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Jul 2019
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Evaluation of the Efficiency of Genome Editing Tools by a Frameshift Fluorescence Protein Reporter
通过移码荧光蛋白报告基因评估基因组编辑工具的效率   

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Abstract

In the last decade, genome editing has been the center of attention as a novel tool for mechanistic investigations and for potential clinical applications. Various genome editing tools like meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes (Cas), have been developed in recent years. For the optimal use as well as continued developments of these genome editing tools, the evaluation of their efficiencies and accuracies is vital. Here, we present a protocol for a reporter based on frameshift fluorescence protein which we recently developed to evaluate the efficiency and accuracy of genome editing tools. In this method, a ~20 bp target sequence containing frame-shifting is inserted after the start codon of a cerulean fluorescence protein (CFP) to inactivate its fluorescence, and only a new insertion/deletion event in the target sequence will reactivate the CFP fluorescence. To increase the traceability, an internal ribosome entry site and a red fluorescence protein, mCherryFP, are placed downstream of the reporter. The percentage of CFP-positive cells resulted from in/del mediated fluorescence restoration can be quantified by fluorescence measuring devices as the readout for genome editing frequency. As a demonstration, we present the usage for CRISPR-Cas9 technique here with flow cytometer as the readout for fluorescence changes.

Keywords: Insertion-deletion (碱基插入缺失), In-del (In-del), Reporter (报告), CRIPSR-Cas9 (CRIPSR-Cas9), Genome editing (基因组编辑), NHEJ (NHEJ)

Background

Genome editing tools are very important for the investigations of biological mechanisms and prevention and/or treatment of genetic diseases (Maeder and Gersbach, 2016). In the last couple of decades, several genome editing tools have been introduced, which include the meganucleases (Epinat et al., 2003), zinc finger nucleases (ZFNs) (Kim et al., 1996), transcription activator-like effector-based nucleases (TALEN) (Christian et al., 2010), and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes (Cas) (Jinek et al., 2012; Cong et al., 2013; Sander and Joung, 2014). In general, these tools create DNA double stranded breaks (DSB) to trigger genome editing in vivo (Maeder and Gersbach, 2016). The evaluation of the efficiencies and specificities of genome editing tool is essential for their applications and further developments. In our recent published study, we described a reporter that can generate quantitative readout for genome editing efficiency (Kumar et al., 2019). In this system, a ~20 bp target sequence is placed in a multiple cloning site (MCS) which is right after the start codon of Cerulean fluorescence protein (CFP) to generate a frame-shift of the open reading frame (ORF). This frameshifted-CFP (FsCFP) can be used as a reporter of genome editing because only when there is a successful DNA-double strand break (DSB) event on the target sequence followed with a non-homologous end joining (NHEJ) to generate an in/del event to shift the reading frame to a correct order (by a chance of up to 1/3), the CFP fluorescence will be reactivated as a positive readout. To facilitate the quantification, an internal ribosome entry site (IRES) and a red fluorescence protein, mCherryFP, is placed after the reporter. In principle, this reporter can be applied to any genome editing system as long as a DSB and NHEJ are expected from the editing. This approach can effectively detect low-efficiency editing in a population of cells with very low false negative or false positive. Furthermore, in this method, the positive cells can be conveniently identified and enriched for the examination or validation of the in/del event in the genome. Also, this method can be easily adapted for screening to optimize the genome-editing enzyme or the other components (such as guide-RNA) in the positive cells. Here, we used the CRISPR-Cas9 technique as a demonstration and the flow cytometry as the readout of the fluorescence events.

In this protocol, the target sequence is inserted between restriction sites of NotI and XhoI before CFP reporter together with sequence for the optimal recognition of Cas9 and a premature STOP codon to create a frame shift. The reporter region is then integrated in the nuclear genome of the target cell by the assistance of lentivirus. The target cells expressing the red fluorescence protein are then isolated by fluorescence-activated cell sorting (FACS), before vectors containing the Cas9 and gRNA are introduced into these cells. After incubation, the ratio of the CFP over mCherryFP was measured in flow cytometry to provide quantitative measurement for the efficiency of the genome editing.

Materials and Reagents

Materials

  1. Cell Culture dish 150 x 25 mm (Asi, catalog number: TD0150 )
  2. Cell Culture dish 90 x 20 mm (Asi, catalog number: TD0100 )
  3. 5 ml serological pipet (Asi, catalog number: SP205 )
  4. 10 ml serological pipet (Asi, catalog number: SP210 )
  5. Cell Culture Flask 75 cm2, filter cap (Asi, catalog number: TV0075 )
  6. Cell Culture Flask 25 cm2, filter cap (Asi, catalog number: TV0025 )
  7. Syringe filter, PES 25 mm, 0.45 μm (Asi, catalog number: TE45-5 )
  8. 10 ml syringes (BD, catalog number: 309604 )
  9. 15 ml and 50 ml conical tubes (Denville, catalog number: C1062-P )
  10. 18 G x 1 ½ needles (BD, catalog number: 305196 )
  11. Bottle top filtration–2 μm PES (VWR, catalog number: 97066-202 )
  12. 15 ml centrifuge tubes (CellPro, catalog number: CN5600 )
  13. 50 ml centrifuge tubes (CellPro, catalog number: CN5603 )
  14. Filter Pipet Tips 1,250 μl (TruPoint, catalog number: FT1250 )
  15. Filter Pipet Tips 200 μl (TruPoint, catalog number: FT1200 )
  16. Filter Pipet Tips 20 μl (TruPoint, catalog number: FT1020 )
  17. Filter Pipet Tips 10 μl (TruPoint, catalog number: FT1010 )
  18. Falcon FACS tubes with 35 μMcell strainer (BD, catalog number: 352235 )
  19. Falcon FACS collection tubes (BD, catalog number: 352063 )
  20. Ice buckets
  21. T4 DNA ligase (New England Biolabs, catalog number: M0202S )
  22. Gel extraction kit (EZ, catalog number: M1002-50 )
  23. One ShotTM TOP10 Chemically Competent E. coli (Invitrogen, catalog number: C404003 )
  24. ZymoPURETM II Plasmid Midiprep Kit (Genesee Scientific, catalog number: 11-550B )
  25. QIAprep Spin Miniprep Kit (50) (QIAGEN, catalog number: 27104 )

Cells and Plasmids
  1. Human embryonic kidney cells (HEK 293T, clone 17) (ATCC, catalog number: CRL-11268 )
  2. Plasmid pQC-XIG (Addgene, catalog number: w497-1 ), deposited by Dr. Eric Campeau, who is currently at Zenith Epigenetics Ltd, Calgary, Canada
  3. Plasmid pCMV-Delta R8.2 (Addgene, catalog number: 12263 ), deposited by Dr. Didier Trono at EPFL
  4. Plasmid pCMV-VSV-G (Addgene, catalog number: 8454 ), deposited by Dr. Robert Weinberg at MIT
  5. gRNAs custom ordered from Vector builder (https://en.vectorbuilder.com/)

Reagents
  1. High Glucose DMEM (1x) (Life Technologies, catalog number: 11995-065 )
  2. F-10 media (1x) (Life Technologies, catalog number: 11550-043 )
  3. 0.25% Trypsin-EDTA (1x) (Life technologies, catalog number: 25200-072 )
  4. 10% FBS (Hyclone, catalog number: SH30910.03 )
  5. Polybrene Transfection Reagent (Millipore, catalog number: TR-1003-G )
  6. Antibiotic-Antimycotic (100x) (Life Technologies, catalog number: 15240062 )
  7. Polyethylenimine (PEI) (Sigma-Aldrich, catalog number: 408727 )
  8. T4-ligase Buffer (New England Biolabs, containing 50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, pH 7.5)
  9. DMEM+F10 culture media (45% DMEM + 45% F-10 + 10% FBS, with 1x Antibiotic-Antimycotic)
  10. Nucleotide oligos/primers:

    Table 1. List of primers used in this protocol

Equipment

  1. Centrifuge 5424 R (Eppendorf, catalog number: 540400138 )
  2. Labnet Accublock Digital Dry Bath (Labnet, catalog number: 19-41620 )
  3. Gene Mote Vortex Mixer (Bioexpress, catalog number: S-3200-1 )
  4. CO2 incubator MCO-19AIC (UV) (Panasonic, catalog number: 13010002 )
  5. Centrifuge 5702 (Eppendorf, catalog number: 0 22626205 )
  6. Fluorescent microscope
  7. Aria-IIU flow cytometer (BD)

Software

  1. FCS Express 6 (Denovo software–https://denovosoftware.com/)

Procedure

  1. Generation of reporter and gRNA constructs
    1. Construction of the Frameshift(Fs) CFP-mCherryFP reporter
      1. The nucleotide sequence consisting of CFP, IRES and mCherryFP and flanked with NotI and EcoRV restriction sites was synthesized by using the service of Genscript, NJ. See Figure 1 for the illustration of the vector map and the nucleotide sequence.
        Note: The CFP was chosen as the frameshift reporter due to its lack of internal starting codons (ATG) near the 5′-end, which is essential to prevent the generation of smaller proteins that are potentially still fluorescent (such as in the case of mCherryFP). Although GFP also lacks internal ATG near the 5′-end, it is often used as a marker in many vectors containing the genome editing machineries. In addition to fluorescence proteins, proteins that don’t have internal ATG near the 5’-end and can be specifically detected or selected may also be designed as frameshifting reporters. These may include luciferases, β-galactosidase, or aminoglycoside 3′-phosphotransferase.


        Figure 1. Construction of the reporter plasmids. In (A), there is a schematic representation of the vector map of pQC-FSCFP-mCherryFP, which is modified from pQC-XIG vector (from Addgene) with a synthesized nucleotide sequence as shown in (B). The synthesized sequence (5′-strand shown) is to be cloned into the pQC-XIG vector by restriction sites NotI and EcoRV. This cloning will replace the original IRES and GFP regions. It will also introduce the CFP coding sequence and additional cloning sites. In the demonstrated example here it also included the 20 nt target sequence.

      2. The above-described nucleotide sequence is cloned into a template vector, pQC-XIG, using the restriction sites NotI and EcoRV (5′ end and 3′ end, respectively).
        Note: This replaces the original IRES and GFP sequences in the pQC-XIG vector for the sake of introducing multiple cloning sites flanking the CFP and mCherryFP sequences for future cloning purposes.
      3. Digest the FsCFP-mCherryFP reporter plasmid generated from above with Not1 and Xho1 (5′ end and 3′ end respectively). Gel purify the digested vector using Gel extraction kit (EZ) and store the product at -20 °C.
        Note: Do not perform phosphatase treatment during or after the digestion, unless the custom ordered oligos were 3′-phosphorylated.
    2. Introducing the target sequence in FsCFP-mCherryFP reporter (see also Table 1 for the nucleotide sequences of primer/oligos)
      1. Synthesizing the target sequence–For example, the target sequence for VEGF-A gene in human is 5′-GGGTGGGGGGAGTTTGCTCC-3′. The targeting sequence is placed after the START codon, followed by a protospacer adjacent motif (PAM) site and a premature STOP codon. If necessary, a few (1-5) extra nucleotides can be added to ensure that the CFP is out of frame. In the shown example, the inclusion of a premature STOP codon is to prevent the production of a long-length protein resulting from the frameshifted coding sequence of CFP, which may be harmful to the cell (Figure 2). The nucleotide oligos (both 5’ and 3’ strand) containing the above (target) sequence as well as overhanging nucleotides which would result from NotI (5′) and XhoI (3′) digestion were then synthesized by using the service of Genscript, NJ:
        Top-strand target oligo: 5′-ggccgcCATATGTGGGTGGGGGGAGTTTGCTCCAGGTGAAc-3′
        Bottom-strand target oligo: 5′-tcgagTTCACCTGGAGCAAACTCCCCCCACCCACATATGGcg-3′
        Notes: The two oligos, if annealed, represent the double-digestion product of NotI (5′) and XhoI (3′). (Figure 3)


        Figure 2. Schematic representation of the nucleotide sequence flanking the target insert region. The position of the 20-nucleotide (nt) gRNA-matching site, the 3nt-protospacer adjacent motif (PAM) sequence for Cas9 binding is indicated along with the START and STOP codon. The two extra nucleotides in this particular example are included to ensure the out-of-frame CFP. The premature STOP codon is included to prevent the translation of a long-length product from the START codon on the shifted frame of the CFP coding sequence.


        Figure 3. Annealing of 5′-strand and 3′-strand oligos. The oligo sequences containing an example with 20-nt target sequence (in bold red font) derived from human VEGF gene, the START codon, PAM site, and premature STOP codon, as well as overhanging nucleotides which would result from NotI (5′) and XhoI (3′) digestion (shown in lower case). After annealing, the 5′ and 3′ strands together represent the digested product of Not1 and Xho1.

      2. Annealing–In an Eppendorf tube, mix the two oligos from above (Step A2a) in 1:1 ratio to a final concentration of 20 μM (each) in 1x T4 ligase buffer. Place the tube in the boiling water (100 °C) for 10-20 min and allow the water to cool down at room temperature for 8-12 h for the annealing process. The product can then be used freshly or stored at -20 °C.
      3. Ligation–Mix the annealed oligos with the digested vector from Step A1c. The oligo would be 3-10 times (5 time is recommended) in excess compared to the vector in molecules. Ligation is carried out by the addition of T4 DNA ligase (NEB) in the presence of 1x T4 ligase buffer (NEB). For each 20 μl reaction which typically contains 1 μg of digested vector and 0.05 μg of annealed oligos, 1 μl of ligase was added. The ligation reaction is performed at 16 °C for 2 h followed by 8 °C overnight
      4. Bacterial transformation–The ligated product from above is introduced into Top10 cells (Invitrogen), according to the manufacturer’s instructions. In general, 2~3 μl of the ligation product was used to transform 50 μl of Bacteria. The transformed bacteria is plated on to LB + Ampicillin (50 μg/ ml) plates for incubation at 37 °C overnight. Grown colonies are picked from the plate to grow in 5 ml liquid LB + Ampicillin (50 μg/ ml) overnight. Plasmid is then isolated using Qiagen QiaPrep Spin Miniprep kit and then submitted for Sanger sequencing with primers specifically targeting the CMV promoter, the CFP region (without cross-reacting with the mCherryFP region), and the IRES region of the plasmid (Figure 1A).
        Primers used in this protocol are listed in Table 1.
        Forward primer targeting CMV promoter:
        5′-AGAGCTCGTTTAGTGAACCGTC-3′
        Reverse primer targeting IRES:
        5′-GACGGCAATATGGTGGAAAATAACATATAGACAAACGCACACCGG-3′
        Forward primer targeting the border between the CMV promoter and the cloning sites:
        5′-GAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACG-3′
        Reverse primer specifically targeting CFP:
        5′-TAGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCTCCTGGACGTAGCC-3′
      5. Colonies carrying the plasmid containing the expected insertion sequence in the FsCFP-mCherryFP reporter is then grown in 100 ml liquid LB + Ampicillin (50 μg/ml) overnight. Plasmid is then isolated with the Midiprep Kit (ZymoPURETM II Plasmid Midiprep Kit). This product can be stored at 20 °C.
    3. Guide RNA (gRNA) construction
      The plasmids for gRNA and Cas9 can be designed and purchased from Vector Builder INC (https://en.vectorbuilder.com/), with GFP as a selection marker. For example, the gRNA sequence for VEGF will be –GGGTGGGGGGAGTTTGCTCC. As a control, scramble gRNA should also be ordered.

  2. Creation of cell lines with FsCFP-mCherryFP reporter and gRNA
    Preparation of cell lines with reporter and gRNA is a 2-step sequential process.
    Step B1: Cell line of interest + FsCFP-mCherryFP (reporter plasmid) → sort for mCherry positive cells
    Step B2: Cell line with FsCFP-mCherryFP + (target) gRNA-GFP → sort for mCherry and GFP double positive cells.

    Step B1: Generation of cell line with FsCFP-mCherryFP reporter
    1. Production of lentiviral particles in HEK 293T cell line
      To begin with, seed the HEK 293T cells so that they will reach ~75% confluency in a T-25 culture flask, which can be estimated by microscopic observations. Change the DMEM+ F10 culture just before the transfection steps below.
      1. In a 1.5 ml Eppendorf tube (tube 1), add 1 μg of VSV-G, 6 μg of Delta R8.2, and 6.5 μg of reporter plasmid containing the targeting sequence (from Step A2e) to 0.5 ml of DMEM + F10 media. Vortex for 20-30 s for mixing and spin down briefly (less than 10 s) in mini-spinner or in a common desktop centrifuge at 2,000 x g.
      2. Add 0.5 ml of DMEM to another Eppendorf tube (tube 2), then add 30 μl of activated Polyethylenimine reagent (Sigma) or other types of transfection reagents such as Lipofectamine (Thermo-Fisher) or JetPrime (Polyplus-Transfection). Vortex immediately for 30 s. Spin down the components briefly as above. Add the contents from tube 1 to tube 2. Vortex the tube immediately for 20-30 s and then spin down briefly again. Incubate at room temperature (RT) for 20-30 min.
      3. After the incubation, add the transfection mix drop by drop to the flask containing the HEK 293T cells and the fresh media. Mix gently and place the flask at 37 °C, 5% CO2 incubator for the production of viral particles in the next three days. The majority of these cells (> 50%) are expected to express mCherryFP (excitation 587 nm, emission 610 nm) after 24 h, which can be visually verified by the presence of red fluorescence under a fluorescence microscope.
      4. Harvest the media from the virus-producing HEK 293T cells after 24 h, 48 h and 72 h (if necessary). Replace with fresh culture media.
      5. Place the harvested media in a sterile 50 ml conical tube. Add polybrene to the final concentration of 30 μg/ml. Vortex briefly to mix.
      6. [Optional] Spin down at a swing-bucket rotor at 1,000 x g for 2 min to remove cell debris.
      7. Filter the supernatant using syringe and a 0.45-micrometer syringe filter. The supernatant should be used immediately on the target cell, although it can also be stored at 4 °C for up to three days. If a longer storage is required, the supernatant should be distributed into aliquots and snap frozen in liquid nitrogen and then stored at -80 °C.
    2. Transduction of target cells
      As a demonstration, HEK293T cells are used as target cells to receive the lentivirus produced from above (Procedure B, Step B1-1).
      1. Grow HEK 293T cells in T-75 flask. The density of the cells should be under 30% confluent before the 1st viral transduction, as visually estimated under an optical microscope.
      2. Dilute the virus-containing supernatant harvested from the T-25 of cells at Step B1-1 with 2x volume of fresh culture media. Replace the media in the T-75 flask containing the target cells with this mix containing the virus and polybrene (final concentration 10 μg/ml).
      3. Repeat the above step for one more time in the next day.
      4. Check the mCherryFP fluorescence under fluorescence microscope to ensure that the fluorescence positive cells are less than 20%, in order to avoid over-infection.
        Note: It is important to keep the target cells less than 75% confluence at any time during the viral induction stage.
    3. Sorting of target cells carrying the reporter plasmid
      1. Trypsinize and collect the target cells by flushing with fresh culture media (at least 5 volume of the trypsin solution used). Transfer the mix to a sterile 50 ml conical tube. Collect the cells by centrifugation in a swing bucket rotor at 1,500 x g for 3 min at room temperature.
        Note: Create a backup of these unsorted cells by freezing them in -80 °C.
      2. Resuspend the cells in 1 ml of sorting medium (DMEM + antibiotic-antimycotic), which should generate a density of ~10 million cells/ml.
      3. Pass the cells through the FACS tubes with 35-micrometer cell strainer.
      4. Add 1 ml of sorting medium in the FACS collection tubes.
      5. Use target cells without any mCherry plasmid as negative control for gating purposes in flow cytometry.
      6. Sort the cells for mCherry positive using FACS–BD Aria-Ilu flow cytometer.
        Note: For success of subsequent culture, we recommend a minimum of 20,000 cells should be acquired for each sample.
      7. Culture the mCherry positive cells in T-25 flask with 5 ml fresh media. Expand the culture to bigger flasks (such as T-75) to generate cells carrying the reporter for the next step and keep aliquots for storages at -80 °C.
        Note: The quality of the cells expressing the reporter should be periodically inspected, which can be verified by the presence of red fluorescence in at least 90% of the cells under fluorescence microscope.

    Step B2: Generation of cell line co-expressing FsCFP-mCherryFP and gRNA-GFP
    1. Packaging of lentiviral particles in HEK 293T cell line
      This step is similar to the one mentioned above (Step B1), except that the gRNA-GFP plasmid (the VEGF-gRNA-GFP or the scrambled-sequence gRNA-GFP) will be used with these plasmids VSV-G (coding for viral envelop protein) and DeltaR8.2 (coding for reverse transcriptase HIV1-pol and packaging factor HIV1-gag suitable for lentivirus).
    2. Transduction of target cells expressing reporter plasmid with the lentivirus containing gRNA-GFP.
      Before proceeding, do a quick visual verification under the fluorescence microscope for the target cells expressing reporter plasmid to make sure more than 90% of cells are mCherry-positive.
      Note: The target cells (expressing reporter plasmid) grown in the T-25 flask should be around 30% confluent right before the 1st viral induction (24 h).
      The target cells can be repeatedly induced for 2-3 days. In the end of the induction, the GFP-producing cells should be less than 50% of the population to avoid over-induction.
    3. Sorting of target cells carrying both the reporter and gRNA
      After 72 h, check the transfection efficiency of target cells under fluorescence microscope for the presence of both mCherry and GFP fluorescence.
      Note: Create a backup of these unsorted cells by freezing them in -80 °C.
      1. Process the cells for sorting as described above (Steps B3a-B3d)
      2. Use appropriate positive controls for gating purposes while sorting. Cells don’t express any fluorescence proteins, or expressing GFP-only, mCherryFP-only, or GFP plus mCherryFP should be used as negative controls to set the gates for CFP-positive events. The detection threshold in general can be set at two folds above the maximum signal generated by the negative controls. If necessary, cells expressing functional GFP, mCherryFP, and CFP can also be used as the positive control (although this is usually not necessary).
      3. Acquire a minimum of 100,000 events for the double-positive cells to ensure reliable detection of any triple-positive cells.
      4. Sort the target cells for mCherry and GFP double-positive and any mCherryFP, GFP, and CFP triple-positive populations using FACS–BD Aria-Ilu flow cytometer. The setting of the sorting gates for CFP-positive events should be adjusted so that the collection window is at least by 2-folds higher than the edge of the main population at the CFP channel from any of the three negative controls. Conversely, the collected double-positive cells should not contain CFP signal higher than that of the negative controls.
      5. Grow and expand the double-positive and triple-positive cells for further analysis.
      Note: Create a backup of these sorted cells by freezing them at -80 °C.
    4. FACS data analysis
      1. Analyze the FACS data by using either FCS Express 6 or FlowJo. The percentage of CFP-positive cells among the double-positive cells can be quantified using FCS Express 6 and MS-Excel.
      2. Forward and side scattering should be used to exclude cell fragments/debris or cell clumps/clusters. The threshold for detection of the GFP and mCherryFP signals should be established with parental cells that were either not treated, or stably transduced with GFP-only or mCherryFP-only expression vectors. The gating threshold for the double-positive cells (GFP and mCherryFP) should be at least one fold away from the individual positives (GFP-only and mCherry-only) (Figure 4).


        Figure 4. A schematic showing flow cytometry gating strategy. Apply the gating for single and live cells by using forward and side scattering. For the individual fluorescence (GFP and mCherry), gating was applied based on the negative control, GFP-only control, and mCherry-only controls. The gates for selecting GFP and mCherryFP double-positive cells should be at least one fold above the upper edge of the signals from the GFP-only and mCherryFP-only cells.

      3. Apply the gating for GFP and mCherryFP double-positive cells in the sampling group and then look for the CFP positive cells in this population. The gate for CFP should be established by using a CFP-negative control cell expressing GFP plus mCherryFP but not CFP. In our experience, this threshold can also be set by using cells carrying the reporter (with mCherryFP marker) and pQC-XIG (with GFP marker). The gating should be set up so that it is at least two folds higher than the border of the main population of the CFP signal generated from the CFP-negative cells.
      4. The same set of gatings should be universally applied to all sample groups. The presence of CFP fluorescence indicates the occurrence of in/del events and thereby successful genome editing (See examples in Figure 5). The non-specific (ns) scramble-sequence gRNA-GFP (Figure 5) reflects the background signals of spontaneously generated CFP events by non-specific gene editing.


        Figure 5. Examples of data to depict the CFP-positive events using this reporter tool in HEK293T cells. Data showing the absence of CFP fluorescence (FsCFP-reporter with target sequence derived from human VEGF gene + pQC-XIG); background CFP fluorescence due to spontaneous mutageneis (reporter + non-specific (ns) gRNA); and presence of CFP-positive events occurred by in/del events (reporter + VEGF gRNA). GFP+mCh+ indicates the gating of double-positive cells (GFP and mCherry). GFP+mCh+CFP+ indicates the presence of CFP+ positive events in the double-positive cells (GFP and mCherry).

      5. The number or the percentage of CFP events among the double positives in each cell population, which is the readout of the genome editing efficiency, can be calculated using MS-Excel or the build-in function of the software (FCS Express 6 in this case) .
    5. Analysis of genome sequence cell colonies
      If desirable, single-cell colonies can be produced by dilution and spreading of the sorted cells in 96-well plates for DNA analysis. See Figure 6 for a summary of all steps in the test.


      Figure 6. Diagram representing workflow and estimated times to establish a cell line carrying the FsCFP-mCherryFP reporter and perform the genomic editing assay (with Cas9 as an example)

    Notes:
    1. If the target sequence contains an internal START codon, care should be taken to ensure that it would not lead to in-frame translation of the CFP. If this is the case, one additional nucleotide can be inserted after the PAM site (preferably after the premature STOP codon) to further shift the reading frame.
    2. This method is sensitive for low-frequency in/del events too. We have tested this by mutating two central nucleotides in the targeting gRNA, which is expected to reduce the targeting efficiency significantly. When this mutated gRNA is co-expressed along with the reporter, a much reduced yet still statistically significant CFP signal was detected in the cells expressing both GFP and mCherryFP.
    3. Spontaneous mutagenesis, such as those resulted from base excision, may also lead to the reactivation of the CFP signal. However, in our experience, the magnitude of such spontaneous events (as demonstrated cells with the reporter and pQC-XIG vector) are at least one order lower than the non-specific action of Cas9 supplemented with a non-specific gRNA. Therefore, in our experience, it is safe to use the cells carrying the reporter (with mCherryFP marker) and the pQC-XIG vector (with GFP marker) to set up the detection gate for CFP. However, if a lower background is desired for setting up the CFP detection gate, a cell that only expressing GFP and mCherryFP and not carrying any coding sequence of CFP can be used as the control.
    4. The gate for CFP detection can be moved up or down for a few (1-5) folds, as long as the threshold is above the border of the main population in CFP-negative control cells (with GFP and mCherryFP double-positive) and as long as the gate is applied universally to all sampling groups, the differences between each group will still exist.

Acknowledgment

We thank the flow cytometry core facility at the Sylvester Comprehensive Cancer Center for providing the service. We also thank the funding resources–NIGMS/NIH, R01#GM107333; DoD (CDMRP), Idea Award, PC140622. The salary of the authors and the cost of experiments are supported in part by these funding resources. These funding bodies were not directly involved in any part of the design of study, the collection, analysis, or interpretation of the data, or writing of the manuscript. This protocol was adapted from this published work ( Kumar et al., 2019).

Competing interests

The authors declare that they have no competing financial interests.

References

  1. Christian, M., Cermak, T., Doyle, E. L., Schmidt, C., Zhang, F., Hummel, A., Bogdanove, A. J. and Voytas, D. F. (2010). Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186(2): 757-761. 
  2. Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A. and Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121): 819-823. 
  3. Epinat, J. C., Arnould, S., Chames, P., Rochaix, P., Desfontaines, D., Puzin, C., Patin, A., Zanghellini, A., Paques, F. and Lacroix, E. (2003). A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells. Nucleic Acids Res 31(11): 2952-2962.
  4. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A. and Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096): 816-821.
  5. Kim, Y. G., Cha, J. and Chandrasegaran, S. (1996). Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci U S A 93(3): 1156-1160.
  6. Kumar, A., Birnbaum, M. D., Moorthy, B. T., Singh, J., Palovcak, A., Patel, D. M. and Zhang, F. (2019). Insertion/deletion-activated frame-shift fluorescence protein is a sensitive reporter for genomic DNA editing. BMC Genomics 20(1): 609. 
  7. Maeder, M. L. and Gersbach, C. A. (2016). Genome-editing technologies for gene and cell therapy. Mol Ther 24(3): 430-446. 
  8. Sander, J. D. and Joung, J. K. (2014). CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32(4): 347-355.

简介

[摘要] 在过去的十年中,基因组编辑作为一种机制研究和潜在临床应用的新工具已成为关注的焦点。近年来,已开发出各种基因组编辑工具,例如大范围核酸酶,锌指核酸酶(ZFN),转录激活子样基于效应子的核酸酶(TALEN)以及成簇的规则间隔的短回文重复序列(CRISPR)相关基因(Cas)。 。对于这些基因组编辑工具的最佳使用和持续发展,评估其效率和准确性至关重要。在这里,我们介绍了一种基于移码荧光蛋白的报告子方案,我们最近开发了该方案以评估效率和 基因组编辑工具的实用性。在这种方法中,在天蓝色荧光蛋白(CFP)的起始密码子后插入一个约20 bp的包含移码的靶序列,以使其荧光失活,并且只有新的插入/缺失事件会重新激活CFP 荧光。 。为了增加可追溯性,将内部核糖体进入位点和红色荧光蛋白mCherryFP 放置在报告子的下游。由in / del介导的荧光恢复产生的CFP阳性细胞的百分比可以通过荧光测量装置定量,作为基因组编辑频率的读数。作为演示,我们在这里介绍CRISPR-Cas9技术的使用以及流式细胞仪作为荧光变化的读数。

[背景] 基因组编辑工具对于生物学机制的研究以及遗传疾病的预防和/或治疗非常重要(Maeder和Gersbach,2016)。在最近的几十年中,引入了几种基因组编辑工具,包括大范围核酸酶(Epinat 等,2003),锌指核酸酶(ZFN)(Kim 等,1996),基于转录激活子的效应子。核酸酶(TALEN)(Christian 等,2010)和成簇的规则间隔的短回文重复序列(CRISPR)相关基因(Cas)(Jinek 等,2012; Cong 等,2013; Sander和Joung,2014 )。通常,这些工具会产生DNA双链断裂(DSB),以在体内触发基因组编辑(Maeder and Gersbach,2016)。基因组编辑的效率和特异性的评估工具是必要的红外应用和进一步发展。在我们最近发表的研究中,我们描述了一个记者,可以产生GENOM定量读出Ë 编辑效率(库马尔等,2019) 。在该系统中,〜20 个碱基的靶序列被放置在一个多克隆位点(MCS) ,其蔚蓝荧光蛋白(CFP)的起始密码子之后是产生的开放阅读框(ORF)的移码。此移码后的CFP(FsCFP )可以用作基因组编辑的报告子,因为仅当在目标序列上成功发生DNA双链断裂(DSB )事件并随后进行非同源末端连接(NHEJ)时,该序列才可以用作基因组编辑如果发生in / del事件,以将阅读框移动到正确的顺序(最多1/3),CFP荧光将重新激活为阳性读数。为便于定量,将内部核糖体进入位点(IRES)和红色荧光蛋白mCherryFP 置于报告基因之后。原则上,只要从编辑中获得期望的DSB和NHEJ,该报告基因就可以应用于任何基因组编辑系统。这种方法可以有效地检测出假阴性或假阳性率极低的细胞群中的低效率编辑。此外,在这种方法中,可以方便地鉴定和富集阳性细胞,以检查或验证基因组中的in /​​ del事件。而且,该方法可以很容易地用于筛选,以优化阳性细胞中的基因组编辑酶或其他成分(例如指南RNA)。在这里,我们使用CRISPR-Cas9技术进行演示,并使用流式细胞仪作为荧光事件的读数。

在此协议中,将目标序列与CFP报告基因之前的NotI 和XhoI 限制性酶切位点之间插入,以最佳识别Cas9的序列和过早的STOP密码子以产生移码。然后借助慢病毒将报告基因区域整合到靶细胞的核基因组中。然后,在将包含Cas9和gRNA的载体引入这些细胞之前,通过荧光激活细胞分选(FACS)分离表达红色荧光蛋白的靶细胞。孵育后,在流式细胞仪中测量CFP与mCherryFP 的比例,以定量测量基因组编辑的效率。

关键字:碱基插入缺失, In-del, 报告, CRIPSR-Cas9, 基因组编辑, NHEJ

材料和试剂


 


用料


细胞培养皿150 x 25 mm(Asi ,目录号:TD0150)
细胞培养皿90 x 20 mm(Asi ,目录号:TD0100)
5 ml血清移液器(Asi ,目录号:SP205)
10 ml血清移液管(Asi ,目录号:SP210)
细胞培养瓶75 cm 2 ,过滤帽(Asi ,目录号:TV0075)
细胞培养瓶25 cm 2 ,过滤帽(Asi ,目录号:TV0025)
注射器过滤,PES 25mm时,0.45 μ 米(阿西,目录号:TE45-5)
10 ml注射器(BD,货号:309604)
15 ml和50 ml锥形管(Denville,目录号:C1062-P)
18 G x 1½针(BD,货号:305196)
瓶顶过滤- 2 μ 米PES(VWR,CATAL OG号:97066-202)
15 ml离心管(CellPro ,目录号:CN5600)
50 ml离心管(CellPro ,目录号:CN5603)
过滤移液管尖端1250 微升(TRUpoint的,目录号:FT1250)
过滤移液管尖端200 微升(TRUpoint的,目录号:FT1200)
过滤移液管尖端20 微升(TRUpoint的,目录号:FT1020)
过滤移液管尖端10 微升(TRUpoint的,目录号:FT1010)
猎鹰FACS管35 μ MCELL 滤网(BD,目录号:352235)
猎鹰FACS收集管(BD,货号:352063)
冰桶
T4 DNA连接酶(新英格兰生物实验室,目录号:M0202S)                                         
凝胶提取试剂盒(EZ,目录号:M1002-50)
一个铅球TM TOP10化学的COM 享有管辖权大肠杆菌(Invitrogen,目录号:C404003)
ZymoPURE TM II质粒Midiprep 试剂盒(Genesee Scientific,目录号:11-550B)                                                       
QIAprep Spin Miniprep 试剂盒(50)(QIAGEN,目录号:27104)                                                                     
 


细胞和质粒


人胚肾细胞(HEK 293T,克隆17)(ATCC ,目录号:CRL-11268)
质粒pQC -XIG (Addgene ,目录号:w497-1 ),由Eric Campeau博士保藏,他目前在加拿大卡尔加里的Zenith EpigeneticsLtd。
质粒pCMV -Delta R8.2 (Addgene ,目录号:12263 ),Didier Trono 博士保藏在EPFL
质粒pCMV -VSV-G (Addgene ,目录号:8454 ),由麻省理工学院的Robert Weinberg博士保藏
从Vector builder(https://en.vectorbuilder.com/)订购的gRNA自定义
 


试剂种类


高葡萄糖DMEM(1x)(Life Technologies,目录号:11995-065)
F-10介质(1x)(Life Technologies,目录号:11550-043)
0.25%胰蛋白酶-EDTA(1x)(生活技术,目录号:25200-072)
10%FBS(Hyclone ,目录号:SH30910.03)
聚乙烯转染试剂(Millipore,目录号:TR-1003-G)                           
抗生素-抗真菌药(100x)(生活技术,目录号:15240062)             
聚乙烯亚胺(PEI)(Sigma-Aldrich,目录号:408727)
T4连接酶缓冲液(New England Biolabs公司,含有50mM的Tris-HCl,10 毫的MgCl 2 ,1 mM的ATP,10 毫DTT,pH 7.5)中
DMEM + F10培养基(45%DMEM + 45%F-10 + 10%FBS,含1x抗生素-抗真菌药)
核苷酸寡核苷酸/引物:




钽BLE 1. 在本协议中使用的引物列表


目的


名称


序列(5 ' -3 ' )


报告者的靶序列


顶链靶寡核苷酸


ggccgcCATATGTGGGTGGGGGGAGTTTGCTCCAGGTGAAc


底链靶寡核苷酸


tcgagTTCACCTGGAGCAAACTCCCCCCACCCACATATGGcg


报道载体的测序引物


靶向CMV启动子的正向引物


AGAGCTCGTTTAGTGAACCGTC


反向引物靶向IRES


GACGGCAATATGGTGGAAAATAACATATAGACAAACGCACACCGG


靶向CMV启动子和克隆位点之间边界的正向引物


GAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACG


专门针对CFP的反向引物:


TAGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCTCCTGGACGTAGCC


 


设备


 


5424 R离心机(Eppendorf,货号:540400138)
Labnet Accublock 数字干浴锅(Labnet ,目录号:19-41620)
Gene Mote涡旋混合器(Bioexpress ,目录号:S-3200-1)
CO 2 培养箱MCO-19AIC(UV)(Panasonic,目录号:13010002)
5702离心机(埃彭多夫(Eppendorf),目录号:022626205)
荧光显微镜
Aria-IIU流式细胞仪(BD)
 


软件


 


FCS Express 6(Denovo 软件– https://denovosoftware.com/)
 


程序


 


报告基因和gRNA构建体的生成
Frameshift(Fs)CFP- mCherryFP 报告基因的构建
由CFP,IRES和的核苷酸序列mCherryFP 和与侧接NotI位和EcoRV位的限制性位点是通过使用的服务合成金斯瑞,NJ。参见图1的图示中的矢量地图和所述核苷酸序列。
注意:由于CFP在5'端附近缺少内部起始密码子(ATG),因此被选为移码报告基因,这对于防止生成可能仍具有荧光性的较小蛋白质(例如在mCherryFP )。尽管GFP在5'端附近也缺乏内部ATG,但它经常被用作许多包含基因组编辑机制的载体的标记。除了荧光蛋白外,在5'端附近没有内部ATG且可以被特异性检测或选择的蛋白也可以设计为移码报告基因。这些可以包括荧光素酶,β-半乳糖苷酶或氨基糖苷3'-磷酸转移酶。


 






图1。报告质粒的构建。在(A)中,存在pQC- FSCFP- mCherryFP 的载体图的示意图,其由pQC- XIG载体(来自Addgene )修饰,具有如图(B)所示的合成核苷酸序列。将合成的序列(5 ' -链示出)将被克隆到PQC 通过限制性位点-XIG矢量的NotI 和EcoRV位。该克隆将取代原始的IRES和GFP区域。它还将介绍CFP编码序列和其他克隆位点。在此处展示的示例中,它还包括20 nt 靶序列。


 


上述- 描述的核苷酸序列被克隆到一个模板矢量,PQC -XIG,使用限制位点的NotI 和EcoRV位(5分别'端和3'端)。
注意:为了在以后的克隆中引入多个位于CFP和mCherryFP 序列两侧的克隆位点,这替代了pQC -XIG载体中的原始IRES和GFP序列。


用Not1和Xho1(分别为5'端和3'端)消化从上方生成的FsCFP-mCherryFP 报告质粒。凝胶使用凝胶提取试剂盒(EZ)纯化消化的载体,并将产物存储在-20 ℃。
注意:不要期间或消化后进行磷酸酶处理,除非定制订购寡聚物3 ' 磷酸化的。


在FsCFP-mCherryFP 报告基因中引入靶序列(引物/寡核苷酸的核苷酸序列也请参见表1)
合成靶序列- 例如,在人类对VEGF-A基因的靶序列为5' - GGGTGGGGGGAGTTTGCTCC - 3'。靶向序列位于START密码子之后,然后是原间隔子相邻基序(PAM)位点和过早的STOP密码子。如有必要,可以添加几个(1-5)额外的核苷酸,以确保CFP不符合标准。在所示的示例中,包含未成熟的STOP密码子是为了防止由于CFP的移码编码序列而产生长蛋白,这可能对细胞有害(图2)。然后,使用Genscript 服务合成包含上述(目标)序列以及NotI (5')和XhoI (3')消化产生的突出核苷酸的核苷酸寡核苷酸(5'和3'链)。新泽西州:
上链目标寡核苷酸:5'-ggccgcCATATGTGGGTGGGGGGAGTTTGCTCCAGGTGAAc-3'


底部链目标寡核苷酸:5'-tcgagTTCACCTGGAGCAAACTCCCCCCACCCACATATGGcg-3'


注意:如果退火,则两个寡核苷酸代表NotI (5')和XhoI (3 ' )的双消化产物。(图3)


 






图2示意图中的核苷酸序列侧翼的目标插入区。20核苷酸(位置NT )gRNA匹配站点,Cas9结合3NT-protospacer相邻基序(PAM)序列是在与START和STOP密码子沿dicated。的总重量○在该特定示例中额外的核苷酸被包括,以确保出框外的CFP。包括过早的STOP密码子,以防止CFP编码序列移位帧上的START密码子翻译出长产物。


 






图3的5退火' -链和3 ' -链寡核苷酸。在含有从例如人VEGF基因的寡核苷酸序列与20-nt的靶序列(粗体红色字体),起始密码子,PAM站点,提前终止密码子,以及突出核苷酸,这将导致从NotI位(5')和XhoI (3')消化(以小写字母显示)。退火后,5 ' 和3 ' 链一起代表Not1和Xho1的消化产物。


 


退火- 在Eppendorf管中,在1从上方(步骤A2A)混合两种寡核苷酸:1倍的比例至20的最终浓度μ 中号(各)在1×T4连接酶缓冲液。将试管放在沸水(100°C)中10-20分钟,然后让水在室温下冷却8-12小时,以进行退火处理。该产品可以新鲜使用,也可以在-20°C下保存。
连接– 将退火的寡核苷酸与步骤A1c中消化的载体混合。与分子中的载体相比,该寡核苷酸将过量3-10倍(建议5倍)。在1x T4连接酶缓冲液(NEB)存在下,通过添加T4 DNA连接酶(NEB)进行连接。对于每个20 μ 升通常含有1反应μ 克消化的载体和0.05的μ 克退火的寡核苷酸,1 μ 升溶液中加入连接酶。连接反应在16°C下进行2小时,然后在8°C下过夜
细菌转化-连接后从上述制品被引入到TOP10细胞(Invitrogen)中,根据制造商“ 小号指令。一般情况下,2〜3 μ 升连接产物的用于转化50 μ 升细菌。将转化的细菌铺板于LB到+氨苄青霉素(50 μ 克/毫升)在37℃下过夜平板孵育。生长的菌落从平板上挑取在5毫升(50生长液体LB +氨苄青霉素μ 克/毫升)过夜。然后质粒使用分离Qiagen公司的QIAprep 旋转小量制备试剂盒,然后提交Sanger测序用引物特异性地靶向CMV启动子,CFP区域(不与交叉反应mCherryFP 区域),和质粒的IRES区(图URE 1 甲)。
表1列出了该协议中使用的引物。


 


靶向CMV启动子的正向引物:


5 ' -AGAGCTCGTTTAGTGAACCGTC-3 '


靶向IRES的反向引物:


5 ' - GACGGCAATATGGTGGAAAATAACATATAGACAAACGCACACCGG-3 '


靶向CMV启动子和克隆位点之间边界的正向引物:


5 ' -GAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACG-3 '


专门针对CFP的反向引物:


5 ' -TAGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCTCCTGGACGTAGCC-3 '


携带含有在预期的插入序列的质粒的集落FsCFP-mCherryFP 记者然后在100ml生长液体LB +氨苄青霉素(50 μ 克/毫升)过夜。然后用Midiprep 试剂盒(ZymoPURE TM II质粒Midiprep 试剂盒)分离质粒。该产品可在20°C下保存。
指导RNA(gRNA)的构建
可以使用GFP作为选择标记,从Vector Builder INC(https://en.vectorbuilder.com/)设计和购买gRNA和Cas9的质粒。例如,VEGF的gRNA序列将为–GGGTGGGGGGAGAGTTTGCTCC。作为对照,也应订购加扰的gRNA。


 


用FsCFP-mCherryFP 报告基因和gRNA 创建细胞系
用报告基因和gRNA制备细胞系的过程分为两步。


步骤B1:感兴趣+细胞系FsCFP-mCherryF P (报道质粒)→ 排序为的mCherry 阳性细胞


步骤B2:用细胞系FsCFP-mCherryFP +(目标)gRNA-GFP → 排序为mCherry的和GFP双阳性细胞。


 


步骤B1:用FsCFP-mCherryFP 报告基因生成细胞系


HEK 293T细胞系中慢病毒颗粒的产生
首先,播种HEK 293T细胞,使其在T-25培养瓶中达到约75%的融合度,这可以通过显微镜观察来估计。在以下转染步骤之前更改DMEM + F10培养物。


在1.5ml Eppendorf管(管1),加入1 μ 克VSV-G,6 μ 克德尔塔R8.2的,和6.5 μ 克含有靶向序列(来自步骤A2E)报告质粒至0.5 毫升的DMEM + F10媒体。在微型旋转器或普通台式离心机中以2000 xg的速度涡旋20-30 s混合并短暂旋转(少于10 s)。
0.5毫升DMEM添加到另一Eppendorf管(管2),再加入30 μ 升活化的聚乙烯亚胺试剂(Sigma)或其它类型的转染试剂如脂质体(热-费希尔)或JetPrime (Polyplus -Transfection)。立即涡旋30 s。如上简要旋转组件。将试管1中的内容物加入试管2。立即使试管涡旋20到30秒,然后再次短暂旋转。在室温(RT)下孵育20-30分钟。
孵育后,将转染混合物逐滴添加至装有HEK 293T细胞和新鲜培养基的烧瓶中。轻轻混合,然后将烧瓶置于37°C,5%CO 2的培养箱中,以在接下来的三天内产生病毒颗粒。这些细胞中的大多数(> 50%)有望在24小时后表达mCherryFP (激发587nm,发射610nm),这可以通过在荧光显微镜下红色荧光的存在来目视验证。
24小时,48小时和72小时后(如有必要)从产生病毒的HEK 293T细胞中收获培养基。用新鲜的培养基代替。
将收获的培养基放入无菌的50 ml锥形管中。加入聚凝胺至30的最终浓度μ 克/毫升。短暂涡旋混合。
[可选]以1,000 xg 的旋转桶转子旋转2分钟,以除去细胞碎片。
使用注射器和0.45微米注射器过滤器过滤上清液。上清液应立即用于靶细胞,尽管也可以在4°C下保存三天。如果需要更长的存储时间,则应将上清液等份分配并在液氮中速冻,然后在-80°C下存储。
靶细胞转导
作为说明,将HEK293T细胞用作靶细胞,以接受从上方产生的慢病毒(步骤B,步骤B1-1)。


在T-75烧瓶中培养HEK 293T细胞。如在光学显微镜下目测估计,在第一次病毒转导之前,细胞的密度应低于30%融合。
稀释含有病毒的上清液收获版用2x体积的新鲜培养基的从T-25细胞中的步骤B1-1。用含有病毒和聚凝胺(最终浓度10该混合物代替在T-75烧瓶中含有靶细胞的培养基μ 克/毫升)中。
第二天再重复一次以上步骤。
在荧光显微镜下检查mCherryFP 荧光,以确保荧光阳性细胞少于20%,以避免过度感染。
注意:重要的是在病毒诱导阶段的任何时候,使靶细胞的融合度保持在75%以下。


携带报告质粒的靶细胞分选
胰蛋白酶化,并通过与新鲜培养基(使用的胰蛋白酶溶液中的至少5体积)冲洗收集靶细胞。将混合物转移到无菌的50 ml锥形管中。通过在室温下以1,500 xg的离心斗转子离心3分钟来收集细胞。
注意:将未分类的细胞在-80°C下冷冻,以创建备份。


将细胞重悬于1 ml分选培养基(DMEM + 抗生素-抗真菌剂)中,该分选培养基应产生约1000万个细胞/ ml的密度。
使细胞通过带有35微米细胞过滤器的FACS管。
在FACS收集管中加入1 ml分选培养基。
使用没有任何mCherry 质粒的靶细胞作为流式细胞术中门控目的的阴性对照。
使用FACS-BD Aria-Ilu流式细胞仪对细胞进行mCherry 阳性分选。
注意:为了成功进行后续培养,我们建议每个样品至少采集20,000个细胞。


培养的mCherry 阳性细胞在T-25烧瓶中,用5 米升新鲜的培养基。将培养物扩大到更大的烧瓶(例如T-75)中,以生成携带报告基因的细胞用于下一步,并将等分试样保存在-80°C下。
注意:应定期检查表达报告基因的细胞的质量,这可以通过在荧光显微镜下至少90%的细胞中存在红色荧光来验证。


 


步骤B2:产生共表达FsCFP-mCherryFP 和gRNA-GFP 的细胞系


HEK 293T 细胞系中慢病毒颗粒的包装
除了将gRNA-GFP质粒(VEGF-gRNA-GFP或加扰序列gRNA-GFP)与这些质粒VSV-G(编码病毒)一起使用外,此步骤与上述步骤(步骤B1)相似。包被蛋白)和DeltaR8.2(编码逆转录酶HIV1-pol和包装因子HIV1-gag适用于慢病毒)。


用含gRNA-GFP的慢病毒转导表达报告质粒的靶细胞。
在继续操作之前,请在荧光显微镜下对表达报告质粒的靶细胞进行快速视觉验证,以确保90%以上的细胞为mCherry 阳性。


注意:在第一次病毒诱导(24小时)之前,在T-25烧瓶中生长的靶细胞(表达报告质粒)应汇合约30%。


靶细胞可以被重复诱导2-3天。在诱导结束时,产生GFP的细胞应少于种群的50%,以避免过度诱导。


携带报告基因和gRNA的靶细胞的分选
72小时后,在荧光显微镜下检查靶细胞的转染效率,以确定是否存在mCherry 和GFP荧光。


注意:将未分类的细胞在-80°C下冷冻,以创建备份。


如上所述处理细胞以进行分类(步骤s B3a- B 3d)
排序时,使用适当的阳性对照进行门控。细胞不表达任何荧光蛋白,或仅表达GFP,仅mCherryFP 或表达GFP 加mCherryFP的细胞应用作阴性对照,以检测CFP阳性事件的门。通常,检测阈值可以设置为高于阴性对照产生的最大信号的两倍。如有必要,表达功能性GFP,mCherryFP 和CFP的细胞也可以用作阳性对照(尽管通常不是必需的)。
为双阳性细胞至少采集100,000个事件,以确保可靠检测任何三阳性细胞。
使用FACS-BD Aria-Ilu流式细胞仪对mCherry 和GFP双阳性以及任何mCherryFP ,GFP和CFP三阳性人群的靶细胞进行排序。应调整CFP阳性事件的分类门设置,以使收集窗口比三个阴性对照中任何一个的CFP通道的主要种群边缘至少高2倍。相反,所收集的双阳性细胞不应包含高于阴性对照的CFP信号。
生长并扩展双阳性和三阳性细胞以进行进一步分析。
注意:将分类的细胞冷冻在-80°C下,以创建备份。


FACS数据分析
通过使用FCS Express 6或FlowJo 分析FACS数据。CFP阳性细胞的双重之间的百分比- 阳性细胞可以使用FCS Express 6中和MS-Excel中量化。
前向和侧面散射应用于排除细胞碎片/碎片或细胞团块/团块。GFP和mCherryFP 信号的检测阈值应通过未处理的亲代细胞或用仅GFP或仅mCherryFP的表达载体稳定转导的亲代细胞建立。为双选通阈值- 阳性细胞(GFP和mCherryFP )应当被至少一个从各个阳性(GFP-只和折叠起来的mCherry -only)(图4)。
 






图4 。示意图显示流式细胞术门控策略。通过使用前向散射和侧面散射将门控应用于单个细胞和活细胞。对于单个荧光(GFP和mCherry ),基于阴性对照,仅GFP对照和仅mCherry 对照应用门控。选择GFP和mCherryFP 双阳性细胞的门应至少比仅GFP和mCherryFP 细胞的信号的上边缘高出一倍。


 


在采样组中对GFP和mCh erryFP 双阳性细胞应用门控,然后在该群体中寻找CFP阳性细胞。CFP的门控应使用表达GFP加mCherryFP 但不表达CFP的CFP阴性对照细胞建立。根据我们的经验,也可以使用携带报告基因(带有mCherryFP 标记)和pQC -XIG(带有GF P标记)的细胞来设置此阈值。选通应设置为至少比CFP阴性细胞生成的CFP信号主要种群的边界高两倍。
同一组门控应普遍应用于所有样品组。CFP荧光的存在指示in / del事件的发生,从而成功进行了基因组编辑(请参见图5中的示例)。非特异性(ns)加扰序列gRNA-GFP(图5)反映了通过非特异性基因编辑自发产生的CFP事件的背景信号。
 






图5 。使用此报告工具在HEK293T细胞中描述CFP阳性事件的数据示例。数据显示没有CFP荧光(FsCFP- 具有来自人VEGF基因+ pQC -XIG的靶序列的报告子);自发诱变引起的背景CFP荧光(报告基因+ 非特异性(ns)gRNA);in / del事件(报告+ VEGF gRNA)发生了CFP阳性事件。GFP + MCH +表示双重的选通- 阳性细胞(GFP和mCherry的)。GFP + MCH + CFP +表示CFP +阳性事件在第存在E双击- 阳性细胞(GFP和mCherry的)。


 


可以使用MS-Excel或软件的内置功能(在这种情况下为FCS Express 6)来计算每个细胞群体中双阳性中CFP事件的数量或百分比,即基因组编辑效率的读数。)。
基因组序列细胞集落分析
如果需要,单- 细胞集落可通过稀释来生产和在DNA分析96孔板分选的细胞的扩散。有关测试中所有步骤的摘要,请参见图6。


 






图6.图表示工作流和估计时间来建立携带的细胞系FsCFP-mCherryFP 记者和执行的基因组编辑测定(与Cas9作为一个例子)


 


笔记:


                            如果靶序列含有内部的START密码子,则应注意确保其不会导致CFP的框内翻译。如果是这种情况,可以在PAM位点之后(最好在过早的STOP密码子之后)插入一个额外的核苷酸,以进一步移动阅读框。
该方法对低频输入/删除事件也很敏感。我们已经通过突变靶向gRNA中的两个中心核苷酸来测试了这一点,这有望显着降低靶向效率。当这种突变的gRNA与报告基因共同表达时,在表达GFP和mCherryFP 的细胞中检测到CFP信号大大降低但仍具有统计学意义。
自发诱变,例如碱基切除引起的诱变,也可能导致CFP信号的重新激活。但是,根据我们的经验,这种自发事件的大小(如报告基因和pQC -XIG载体所证实的细胞)比补充有非特异性gRNA的Cas9的非特异性作用低至少一个数量级。因此,根据我们的经验,使用携带报告基因(带有mCherryFP 标记)和pQC -XIG载体(带有GFP标记)的细胞来建立CFP检测门是安全的。但是,如果需要较低的背景来设置CFP检测门,则可以将仅表达GFP和mCherryFP 且不携带任何CFP编码序列的细胞用作对照。
为CFP检测栅极可以或者上下移动几个(1-5)折叠,只要阈值在CFP-阴性对照细胞的主要群体的边界(与GFP和上述mCherryFP 双- 阳性)和只要将通用门应用于所有采样组,每个组之间的差异仍将存在。
 


致谢


 


我们感谢Sylvester综合癌症中心的流式细胞仪核心设施提供的服务。我们还要感谢资金资源– NIGMS / NIH,R01#GM107333;国防部(CDMRP),创意奖,PC140622。这些资助资源部分支持作者的薪水和实验费用。这些资助机构没有直接参与研究设计,收集,分析,数据解释或手稿撰写的任何部分。该协议改编自已发表的工作(Kumar 等人,2019)。              


 


利益争夺


 


作者宣称他们没有竞争的财务利益。






参考文献


 


克里斯蒂安·M。,塞尔马克·T。,多伊尔·EL,施密特·C。,张·F,胡默尔·A。,博格达诺夫,AJ和沃伊塔斯(DF)(2010)。TAL效应子核酸酶靶向DNA双链断裂。遗传学186(2):757-761。              
Cong,L.,Ran,FA,Cox,D.,Lin,S.,Barretto,R.,Habib,N.,Hsu,PD,Wu,X.,Jiang,W.,Marraffini,LA和Zhang,F (2013)。使用CRISPR / Cas系统进行多重基因组工程。科学339(6121):819-823。              
埃皮纳特·J·C,阿诺尔德·S。,查姆斯·P。,罗凯克斯·P。,德方丹斯·D。,普赞C.,帕丁·A。,赞格里尼·A。,帕克斯·F。和拉克鲁瓦E.(2003 )。一种新颖的工程化大范围核酸酶在酵母和哺乳动物细胞中诱导同源重组。Nucleic Acids Res 31(11):2952-2962。
Jinek,M.,Chylinski,K.,Fonfara,I.,Hauer,M.,Doudna,JA和Charpentier,E.(2012)。可编程双RNA指导的DNA核酸内切酶在适应性细菌免疫中的作用。科学337(6096):816-821。
Kim,YG,Cha,J.和Chandrasegaran,S.(1996)。杂交限制酶:锌指与Fok I切割域的融合体。美国国家科学院院刊93(3):1156-1160。
Kumar,A.,Birnbaum,MD,Moorthy,BT,Singh,J.,Palovcak,A.,Patel,DM和Zhang F.(2019)。插入/缺失激活移码荧光蛋白是基因组DNA编辑的敏感报告基因。BMC基因组学20(1):609。              
Maeder,ML和Gersbach,CA(2016)。基因和细胞疗法的基因组编辑技术。分子24(3):430-446。              
Sander,JD和Joung,JK(2014)。用于编辑,调节和靶向基因组的CRISPR-Cas系统。Nat Biotechnol 32(4):347-355。
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引用:Moorthy, B. T., Kumar, A., Lotenfoe, L. X. and Zhang, F. (2020). Evaluation of the Efficiency of Genome Editing Tools by a Frameshift Fluorescence Protein Reporter. Bio-protocol 10(10): e3622. DOI: 10.21769/BioProtoc.3622.
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