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Efficient Generation of Multi-gene Knockout Cell Lines and Patient-derived Xenografts Using Multi-colored Lenti-CRISPR-Cas9
使用多色Lenti-CRISPR-Cas9高效生成多基因敲除细胞系和患者源性异种移植物   

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Abstract

CRISPR-Cas9 based knockout strategies are increasingly used to analyze gene function. However, redundancies and overlapping functions in biological signaling pathways can call for generating multi-gene knockout cells, which remains a relatively laborious process. Here we detail the application of multi-color LentiCRISPR vectors to simultaneously generate single and multiple knockouts in human cells. We provide a complete protocol, including guide RNA design, LentiCRISPR cloning, viral production and transduction, as well as strategies for sorting and screening knockout cells. The validity of the process is demonstrated by the simultaneous deletion of up to four programmed cell death mediators in leukemic cell lines and patient-derived acute lymphoblastic leukemia xenografts, in which single cell cloning is not feasible. This protocol enables any lab with access to basic cellular biology equipment, a biosafety level 2 facility and fluorescence-activated cell sorting capabilities to generate single and multi-gene knockout cell lines or primary cells efficiently within one month.

Keywords: CRISPR(CRISPR), Cas9(Cas9), Multiple gene knockout(多基因敲除), Lentivirus(慢病毒), Primary human leukemia(原发性人白血病), Xenograft(异种移植物), sgRNA design(sgRNA设计)

Background

Starting with curious initial observations of genetic elements known as clustered regularly interspaced short palindromic repeats (CRISPRs) within bacterial genomes (Ishino et al., 1987; Mojica et al., 2000) and subsequent gene editing in mammalian cells (Cong et al., 2013; Mali et al., 2013), CRISPR-Cas9 has become the cutting edge option for inexpensive and efficient gene editing. With successful application in cellular systems ranging from tobacco plant cells to zebrafish and primary human cells (Hsu et al., 2014), CRISPR-Cas9 can be directed by design of a short 20 nucleotide RNA sequence to create targeted DNA double strand breaks (DSB) within large genomes (Park et al., 2016). After DSBs occur, cells can initiate repair either through high fidelity homologous recombination (HR) or error-prone non-homologous end joining (NHEJ), often leading to small insertion and deletion (indel) mutations resulting in gene knockout (Gaj et al., 2013; Bétermier et al., 2014) (Figure 1).


Figure 1. Principle of genome editing by CRISPR Cas9. The principle of a gene knockout by CRISPR-Cas9 is shown exemplarily for the RIP1 sequence. A. Single guided RNA (sgRNA) consists of the target sequence specific crRNA (CRISPR RNA) and the constant tracrRNA (trans-activating crRNA) (Jinek et al., 2012). crRNA is binding to the genomic DNA adjacent to the PAM motif and tracrRNA guides the Cas9 enzyme to the locus. B. Cas9 mediated DNA double strand breaks (DSB) activate non-homologous end joining (NHEJ). C. Imprecise DSB repair leads to gain or loss of nucleotides (indels) with a two-thirds chance of causing frameshift mutations that may result in the generation of premature stop codons.

A number of different strategies have emerged to deliver Cas9 protein and targeting RNA into cells, including electroporation or transfection of Cas9/sgRNA ribonucleoprotein complexes, mRNA, plasmid or lentiviral vectors carrying sgRNA and Cas9 payloads (Sander and Joung, 2014; Shalem et al., 2014). Previously these LentiCRISPR plasmids carried a resistance gene to allow selection of cells with constitutive expression of the machinery necessary for CRISPR-Cas9-directed gene disruption.

As shown in our recent publication (McComb et al., 2016), we have adapted the LentiCRISPR protocol for directed disruption of several genes simultaneously in cell lines and primary leukemia cells based on selection by fluorescence combined with fluorescence-activated cell sorting (FACS). By swapping the puromycin resistance gene for fluorescent protein markers (EGFP, mCherry, tagBFP, or RFP657), up to four genes can be simultaneously targeted for CRISPR-Cas9-mediated gene disruption. Fluorescence-activated cell sorting enables isolation of cell lines or primary human cells bearing sgRNAs targeting one to four genes in one single experimental step. Our multi-color LentiCRISPR technique thus allows the simultaneous generation of knockout cells bearing anywhere between one and four gene knockouts, allowing rapid testing of gene-gene interaction within a set of genes of interest. The backbone vectors with the four different fluorescence markers as well as the herein described target constructs including cloning information have been deposited at Addgene.

Here we provide a complete step-by-step guide protocol to generate single and multi-gene knockout cells by multicolor LentiCRISPR (see Figure 2 for schematic overview).


Figure 2. Schematic overview of the procedure to generate multicolor LentiCRISPR knockout cell lines and patient derived xenografts

Development of the protocol
To study the regulation of cell death in leukemia, we developed multi-color LentiCRISPR as a tool to target proteins essential for two divergent pathways of programmed cell death, apoptosis and necroptosis (McComb et al., 2016). Efficient deletion of the respective targets, like RIP1, RIP3, MLKL, FADD and CASP8, was demonstrated by Western blot analysis of protein in targeted compartments (see Data analysis). Through simultaneous gene disruption, we showed that it is necessary to inactivate both apoptosis and necroptosis within leukemic cell lines and patient-derived xenografts in order to render cells resistant to SMAC mimetics, a specific class of chemotherapeutic compounds targeting the inhibitor of apoptosis proteins, IAPs. These data provide convincing evidence that both apoptosis and necroptosis can independently kill leukemia cells in vivo, and are a strong proof of concept for the multicolor LentiCRISPR technique as a means to investigate gene redundancy.

Experimental design
sgRNA design and preparation. We first describe a fast and easy way to design and clone sgRNAs for any target gene with a single pot reaction for restriction and ligation (Figure 3). We have had good success utilizing the CRISPR design online tool (http://cripr.mit.edu) from the Massachusetts Institute of Technology, developed by the lab of Feng Zhang to predict binding sites for Cas9 with minimal risk of off-target activity (Hsu et al., 2013). Alternative sgRNA prediction software (such as http://crispor.tefor.net/) can also be used to provide in silico prediction of sgRNA-specific cleavage activity based on a number of different algorithms. However, we still recommend the design of three sgRNAs per target gene and assessing their gene knockout activity in cell lines before moving on to more challenging applications. Strategies targeting only the 5’ exon of candidate genes might induce in-frame mutations that can retain the full protein functionality. A recent publication showed that targeting particular exonic regions with key functional protein domains increases the chance of null mutations without a full protein knockout (Shi et al., 2015). For this reason, we suggest spreading sgRNA candidates among different exons to increase the probability of achieving a potent gene deletion.


Figure 3. Principle of primer design and cloning for LentiCRISPR-Cas9 mediated gene knockout. Primer design is shown exemplarily for RIP1. A. From the genomic sequence the target locus for CRISPR editing was chosen and screened by http://crispr.mit.edu for sgRNA binding sites. B. After choosing a guide by score and location, complement primer sequence can be generated. C and D. For ligation into the Esp3I restriction site of the pLentiCRISPR plasmid sticky ends (CACC/CAAA) and the U6 transcriptional start site (TSS), (G) has to be added to the oligonucleotide sequence. U6, RNA Pol III promoter; EFS, EF1 short promoter.

Lentiviral production and infection. This protocol makes use of multicolor LentiCRISPR plasmids cloned from one-vector LentiCRISPR system developed by the Zhang lab (Shalem et al., 2014). Protocol conditions have been optimized for the transduction of acute lymphoblastic leukemia (ALL) cell lines and patient derived xenograft ALL cells from previously published protocols (Tiscornia et al., 2006; Kutner et al., 2009; Weber et al., 2012). Optimized conditions are recommended for every cell line or patient-derived sample. Here, we describe the production of lentiviral particles with the VSV-G envelope, because it is known for its high titers and broad tropism. Depending on the target cells the pseudotyping can be exchanged. For murine applications, the exchange to a mouse ecotropic envelope protein enhances safety and enables the use of the lentiviral vectors under biosafety level 1 conditions.

Analysis of knockout efficiency
After purification of cells transduced with Cas9/sgRNA targeted against a gene of interest, it is straightforward to confirm knockout at protein level (measurement via flow cytometry, ELISA or Western blot). Thus it is essential that a specific antibody for your protein of interest is available (see Figure 4 for further discussion and considerations for single cell cloning). Regardless of the viral transduction efficiency and the gene-editing efficacy of the selected sgRNA/CRISPR-Cas9 construct, DNA mutations do not invariantly lead to a loss of protein. Based on the triplet coding sequences the ratio of a frameshift after NHEJ is 2:1 resulting in possible indels without frameshift. Depending on the location of indel formation, this may also lead to unpredictable effects on mRNA or protein stability. Thus, we do not recommend knockout confirmation at DNA or RNA level by sequencing or SURVEYOR nuclease assay since this does not confirm the loss of the protein expression and function. Lesions can lead to a loss of amino acids and a change in protein functionality, but can also result in a slightly impaired protein with a near wild type function. Depending on the target gene a functional assay can be performed (i.e., enzyme activity) or cellular localization can be visualized (i.e., for nuclear receptors) but we recommend performing Western blots or ELISA to quantify protein level.

Limitations of the technique
Lentiviral vector efficiently delivers the CRISPR machinery to a wide range of cell lines and primary cells. However, as with any viral approach, there is a risk of insertional mutagenesis, although this can be minimized by transducing cells at a low multiplicity of infection (MOI) to limit the number of integration events. Constitutive expression of Cas9/sgRNA may also lead to accumulation of mutations at off-target sites, stressing the need for good sgRNA design to limit off-target binding. 

Efficiency of knockouts using LentiCRISPR can vary significantly depending on the specific genes targeted, especially for targets that confer a selective advantage/disadvantage to knockout cells. It might thus be a benefit to utilize an inducible CRISPR plasmid for such applications. In this case, Cas9 or sgRNA expression would be controllable in vitro and in vivo by administration of i.e., doxycycline. 
  Our strategy also depends on the reliability of the detection of reporter fluorescence. There is some evidence for silencing of expression over time for fluorescent proteins of the GFP family when expressed from lentiviral vectors. The populations should therefore be continuously monitored for expression. While lentiviral delivery has been proven to be very efficient in many different cellular systems, transduction efficiency heavily depends on the size of the delivered plasmids (Canté-Barrett et al., 2016). The large size of the LentiCRISPR vector is known to lead to low viral titers, nonetheless, viral concentration and other optimizations in the protocol below have allowed us to successfully apply these vectors in hard to transduce leukemic cell lines and primary cells.


Figure 4. Considerations for single cell cloning. We recommend single cell cloning if a clonal cell population with a constant genetic background is desired for long term experimentation. Here we present a Western blot confirmation of the knockout of RIP1 in either (A) double sorted or (B) single cell cloned Jurkat cells. Irrespective the purity of the sorted cell population, a minor RIP1 signal remains in the double sorted population, whereas in single cell clones a pure knockout can be achieved. Proteins were detected with mouse anti-RIP1 (1:1,000) and goat anti-mouse-HRP (1:5,000).

Materials and Reagents

  1. Filtered sterile pipette tips
    10 µl (STARLAB INTERNATION, catalog number: S1121-3810 )
    200 µl (STARLAB INTERNATION, catalog number: S1120-8810 )
    1,000 µl (STARLAB INTERNATION, catalog number: S1126-7810 )
  2. TC plate
    6 well (SARSTEDT, catalog number: 83.3920 )
    24 well (SARSTEDT, catalog number: 83.3922 )
    96 well R (SARSTEDT, catalog number: 83.3925 )
  3. Filters: Filtropur S
    0.22 µm (SARSTEDT, catalog number: 83.1826.102 )
    0.45 µm (SARSTEDT, catalog number: 83.1826 )
  4. Serological pipettes
    5 ml (SARSTEDT, catalog number: 86.1253.001 )
    10 ml (SARSTEDT, catalog number: 86.1254.001 )
    25 ml (SARSTEDT, catalog number: 86.1685.001 )
  5. Amicon Ultra-15 centrifugal filter units (EMD Millipore, catalog number: UFC900308 )
  6. Tube
    15 ml (SARSTEDT, catalog number: 62.554.502 )
    50 ml (SARSTEDT, catalog number: 62.547.254 )
    13 ml for bacteria culture (SARSTEDT, catalog number: 62.515.006 )
  7. SafeSeal tubes 1.5 ml (SARSTEDT, catalog number: 72.706 )
  8. Nitrocellulose membranes (Trans-Blot Turbo transfer pack) (Bio-Rad Laboratories, catalog number: 170-4159 )
  9. Disposable syringe 10 ml (CODAN, catalog number: 62.6616 )
  10. Injectomat syringe 50 ml (Fresenius Kabi, catalog number: 9000711 )
  11. Petri dishes (92 x 16 mm) for LB plates (SARSTEDT, catalog number: 82.1472.001 )
  12. Round-bottom tube, 5 ml (Corning, Falcon®, catalog number: 352052 )
  13. Round-bottom tube with cell strainer cap, 5 ml (Corning, Falcon®, catalog number: 352235 )
  14. TC flask T175, vent. cap (SARSTEDT, catalog number: 83.3912.002 )
  15. Microvette 100 LH, Lithium-Heparin (SARSTEDT, catalog number: 20.1282 )
  16. NSG (NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ) mice (RRID:IMSR_ARC:NSG), age ~4-6 weeks
    Caution: Institutional animal care guidelines must be followed. Here the animal experiments were approved by the veterinary office and the ethics commission of the Canton of Zurich, Switzerland.
  17. HEK293T cells (ATCC, catalog number: CRL-3216 )
    Note: Cells should be growing actively in culture with DMEM medium with 10% (v/v) FBS. Cells should be maintained between 10-90% confluence, splitting at least twice per week.
  18. Target cells
    Note: Cells should be growing in healthy culture with appropriate medium. For most ALL cell lines RPMI with 10% (v/v) FBS is suitable. Primary cells should be thawed a day before transduction.
    Caution: Primary human cells have to be considered potentially hazardous. Primary derived xenografts were obtained from human ALL samples recovered from cryopreserved bone marrow aspirates. Here patients were enrolled in the ALL-BFM 2000 and ALL-BFM 2009 studies. Informed consent was given in accordance with the Declaration of Helsinki and approval was granted by the Ethics Commission of the Kanton Zurich.
  19. One Shot TOP10 Chemically Competent E. coli (Thermo Fisher Scientific, InvitrogenTM, catalog number: C404010 or equivalent)
  20. CRISPR target plasmid with BsmBI (Esp3I) insert site, e.g.,
    pLentiCRISPR-EGFP (Addgene, catalog number: 75159 )
    RFP657 (Addgene, catalog number: 75162 )
    BFP (Addgene, catalog number: 75160 )
    mCherry (Addgene, catalog number: 75161 )
  21. psPAX2 plasmid (Addgene, catalog number: 12260 )
  22. p.CMV.VSV.G plasmid (Addgene, catalog number: 8454 )
    Caution: It may cause an allergic skin reaction or asthma symptoms.
  23. Oligonucleotides 100 µM, standard synthesis, desalted purification (for example sequences see Table 1)

    Table 1. Sequences of oligonucleotides for pLentiCRISPR cloning


  24. Tango buffer (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: BY5 )
  25. T4 DNA ligase (New England Biolabs, catalog number: M0202M ) make aliquots of T4 DNA ligase buffer and use only freshly thawed aliquots for reaction
  26. Esp3I restriction enzyme (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: ER0451 )
    Note: NOT NEB BsmBI, BsmBI requires 55 °C for efficient cleavage – not compatible with this protocol.
  27. SOC medium (Sigma-Aldrich, catalog number: S1797 or equivalent)
  28. Ampicillin (Sigma-Aldrich, catalog number: A9518 or equivalent)
  29. RED Taq Ready Mix PCR Reaction mix (Sigma-Aldrich, catalog number: R2523 or equivalent)
  30. Mini- and Midi-plasmid Preparation Kit (QIAGEN, catalog number: 27106 or equivalent)
  31. Tris-acetate-EDTA buffer (TAE, 25x) (Thermo Fisher Scientific, AmbionTM, catalog number: AM9870 or equivalent)
  32. Polyethylenimin (PEI transfection reagent) (Sigma-Aldrich, catalog number: 408727 )
    Note: You must test your preparation of PEI prior to use to establish the concentration, which is necessary to get the best transduction efficiency. We generally use ~20 µg/ml final concentration.
    Caution: It is toxic if swallowed.
  33. ProFection Mammalian Transfection System (Promega, catalog number: E1200 )
  34. Chloroquine diphosphate salt (Sigma-Aldrich, catalog number: C6628 or equivalent)
    Caution: It is harmful if swallowed.
  35. Calcium chloride (CaCl2)
  36. HBS
  37. Polybrene (hexadimethrine bromide) (Sigma-Aldrich, catalog number: H9268 ) stock solution 8 mg/ml in ddH2O
    Caution: It is harmful if swallowed.
  38. Dulbecco’s phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: D1408 or equivalent)
  39. Trypsin (0.05% in PBS) with EDTA (BioConcept, catalog number: 5-51F00-I or equivalent)
  40. Formalin (4% formaldehyde) (Formafix, catalog number: 01-1010 )
  41. Polyethylene glycol (PEG 6000) (Sigma-Aldrich, catalog number: 81253 )
  42. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 or equivalent)
  43. RetroNectin Recombinant Human Fibronectin Fragment (Takara Bio, Clontech, catalog number: T100B )
    Note: Prepare Retronectin according to manufacturer’s protocol, aliquot and store at -20 °C. Retronectin can be reused up to 4 times without quality decrease.
  44. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418 or equivalent)
  45. Hank’s balanced salt solution (HBSS) (Thermo Fisher Scientific, catalog number: 14170088 or equivalent)
  46. HEPES, 1 M (Thermo Fisher Scientific, catalog number: 15630056 or equivalent)
    Caution: It can cause skin irritation and serious eye irritation.
  47. Flow cytometry antibody (PE-Cy7 anti-human CD19) (BioLegend, catalog number: 302216 , RRID:AB_314246)
  48. NuPage MES SDS 20x running buffer (Thermo Fisher Scientific, NovexTM, catalog number: NP0002-02 ) dilute 1:20 with ddH2O for final concentration
  49. Pageruler Prestained Protein ladder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 26616 or equivalent)
  50. Precast gels (Criterion XT 4-12% Bis-Tris 1.0 mm gels)
    18 well (Bio-Rad Laboratories, catalog number: 3450124 )
    26 well (Bio-Rad Laboratories, catalog number: 3450125 )
  51. Nonfat-dried milk (Sigma-Aldrich, catalog number: M7409 or equivalent)
    Note: Prepare 5% (w/v) working solution in 1x TBS-T. Prepare fresh and store at 4 °C for short term.
  52. Sodium azide (NaN3) (Sigma-Aldrich, catalog number: S2002 or equivalent)
    Caution: It is fatal if swallowed or in contact with skin. May cause damage to organs if swallowed. Very toxic to aquatic life.
  53. Secondary HRP-conjugated antibody
  54. Western blot primary antibodies
    Mouse anti-RIP1 (BD, BD Biosciences, catalog number: 551042 , RRID:AB_394015)
    Rabbit anti-FADD (Cell Signaling Technology, catalog number: 2782 , RRID:AB_2100484)
    Mouse anti-Tubulin (Sigma-Aldrich, catalog number: T9026 , RRID:AB_477593)
    Rat anti-MLKL (EMD Millipore, catalog number: MABC604 )
    Mouse anti-CASP8 (Cell Signaling Technology, catalog number: 9746 , RRID:AB_2068482)
    Rabbit anti-RIP3 (Abnova, catalog number: PAB0287 , RRID:AB_1019004)
  55. Western blot secondary antibodies conjugated to horseradish peroxidase
    Goat anti-mouse (Cell Signaling Technology, catalog number: 7076 , RRID:AB_330924)
    Goat anti-rabbit (Cell Signaling Technology, catalog number: 7074 , RRID:AB_10697506)
    Goat anti-rat (Cell Signaling Technology, catalog number: 7077 , RRID:AB_10694715)
  56. Baytril, Bayer, 0.5 ml in 250 ml autoclaved drinking water (stock 2.5%)
  57. Gene Ruler 1 kb Plus DNA ladder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: SM1331 or equivalent)
  58. GelRed, 10,000x (Biotium, catalog number: 41003 )
    Note: GelRed is superior to ethidium bromide by having low toxicity and high sensitivity.
  59. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 221465 or equivalent)
    Caution: It is dangerous. Causes severe skin burns and eye damage.
  60. SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 34096 )
  61. Benzonase Nuclease (Sigma-Aldrich)
  62. Bacto tryptone (BD, BactoTM, catalog number: 211705 or equivalent)
  63. Yeast extract (BD, BactoTM, catalog number: 212750 or equivalent)
  64. Agar (BD, BactoTM, catalog number: 214010 or equivalent)
  65. Agarose (Eurogentec, catalog number: EP-0010-05 or equivalent)
  66. DMEM medium (Sigma-Aldrich, catalog number: D5546 or equivalent)
  67. Fetal calf serum (FCS) (Sigma-Aldrich, catalog number: 12133C or equivalent)
  68. L-glutamine, 200 mM (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 or equivalent)
  69. Sodium pyruvate, 100 mM (Thermo Fisher Scientific, GibcoTM, catalog number: 11360070 or equivalent)
  70. RPMI medium (Sigma-Aldrich, catalog number: R8758 or equivalent)
  71. Penicillin-streptomycin (Pen/Strep) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 or equivalent)
  72. FBS
  73. Dimethylsulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 or equivalent)
  74. Trizma HCl (Sigma-Aldrich, catalog number: T5941 or equivalent)
  75. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 or equivalent)
    Caution: It is harmful if swallowed and toxic in contact with skin. Flammable solid. Can cause eye and skin irritation and may cause respiratory irritation.
  76. Bromophenol blue (Bio-Rad Laboratories, catalog number: 161-0404 or equivalent)
  77. Glycerol (Sigma-Aldrich, catalog number: G5516 or equivalent)
  78. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 or equivalent)
    Caution: It is toxic if swallowed or if inhaled. Fatal in contact with skin. Causes skin irritations and eye damage.
  79. Ammonium chloride (NH4Cl) (Carl Roth, catalog number: K298.1 or equivalent)
    Caution: It is harmful if swallowed. It causes serious eye irritation.
  80. Potassium chlorate (KClO3) (EMD Millipore, catalog number: 104854 or equivalent)
    Caution: It is dangerous and harmful if swallowed or inhaled. May cause fire, is a strong oxidizer.
  81. Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) (Sigma-Aldrich, catalog number: E5134 or equivalent), prepare 0.5 M stock solution with ddH2O and pH 8.0 with NaOH
    Caution: It is harmful if inhaled.
  82. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 or equivalent)
  83. Trizma base (Sigma-Aldrich, catalog number: 93352 or equivalent)
  84. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H1758 or equivalent)
    Caution: It is toxic if inhaled. Causes severe skin burns and eye damage.
  85. Tween 20 (Sigma-Aldrich, catalog number: P9416 or equivalent)
  86. Ponceau S (Sigma-Aldrich, catalog number: P3504 )
  87. Acetic acid (Sigma-Aldrich, catalog number: A6283 or equivalent)
    Caution: It is flammable and causes severe skin burns and eye damage.
  88. LB agar plates Combine (see Recipes)
  89. LB medium Combine (see Recipes)
  90. DMEM complete (see Recipes)
  91. RPMI medium (see Recipes)
  92. Freezing medium (see Recipes)
  93. 3x SDS lysis buffer (see Recipes)
  94. 1x SDS loading buffer (see Recipes)
  95. Red blood lysis buffer (RBC buffer) (see Recipes)
  96. 10x TBS (see Recipes)
  97. 1x TBS-T (see Recipes)
  98. Ponceau S solution (see Recipes)
  99. SDS Glycine stripping buffer (see Recipes)

Equipment

  1. Pipettes (Gilson)
  2. Pipetus pipetting aid (Hirschmann Laborgeräte)
  3. Shaker (Thomas Scientific, model: Rocker Gyratory Lab Scale TSSL3, catalog number: 51900-27 )
  4. Thermocycler
  5. Microbiological incubator with 37 °C, atmospheric CO2 (Thermo Fisher Scientific, Heraeus)
  6. HeraCell 150 incubator with 37 °C, 5% CO2 (Thermo Fisher Scientific, model: HeraCell 150 incubator )
  7. MUPID-One electrophoresis system (LABGENE Scientific, model: MUPID-One electrophoresis system )
  8. Bench-top centrifuge (Eppendorf, model: 5417 C )
  9. Nalgene Oak Ridge centrifuge tube (Lab Depot, catalog number: 21009-386-PK )
    Note: These tubes are appropriate for ultracentrifugation and can’t be exchanged by standard tubes.
  10. Water bath (Thermo Fisher Scientific)
  11. Thermomixer comfort (Eppendorf)
  12. Vortex mixer Vortex-Genie 2 (Scientific Industries, model: Vortex-Genie 2 )
  13. FACSARIA III flow cytometry sorter (BD, model: FACSAria III )
  14. FACS Canto II flow cytometer (BD, BD Biosciences, model: FACSCANTO II )
  15. Gel documentation (Syngene)
  16. Heraeus Multifuge 3S (Thermo Fisher Scientific, model: HeraeusTM MultifugeTM 3S )
  17. Laminar flow hood for sterile tissue culture, biosafety level 2 approved (Thermo Fisher Scientific)
  18. Mastercycler nexus (Eppendorf)
  19. Mr. Frosty freezing container (Thermo Fisher Scientific)
  20. Neubauer chamber (BRAND)
  21. TransBlot Turbo transfer system (Bio-Rad Laboratories, model: Trans-Blot® TurboTM transfer system )
  22. Western blot chamber (Criterion Cell) and PowerPac basic power supply (Bio-Rad Laboratories)
  23. Spatula

Software

  1. A Plasmid Editor (http://biologylabs.utah.edu/jorgensen/wayned/ape/) (Optional)
  2. Oligonucleotide Properties Calculator (http://biotools.nubic.northwestern.edu) (Optional)

Procedure

Part I. sgRNA design and preparation


  1. Design of sgRNA sequence (Timing 1-2 h)
    1. Download genomic sequence from genome database (e.g., NCBI in FASTA format).
    2. Open your sequence with ‘A plasmid Editor’ (http://biologylabs.utah.edu/jorgensen/wayned/ape/) or similar sequence analysis software.
    3. Choose a target locus for CRISPR editing. Due to the variability of CRISPR knockout efficiency we generally choose 3 different exonic regions if possible. See introduction for discussion of additional considerations for target locus selection.
    4. Screen your target genomic locus for CRISPR guide RNA binding sites by inputting specific sequences into an online tool such as http://crispr.mit.edu.
      Note: Initial work was done with the crispr.mit.edu online tool. But alternative CRIPSPR calculators like http://crispor.tefor.net/ may offer additional prediction efficiency.
    5. Choose guides by score and location in the genome to determine targeting sites. Cas9 will cut 3 bp upstream of PAM site. We recommend choosing three different sgRNAs to improve the probability of achieving a full knockout.
    6. Copy the 20 nucleotide sequence of the selected guide RNA(s) without PAM and generate the complement sequence by using a bioinformatics software tool such as e.g., the Oligonucleotide Properties Calculator (http://biotools.nubic.northwestern.edu).
    7. For ligation into pLentiCRISPR plasmids add additional nucleotide to the sequence to generate sticky ends complement to the Esp3I restriction site (CACC/AAAC) and the transcriptional start site for RNA polymerase III (single G/C).
      Oligonucleotide
      Additional sequence (5’-3’)
      Fw_sg
      CACCG(N20)
      Rev_sg
      AAAC(N20)C

    8. Order the forward and reverse oligonucleotides (simple desalted oligonucleotides are adequate for this protocol).
    9. See example oligonucleotide design strategy for RIP1 in Figure 3.

  2. Annealing of single-stranded oligonucleotides (Timing 2.5 h)
    1. For lyophilized oligonucleotides, add an appropriate volume of ddH2O to obtain stock concentration of 100 µM.
    2. Pipette the following reaction for each pair of forward and reverse oligonucleotide.
      Component
      Amount
      Fw_sg oligo
      10 µl
      Rev_sg oligo
      10 µl
      Tango buffer
      10 µl
      ddH2O
      70 µl

    3. Bring up to 95 °C for 5 min in a thermocycler.
    4. Anneal the oligonucleotides by cooling to room temperature over 1-2 h.

  3. Ligation of double-stranded oligonucleotides into pLentiCRISPR (Timing 6 h)
    1. Set up following single pot reaction for plasmid restriction and ligation of annealed double-stranded oligonucleotides into pLentiCRISPR.
      Component
      Amount
      pLentiCRISPR
      150 ng
      double-stranded oligos (from step B4, Part I)
      1 µl
      NEB T4 ligase buffer (10x)
      2 µl
      ddH2O
      up to 18 µl
      Esp3I
      0.5 µl
      T4 DNA ligase
      0.5 µl

    2. Run the following reaction in a thermocycler.
       
      Cycle
      Temperature
      Time
      Ligation (15 cycles)
      37 °C
      5 min
      16 °C
      10 min
      Final
      37 °C
      15 min
      80 °C
      5 min
      Hold
      4 °C

      Note: There is an additional BsmBI (Esp3I) site in the tagBFP sequence in the pLentiCRISPR-BFP vector. For this vector the final 37 °C step should be removed from the cycle protocol to improve yield.
      PAUSE POINT: After heat inactivation of Esp3I the plasmid can be immediately transformed into bacteria or stored at -20 °C.

  4. Transformation and identification of bacterial clones with pLentiCRISPR and double-stranded oligonucleotide insert (Timing 3 d)
    Day 1:
    1. Transform ligation mix into competent bacteria.
    2. Add 2 µl ligation mix into ice-cold competent bacteria (50 µl).
      Note: Do not use more than 5 µl ligation product for transformation of 50 µl competent bacteria.
    3. Incubate mixture on ice for 20 min and heat-shock it at 42 °C for 90 sec.
    4. Cool down on ice for 2 min.
    5. Add 200 µl SOC medium (without antibiotics) and incubate for 45 min at 37 °C with gentle shaking.
    6. Plate 100 µl bacteria suspension on LB plate containing 100 µg/ml ampicillin.
    7. Incubate overnight at 37 °C in the microbiological incubator.

    Day 2:

    1. Run a colony PCR for 3-5 colonies per transformed pLentiCRISPR.
      Note: No colonies? See notes in Table 2.
    2. Prepare separate LB-agar plate with a number for each clone that will be picked.
    3. Prepare PCR reaction mix. Use Fw_U6 as forward primer and the sequence specific Rev_sg oligonucleotide as reverse primer.
      Component
      Amount
      RED Taq Ready mix (2x)
      5 µl
      ddH2O
      4 µl
      Fw_U6 (10 µM)
      0.5 µl
      Rev_sg oligo (10 µM)
      0.5 µl

    4. Pick single colonies with pipette tip, streak on separate numbered plate and subsequently submerge pipette tip in one well of PCR reaction buffer.
    5. After picking all colonies, remove pipette tips, run PCR and put numbered plate with streaks at 37 °C for 2-5 h in the microbiological incubator.
    6. Colony PCR program:
      Cycle
      Temperature
      Time
      Initial 
      95 °C
      3 min
      Cycle (25 cycles)
      95 °C
      30 sec
      55 °C
      30 sec
      72 °C
      10 sec
      Final
      72 °C
      10 min
      Hold
      4 °C


    7. Load PCR reactions on an agarose gel (2% [w/v] agarose/TAE buffer) (see Figure 5 for estimated product).
      Note: No positive colonies? Too many colonies? See notes in Table 2.


      Figure 5. Example of colony PCR products. pLentiCRISPR clones with inserted double-stranded oligonucleotides give a band around 100 bp. Empty vectors should give no band and will show only the primer dimers.

    8. Pick positive clone(s) from numbered plates to start overnight cultures in LB medium at 37 °C in the microbiological incubator.

    Day 3:

    1. Isolate plasmid DNA by mini or midi prep according to manufacturer’s protocol.
      PAUSE POINT: Plasmids can be immediately transfected into HEK293T cells or stored at 4 °C or -20 °C.

Part II. Lentiviral production and infection

  1. Production of pLentiCRISPR lentiviral particles (Timing 3-5 d)
    Day 1:
    1. In the afternoon seed 500,000 HEK293T cells per 6 well plate in 2 ml of DMEM complete.
      Note: Keep HEK293T cells always subconfluent. We also recommend working without antibiotics as we generally achieve higher titers without antibiotics in the medium. This protocol details the production of pilot-scale batches of viral supernatant (approximately 6-9 ml) appropriate for cell line applications but can be scaled up using T175 flasks to produce larger batches.
    2. Incubate overnight to allow HEK293T cells to adhere and equilibrate.

    Day 2

    1. Transfect HEK293T cells with lentiviral plasmids (pLentiCRISPR, psPAX2 and pVSV.G). We recommend using one of the two following techniques.
      Notes:
      a. Be careful not to disturb the cells while you add the transfection reagents.
      b. We use a ratio of 8:3:3 for LentiCRISPR:psPAX2:pVSV.G resulting in 4 µg pLentiCRISPR, and 1.5 µg of each of psPAX2 and p.CMV.VSV.G per well. But plasmid ratio should be titrated.
      1. PEI transfection reagent
        1. Thaw all reagents to room temperature (RT, about 22 °C).
        2. Dilute the plasmids in 1 ml of serum free, antibiotic free DMEM.
        3. Add 20 µl of PEI reagent and vortex immediately.
        4. Incubate the PEI/plasmid mixture for exactly 20 min at RT.
        5. Add the mixture dropwise directly to the HEK293T cells.
      2. ProFection mammalian transfection system
        1. Thaw all reagents to RT.
        2. Replace the cell culture medium with 3 ml fresh antibiotic free DMEM complete and add 3 µl chloroquine (final concentration 25 µM).
        3. Mix plasmids with 2 M CaCl2 and add ddH2O up to 150 µl.
        4. Gently add DNA-CaCl2 solution dropwise to 150 µl HBS while vortexing.
        5. Incubate for 30 min at room temperature.
        6. Add the mixture dropwise directly to the HEK293T cells.

    Note: All subsequent steps are subject to biosafety level 2 regulations. Perform all work in a biosafety cabinet and do not use vacuum to suction supernatants to avoid aerosolizing virus.

    1. After 4-6 h, remove the transfection medium and carefully replace by 3 ml pre-warmed DMEM complete. For PEI transfection, medium change is not strictly necessary, no obvious toxicity to cells could be observed.

    Day 3:

    1. The next morning examine the transfection efficiency by fluorescence microscopy. Some fluorescent signal should be visible in the majority of cells, although signal can be expected to increase for up to 72 h.
      Note: No fluorescence? See notes in Table 2.
    2. Collect the supernatant from the HEK293T cells and replace it with 3 ml pre-warmed DMEM complete medium. Keep viral supernatant on ice.
    3. Put HEK293T cells back to incubator (5% CO2) until next day.
    4. Filter the supernatant through a 0.45 µm filter to clear any remaining cells and freeze an aliquot of 100 µl for titer test at -80 °C.
    5. Supernatant can be kept at 4 °C in order to combine supernatant collected on subsequent days.
      Note: Do not user smaller filter size as it will decrease virus titer.

    Day 4:

    1. Repeat steps A6-A9 (Part II).

    Day 5:

    1. Repeat steps A6-A9 (Part II).

    Note: You can harvest viral supernatant up to 72 h after transfection, but the viral titer is decreasing after 48 h. Control the appearance of the HEK293T cells, cells should not overgrow massively. The highest titer is normally reached 24 h after transfection.
    PAUSE POINT: Supernatant can be immediately concentrated (see step C1, Part II) or aliquoted and stored at 4 °C for up to 1 week, or at -80 °C with a marginal decrease in viral titer (> 1 year). Freeze and thaw cycles should be avoided, as it will reduce titer.

  2. Titration of pLentiCRISPR lentiviral particles (Timing 3 d)
    Note: For initial screening of sgRNA functionality viral supernatants can be used directly on target cells without titration, however it is recommended to still perform titrations as viral aliquots can be stored and used for later experiments.
    Day 1:
    1. Plate 50,000 HEK293T cells in 0.5 ml DMEM complete per well in a 24 well plate.
    2. Work in triplicates.
      Note: HEK293T titers are a relative measurement of infectious units, thus we recommend working always with HEK293T cells under the same conditions (incubation times, volumes etc.). Stable processes enables to compare the titers between different productions.
    3. Add 0.5 µl polybrene (final concentration of 8 µg/ml).
    4. Incubate HEK293T cells for 6 h in the incubator (5% CO2).
    5. Add viral supernatant in increasing amounts to the medium (20 µl-100 µl of unconcentrated virus).

    CAUTION: All subsequent steps are subject to biosafety level 2 regulations. Do not use the vacuum pump to suction supernatants to avoid aerosolizing virus.
    Note: If this is done for the first time use a broad range of concentrations (1/10 to 1/100,000 relative dilutions of viral supernatant) to ensure that the data will be evaluable.

    1. Centrifuge the 24 well plate for 1 h, 750 x g, RT and keep overnight in the incubator (5% CO2).

    Day 2:

    1. Replace medium by 0.5 ml fresh DMEM complete and incubate for 48 h.

    Day 3:

    1. Remove supernatant and wash cells with PBS.
    2. Add 0.5 ml trypsin/EDTA and incubate for 5 min at 37 °C until the cells detach from the surface.
    3. Add 0.5 ml DMEM complete, resuspend the cells and transfer into FACS tubes.
    4. Spin for 5 min, 750 x g, RT.
    5. Remove supernatant and resuspend the cells in 0.5 ml PBS with 2% (v/v) formalin.
    6. Measure the transduction efficiency via flow cytometry.
    7. The linear range of viral transduction is in between 5-30%. Calculate the titer only for those wells that are comprised. The titer is calculated with the following formula. For more details refer to Weber et al. (2012).



      Notes:
      1. Under the procedure described in this protocol we generally expect to achieve viral supernatants that show cfu/ml above 1 x 105.
      2. Low titer? See notes in Table 2.

  3. Concentration of lentiviral particles (Timing 1-8 h)
    1. To increase the titer and therefore the transduction potential you can concentrate the virus by (a) column, (b) ultracentrifugation or (c) PEG 6000 precipitation.
      1. Concentration by Amicon centrifugal filter units
        1. Centrifugation speed and time should be optimized.
        2. We recommend loading 15 ml viral supernatant into the upper chamber and centrifuge for 10 min at 1,800 x g at 4 °C.
        3. Volume of viral supernatant should be decreased to 1 ml.
        4. Concentration index 1:15.
      2. Concentration by ultracentrifugation
        1. Load up to 40 ml viral supernatant to a Nalgene Oak Ridge centrifuge tube.
        2. Centrifuge for 6 h, at 8,000 x g, 4 °C.
        3. Afterwards virus is visible as a transparent jelly pellet.
        4. Discard supernatant carefully and resuspend pellet by vigorously pipetting in 1 ml medium of your choice.
        5. Concentration index: 1:40.
      3. Concentration by PEG 6000 precipitation
        1. Day 1. To 5 ml virus supernatant, add 1.3 ml of 50% PEG 6000 solution, 0.54 ml 4 M NaCl and 0.59 ml PBS. The final PEG 6000 concentration will be 8.5% and the final NaCl concentration will be ~0.3 M.
        2. Mix contents and store at 4 °C overnight.
        3. Day 2. Centrifuge mixture for 30 min at 2,000 x g, 4 °C.
        4. After centrifugation a white virus pellet is visible.
        5. Carefully decant the supernatant and resuspend the pellet in 100-500 µl medium by vigorously pipetting liquid up and down.
        6. Concentration index 1:50.

  4. Transduction of cell lines with pLentiCRISPR lentiviral particles (Timing 4 d)
    Note: This protocol is optimized for human leukemia cell lines (e.g., Jurkat). See below for optimized protocol for primary leukemic cells (step E1, Part II).
    CAUTION: All subsequent steps are subject to biosafety level 2 regulations. Do not use the vacuum pump to suction supernatants to avoid aerosolizing virus.
    Day 1:
    1. Count target cells and place 1 million cells in 150 µl RPMI medium per well in a 24 well plate.
    2. Add polybrene to the target cells at a concentration of 8 µg/ml.
    3. Thaw viral supernatant quickly in a water bath and keep it on ice as soon as it is melted.
    4. Add 50 µl-1 ml viral supernatant(s) on top of the cells, depending on desired MOI (multiplicity of infection).
      Note: For multiple target knockouts mix virus particles to transduce with the mixture in one step or perform serial transduction. In particular for triple and quadruple knockouts serial transduction can improve efficiencies.
    5. Gently shake the plate to mix the cells with the viral supernatant.
    6. After 2 h bring up to 2 ml with fresh RPMI medium.
    7. Incubate overnight at 37 °C.

    Day 2:

    1. Centrifuge the cells and gently remove the supernatant by pipetting and replace with 1 ml fresh RPMI.

    Day 3:

    1. Repeat steps D1-D8 (Part II). You can repeat viral transduction up to three times if higher transduction rates are desired.

    Day 4:

    1. Take a small aliquot of cells and fix by adding the same volume of 4% formalin (final concentration 2%) and incubate on ice for 10 min. Proceed to determine the transduction efficiency via flow cytometry.
    2. For a pure knockout cell line we recommend performing single cell clones either by limiting dilution or flow cytometry sort. But for a first validation of knockout efficiency and biological phenotype of the knockout we recommend using the bulk population after one or two rounds of flow cytometry sorting.
      Notes:
      1. Cells should not be transduced with more than 70-80% efficiency because this leads to multiple insertion, toxicity and higher risk of insertional mutagenesis.
      2. Low transduction efficiency? Too high transduction? See notes in Table 2.

  5. Transduction of patient derived xenograft cells with pLentiCRISPR lentiviral particles (Timing 4 d)
    Notes:
    1. This protocol is optimized for human patient derived xenograft ALL cells (PDX). For some PDX it might be sufficient to use the transduction protocol with polybrene.
    2. Work with PDX is regulated by the Declaration of Helsinki. You should seek approval for animal experiments from your local animal care authority.
    Day 1:
    1. Thaw frozen PDX cells quickly in the water bath and transfer to a 15 ml Falcon tube as soon as the suspension is solvent.
    2. Add 10 times volume of cold RPMI medium drop by drop to thawed cells suspension, gently shake the tube when adding.
    3. Wash cells by centrifugation and resuspension in fresh medium.
    4. Count and seed 1 million cells per well in a 24 well plate in 1 ml medium and incubate at 37 °C overnight.
      Note: Low viability? See notes in Table 2.
    5. Dilute RetroNectin to a final concentration of 0.05 mg/ml in PBS. RetroNectin can be aliquoted and frozen at -20 °C. Solution can be reused for 5 times. Thaw solution quickly in the water bath and put on ice afterwards.
    6. Coat a 6 well plate with 2 ml RetroNectin solution for either 2 h at room temperature or at 4 °C overnight.

    Day 2:

    1. Remove the RetroNectin solution and block for 30 min with 2 ml 2% (v/v) BSA/PBS at room temperature. You can transfer the RetroNectin to a fresh plate to coat for the second transduction round.
    2. Wash the plate with 3 ml HBSS/2.5% (v/v) HEPES per well.
    3. Thaw viral supernatant quickly in the water bath and keep it on ice as soon as it is melted.
      Note: For multiple target knockouts mix viral particles to transduce with the mixture in one step or perform serial transduction/transplantation cycles. In particular for triple and quadruple knockouts serial transduction can improve efficiencies.
    4. Discard the washing buffer from the plate and add 2 ml viral supernatant.
    5. Centrifuge the plate for 20 min at 4 °C and 750 x g.
    6. Discard the viral supernatant but do not allow the plate to dry.
    7. Repeat steps E10-E12 (Part II) for 3 times.
    8. Transfer target cells to virus coated plate and centrifuge for 2 min at 4 °C and 750 x g.
    9. Incubate at 37 °C overnight.

    Day 3:

    1. Wash the cells 3 times with 10 ml of PBS by centrifugation for 5 min at 750 x g, RT.
    2. Transplant the cells directly into immunocompromised mice.
    3. An aliquot of primary PDX cells can also be transferred onto mesenchymal stroma feeder cells to maintain their survival in vitro for later analysis.

    Day 4:

    1. 48 h after transduction cells can be tested by flow cytometry for fluorescent protein expression to determine the transduction efficiency.
      Note: Fluorescence maximum might be reached between Day 3 and Day 5, but the life span of primary cells in vitro can be shorter. We recommend transplanting cells before the transduction efficiency is directly measured via flow cytometry.


Part III. Expansion and confirmation of knockout clones

  1. Expansion of knockout clones in NSG mice (Timing transplantation 2 h, engraftment weeks to months)
    1. Count the cells and spin down the required number. Good transplantation rates can be obtained with 1 million cells, but as few as 100 cells can successfully engraft.
    2. Resuspend cells in 100 µl PBS per injection.
    3. Transplant cells via intravenous or intrafemural transplantation (for more details see Schmitz et al., 2011).
      Note: Institutional animal care guidelines must be followed. Animal experiments performed in the development of this protocol were approved by the veterinary office and the ethics commission of the Canton of Zurich, Switzerland.
    4. Keep mice under antibiotic treatment until termination of the experiment.
    5. Follow up the engraftment by regular bleeding, according to local legal regulations. We use approximately 20 µl of blood taken via tail vein puncture and a Microvette tube with lithium heparin.
    6. Dilute blood with PBS to 100 µl and add 900 µl red blood cell lysis buffer.
    7. Incubate for 5 min at 4 °C.
    8. Centrifuge at 750 x g for 5 min at 4 °C.
    9. Wash cell pellet with 500 µl PBS and centrifuge again.
    10. Resuspend in 100 µl PBS with anti-human CD19-PECy7 antibody, as it will not interfere with any of the fluorescent proteins used here (dilution 1:1,000).
    11. Incubate for 20-30 min at 4 °C.
    12. Add 500 µl PBS and centrifuge again.
    13. Resuspend in 150 µl PBS and analyse by flow cytometry.
    14. Calculate the engraftment with following formula.



      CAUTION: Criteria for euthanasia have to be applied according to the animal experimentation approval as approved by the responsible authorities.
      Notes:
      1. Depending on the PDX human engraftment can be detected in the peripheral blood already after a few days, but spleen is small. Other patients show high engraftment in the bone marrow and spleen but only low levels in the blood. For each PDX we recommend checking the blood regularly and to palpate the spleen.
      2. No engraftment? See notes in Table 2.
    15. To generate pure patient-derived knockout ALL cells, we suggest to do serial transplantations after sort amplification.
    16. To harvest the PDX cells from the spleen, sacrifice the mouse according to institutional standards and isolate the spleen from the abdomen.
    17. Transfer the tissue to a cell strainer, force spleen through the strainer and wash with 10 ml PBS to create a single cell suspension.
    18. Dilute cell suspension 1:1 with RBC buffer and incubate for 5 min at 4 °C.
    19. Centrifuge at 750 x g for 5 min at 4 °C and resuspend pellet in PBS.
    20. Count the cells and prepare for flow cytometry sort.
      PAUSE POINT: PDX cells can be stored at -80 °C (short term) or in liquid nitrogen (long term). For this spin down the cells and resuspend up to 50 million cells in freezing medium and freeze carefully in a freezing container.

  2. Flow cytometry sorting of single and multi-knockout cells (Timing 2 h)
    1. Pellet cells by centrifugation at 750 x g for 5 min at RT and resuspend at 5 million cells per ml in PBS, or a cell concentration recommended by flow facility.
    2. Filter cell suspension through cell strainer cap of a round-bottom tube.
    3. Use hierarchical gating to sort populations of single, double, triple, and/or quadruple positive cells (see below Data analysis).
    4. In case of low transduction efficiencies follow a serial sorting strategy to first enrich single or double positive populations with an inclusive gating strategy. Cells can then be regrown and later sorted to isolate multi-knockout cells (Figure 6).


      Figure 6. Sorting strategy for double and triple positive CRISPR cells. A. To purify single, double and triple transduced cells that are represented with a low frequency, first sort for one color. This population will include single, double and triple positive cells. B and C. After expansion in vitro in cell culture or in vivo in the mouse, cells can be resorted by flow cytometry specifically for the double and triple positive populations.

    5. Return cells to culture conditions, or re-transplant into mice for later resorting and/or confirmation of knockout.
    6. For cell lines we recommend sorting single cell clones.
    7. For this prepare a 96 well plate with 100 µl medium in each well and sort one cell per well.
    8. Culture the cells until cell numbers are high enough to confirm the knockout by Western blot. Note that we have observed better knockout frequencies in sorted CRISPR containing cells after at least 3 weeks (and as long as 6 weeks) in culture, although this likely varies between different cell types.

  3. Confirmation of gene knockout by Western blot (Timing 2 d)
    Day 1:
    1. Count the cells and collect them in a 1.5 ml tube. Centrifuge at 750 x g for 5 min at RT and wash the pellet with PBS.
    2. Pellet the cells again and add 1x SDS loading buffer at a concentration of 300,000 cells in 80 µl.
      Note: It is not recommended lysing less than 100,000 or more than 1 million cells.
    3. Completely lyse cells by vortexing and denature the proteins by incubating the samples at 95 °C for 5 min in a thermomixer.
      PAUSE POINT: After lysis samples can be stored at -20 °C for up to several months before later analysis.
    4. Assemble a Western blot chamber with a precast gel according to the manufacturer’s instructions and remove the comb.
    5. Pour fresh 1x MES running buffer to the top posterior chamber and fill it completely. Pour 1x MES buffer to the front chamber until the mark or until the lower end of the comb.
      Note: The 1x MES running buffer used in the top chamber must be fresh. For the front chamber the buffer can be reused for up to 3 times.
    6. Wash the wells to remove excess of storage buffer and loose pieces of gel by pipetting up and down the running buffer inside each well with a volume of around 100 µl.
    7. Load the cell lysates on the gel.
      1. For a 26 well gel load 10-15 µl of lysate per well and 3 µl of protein ladder.
      2. For an 18 well gel load 20-30 µl of lysate per well and 5 µl of protein ladder.
    8. Load the same volume of 1x SDS loading buffer in the empty wells to make the samples run straighter.
    9. Cover the Western blot chamber and run the gel at a constant voltage of 120 to 140 V (it will take 1 h 30 min to 45 min approximately) until the blue mark of the samples reaches the end of the gel.
    10. Remove the gel from the chamber and open the plastic covers carefully with a spatula.
    11. Remove the top part including the wells until over the top band of the marker. Assemble the transfer stack according to manufacturer’s instructions.
    12. Transfer the proteins from the gel to the membrane. We routinely use a Bio-Rad protocol optimized for high molecular weight proteins: 2.5 A and 25 V for 10 min.
      Note: Avoid touching the membrane with your hands.
    13. To check that the loading was even and the transfer was complete, incubate the membrane with a Ponceau S solution for 1 min and wash it with tap water.
    14. Wash the membrane once with TBS-T until most of the Ponceau S stain is removed and block the membrane for 1 h with 5% milk in TBS-T.
    15. Incubate the membrane with the primary antibody diluted according to manufacturer’s instructions under agitation either overnight at 4 °C or for 2 h at RT. You can add sodium azide (0.01%, v/v) to store it at 4 °C and reuse it.
    16. Wash the membrane with TBS-T 4 x for 5 min each with agitation at RT.
    17. Incubate with the appropriate secondary HRP-conjugated antibody diluted 1:5,000 in 5% milk in TBS-T for 1 h at RT with agitation.

    Day 2:

    1. Wash the membrane with TBS-T 4 x for 5 min each with agitation at RT.
    2. To acquire the images, cover the membrane with super signal and incubate it for 1 min. Remove excess super signal and place the membrane on a plastic cover. Acquire the chemiluminescence and the white light for the ladder without moving the membrane.
    3. To probe for other proteins in the same membrane, wash it once with TBST-T and strip it with SDS glycine solution for 45 min to 1 h under agitation at RT. Wash it 4 x for 5 min with TBS-T and repeat steps C14-C19 (Part III).
      Note: No protein signal in positive control? No knockout? See notes in Table 2.

Data analysis

The protocol described herein details an efficient method to knockout target genes in a time and cost efficient manner. We have successfully used the pLentiCRISPR Cas9 system to generate up to four knockouts simultaneously. First, a quick screen should be performed to select a sgRNA targeting sequence with good activity for generating a knockout (see introduction for considerations in sgRNA design). Here we have selected three different sgRNAs targeting the RIP1 gene, which were tested in an easily transducable cell line (Figure 7A). Sorting the fluorescent cells by flow cytometry with a subsequent Western blot will show which sgRNA presents the strongest knockout potential (Figure 7B). Here, the knockout inside of exon 6 was more efficient than knockouts in exon 9. Based on this result, a candidate sgRNA can then be used for knockout with target cell lines or PDX material. By freezing viral aliquots at -80 °C, those vectors with the highest activity can be immediately applied after initial selection of a sgRNA sequence with good activity. Four different pLentiCRISPR viral particles can be mixed and matched to enable the knockout of up to four target genes simultaneously (Figure 8). In our example, we were able to show that certain double knockout cells are resistant to treatment with SMAC mimetic birinapant in vivo (for more details see McComb et al., 2016). Figures 8A and 8B shows the distribution of single, double, triple and quadruple knockouts before (A) and after (B) selection with chemotherapeutic drug in vivo. Populations of knockout cells were isolated by fluorescence activated cell sort and retransplanted into NSG mice. After expansion in vivo cells were harvested and lysed for Western blot analysis. Here the knockout of up to four targeted genes in primary leukemia xenografts could be confirmed (Figure 8C).


Figure 7. Validation of sgRNA screen for RIP1 knockout in leukemia cell lines. A. Three different sgRNAs were designed for the RIP1 gene. RIP1.2 is located in exon 6, RIP1.1 and RIP1.3 are located in exon 9. Each sgRNA was cloned and transduced via LentiCRISPR viral particles into 1 million cells at an MOI of < 0.1. B. 5 d after viral transduction, cells were sorted for fluorescent signal and cultured for > 2 weeks in vitro to allow gene knockout to occur and generate enough cell material for Western blot. Western blot was developed with mouse anti-RIP1 (1:1,000) and goat anti-mouse-HRP (1:5,000).


Figure 8. Quadruple knockout in primary human PDX. Primary human acute lymphoblastic leukemia xenografts were transduced either in double or in quadruple combination with RIP3, FADD, MLKL and Caspase8 (CASP8) targeting pLentiCRISPR lentiviral supernatants and directly transplanted into NSG mice. To select for knockout cells mice were treated with birinapant (30 mg/kg) daily (for more details see McComb et al., 2016). A and B. Engraftment was controlled by flow cytometry of peripheral blood. Here we present examples of leukemic engraftment (A) without or (B) with selective birinapant treatment, showing enriched knockout populations. Dot plots show the gating strategy for single and multi-gene knockout cells. First living lymphocytes were defined by forward scatter (FCS) and sideward scatter (SSC). Human engraftment is detected by hCD19 and autofluorescence can be excluded by gating negative cells in an unused channel such as PerCP-Cy5.5 (shown here). Removing autofluorescent cells in this way is extremely helpful for examining rare populations, such as the quadruple positive cells seen here. Subsequently the single or multi-positive cells can be visualized according to their fluorescence (mCherry, BFP, GFP and RFP647). C. To confirm the gene knockout, respective populations were sorted and expanded separately in NSG mice. After harvest, knockout cell lysates were examined via Western blot. Lysates were loaded on two gels in parallel and Western blot was developed in serial detection/stripping steps with following antibodies: rabbit anti-FADD, rat anti-MLKL, rabbit anti-RIP3, mouse anti-CASP8, mouse anti-tubulin, goat anti-mouse-HRP, goat anti-rabbit-HRP, goat anti-rat-HRP. Primary antibody dilution 1:1,000, secondary antibody dilution 1:5,000. *RIP3-specific band.

Notes

Table 2. Troubleshooting advice

Recipes

  1. LB agar plates Combine
    5 g NaCl
    5 g Bacto tryptone
    2.5 g yeast extract
    7.5 g agar and fill up to 500 ml with ddH2O
    Autoclave the solution, cool down, add antibiotics (e.g., Ampicillin 100 µl/ml) and pour into Petri dishes
    Wait until the agar solidifies and store at 4 °C
  2. LB medium Combine
    5 g NaCl
    5 g Bacto tryptone
    2.5 g yeast extract and fill up to 500 ml with ddH2O
    Autoclave solution, cool down and add antibiotics (e.g., Ampicillin 100 µl/ml)
    Store at 4 °C
  3. DMEM complete
    Supplement DMEM with 10% (v/v) FCS, 2% (v/v) L-glutamine, 1% (v/v) sodium pyruvate and 2% (v/v) HEPES
    Store at 4 °C
  4. RPMI medium
    Supplement RPMI with 10% (v/v) FCS, 1% (v/v) L-glutamine and 1% (v/v) Pen/Strep
    Store at 4 °C
  5. Freezing medium
    Supplement FBS with 10% (v/v) DMSO
  6. 3x SDS lysis buffer
    250 mM Trizma HCl pH 6.8
    4% (w/v) SDS
    0.02% (w/v) bromophenol blue
    40% (v/v) glycerol
    4% (v/v) 2-mercaptoethanol
    To make 1x SDS loading buffer dilute 1:3 in PBS
    Aliquots can be stored at -20 °C
  7. Red blood lysis buffer (RBC buffer)
    150 mM NH4Cl
    8 mM KClO3
    0.2 mM EDTA
    Mix and filter solution sterile with 0.22 µm filter and aliquot for freezing at -20 °C
    CAUTION: Avoid freeze and thaw cycles. Thawed aliquots can be stored at 4 °C for up to one month.
  8. 10x TBS
    140 mM NaCl
    26 mM KCl
    250 mM Trizma base
    Adjust pH to 7.4 with HCl
    Store at RT
  9. 1x TBS-T
    Dilute 10x TBS stock with distilled water and add Tween-20 to 0.1% (v/v)
    Store at RT
  10. Ponceau S solution
    Dissolve 0.1% (w/v) Ponceau S in 5% (v/v) acetic acid
    Store at RT
    Solution can be reused
  11. SDS glycine stripping buffer
    0.1 M glycine
    0.5% (w/v) SDS
    Adjust pH to 2.5 (use 12 N HCl)
    Store at RT

Acknowledgments

We are indebted to many colleagues for their kind support. Particularly we want to thank B. Marovca for mouse transplantation support, D. Morf and S. Jenni for flow cytometry sorting, C. Stocking (Heinrich-Pette-Institute) for providing her virus production and transduction protocols. This work was supported by the ‘Stiftung Kinderkrebsforschung Schweiz’, the MAM-Fonds of the Children’s Research Centre of the University Children’s Hospital Zurich, the Empiris foundation, the clinical research focus program ‘human hemato-lymphatic diseases’ of the University of Zurich, the Swiss Cancer League (KFS 3609-02-2015), the Novartis Foundation for Biomedical Research, the Swiss national Science Foundation SNF (310030- 133108), the Canadian institutes for health research CIHR, the Forschungskredit of the University of Zurich (FK-14-016) and the Fondation Panacée.
L.H. and S.M. contributed equally to this manuscript. L.H. and S.M. conceived and designed protocols. S.M., J.A. and L.H. performed experiments. Y.H. contributed to the protocol optimization. M.H., A.A. and P.P. developed the single-pot restriction/insertion protocol. L.H., S.M., J.A., JP.B. and B.B. wrote the manuscript with input from all authors. The authors declare that they have no competing financial interests.
The protocol we describe here is based on methods used in our article entitled ‘Activation of concurrent apoptosis and necroptosis by SMAC mimetics for the treatment of refractory and relapsed ALL’ (McComb et al., 2016).

References

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简介

基于CRISPR-Cas9的敲除策略越来越多地用于分析基因功能。然而,生物信号通路中的冗余和重叠功能可能需要产生多基因敲除细胞,这仍然是一个相对费力的过程。在这里,我们详细介绍了多色LentiCRISPR载体在人体细胞中同时产生单次和多次敲除的应用。我们提供了一个完整的方案,包括指导RNA设计,LentiCRISPR克隆,病毒生产和转导,以及排序和筛选敲除细胞的策略。该过程的有效性通过同时删除白血病细胞系中多达四个程序性细胞死亡介质和来自患者来源的急性淋巴细胞白血病异种移植物,其中单细胞克隆是不可行的。该协议允许任何具有基本细胞生物学设备的实验室,生物安全2级设备和荧光激活细胞分选功能,可在一个月内有效产生单基因和多基因敲除细胞系或原代细胞。

从对细菌基因组中被称为聚簇定期交织的短回文重复(CRISPR)的遗传元件的好奇的初步观察开始(Ishino等人,1987; Mojica等人,2000 )和随后在哺乳动物细胞中的基因编辑(Cong等人,2013; Mali等人,2013),CRISPR-Cas9已经成为廉价和有效的基因编辑。随着从烟草植物细胞到斑马鱼和原代人类细胞(Hsu等人,2014)的细胞系统的成功应用,CRISPR-Cas9可以通过短的20个核苷酸RNA序列的设计来引导在大基因组内的靶向DNA双链断裂(DSB)(Park等人,2016)。 DSB发生后,细胞可以通过高保真同源重组(HR)或易出错的非同源末端连接(NHEJ)启动修复,通常导致导致基因敲除的小插入和缺失(indel)突变(Gaj et al。,2013;Bétermieret al。,2014)(图1)。


图1. CRISPR Cas9的基因组编辑原理。 CRISPR-Cas9基因敲除的原理显示为RIP1序列的示例。 A.单引导RNA(sgRNA)由靶序列特异性crRNA(CRISPR RNA)和恒定tracrRNA(反式激活性crRNA)组成(Jinek等人,2012)。 crRNA与邻近PAM基序的基因组DNA结合,tracrRNA引导Cas9酶到基因座。 B. Cas9介导的DNA双链断裂(DSB)激活非同源末端连接(NHEJ)。 C.不精确的DSB修复导致核苷酸(indel)的增加或减少,其三分之二的机会引起可能导致产生过早终止密码子的移码突变。

已经出现了许多不同的策略来提供Cas9蛋白并将RNA靶向细胞,包括电穿孔或转染携带sgRNA和Cas9有效载荷的Cas9 / sgRNA核糖核蛋白复合物,mRNA,质粒或慢病毒载体(Sander和Joung,2014; Shalem 等,,2014)。以前,这些LentiCRISPR质粒携带抗性基因以允许选择具有CRISPR-Cas9定向基因破坏所必需的机构的组成型表达的细胞。
如我们最近的出版物(McComb等人,2016)所示,我们已经修改了LentiCRISPR方案,用于基于通过荧光组合的选择在细胞系和原代白血病细胞中同时定向破坏若干基因用荧光激活细胞分选(FACS)。通过交换荧光蛋白标记(EGFP,mCherry,tagBFP或RFP657)的嘌呤霉素抗性基因,最多可以同时靶向四个基因用于CRISPR-Cas9介导的基因破坏。荧光激活细胞分选使得能够在一个单一实验步骤中分离出靶向一至四个基因的具有sgRNA的细胞系或原代人细胞。我们的多色LentiCRISPR技术因此允许同时产生携带1到4个基因敲除之间任何地方的敲除细胞,允许在一组感兴趣的基因内快速测试基因 - 基因相互作用。具有四种不同荧光标记的骨架载体以及包括克隆信息在内的本文描述的靶构建体已经沉积在附加物中。
&nbsp;这里我们提供了一个完整的分步指导方案,通过多色LentiCRISPR生成单基因和多基因敲除细胞(参见图2的原理图)。


图2.生成多色LentiCRISPR敲除细胞系和患者衍生异种移植物的程序的示意图

关键字:CRISPR, Cas9, 多基因敲除, 慢病毒, 原发性人白血病, 异种移植物, sgRNA设计

材料和试剂

  1. 过滤的无菌移液器吸头
    10μl(STARLAB INTERNATION,目录号:S1121-3810)
    200μl(STARLAB INTERNATION,目录号:S1120-8810)
    1000μl(STARLAB INTERNATION,目录号:S1126-7810)
  2. TC板
    6井(SARSTEDT,目录号:83.3920)
    24井(SARSTEDT,目录号:83.3922)
    96孔R(SARSTEDT,目录号:83.3925)
  3. 过滤器:Filtropur S
    0.22μm(SARSTEDT,目录号:83.1826.102)
    0.45μm(SARSTEDT,目录号:83.1826)
  4. 血清移液管
    5毫升(SARSTEDT,目录号:86.1253.001)
    10毫升(SARSTEDT,目录号:86.1254.001)
    25毫升(SARSTEDT,目录号:86.1685.001)
  5. Amicon Ultra-15离心过滤器(EMD Millipore,目录号:UFC900308)

  6. 15毫升(SARSTEDT,目录号:62.554.502)
    50毫升(SARSTEDT,目录号:62.547.254)
    13 ml用于细菌培养(SARSTEDT,目录号:62.515.006)
  7. SafeSeal管1.5 ml(SARSTEDT,目录号:72.706)
  8. 硝酸纤维素膜(Trans-Blot Turbo transfer pack)(Bio-Rad Laboratories,目录号:170-4159)
  9. 一次性注射器10 ml(CODAN,目录号:62.6616)
  10. 注射器注射器50ml(Fresenius Kabi,目录号:9000711)
  11. LB板的培养皿(92 x 16毫米)(SARSTEDT,目录号:82.1472.001)
  12. 圆底管,5毫升(康宁,Falcon ®,目录号:352052)
  13. 带有细胞过滤器盖的圆底管,5ml(Corning,Falcon ®,目录号:352235)
  14. TC烧瓶T175,排气。帽(SARSTEDT,目录号:83.3912.002)
  15. Microvette 100 LH,锂 - 肝素(SARSTEDT,目录号:20.1282)
  16. NSG(NOD.Cg- /szJ)小鼠(RRID:IMSR_ARC:NSG),年龄〜4-6周
    注意:必须遵守体制动物护理指南。动物实验由兽医办公室和瑞士苏黎世州伦理委员会批准。
  17. HEK293T细胞(ATCC,目录号:CRL-3216)
    注意:用10%(v/v)FBS的DMEM培养基培养细胞应积极生长。细胞应保持在10-90%汇合之间,每周至少分裂两次。
  18. 靶细胞
    注意:细胞应该在适当的培养基中生长在健康的培养基中。对于大多数ALL细胞系,具有10%(v/v)FBS的RPMI是合适的。原代细胞应在转导前一天解冻。
    注意:原代人细胞必须被认为是潜在的危险。原代衍生的异种移植物从从冷冻保存的骨髓抽吸物回收的人ALL样品获得。这些患者参加了ALL-BFM 2000和ALL-BFM 2009研究。根据"赫尔辛基宣言"通知知情同意书,苏黎世康德伦理委员会批准了批准。
  19. 一击TOP10化学能力大肠杆菌(Thermo Fisher Scientific,Invitrogen TM,目录号:C404010或等同物)
  20. CRISPR靶标质粒,具有Bsm(BI)3I插入位点,例如,
    pLentiCRISPR-EGFP(Addgene,目录号:75159)
    RFP657(Addgene,目录号:75162)
    BFP(Addgene,目录号:75160)
    mCherry(Addgene,目录号:75161)
  21. psPAX2质粒(Addgene,目录号:12260)
  22. p.CMV.VSV.G质粒(Addgene,目录号:8454)
    注意:可能会导致皮肤过敏反应或哮喘症状。
  23. 寡核苷酸100μM,标准合成,脱盐纯化(例如序列参见表1)

    表1. pLentiCRISPR克隆的寡核苷酸序列


  24. 探针缓冲液(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:BY5)
  25. T4 DNA连接酶(New England Biolabs,目录号:M0202M)制备T4 DNA连接酶缓冲液等分试样,仅使用新鲜解冻的等分试样进行反应
  26. 3I限制酶(Thermo Fisher Scientific,Thermo Scientific TM,目录号:ER0451)
    注意:不是NEB BsmBI,BsmBI需要55°C才能有效地切割 - 与本协议不兼容。
  27. SOC培养基(Sigma-Aldrich,目录号:S1797或等同物)
  28. 氨苄青霉素(Sigma-Aldrich,目录号:A9518或等同物)
  29. RED Taq 准备混合PCR反应混合物(Sigma-Aldrich,目录号:R2523或等同物)
  30. 微量和半质粒制备试剂盒(QIAGEN,目录号:27106或等同物)
  31. Tris-acetate-EDTA缓冲液(TAE,25x)(Thermo Fisher Scientific,Ambion TM,目录号:AM9870或等同物)
  32. 聚乙烯亚胺(PEI转染试剂)(Sigma-Aldrich,目录号:408727)
    注意:您必须先测试PEI的准备,才能确定浓度,以获得最佳的转导效率。我们通常使用〜20μg/ml终浓度。
    注意:吞咽有毒。
  33. ProFection哺乳动物转染系统(Promega,目录号:E1200)
  34. 二氯磷酸盐(Sigma-Aldrich,目录号:C6628或等同物)
    注意:吞咽有害。
  35. 氯化钙(CaCl 2)
  36. HBS
  37. 聚己二烯(溴化己二胺)(Sigma-Aldrich,目录号:H9268)储存液在ddH 2 O中8mg/ml
    注意:吞咽有害。
  38. Dulbecco的磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:D1408或等同物)
  39. 使用EDTA的胰蛋白酶(0.05%PBS)(BioConcept,目录号:5-51F00-I或等同物)
  40. 福尔马林(4%甲醛)(Formafix,目录号:01-1010)
  41. 聚乙二醇(PEG 6000)(Sigma-Aldrich,目录号:81253)
  42. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653或等同物)
  43. RetroNectin重组人纤连蛋白片段(Takara Bio,Clontech,目录号:T100B)
    注意:根据制造商的方案准备Retronectin,等分并储存在-20°C。再次使用Retronectin可以重复使用4次而不会降低质量。
  44. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A9418或等同物)
  45. Hank的平衡盐溶液(HBSS)(Thermo Fisher Scientific,目录号:14170088或等同物)
  46. HEPES,1 M(Thermo Fisher Scientific,目录号:15630056或同等品)
    注意:可能会引起皮肤刺激和眼睛刺激。
  47. 流式细胞术抗体(PE-Cy7抗人CD19)(BioLegend,目录号:302216,RRID:AB_314246)
  48. NuPage MES SDS 20x运行缓冲液(Thermo Fisher Scientific,Novex TM,目录号:NP0002-02)用ddH 2 O稀释1:20用于最终浓度
  49. Pageruler预染蛋白梯(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:26616或等同物)
  50. 预制凝胶(标准XT 4-12%Bis-Tris 1.0 mm凝胶)
    18孔(Bio-Rad Laboratories,目录号:3450124)
    26孔(Bio-Rad Laboratories,目录号:3450125)
  51. 非脱脂乳(Sigma-Aldrich,目录号:M7409或等同物)在1x TBS-T中制备5%(w/v)工作溶液。准备新鲜并在4°C短期储存
  52. 叠氮化钠(NaN 3 3)(Sigma-Aldrich,目录号:S2002或等同物)
    注意:吞咽或与皮肤接触是致命的。吞咽会造成器官损伤。对水生生物毒性极大。
  53. 次级HRP结合抗体
  54. 蛋白印迹一抗抗体
    小鼠抗RIP1(BD,BD Biosciences,目录号:551042,RRID:AB_394015)
    兔抗FADD(Cell Signaling Technology,目录号:2782,RRID:AB_2100484)
    小鼠抗微管蛋白(Sigma-Aldrich,目录号:T9026,RRID:AB_477593)
    大鼠抗MLKL(EMD Millipore,目录号:MABC604)
    小鼠抗CASP8(Cell Signaling Technology,目录号:9746,RRID:AB_2068482)
    兔抗RIP3(Abnova,目录号:PAB0287,RRID:AB_1019004)
  55. 与辣根过氧化物酶缀合的Western印迹二抗 山羊抗鼠(Cell Signaling Technology,目录号:7076,RRID:AB_330924)
    山羊抗兔(Cell Signaling Technology,目录号:7074,RRID:AB_10697506)
    山羊抗大鼠(Cell Signaling Technology,目录号:7077,RRID:AB_10694715)
  56. Baytril,拜耳,0.5毫升250毫升高压消毒饮用水(库存2.5%)
  57. Gene Ruler 1 kb Plus DNA梯(Thermo Fisher Scientific,Thermo Scientific TM,目录号:SM1331或等效物)
  58. GelRed,10,000x(Biotium,目录号:41003)
    注意:GelRed通过具有低毒性和高灵敏度优于溴化乙锭。
  59. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:221465或等同物)
    注意:这是危险的。造成严重的皮肤灼伤和眼睛损伤。
  60. SuperSignal ® West Femto最大灵敏度基板(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:34096)
  61. Benzonase Nuclease(Sigma-Aldrich)
  62. Bacto胰蛋白胨(BD,Bacto TM ,目录号:211705或同等物)
  63. 酵母提取物(BD,Bacto TM ,目录号:212750或同等物)
  64. 琼脂(BD,Bacto TM ,目录号:214010或同等品)
  65. 琼脂糖(Eurogentec,目录号:EP-0010-05或等同物)
  66. DMEM培养基(Sigma-Aldrich,目录号:D5546或等同物)
  67. 胎牛血清(FCS)(Sigma-Aldrich,目录号:12133C或等同物)
  68. L-谷氨酰胺,200mM(Thermo Fisher Scientific,Gibco TM,目录号:25030081或等同物)
  69. 丙酮酸钠,100mM(Thermo Fisher Scientific,Gibco TM,目录号:11360070或等同物)
  70. RPMI培养基(Sigma-Aldrich,目录号:R8758或等同物)
  71. 青霉素 - 链霉素(Pen/Strep)(Thermo Fisher Scientific,Gibco TM,目录号:15140122或等同物)
  72. FBS
  73. 二甲亚砜(DMSO)(Sigma-Aldrich,目录号:D8418或等同物)
  74. Trizma HCl(Sigma-Aldrich,目录号:T5941或等同物)
  75. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771或等同物)
    注意:吞咽有毒,与皮肤接触有毒。易燃固体。可能导致眼睛和皮肤刺激,并可能引起呼吸道刺激。
  76. 溴苯酚蓝(Bio-Rad Laboratories,目录号:161-0404或等同物)
  77. 甘油(Sigma-Aldrich,目录号:G5516或等同物)
  78. 2-巯基乙醇(Sigma-Aldrich,目录号:M6250或等同物)
    注意:吞咽或吸入有毒。致命与皮肤接触。造成皮肤刺激和眼睛损伤。
  79. 氯化铵(NH 4 Cl)(Carl Roth,目录号:K298.1或等同物)
    注意:吞咽有害。它会引起严重的眼睛刺激。
  80. 氯酸钾(KClO 3)(EMD Millipore,目录号:104854或等同物)
    注意:吞咽或吸入是危险的,有害的。可能引起火灾,是强氧化剂。
  81. 乙二胺四乙酸二钠盐二水合物(EDTA)(Sigma-Aldrich,目录号:E5134或等同物),制备具有ddH 2 O的0.5M储备溶液,用NaOH调节pH 8.0。 注意:吸入有害。
  82. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541或等同物)
  83. Trizma碱(Sigma-Aldrich,目录号:93352或等同物)
  84. 盐酸(HCl)(Sigma-Aldrich,目录号:H1758或等同物)
    注意:吸入时有毒。造成严重的皮肤灼伤和眼睛损伤。
  85. 吐温20(Sigma-Aldrich,目录号:P9416或等同物)
  86. Ponceau S(Sigma-Aldrich,目录号:P3504)
  87. 乙酸(Sigma-Aldrich,目录号:A6283或等同物)
    注意:易燃,造成严重的皮肤灼伤和眼睛损伤。
  88. LB琼脂板结合(见食谱)
  89. LB培养基组合(参见食谱)
  90. DMEM完成(见配方)
  91. RPMI培养基(见食谱)
  92. 冷冻介质(参见食谱)
  93. 3x SDS裂解缓冲液(参见食谱)
  94. 1x SDS加载缓冲液(参见食谱)
  95. 红血液裂解缓冲液(RBC缓冲液)(参见食谱)
  96. 10x TBS(见配方)
  97. 1x TBS-T(见食谱)
  98. Ponceau S解决方案(见配方)
  99. SDS甘氨酸剥离缓冲液(参见食谱)

设备

  1. 移液器(Gilson)
  2. Pipetus移液助剂(HirschmannLaborgeräte)
  3. 振荡器(Thomas Scientific,型号:Rocker旋转实验室秤TSSL3,目录号:51900-27)
  4. 热循环仪
  5. 微生物培养箱,37℃,大气CO 2(Thermo Fisher Scientific,Heraeus)
  6. HeraCell 150培养箱,37℃,5%CO 2(Thermo Fisher Scientific,型号:HeraCell 150培养箱)
  7. MUPID-One电泳系统(LABGENE Scientific,型号:MUPID-One电泳系统)
  8. 台式离心机(Eppendorf,型号:5417 C)
  9. Nalgene Oak Ridge离心管(Lab Depot,目录号:21009-386-PK)
    注意:这些管适用于超速离心,不能用标准管更换。
  10. 水浴(赛默飞世尔科技)
  11. Thermomixer舒适(Eppendorf)
  12. 涡旋混合器Vortex-Genie 2(Scientific Industries,型号:Vortex-Genie 2)
  13. FACSARIA III流式细胞仪分选仪(BD,型号:FACSAria III)
  14. FACS Canto II流式细胞仪(BD,BD Biosciences,型号:FACSCANTO II)
  15. 凝胶文件(Syngene)
  16. Heraeus Multifuge 3S(Thermo Fisher Scientific,型号:Heraeus TM Multifuge TM 3S)
  17. 用于无菌组织培养的层流罩,生物安全2级认证(Thermo Fisher Scientific)
  18. Mastercycler nexus(Eppendorf)
  19. Frosty先生冰箱(Thermo Fisher Scientific)
  20. Neubauer室(BRAND)
  21. TransBlot Turbo传输系统(Bio-Rad Laboratories,型号:Trans-Blot Turbo TM 传输系统)
  22. Western印迹室(Criterion Cell)和PowerPac基本电源(Bio-Rad Laboratories)
  23. Spatula

软件

  1. 质粒编辑器( http://biologylabs.utah.edu/jorgensen/wayned/ape/)(可选)
  2. 寡核苷酸属性计算器( http://biotools.nubic.northwestern.edu ) (可选)

程序

第一部分。sgRNA设计和准备


  1. sgRNA序列设计(定时1-2小时)
    1. 从基因组数据库(例如,,以FASTA格式的NCBI)下载基因组序列。
    2. 用"质粒编辑"打开你的序列( http://biologylabs .utah.edu/jorgensen/wayned/ape/)或类似的序列分析软件。
    3. 选择CRISPR编辑的目标轨迹。由于CRISPR敲除效率的变化,我们通常可以选择3个不同的外显子区域。有关目标轨迹选择的其他注意事项的讨论,请参见引言
    4. 通过将特定序列输入到在线工具(如 http)中,筛选CRISPR指导RNA结合位点的靶基因座://crispr.mit.edu
      注意:初步工作是用crispr.mit.edu在线工具完成的。但是另一种CRIPSPR计算器,例如 http://crispor.tefor。 net/ 可能会提供额外的预测效果。
    5. 通过基因组中的分数和位置选择指南,以确定目标网站。 Cas9将在PAM位点上游切断3 bp。我们建议选择三种不同的sgRNA来提高实现完全敲除的概率
    6. 复制所选导向RNA的20个核苷酸序列而不使用PAM,并通过使用生物信息学软件工具(如,Oligonucleotide Properties Calculator( http://biotools.nubic.northwestern.edu )。
    7. 对于pLentiCRISPR质粒的连接,向该序列添加另外的核苷酸以产生针对3'端限制性位点(CACC/AAAC)的粘性末端和RNA聚合酶III(单G/C)的转录起始位点。
      寡核苷酸
      附加顺序(5'-3')
      Fw_sg
      CACCG(N 20)
      Rev_sg
      AAAC(N 20)C

    8. 订购正向和反向寡核苷酸(简单的脱盐寡核苷酸对于该方案是足够的)
    9. 参见图3中RIP1的寡核苷酸设计策略示例。

  2. 单链寡核苷酸的退火(定时2.5小时)
    1. 对于冻干的寡核苷酸,加入适当体积的ddH 2 O以获得100μM的储备浓度。
    2. 为每对正向和反向寡核苷酸移取以下反应
      组件
      金额
      Fw_sg oligo
      10μl
      Rev_sg oligo
      10μl
      探戈缓冲区
      10μl
      ddH 2 O
      70μl

    3. 在热循环仪中达到95°C 5分钟。
    4. 通过1-2小时冷却至室温退火寡核苷酸
  3. 将双链寡核苷酸连接到pLentiCRISPR(定时6小时)
    1. 设置用于质粒限制的单釜反应并将退火的双链寡核苷酸连接到pLentiCRISPR中。
      组件
      金额
      pLentiCRISPR
      150 ng
      双链寡核苷酸(来自步骤B4,第一部分)
      1μl
      NEB T4连接酶缓冲液(10x)
      2μl
      ddH 2 O
      高达18μl
      E E E E E E s s>>>> p>>>>>。。。。。。。。。。 0.5μl
      T4 DNA连接酶
      0.5μl

    2. 在热循环仪中运行以下反应。
       
      Cy cle
      Temperat u re
      蒂姆 e
      连接(15个周期)
      37°C
      5分钟
      16°C
      10分钟
      最终
      37°C
      15分钟
      80°C
      5分钟
      握住
      4°C

      注意:pLentiCRISPR-BFP载体中tagBFP序列中还有一个额外的BsmBI(Esp3I)位点。对于该载体,最终的37°C步骤应从循环方案中移除,以提高产量。
      暂停点:在Esp3I热灭活后,质粒可以立即转化成细菌或储存在-20°C。

  4. 用pLentiCRISPR和双链寡核苷酸插入片段转化和鉴定细菌克隆(定时3 d)
    第1天:
    1. 将结扎混合物转化为感染性细菌
    2. 将2μl连接混合物加入冰冷的感受态细菌(50μl)中 注意:不要使用超过5μl的连接产物转化50μl感受态细菌。
    3. 将混合物在冰上孵育20分钟,并在42℃下热休克90秒
    4. 在冰上冷却2分钟。
    5. 加入200μlSOC培养基(无抗生素),37℃温和摇动孵育45分钟
    6. 在含有100μg/ml氨苄青霉素的LB平板上铺板100μl细菌悬浮液。
    7. 在微生物培养箱中37℃孵育过夜。

    第二天:

    1. 每转化的pLentiCRISPR对3-5个菌落进行菌落PCR。
      注意:没有殖民地?见表2中的注释。
    2. 准备单独的LB琼脂平板,每个克隆的数字将被挑选。
    3. 准备PCR反应混合物。使用Fw_U6作为正向引物,使用序列特异性的Rev_sg寡核苷酸作为反向引物
      通讯 ponent
      Amo unt
      RED Taq Ready混音(2x)
      5μl
      ddH 2 O
      4μl
      Fw_U6(10μM)
      0.5μl
      Rev_sg oligo(10μM)
      0.5μl

    4. 用移液管尖端挑取单个菌落,在单独的编号板上条纹,随后将移液管尖端浸入PCR反应缓冲液的一个孔中。
    5. 摘取所有菌落后,取出移液管吸头,进行PCR,并在37℃下将带有条纹的编号板在微生物培养箱中放置2-5小时。
    6. 菌落PCR程序:
      Cy cle
      Temp erature
      时间
      初始 
      95°C
      3分钟
      周期(25个周期)
      95°C
      30秒
      55°C
      30秒
      72°C
      10秒
      最终
      72°C
      10分钟
      握住
      4°C


    7. 在琼脂糖凝胶(2%[w/v]琼脂糖/TAE缓冲液)上加载PCR反应(参见图5的估计产物)。
      注意:没有阳性菌落?殖民地太多了?见表2中的注释。


      图5.集落PCR产物的实例具有插入的双链寡核苷酸的pLentiCRISPR克隆产生约100bp的条带。空载体不应该没有带,只会显示引物二聚体。

    8. 从编号的平板上挑取阳性克隆,在微生物培养箱中于37℃在LB培养基中开始过夜培养。

    第3天:

    1. 根据制造商的方案,通过微型或半制备分离质粒DNA。
      暂停点:质粒可以立即转入HEK293T细胞中o > 在4°C或-20°C

第二部分高级病毒 pro 功能 和感染

  1. 生产pLentiCRISPR慢病毒颗粒(定时3-5天)
    第1天:
    1. 下午种子500,000 HEK293T细胞每6孔板在2ml DMEM中完成。
      注意:保持HEK293T细胞始终 我们也建议无抗生素工作,因为我们通常在介质中达到较高的抗生素效价。该协议详细说明适用于细胞系应用的中试规模的病毒上清液(约6-9ml)的生产,但可以使用T175烧瓶扩大以产生较大的批次
    2. 孵育过夜以使HEK293T细胞粘附并平衡。

    第二天

    1. 用慢病毒质粒(pLentiCRISPR,psPAX2和pVSV.G)转染HEK293T细胞。我们建议使用以下两种技术之一。
      注意:
      a。在加入转染试剂时,请小心不要打扰细胞。
      b。对于LentiCRISPR:psPAX2:pVSV.G,我们使用8:3:3的比例,得到4μgpLentiCRISPR和每个psPAX2和p.CMV.VSV.G每孔1.5μg。但是应该滴定质粒比。
      1. PEI转染试剂
        1. 将所有试剂解冻至室温(RT,约22°C)。
        2. 将质粒稀释在1毫升无血清,无抗生素的DMEM中
        3. 加入20μlPEI试剂,立即涡旋
        4. 在室温下孵育PEI /质粒混合物20分钟。
        5. 将混合物直接滴加到HEK293T细胞中。
      2. ProFection哺乳动物转染系统
        1. 将所有试剂解冻至RT。
        2. 用3ml新鲜无抗生素的DMEM完全替换细胞培养基,加入3μl氯喹(终浓度25μM)。
        3. 将质粒与2M CaCl 2混合,并加入高达150μl的ddH 2 O。
        4. 轻轻加入DNA-CaCl 2水溶液至150μlHBS,同时涡旋
        5. 在室温下孵育30分钟。
        6. 将混合物直接滴加到HEK293T细胞中。

    te:所有后续步骤均符合生物安全二级法规。在生物安全柜内进行所有工作,不要使用真空吸吸上清液,以免气雾化病毒

    1. 4-6小时后,取出转染培养基,小心地用3ml预热的DMEM置换。对于PEI转染,不需要中等程度的变化,对细胞无明显毒性。

    第3天:

    1. 第二天早上通过荧光显微镜检查转染效率。在大多数细胞中,一些荧光信号应该是可见的,尽管信号可以增加长达72小时 注意:无荧光?见表2中的注释。
    2. 从HEK293T细胞收集上清液,并用3ml预热的DMEM完全培养基代替。将病毒上清液保留在冰上
    3. 将HEK293T细胞放回培养箱(5%CO 2)至第二天。
    4. 通过0.45μm过滤器过滤上清液,以清除任何剩余的细胞,并冷冻100μl的等分试样,以在-80°C下进行滴定试验。
    5. 上清液可以保存在4°C,以便将以后收集的上清液合并 注意:不要使用较小的过滤器尺寸,因为它会降低病毒滴度

    第4天:

    1. 重复步骤A6-A9(第二部分)。

    第5天:

    1. 重复步骤A6-A9(第二部分)。

    你可以在转染后72小时收获病毒上清液,但48小时后病毒滴度会下降。控制HEK293T细胞的外观,细胞不应大量过量。转染后24 h通常达到最高滴定度。
    暂停点:上清液可立即浓缩(参见步骤C1,第II部分)或等分并在4℃下储存长达1周,或在-80℃下以病毒滴度(> 1年)略有降低。应避免冷冻和解冻循环,因为它会降低滴定度。

  2. 滴定pLentiCRISPR慢病毒颗粒(定时3 d)
    注意:对于sgRNA功能的初步筛选,病毒上清液可以直接用于靶细胞而不进行滴定,但是建议仍然进行滴定,因为病毒等分试样可以储存并用于后续实验。
    第1天:
    1. 在50孔板中每孔用0.5ml DMEM板中的50,000个HEK293T细胞板
    2. 一式两份。
      注意:HEK293T滴度是感染单位的相对测量,因此我们建议在相同条件下(孵育时间,体积等)与HEK293T细胞一起工作。稳定的过程可以比较不同生产之间的滴度。
    3. 加入0.5μl聚凝胺(终浓度为8μg/ml)
    4. 在孵育器(5%CO 2)中孵育HEK293T细胞6小时。
    5. 将增加的病毒上清液加入培养基(20μl-100μl未浓缩的病毒)中

    注意:所有后续步骤均符合生物安全二级法规。不要使用真空泵抽吸上清液以避免气雾化病毒。
    注意:如果这是首次使用广泛的浓度(病毒上清液的1/10至1/100,000相对稀释度),以确保数据可评估。

    1. 将24孔板离心1小时,750×RT,并在培养箱(5%CO 2)中保持过夜。

    第二天:

    1. 用0.5ml新鲜DMEM置换培养基,孵育48h。

    第3天:

    1. 去除上清液并用PBS洗涤细胞。
    2. 加入0.5 ml胰蛋白酶/EDTA,37℃孵育5 min,直至细胞从表面脱离。
    3. 加入0.5ml DMEM完成,重悬细胞并转移到FACS管中。
    4. 旋转5分钟,750 g ,RT。
    5. 去除上清液并将细胞重悬于含有2%(v/v)福尔马林的0.5ml PBS中。
    6. 通过流式细胞术测量转导效率。
    7. 病毒转导的线性范围在5-30%之间。仅对所包含的井计算滴定度。滴定度用下式计算。有关详细信息,请参阅Weber e 。 (2012)。



      注意:
      1. 在本方案中描述的程序中,我们通常希望实现显示超过1×10 5 cfu/ml的cfu/ml的病毒上清液。 /em>
      2. 低滴度?见表2中的注释。

  3. 慢病毒颗粒浓度(定时1-8小时)
    1. 为了提高滴度,因此可以通过(a)柱,(b)超速离心或(c)PEG 6000沉淀来浓缩病毒。
      1. 浓度由Amicon离心过滤器单位
        1. 应优化离心速度和时间。
        2. 我们建议将15ml病毒上清液加载到上腔室中,并在4℃下以1800°K离心10分钟。
        3. 病毒上清液体积应降至1 ml
        4. 浓度指数1:15。
      2. 浓缩超离心
        1. 将最多40ml病毒上清液加载到Nalgene Oak Ridge离心管中
        2. 离心6小时,8,000℉,4℃。
        3. 之后,透明果冻颗粒可见病毒。
        4. 仔细弃去上清液,并通过大力吸取重悬浮颗粒在您选择的1ml培养基中
        5. 浓度指数:1:40
      3. PEG 6000沉淀浓度
        1. 第1天。向5ml病毒上清液中加入1.3ml 50%PEG 6000溶液,0.54ml 4M NaCl和0.59ml PBS。最终的PEG 6000浓度将为8.5%,最终的NaCl浓度将为〜0.3M
        2. 混合内容并在4℃下保存过夜。
        3. 第二天离心混合物在2,000℉,4℃下离心30分钟。
        4. 离心后可见白色病毒颗粒。
        5. 小心地倒出上清液,并将沉淀物重悬于100-50通过大量吸取液体上下液体培养基。
        6. 浓度指数1:50。

  4. 用pLentiCRISPR慢病毒颗粒转导细胞系(定时4 d)
    注意:本协议针对人类白血病细胞系(如Jurkat)进行了优化。请参阅下面的原始白血病细胞的优化协议(st
    ep E1,Part II)。
    注意:所有后续步骤均符合生物安全二级法规。不要使用真空泵抽吸上清液以避免气雾化病毒。
    第1天:
    1. 计数靶细胞,并将100万个细胞置于150μlRPMI培养基中,每孔在24孔板中。
    2. 以8μg/ml的浓度将聚凝胺加入靶细胞。
    3. 在水浴中快速解冻病毒上清液,一旦融化即可将其保存在冰上
    4. 根据所需的MOI(多重感染),在细胞顶部加入50μl-1ml的病毒上清液。
      注意:对于多个目标敲除,将病毒颗粒与混合物一步转化或进行连续转导。特别是对于三重和四重敲除序列转导可以提高效率。
    5. 轻轻摇动板,将细胞与病毒上清液混合
    6. 2小时后,用新鲜的RPMI培养基升至2毫升
    7. 在37°C孵育过夜。

    第二天:

    1. 离心细胞并通过移液轻轻去除上清液,并用1ml新鲜RPMI替代。

    第3天:

    1. 重复步骤D1-D8(第二部分)。如果需要更高的转导率,您可以重复病毒转导三次。

    第4天:

    1. 取一小部分细胞并加入相同体积的4%福尔马林(终浓度2%)并在冰上孵育10分钟来固定。继续通过流式细胞术确定转导效率。
    2. 对于纯的敲除细胞系,我们建议通过限制稀释或流式细胞术分类进行单细胞克隆。但是对于敲除效率和敲除生物表型的第一次验证,我们建议在一轮或两轮流式细胞术分选后使用大量群体。
      注意:
      1. 细胞不应该以超过70-80%的效率转导,因为这会导致多次插入,毒性和更高的插入突变风险。
      2. 低传导效率?转导过高?见表2中的注释。

  5. 用pLentiCRISPR慢病毒颗粒转导患者衍生的异种移植细胞(时间4 d)
    注意:
    1. 该方案针对人类患者衍生的异种移植物ALL细胞(PDX)进行了优化。对于某些PDX,使用polybrene的转导方案可能就足够了。
    2. 与PDX合作由赫尔辛基宣言所规定。您应该从当地的动物保健机构获得动物实验的批准。
    第1天:
    1. 解冻冷冻的PDX细胞在水浴中快速转移到一个15毫升的Falcon管,一旦悬浮液溶剂。
    2. 向解冻的细胞悬液中逐滴加入10倍体积的冷RPMI培养基,加入时轻轻摇动管。
    3. 通过离心清洗细胞并重新悬浮于新鲜培养基中
    4. 在1ml培养基中的24孔板中计数并种胚100万个细胞,并在37℃下孵育过夜。
      注意:生存能力低下见表2中的注释。
    5. 在PBS中稀释RetroNectin至终浓度为0.05mg/ml。 RetroNectin可以在-20℃下等分并冷冻。解决方案可以重复使用5次。在水浴中迅速解冻,然后放在冰上。
    6. 将6孔板用2ml RetroNectin溶液在室温或4℃过夜2小时。

    第二天:

    1. 去除RetroNectin溶液,并在室温下用2ml 2%(v/v)BSA/PBS封闭30分钟。您可以将RetroNectin转移到新鲜的盘子上以进行第二次转导。
    2. 用3ml HBSS/2.5%(v/v)HEPES /孔清洗板。
    3. 在水浴中快速解冻病毒上清液,一旦融化即可将其保存在冰上 注意:对于多个目标敲除,将病毒颗粒在一个步骤中混合混合物,或进行连续转导/移植循环。特别是对于三重和四重敲除序列转导可以提高效率。
    4. 从板中弃去洗涤缓冲液,加入2ml病毒上清液。
    5. 在4℃和750×g/g下将板离心20分钟。
    6. 丢弃病毒上清液,但不要让其干燥。
    7. 重复步骤E10-E12(Part II)3次。
    8. 将靶细胞转移到病毒包被的平板上,并在4℃和750×g/g下离心2分钟。
    9. 在37°C孵育过夜。

    第3天:

    1. 通过在750×RT,RT下离心5分钟,用10ml PBS洗涤细胞3次。
    2. 将细胞直接移植到免疫受损小鼠中。
    3. 原始PDX细胞的等分试样也可以转移到间充质基质饲养细胞上以维持其生存。我我 > t r o 供以后分析。

    第4天:

    1. 通过流式细胞仪检测转导细胞48 h后的荧光蛋白表达,以确定转导效率 注意:在第3天和第5天之间可能会达到荧光最大值,但是体外原代细胞的寿命可以更短。我们建议在转导之前移植细胞通过流式细胞仪直接测量效率。


第三部分。敲除克隆的扩增和确认

  1. NSG小鼠中敲除克隆的扩增(定时移植2 h,移植周至数月)
    1. 计数细胞并旋转所需数量。可以用100万个细胞获得良好的移植率,但可以成功移植至少100个细胞。
    2. 每次注射后将细胞重悬于100μlPBS
    3. 移植细胞通过静脉内或心内移植(更多细节参见Schmitz等人,2011)。
      注意:必须遵守机构动物保健指南。在该协议的开发中进行的动物实验得到了瑞典苏黎世州的兽医办公室和伦理委员会的批准。
    4. 将小鼠置于抗生素治疗下,直到实验终止。
    5. 根据当地法律规定,定期出血,随访入院。我们使用约20μl通过尾静脉穿刺的血液和一个含有肝素肝素的微卫生管。
    6. 用PBS将血液稀释至100μl,加入900μl红细胞裂解缓冲液。
    7. 在4°C孵育5分钟。
    8. 在4℃下以750×离心机离心5分钟。
    9. 用500μlPBS洗涤细胞沉淀,再次离心
    10. 用抗人CD19-PECy7抗体重悬于100μlPBS中,因为它不会干扰任何这里使用的荧光蛋白(稀释度1:1,000)。
    11. 在4°C孵育20-30分钟。
    12. 加入500μlPBS,再次离心
    13. 重悬于150μlPBS中,用流式细胞术分析
    14. 用以下公式计算植入:



      注意:安乐死的标准必须根据负责当局批准的动物实验批准进行。
      注意:
      1. 其他患者在骨髓和脾脏中显示高植入,但在血液中仅显示低水平。对于每个 PDX ,我们建议定期检查血液并触诊脾脏。
      2. 没有移植?见表2中的注释。
    15. 为了产生纯粹的患者来源的敲除ALL细胞,我们建议在分类扩增后进行连续移植。
    16. 从脾脏收获PDX细胞,按照制度标准处死小鼠,并从腹部分离脾脏。
    17. 将组织转移至细胞过滤器,强制脾穿过过滤器,并用10ml PBS洗涤以产生单细胞悬浮液。
    18. 用RBC缓冲液稀释细胞悬液1:1,4℃孵育5分钟
    19. 在4℃离心5分钟,并将沉淀重悬于PBS中。
    20. 计数细胞并准备流式细胞术排序。
      暂停点:PDX细胞可以储存在-80°C(短期)或液氮(长期)。为了将细胞旋转下来,在冷冻介质中重新悬浮多达5000万个细胞,并在冷冻容器中小心地冷冻。

  2. 单次和多次敲除细胞的流式细胞术分选(定时2小时)
    1. 通过在750℃下离心5分钟并在PBS中以500万个细胞/ml重悬,或由流动设备推荐的细胞浓度重悬。 >
    2. 通过圆底管的细胞过滤器盖过滤细胞悬浮液。
    3. 使用分层门控来排序单,双,三和/或四倍阳性细胞的群体(参见下面的数据分析)。
    4. 在传导效率低的情况下,采用序列分选策略,首先用包含门控策略丰富单个或双重阳性人群。然后可以再培养细胞,然后再分选细胞以分离多重敲除细胞(图6)

      图6.双重和三重阳性CRISPR细胞的分选策略。 A.为了纯化用低频率表示的单次,双重和三次转导的细胞,首先分选一种颜色。这个人群将包括单一,双重和三重阳性细胞。 B和C.扩展后 > v o ,细胞可以通过流式细胞仪专门用于双重和三重阳性人群。

    5. 将细胞返回到培养条件,或者重新移植到小鼠中以备后续使用和/或确认敲除
    6. 对于细胞系,我们建议对单细胞克隆进行排序。
    7. 为此,准备96孔板,每个孔中加入100μl培养基,并对每孔一个细胞进行分选
    8. 培养细胞直到细胞数量足够高以通过蛋白质印迹确认敲除。请注意,在培养至少3周(长达6周)后,我们已经观察到在含CRISPR的细胞中有更好的敲除频率,尽管这可能因不同的细胞类型而异。

  3. 通过蛋白质印迹确认基因敲除(定时2 d)
    第1天:
    1. 计数细胞并将其收集在1.5 ml管中。在室温下离心750分钟5分钟,并用PBS洗涤沉淀。
    2. 再次沉淀细胞,加入浓度为30万细胞的1×SDS加载缓冲液(80μl) 注意:不建议使用小于100,000或超过100万个细胞裂解。
    3. 通过在95℃在温热混合器中温育样品5分钟来涡旋和变性蛋白质来完全裂解细胞。
      暂停点:溶解后的样品可以在-20°C下储存多达几个月,然后再分析。
    4. 按照制造商的说明装配带有预制凝胶的Western印迹室,并取出梳子
    5. 将新鲜的1x MES运行缓冲液倒入顶部后室,并将其完全填充。将1x MES缓冲液倒入前室,直到标记或直到梳子的下端。
      注意:顶部室中使用的1x MES运行缓冲区必须是新鲜的。对于前室,缓冲液可重复使用3次。
    6. 通过用大约100μl的体积吸取每个孔内的运行缓冲液,洗涤孔以除去过量的储存缓冲液和松散的凝胶块。
    7. 将细胞裂解液装入凝胶上
      1. 对于26孔凝胶负载每孔10-15微升裂解物和3微升蛋白质梯
      2. 对于18孔凝胶载体,每孔20-30μl裂解物和5μl蛋白质梯度。
    8. 在空井中加载相同体积的1x SDS加载缓冲液,使样品运行更直。
    9. 覆盖Western印迹室,并以120至140V的恒定电压运行凝胶(约1小时30分钟至45分钟),直到样品的蓝色标记达到凝胶的末端。
    10. 从室中取出凝胶,并用刮刀小心地打开塑料盖。
    11. 移除包括孔在内的顶部,直到标记的顶部带。按照制造商的说明组装转移堆栈。
    12. 将蛋白质从凝胶转移到膜上。我们通常使用针对高分子量蛋白质优化的Bio-Rad方案:2.5A和25V 10分钟 注意:避免用手接触膜。
    13. 为了检查负载是否均匀,转移是否完成,用Ponceau S溶液孵育膜1分钟,并用自来水冲洗。
    14. 用TBS-T清洗膜一次,直至去除大部分的Ponceau S染色,并用TBS-T中的5%牛奶将膜封闭1小时。
    15. 根据制造商的说明书,在4℃下搅拌过夜或在室温下搅拌2小时,将膜与初级抗体一起孵育。您可以加入叠氮化钠(0.01%,v/v)将其储存在4°C并重新使用。
    16. 用TBS-T 4 x洗涤膜5分钟,同时在室温下搅拌
    17. 与TBS-T中的5%乳中1:5,000稀释的合适的次级HRP缀合的抗体在室温下搅拌孵育1小时。

    第二天:

    1. 用TBS-T 4 x洗涤膜5分钟,同时在室温下搅拌
    2. 要获取图像,用超级信号覆盖膜并孵育1分钟。去除多余的超级信号,并将膜放在塑料盖上。获取梯子的化学发光和白光,而不移动膜。
    3. 为了探测同一膜中的其他蛋白质,用TBST-T洗涤一次,并在室温搅拌下用SDS甘氨酸溶液洗脱45分钟至1小时。用TBS-T洗涤4×5分钟,重复步骤C14-C19(第三部分)。
      注意:阳性对照中没有蛋白质信号?没有淘汰赛见表2中的注释。

数据分析

本文描述的方案详细描述了以时间和成本有效的方式敲除靶基因的有效方法。我们已经成功地使用pLentiCRISPR Cas9系统同时生成多达四个敲除。首先,应该进行快速筛选以选择具有良好活性以产生敲除的sgRNA靶向序列(参见关于sgRNA设计中的注意事项的介绍)。在这里,我们选择了三种不同的靶向RIP1基因的sgRNA,它们在易于转导的细胞系中进行了测试(图7A)。通过流式细胞术用随后的蛋白质印迹法分选荧光细胞将显示哪种sgRNA具有最强的敲除电位(图7B)。在这里,外显子6内的敲除比外显子9中的敲除更有效。基于这个结果,候选sgRNA可以用于与靶细胞系或PDX材料敲除。通过在-80℃冷冻病毒等分试样,可以在初始选择具有良好活性的sgRNA序列之后立即施用具有最高活性的那些载体。可以混合和匹配四种不同的pLentiCRISPR病毒颗粒,以使得能够同时敲除多达四个靶基因(图8)。在我们的例子中,我们能够证明某些双重敲除细胞对体内SMAC模拟双联蛋白的治疗具有抗性(更多细节参见McComb等人,2016) 。图8A和8B显示了在体内化疗药物(A)和(B)选择之后的单,双,三重和四重敲除物的分布。通过荧光激活细胞分选分离敲除细胞的种群并将其重新移植入NSG小鼠。在体内扩增后,收获细胞并裂解以进行蛋白质印迹分析。这可以证实原代白血病异种移植物中多达四个靶基因的敲除(图8C)。


图7.在白血病细胞系中RIP1敲除的sgRNA屏幕的验证A.为RIP1 基因设计了三种不同的sgRNA。 RIP1.2位于外显子6中,RIP1.1和RIP1.3位于外显子9中。通过LentiCRISPR病毒颗粒将每个sgRNA克隆并转导至100万个细胞, 0.1。在病毒转导后5天,将细胞分选荧光信号,并培养> 2周以允许基因敲除发生并产生足够的用于Western印迹的细胞材料。用小鼠抗RIP1(1:1,000)和山羊抗小鼠HRP(1:5000)开发蛋白质印迹。


图8.原代人PDX中的四重敲除。
原代人急性淋巴细胞白血病异种移植物以针对pLentiCRISPR慢病毒上清液的RIP3,FADD,MLKL和Caspase8(CASP8)双重或四重组合转导,并直接移植进入NSG小鼠。为了选择敲除细胞,每天用birinapant(30mg/kg)处理小鼠(更多细节参见McComb等人,2016)。 A和B.通过外周血的流式细胞术控制植入。在这里,我们提出没有或(B)的选择性birinapant治疗的白血病植入(A)的例子,显示富集的敲除种群。点图显示单基因和多基因敲除细胞的门控策略。第一个活体淋巴细胞由前向散射(FCS)和侧向散射(SSC)定义。通过hCD19检测到人类移植物,并且可以通过在未使用的通道(例如PerCP-Cy5.5)中门控阴性细胞来排除自体荧光(如图所示)。以这种方式去除自体荧光细胞对于检查稀有群体非常有帮助,例如这里看到的四倍体阳性细胞。随后,可以根据其荧光(mCherry,BFP,GFP和RFP647)显现单个或多个阳性细胞。 C.为了确认基因敲除,分别在NSG小鼠中分选和扩增各个群体。收获后,通过蛋白质印迹检查敲除细胞裂解物。将裂解物平行装载在两个凝胶上,并在以下抗体的连续检测/剥离步骤中开发蛋白质印迹:兔抗FADD,大鼠抗MLKL,兔抗RIP3,小鼠抗CASP8,小鼠抗微管蛋白,山羊抗 - 鼠-HRP,山羊抗兔-HRP,山羊抗大鼠HRP。初级抗体稀释1:1,000,二抗稀释1:5000。 * RIP3特定频段。

笔记

表2. T 故障排除 建议

食谱

  1. LB琼脂板结合
    5克NaCl
    5克Bacto胰蛋白胨
    2.5克酵母提取物
    7.5g琼脂,并用ddH 2 O +/
    填充至500ml 将溶液高压灭菌,冷却,加入抗生素(例如,,氨苄青霉素100μl/ml),倒入培养皿中
    等待琼脂凝固并储存在4°C
  2. LB培养基组合
    5克NaCl
    5克Bacto胰蛋白胨
    2.5克酵母提取物,并用ddH 2 O +/
    填充至500毫升 高压灭菌溶液,冷却并加入抗生素(例如,氨苄青霉素100μl/ml)
    储存于4°C
  3. DMEM完成
    补充DMEM与10%(v/v)FCS,2%(v/v)L-谷氨酰胺,1%(v/v)丙酮酸钠和2%(v/v)HEPES
    储存于4°C
  4. RPMI媒体
    补充具有10%(v/v)FCS,1%(v/v)L-谷氨酰胺和1%(v/v)Pen/Strep的RPMI
    储存于4°C
  5. 冷冻介质
    补充FBS与10%(v/v)DMSO
  6. 3x SDS裂解缓冲液
    250mM Triszma HCl pH 6.8
    4%(w/v)SDS
    0.02%(w/v)溴酚蓝
    40%(v/v)甘油 4%(v/v)2-巯基乙醇
    使1x SDS加载缓冲液在PBS中稀释1:3
    等分试样可以保存在-20°C
  7. 红血液裂解缓冲液(RBC缓冲液)
    150mM NH 4 Cl
    8mM KClO 3
    0.2 mM EDTA
    混合和过滤溶液无菌,0.22μm过滤器,等分,冷冻-20°C 注意:避免冻融循环。解冻的等分试样可以在4℃下储存长达一个月。
  8. 10x TBS
    140 mM NaCl
    26 mM KCl
    250毫升Trizma基地
    用HCl调节pH至7.4 存储在RT
  9. 1x TBS-T
    用蒸馏水稀释10倍TBS原液,加入Tween-20〜0.1%(v/v) 存储在RT
  10. Ponceau S解决方案
    在5%(v/v)乙酸中溶解0.1%(w/v)Ponceau S 存储在RT
    解决方案可以重复使用
  11. SDS甘氨酸剥离缓冲液
    0.1 M甘氨酸
    0.5%(w/v)SDS
    将pH调节至2.5(使用12 N HCl)
    存储在RT

致谢

我们感谢许多同事的善意支持。特别是我们要感谢B.Marovca的小鼠移植支持,D. Morf和S. Jenni进行流式细胞分选,C.放养(Heinrich-Pette研究所),用于提供她的病毒生产和转导方案。苏黎世大学儿童医院儿童研究中心的MAM-Fonds,苏黎世大学的Empiris基金会,临床研究重点项目"人体血液淋巴系疾病","基督教基金会","瑞士癌症联盟(KFS 3609-02-2015),诺华生物医学研究基金会,瑞士国家科学基金会SNF(310030-133108),加拿大卫生研究所CIHR,苏黎世大学Forschungskredit(FK- 14-016)和Panacée基金会。
L.H.和S.M.对这份手稿作了同样的贡献。 L.H.和S.M.构思和设计的协议。 S.M.,J.A.和L.H.进行实验。 。。有助于协议优化。 M.A.A.A.和P.P.开发了单锅限制/插入方案。 L.H.,S.M.,J.A.,JP.B.和B.B.写了手稿与所有作者的输入。作者宣称他们没有竞争的经济利益。
我们在这里描述的方案是基于我们的文章中使用的方法,标题为"Activation of concurrent apoptosis and necroptosis by SMAC mimetics for the treatment of refractory and relapsed ALL"(McComb等人,2016)。

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Behrmann, L., McComb, S., Aguadé-Gorgorió, J., Huang, Y., Hermann, M., Pelczar, P., Aguzzi, A., Bourquin, J. and Bornhauser, B. C. (2017). Efficient Generation of Multi-gene Knockout Cell Lines and Patient-derived Xenografts Using Multi-colored Lenti-CRISPR-Cas9. Bio-protocol 7(7): e2222. DOI: 10.21769/BioProtoc.2222.
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