makarova, K. S., Koonin, E. V. and van der Oost, J. (2008). Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321(5891): 960-964.
  4. Citorik, R. J., Mimee, M. and Lu, T. K. (2014). Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Nat Biotechnol 32(11): 1141-1145.
  5. Edgar, R., Friedman, N., Molshanski-Mor, S. and Qimron, U. (2012). Reversing bacterial resistance to antibiotics by phage-mediated delivery of dominant sensitive genes. Appl Environ Microbiol 78(3): 744-751.
  6. Harper, D. R. and Enright, M. C. (2011). Bacteriophages for the treatment of Pseudomonas aeruginosa infections. J Appl Microbiol 111(1): 1-7.
  7. 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.
  8. Lemay, M. L., Tremblay, D. M. and Moineau, S. (2017). Genome engineering of virulent lactococcal phages using CRISPR-Cas9. ACS Synth Biol.
  9. Marinelli, L. J., Piuri, M., Swigonova, Z., Balachandran, A., Oldfield, L. M., van Kessel, J. C. and Hatfull, G. F. (2008). BRED: a simple and powerful tool for constructing mutant and recombinant bacteriophage genomes. PLoS One 3(12): e3957.
  10. Martel, B. and Moineau, S. (2014). CRISPR-Cas: an efficient tool for genome engineering of virulent bacteriophages. Nucleic Acids Res 42(14): 9504-9513.
  11. Merril, C. R., Scholl, D. and Adhya, S. L. (2003). The prospect for bacteriophage therapy in Western medicine. Nat Rev Drug Discov 2(6): 489-497.
  12. 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.
  13. Selick, H. E., Kreuzer, K. N. and Alberts, B. M. (1988). The bacteriophage T4 insertion/substitution vector system. A method for introducing site-specific mutations into the virus chromosome. J Biol Chem 263(23): 11336-11347.
  14. Suttle, C. A. (2007). Marine viruses--major players in the global ecosystem. Nat Rev Microbiol 5(10): 801-812.
  15. Weber-Dabrowska, B. Mulczyk, M. and Górski, A. (2001). Bacteriophage therapy for infections in cancer patients. Clin Appl Immunol Rev 1(3-4): 131-134.
  16. Yanisch-Perron, C., Vieira, J. and Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33(1): 103-119.
  17. Yosef, I., Kiro, R., Molshanski-Mor, S., Edgar, R. and Qimron, U. (2014). Different approaches for using bacteriophages against antibiotic-resistant bacteria. Bacteriophage 4(1): e28491.
  18. Yosef, I., Manor, M., Kiro, R. and Qimron, U. (2015). Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria. Proc Natl Acad Sci U S A 112(23): 7267-7272.

简介

我们提出了一种用于从T7噬菌体基因组中删除基因的基于CRISPR-Cas的技术。 首先将编码与待缺失的靶基因的同源臂的DNA片段克隆到质粒中。 然后将T7噬菌体在携带该质粒的大肠杆菌中繁殖。 在这种繁殖期间,一些噬菌体基因组与质粒进行同源重组,从而缺失靶基因。 为了选择这些基因组,CRISPR-Cas系统用于切割未编辑的基因组,从而能够分离所需的重组噬菌体。 该协议允许在T7噬菌体中无缝地删除所需的基因,并且可以扩展到其它噬菌体和其他类型的遗传操作。
【背景】噬菌体(噬菌体)是生物圈中最普遍和广泛分布的生物实体,突出了它们的生态重要性(Suttle,2007)。许多研究还提出将噬菌体用于医疗目的(Weber-Dabrowska等人,2001; Merril等人,2003; Harper和Enright,2011; Edgar ,2012; Bikard等人,2014; Citorik等人,2014; Yosef等人, 2014年和2015年)。不幸的是,仅有少数公开的方法详细描述了噬菌体基因组学的基因工程(Selick等人,1988; Marinelli等人,2008; Pires等人,,2016),此外,其中一些方法是乏味的,有些无法实现期望的结果,如无缝删除。因此,需要一种用于噬菌体无缝基因工程的简单有效的技术。在本协议中,我们介绍了2014年(Kiro等人,2014年)中描述的用于删除E基因的技术。大肠杆菌噬菌体T7。我们首先设计了促进所需同源重组事件的遗传构建体。然后使用CRISPR-Cas型I-E系统选择所需的工程化噬菌体。还有一些其他研究报告还报道了使用CRISPR-Cas系统来设计噬菌体,读者可以参考这些系统,为特定要求选择最合适和最适合的方案(Martel和Moineau,2014; Box等人,,2015; Lemay等人,2017)。

关键字:噬菌体, 大肠埃希杆菌, 同源重组, 正向选择

材料和试剂

  1. 物料
    1. 1.7ml微量离心管(Corning,Axygen ,目录号:MCT-175-C)
    2. 15 ml管(Corning,目录号:430052)
    3. PCR管(Corning,Axygen ,目录号:PCR-0208-C)
    4. 移液器提示
  2. 细菌菌株
    1. (rB-,mB- )gal gal a a a
    2. 电能NEB5α(New England Biolabs)
  3. 噬菌体
    1. WT T7噬菌体(实验室收集,可在ATCC,目录号:BAA-1025-B2)
  4. 质粒
    1. pUC19(Yanisch-Perron等人,1985)
    2. pWUR397(Brouns等人,2008,T7启动子下的cas3,Kan R )
    3. pWUR400(Brouns等人,2008,T7启动子下的级联基因,Str R )
    4. pWUR477(Brouns等人),2008年。在T7启动子下,用对照间隔基克隆的pACYCDuet-1(Novagen),cam / sup> R /
    1. 限制性内切酶(New England Biolabs,目录号:R0176S)
    2. 与T4 DNA连接酶缓冲液(New England Biolabs,目录号:M0202S)一起使用的T4多核苷酸激酶(New England Biolabs,目录号:M0201S)
  5. 套件
    1. 凝胶和PCR清除试剂盒(MACHEREY-NAGEL,目录号:740609.50)
    2. KAPA HiFi PCR试剂盒(Roche Diagnositics,目录号:07958935001)
    3. Lamda Taq PCR Master mix(Lamda Biotech,目录号:D123P-200)
    4. 快速连接试剂盒(New England Biolabs,目录号:M2200L)
  6. 试剂
    1. 琼脂(BD,Difco TM ,目录号:214010)
    2. 氨苄青霉素(Merck,目录号:171254;在双蒸水中储存100毫克/毫升,过滤,-20℃)
    3. 氯霉素(Merck,目录号:220551;在乙醇中储存35mg / ml,过滤,-20℃)
    4. 异丙基-β-D-硫代吡喃半乳糖苷(IPTG)(Bio-Lab,目录号:16242352;在双蒸水中储存1M,过滤,-20℃)
    5. 卡那霉素(Merck,目录号:420311;在50%甘油和双蒸水中储存50mg / ml,过滤,-20℃)
    6. L-阿拉伯糖(Gold Bio,目录号:A-300-1;在双蒸水中储存20%,过滤,RT)
    7. LB(Luria-Bertani)培养基(10g / L胰蛋白胨,5g / L酵母提取物和5g / L NaCl)(Acumedia)
    8. 分子生物学水(Bio-Lab,目录号:232123)
    9. PEG 8000(聚乙二醇)(Promega,目录号:V3011;在双蒸水中储存50%,过滤,RT)
    10. 链霉素(EMD Millipore,目录号:5711;在双蒸水中储存50mg / ml,过滤,-20℃)
    11. TAE缓冲液(Bio-Lab,目录号:20502323)
  7. LB培养基(参见食谱)
  8. LB琼脂平板(参见食谱)
  9. 软琼脂(见食谱)
  10. TAE缓冲区(见配方)

设备

  1. 细胞密度计(OD 600读数器)(Amersham Biosciences,型号:Ultrospec 10)
  2. 电穿孔装置(Bio-Rad Laboratories,型号:MicroPulser TM )
  3. 凝胶电泳仪(Cleaver Scientific,型号:MultiSUB Choice)
  4. 微型离心机(Eppendorf,型号:MiniSpin ®)
  5. NanoDrop分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM 2000c)
  6. 移液器
  7. 振荡器(对于50ml管,在37℃下)(Thermo Fisher Scientific,Thermo Scientific& TM,型号:MaxQ TM 2000)
  8. 热循环仪(Bio-Rad Laboratories,型号:C1000 Touch TM )
  9. Thermoblock(Labnet International,型号:AccuBlock TM Digital Dry Baths)

程序

  1. 质粒构建
    1. pGeneX的构建
      该质粒编码与目标遗传基因座的上游和下游序列同源的DNA。这些DNA将与所需的基因座重组,从而产生所需的重组噬菌体(图1)。
      1. 使用与pUC19质粒作为模板的PCR扩增反应,使用与待缺失的靶基因侧翼序列同源的60bp 5'突出端的引物(以下称为“基因X”)。正向引物应携带与基因X 下游60bp相同的60bp突出序列。反向引物应携带与上游序列匹配的60bp突出端。见图1中的设计




        图1. pGeneX质粒的示意图。 :一种。编码序列退火到pUC19骨架(灰色)的正向和反向引物的实例,其具有与基因X的上游(红色)和下游(蓝色)侧翼序列相同的60bp 5'突出序列。 B.用这些引物在质粒上进行PCR扩增并自连接得到pGeneX质粒
      2. 向PCR混合物中直接加入1μl限制性内切酶Dpn以消除pUC19的残基。
      3. 在37°C孵育1小时。
      4. 使用凝胶电泳根据大小分离DNA产物
      5. 使用Gel和PCR清洁试剂盒根据制造商的说明书净化产品。用20μl洗脱。
      6. 磷酸化纯化的DNA:
        1. 将纯化的DNA在70℃孵育5分钟
        2. 在冰上冷却5分钟。
        3. 加入20μl纯化的DNA:
          3μlT4 DNA连接酶缓冲液
          1μlT4多核苷酸激酶
          3μlPEG-800(50%)
          3μl分子生物学水
        4. 在37°C孵育30分钟。

          注意:也可以用5'-磷酸合成DNA寡核苷酸。在这种情况下,应跳过步骤A1f 。

      7. 使用Gel和PCR清洁试剂盒根据制造商的说明书净化产品。用20μl洗脱。
      8. 自身连接磷酸化的DNA:
        1. 加入10μl磷酸化DNA:
          10μl快速连接缓冲液
          1μl快速连接酶
        2. 在25°C孵育15分钟。
      9. 使用Gel和PCR清洁试剂盒根据制造商的说明书净化产品。用20μl洗脱。
    2. pAnti-GeneX的构建
      该质粒编码用于靶向未修饰的噬菌体的特异性间隔物,从而能够选择经修饰的噬菌体。
      1. 使用具有5'突出端的引物,以pWUR477质粒作为模板进行PCR扩增反应,在连接时形成新的间隔基。见下图2


        注意:最小反应体积为30μl。


        图2. pAnti-GeneX构建体的示意图。A.产生靶向基因X的间隔物的引物设计的实例。靶向的DNA是粉红色的。该原始样品是感兴趣的基因中的一个33bp的序列,之前是一个AA,产生一个功能性原始相邻基序(PAM)。正向和反向引物编码pWUR477模板的退火序列和一起形成所需间隔序列的5'突出端。 B.使用正向和反向引物对pWUR477质粒进行PCR扩增,并自连接构建pAnti-GeneX质粒。灰色矩形表示重复序列,粉红色代表新的间隔物,橙色代表间隔物2,红色代表间隔物3,黑色代表质粒骨架。
      2. 向PCR混合物中直接加入1μl限制酶Dpn,以消除pWUR477的残留。
      3. 在37°C孵育1小时。
      4. 使用凝胶电泳根据大小分离DNA产物
      5. 使用Gel和PCR清洁试剂盒根据制造商的说明书净化产品。用20μl洗脱。
      6. 磷酸化纯化的DNA:
        1. 将纯化的DNA在70℃孵育5分钟
        2. 在冰上冷却5分钟。
        3. 加入20μl纯化的DNA:
          3μlT4 DNA连接酶缓冲液 1μlT4多核苷酸激酶 3μlPEG-800(50%) 3μl分子生物学水
        4. 在37°C孵育30分钟。

        注意:也可以用5'-磷酸合成DNA寡核苷酸。在这种情况下,应跳过步骤A2f

      7. 使用Gel和PCR清洁试剂盒,根据制造商的说明书对产品进行纯化。用20μl洗脱。
      8. 自身连接磷酸化的DNA:
        1. 加入10μl磷酸化DNA:
          10μl快速连接缓冲液
          1μl快速连接酶
        2. 在25°C孵育15分钟。
      9. 使用Gel和PCR清洁试剂盒根据制造商的说明书净化产品。洗脱20μl。

  2. 重组T7裂解液的制备
    1. 将pGeneX和pUC19质粒(缺少同源序列)作为对照转化到NEB5α细胞中。
    2. 将每个转化体的一个菌落接种到补充有100μg/ ml氨苄青霉素的3ml LB中,并在37℃下摇动过夜。
    3. 将过夜培养物1:100稀释至6ml新鲜的LB中,补充有100μg/ ml氨苄青霉素
    4. 在37°C下振荡至600°〜1°。
    5. 用WT T7噬菌体以约0.1μg(10μg/天〜10μg/ PFU)的MOI感染5ml培养物。将剩下的1ml培养物作为未感染的对照物。
    6. 在37℃下摇动感染的细菌,直到发生裂解(通常为2小时)。
      注意:可以通过“清除”的文化,细胞碎片串或半透明的外观来识别裂解。

  3. 选择T7工程噬菌体
    1. 将pWUR397,pWUR400和靶向质粒(pAnti-GeneX)或对照质粒(pWUR477)转化到BL21-AI细胞中。
    2. 将每个转化体的一个菌落接种到补充有50μg/ ml链霉素,50μg/ ml卡那霉素和35μg/ ml氯霉素的3ml LB中,并在37℃下摇动过夜。
    3. 制备补充有50μg/ ml链霉素,50μg/ ml卡那霉素和35μg/ ml氯霉素的至少4个LB琼脂平板(参见食谱)。
    4. 将过夜培养物1:100稀释至5ml补充有适当抗生素的新鲜LB中
    5. 在37℃(无诱导剂)下振荡直至OD 600〜1。
    6. 向培养物中加入0.2%L-阿拉伯糖和0.1mM IPTG(终浓度)
    7. 在37°C下摇一个小时。
    8. 预热软琼脂(0.7%琼脂,见食谱)到液相。向琼脂中加入0.2%L-阿拉伯糖和0.1mM IPTG,并保持在50℃
    9. 预热LB琼脂平板与步骤C3中制备的抗生素在37℃下至少1小时。
    10. 对于噬菌体和细菌的每种组合(参见表1),在15ml管中混合:
      100μl细菌(来自步骤C7) 在“重组T7裂解液的制备”中制备的10μl噬菌体裂解物 3.5ml预热的软琼脂(0.7%),含有诱导剂
      表1.噬菌体与细菌的组合所有四种不同的组合:用重组/非重组裂解物靶向/非靶向细菌。


    11. 短暂地涡旋,立即倒在预热板上。轻轻倾斜并旋转平板以均匀铺展顶部琼脂。
    12. 使板冷却5分钟,倒置,并在室温下孵育过夜或在37℃下孵育4-6小时(直到斑块可见)。

  4. 隔离和验证
    1. 将一个靶向细菌菌落(BL21-AI / pWUR397,pWUR400,pAnti-GeneX)接种到补充有50μg/ ml链霉素,50μg/ ml卡那霉素和35μg/ ml氯霉素的3ml LB中。
    2. 在37℃下过夜。
    3. 将稀释的斑块大于0.4厘米,加入100微升LB 注意:即使在控制板上也可以使用小斑块。希望的斑块(即具有所需缺失突变的斑块)应该比背景斑块大3-5倍。
    4. 对稀释的斑块进行PCR扩增反应以进行验证:使用引物上游和下游的引物。根据PCR产物大小确定基因缺失。使用野生型T7作为阳性对照的模板。见图3.
      注意:删除编码相应原始样本的片段可能导致不编码所需删除(通过转义CRISPR-Cas定位)的孤立噬菌体。





      图3. PCR验证的图示。正向和反向引物退火到上游和下游到基因X(绿色)。编辑的噬菌体的PCR扩增产生较小的DNA产物。通过缺失编码相应原始样品的片段的WT噬菌体或噬菌体的PCR扩增产生全长DNA产物。黑色方形代表凝胶电泳,白色条纹代表DNA产品
    5. 预热软琼脂(0.7%琼脂)到液相。向琼脂中加入0.2%L-阿拉伯糖和0.1mM IPTG(终浓度),并保持在50℃。
    6. 预热LB琼脂平板在37℃至少1小时。每个修改的噬菌体一个板。
    7. 在预热的LB琼脂平板上条纹阳性删除噬菌体。
    8. 对于每个修饰的噬菌体,混合在15ml管中:
      200μl目标细菌
      3毫升预热的软琼脂与诱导剂
    9. 将细菌轻柔倒入条纹板上(步骤D6)。
      注意:将较低浓度区域的细菌倒入软琼脂中,使液体扩散到高浓度端。
    10. 使板冷却5分钟,倒置,并在室温下孵育过夜或在37℃下孵育4-6小时(直到斑块可见)。
    11. 从每个板上挑一个斑块,重复步骤D3-D9,3次。
    12. 在分离的噬菌体上进行PCR扩增,并用DNA测序验证所需的序列
    13. 稀释1:50将细菌定向到补充有50μg/ ml链霉素,50μg/ ml卡那霉素和35μg/ ml氯霉素,0.2%L-阿拉伯糖和0.1mM IPTG的5ml LB中。
    14. 在37°C振荡至600°至0.5°。
    15. 用10μl经过验证的工程化噬菌体感染培养物。
    16. 在37℃下摇动感染的细菌,直到发生裂解(通常为2小时)

数据分析

方案的流程图如图4所示。分离所需重组噬菌体和工程细胞百分比的方法的概述可以在原始论文(Kiro等人,2014)中找到。


图4.协议流程图

食谱

  1. LB培养基
    1. 测量20 g LB(Luria-Bertani)中等粉末,并用去离子水填充至1 L
    2. 高压灭菌解决方案并冷却
    3. 存储在RT
  2. LB琼脂平板
    1. 测量7.5 g琼脂,加入无菌LB培养基至500 ml
    2. 高压灭菌溶液,冷却并加入抗生素
    3. 倒入培养皿
    4. 等到琼脂凝固了
    5. 储存于4°C
  3. 软琼脂
    1. 测量1.75克琼脂,加入无菌LB培养基至250毫升
    2. 高压灭菌解决方案并冷却
    3. 存储在RT
  4. TAE缓冲液
    1. 倒入200毫升TAE缓冲液(储备),加入去离子水达10升
    2. 彻底混合
    3. 存储在RT

致谢

导致这些成果的研究由欧洲研究理事会根据欧洲共同体第七框架计划(FP7 / 207-2013)/ ERC拨款协议第336079号(EcCRISPR)提供资金。这里描述的协议来自(Kiro等人,2014)。

参考

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Manor, M. and Qimron, U. (2017). Selection of Genetically Modified Bacteriophages Using the CRISPR-Cas System. Bio-protocol 7(15): e2431. DOI: 10.21769/BioProtoc.2431.
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