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CRISPR/Cas9 Editing of the Bacillus subtilis Genome
枯草芽孢杆菌基因组的CRISPR/Cas9编辑   

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Abstract

A fundamental procedure for most modern biologists is the genetic manipulation of the organism under study. Although many different methods for editing bacterial genomes have been used in laboratories for decades, the adaptation of CRISPR/Cas9 technology to bacterial genetics has allowed researchers to manipulate bacterial genomes with unparalleled facility. CRISPR/Cas9 has allowed for genome edits to be more precise, while also increasing the efficiency of transferring mutations into a variety of genetic backgrounds. As a result, the advantages are realized in tractable organisms and organisms that have been refractory to genetic manipulation. Here, we describe our method for editing the genome of the bacterium Bacillus subtilis. Our method is highly efficient, resulting in precise, markerless mutations. Further, after generating the editing plasmid, the mutation can be quickly introduced into several genetic backgrounds, greatly increasing the speed with which genetic analyses may be performed.

Keywords: Genome editing(基因组编辑), CRISPR(CRISPR), Cas9(Cas9), Bacillus subtilis(枯草芽孢杆菌), Gene deletion(基因删除), Point mutation(点突变)

Background

Bacillus subtilis is a highly tractable, Gram-positive bacterium. It is amenable to genetic studies, using a variety of vectors to quickly and efficiently introduce mutations by homologous recombination. Although there are many different methods to introduce mutations in B. subtilis, each method has its limitations. A simple and straightforward method to make a mutation in B. subtilis is gene disruption, wherein a plasmid is integrated within a gene of interest (Vagner et al., 1998). The major limitations include: 1) the potential for polar effects by disrupting an operon; 2) introduction and retention of foreign DNA; 3) once an antibiotic resistance cassette is used, the researcher has to use a different cassette if a given mutation is to be studied in the context of other mutations; and 4) the method is limited to targeting an entire gene and cannot yield more precise point mutations. Another method employed in B. subtilis genetic studies is allelic replacement, wherein a gene of interest is replaced with an antibiotic resistance cassette (Guerout-Fleury et al., 1996). Although polar effects should be reduced by simply replacing one gene with another, this method still suffers from several limitations described above. Recently, a gene deletion library was constructed which allows for the removal of the antibiotic resistance cassette (strains are available from the Bacillus genetic stock center). As a result, researchers can use the same method for many mutations because the resistance cassette is removed after each allelic replacement. The method is an improvement, although it is still limited to gene deletions and cannot be used for point mutations. Finally, there are two methods to introduce markerless mutations in B. subtilis including point mutations. One method utilizes the upp gene as a counter-selectable marker (Fabret et al., 2002), and the other uses a plasmid called pMad (Arnaud et al., 2004) or its derivative pMiniMad which allows for mutation integration after removal of the integrating vector (Patrick and Kearns, 2008). Although these methods can introduce precise point mutations, our experience (making four gene deletions and inserting gfp at one genetic locus) using the latter method (Arnaud et al., 2004; Patrick and Kearns, 2008) is that it is quite time consuming with a success rate that is not very high (on average, about 12% success). Although we do not have experience with the upp counter selection method, the authors engineered a GGA→GAC change in the lexA gene and reported the intended change in sequence for three out of four screened isolates with the incorrect isolate yielding multiple mutations in the targeted lexA gene (Fabret et al., 2002). A major drawback, though, is that the method requires deletion of the endogenous upp gene in B. subtilis, which requires that the Δupp strain be used as the new ‘wild-type’ control. Therefore, although the methods described above work, we were in search of a genome editing method with a higher efficiency that also required less time at the bench. These criteria prompted us to adapt a CRISPR/Cas9 genome editing system (Jiang et al., 2013) to B. subtilis (Burby and Simmons, 2017). CRISPR/Cas9 can be used to introduce a variety of mutations including gene deletions, fusions, and even point mutations (Sternberg and Doudna, 2015). Further, by constructing the editing system on a single broad host-range plasmid with a temperature sensitive origin of replication, all vector DNA introduced during the procedure can easily be removed. Success rates have proven to be much higher for point mutations and small, gene-sized deletions (often over 80% success, but 100% success is not atypical), reducing the number of isolates that need to be screened. Although this method solves many of the limitations of the contemporary methods, our CRISPR/Cas9 genome editing system is limited by the requirement of a proto-spacer adjacent motif or PAM sequence (NGG in our system), and the requirement of two cloning steps. Nonetheless, the ability to make a variety of markerless mutations, coupled with the rapidity with which the mutations can be transferred to different genetic backgrounds still provides a significant improvement over current methods for genome editing in B. subtilis. The system we have developed may also be applicable to other Gram-positive bacteria with little or no manipulation of the DNA reagents described herein.

Materials and Reagents

  1. Pipette tips :
    with boxes (USA Scientific, catalog numbers: 1161-3800 ; 1161-1800 and 1161-1820 )
    for refills (USA Scientific, catalog numbers: 1161-3700 ; 1161-1700 and 1161-1720 )
  2. Microfuge tubes (Fisher Scientific, catalog number: 02-681-320 )
  3. PCR tubes (Fisher Scientific, catalog number: 14-230-225 )
  4. Wooden sticks for colony purification/bacteria re-streaking (Ted Pella, catalog number: 1282 )
  5. Round bottom culture tube (Fisher Scientific, catalog number: 14-956-6D )
  6. Cryo-vials (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 368632 )
  7. Cryo-vial caps (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 375930 )
  8. Silica spin columns for gel extraction and plasmid mini-prep (Epoch Life Science, catalog number: 1920250 )
  9. Petri dishes (Fisher Scientific, catalog number: FB0875712 )
  10. pPB41 plasmid (plasmid and sequence available from the Bacillus Genetic Stock Center upon request; http://www.bgsc.org)
  11. pPB105 plasmid (plasmid and sequence available from the Bacillus Genetic Stock Center upon request; http://www.bgsc.org)
  12. TOP10 Escherichia coli competent cells (Thermo Fisher Scientific, InvitrogenTM, catalog number: C404010 )
  13. MC1061 Escherichia coli competent cells (Strain number PEB336, available upon request)
  14. BsaI-HF with accompanying 10x Cutsmart buffer (New England Biolabs, catalog number: R3535L )
  15. Calf intestinal alkaline phosphatase (CIP) (New England Biolabs, catalog number: M0290L )
  16. Isopropanol (Fisher Scientific, catalog number: A451-4 )
  17. Ultra-pure distilled H2O (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977-15 )
  18. T4 DNA ligase with accompanying 10x T4 ligase buffer (New England Biolabs, catalog number: M0202L )
  19. T4 polynucleotide kinase (New England Biolabs, catalog number: M0201S )
  20. Q5 DNA polymerase (New England Biolabs, catalog number: M0491L )
  21. PCR primers (synthesized by Integrated DNA Technologies)
    1. Primers to amplify pPB41
      1. oPEB217: 5’-GAACCTCATTACGAATTCAGCATGC
      2. oPEB218: 5’-GAATGGCGATTTTCGTTCGTGAATAC
    2. Primers to amplify CRISPR/Cas9
      1. oPEB232: 5’-GCTGTAGGCATAGGCTTGGTTATG
      2. oPEB234:
        5’-GTATTCACGAACGAAAATCGCCATTCCTAGCAGCACGCCATAGTGACTG
    3. Primer to sequence CRISPR insert
      oPEB253: 5’-GAAGGGTAGTCCAGAAGATAACGA
    4. Primer to sequence 5’ side of editing template
      oPEB227: 5’-CCGTCAATTGTCTGATTCGTTA
    5. Example upstream editing template primers
      1. oPEB237:
        5’-GCATGCTGAATTCGTAATGAGGTTCAAAACGGCAGAGTATACAGAGGAG
      2. oPEB238: 5’-CCGGTTCCTTTTCCAGCGATGATTGACACTCTTGGATATCCG
    6. Example downstream editing template primers
      1. oPEB239: 5’-AAGAGTGTCAATCATCGCTGGAAAAGGAACCGGCGCTTTAAG
      2. oPEB240: 5’-GCATAACCAAGCCTATGCCTACAGCtaggaagaagaatcatttcgaagc
    7. Example genotyping primer for H743A mutation in mutS2
      1. oPEB262: 5’- GGATATCCAAGAGTGTCAATCATCGCT
  22. Glycerol (Fisher Scientific, catalog number: BP229-4 )
  23. Tris base (Fisher Scientific, catalog number: BP152-5 )
  24. Glacial acetic acid (Fisher Scientific, catalog number: A38-212 )
  25. EDTA (Fisher Scientific, catalog number: BP120-1 )
  26. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-10 )
  27. Tryptone (BD, BactoTM, catalog number: 211699 )
  28. Yeast extract (BD, BactoTM, catalog number: 212720 )
  29. Ampicillin (Fisher Scientific, catalog number: BP1760-25 )
  30. Spectinomycin (MP Biomedicals, catalog number: 0 215206725 )
  31. Chloramphenicol (Fisher Scientific, catalog number: BP904-100 )
  32. Agar (Acros Organics, catalog number: 400400050 )
  33. Agarose (Fisher Scientific, catalog number: BP1356-500 )
  34. Ethidium bromide (Sigma-Aldrich, catalog number: E8751-25G )
  35. Guanidine thiocyanate (Fisher Scientific, catalog number: BP221-1 )
  36. Guanidine HCl (Fisher Scientific, catalog number: BP178-1 )
  37. Ethanol (Decon Labs, catalog number: 2701 )
  38. Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A144-212 )
  39. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266-100G )
  40. 100 mM dNTP set (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10297018 )
  41. Dithiothreitol (DTT) (Fisher Scientific, catalog number: BP172-25 )
  42. β-nicotinamide adenine dinucleotide (NAD+) (Acros Organics, catalog number: 124530010 )
  43. T5 exonuclease (New England Biolabs, catalog number: M0363S )
  44. Phusion DNA polymerase (New England Biolabs, catalog number: M0530L )
  45. Taq DNA ligase (New England Biolabs, catalog number: M0208L )
  46. Magnesium sulfate (MgSO4) (Fisher Scientific, catalog number: M65-500 )
  47. Potassium phosphate dibasic (K2HPO4) (Fisher, catalog number: BP363-1 )
  48. Potassium phosphate monobasic (KH2PO4) (Fisher Scientific, catalog number: BP362-1 )
  49. Trisodium citrate dihydrate (C6H5Na3O7·2H2O) (Fisher Scientific, catalog number: BP327-1 )
  50. Glucose (Sigma-Aldrich, catalog number: G8270-1KG )
  51. Tryptophan (Fisher Scientific, catalog number: BP395-100 )
  52. Phenylalanine (Fisher Scientific, catalog number: BP391-100 )
  53. Potassium hydroxide (KOH) (Fisher Scientific, catalog number: P250-500 )
  54. Ferric ammonium citrate (Sigma-Aldrich, catalog number: F5879 )
  55. Potassium aspartate (Sigma-Aldrich, catalog number: A6558 )
  56. PEG-8000 (Dot Scientific, catalog number: DSP48080-500 )
  57. 50x TAE (see Recipes)
  58. 1x TAE (see Recipes)
  59. LB media (see Recipes)
  60. LB agar plates (see Recipes)
  61. 100 mg/ml ampicillin stock (see Recipes)
  62. 100 mg/ml spectinomycin stock (see Recipes)
  63. 5 mg/ml chloramphenicol stock (see Recipes)
  64. 70% (v/v) ethanol (see Recipes)
  65. 1% agarose gel (see Recipes)
  66. QG buffer (see Recipes)
  67. PB buffer (see Recipes)
  68. Tris HCl , pH 7.5 (see Recipes)
  69. PE buffer (see Recipes)
  70. 10x annealing buffer (see Recipes)
  71. 5x isothermal reaction buffer (Gibson, 2011) (see Recipes)
  72. 2x Gibson master mix (Gibson, 2011) (see Recipes)
  73. 1 M MgSO4 (see Recipes)
  74. LM media (LB media + 3 mM MgSO4) (see Recipes)
  75. 10x PC buffer (see Recipes)
  76. MD media (see Recipes)
  77. 0.1 N KOH (see Recipes)
  78. 100 mg/ml tryptophan stock (see Recipes)
  79. 100 mg/ml phenylalanine stock (see Recipes)
  80. 2.2 mg/ml ferric ammonium citrate stock (see Recipes)
  81. 100 mg/ml potassium aspartate stock (see Recipes)

Equipment

  1. Incubator (Napco, model: 320 )
  2. Dry heat block (Fisher Scientific, model: Fisher ScientificTM IsotempTM Digital Dry Baths/Block Heaters , catalog number: 11-718-2)
  3. Pipettes (Eppendorf, catalog number: 022575442 )
  4. Centrifuge (Eppendorf, model: 5424 )
  5. Thermocycler (Eppendorf, model: 6325 )
  6. Electrophoresis apparatus (Bio-Rad Laboratories, model: Mini-Sub® Cell GT Horizontal Electrophoresis System , catalog number: 1704406)
  7. Power source for electrophoresis (Bio-Rad Laboratories, model: PowerPacTM Basic Power Supply , catalog number: 1645050)
  8. Roller drum (Eppendorf, New Brunswick, model: TC-7 )
  9. Milli-Q H2O dispenser (Thermo Fisher Scientific, Thermo ScientificTM, model: BarnsteadTM GenPureTM Pro , catalog number: 50131950)
  10. Microwave (Panasonic, catalog number: NNS954WFR )
  11. Autoclave

Software

  1. Online tool Primer3 (Koressaar and Remm, 2007; Untergasser et al., 2012)
  2. Oligocalc (Kibbe, 2007)

Procedure

  1. Construction of plasmid containing spacer used to target the B. subtilis genome
    1. Prepare pPB41 digested with the restriction endonuclease BsaI (Figure 1)
      Assemble 50 μl restriction digest reaction: 1x Cutsmart buffer (NEB), 2 μl BsaI-HF (20,000 U/ml; NEB), and 5 μg pPB41. Incubate reaction at 37 °C in an incubator for 3 to 6 h. Add 1 μl of calf intestinal phosphatase (CIP; 10,000 U/ml; NEB), and incubate at 37 °C for 1 h. Perform 1% agarose gel electrophoresis in 1x TAE buffer (see Recipes), followed by gel extraction of the digested plasmid.
      Notes:
      1. The plasmid pPB41 is typically used, but we also generated a derivative, pPB105, wherein the spectinomycin resistance cassette was replaced with a chloramphenicol resistance cassette (see Figure 1). All steps of the procedure are identical for pPB105, except for E. coli clones are selected using LB agar plates containing 100 μg/ml ampicillin or 20 μg/ml chloramphenicol (chloramphenicol selection works but colonies grow slower), and B. subtilis transformants are selected using LB agar plates containing 5 μg/ml chloramphenicol.
      2. Gel extractions are performed by excising the appropriate DNA band from the agarose gel from two 50 μl PCR reactions and placing in a microfuge tube. The gel slice is dissolved by adding 350 μl QG buffer (see Recipes) and incubating at 65 °C in a heat block. After the gel slice is fully dissolved, 200 μl of isopropanol is added and mixed by pipetting. The entire gel extraction is loaded onto a silica spin column. The flow-through is collected via centrifugation (12,000 x g for 1 min at room temperature) and discarded. The column is washed and the flow-through discarded first with 500 μl PB buffer (see Recipes), then with 750 μl PE buffer (see Recipes). The column is dried by centrifugation as above. The DNA is eluted from the column with 75 μl ultra-pure H2O via centrifugation as above.


        Figure 1. Digestion of pPB41 or pPB105 with BsaI-HF

    2. Prepare phosphorylated proto-spacer (Figure 2)
      1. The proto-spacer is designed by searching the B. subtilis genome for a proto-spacer adjacent motif (PAM) site, consisting of the nucleotide sequence 5’-NGG-3’ (where N stands for any nucleotide), at the location that the edit is to be made (Figure 2A). The thirty nucleotides 5’ of the PAM site are used for the proto-spacer (Figure 2A). Oligonucleotides for the proto-spacer are ordered from Integrated DNA Technologies (IDT) as shown in Figure 2B. The two oligonucleotides are reverse complementary to each other with the exception of the overhangs (Figure 2B). Prepare oligonucleotide stocks at a concentration of 100 μM using ultra-pure distilled H2O (Invitrogen Life Technologies).
      2. Assemble annealing reaction: 1x annealing buffer (see Recipes), 10 μM oligonucleotide 1, 10 μM oligonucleotide 2. Incubate reaction at 100 °C in a heat block for 5 min. The reaction is transferred to a beaker of water, pre-heated to 100 °C. The entire beaker is placed at room temperature and allowed to cool slowly to room temperature.
      3. Prepare phosphorylation reaction: 1x T4 DNA ligase buffer (NEB), 1 μl T4 polynucleotide kinase (PNK; 10,000 U/ml; NEB), 1 μM annealed oligonucleotides from above. Incubate at 37 °C in an incubator for 30 min, and then heat inactivate T4 PNK at 65 °C in a heat block for 20 min. Store annealed and phosphorylated oligonucleotides at -20 °C until ready to use in ligation reaction.
        Notes:
        1. Although we typically phosphorylate the proto-spacer using T4 PNK, it is also possible to order the oligonucleotides with a 5’-phosphate modification from IDT.
        2. Given that the T4 PNK inactivation is carried out at 65 °C, it is possible that the proto-spacer could denature. Although we have not had a need to change the order of the protocol (and we have not tried), T4 PNK is active on both single stranded and double stranded DNA, and therefore, we see no theoretical concern with reversing the order, and phosphorylating prior to annealing.


          Figure 2. Proto-spacer design and preparation. A. A proto-spacer (highlighted blue and capital letters) and proto-spacer adjacent motif (PAM site; highlighted red); B. Oligonucleotides with proper overhangs (lower case letters) necessary to ligate into pPB41 (Jiang et al., 2013; Burby and Simmons, 2017); C. Annealing and phosphorylation reaction to prepare a dsDNA proto-spacer for ligation into pPB41.

    3. Ligate proto-spacer and pPB41 and isolate new plasmid (Figure 3)
      Assemble 20 μl ligation reaction: 1x T4 DNA ligase buffer (NEB), 40 to 100 ng pPB41 (digested with BsaI-HF and CIP in previous step), 25 nM proto-spacer (annealed and phosphorylated in previous step), 1 μl T4 DNA ligase (400,000 U/ml NEB). Let reaction stand at room temperature for 2-3 h. Use 10 μl of ligation reaction to transform 100 μl of chemically competent TOP10 Escherichia coli. Clones can be selected using LB agar plates (see Recipes) containing 100 μg/ml ampicillin or 100 μg/ml spectinomycin. Clones are verified via Sanger sequencing using oPEB253.
      Note: The first cloning step, wherein the proto-spacer is ligated into pPB41, does not require a certain E. coli strain. We use TOP10 cells as our general cloning strain.


      Figure 3. Ligation of phosphorylated proto-spacer and pPB41 to generate a ‘targeting plasmid’

  2. Construction of editing plasmid
    1. PCR amplify pPB41 to linearize (Figure 4A)
      Amplify pPB41 via PCR using Q5 DNA polymerase (NEB) and primers oPEB217 and oPEB218. Perform 1% agarose gel electrophoresis in 1x TAE buffer (see Recipes) followed by gel extraction. PCR product is stored at -20 °C until used.
      Notes:
      1. The template used for this step could be either pPB41 or the plasmid containing the spacer generated in step B1.
      2. The PCR program used to linearize pPB41 is:



        Figure 4. Construction of editing plasmid. A. Schematic representation of pPB41 PCR amplicon; B. Schematic representation of CRISPR/Cas9 PCR amplicon; C. Schematic representation of upstream and downstream portions of editing template PCR amplicons; D. Gibson Assembly of editing plasmid using PCR products from A-C.

    2. PCR amplify CRISPR/Cas9 using plasmid generated in step B1 (Figure 4B)
      Amplify plasmid generated in step B1 (pPB43 in the example) via PCR using Q5 DNA polymerase (NEB) and primers oPEB232 and oPEB234. Perform 1% agarose gel electrophoresis in 1x TAE buffer (see Recipes) followed by gel extraction. PCR product is stored at -20 °C until used.
      Note: The PCR program used to amplify CRISPR/Cas9 is the same used in step B1 above.
    3. PCR amplify editing template (Figure 4C)
      Amplify upstream and downstream portions of editing template separately via PCR using Q5 DNA polymerase (NEB) (primers used in the example are oPEB237/oPEB238 and oPEB239/oPEB240 for the upstream and downstream portions, respectively). Primers are designed using the online tool Primer3 (Koressaar and Remm, 2007; Untergasser et al., 2012) and Oligocalc (Kibbe, 2007). The primers specific to the editing template are combined with the overhangs for pPB41 (upstream forward primer starts 5’-GCATGCTGAATTCGTAATGAGGTTC, followed by the editing template specific sequence; downstream reverse primer starts 5’-GCATAACCAAGCCTATGCCTACAGC, followed by editing template specific sequence), which will allow the editing template to be incorporated into the editing plasmid. The editing template is designed to be approximately 2 kilobases, with the upstream and downstream portions consisting of about 1 kb each. The critical aspect of editing template design is ensuring that the guided Cas9 can no longer target the genome after the desired mutation is introduced. For deletions, the entire sequence of the spacer and the PAM site can be removed. For point mutations, it is more complicated. In Jiang et al. (2013), it was found that mutating the PAM site or the first few bases of the spacer is optimal (Jiang et al., 2013). The overlap at the center of the editing template should be 25 bases on each portion of the template (the result is that there are 50 bp that overlap in total). After PCR, perform 1% agarose gel electrophoresis in 1x TAE buffer (see Recipes) followed by gel extraction. PCR products are stored at -20 °C until used.
      Notes: 
      1. Editing template primers are designed using the online tool Primer3 (Koressaar and Remm, 2007; Untergasser et al., 2012). Approximately 250 bp is selected at least 750 bp from the center of the editing template and a primer list is generated using default parameters with the following exceptions: 1) primer size used is min: 22, opt: 24, max: 28; 2) primer GC% min: 35; 3) Max Self Complementarity: 6.0; and 4) Max 3’ Self Complementarity: 2.0. A primer is then selected and cross examined using a second online tool, Oligocalc (Kibbe, 2007). The purpose is to ensure that potential hairpin formation, 3’ complementarity, and self-annealing are all undetectable using this program.
      2. The editing template used in the example in the figures is 3 kb (about 1.5 kb each for upstream and downstream portions of the editing template), but we routinely use 2 kb. We also present four different gene deletions made using editing templates of about 2 kb.
      3. The PCR program used to amplify each portion of the editing template is:


    4. Construct editing plasmid using Gibson Assembly (Gibson, 2011) (Figure 4D)
      Assemble 10 μl Gibson Assembly reaction: 1x Gibson master mix (see Recipes), 40-100 ng of linearized pPB41 from above, 40-100 ng of CRISPR/Cas9 PCR product containing spacer from above, and 20-40 ng of each portion of the editing template. Incubate reaction at 50 °C in a thermocycler for 90 min. Use 5 μl of Gibson Assembly reaction to transform 80 μl chemically competent E. coli strain MC1061. Clones can be selected as in step A3. Clones are sequenced using primers oPEB253, oPEB227, and editing template specific primers.
      Notes:
      1. It is absolutely critical to use a cloning strain that is recA+, because transformation of naturally competent B. subtilis is much more efficient using plasmids that are multimeric. The E. coli strain we use for plasmid propagation is MC1061. We have also encountered difficulties in obtaining certain editing plasmids in MC1061 for reasons that are not clear. In these instances, we scaled up the Gibson Assembly reaction to a total volume of 20 μl and we used 20 μl to transform B. subtilis as in Procedure C. The efficiency of this procedure was lower, with some spectinomycin sensitive isolates retaining the wild-type genotype, so typically at least twelve isolates were screened.
      2. The editing template is sequenced using oPEB227, which sequences from the 5’ end of pPB41. Additional primers are designed within the editing template to verify the remaining sequence.

  3. Editing genome of B. subtilis
    1. Transform B. subtilis with editing plasmid obtained from previous step
      1. Prepare naturally competent B. subtilis cultures (Burby and Simmons, 2017) (Figure 5A).
        Streak out strain from frozen 20% glycerol stock on LB agar plate for single colonies using a wooden stick and incubate plate at 30 or 37 °C overnight in an incubator.
      2. In the morning, inoculate 2 ml LM media (see Recipes) in a 14 ml disposable, round-bottom culture tube. Incubate culture at 37 °C on a roller drum in an incubator until the OD600 is between 0.8 and 1.5 (approximately 2 h growth).
      3. Transfer 20 μl of LM culture to 0.5 ml pre-warmed MD media (see Recipes). Incubate MD culture at 37 °C for 4 to 6 h on a roller drum.
      4. Add editing plasmid DNA (approximately 200 to 600 ng) and incubate at 37 °C on roller drum for 60 to 90 min. Transformants are selected by plating 200 μl on LB agar plates containing 100 μg/ml spectinomycin and incubated at 30 °C overnight.
        Notes:
        1. We have found 5 h in MD media to work very well, and is used most often, but adding DNA up to an hour early or an hour late is also successful.
        2. Plating 90 min after DNA addition works very well, but plating at 60 min will also yield transformants.
    2. Several isolates (6-12) are established by colony purifying transformants (Figure 5B)
      1. Colony purifying refers to restreaking isolated colonies three to four consecutive times for single colonies.
      2. Single colonies from preceding step are re-streaked on LB agar plates for single colonies containing 100 μg/ml spectinomycin and incubated at 30 °C overnight in an incubator.
    3. Cure isolates of plasmid (Figure 5C)
      Single colonies from preceding step are re-struck on LB agar plates for single colonies and incubated at 42-45 °C overnight.
      Note: We have an incubator set to 45 °C, but we have used 42 °C successfully.


      Figure 5. Outline of genome editing procedure in B. subtilis (Adapted from Figure S2 [Burby and Simmons, 2017]). A. The first step is to transform B. subtilis with the editing plasmid. Inset shows wild-type mutS2 locus. B. After establishing isolates using colony purification, the isolates are cured of the editing plasmid. Inset depicts homology directed repair using the editing template on the plasmid. C. The final step is to verify loss of the editing plasmid and genotype isolates to verify the mutation was successfully introduced. Inset shows edited mutS2 locus.

    4. Screen isolates for loss of plasmid
      Single colonies from preceding step are re-streaked onto LB agar plates containing 100 μg/ml spectinomycin, and then onto LB agar plates for single colonies. Plates are incubated at 45 °C overnight in an incubator. If the isolates lost the plasmid, there would be no growth on the LB agar plates with spectinomycin.
    5. Screen for intended mutation (Figure 6)
      The isolates that were unable to grow on plates containing spectinomycin are further screened for the presence of the desired mutation. In the case of a point mutation, a primer can be designed to anneal at the point mutation (Figure 6A), or the genetic locus may be amplified using primers outside the editing template as done for a deletion (see below), followed by Sanger sequencing to verify the point mutation is present and no other mutations were introduced. In the case of a gene deletion, PCR primers that anneal outside of the recombination template within the genomic DNA are used to PCR amplify the genetic locus (Figure 6B). The migration of the PCR product will be faster relative to the product from wild-type locus.


      Figure 6. PCR based verification of genome edit. A. A schematic representation showing the locations of PCR primers used in panel B; B. Agarose gel stained with ethidium bromide of amplified PCR products using primers specific to the point mutation (H743A, for which we changed CAC→GCT) and the B. subtilis chromosome (left panel), and using primers specific to the editing plasmid (right panel). Of twelve isolates screened all were found to have the predicted point mutation, and two of twelve retained resistance to spectinomycin (asterisks). The slow migrating band in the PY79 lane corresponds to nonspecific PCR amplification in the wild type background. The editing plasmid was detectable in spectinomycin resistant isolates, but not in the remaining ten. C. Agarose gel stained with ethidium bromide of PCR reactions using primers specific for deletion of four B. subtilis genes. Primers were designed outside of the editing template, and are therefore specific to chromosomal DNA. For all four deletions, all isolates were found to be spectinomycin sensitive, and PCR genotyping of three isolates for each deletion found that all isolates tested had the intended deletion. Note that for the control reaction for ponA, the PCR product is faint because the amplicon is approximately 5 kb and the extension time of the PCR was 3 min, which is near the limit for complete extension for Q5 DNA polymerase; the other three control amplicons are no larger than 3.4 kb.

    6. Save frozen stock of new strain
      Separate LB cultures of at least two isolates that are confirmed to have the intended mutation are grown to an OD600 of approximately 1 to 1.5. A frozen stock is saved by adding glycerol to a final concentration of 20% in a cryo-vial. A cryo-vial cap is added to the lid and labeled appropriately. Cryo-vials are placed directly into a freezer at -80 °C.

Data analysis

There are two steps where data analysis is required for constructing a mutant strain. The first is assaying for loss of the editing plasmid using antibiotic sensitivity, and the second is to assay the genetic locus for the intended mutation using PCR genotyping. In order to determine if the isolates have been cured of the editing plasmid, six to twelve isolates are streaked onto LB agar plates with and without 100 μg/ml spectinomycin and grown overnight (see step C4 of the protocol). The isolates that do not grow on LB agar plates containing spectinomycin have been cured of the editing plasmid. The isolates that have been cured of the plasmid are further genotyped via PCR. Data analysis of PCR genotyping will depend on the type of edit being introduced. For a deletion, PCR amplification of the genetic locus will result in a product with a migration that is faster than that of the wild-type locus, resulting in the PCR product appearing lower on the agarose gel. For an insertion, the PCR product would migrate slower than the wild-type product on the agarose gel. For a point mutation, there will be no change in the size of the PCR product. As a result, a mutation specific primer or Sanger sequencing must be used to confirm the correct genotype. For a mutation specific primer (see Figure 6B), the PCR product will be present or absent. If the PCR product is present, that would indicate the mutation is present. If Sanger sequencing is used, the sequence of the mutation will be detected instead of the wild-type sequence.

Recipes

  1. 50x TAE
    242 g Tris base
    57.1 ml glacial acetic acid
    100 ml EDTA, pH 8.0
    Add Milli-Q H2O to 1 L
    Store at room temperature
  2. 1x TAE
    20 ml 50x TAE
    980 ml Milli-Q H2O
    Store at room temperature
  3. LB media
    10 g NaCl
    10 g tryptone
    5 g yeast extract
    Add Milli-Q H2O to a total volume of 1 L
    Autoclave to sterilize
  4. LB agar plates
    1 L LB media
    18 g agar
    Autoclave to sterilize
    Allow to cool to about 60 °C; if antibiotics are required, they are added at this point
    Spectinomycin is added to a final concentration of 100 μg/ml
    Ampicillin is added to a final concentration of 100 μg/ml
    Chloramphenicol is added to a final concentration of 20 μg/ml for E. coli and 5 μg/ml for B. subtilis
    Pour plates
  5. 100 mg/ml ampicillin stock
    1 g ampicillin
    Add Milli-Q water to 10 ml
    Filter sterilize
    Aliquot and store at -20 °C
  6. 100 mg/ml spectinomycin stock
    1 g spectinomycin
    Add Milli-Q water to 10 ml
    Filter sterilize
    Aliquot and store at -20 °C
  7. 5 mg/ml chloramphenicol stock
    50 mg chloramphenicol
    Add 70% (v/v) ethanol to 10 ml
    Filter sterilize
    Store at -20 °C
  8. 70% (v/v) ethanol
    70 ml ethanol
    30 ml Milli-Q H2O
    Store at room temperature
  9. 1% agarose gel
    1 g agarose
    100 mL 1x TAE
    Heat solution to dissolve agarose in microwave
    Add ethidium bromide to a final concentration of 0.2 μg/ml
    Pour gel
  10. QG buffer
    5.5 M guanidine thiocyanate
    20 mM Tris HCl, pH 6.6
  11. PB buffer
    5 M guanidine HCl
    30% (v/v) isopropanol
  12. Tris HCl , pH 7.5
    Tris HCl is prepared by dissolving Tris base in Milli-Q H2O and adjusting the pH to 7.5 using HCl
  13. PE buffer
    10 mM Tris HCl, pH 7.5
    80% (v/v) ethanol
  14. 10x annealing buffer
    100 mM Tris HCl, pH 7.5
    1 M NaCl
    1 mM EDTA, pH 8.0
    Ultra-pure H2O
    Filter sterilize
  15. 5x isothermal reaction buffer (Gibson, 2011)
    0.5 M Tris HCl, pH 7.5
    25% (w/v) PEG-8000
    50 mM MgCl2
    50 mM DTT
    1 mM each of dNTPs
    5 mM NAD+
    Store at -20 °C
  16. 2x Gibson master mix (Gibson, 2011)
    320 μl 5x isothermal reaction buffer
    0.64 μl T5 exonuclease (10,000 U/ml)
    20 μl Phusion DNA polymerase (2,000 U/ml)
    160 μl Taq DNA ligase (40,000 U/ml)
    Store at -20 °C
  17. 1 M MgSO4
    120.37 g MgSO4 (F.W. 120.37 g)
    Add Milli-Q H2O to 1 L
    Autoclave to sterilize
    Store at room temperature
  18. LM media (LB media + 3 mM MgSO4)
    2 ml LB media
    6 μl 1 M MgSO4
  19. 10x PC buffer
    10.7 g K2HPO4
    6 g KH2PO4
    1.18 g trisodium citrate dehydrate (C6H5Na3O7·2H2O)
    Add Milli-Q H2O to 100 ml
    Filter sterilize
    Store at room temperature
  20. MD media
    1x PC buffer
    2% (w/v) glucose
    50 μg/ml tryptophan
    50 μg/ml phenylalanine
    11 μg/ml ferric ammonium citrate
    2.5 mg/ml potassium aspartate
    3 mM MgSO4
    Filter sterilize
    Store protected from light at 4 °C
  21. 0.1 N KOH
    56.1 mg KOH (F.W. 56.11 g)
    Add Milli-Q H2O to 10 ml
    Prepare fresh for each use
  22. 100 mg/ml tryptophan stock
    1 g tryptophan
    Add 0.1 N KOH to 10 ml (prepare fresh)
    Filter sterilize
    Store protected from light at 4 °C
  23. 100 mg/ml phenylalanine stock
    1 g phenylalanine
    Add 0.1 N KOH to 10 ml (prepare fresh)
    Filter sterilize
    Store protected from light at 4 °C
  24. 2.2 mg/ml ferric ammonium citrate stock
    110 mg ferric ammonium citrate
    Add Milli-Q H2O to 50 ml
    Filter sterilize
    Store protected from light at 4 °C
  25. 100 mg/ml potassium aspartate stock
    5 g potassium aspartate
    Add Milli-Q H2O to 50 ml
    Filter sterilize
    Store at 4 °C

Acknowledgments

This work was supported by NIH grant R01 GM107312 to L.A.S. and a pre-doctoral fellowship from the National Science Foundation #DGE 1256260 to P.E.B.

References

  1. Arnaud, M., Chastanet, A. and Debarbouille, M. (2004). New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, Gram-positive bacteria. Appl Environ Microbiol 70(11): 6887-6891.
  2. Burby, P. E. and Simmons, L. A. (2017). MutS2 promotes homologous recombination in Bacillus subtilis. J Bacteriol 199(2).
  3. Fabret, C., Ehrlich, S. D. and Noirot, P. (2002). A new mutation delivery system for genome-scale approaches in Bacillus subtilis. Mol Microbiol 46(1): 25-36.
  4. Gibson, D. G. (2011). Enzymatic assembly of overlapping DNA fragments. Methods Enzymol 498: 349-361.
  5. Guerout-Fleury, A. M., Frandsen, N. and Stragier, P. (1996). Plasmids for ectopic integration in Bacillus subtilis. Gene 180(1-2): 57-61.
  6. 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.
  7. Kibbe, W. A. (2007). OligoCalc: an online oligonucleotide properties calculator. Nucleic Acids Res 35(Web Server issue): W43-46.
  8. Koressaar, T. and Remm, M. (2007). Enhancements and modifications of primer design program Primer3. Bioinformatics 23(10): 1289-1291.
  9. Patrick, J. E. and Kearns, D. B. (2008). MinJ (YvjD) is a topological determinant of cell division in Bacillus subtilis. Mol Microbiol 70(5): 1166-1179.
  10. Sternberg, S. H. and Doudna, J. A. (2015). Expanding the biologist's toolkit with CRISPR-Cas9. Mol Cell 58(4): 568-574.
  11. Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B. C., Remm, M. and Rozen, S. G. (2012). Primer3--new capabilities and interfaces. Nucleic Acids Res 40(15): e115.
  12. Vagner, V., Dervyn, E. and Ehrlich, S. D. (1998). A vector for systematic gene inactivation in Bacillus subtilis. Microbiology 144: 3097-3104.

简介

大多数现代生物学家的基本过程是研究生物体的遗传操作。尽管许多不同的方法用于编辑细菌基因组已经在实验室中使用了数十年,但CRISPR / Cas9技术对细菌遗传学的适应使得研究人员能够以无与伦比的设施来操纵细菌基因组。 CRISPR / Cas9允许基因组编辑更精确,同时也提高将突变转移到各种遗传背景的效率。因此,在遗传操作难以处理的易处理生物和生物体中实现了这些优点。在这里,我们描述了我们编辑枯草芽孢杆菌细菌基因组的方法。我们的方法是高效的,导致精确,无标记的突变。此外,在产生编辑质粒之后,可以将突变快速导入几个遗传背景,大大增加可进行遗传分析的速度。

枯草芽孢杆菌是高度易处理的革兰氏阳性菌。遗传研究适用于使用多种载体通过同源重组快速有效地引入突变。尽管有许多不同的方法来引入B突变。 subtilis,每种方法都有其局限性。一种简单而简单的方法,用于在B中进行突变。枯草芽孢杆菌是基因破坏,其中将质粒整合到感兴趣的基因内(Vagner等人,1998)。主要的局限性包括:1)扰乱操纵子的极地作用的潜力; 2)引进和保留外来DNA; 3)一旦使用抗生素耐药性盒,如果在其他突变的背景下研究给定的突变,则研究者必须使用不同的盒;和4)该方法限于靶向整个基因,并且不能产生更精确的点突变。 B中采用的另一种方法。枯草芽孢杆菌基因研究是等位基因替换,其中感兴趣的基因被抗生素抗性盒替代(Guerout-Fleury等人,1996)。虽然通过简单地用另一个替换一个基因来减少极性效应,但是该方法仍然受到上述几个限制。最近,构建了一种能够去除抗生素抗性盒的基因缺失文库(菌株可从芽孢杆菌属遗传物质中心获得)。因此,研究人员可以使用相同的方法进行许多突变,因为每次等位基因置换后,电阻盒被去除。该方法是一种改进,尽管它仍然限于基因缺失,不能用于点突变。最后,有两种方法在B中引入无标记突变。 subtilis 包括点突变。一种方法利用上调基因作为反选择标记(Fabret等人,2002),另一种方法使用称为pMad(Arnaud等)的质粒2004)或其衍生的pMiniMad,其允许在去除积分载体之后进行突变整合(Patrick和Kearns,2008)。虽然这些方法可以引入精确的点突变,但我们的经验(使用四种基因缺失并在一个遗传基因座插入gfp )使用后一种方法(Arnaud等人,2004; Patrick和Kearns,2008)认为这是非常耗时的成功率不是很高(平均而言,大约12%的成功)。尽管我们没有使用upp 计数器选择方法的经验,作者在“lexA”基因中设计了一个GGA→GAC变化,并报告了3个四个筛选的分离物与不正确的分离物在靶向的lexA基因中产生多个突变(Fabret等人,2002)。但是,主要缺点是该方法需要删除B中的内源性上调基因。 subtilis,其要求使用Δupp菌株作为新的“野生型”对照。因此,尽管上述方法起作用,但是我们正在寻找一种更高效率的基因组编辑方法,而且在长凳上也需要更少的时间。这些标准促使我们将CRISPR / Cas9基因组编辑系统(Jiang等人,2013)适应于B。 subtilis(Burby and Simmons,2017)。 CRISPR / Cas9可用于引入各种突变,包括基因缺失,融合,甚至点突变(Sternberg和Doudna,2015)。此外,通过在具有温度敏感复制起点的单个宽宿主范围质粒上构建编辑系统,可以容易地去除在该过程中引入的所有载体DNA。成功率已被证明是高得多的点突变和小的,基因大小的删除(通常超过80%的成功,但100%的成功不是非典型的),减少需要筛选的分离株的数量。虽然这种方法解决了当代方法的许多局限性,但我们的CRISPR / Cas9基因组编辑系统受到原间隔相邻基序或PAM序列(我们系统中NGG)的要求以及两个克隆步骤的要求的限制。尽管如此,制作各种无标记突变的能力,加上突变可以转移到不同遗传背景的快速度仍然比现有的基因组编辑方法提供了显着的改进。枯草芽。我们开发的系统也可以适用于其中对本文所述的DNA试剂很少或不进行操作的其它革兰氏阳性细菌。

关键字:基因组编辑, CRISPR, Cas9, 枯草芽孢杆菌, 基因删除, 点突变

材料和试剂

  1. 移液器提示:
    (USA Scientific,目录号:1161-3800; 1161-1800和1161-1820)
    用于补充(USA Scientific,目录号:1161-3700; 1161-1700和1161-1720)
  2. Microfuge管(Fisher Scientific,目录号:02-681-320)
  3. PCR管(Fisher Scientific,目录号:14-230-225)
  4. 用于菌落纯化/细菌再条纹的木棍(Ted Pella,目录号:1282)
  5. 圆底培养管(Fisher Scientific,目录号:14-956-6D)
  6. 冷冻小瓶(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:368632)
  7. 冷冻瓶盖(Thermo Fisher Scientific,Thermo Scientific TM,目录号:375930)
  8. 硅胶旋转柱用于凝胶提取和质粒微量制备(Epoch Life Science,目录号:1920250)
  9. 培养皿(Fisher Scientific,目录号:FB0875712)
  10. pPB41质粒(质粒和序列可得自 Genetic Stock Center根据要求; http://www.bgsc.org
  11. pPB105质粒(质粒和序列可从 Genetic Stock Center获得; http://www.bgsc.org
  12. TOP10大肠杆菌感受态细胞(Thermo Fisher Scientific,Invitrogen TM,目录号:C404010)
  13. MC1061大肠杆菌感受态细胞(菌株编号PEB336,可根据要求提供)
  14. Bsa 伴随10x Cutsmart缓冲液的I-HF(New England Biolabs,目录号:R3535L)
  15. 小牛肠碱性磷酸酶(CIP)(New England Biolabs,目录号:M0290L)
  16. 异丙醇(Fisher Scientific,目录号:A451-4)
  17. 超纯蒸馏H 2 O(Thermo Fisher Scientific,Invitrogen TM,目录号:10977-15)
  18. T 4 DNA连接酶与伴随的10xT4连接酶缓冲液(New England Biolabs,目录号:M0202L)
  19. T 多核苷酸激酶(New England Biolabs,目录号:M0201S)
  20. Q5 DNA聚合酶(New England Biolabs,目录号:M0491L)
  21. PCR引物(由Integrated DNA Technologies合成)
    1. 引物扩增pPB41
      1. oPEB217:5'-GAACCTCATTACGAATTCAGCATGC
      2. oPEB218:5'-GAATGGCGATTTTCGTTCGTGAATAC
    2. 引物扩增CRISPR/Cas9
      1. oPEB232:5'-GCTGTAGGCATAGGCTTGGTTATG
      2. oPEB234:
        5'-GTATTCACGAACGAAAATCGCCATTCCTAGCAGCACGCCATAGTGACTG
    3. 引物序列CRISPR插入物
      oPEB253:5'-GAAGGGTAGTCCAGAAGATAACGA
    4. 编辑模板的序列5'的引物
      oPEB227:5'-CCGTCAATTGTCTGATTCGTTA
    5. 示例上游编辑模板引物
      1. oPEB237:
        5'-GCATGCTGAATTCGTAATGAGGTTCAAAACGGCAGAGTATACAGAGGAG
      2. oPEB238:5'-CCGGTTCCTTTTCCAGCGATGATTGACACTCTTGGATATCCG
    6. 示例下游编辑模板引物
      1. oPEB239:5'-AAGAGTGTCAATCATCGCTGGAAAAGGAACCGGCGCTTTAAG
      2. oPEB240:5'-GCATAACCAAGCCTATGCCTACAGCtaggaagaagaatcatttcgaagc
    7. MutS2中H743A突变的实例基因分型引物
      1. oPEB262:5'-GGATATCCAAGAGTGTCAATCATCGCT
  22. 甘油(Fisher Scientific,目录号:BP229-4)
  23. Tris碱(Fisher Scientific,目录号:BP152-5)
  24. 冰醋酸(Fisher Scientific,目录号:A38-212)
  25. EDTA(Fisher Scientific,目录号:BP120-1)
  26. 氯化钠(NaCl)(Fisher Scientific,目录号:S271-10)
  27. Tryptone(BD,Bacto TM ,目录号:211699)
  28. 酵母提取物(BD,Bacto TM ,目录号:212720)
  29. 氨苄青霉素(Fisher Scientific,目录号:BP1760-25)
  30. 壮观霉素(MP Biomedicals,目录号:0215206725)
  31. 氯霉素(Fisher Scientific,目录号:BP904-100)
  32. 琼脂(Acros Organics,目录号:400400050)
  33. 琼脂糖(Fisher Scientific,目录号:BP1356-500)
  34. 溴化乙锭(Sigma-Aldrich,目录号:E8751-25G)
  35. 硫氰酸胍(Fisher Scientific,目录号:BP221-1)
  36. 胍盐酸盐(Fisher Scientific,目录号:BP178-1)
  37. 乙醇(Decon Labs,目录号:2701)
  38. 盐酸(HCl)(Fisher Scientific,目录号:A144-212)
  39. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266-100G)
  40. 100mM dNTP组(Thermo Fisher Scientific,Invitrogen TM,目录号:10297018)
  41. 二硫苏糖醇(DTT)(Fisher Scientific,目录号:BP172-25)
  42. β-烟酰胺腺嘌呤二核苷酸(NAD + )(Acros Organics,目录号:124530010)
  43. T 外切核酸酶(New England Biolabs,目录号:M0363S)
  44. Phusion DNA polymerase(New England Biolabs,目录号:M0530L)
  45. DNA连接酶(New England Biolabs,目录号:M0208L)
  46. 硫酸镁(MgSO 4)(Fisher Scientific,目录号:M65-500)
  47. 磷酸氢二钾(K 2)HPO 4(Fisher,目录号:BP363-1)
  48. 磷酸二氢钾(KH 2 PO 4)(Fisher Scientific,目录号:BP362-1)
  49. 柠檬酸三钠脱水(C 6 H 5 Na 3 O 3·2H 2·2H 2/O)(Fisher Scientific,目录号:BP327-1)
  50. 葡萄糖(Sigma-Aldrich,目录号:G8270-1KG)
  51. 色氨酸(Fisher Scientific,目录号:BP395-100)
  52. 苯丙氨酸(Fisher Scientific,目录号:BP391-100)
  53. 氢氧化钾(KOH)(Fisher Scientific,目录号:P250-500)
  54. 柠檬酸铁铵(Sigma-Aldrich,目录号:F5879)
  55. 天门冬氨酸钾(Sigma-Aldrich,目录号:A6558)
  56. PEG-8000(Dot Scientific,目录号:DSP48080-500)
  57. 50x TAE(见配方)
  58. 1x TAE(见食谱)
  59. LB媒体(见配方)
  60. LB琼脂平板(参见食谱)
  61. 100毫克/毫升氨苄青霉素(见配方)
  62. 100毫克/毫升壮观霉素原料(见食谱)
  63. 5毫克/毫升氯霉素原料(见食谱)
  64. 70%(v/v)乙醇(参见食谱)
  65. 1%琼脂糖凝胶(参见食谱)
  66. QG缓冲区(见配方)
  67. PB缓冲(见配方)
  68. Tris HCl,pH 7.5(参见食谱)
  69. PE缓冲区(见配方)
  70. 10x退火缓冲液(见配方)
  71. 5x等温反应缓冲液(Gibson,2011)(见配方)
  72. 2x吉布森大师组合(Gibson,2011)(见食谱)
  73. 1 M MgSO 4(参见食谱)
  74. LM培养基(LB培养基+ 3mM MgSO 4)(参见食谱)
  75. 10x PC缓冲(见配方)
  76. MD媒体(见配方)
  77. 0.1 N KOH(参见食谱)
  78. 100毫克/毫升色氨酸储备(见食谱)
  79. 100毫克/毫升苯丙氨酸(见配方)
  80. 2.2毫克/毫升柠檬酸铁铵储备(见食谱)
  81. 100毫克/毫升天门冬氨酸钾股票(见食谱)

设备

  1. 孵化器(Napco,型号:320)
  2. 干热块(Fisher Scientific,型号:Fisher Scientific TM Isotemp TM数字干浴/块加热器,目录号:11-718-2)
  3. 移液器(Eppendorf,目录号:022575442)
  4. 离心机(Eppendorf,型号:5424)
  5. 热循环仪(Eppendorf,型号:6325)
  6. 电泳装置(Bio-Rad Laboratories,型号:Mini-Sub Cell GT卧式电泳系统,目录号:1704406)
  7. 电泳用电源(Bio-Rad Laboratories,型号:PowerPac TM 基本电源,目录号:1645050)
  8. 滚筒(Eppendorf,New Brunswick,型号:TC-7)
  9. Milli-Q H 2 O分配器(Thermo Fisher Scientific,Thermo Scientific TM,型号:Barnstead TM GenPure TM Pro,目录号:50131950)
  10. 微波(Panasonic,目录号:NNS954WFR)
  11. 高压灭菌器

软件

  1. 在线工具Primer3(Koressaar和Remm,2007; Untergasser等人,2012)
  2. Oligocalc(Kibbe,2007)

程序

  1. 构建含有用于靶向B的间隔物的质粒。枯草芽孢杆菌基因组
    1. 准备用限制性内切核酸酶Bsa消化的pPB41(图1)
      组装50μl限制性消化反应:1x Cutsmart缓冲液(NEB),2μlBsa I-HF(20,000U/ml; NEB)和5μgpPB41。在培养箱中37℃孵育3至6小时。加入1μl小牛肠道磷酸酶(CIP; 10,000 U/ml; NEB),37℃孵育1 h。在1×TAE缓冲液中进行1%琼脂糖凝胶电泳(参见食谱),然后凝胶提取消化的质粒。
      注意:
      1. 通常使用质粒pPB41,但是我们还产生了一种衍生物pPB105,其中壮观霉素抗性盒被氯霉素抗性盒替代(参见图1)。程序的所有步骤对于pPB105是相同的,除了使用含有100μg/ml氨苄青霉素或20μg/ml氯霉素的LB琼脂平板(选择氯霉素,但是菌落生长较慢)来选择大肠杆菌克隆,并且枯草芽孢杆菌转化体使用含有5μg/ml氯霉素的LB琼脂平板选择。//em>
      2. 凝胶提取通过从两个50μlPCR反应中从琼脂糖凝胶中切出合适的DNA条带并置于微量离心管中进行。通过加入350μlQG缓冲液(参见食谱)并在65℃下在加热块中温育来溶解凝胶切片。凝胶切片完全溶解后,加入200μl异丙醇并通过移液混合。将整个凝胶提取物加载到硅胶柱上。通过离心(室温下12,000×g,1分钟)收集流出物并丢弃。将柱洗涤并首先用500μlPB缓冲液(参见食谱),然后用750μlPE缓冲液(参见食谱)丢弃。如上所述通过离心干燥柱。通过如上所述的离心将DNA从柱中以75μl超纯H 2 O洗脱。


        图1.用Bsa I-HF 消化pPB41或pPB105

    2. 准备磷酸化原间隔(图2)
      1. 原始间隔是通过搜索 B来设计的。枯草芽孢杆菌基因组,在编辑的位置由核苷酸序列5'-NGG-3'(其中N代表任何核苷酸)组成的原间隔相邻基序(PAM)位点图2A)。 PAM位点的30个核苷酸5'用于原间隔(图2A)。原始间隔区的寡核苷酸由Integrated DNA Technologies(IDT)排序,如图2B所示。两个寡核苷酸彼此是反向互补的,但突出端除外(图2B)。使用超纯蒸馏H 2 O(Invitrogen Life Technologies)制备浓度为100μM的寡核苷酸库存。
      2. 组装退火反应:1×退火缓冲液(参见食谱),10μM寡核苷酸1,10μM寡核苷酸2.在100℃下在加热块中孵育5分钟。将反应物转移到预烧至100℃的水烧杯中。将整个烧杯置于室温,并缓慢冷却至室温
      3. 准备磷酸化反应:1×T 4 DNA连接酶缓冲液(NEB),1μlT 4个多核苷酸激酶(PNK; 10,000U/ml; NEB),1μM退火的寡核苷酸以上。在37℃下在培养箱中孵育30分钟,然后在65℃下在热块中加热灭活T 4 N PNK 20分钟。将退火和磷酸化的寡核苷酸在-20℃储存,直至准备用于连接反应 注意:
        1. 尽管我们通常使用T4 PNK对原间隔物进行磷酸化,但也可以从IDT中对具有5'-磷酸修饰的寡核苷酸进行排序。
        2. 考虑到T4 PNK灭活在65℃下进行,原始间隔物可能变性。虽然我们没有需要改变方案的顺序(我们还没有尝试过),但T4 PNK对单链和双链DNA都是活跃的,因此,我们看不到逆转顺序的理论问题,磷酸化退火前。


          图2.原始间隔设计和准备。A.原始间隔(突出显示的蓝色和大写字母)和原间隔相邻基序(PAM位点;突出显示红色); B.与pPB41连接所需的具有适当突出端(小写字母)的寡核苷酸(Jiang等人,2013; Burby和Simmons,2017); C.退火和磷酸化反应以制备用于连接到pPB41中的dsDNA原间隔物。

    3. Ligate proto-spacer和pPB41并分离新质粒(图3) 组装20μl连接反应:1×T 4 DNA连接酶缓冲液(NEB),40至100ng pPB41(在前一步骤中用Bsa I-HF消化和CIP),25 nM原间隔(在前一步骤中退火并磷酸化),1μlT 4 DNA连接酶(400,000U/ml NEB)。让反应在室温下静置2-3小时。使用10μl连接反应来转化100μl化学感受态的TOP10大肠杆菌。可以使用含有100μg/ml氨苄青霉素或100μg/ml壮观霉素的LB琼脂平板(参见食谱)来选择克隆。通过使用oPEB253的Sanger测序验证克隆。
      注意:其中原间隔物连接到pPB41中的第一克隆步骤不需要某种大肠杆菌菌株。我们使用TOP10细胞作为我们的一般克隆株。


      图3.磷酸化原间隔蛋白和pPB41的连接以产生"靶向质粒"


  2. 编辑质粒的构建
    1. PCR扩增pPB41线性化(图4A)
      使用Q5 DNA聚合酶(NEB)和引物oPEB217和oPEB218通过PCR扩增pPB41。在1×TAE缓冲液(参见食谱)中进行1%琼脂糖凝胶电泳,然后进行凝胶提取。 PCR产物储存在-20°C直到使用。
      注意:
      1. 用于该步骤的模板可以是pPB41或在步骤B1中产生的含有间隔区的质粒。
      2. 用于线性化pPB41的PCR程序是:



        图4.编辑质粒的构建。 A.PPB41 PCR扩增子的示意图; B.CRISPR/Cas9 PCR扩增子的示意图; C.编辑模板PCR扩增子的上游和下游部分的示意图; D.Gibson使用A-C的PCR产物编辑质粒的大会。

    2. PCR使用在步骤B1(图4B)中生成的质粒
      扩增CRISPR/Cas9 使用Q5 DNA聚合酶(NEB)和引物oPEB232和oPEB234通过PCR扩增步骤B1中产生的质粒(本实施例中为pPB43)。在1×TAE缓冲液(参见食谱)中进行1%琼脂糖凝胶电泳,然后进行凝胶提取。 PCR产物储存在-20°C直到使用。
      注意:用于扩增CRISPR/Cas9的PCR程序与上述步骤B1中使用的相同。
    3. PCR扩增编辑模板(图4C)
      使用Q5 DNA聚合酶(NEB)通过PCR分别扩增编辑模板的上游和下游部分(本实施例中使用的引物分别用于上游和下游部分是oPEB237/oPEB238和oPEB239/oPEB240)。引物是使用在线工具Primer3(Koressaar和Remm,2007; Untergasser等人,2012)和Oligocalc(Kibbe,2007)设计的。编辑模板特异性引物与pPB41突变(上游正向引物起始5'-GCATGCTGAATTCGTAATGAGGTTC,接着是编辑模板特异性序列;下游反向引物开始5'-GCATAACCAAGCCTATGCCTACAGC,随后编辑模板特异性序列)将允许将编辑模板并入编辑质粒。编辑模板设计为大约2千碱基,其上游和下游部分由每个约1kb组成。编辑模板设计的关键方面是确保引导的Cas9在引入所需的突变后不能再靶向基因组。对于缺失,可以去除间隔物和PAM位点的整个序列。对于点突变,它更复杂。江泽民等人。 (2013)发现,突变PAM位点或间隔区的前几个碱基是最优的(Jiang等人,2013)。编辑模板中心的重叠应该在模板的每一部分上有25个碱基(结果是总共有50bp重叠)。 PCR后,在1×TAE缓冲液(参见食谱)中进行1%琼脂糖凝胶电泳,然后进行凝胶提取。 PCR产物储存在-20°C直到使用。
      注意:
      1. 使用在线工具Primer3设计编辑模板引物(Koressaar和Remm,2007; Untergasser等,2012)。从编辑模板的中心选择大约250 bp的bp,并使用默认参数生成引物列表,但以下情况除外:1)引物尺寸为最小值:22,选择:24,最大值:28; 2)底漆GC%min:35; 3)最大自我互补性:6.0;和4)最大3'自身互补性:2.0。然后选择引物并使用第二个在线工具Oligocalc(Kibbe,2007)进行交叉检验。目的是确保潜在的发夹形成,3'互补性和自退火均不能使用该程序检测到。
      2. 在图中示例中使用的编辑模板为3 kb(每个编辑模板的上游和下游部分约为1.5 kb),但我们通常使用2 kb。我们还提供使用约2 kb的编辑模板制作的四种不同的基因缺失。
      3. 用于放大编辑模板的每个部分的PCR程序是:


    4. 使用Gibson Assembly(Gibson,2011)构建编辑质粒(图4D)
      组装10μl吉布森装配反应:1次吉布森主混合物(参见食谱),40-100ng来自上述的线性化pPB41,40-100ng含上述间隔物的CRISPR/Cas9 PCR产物和20-40ng编辑模板。在50℃下在热循环仪中孵育反应90分钟。使用5μlGibson装配反应转化80μl化学有效的E。大肠杆菌菌株MC1061。可以像步骤A3那样选择克隆。使用引物oPEB253,oPEB227和编辑模板特异性引物对克隆进行测序。
      注意:
      1. 使用recA +的克隆菌株是绝对关键的,因为天然感受态枯草芽孢杆菌的转化使用多聚体的质粒更为有效。我们用于质粒繁殖的大肠杆菌菌株是MC1061。我们在MC1061获得某些编辑质粒时也遇到困难,原因不明确。在这些情况下,我们将吉布森装配反应扩大到总体积20μl,我们使用20μl转化枯草芽孢杆菌,如方法C.该方法的效率较低,一些壮观霉素敏感的分离株保留野生型,类型基因型,因此通常至少筛选12个分离株。
      2. 使用oPEB227(pPB41的5'末端的序列)对编辑模板进行测序。在编辑模板中设计其他引物,以验证剩余序列。

  3. 编辑B的基因组。 subtilis
    1. 转换B。枯草芽孢杆菌与前一步骤获得的编辑质粒
      1. 准备自然胜任的枯草芽孢杆菌培养物(Burby and Simmons,2017)(图5A) 使用木棒将培养板在30℃或37℃孵育的LB琼脂平板上的冷冻的20%甘油基质上分离出菌株过夜。
      2. 早晨,在14ml一次性圆底培养管中接种2ml LM培养基(参见食谱)。在37℃下在培养箱中的滚筒上培养培养物,直到OD 600在0.8和1.5之间(约2小时生长)。
      3. 将20μl的LM培养物转移到0.5ml预热的MD培养基中(参见Recipes)。在滚筒上37℃下培养MD培养4至6小时。
      4. 加入编辑质粒DNA(约200至600ng),并在37℃下在滚筒上孵育60至90分钟。通过在含有100μg/ml壮观霉素的LB琼脂平板上电镀200μl来选择转化体,并在30℃温育过夜。
        注意:
        1. 我们已经在MD媒体上找到了5小时,工作得很好,并且最常用,但是在一小时之内或之后添加DNA也是成功的。
        2. 在DNA加入后90分钟电镀非常好,但60分钟的电镀也将产生转化体
    2. 通过菌落纯化转化体(图5B)
      建立几个分离株(6-12)
      1. 殖民地纯化是指为单个殖民地连续三至四次重建隔离的殖民地。
      2. 将来自前述步骤的单个菌落在含有100μg/ml壮观霉素的单个菌落的LB琼脂平板上重新条纹,并在30℃下在培养箱中孵育过夜。
    3. 固定质粒分离株(图5C)
      将来自前述步骤的单个菌落重新置于单个菌落的LB琼脂平板上,并在42-45℃温育过夜。
      注意:我们的孵化器设置为45°C,但成功使用了42°C。


      图5. B中基因组编辑程序的概要。 subtilis (摘自图S2 [Burby and Simmons,2017])。 A.第一步是转换B。枯草芽孢杆菌与编辑质粒。插入显示野生型mutS2 轨迹。 B.使用菌落纯化建立分离株后,分离物固化编辑质粒。插图描述使用质粒上的编辑模板进行同源性定向修复。 C.最后一步是验证编辑质粒和基因型分离株的损失,以验证突变是否成功引入。插入显示编辑的mutS2 轨迹。

    4. 筛选分离物用于质粒损失
      将来自前面步骤的单个菌落重新划分到含有100μg/ml壮观霉素的LB琼脂平板上,然后在LB琼脂平板上重新分成单个菌落。将板在45℃下在培养箱中孵育过夜。如果分离物丢失了质粒,则在具有壮观霉素的LB琼脂平板上不会生长
    5. 预期突变筛选(图6)
      在含有壮观霉素的平板上不能生长的分离物进一步筛选出所需的突变的存在。在点突变的情况下,可以将引物设计为在点突变处退火(图6A),或者可以使用编辑模板外的引物扩增遗传基因座,如删除(见下文),随后是Sanger测序以验证点突变是否存在,并且不引入其他突变。在基因缺失的情况下,使用在基因组DNA内退火重组模板之外的PCR引物进行PCR扩增遗传基因座(图6B)。相对于来自野生型基因座的产物,PCR产物的迁移将更快

      图6.基于基因组编辑的基于PCR的验证。 A.显示了B组中使用的PCR引物的位置的示意图; B.使用扩增PCR产物的溴化乙锭染色琼脂糖凝胶,使用特异于点突变的引物(H743A,我们改变了CAC→GCT)和B。 (左图),并使用特异于编辑质粒的引物(右图)。筛选的12个分离株全部被发现具有预测点突变,其中2个保守对壮观霉素(星号)的抵抗。 PY79泳道中的缓慢迁移带对应于野生型背景中的非特异性PCR扩增。编码质粒在壮观霉素抗性分离物中可检测到,但在其余十个中不能检测到。 C.琼脂糖凝胶用溴化乙锭染色PCR反应,使用特异性缺失四个B的引物。枯草芽孢杆菌基因。引物设计在编辑模板之外,因此特异于染色体DNA。对于所有四个缺失,发现所有分离株都是壮观霉素敏感的,每个缺失的三个分离株的PCR基因分型发现所有测试的分离株具有预期的缺失。注意,对于ponA的对照反应,PCR产物是微弱的,因为扩增子约为5kb,PCR的延伸时间为3分钟,这接近Q5 DNA完全延伸的极限聚合酶其他三个对照扩增子不大于3.4kb
    6. 保存冻结库存的新菌株
      将确认具有预期突变的至少两个分离株的分离的LB培养物生长至约1至1.5的OD 600。通过在冷冻小瓶中加入甘油至最终浓度为20%来节省冷冻原料。将一个冷冻小瓶盖加入盖子并贴上标签。将冷冻小瓶直接放入-80℃的冷冻箱中

数据分析

有两个步骤需要数据分析来构建突变菌株。第一个是使用抗生素敏感性测定编辑质粒的损失,第二个是使用PCR基因分型来测定用于预期突变的遗传基因座。为了确定分离物是否已被修饰的编辑质粒,将6至12个分离株在含有和不含有100μg/ml壮观霉素的LB琼脂平板上划线并生长过夜(参见方案的步骤C4)。在含有壮观霉素的LB琼脂平板上不生长的分离物已经被修复了编辑质粒。通过PCR进一步对已经固化的质粒的分离物进行基因分型。 PCR基因分型的数据分析将取决于正在引入的编辑类型。对于缺失,遗传基因座的PCR扩增将导致迁移的产物比野生型基因座的迁移快,导致PCR产物在琼脂糖凝胶上显现较低。对于插入,PCR产物将迁移比琼脂糖凝胶上的野生型产物慢。对于点突变,PCR产物的大小将不会改变。因此,必须使用突变特异性引物或Sanger测序来确认正确的基因型。对于突变特异性引物(参见图6B),PCR产物将存在或不存在。如果存在PCR产物,则表明存在突变。如果使用Sanger测序,将检测突变的序列,而不是野生型序列。

食谱

  1. 50倍TAE
    242克Tris碱
    57.1毫升冰醋酸
    100ml EDTA,pH 8.0
    将Milli-Q H 2 O加入1L
    在室温下存放
  2. 1x TAE
    20 ml 50x TAE
    980ml Milli-Q H 2 O O
    在室温下存放
  3. LB媒体
    10克NaCl
    10克胰蛋白胨
    5克酵母提取物
    将Milli-Q H 2 O添加到总体积为1L的
    高压釜灭菌
  4. LB琼脂板
    1 L LB媒体
    18克琼脂
    高压釜灭菌
    允许冷却至约60℃;如果需要抗生素,那么现在就加入它们 加入壮观霉素至终浓度为100μg/ml 加氨苄青霉素至终浓度为100μg/ml 加入氯霉素至终浓度为20μg/ml。大肠杆菌,B组为5μg/ml。 subtilis
    倒盘
  5. 100毫克/毫升氨苄青霉素库存
    1克氨苄青霉素
    加入Milli-Q水至10 ml 过滤灭菌
    等分并储存于-20°C
  6. 100毫克/毫升壮观霉素库存
    1g壮观霉素
    加入Milli-Q水至10 ml 过滤灭菌
    等分并储存于-20°C
  7. 5毫克/毫升氯霉素库存
    50毫克氯霉素
    加入70%(v/v)乙醇至10 ml
    过滤灭菌
    储存于-20°C
  8. 70%(v/v)乙醇 70毫升乙醇
    30毫升Milli-Q H 2 O O
    在室温下存放
  9. 1%琼脂糖凝胶 1克琼脂糖 100 ml 1x TAE
    微波溶解琼脂糖的热溶液
    加入溴化乙锭至终浓度为0.2μg/ml 倾斜凝胶
  10. QG缓冲区
    5.5 M硫氰酸胍
    20mM Tris HCl,pH6.6
  11. PB缓冲区
    5 M盐酸胍
    30%(v/v)异丙醇
  12. Tris HCl,pH 7.5
    通过将Tris碱溶解在Milli-Q H 2 O中并使用HCl将pH调节至7.5来制备Tris HCl
  13. PE缓冲区
    10mM Tris HCl,pH7.5
    80%(v/v)乙醇
  14. 10x退火缓冲液
    100mM Tris HCl,pH7.5
    1 M NaCl
    1mM EDTA,pH 8.0
    超纯H 2 O O
    过滤灭菌
  15. 5x等温反应缓冲液(Gibson,2011)
    0.5M Tris HCl,pH7.5
    25%(w/v)PEG-8000
    50mM MgCl 2
    50 mM DTT
    每个dNTPs 1 mM 5mM NAD +
    储存于-20°C
  16. 2x吉布森大师组合(Gibson,2011)
    320μl5x等温反应缓冲液
    0.64μlT 5外切核酸酶(10,000U/ml)
    20μlPhusion DNA聚合酶(2,000 U/ml)
    160μlTaq DNA连接酶(40,000U/ml)
    储存于-20°C
  17. 1 M MgSO 4
    120.37g MgSO 4(F.W.120.37g)
    将Milli-Q H 2 O加入1L
    高压釜灭菌
    在室温下存放
  18. LM培养基(LB培养基+ 3mM MgSO 4)
    2 ml LB培养基 6μl1 M MgSO 4
  19. 10x PC缓冲区
    10.7g K 2 HPO 4
    6g KH 2 PO 4
    1.18g柠檬酸三钠脱水(C 6 H 5 Na 3 O 3·2H 2·2H 2 O) sub> O)
    将Milli-Q H 2 O加入到100 ml
    中 过滤灭菌
    在室温下存放
  20. MD媒体
    1x PC缓冲区
    2%(w/v)葡萄糖
    50μg/ml色氨酸
    50μg/ml苯丙氨酸 11μg/ml柠檬酸铁铵// 2.5mg/ml天冬氨酸钾
    3mM MgSO 4
    过滤灭菌
    商店防止光线在4°C
  21. 0.1 N KOH
    56.1mg KOH(F.W.56.11g)
    将Milli-Q H 2 O 2加入到10ml
    中 为每次使用准备新鲜
  22. 100毫克/毫升色氨酸库存
    1克色氨酸
    加入0.1N KOH至10ml(准备新鲜)
    过滤灭菌
    商店防止光线在4°C
  23. 100毫克/毫升苯丙氨酸股票
    1g苯丙氨酸
    加入0.1N KOH至10ml(准备新鲜)
    过滤灭菌
    商店防止光线在4°C
  24. 2.2毫克/毫升柠檬酸铁铵矿物质
    110毫克柠檬酸铁铵
    将Milli-Q H 2 O加入到50ml的
    中 过滤灭菌
    商店防止光线在4°C
  25. 100毫克/毫升天门冬氨酸钾股票
    5g天冬氨酸钾
    将Milli-Q H 2 O加入到50ml的
    中 过滤灭菌
    储存于4°C

致谢

这项工作得到NIH授权R01 GM107312对L.A.S.的支持。和国家科学基金会#DGE 1256260至P.E.B.的博士后研究金。

参考文献

  1. Arnaud,M.,Chastanet,A.and Debarbouille,M。(2004)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/15528558" target ="_ blank">在天然不可转化,低GC含量,革兰氏阳性菌中进行有效等位基因置换的新载体。"应用环境微生物"70(11):6887-6891。
  2. Burby,PE和Simmons,LA(2017)。 MutS2促进枯草芽孢杆菌中的同源重组。 199菌株199(2)。
  3. Fabret,C.,Ehrlich,SD和Noirot,P。(2002)。用于枯草芽孢杆菌的基因组规模方法的新的突变传递系统。 Mol Microbiol 46(1):25-36。 />
  4. Gibson,DG(2011)。重叠DNA的酶装配片段。 方法Enzymol 498:349-361。
  5. Guerout-Fleury,AM,Frandsen,N.和Stragier,P。(1996)。用于在枯草芽孢杆菌中异位整合的质粒。基因 180(1-2):57-61。
  6. Jiang,W.,Bikard,D.,Cox,D.,Zhang,F.and Marraffini,LA(2013)。  使用CRISPR-Cas系统的RNA编码细菌基因组。生物技术31(3):233-239。
  7. Kibbe,WA(2007)。 OligoCalc:一种在线寡核苷酸属性计算器。 35核心酸Res Res。35(Web服务器问题):W43-46。
  8. Koressaar,T.和Remm,M。(2007)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/17379693"target ="_ blank" >引物设计程序的增强和修改Primer3。生物信息学 23(10):1289-1291。
  9. Patrick,JE和Kearns,DB(2008)。  MinJ (YvjD)是枯草芽孢杆菌中细胞分裂的拓扑决定因子。 Mol Microbiol 70(5):1166-1179。
  10. Sternberg,SH和Doudna,JA(2015)。  扩展 生物学家与CRISPR-Cas9的工具包。分子细胞 58(4):568-574。
  11. Untergasser,A.,Cutcutache,I.,Koressaar,T.,Ye,J.,Faircloth,BC,Remm,M.and Rozen,SG(2012)。< a class ="ke-insertfile"href = http://www.ncbi.nlm.nih.gov/pubmed/22730293"target ="_ blank"> Primer3 - 新功能和界面。 核酸研究 40(15) :e115。
  12. Vagner,V.,Dervyn,E.和Ehrlich,SD(1998)。< a class ="ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pubmed/?term= A + vector + for +系统+基因+灭活+在+芽孢杆菌+枯草芽孢杆菌中。 target ="_ blank">用于枯草芽孢杆菌中的系统基因失活的载体 微生物学 144:3097-3104。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Burby, P. E. and Simmons, L. A. (2017). CRISPR/Cas9 Editing of the Bacillus subtilis Genome. Bio-protocol 7(8): e2272. DOI: 10.21769/BioProtoc.2272.
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