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Robust Generation of Knock-in Cell Lines Using CRISPR-Cas9 and rAAV-assisted Repair Template Delivery

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Scientific Reports
Jun 2016



The programmable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease 9 (Cas9) technology revolutionized genome editing by providing an efficient way to cut the genome at a desired location (Ledford, 2015). In mammalian cells, DNA lesions trigger the error-prone non-homologous end joining (NHEJ) DNA repair mechanism. However, in presence of a DNA repair template, Homology-Directed Repair (HDR) can occur leading to precise repair of the lesion site. This last process can be exploited to enable precise knock-in changes by introducing the desired genomic alteration on the repair template. In this protocol we describe the delivery of long repair templates (> 200 nucleotides) using recombinant Adeno Associated Virus (rAAV) for CRISPR-Cas9-based knock-in of a C-terminal tag sequence in a human cell line.

Keywords: CRISPR-Cas9 (CRISPR-Cas9), Recombinant adeno-associated virus (rAAV) (重组腺相关病毒(rAAV)), Genome engineering (基因组工程), Epitope tagging (表位附加)


Despite numerous reports on knock-out model systems generated by CRISPR-Cas9, knock-in reports are still lagging behind. Because of the many applications, generating knock-in cell lines remains an obvious goal of genome editing. The introduction of knock-in alterations generally relies on the presence of a repair template DNA and activation of the HDR repair mechanism after a site-specific double strand (ds)DNA break is introduced in the genome close to the site of alteration. Different templates can be delivered to the repair machinery ranging from a classical linearized vector containing extensive homology regions and an optional selection cassette, to single strand (ss)DNA oligonucleotides of about 200 nucleotides (Chen et al., 2011). Although ssDNA oligonucleotides are a popular tool, they can only be used to introduce small alterations such as mutations or epitope tags because of DNA synthesis limitations. In addition, the lack of a selection cassette requires robust screening strategies to identify correct clones as no selective pressure is applied on the HDR process. Successful use of integration-deficient rAAV for homologous recombination was already shown before the availability of tailored nucleases (Khan et al., 2011). Both its efficient delivery and ssDNA genome make rAAV a powerful tool to deliver donor repair templates for homologous recombination. Moreover, the secondary structures at the end of the ssDNA molecule block exonuclease activity and stabilize the donor DNA. Even without the use of specific nucleases, knock-in efficiencies of up to 0.7% were obtained in fibroblasts cells (Russell and Hirata, 1998), which was further increased by introducing selection cassettes.

By combining CRISPR-Cas9 with rAAV-mediated repair template delivery, knock-in cell lines can be generated in a robust manner with efficiencies well beyond 50% when selection cassettes are used. This protocol describes the complete procedure for epitope tagging of a gene of choice in the HCT116 colon carcinoma cell line using CRISPR-Cas9 and rAAV. A timeline for the complete experimental procedure is shown in Figure 1.

Figure 1. Timeline for the generation of a knock-in cell line using CRISPR-Cas9 and rAAV-assisted repair template delivery. Dotted timespans indicate periods of incubation or expansion, requiring limited to no hands-on time.

Materials and Reagents

  1. T25 cell culture treated flasks with filter caps (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 136196 )
  2. Eppendorf Safe-Lock microcentrifuge tubes (Eppendorf, catalog number: 022363204 )
  3. 5Prime Phase Lock gel tubes heavy 2 ml (Quantabio, catalog number: 2302830 )
  4. T75 cell culture treated flasks with filter caps (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 178905 )
  5. Greiner 50 ml centrifuge tubes (Greiner Bio One International, catalog number: 227261 )
  6. Greiner cell scrapers (Greiner Bio One International, catalog number: 541081 )
  7. 24-well cell culture-treated multidish (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 142475 )
  8. 96-well microplates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 156545 )
  9. 96-well PCR plate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: AB0700 )
  10. 6-well cell culture-treated multidish (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 140675 )
  11. Aluminium foil tape (3M, catalog number: 425DWB )
  12. Illustra Microspin S-400 HR columns (GE Healthcare, catalog number: 27-5140-01 )
  13. Immobilon-FL PVDF transfer membrane (EMD Millipore, catalog number: IPFL00010 )
  14. Whatman 3 MM chr cellulose blotting sheets (GE Healthcare, catalog number: 3030-917 )
  15. 0.22 µm filter (EMD Millipore, catalog number: SLGV033RS )
  16. AAV-293 cell line (Agilent Technologies, catalog number: 240073 )
  17. HCT 116 cell line (ATCC, catalog number: CCL-247 )
  18. Cas9 D10A nickase mutant (nCas9) (Addgene, catalog numbers: 48140 and 62987 )
  19. pAav-MCS-PQS1-3xFLAG or pAav-MCS-PQS2-3xHA (Addgene, catalog numbers: 84883 and 84917 respectively)
  20. pDG rAAV packaging plasmid (PlasmidFactory, catalog number: PF421 )
  21. Wild-type Cas9 expression vectors (Cas9) (Addgene, catalog numbers: 48138 and 62988 )
  22. Competent bacterial cells of choice (e.g., TOP10 or DH5α)
  23. Surveyor Mutation Detection Kit (Integrated DNA Technologies, catalog number: 706025 )
  24. UltraPure phenol:chloroform:isoamyl alcohol (25:24:1, v/v) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15593031 )
  25. Sodium chloride (NaCl) molecular biology grade (EMD Millipore, catalog number: 567441 )
  26. 2-propanol (Sigma-Aldrich, catalog number: 278475 )
  27. Ethanol (EMD Millipore, catalog number: 100983 )
  28. AccuPrime Pfx DNA polymerase (Thermo Fisher Scientific, InvitrogenTM, catalog number: 12344-024 )
  29. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: 449709 )
  30. DMEM, high glucose GlutaMAX medium (Thermo Fisher Scientific, GibcoTM, catalog number: 31966047 )
  31. Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  32. Benzonase nuclease (Sigma-Aldrich, catalog number: E1014 )
  33. AAV-purification Vira Kit 3-use (Virapur, catalog number: 003063 )
  34. AAV helper-free system (Agilent Technologies, catalog number: 240071 )
  35. McCoy’s 5A medium, modified (Thermo Fisher Scientific, GibcoTM, catalog number: 16600082 )
  36. Opti-MEM I reduced serum medium, GlutaMAX supplement (Thermo Fisher Scientific, GibcoTM, catalog number: 51985026 )
  37. Fugene HD transfection reagent (Promega, catalog number: E2311 )
  38. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10500064 )
  39. Trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25300096 )
  40. Puromycin (Sigma-Aldrich, catalog number: P8833 )
  41. Crystal violet solution (Sigma-Aldrich, catalog number: HT90132 )
  42. Geneticin/G418 (Thermo Fisher Scientific, GibcoTM, catalog number: 11811031 )
  43. GoTaq G2 Hot Start polymerase (Promega, catalog number: M7405 )
  44. dNTP 100 mM PCR grade (Agilent Technologies, catalog number: 200415 )
  45. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  46. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650 )
  47. TAT-Cre recombinase (Excellgen, catalog number: EG-1001 )
  48. NucleoSpin Gel and PCR Clean-up Kit (Machery-Nagel, catalog number: 740609 )
  49. Random Primer DNA Labeling Kit (Takara Bio, catalog number: 6045 )
  50. [α-32P] dCTP, 50 µCi (PerkinElmer, catalog number: BLU013H250UC )
  51. Exo-free Klenow enzyme (New England Biolabs, catalog number: M0212S )
  52. SuRE/Cut buffer H (Roche Diagnostics, catalog number: 11417991001 )
  53. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A4503 )
  54. EcoRI 40 U/µl (Roche Diagnostics, catalog number: 10200310001 )
  55. Nytran SuPerCharge TurboBlotter Kit (Sigma-Aldrich, catalog number: Z613924 )
    Note: This product has been discontinued.
  56. PerfectHyb plus hybridization buffer (Sigma-Aldrich, catalog number: H7033 )
  57. 4-12% criterion XT Bis-Tris protein gel 18 well, 30 µl (Bio-Rad Laboratories, catalog number: 3450124 )
  58. Mouse monoclonal anti-FLAG M2 antibody (Sigma-Aldrich, catalog numbers: F3165 )
    Or rat monoclonal anti-HA high affinity (Roche Diagnostics, catalog number: 11867423001 )
  59. Zero Blunt PCR Cloning Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: K270020 )
  60. NucleoSpin Plasmid EasyPure (Machery-Nagel, catalog number: 740727 )
  61. Tris ultra-pure grade (MP Biomedicals, catalog number: 02103133 )
  62. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E5134 )
  63. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: 436143 )
  64. Proteinase K from Tritirachium album > 800 U/ml (Sigma-Aldrich, catalog number: P4850 )
  65. HEPES (Sigma-Aldrich, catalog number: H4034 )
  66. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: 255793 )
  67. Direct lysis reagent (cell) (VIAGEN BIOTECH, catalog number: 301-C )
  68. CHAPS hydrate (Sigma-Aldrich, catalog number: C5070 )
  69. cOmplete protease inhibitor (Roche Diagnostics, catalog number: 11697498001 )
  70. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 30721 )
  71. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 221465 )
  72. Sodium citrate tribasic dehydrate [HOC(COONa)(CH2COONa)2·2H2O] (Sigma-Aldrich, catalog number: C8532 )
  73. SmartLadder (Eurogentec, catalog number: MW-1700-10 )
  74. Phase Lock lysis buffer (see Recipes)
  75. TE-buffer (see Recipes)
  76. 2x HEBS (HEPES-buffered saline) (see Recipes)
  77. rAAV cell lysis buffer (see Recipes)
  78. Direct PCR lysis buffer (see Recipes)
  79. CHAPS lysis buffer (see Recipes)
  80. Depurination buffer (see Recipes)
  81. Denaturation buffer (see Recipes)
  82. Neutralization buffer (see Recipes)
  83. 20x SSC (see Recipes)
  84. Wash buffer 1 (see Recipes)
  85. Wash buffer 2 (see Recipes)
  86. Wash buffer 3 (see Recipes)


  1. BSL2 cell culture facilities
  2. Thermoshaker
  3. Vortex (e.g., IKA, model: MS2 minishaker )
  4. Inverted microscope for cell culture use (e.g., Carl Zeiss, model: Axiovert 25 )
  5. Non-circulating water bath
  6. 250 ml sterile storage bottle (Corning, catalog number: 430281 )
  7. Thermal cycler instrument (e.g., Bio-Rad Laboratories, model: T100TM Thermal Cycler , catalog number: 1861096)
  8. Western blotting equipment, e.g.,
    Criterion vertical electrophoresis cell (Bio-Rad Laboratories, catalog number: 1656001 )
    Criterion blotter with plate electrodes (Bio-Rad Laboratories, catalog number: 1704070 )
  9. GS gene linker UV chamber (Bio-Rad Laboratories, model: GS Gene LinkerTM UV Chamber )
  10. Phosphor imager device (e.g., GE Healthcare, model: Typhoon 9200 )
  11. Required facilities and procedures (e.g., waste disposal procedures) should be in place for using radioactively labeled nucleotides ([α-32P] dCTP)
  12. Tabletop centrifuge (Eppendorf, model: 5430 R )


  1. Design of guide RNA (gRNA) constructs for the target gene of interest
    1. Insert the genomic sequence coding for the C-terminal region of the protein of interest in a guide design tool of choice (e.g., crispr.mit.edu). In most cases, an input sequence with a total length of 250 nucleotides surrounding the stop codon harbors enough PAM motifs for the design tool to generate multiple guide sequences.
    2. Select guides in close proximity of the C-terminus. Bear in mind potential off-target effects when defining the guides. Most design tools provide this information directly by ranking potential guides based on off-target hit-scores in the target genome.
    3. Clone guides in a Cas9 expression vector of choice. Use of the Cas9 D10A nickase mutant (nCas9, Addgene plasmids #48140 and #62987) is advised to minimize off-target cleavage events. Alternatively, wild-type Cas9 expression vectors are also available on Addgene (plasmids #48138 and #62988). Detailed cloning procedures for the constructs are described by Ran et al. (2013). When using the Cas9 D10A nickase (nCas9) maintain a minimal offset between the guide pairs to ensure optimal efficiency.
      Note: It is recommended to assess the cleavage efficiency of multiple gRNAs in a separate experiment. This can be done by transfecting the constructs and performing a mismatch cleavage assay (e.g., Surveyor mismatch cleavage assay) on the selected cell population (Qiu et al., 2004). We usually test 2-3 different gRNAs for each genomic region of interest. In case of using nCas9 it is recommended to test 2-3 pairs of gRNAs.

  2. Generation of the rAAV targeting construct by PCR amplification of homology regions
    A backbone plasmid for C-terminal tagging with a combinatorial tag consisting of the PQS1 peptide (Vandemoortele et al., 2004) and a 3xFlag tag has been deposited in Addgene (plasmid #84883). An alternative version of this targeting construct containing the PQS2 peptide and a 3xHA-tag is also available on Addgene (plasmid #84917). 5’ and 3’ homology regions (HRs) can be inserted in multiple cloning sites present in these plasmids via standard cloning procedures. 
    1. Prepare genomic DNA (gDNA) using a phenol extraction and ethanol precipitation protocol
      1. Detach and pellet 2.5 x 106 cells corresponding to a subconfluent T25 flask in an Eppendorf tube by centrifugation at 500 x g for 5 min at room temperature (RT).
      2. Add 500 µl gDNA Phase Lock lysis buffer (see Recipes) to the pellet and incubate overnight at 37 °C.
      3. Prior to adding the lysate, spin down the Phase Lock tubes by short centrifugation. Shake the lysate samples well before transferring to the Phase Lock tube.
      4. Add 500 µl phenol:chloroform:isoamyl alcohol (25:24:1, v/v) and shake vigorously for 1 min.
      5. Centrifuge Phase Lock tube for 3 min at 16,000 x g at RT. Transfer aqueous phase into a fresh Eppendorf tube and add 50 µl 3 M NaCl and 500 µl 2-propanol. The genomic DNA can be observed after inverting the tubes several times.
      6. Centrifuge for 2 min at 16,000 x g at RT. Discard supernatant and add 1 ml 70% ethanol to the DNA pellet. Invert several times and incubate for 5 min at RT. Centrifuge for 2 min at 16,000 x g at RT.
      7. Discard supernatant. Air dry the pellet at 52 °C for 5 min prior to re-suspending the pellet in 100 µl 10 mM TE-buffer pH 7.5. Allow pellet to dissolve for 1 h by incubating at 65 °C in a thermoshaker. Measure genomic DNA concentration.
    2. Design primers to amplify the HRs with appropriate overhanging restriction sites for insertion in the backbone plasmid. 
      Note: It is recommended to use HRs of at least 650 bp. Shorter HRs are reported to substantially reduce homologous recombination potential (Vasileva et al., 2006). Targeting constructs exceeding 4.7 kb in total length between inverted terminal repeat (ITR) sequences will hamper packaging efficiency of rAAV (Wu et al., 2010). 
    3. Prepare PCR mix
      10x AccuPrime Pfx reaction mix
      5 µl
      Forward primer (10 µM)
      1.5 µl
      Reverse primer (10 µM)
      1.5 µl
      AccuPrime Pfx DNA polymerase
      0.5 µl
      gDNA (100 ng/µl)
      1 µl
      40.5 µl

    4. Amplify the HRs using a touchdown PCR program. Elongation times in italics are adjusted according to the length of the HR (1 min/kb).

    5. Verify and purify the PCR product by agarose gel electrophoresis before proceeding with cloning steps.
      Note: As the backbone constructs mentioned above contain multiple cloning sites (MCS) flanking the neomycin selection cassette, cloning of the final targeting constructs can be performed using different restriction enzyme sites. Alternative cloning methods (e.g., Gibson assembly, In-Fusion) are also compatible with these vectors.

  3. rAAV production and purification
    1. AAV-293 transfection
      1. Seed 10 T75 flasks with 4 x 106 AAV-293 cells 24 h before transfection.
      2. Aspirate and add fresh growth medium at least 30 min prior to transfection.
      3. Prepare DNA transfection mix for every T75.
        pAAV targeting construct (1 µg/µl)
        15 µl
        pDG (1 µg/µl)
        30 µl
        2.5 M CaCl2
        75 µl
        630 µl

        Note: The pDG plasmid serves as a helper plasmid for rAAV packaging. pDG contains all the required AAV and adenoviral functions to ensure amplification and packaging of (r)AAV vectors.
      4. Add DNA transfection mix dropwise to 750 µl 2x HEBS (see Recipes) while using a vortex to ensure thorough mixing. Incubate mixture for 10 min at RT.
      5. Transfer transfection mix to cells in a dropwise fashion.
        Note: Transfected AAV-293 cells and purified rAAV virus should be handled in a BSL-2 facility from this point onwards.
      6. Replace growth medium with 12 ml of fresh DMEM growth medium 24 h after transfection and incubate for an additional 48 h.
        Note: Before replacing the growth medium a fine precipitate should be found between the transfected cells by visual inspection using a light microscope. The calcium-phosphate-DNA precipitate aids binding of the DNA to the cell surface before entering the cells by endocytosis. Suboptimal transfection buffers (e.g., 2x HEBS) or DNA qualities can affect precipitate formation leading to low transfection efficiencies. Growth impairment may occur due to transfection.
    2. Harvest and purification of rAAV particles
      1. Collect growth medium with detached cells in 50 ml tubes.
        Note: Cells may detach during rAAV production. The total amount of growth medium of all T75 Falcons used for production is equally divided over 4 50 ml tubes for convenience in further steps of the purification procedure.
      2. Collect cells in 1.5 ml PBS/T75 flask using a cell scraper and add cell suspensions to the 50 ml tubes. Centrifuge the cells for 3 min at 500 x g and transfer the supernatants to new 50 ml tubes.
      3. Add 2.5 ml PBS to the obtained cell pellets, re-suspend the cell pellets and combine all cell suspensions in one new tube. Centrifuge for 3 min at 500 x g and re-suspend the cell pellet in 3 ml rAAV lysis buffer.
      4. Subject pellets to 3 freeze-thaw cycles with brief vortexing between cycles. Transfer the lysate to the supernatant of step C2b.
        Note: Unpurified virus can be stored at -80 °C. Tubes should be thawed prior to purification using a 37 °C water bath while swirling the tube to maintain low temperature.
      5. Centrifuge the tubes at 900 x g for 30 min at 4 °C. Collect supernatant in a 250 ml sterile bottle and add Benzonase nuclease (final conc. 50 U/ml).
      6. Purify rAAV particles using the VIRAPUR AAV Purification Kit according to the manufacturer’s protocol. Purified virus should be stored in aliquots at -80 °C until use.

  4. Transfection and infection of HCT116 cells
    1. Seed 2.7 x 105 HCT116 cells/well in a 24-well plate containing 1 ml McCoys growth medium.
    2. Prepare following mixes in separate tubes.
      FugeneHD mix

      DNA mix

      11.7 µl
      11.7 µl
      Fugene HD transfection reagent
      1.4 µl
      Cas9 + gRNA plasmid DNA
      0.5 µg

      Gently add the Fugene HD mix to the DNA mix, tap the tube gently and incubate the mixture for 10 min at RT. During this incubation step, replace the medium on the HCT116 cells with 250 µl Opti-MEM.
      Note: McCoys growth medium is replaced by Opti-MEM during transfection since cytotoxicity of Fugene-based transfections can increase due to the presence of antibiotics in the growth medium (e.g., penicillin and streptomycin).
    3. Add the Fugene HD-DNA mix dropwise to the HCT116 cells.
    4. Add 250 µl additional McCoys medium containing 20% FBS to the cells 6 to 8 h after transfection.
    5. Detach cells with trypsin 24 h post transfection. Transfer half of the cell suspension to a T25 flask. The remainder of the cells can be either discarded or used as non-infected G418 control population described in step D7.
    6. Dilute 50 µl of rAAV virus in 1 ml McCoys growth medium and add the mixture to the transfected cells 24 h after seeding in T25 flasks.
      Note: If colony numbers are markedly lower than expected (Figure 2), infection volumes can be increased to 150 µl or even 500 µl. Selection of cells transfected with CRISPR-Cas9 plasmids by the Zhang lab (see Materials) using puromycin or EGFP-based cell sorting prior to infection is optional. In most experiments, it is sufficient to solely rely on Geneticin/G418 selection for enrichment as this is a direct indicator of rAAV cassette insertion in the target genome. Moreover, treatment of the cells with puromycin or FACS sorting before G418 selection may have a detrimental effect on survival.

      Figure 2. Crystal violet staining of rAAV-infected (left) and non-infected HCT116 cells (right) after 14 days of G418 selection. The crystal violet stain visualizes cell foci resistant to G418.

    7. Add G418 to the cells at a concentration of 1 mg/ml 72 h after transfection.
      Note: An additional infected HCT116 population and non-infected HCT116 population is subjected to G418 selection to assess infection efficiency by crystal violet staining of the G418-resistant cell foci or colonies. The expected staining outcome is shown in Figure 2.
    8. Split cells and add fresh G418-supplemented growth medium when culture reaches 70% confluency. After 14 days of selection, seed single HCT116 cells in 96-well plates by manual dilution. Maintain 1 mg/ml G418 selection in the 96-well plates.
      Note: In a typical manual dilution step the cells are detached, counted and diluted to 10, 20 and 40 cells/ml. For each density (1, 2 or 4 cells/well) one plate is seeded by pipetting 100 µl of diluted cell suspension in each well of a 96-well tissue culture plate prefilled with 100 µl growth medium. It is recommended to seed different densities to ensure sufficient wells with single cells.
    9. Screen every well for presence of clonal cell populations one week after seeding by visual inspection using light microscopy.
    10. Once the majority of clonal populations reach approximately 70% confluence (Figure 3) PCR screening of the clonal cell populations can start (Figure 4).

      Figure 3. Typical view of an HCT116 clonal population. Once most identified populations reach 70% confluence PCR-based screening can be started. Left: expanding colony; right: colony ready for screening. Scale bars = 100 µm.

  5. PCR-based screening for homology-directed repair events
    The different primers used for PCR screening are depicted in Figure 4. It is recommended to design HR screening PCR reactions containing the junction of the HR with the flanking genomic DNA sequence in order to avoid the detection of random integration events (false positives). Random integration events can be detected by Southern blotting (step G2 and Data analysis section).  

    Figure 4. rAAV-mediated homology-directed repair of CRISPR-induced double stranded breaks on a single allele. Different primer pairs used for screening throughout this protocol are indicated in separate colors. Blue primers: 5’HR screening primers; green primers: 3’HR screening primers; black primers: selection cassette removal (CR) screening primer pair; red primers: primer pair for generation of the Southern blot DNA template (SBDT); grey arrows: inverted terminal repeats (ITR); blue striped triangles: loxP sequences; TAG: epitope tag; selection cassette: neomycin-resistance gene under control of the phosphoglycerate kinase (PGK) promotor; purple elements: nickase Cas9 (nCas9) pair.

    1. Generation and lysis of PCR screening plate
      1. Pipet 20 µl of direct PCR lysis buffer (see Recipes) to a new 96-well PCR plate.
      2. Remove growth medium from the 96-well plate containing the clonal cell populations. Wash once with PBS before detaching cells in 25 µl trypsin. Transfer 5 µl of the detached cells to the PCR plate prefilled with PCR lysis buffer and cover with aluminium foil tape. Rapidly add 200 µl of standard growth medium to the remaining cell suspension in the 96-well tissue culture plate for further culturing. Place the 96-well PCR plate in a thermal cycler and subject samples to the following program:
        15 min
        55 °C
        45 min
        85 °C

        4 °C

        Note: After lysis the 96-well PCR plate can be stored at 2-8 °C until PCR screening.
    2. PCR amplification.
      1. Perform a PCR reaction spanning the 5’HR using the lysis material of each clone as input DNA to assess rAAV-mediated integration of the repair template using the program described in step B4.
        5x Colorless GoTaq Flexi buffer
        4 µl
        5’HR Fwd primer (100 µM)
        0.15 µl
        5’HR Rev primer (100 µM)
        0.15 µl
        dNTP’s (25 mM each)
        0.2 µl
        MgCl2 (25 mM)
        1.2 µl
        1.2 µl
        10.9 µl
        GoTaq (5 U/µl)
        0.2 µl
        Lysis material

      2. Assess amplification by agarose electrophoresis. Repeat reaction with 3’HR primers for every positive clone.
        5x Colorless GoTaq Flexi buffer
        4 µl
        3’HR Fwd primer (100 µM)
        0.15 µl
        3’HR Rev primer (100 µM)
        0.15 µl
        dNTP’s (25 mM each)
        0.2 µl
        MgCl2 (25 mM)
        1.2 µl
        1.2 µl
        10.9 µl
        GoTaq (5 U/µl)
        0.2 µl
        Lysis material
        2 µl

      3. Analyze amplification by agarose electrophoresis.
      4. Positive clones are expanded and frozen.

  6. Tat-Cre mediated selection cassette removal
    1. Seed PCR-positive clones from Procedure E in separate wells of a 96-well plate at a density of 1 x 103 cells/well.
    2. Add TAT-Cre recombinase at a final concentration of 2.5 µM to each well 24 h after seeding.
    3. Wash twice with DPBS before adding fresh growth medium after 24 h of TAT-Cre incubation. Cells are ready for single cell seeding by manual dilution in 96-well plates 4 days after TAT-Cre treatment.

  7. Screening and validation of final clones
    Note: Assays described in this section are typically performed in a sequential manner.  
    1. PCR amplification
      1. Prepare lysis plate as described in step E1. 
        Verify excision of the selection cassette by PCR using the program described in step B4. Run this PCR reaction also on untreated positive clones to obtain a reference for clones without successful recombination (see note in step G1c below).
        5x Colorless GoTaq Flexi buffer
        4 µl
        CR Fwd primer (100 µM)
        0.15 µl
        CR Rev primer (100 µM)
        0.15 µl
        dNTP’s (25 mM each)
        0.2 µl
        MgCl2 (25 mM)
        1.2 µl
        1.2 µl
        10.9 µl
        GoTaq (5 U/µl)
        0.2 µl
        Lysis material
        2 µl

      2. Analyze amplicon size by agarose electrophoresis. In silico removal of the cassette using software for manipulation of DNA sequences can be used to define the expected length of the amplicon.
        Note: Successful PCR for the clones without recombination can be difficult to obtain due to size of the amplicon and GC content of the promoter in the neomycin selection cassette.
    2. Southern blot
      1. PCR amplify a part of the neomycin resistance cassette using the PCR program described in step B4. Purify PCR product with NucleoSpin Gel and PCR Clean-up Kit.
        10x AccuPrime Pfx reaction mix
        5 µl
        SBDT forward primer (10 µM)
        1.5 µl
        SBDT reverse primer (10 µM)
        1.5 µl
        AccuPrime Pfx DNA polymerase (2.5 U/μl)
        0.5 µl
        rAAV targeting construct (1 ng/µl)
        10 µl
        31.5 µl

        SBDT Fwd primer
        SBDT Rev primer

      2. Denature template DNA using Random Primer DNA Labeling Kit.
        Template DNA (50 ng/µl)
        1 µl
        Random primer
        2 µl
        11 µl

      3. Boil for 5 min. Snap cool sample on ice for 5 min afterwards. Add following mix to the sample:
        10x buffer
        2.5 µl
        dNTP mix (0.2 mM each)
        2.5 µl
        [α-32P] dCTP (50 µCi)
        5 µl
        Exo-free Klenow enzyme
        1 µl

      4. Incubate for 20 min at 37 °C. Inactivate the Exo-free Klenow enzyme by incubating at 65 °C for 5 min. Boil sample for 3 min.
        Note: Appropriate protective measurements should be taken to work with [α-32P] dCTP and the labeled probe generated using this reagent.
      5. Purify the probe using an Illustra Microspin S-400 HR column according to the protocol provided by the manufacturer. 
      6. Prepare 10 µg 500 ng/µl gDNA of the selected clones as described in step B1. Digest overnight with EcoRI at 37 °C.
        gDNA (500 ng/µl)
        20 µl
        SuRE/Cut buffer H
        3 µl
        0.1% BSA
        0.3 µl
        EcoRI (40 U/µl)
        0.5 µl
        6.2 µl

      7. Separate digested gDNA by 0.7% agarose gel electrophoresis. Incubate gel in the different buffers (see Recipes) according to following scheme:
        Depurination buffer
        10 min
        Denaturation buffer
        2 x 15 min
        Neutralization buffer
        2 x 15 min
        20x SSC
        10 min

        Note: Thoroughly rinse both the electrophoresis tank and gel tray with 0.5% SDS and hot water to minimize background signal on Southern blot. Include genomic DNA of clones prior to TAT-Cre treatment for Southern blot as control samples.
      8. Blot overnight using the Nytran SuPerCharge TurboBlotter Kit.
      9. Wash membrane for 5 min in 2x SSC (see Recipes). Crosslink on Whatman paper (pre-wetted in 2x SSC) in a GS gene linker UV chamber using program C3 (150 mJ by 254 nm UV irradiation).
      10. Transfer membrane to a tube containing PerfectHyb plus hybridization buffer preheated to 68 °C. Incubate for 1 h at 68 °C.
      11. Denature radioactive probe by boiling for 10 min. Snap cool on ice for 2 min.
      12. Add 25 µl of denatured probe to the membrane in fresh preheated PerfectHyb plus buffer. Incubate overnight at 68 °C.
      13. Rinse membrane three times with wash buffer 1 (see Recipes). Wash membrane according to following scheme.
        Wash buffer 1
        10 min
        Wash buffer 2
        15 min
        Wash buffer 3
        10 min

        Note: Wash buffers 2 and 3 (see Recipes) should be preheated to 65 °C.
      14. Image on phosphorimager device after overnight exposure on a phosphor screen.
    3. Western blot
      1. Lyse a subconfluent T25 flask of each clone in 100 µl CHAPS lysis buffer (see Recipes). Incubate for 5 min on ice before centrifuging suspension for 10 min > 16,000 x g in a tabletop centrifuge. Transfer supernatant to new tube.
      2. Measure protein concentration by Bradford assay. Load 50 µg of protein material on SDS-PAGE. After blotting of the proteins on a membrane, modified endogenous proteins can be visualized by (tag-)specific primary antibodies (i.e., anti-FLAG or anti-HA, depending on the chosen backbone plasmid).
    4. Sanger sequencing.
      1. Prepare gDNA of every clone as described in step B1.
      2. PCR amplify region of interest using the PCR mix and program described in steps B3 and B4 respectively. Purify PCR fragment and ligate in pCR-Blunt vector using Zero Blunt PCR Cloning Kit.
      3. Transform in competent bacterial cells of choice (e.g., TOP10 or DH5α) and prepare plasmid DNA using the NucleoSpin Plasmid EasyPure Kit. pCR-Blunt inserts can be sequenced with M13 forward and reverse primers.
        M13 forward
        M13 reverse

        Note: As most of the clones are heterozygous for the desired modification, multiple plasmids of each clone should be sent for sequencing since the pCR-Blunt plasmids will also contain amplicons for the unmodified allele. 

Data analysis

  1. The different analysis options for validation of the selected clones are straightforward assays typically resulting in non-ambiguous data. The PCR step after G418 selection of clones ensures the integration of the epitope tag and the selection cassette in the correct position of the genome. The PCR results should be positive for both homology arms to ascertain lack of aberrant recombination events. These clones are typically also analyzed by Southern blotting to confirm the presence of a single cassette. Random integrations can occur, leading to the presence of an additional construct in the selected PCR-positive clones. Cell clones showing extra bands on the Southern analysis should be eliminated for further use.
  2. After Cre-based removal of the selection cassette, PCR analysis spanning the modified site and the LOX scar should result in a band of the correct size. As mentioned above, PCR for clones wherein removal of the selection was not successful can fail due to size of the insert and the GC content of the promoter in the selection cassette. Clones with failed PCR reactions for the modified site should be eliminated for further analysis.
  3. Southern analysis further confirms removal of the cassette as the probe is directed against the Neomycin resistance gene. Clones showing multiple bands prior to TAT-Cre treatment are omitted for further validation, as these additional bands point to (a) random integration event(s). Selected Cre-treated clones with remaining bands after Southern blotting are not used for further analysis.
  4. Western analysis should confirm the presence of the epitope tag on the protein of interest. However, this implies that the protein is sufficiently expressed to allow detection by Western blotting. In some cases, a stimulus or a stabilizing agent (e.g., proteasome inhibitor) can be added to the cells to increase the levels of the protein (e.g., MDM2, Vandemoortele et al., 2016). Failure to detect the tagged protein by Western blotting is not necessarily an indication to exclude the cell line for further analysis but may complicate further downstream analysis. Note also that actual protein expression may vary between different clones. The testing of additional clones can still reveal clones with positive Western blot results. Note also that the size of the protein can deviate from the major protein isoform described in the database due to alternative splicing, protein processing, posttranslational modifications, incorrect annotation, etc. Size confirmation of the protein can be obtained with a specific antibody.
  5. Sequence verification is required to ensure correct integration of the tag and eliminates rare clones with frameshifts or other mutations that may have occurred during the engineering process.
  6. Only clones passing all previous assays are withheld for further gene-specific validation assays (such as modulation by a stimulus or protein interaction profiling).


  1. Phase Lock lysis buffer
    20 mM Tris-HCl, pH 7.5
    5 mM EDTA
    0.15 M NaCl
    0.2% SDS
    6 U proteinase K from Tritirachium album
    Add ddH2O to 500 µl
  2. TE-buffer
    10 mM Tris-HCl, pH 7.5
    1 mM EDTA
  3. 2x HEBS (HEPES-buffered saline)
    0.28 M NaCl
    50 mM HEPES
    1.5 mM Na2HPO4
    800 ml ddH2O
    Adjust to pH 7.05
    Add ddH2O to 1 L
    Note: The exact pH is critical for transfection efficiency. 2x HEBS can be stored at -20 °C. Avoid repeated freeze thaw cycles.
  4. rAAV cell lysis buffer
    0.15 M NaCl
    50 mM Tris-HCl, pH 8.5
    Filter sterilize using 0.22 μm filter
  5. Direct PCR lysis buffer
    2.1 ml Direct PCR lysis reagent (cell)
    17 U proteinase K from Tritirachium album
  6. CHAPS lysis buffer
    50 mM HEPES, pH 7.4
    100 mM NaCl
    0.8% CHAPS (w/v)
    cOmplete protease inhibitor
  7. Depurination buffer
    0.25 N HCl
  8. Denaturation buffer
    1.5 M NaCl
    0.5 N NaOH
  9. Neutralization buffer
    0.5 M Tris, pH 7.4
    1.5 M NaCl
  10. 20x SSC
    3 M NaCl
    0.3 M sodium citrate tribasic dehydrate
    Adjust to pH 7.0
    Add ddH2O to 1 L
  11. Wash buffer 1
    200 ml 10x SSC
    10 ml 10% SDS
    790 ml ddH2O
  12. Wash buffer 2
    100 ml 10x SSC
    10 ml 10% SDS
    890 ml ddH2O
  13. Wash buffer 3
    10 ml 10x SSC
    10 ml 10% SDS
    980 ml ddH2O


G.V. is a PhD student supported by the Fund for Scientific Research – Flanders (FWO) (grant 3G050913N). S.E. acknowledges support from FWO (grants G011312N and 3G050913N). Cell lines constructed using this protocol were described in ‘An extra dimension in protein tagging by quantifying universal proteotypic peptides using targeted proteomics’ by Vandemoortele et al. (2016) and ‘Intelligent mixing of proteomes for elimination of false positives in affinity purification-mass spectrometry’ by Eyckerman et al. (2016), doi:10.1038/srep27220 and doi: 10.1021/acs.jproteome.6b00517 respectively.


  1. Chen, F., Pruett-Miller, S. M., Huang, Y., Gjoka, M., Duda, K., Taunton, J., Collingwood, T. N., Frodin, M. and Davis, G. D. (2011). High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases. Nat Methods 8(9): 753-755.
  2. Eyckerman, S., Impens, F., Van Quickelberghe, E., Samyn, N., Vandemoortele, G., De Sutter, D., Tavernier, J. and Gevaert, K. (2016). Intelligent mixing of proteomes for elimination of false positives in affinity purification-mass spectrometry. J Proteome Res 15(10): 3929-3937.
  3. Khan, I. F., Hirata, R. K. and Russell, D. W. (2011). AAV-mediated gene targeting methods for human cells. Nat Protoc 6(4): 482-501.
  4. Ledford, H. (2015). CRISPR, the disruptor. Nature 522(7554): 20-24.
  5. Qiu, P., Shandilya, H., D'Alessio, J. M., O'Connor, K., Durocher, J. and Gerard, G. F. (2004). Mutation detection using Surveyor nuclease. Biotechniques 36(4): 702-707.
  6. Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A. and Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8(11): 2281-2308.
  7. Russell, D. W. and Hirata, R. K. (1998). Human gene targeting by viral vectors. Nat Genet 18(4): 325-330.
  8. Vandemoortele, G., Staes, A., Gonnelli, G., Samyn, N., De Sutter, D., Vandermarliere, E., Timmerman, E., Gevaert, K., Martens, L. and Eyckerman, S. (2016). An extra dimension in protein tagging by quantifying universal proteotypic peptides using targeted proteomics. Sci Rep 6: 27220.
  9. Vasileva, A., Linden, R. M. and Jessberger, R. (2006). Homologous recombination is required for AAV-mediated gene targeting. Nucleic Acids Res 34(11): 3345-3360.
  10. Wu, Z., Yang, H. and Colosi, P. (2010). Effect of genome size on AAV vector packaging. Mol Ther 18(1): 80-86.


可编程集群定期间隔短回归度(CRISPR)相关核酸酶9(Cas9)技术通过提供在所需位置切割基因组的有效方式,彻底改变了基因组编辑(Ledford,2015)。 在哺乳动物细胞中,DNA损伤触发易发生非同源末端连接(NHEJ)DNA修复机制。 然而,在DNA修复模板的存在下,可以发生同源性定向修复(HDR),导致病变部位的精确修复。 可以利用最后的方法,通过在修复模板上引入所需的基因组改变来实现精确的敲入变化。 在本协议中,我们描述了使用重组腺相关病毒(rAAV)在人细胞系中进行基于CRISPR-Cas9的C-末端标签序列敲入的长修复模板(> 200个核苷酸)的递送。

尽管有关CRISPR-Cas9产生的敲门模型系统的大量报告,敲门砖报告仍然落后。由于许多应用,产生敲入细胞系仍然是基因组编辑的明显目标。敲入改变的引入通常依赖于修复模板DNA的存在,并且在位点特异性双链(ds)DNA断裂被引入接近改变位点的基因组中后,HDR修复机制的激活。不同的模板可以传送到修复机器,范围从含有广泛同源区域和可选选择盒的经典线性化载体到约200个核苷酸的单链(ss)DNA寡核苷酸(Chen等人, 2011)。尽管ssDNA寡核苷酸是一种受欢迎的工具,但由于DNA合成限制,它们只能用于引入小的改变,如突变或表位标签。此外,缺少选择盒式磁带需要强大的筛选策略来识别正确的克隆,因为没有选择压力应用于HDR过程。在定制的核酸酶的可用性之前已经显示成功使用整合缺陷型rAAV进行同源重组(Khan等人,2011)。它的有效递送和ssDNA基因组使rAAV成为用于同源重组的供体修复模板的强大工具。此外,ssDNA分子末端的二级结构阻断核酸外切酶活性并稳定供体DNA。即使没有使用特定的核酸酶,在成纤维细胞(Russell和Hirata,1998)中获得高达0.7%的敲入效率,通过引入选择盒进一步增加。


关键字:CRISPR-Cas9, 重组腺相关病毒(rAAV), 基因组工程, 表位附加


  1. 带有过滤帽的T25细胞培养处理过的烧瓶(Thermo Fisher Scientific,Thermo Scientific TM,目录号:136196)
  2. Eppendorf Safe-Lock微量离心管(Eppendorf,目录号:022363204)
  3. 5Prime Phase Lock凝胶管重2毫升(Quantabio,目录号:2302830)
  4. 具有过滤帽的T75细胞培养处理的烧瓶(Thermo Fisher Scientific,Thermo Scientific TM,目录号:178905)
  5. Greiner 50ml离心管(Greiner Bio One International,目录号:227261)
  6. Greiner细胞刮刀(Greiner Bio One International,目录号:541081)
  7. 24孔细胞培养处理的多片(Thermo Fisher Scientific,Thermo Scientific TM,目录号:142475)
  8. 96孔微孔板(Thermo Fisher Scientific,Thermo Scientific TM,目录号:156545)
  9. 96孔PCR板(Thermo Fisher Scientific,Thermo Scientific TM,目录号:AB0700)
  10. 6孔细胞培养处理多片(Thermo Fisher Scientific,Thermo Scientific TM,目录号:140675)
  11. 铝箔胶带(3M,目录号:425DWB)
  12. Illustra Microspin S-400 HR柱(GE Healthcare,目录号:27-5140-01)
  13. Immobilon-FL PVDF转移膜(EMD Millipore,目录号:IPFL00010)
  14. Whatman 3 MM chr纤维素吸印片(GE Healthcare,目录号:3030-917)
  15. 0.22μm过滤器(EMD Millipore,目录号:SLGV033RS)
  16. AAV-293细胞系(Agilent Technologies,目录号:240073)
  17. HCT 116细胞系(ATCC,目录号:CCL-247)
  18. Cas9 D10A切口酶突变体(nCas9)(Addgene,目录号:48140和62987)
  19. pAav-MCS-PQS1-3xFLAG或pAav-MCS-PQS2-3xHA(Addgene,目录号:分别为84883和84917)
  20. pDG rAAV包装质粒(PlasmidFactory,目录号:PF421)
  21. 野生型Cas9表达载体(Cas9)(Addgene,目录号:48138和62988)
  22. 选择的感染细菌细胞(例如,,TOP10或DH5α)
  23. Surveyor Mutation Detection Kit(综合DNA技术,目录号:706025)
  24. UltraPure苯酚:氯仿:异戊醇(25:24:1,v/v)(Thermo Fisher Scientific,Invitrogen公司,目录号:15593031)
  25. 氯化钠(NaCl)分子生物学级(EMD Millipore,目录号:567441)
  26. 2-丙醇(Sigma-Aldrich,目录号:278475)
  27. 乙醇(EMD Millipore,目录号:100983)
  28. AccuPrime Pfx DNA聚合酶(Thermo Fisher Scientific,Invitrogen TM,目录号:12344-024)
  29. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:449709)
  30. DMEM,高葡萄糖GlutaMAX培养基(Thermo Fisher Scientific,Gibco TM,目录号:31966047)
  31. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:10010023)
  32. Benzonase核酸酶(Sigma-Aldrich,目录号:E1014)
  33. AAV纯化Vira Kit 3使用(Virapur,目录号:003063)
  34. AAV无辅助系统(Agilent Technologies,目录号:240071)
  35. McCoy的5A培养基(Thermo Fisher Scientific,Gibco TM,目录号:16600082)
  36. Opti-MEM I减少血清培养基,GlutaMAX补充剂(Thermo Fisher Scientific,Gibco TM,目录号:51985026)
  37. Fugene HD转染试剂(Promega,目录号:E2311)
  38. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10500064)
  39. 胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM,目录号:25300096)
  40. 嘌呤霉素(Sigma-Aldrich,目录号:P8833)
  41. 结晶紫溶液(Sigma-Aldrich,目录号:HT90132)
  42. 遗传霉素/G418(Thermo Fisher Scientific,Gibco TM,目录号:11811031)
  43. Go Taq G2热启动聚合酶(Promega,目录号:M7405)
  44. dNTP 100mM PCR级(Agilent Technologies,目录号:200415)
  45. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  46. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D2650)
  47. TAT-Cre重组酶(Excellgen,目录号:EG-1001)
  48. NucleoSpin凝胶和PCR清除试剂盒(Machery-Nagel,目录号:740609)
  49. 随机引物DNA标记试剂盒(Takara Bio,目录号:6045)
  50. [α- 32 P] dCTP,50μCi(PerkinElmer,目录号:BLU013H250UC)
  51. 不含Klenow酶(New England Biolabs,目录号:M0212S)
  52. SuRE/Cut缓冲液H(Roche Diagnostics,目录号:11417991001)
  53. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A4503)
  54. 生态 RI 40 U /μl(Roche Diagnostics,目录号:10200310001)
  55. Nytran SuPerCharge TurboBlotter Kit(Sigma-Aldrich,目录号:Z613924)
  56. PerfectHyb加杂交缓冲液(Sigma-Aldrich,目录号:H7033)
  57. 4-12%标准XT Bis-Tris蛋白凝胶18孔,30μl(Bio-Rad Laboratories,目录号:3450124)
  58. 小鼠单克隆抗FLAG M2抗体(Sigma-Aldrich,目录号:F3165)
    或大鼠单克隆抗HA高亲和力(Roche Diagnostics,目录号:11867423001)
  59. Zero Blunt PCR Cloning Kit(Thermo Fisher Scientific,Invitrogen TM,目录号:K270020)
  60. NucleoSpin质粒EasyPure(Machery-Nagel,目录号:740727)
  61. Tris超纯级(MP Biomedicals,目录号:02103133)
  62. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E5134)
  63. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:436143)
  64. 来自曲霉属相册的蛋白酶K 800 U/ml(Sigma-Aldrich,目录号:P4850)
  65. HEPES(Sigma-Aldrich,目录号:H4034)
  66. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:255793)
  67. 直接溶解试剂(细胞)(VIAGEN BIOTECH,目录号:301-C)
  68. CHAPS水合物(Sigma-Aldrich,目录号:C5070)
  69. 蛋白酶抑制剂(Roche Diagnostics,目录号:11697498001)
  70. 盐酸(HCl)(Sigma-Aldrich,目录号:30721)
  71. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:221465)
  72. 柠檬酸三钠脱水剂[HOC(COONa)(CH 2 COONa)2/2H 2 O](Sigma-Aldrich,目录号:C8532 )
  73. SmartLadder(Eurogentec,目录号:MW-1700-10)
  74. Phase Lock裂解缓冲液(见配方)
  75. TE缓冲(见配方)
  76. 2x HEBS(HEPES缓冲盐水)(参见食谱)
  77. rAAV细胞裂解缓冲液(参见食谱)
  78. 直接PCR裂解缓冲液(参见食谱)
  79. CHAPS裂解缓冲液(参见食谱)
  80. 去除缓冲液(见配方)
  81. 变性缓冲液(见配方)
  82. 中和缓冲(见配方)
  83. 20x SSC(见配方)
  84. 洗涤缓冲液1(参见食谱)
  85. 洗涤缓冲液2(参见食谱)
  86. 洗涤缓冲液3(参见食谱)


  1. BSL2细胞培养设施
  2. Thermoshaker
  3. 涡流(例如,IKA,型号:MS2 minishaker)
  4. 用于细胞培养的倒置显微镜(例如,Carl Zeiss,型号:Axiovert 25)
  5. 非循环水浴
  6. 250毫升无菌储存瓶(康宁,目录号:430281)
  7. 热循环仪(例如,Bio-Rad Laboratories,型号:T100 热循环仪,目录号:1861096)
  8. 蛋白质印迹设备,例如,
    标准垂直电泳池(Bio-Rad Laboratories,目录号:1656001)
    具有平板电极的标准吸墨纸(Bio-Rad Laboratories,目录号:1704070)
  9. GS基因接头UV室(Bio-Rad Laboratories,model:GS Gene Linker TM UV Chamber)
  10. 荧光成像仪装置(例如,GE Healthcare,型号:Typhoon 9200)
  11. 应使用放射性标记的核苷酸([α- 32 P] dCTP)所需的设施和程序(例如废物处理程序)
  12. 台式离心机(Eppendorf,型号:5430 R)


  1. 设计目标靶基因的引导RNA(gRNA)构建体
    1. 将编码感兴趣蛋白质C末端区域的基因组序列插入到选择的引导设计工具中(例如, crispr.mit.edu )。在大多数情况下,围绕终止密码子的总长度为250个核苷酸的输入序列具有足够的PAM基序用于设计工具以产生多个引导序列。
    2. 选择靠近C-terminal的导游。在定义指南时,请牢记潜在的脱靶效应。大多数设计工具通过基于目标基因组中的目标命中得分来排列潜在的指导,直接提供这些信息
    3. 克隆指南在一个Cas9表达载体的选择。建议使用Cas9 D10A切口突变体(nCas9,Addgene质粒#48140和#62987)以尽量减少脱靶裂解事件。或者,野生型Cas9表达载体也可用于Addgene(质粒#48138和#62988)。构建体的详细克隆程序由Ran等人描述。 (2013)。当使用Cas9 D10A切口(nCas9)时,保持引导对之间的最小偏移,以确保最佳效率。

  2. 通过PCR扩增同源区域来生成rAAV靶向构建体 使用由PQS1肽(Vandemoortele等人,2004)和3xFlag标签组成的组合标签的C-末端标签的骨架质粒已经沉积在Addgene(质粒#84883)上。含有PQS2肽和3xHA标签的该靶向构建体的替代形式也可在Addgene(质粒#84917)上获得。可以通过标准克隆程序将5'和3'同源区(HR)插入到这些质粒中存在的多个克隆位点。
    1. 使用酚提取和乙醇沉淀方案制备基因组DNA(gDNA)
      1. 通过在室温(RT)下以500×g离心5分钟,分离并沉淀相当于Eppendorf管中的亚汇合T25烧瓶的2.5×10 6个细胞。 >
      2. 加入500μlgDNA Phase Lock裂解缓冲液(参见食谱)至沉淀,并在37℃下孵育过夜。
      3. 加入裂解液之前,通过快速离心法将相锁管旋转下来。在转移到锁相管之前摇匀裂解物样品。
      4. 加入500μl苯酚:氯仿:异戊醇(25:24:1,v/v),并剧烈振荡1分钟。
      5. 在室温下以16,000×g离心相锁定管3分钟。将水相转移到新鲜的Eppendorf管中,加入50μl3 M NaCl和500μl2-丙醇。反复管后可以观察到基因组DNA几次
      6. 在室温下以16,000 x g离心2分钟。弃去上清液,加入1 ml 70%乙醇至DNA沉淀。倒置几次,室温孵育5分钟。在室温下以16,000×g离心2分钟。
      7. 丢弃上清液。将沉淀物在52℃下空气干燥5分钟,然后将沉淀重新悬浮在100μl10mM TE缓冲液pH7.5中。允许颗粒溶解1小时通过在65℃下在热心机中孵育。测量基因组DNA浓度。
    2. 设计引物,用适当的突出限制位点扩增HR,以插入骨架质粒 注意:建议使用至少650bp的HR。据报道,较短的HR显着降低同源重组潜力(Vasileva等,2006)。反向末端重复(ITR)序列之间的全长超过4.7kb的靶向构建将阻碍rAAV的包装效率(Wu et al。,2010)。
    3. 准备PCR混合物
      10x AccuPrime

      AccuPrime Pfx DNA聚合酶
      gDNA(100ng /μl)
      ddH 2 O

    4. 使用触地式PCR程序扩大人力资源。根据人力资源的长度(1分钟/kb)调整斜体延长时间。

    5. 在进行克隆步骤之前,通过琼脂糖凝胶电泳验证和纯化PCR产物 注意:由于上述骨架结构包含位于新霉素选择盒侧翼的多个克隆位点(MCS),所以可以使用不同的限制酶位点进行最终靶向构建体的克隆。替代的克隆方法(例如,Gibson装配,In-Fusion)也与这些载体兼容。

  3. rAAV生产和净化
    1. AAV-293转染
      1. 在转染前24小时,加入4×10 6个AAV-293细胞的种子10T75烧瓶。
      2. 在转染前至少30分钟吸出并加入新鲜生长培养基。
      3. 为每个T75准备DNA转染混合物。
        2.5M CaCl 2
        ddH 2 O

        注意:pDG质粒用作rAAV包装的辅助质粒。 pDG包含所有必需的AAV和腺病毒功能,以确保扩增和包装(r)AAV载体。
      4. 将DNA转染混合物滴加到750μl2x HEBS(参见食谱),同时使用涡流以确保充分混合。在室温下孵育10分钟。
      5. 将转染混合液以一种逐滴的方式转移到细胞中 注意:转染的AAV-293细胞和纯化的rAAV病毒应在此之后在BSL-2设施中处理。
      6. 在转染24小时后用12ml新鲜的DMEM生长培养基代替生长培养基,再孵育48小时 注意:在更换生长培养基之前,应使用光学显微镜通过目视检查在转染的细胞之间发现细小的沉淀物。磷酸钙-DNA沉淀有助于通过胞吞作用进入细胞之前DNA与细胞表面的结合。次优转染缓冲液(例如2x HEBS)或DNA质量可影响沉淀物形成,导致低转染效率。由于转染可能会导致生长受损。
    2. rAAV颗粒的收获和净化
      1. 在50ml管中收集分离细胞的生长培养基。
      2. 使用细胞刮刀将细胞收集在1.5ml PBS/T75烧瓶中,并将细胞悬浮液加入到50ml管中。以500×g离心细胞3分钟,并将上清液转移到新的50ml管中。
      3. 将2.5ml PBS加入到获得的细胞沉淀中,重新悬浮细胞沉淀并将所有细胞悬浮液组合在一个新管中。以500×g离心3分钟,并将细胞沉淀重新悬浮于3ml rAAV裂解缓冲液中。
      4. 主题丸至3次冻融循环,循环之间短暂涡旋。将裂解物转移到步骤C2b的上清液。
      5. 在4℃下以900×g离心管30分钟。将上清液收集在250 ml无菌瓶中,加入Benzonase核酸酶(最终浓度为50 U/ml)
      6. 使用VIRAPUR AAV纯化试剂盒根据制造商的方案纯化rAAV颗粒。纯化病毒应储存在-80°C等份,直至使用。

  4. HCT116细胞的转染和感染
    1. 种子2.7×10 5个HCT116细胞/孔在含有1ml McCoys生长培养基的24孔板中。
    2. 在单独的管中准备以下混合物。


      Fugene HD转染试剂
      Cas9 + gRNA质粒DNA

      将Fugene HD混合物轻轻添加到DNA混合物中,轻轻敲打管,并在室温下孵育混合物10分钟。在该孵育步骤中,用250μlOpti-MEM替换HCT116细胞上的培养基 注意:在转染过程中,McCoys生长培养基被Opti-MEM所代替,因为基于Fugene的转染的细胞毒性可能由于生长培养基(例如青霉素和链霉素)中存在抗生素而增加。 >
    3. 将Fugene HD-DNA混合物滴加到HCT116细胞中。
    4. 转染后6〜8 h,向细胞中加入250μl含有20%FBS的McCoys培养基。
    5. 转染后24小时用胰蛋白酶分离细胞。将一半的细胞悬浮液转移到T25烧瓶中。可以将剩余的细胞丢弃或用作步骤D7中描述的未感染的G418对照群体。
    6. 在1 ml McCoys生长培养基中稀释50μlrAAV病毒,并在接种于T25烧瓶后24 h将混合物加入转染细胞。

      图2.在G418选择14天后,rAAV感染(左)和未感染的HCT116细胞(右)的结晶紫染色。结晶紫染色显示耐G418的细胞灶。 >
    7. 转染后72 h,以1 mg/ml的浓度将G418加入细胞 注意:另外感染的HCT116群体和未感染的HCT116群体进行G418选择,以评估G418抗性细胞病灶或菌落的结晶紫染色的感染效率。预期的染色结果如图2所示。
    8. 分离细胞并在培养达到70%融合时添加新鲜的G418补充生长培养基。选择14天后,通过手动稀释在96孔板中种单个HCT116细胞。在96孔板中保持1 mg/ml G418选择。
    9. 通过使用光学显微镜的目视检查,在播种后一周筛选每个孔以克隆细胞群体的存在
    10. 一旦大多数克隆群体达到约70%汇合(图3),可以开始克隆细胞群体的PCR筛选(图4)。

      图3. HCT116克隆群体的典型视图。一旦大多数确定的群体达到70%融合,就可以开始基于PCR的筛选。左:扩大殖民地右:殖民地准备进行筛选。比例尺=100μm
  5. 基于PCR的筛查同源性修复事件

    在本方案中用于筛选的不同引物对以分开的颜色表示。蓝色引物:5'HR筛选引物;绿色引物:3'HR筛选引物;黑色引物:选择盒去除(CR)筛选引物对;红色引物:用于产生Southern印迹DNA模板(SBDT)的引物对;灰色箭头:反向末端重复(ITR);蓝色条纹三角形:loxP序列; TAG:表位标签;选择盒:磷酸甘油酸激酶(PGK)启动子控制下的新霉素抗性基因;紫色元素:切口酶Cas9(nCas9)对。

    1. PCR筛选板的产生和裂解
      1. 将20μl直接PCR裂解缓冲液(见食谱)吸管至新的96孔PCR板。
      2. 从包含克隆细胞群的96孔板中除去生长培养基。在用25μl胰蛋白酶分离细胞之前用PBS洗涤一次。将5μl分离的细胞转移到预先用PCR裂解缓冲液预填充的PCR板上并用铝箔带覆盖。快速地向96孔组织培养板中的剩余细胞悬浮液中加入200μl标准生长培养基用于进一步培养。将96孔PCR板置于热循环仪中,并将样品送至以下程序:


    2. PCR扩增
      1. 使用每个克隆的裂解材料作为输入DNA进行跨越5'HR的PCR反应,以使用步骤B4中描述的程序来评估修复模板的rAAV介导的整合。
        5x无色Go Taq Flexi缓冲区
        5'HR Fwd引物(100μM)
        5'HR Rev引物(100μM)
        dNTP(25 mM each)
        MgCl 2(25mM)
        ddH 2 O
        去 Taq (5 U /μl)

      2. 通过琼脂糖电泳评估扩增。每个阳性克隆与3'HR引物重复反应
        5x无色Go Taq Flexi缓冲区
        3'HR Fwd引物(100μM)
        3'HR Rev引物(100μM)
        dNTP(25 mM each)
        MgCl 2(25mM)
        ddH 2 O
        去 Taq (5 U /μl)

      3. 通过琼脂糖电泳分析扩增
      4. 阳性克隆扩大并冻结。

  6. Tat-Cre介导的选择盒去除
    1. 来自步骤E的种子PCR阳性克隆在96孔板的单独孔中,密度为1×10 3个细胞/孔。
    2. 在接种后24小时向每个孔添加终浓度为2.5μM的TAT-Cre重组酶。
    3. 在加入新鲜生长培养基的TAT-Cre孵育24小时后,用DPBS洗涤两次。在TAT-Cre处理4天后,通过手动稀释96孔板,细胞准备进行单细胞接种。

  7. 最终克隆的筛选和验证
    1. PCR扩增
      1. 按步骤E1所述制备裂解板。
        5x无色Go Taq Flexi缓冲区
        CR  Fwd引物(100μM)
        CR  Rev引物(100μM)
        dNTP(25 mM each)
        MgCl 2(25mM)
        ddH 2 O
        去 Taq (5 U /μl)

      2. 通过琼脂糖电泳分析扩增子大小。使用用于操作DNA序列的软件的盒式计算机移除可用于定义扩增子的预期长度。
    2. Southern印迹
      1. PCR使用步骤B4中所述的PCR程序扩增一部分新霉素抗性盒。用NucleoSpin凝胶和PCR清除试剂盒纯化PCR产物。
        10x AccuPrime

        AccuPrime Pfx DNA聚合酶(2.5U /μl)
        rAAV靶向构建体(1ng /μl)
        ddH 2 O

        SBDT Fwd底漆
        SBDT Rev底漆

      2. 使用随机引物DNA标记试剂盒变性模板DNA
        模板DNA(50 ng /μl)
        ddH 2 O

      3. 煮5分钟将冰冷的样品在冰上保存5分钟。添加以下混合样品:
        [α- 32 P] dCTP(50μCi)

      4. 在37℃下孵育20分钟。通过在65℃下孵育5分钟来灭活无exo-free Klenow酶。沸腾样品3分钟。
        注意:应采取适当的保护措施,使用[α- 32 P] dCTP和使用该试剂产生的标记探针。
      5. 根据制造商提供的方案,使用Illustra Microspin S-400 HR色谱柱纯化探针。 
      6. 如步骤B1所述,制备所选克隆的10μg500 ng /μlgDNA。在37℃下用生态环境消化一夜。
        gDNA(500ng /μl)
        SuRE /剪切缓冲区H
        生态 RI(40 U /μl)
        H 2 O

      7. 通过0.7%琼脂糖凝胶电泳分离消化的gDNA。根据以下方案,在不同的缓冲液中孵育凝胶(参见食谱):
        2 x 15分钟
        2 x 15分钟
        20x SSC

      8. 使用Nytran SuPerCharge TurboBlotter套件过夜。
      9. 在2x SSC中洗涤膜5分钟(参见食谱)。使用程序C3(150mJ×254nm UV照射)在GS基因接头UV室中的Whatman纸上(在2x SSC中预润湿)的交联。
      10. 将膜转移到含有PerfectHyb plus杂交缓冲液的管中预热至68℃。在68°C孵育1小时。
      11. 变性放射性探针沸腾10分钟。在冰上保存2分钟。
      12. 在新鲜预热的PerfectHyb加缓冲液中,将25μl变性探针加入到膜中。在68°C孵育过夜。
      13. 用洗涤缓冲液1冲洗膜三次(参见食谱)。按照以下方案洗膜。

      14. 在磷光体屏幕上过夜曝光后的磷光体装置上的图像。
    3. 蛋白质印迹
      1. 将每个克隆的亚融合T25烧瓶溶解在100μlCHAPS裂解缓冲液(参见Recipes)中。在离心悬浮液10分钟之前,在冰上孵育5分钟> 16,000 x g 在桌面离心机中。将上清液转移至新管。
      2. 通过Bradford测定法测定蛋白质浓度。在SDS-PAGE上载入50μg蛋白质物质。在蛋白质膜上印迹后,修饰的内源蛋白质可以通过(标签)特异性一级抗体(即,根据选择的骨架质粒)来显现,即抗FLAG或抗HA。
    4. 桑格测序。
      1. 按照步骤B1所述准备每个克隆的gDNA。
      2. PCR分别使用步骤B3和B4中描述的PCR混合物和程序扩增感兴趣的区域。纯化PCR片段,并使用Zero Blunt PCR克隆试剂盒在pCR-Blunt载体中连接
      3. 在选择的有效细菌细胞(例如,TOP10或DH5α)中转化,并使用NucleoSpin Plasmid EasyPure Kit制备质粒DNA。可以用M13正向和反向引物测序pCR-Blunt插入片段。

        注意:由于大多数克隆对于所需的修饰是杂合的,所以应该发送每个克隆的多个质粒进行测序,因为pCR-Blunt质粒还将含有未修饰等位基因的扩增子。 >


  1. 用于验证所选克隆的不同分析选项是直接测定,通常导致非模糊数据。 G418选择克隆后的PCR步骤确保了表位标签和选择盒在基因组正确位置的整合。 PCR结果对于同源性臂均应是阳性的,以确定缺乏异常重组事件。通常还通过Southern印迹分析这些克隆以确认存在单个盒。可能发生随机整合,导致在选择的PCR阳性克隆中存在另外的构建体。应该消除在Southern分析中显示额外条带的细胞克隆以供进一步使用。
  2. 在基于Cre的去除选择盒后,跨越修饰位点和LOX瘢痕的PCR分析应该导致正确大小的条带。如上所述,由于插入物的大小和选择盒中的启动子的GC含量,其中除去选择不成功的克隆的PCR可能失败。应该消除用于修饰位点的PCR反应失败的克隆进行进一步分析。
  3. 当探针针对新霉素抗性基因时,Southern分析进一步确认了盒的去除。在TAT-Cre处理之前显示多个频带的克隆被省略用于进一步验证,因为这些附加频带指向(a)随机积分事件。在Southern印迹后,选择经过克隆处理的具有残留条带的克隆不用于进一步分析。
  4. Western分析应该证实目标蛋白上存在表位标签。然而,这意味着蛋白质被充分表达以允许通过Western印迹检测。在一些情况下,刺激或稳定剂(例如,蛋白酶体抑制剂)可以加入到细胞中以增加蛋白质的水平(例如,MDM2, Vandemoortele等人,2016)。通过Western印迹检测未标记的蛋白质不一定表示排除细胞系用于进一步分析,但可能使进一步的下游分析复杂化。还要注意,实际的蛋白质表达可能在不同克隆之间变化。附加克隆的测试仍然可以显示具有阳性Western印迹结果的克隆。还要注意,由于可选的剪接,蛋白质处理,翻译后修饰,不正确的注释,等等,蛋白质的大小可以偏离数据库中描述的主要蛋白质同种型。可以用特异性抗体获得蛋白质的大小确认。
  5. 需要进行序列验证以确保标签的正确集成,并消除在工程过程中可能发生的具有帧特征或其他突变的稀有克隆。
  6. 只有通过所有以前测定的克隆才能进一步进行基因特异性验证测定(例如通过刺激或蛋白质相互作用分析进行调制)。


  1. 锁相裂解缓冲液
    20mM Tris-HCl,pH7.5
    5 mM EDTA
    0.15 M NaCl
    6 U蛋白酶K从Tritirachium相册
    将ddH 2 O加入500μl
  2. TE缓冲区
    10mM Tris-HCl,pH7.5
    1 mM EDTA
  3. 2x HEBS(HEPES缓冲盐水)
    0.28 M NaCl
    50 mM HEPES
    1.5mM Na 2 HPO 4
    800毫升ddH 2 O
    调整至pH 7.05
    将ddH 2 O添加到1 L
    注意:确切的pH对转染效率至关重要。 2x HEBS可储存于-20°C。避免反复冻融循环。
  4. rAAV细胞裂解缓冲液
    0.15 M NaCl
    50mM Tris-HCl,pH8.5
  5. 直接PCR裂解缓冲液
    2.1 ml直接PCR裂解试剂(细胞)

  6. CHAPS裂解缓冲液
    50 mM HEPES,pH 7.4
    100mM NaCl
  7. 去除缓冲液
    0.25 N HCl
  8. 变性缓冲液
    1.5 M NaCl
    0.5 N NaOH
  9. 中和缓冲区
    0.5 M Tris,pH 7.4
    1.5 M NaCl
  10. 20x SSC
    3 M NaCl
    0.3 M柠檬酸三钠脱水剂
    调整至pH 7.0
    将ddH 2 O添加到1 L
  11. 洗涤缓冲液1
    200 ml 10x SSC
    10ml 10%SDS
    790ml ddH 2 O O
  12. 洗涤缓冲液2
    100ml 10x SSC
    10ml 10%SDS
    890毫升ddH 2 O -/-
  13. 洗涤缓冲液3
    10ml 10x SSC
    10ml 10%SDS
    980ml ddH 2 O O


G.V.是由弗兰德斯(FWO)科学研究基金(授权3G050913N)支持的博士生。 S.E.承认FWO的支持(G011312N和3G050913N)。使用该方案构建的细胞系在Vandemoortele等人的"通过使用靶向蛋白质组学量化通用蛋白质肽的蛋白质标记中的额外维度"中有描述。 (2016)和Eyckerman等人的"用于消除亲和纯化 - 质谱法中的假阳性的蛋白质组的智能混合"。 (2016),doi:10.1038/srep27220和doi:10.1021/acs.jproteome.6b00517。


  1. Chen,F.,Pruett-Miller,SM,Huang,Y.,Gjoka,M.,Duda,K.,Taunton,J.,Collingwood,TN,Frodin,M.and Davis,GD(2011) a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/21765410"target ="_ blank">使用具有锌指核酸酶的ssDNA寡核苷酸进行高频基因组编辑。/a> Nat方法 8(9):753-755。
  2. Eyckerman,S.,Impens,F.,Van Quickelberghe,E.,Samyn,N.,Vandemoortele,G.,De Sutter,D.,Tavernier,J.and Gevaert,K。(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27640904"target ="_ blank">智能混合蛋白质组,用于消除亲和纯化 - 质谱中的假阳性。/a> J Proteome Res 15(10):3929-3937。
  3. Khan,IF,Hirata,RK and Russell,DW(2011)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/21455185"target =" AAV介导的人类细胞基因靶向方法。 Nat Protoc 6(4):482-501。
  4. Ledford,H。(2015)。 CRISPR,破坏者 522(7554):20-24。
  5. Qiu,P.,Shandilya,H.,D'Alessio,JM,O'Connor,K.,Durocher,J.and Gerard,GF(2004)。  使用Surveyor核酸酶进行突变检测。生物技术 36(4):702-707。
  6. Ran,FA,Hsu,PD,Wright,J.,Agarwala,V.,Scott,DA and Zhang,F。(2013)。  使用CRISPR-Cas9系统的基因组工程。 Nat Protoc 8(11):2281-2308。 br />
  7. Russell,DW和Hirata,RK(1998)。  Human病毒载体的基因靶向。 Nat Genet 18(4):325-330。
  8. Vandemerma,E.,Gonnelli,G.,Samyn,N.,De Sutter,D.,Vandermarliere,E.,Timmerman,E.,Gevaert,K.,Martens,L。和Eyckerman,S. (2016)。通过量化的蛋白质标签中的额外维度使用靶向蛋白质组学的通用蛋白型肽。 6:27220.
  9. Vasileva,A.,Linden,RM和Jessberger,R。(2006)。同源重组是AAV介导的基因靶向所必需的。核酸研究34(11):3345-3360。
  10. Wu,Z.,Yang,H。和Colosi,P。(2010)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/19904234" target ="_ blank">基因组大小对AAV载体包装的影响。 Mol Ther 18(1):80-86。
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引用:Vandemoortele, G., De Sutter, D. and Eyckerman, S. (2017). Robust Generation of Knock-in Cell Lines Using CRISPR-Cas9 and rAAV-assisted Repair Template Delivery. Bio-protocol 7(7): e2211. DOI: 10.21769/BioProtoc.2211.