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Nov 2021

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Optimized CRISPR-Cas9-based Strategy for Complex Gene Targeting in Murine Embryonic Stem Cells for Germline Transmission
用于生殖系传递的基于 CRISPR-Cas9 的小鼠胚胎干细胞中优化的复杂基因靶向策略   

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Abstract

Although CRISPR-Cas9 genome editing can be performed directly in single-cell mouse zygotes, the targeting efficiency for more complex modifications such as the insertion of two loxP sites, multiple mutations in cis, or the precise insertion or deletion of longer DNA sequences often remains low (Cohen, 2016). Thus, targeting and validation of correct genomic modification in murine embryonic stem cells (ESCs) with subsequent injection into early-stage mouse embryos may still be preferable, allowing for large-scale screening in vitro before transfer of thoroughly characterized and genetically defined ESC clones into the germline. This procedure can result in a reduction of animal numbers with cost effectiveness and compliance with the 3R principle of animal welfare regulations. Here, we demonstrate that after transfection of homology templates and PX458 CRISPR-Cas9 plasmids, EGFP-positive ESCs can be sorted with a flow cytometer for the enrichment of CRISPR-Cas9-expressing cells. Cell sorting obviates antibiotic selection and therefore allows for more gentle culture conditions and faster outgrowth of ESC clones, which are then screened by qPCR for correct genomic modifications. qPCR screening is more convenient and less time-consuming compared to analyzing PCR samples on agarose gels. Positive ESC clones are validated by PCR analysis and sequencing and can serve for injection into early-stage mouse embryos for the generation of chimeric mice with germline transmission. Therefore, we describe here a simple and straightforward protocol for CRISPR-Cas9-directed gene targeting in ESCs.


Graphical abstract:




Keywords: CRISPR-Cas9 (CRISPR-Cas9), Mutagenesis (诱变), Murine embryonic stem cells (鼠胚胎干细胞), mESC (mESC), Gene targeting (基因靶标), Genetically engineered mouse mutants (基因工程小鼠突变体), GEMMs (GEMMs)

Background

The advent of CRISPR-Cas9 technology has led to a surge in interest in gene editing, due to its simplicity, speed, and low cost. CRISPR-Cas9 relies upon RNA-DNA binding, providing a significant simplification over previous gene editing methods like zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), which rely upon protein-DNA binding at the site of interest and are therefore more cumbersome to engineer and not ubiquitously useful (González Castro et al., 2021). Due to its efficacy in vivo, CRISPR-Cas9 has quickly become a standard method for the development of genetically engineered mouse mutants. CRISPR-Cas9 machinery, composed of single guide RNAs (sgRNA), homology templates, and Cas9 protein, can be injected into zygotes for creation of chimeric offspring (Qin et al., 2016; Hall et al., 2018; Muñoz-Santos et al., 2020). However, depending on the nature of the desired genetic modification, the efficacy of correct mutagenesis can vary considerably, with a concomitant increase in mouse numbers and associated burden when creating new mouse lines.


An alternative strategy for utilizing CRISPR-Cas9 technology to create mutant mouse lines is to enhance classical gene targeting in murine embryonic stem cells (ESCs) (Oji et al., 2016). CRISPR-Cas9-mediated DNA double-strand breaks induce homology directed repair (HDR) for more efficient introduction of complex or multiple genetic alterations into the germline. Correctly mutated ESCs can be injected into early-stage mouse embryos to yield chimeric mice (Qin and Wang, 2019). Thus, ex vivo gene editing in ESCs can reduce mouse numbers for generation of specific gene modified lines and is aimed at improving the workflow in accordance with the principles of the 3Rs (replace, reduce and refine).


Here, we report an optimized workflow for the induction of targeted mutations in murine ESCs for subsequent germline transmission in mice. We established this protocol to introduce specific point mutations at two positions in exon 7 and exon 17 in the murine Malt1 locus in one step (O'Neill et al., 2021). The two missense mutations yield glutamate to alanine exchanges at amino acid positions 325 and 814 within the two TRAF6 binding motifs of the MALT1A protein and specifically abolish the interaction with the ubiquitin ligase TRAF6. This simple and straightforward protocol should be generally useful for almost any type of targeted mutagenesis of the mouse germline. In short, murine ESCs are transfected with CRISPR-Cas9 machinery using a lipid-based transfection system. Successfully lipofected cells are enriched via an EGFP marker by cell sorting and screened by qPCR using primers specific to the targeted mutation site. The use of lipofection and cell sorting yields mutant ESC clones more conveniently and faster than with classical protocols utilizing electroporation and subsequent selection by antibiotic resistance. The resulting ESC clones are potentially totipotent and competent for germline transmission upon injection into C57BL/6 mouse embryos, yielding chimeric mice for generation of the desired mouse line. This protocol provides a framework for researchers who seek to develop mouse models with complex, difficult or multiple mutation loci, including deletion of large stretches of DNA, insertion of exon-flanking loxP sites for conditional models, and insertion of reporter genes at specific loci. This workflow allows for high-throughput screening of ESC clones and an overall reduction of mouse numbers and associated burden.

Materials and Reagents

Cell lines and oligonucleotides

  1. R1/E (129S1/X1) murine embryonic stem cells (ESCs)

  2. Murine embryonic feeder (MEF) cells (prepared from wildtype mice)

  3. Top10 chemically competent E. coli (produced in-house (transformation efficiency: 5 × 106) or commercially available cells [e.g., XL10-Gold Ultracompetent cells (Agilent, catalog number: 200317)]

  4. Homology templates - Ultramer DNA Oligos, 2 nanomole scale (IDT)

  5. Single guide (sg) RNA oligonucleotides - forward and reverse (Eurofins, 100 µM)

  6. qPCR primers (Eurofins, 100 µM)

  7. PX458 vector (pSpCas9(BB)-2A-GFP) (Addgene, catalog number: 48138)


Materials

  1. 10 cm Petri dishes (Corning, catalog number: 353003)

  2. Pipette tips - various

  3. 96-well flat-bottom plates (Corning, catalog number: 3997)

  4. 96-well round-bottomed plates (Greiner, catalog number: 6500101)

  5. 3 mL FACS tubes (Falcon, catalog number: 352063)

  6. 6-well plates (ThermoFisher Scientific, catalog number: 140685)

  7. 100 µm cell strainer (Neolab, catalog number: GF-0061)

  8. Lipofectamine 3000 transfection kit (Invitrogen, catalog number: L3000-001)

  9. BbsI restriction enzyme (NEB, catalog number: R0539L, 10 units/µL)

  10. T4 DNA ligase (Thermo Fisher Scientific, catalog number: EL0011)

  11. NEB buffer 2.1 (10×) (NEB, catalog number: B7202S)

  12. Sterile, nuclease-free water (VWR, catalog number: PD092)

  13. NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, catalog number: 7400609.50)

  14. SOC medium (Life Technologies, catalog number: 15544034)

  15. Ampicillin (Gibco, catalog number: 11593027)

  16. LB agar (Carl Roth, catalog number: X9963)

  17. LB (Luria/Miller) medium (Carl Roth, catalog number: X968.3)

  18. Miniprep kit (DNA isolation kit) (Macherey-Nagel, catalog number: 740499.250)

  19. Mitomycin C (MMC) (Sigma-Aldrich, catalog number: M4287-5x2mg)

  20. DPBS (ThermoFisher Scientific, catalog number: 14190144)

  21. OptiMEM medium (ThermoFisher Scientific, catalog number: 31985062)

  22. Proteinase K (ThermoFisher Scientific, catalog number: 26160)

  23. Trypsin-EDTA 0.05% (ThermoFisher Scientific, catalog number: 25300-054)

  24. DMSO (Carl Roth, catalog number: A994.1)

  25. DMEM medium (Gibco, catalog number: 41966-129)

  26. KnockoutTM DMEM medium (Life Technologies, catalog number: 10829018)

  27. Glutamax (Invitrogen, catalog number: 35050061)

  28. NEAA (Gibco, catalog number: 11140-035)

  29. Pen/Strep (Gibco, catalog number: 15140-122)

  30. Fetal bovine serum (FBS) (Gibco, catalog number: 10270-106) – regular charge for MEF cell culture

  31. ESC FBS (tested for ESC culture to maintain totipotency)

  32. β-Mercaptoethanol (Thermo Fisher, catalog number: 31350010)

  33. LIF/ESGRO (Millipore, catalog number: ESG1107)

  34. EDTA (Carl Roth, catalog number: 8043.1)

  35. Sodium chloride (NaCl) (Carl Roth, catalog number: 3957.2)

  36. Tris (Carl Roth, catalog number: AE15.2)

  37. 1× SYBRTM Safe DNA Gel Stain (ThermoFisher Scientific, catalog number: S33102)

  38. Ethidium Bromide solution 0.07% (PanReac AppliChem, catalog number: 1239-45-8)

  39. Glycerol (Carl Roth, catalog number: 4043.3)

  40. Sodium dodecyl sulfate (SDS) (Carl Roth, catalog number: 2326.2)

  41. Bromophenol blue (Sigma-Aldrich, 115-39-9)

  42. Xylene cyanol (Sigma-Aldrich, 2650-17-1)

  43. 10× annealing buffer (see Recipes)

  44. MEF medium (see Recipes)

  45. ESC medium (see Recipes)

  46. 2× MEF Freezing medium (see Recipes)

  47. 2× ESC Freezing medium (see Recipes)

  48. 10× MMC stock (see Recipes)

  49. 10× DNA loading buffer (see Recipes)

Equipment

  1. Microscope (Carl ZeissTM, Axiovert 40 CFL equipped with a ZeissTM A-Plan 2.5×/0.06 objective and a ZeissTM E-PI 10×/20 ocular)

  2. Fluorescence microscope (Life Technologies, EVOS FL)

  3. Vortex (Merck, Vortex Genie 2, catalog number: Z258423-1EA)

  4. Gel imaging system/UV transilluminator (INTAS)

  5. NanodropTM 2000 (ThermoFisher Scientific, ND-2000)

  6. Micropipettes (Eppendorf, variable volumes)

  7. Multi-channel micropipettes (Eppendorf, variable volumes)

  8. Tabletop cooling centrifuge (Eppendorf, 5417R)

  9. Sterile bench (Thermofisher Scientific, HERASafe KS/Telstar, Telstar Bio II A)

  10. LightCycler® Real-time PCR machine (Roche, LightCycler 480 II)

  11. Autoclave (various)

  12. Cell sorter (MoFlo, Cytomation, with Summit 4.3 software)

  13. Electrophoresis chamber (Carl Roth, Midi)

  14. Electrophoresis power supply (Sigma-Aldrich, Consort EV243)

Software

  1. MoFlo® computer software (Summit 4.3 Software, Beckman Coulter); FlowJo (BD)

  2. LightCycler® 480 software (https://lifescience.roche.com/en_de/products/lightcycler14301-480-software-version-15.html)

  3. CRISPR-Cas9 guide design software such as CRISPick by BROAD Institute (numerous other options are available, see Cui et al., 2018)

Procedure

The general aim of this protocol is to manipulate the germline of murine embryonic stem cells (ESCs) by introducing one or more mutations simultaneously and possibly even within the same gene. For this, ESCs are transfected with one or more CRISPR plasmids encoding sgRNA(s) that introduce double-stranded DNA breaks at the site(s) of mutagenesis. Homology templates that contain the desired mutations are co-transfected with the guide(s) to induce homology directed repair (HDR). Afterwards, single cell-derived ESC clones are grown, isolated, and screened for correct genetic manipulation of the genomic sequence(s).


  1. Selection of CRISPR single guide RNA (sgRNA) sequences (CRISPR guides)

    Use a CRISPR-Cas9 sgRNA design program to find optimal spCas9 (S. pyogenes Cas9) cleavage sites and corresponding 20 nucleotide guide sequences near the target gene locus, for example, CRISPick from the Broad Institute based on algorithms developed by Doench and colleagues (Doench et al., 2016; Sanson et al., 2018). It is important that the Cas9 cleavage site (located 3 to 4 base pairs upstream of the S. pyogenes PAM sequence 5’-NGG) be as close as possible to the site of mutation since an inverse correlation between mutation efficiency and cut-to-mutation distance has been described (Paquet et al., 2016). For improved expression of the guide RNA by the U6 RNA polymerase III promoter of the PX458 plasmid, it is recommended to add an extra “G” at the 5‘ of the sgRNA if the 20-nt guide sequence does not already begin with a “G” (Ran et al., 2013). For cloning, a guide oligo (with a BbsI 5’-CACC sticky end) and the corresponding reverse complement guide oligo (with a BbsI 5’-AAAC sticky end) must be annealed to obtain a double-stranded oligonucleotide that can be cloned into the BbsI site of the PX458 vector (pSpCas9 backbone) (Figure 1). Note that the guide sequence does not include the 5’-NGG PAM sequence itself. For further details see Ran et al. (2013).



    Figure 1. sGuide RNA oligonucleotide design example taken from O'Neill et al. (2021), Figure S1, Malt1 exon 17 targeting.

    A 20-bp guide sequence was identified by a CRISPR design algorithm (Doench et al., 2016) and subsequently used for designing the forward oligo. An “extra G” (blue) was added to the 5’ end of the 20-bp guide sequence, followed by the BbsI 5’-CACC sticky end (green). The reverse oligo is the complement of the 20-bp guide sequence plus a “C” complementary to the “extra G (blue)” and has a 5’-AAAC sticky end (green). The annealed oligo has sticky end overhangs (green) for ligation into the BbsI sites of the PX458 CRISPR plasmid.


  2. Design of ssODN Homology Templates

    For the generation of point mutations or the introduction of small insertions or deletions, it is possible to use approximately 200 bp single-stranded oligodesoxyribonucleotides (ssODN) as homology templates for HDR. These can be ordered from companies specializing in high-precision DNA synthesis. The desired alteration(s) should be placed at the center of the ssODN. If possible, the PAM sequence, which directs Cas9 to the target site, should be silently mutated within the homology template sequence to prevent Cas9-directed cleavage of the homology template or the successfully modified target locus. Alternatively, or in addition to PAM mutation, silent mutations within the 20-nt guide sequence can be introduced, which at the same time facilitates the design of a mutant allele-specific PCR primer (there is no specific number of silent mutations that can be universally recommended; in our hands, four silent mutations proved to be appropriate, as shown in Figure 2). Moreover, an artificial restriction site may be generated within the homology template by silent mutagenesis, offering an additional option for screening ESC clones. An example HDR is shown in Figure 2. We want to mention that, for another project, we achieved successful targeting by using a linear double-stranded DNA homology template of approximately 1,400 bp total length with 700 bp homology arms flanking the site of mutagenesis (this might be useful for larger scale genetic manipulations such as deletion or insertion of longer stretches of DNA).



    Figure 2. Homology template design example taken from O'Neill et al. (2021).

    Malt1 exon 17 targeting with the ‘antisense guide’ sequence as shown in Figure 1. The desired targeted mutation is shown in orange. The “CCC” triplet codon (representing the “GGG” PAM sequence since an antisense guide was utilized here) (grey) could not be silently mutated. Instead, silent mutations (blue) were inserted to prevent binding of the sgRNA to the homology template. For comparison, the wildtype sequence of the homology region is shown above with the “GGG” PAM in grey and the 20-nt guide sequence indicated by a box. The approximate expected cleavage site in the wildtype sequence is indicated with a double line.


  3. Cloning CRISPR guide oligos into the PX458 plasmid

    This protocol uses the plasmid PX458, which contains a cassette for transient co-expression of SpCas9 and EGFP and a cassette into which a custom designed CRISPR guide oligo can be ligated to generate a single guide sequence that is expressed under the control of a U6 RNA polymerase III promoter (Ran et al., 2013). When transfected into ESCs, the PX458-guide construct leads to expression of the sgRNA, Cas9, and EGFP. EGFP-positive ESCs can be enriched by FACS for further culture on feeder cells and outgrowth of single ESC clones.

    1. Anneal the forward and reverse sgRNA oligonucleotides. Set up the annealing reaction by mixing:

      Sense guide oligonucleotide (100 µM) 2 µL
      Antisense guide oligonucleotide (100 µM) 2 µL
      Annealing buffer (10×) 2 µL
      Water (nuclease-free) 14 µL

      Heat the mixture to 95°C in a heating block for 5 min. Turn off the heating block and allow mixture to cool to room temperature (approximately 3 h). Dilute annealed oligonucleotides 1:400 in nuclease-free water.

    2. Digest the PX458 vector with BbsI for guide insertion. Set up the following restriction digest reaction:

      PX458 2 µg
      NEB 2.1 buffer (10×) 2 µL
      BbsI (10 units/µL) 1.5µL
      Water (nuclease-free) add to 20 µL total

      Incubate for 1 h at 37°C on a heating block. Mix restriction reaction with 2 µL of 10× DNA loading buffer and separate on a 1% agarose gel (containing 0.5 µg/mL ethidium bromide or 1× SYBRTM Safe DNA Gel Stain) at 120 V for 1 h. Cut out the linearized PX458 vector fragment (9,270 bp), purify with a gel purification kit, and measure concentration on a NanodropTM.

    3. Ligate the annealed oligonucleotides (from step1; 1:400 diluted = 25 nM) into the PX458 vector (from step 2). Set up the following ligation mix:

      PX458 (linearized) 100 ng
      Annealed guide oligos (25 nM) 2 µL
      T4 ligase (5 units/µL) 2 µL
      T4 ligase buffer (5×) 2 µL
      Water (nuclease-free) add to 10 µL total

      Incubate the ligation mix at room temperature for 15 min.

    4. For transformation into E. coli, add 2 µL of ligated sgRNA guides (from step 3) to 25 µL chemically competent E. coli. Incubate mixture on ice for 5 min, heat-shock at 42°C for 40 s, and place on ice for 5 min. Mix E. coli with 150 µL of SOC medium without antibiotics and incubate for 1 h at 37°C. Spread the total cell volume on LB plates containing Ampicillin and incubate overnight at 37°C. Select colonies and inoculate into 5 mL of LB medium with Ampicillin and grow overnight at 37°C and 180 rpm. Isolate plasmid DNA using a DNA isolation kit and quantify DNA via NanodropTM. Following sequencing verification of correct guide insertion, the PX458 CRISPR guide plasmid can be used for transfection into murine ESCs for co-expression of the sgRNA and Cas9, with EGFP as a reporter for successful transfection and enrichment via flow cytometric cell sorting.


  4. Preparation and Culture of Murine Embryonic Fibroblasts (MEFs)

    ESCs need to be cultured on a layer of mouse embryonic fibroblasts (MEFs), which act as feeder cells. MEFs are generated from 12.5 dpc (days post coitum) wildtype mouse embryos and can be passaged four times before being growth arrested by treatment with Mitomycin C (MMC) so that they then can serve as feeder cells for ESC culture.

    1. Harvesting MEFs

      Prepare murine MEF cells from wildtype mouse embryos on day 12.5. Briefly, in a cell culture dish containing PBS, separate each embryo from the yolk sac and remove and discard the head and liver. Subsequently, each embryo is processed separately: mesh remaining tissue through a 100 µm mesh into MEF medium and transfer cells of one embryo to a 6-cm cell culture dish. Let MEF cells grow overnight. On the next day, wash away non-adherent, dead cells and allow cells to grow further until confluent (approximately 2 days).

    2. Passaging MEFs

      Detach adherent MEF cells from the surface of the 6 cm dish by washing cells with PBS, adding 2 mL trypsin, and incubating 5 min at 37°C. Stop trypsinization by adding MEF medium. Transfer harvested MEFs from the 6 cm dish to one 15 cm plate and let grow in the incubator. Upon confluency, perform a second passage from the 15 cm plate (use 8 mL trypsin) to four 15 cm dishes. MEF cells can now be frozen in liquid nitrogen (see step 3) or treated with Mitomycin C (MMC) for use in ESC culture (see step 4).

    3. Freezing and thawing MEFs

      Harvest MEFs from one confluent 15 cm dish (approximately 2 × 107 cells) by trypsinization, as described in step 2. Take up harvested cells in 2 mL of MEF medium and put on ice. Add 2 mL of ice-cold 2× Freezing medium (1:1) and transfer 1 mL aliquots (containing approx. 5 × 106 cells) to cryo-vials, which are placed in a styrofoam box for slow freezing in a -80°C freezer. After 24 h, transfer the frozen cells to liquid nitrogen for long-term storage. For thawing, float a cryo-vial in a 37°C water bath and transfer the thawed cell suspension into 10 mL of PBS. Centrifuge at 350 × g for 5 min, take up the cell pellet in 10 mL of MEF medium, distribute the cells to four 10 cm dishes and incubate at 37°C/5% CO2. Either let cells grow for 2 days for further passaging or use on the next day for treatment with MMC (see step 4).

    4. Treatment of MEFs with Mitomycin (MMC)

      MEF cell density for use as an MMC-treated feeder cell layer is 2.5 × 106 cells per 10 cm dish. Treat MEF cells with MMC (10 µg/mL in MEF medium) and incubate at 37°C for 2 to 3 h to inhibit further proliferation. Remove medium containing MMC, wash cells thoroughly three times with PBS, and add fresh MEF medium. MCC-treated MEFs are now ready for use as a base layer for ESC growth (and should be used within 2 days after MMC treatment).


  5. ESC culture (R1/E cells)

    ESCs must be cultured under conditions that allow for maintenance of totipotency. For this, ESCs are cultured on a layer of feeder cells and must be kept at appropriate cell density in ESC medium containing FBS that has been approved for ESC culture (can be purchased from commercial sources or obtained from labs specializing in ESC culture). Murine R1/E ESCs are always grown on a confluent (but not too dense) feeder layer of MMC-treated MEFs (approximately 40,000 feeder cells/cm2).

    1. Seed approximately 30,000 R1/E ESCs per cm2 on feeder cells in ESC medium. Medium is exchanged daily, and ESCs are usually split after 2 days of culture at appropriate cell density and morphology: this condition is reached when the ESCs form numerous distinct colonies with sharp edges that together cover approximately one-third of the surface of the culture dish without touching each other (Figure 3). For splitting, ESCs are washed with PBS and treated for 5 min with trypsin, and trypsin is inactivated by addition of ESC medium. A single cell suspension is obtained by vigorous pipetting with an electric pipetting device: draw the cell suspension into a 10 mL pipet, press the tip of the pipette onto the bottom of the cell culture dish, and force the cell suspension out of the pipet. Repeat this 10 times. Count cells and seed 30,000 cells/cm2 on fresh feeder cells. With some experience, it may not be necessary to always count the ESCs, and after visual inspection of the cells under the microscope it is possible to estimate an acceptable split factor, usually 1:6 for ESCs at optimal growth conditions and cell density (a split factor of 1:6 means that the cells are harvested from a cell culture plate and 1/6 of the cells are transferred into a new cell culture plate of the same format).



      Figure 3. Morphology of R1/E ESCs.

      R1/E cells form distinct colonies and cover approximately one-third of the plate surface.


    2. ESCs can be frozen in liquid nitrogen: for this, ESCs are trypsinized as described above, cells are counted, spun down at 350 × g, adjusted to 5 × 106 cells/mL in ESC medium, and shortly pre-cooled on ice. An equal volume of ice-cold 2× ESC Freezing medium is added and, after mixing, 1 mL of cell suspension is distributed to cryo-vials for freezing at -80°C within a styrofoam box with subsequent transfer to liquid nitrogen. For later thawing, the cryo-vial is placed in a 37°C water bath and, when content is liquid, the cells are transferred into 10 mL PBS in a 15 mL tube, spun at 350 × g, and the cell pellet is taken up in ESC medium. Cells from each cryo-vial (2.5 × 106 cells) are seeded on feeder cells in a 10 cm dish.


  6. Transfection of ESCs

    Reported protocols typically use electroporation to transfect ESCs with plasmids and homology templates. Electroporation requires large amounts of DNA (20 µg) as well as many cells (commonly approximately 5 × 106 cells grown in a 10 cm dish), and many cells die due to the harsh electroporation conditions. Lipofection proves to be gentler, allowing the use of much lower cell numbers (200,000 cells grown in a 6-well plate) and smaller DNA amounts (2 µg or less). This results in approximately 5% transfection efficiency as measured by the percentage of EGFP-positive cells in flow cytometry, while only a small proportion of transfected cells with high EGFP expression can be seen under a fluorescence microscope (Figure 4). Transfection of ESCs is performed in a 6-well format using lipofection via the Lipofectamine 3000 kit based on the manufacturer’s protocol.

    1. Approximately 16 h prior to transfection, seed 200,000 ESCs per well of a 6-well plate in 2 mL of ESC medium (on feeder cells).

    2. On the day of transfection, prepare mixtures in Tube A and Tube B:

      Tube A: 125 µL OptiMEM medium + 5 µL Lipofectamine 3000 reagent.

      Tube B: 125 µL OptiMEM medium + 6 µL P3000 reagent + 6 µg DNA (PX458 plasmids and homology templates, evenly distributed to the maximum total DNA amount of 6 µg).

    3. Mix the contents of tubes A and B together and incubate at room temperature for 15 min.

    4. Transfer the entire transfection volume to the ESCs in the 6-well (step 1).

    5. Incubate the ESCs with the DNA-liposome complexes for 6 h.

    6. After incubation, replace the medium with fresh ESC medium and let cells grow for 24 h in the incubator before sorting EGFP-positive cells by FACS.


  7. FACS-based cell sorting of ESCs and monoclonal ESC clone growth

    Successfully transfected ESCs express EGFP encoded by the PX458 plasmid and can therefore be enriched to more than 95% by FACS (Figure 4B). Only 2000 sorted EGFP-positive ESCs are needed to be seeded on a feeder layer in a 10 cm dish for the outgrowth of up to 100 ESC clones. In contrast to classical protocols that use DNA constructs with antibiotic resistance cassettes for selection, FACS-sorted ESCs do not need to be treated with antibiotics, and therefore the ESC plates do not contain millions of dying cells in culture, which would have to be carefully washed away. Monoclonal ESC clones grow with ESC medium exchanged every second day and are ready for picking within 7 days, which is approximately 2 days faster than experienced with classical protocols using electroporation and antibiotic selection.

    1. Twenty-four hours after lipofection, wash ESCs in the 6-well plates (from Procedure F, step 6) 1× with PBS.

    2. Add 500 µL of trypsin, incubate 5 min, add 500 µL of ESC medium, and pipette vigorously up and down to generate a single cell suspension containing ESCs and feeder cells.

    3. Transfer the cell suspension to a new 6-well plate. Within 1 h, most feeder cells but not the ESCs will re-adhere. Gently remove the ESCs and transfer to a 3 mL tube for cell sorting.

    4. Using a cell sorter equipped with a 100 µm nozzle, sort EGFP-positive ESCs into ESC medium. Plate 2000 sorted cells onto a 10 cm dish pre-laid with 3 × 106 MMC-treated MEF cells and allow cells to grow for approximately 7 days with medium changes every second day (Figure 5A). Up to 100 ESC colonies can be expected to grow per 10 cm dish. Therefore, it is recommended to seed ten 10 cm dishes per condition to obtain sufficient numbers of colonies for screening (we recommend screening approximately 500 colonies).



    Figure 4. ESCs 24 h after lipofection with PX458.

    (A) Cells with high EGFP expression seen under a fluorescence microscope. (B) Flow cytometry allows for sensitive detection of ESCs with even low to moderate EGFP expression (black curve) when compared to untransfected cells (grey curve). Cell sorting leads to enrichment of EGFP-expressing cells to over 95% (green curve).


  8. Picking ESC clones

    Monoclonal ESC clones can be picked approximately 7 days after cell sorting when distinct 3-dimensional colonies have grown on the 10-cm plates and can be seen under a microscope (Figure 5A).

    1. Prior to picking colonies, seed 2 flat-bottomed 96-well plates with MMC-treated MEF cells in 100 µL of ESC medium (approximately 10,000 cells per well). Keep plates in the incubator until needed.

    2. Wash the 10 cm plates containing ESC colonies with 10 mL of PBS and add 10 mL of fresh PBS to the plates.

    3. Place a microscope under the sterile work bench to visualize the single ESC colonies (Figure 5B). Ensure that there is sufficient space to approach the plate from the side with a 100 µL pipette. Detach each colony from the feeder layer by gently dislodging it from the surface with the pipette tip and drawing it up in a volume of 50 µL. Care should be taken to extract the entire ESC colony to avoid distribution of ESCs to the plate and therefore cross-contamination of other monoclonal colonies.



      Figure 5. Cell picking workflow and set-up.

      (A) EGFP positive cells from cell sorting are plated at a density of 2000 cells per 10 cm dish on a layer of MMC-treated MEF cells. Following a 7-day growth period, 50–100 monoclonal ESC clones can be picked from each 10 cm dish into duplicate 96-well plates for freezing or qPCR screening, respectively. (B) Microscope set-up for clone picking under a sterile cell culture hood.


    4. Transfer the picked colonies to a round-bottomed 96-well plate. Pick 96 clones to fill the plate and as many plates as are needed for screening (we typically pick five 96-well plates). Proceed as quickly as possible to step 5 (it is recommended to work in tandem: one person picking and another processing the picked clones).

    5. Following collection of 96 colonies, add 30 µL of trypsin to each round-bottomed 96-well containing the picked ESC clones and incubate the plate at 37°C for 10 min. Add 100 µL of ESC medium to each well to stop trypsinization.

    6. Using a multichannel micropipette, vigorously pipette up and down in each well to dissociate the ESC clone aggregates into single cells and split the contents of each well into the two flat-bottomed plates containing MEF cells (prepared in step 1). Incubate both plates for 2 days. Plate #1 is used for screening, and plate #2 is frozen for later recovery of positive clones identified by qPCR screening and sequence analysis.

    7. Before freezing ESCs in 96-well plates it can be of value to inspect every single 96-well for judging cell density and take notes about the cell culture plate format into which the cells should be seeded upon defrosting (this can be estimated by the 1:6 split factor as described in Procedure E, step 1). For freezing ESCs in 96-well plates, wash cells in Plate #2 (from step 6) 1× with PBS, trypsinize cells by adding 30 µL of trypsin per well, incubate for 5 min in the incubator, mix with 70 µL of ESC medium to stop the trypsinization reaction, and pipette vigorously to resuspend cells. Pre-cool the plate shortly on ice, then add 100 µL of ice-cold 2× ESC freezing medium, mix and freeze plate within a styrofoam box at -80°C, and later transfer to liquid nitrogen. For thawing cells from a 96-well plate, thaw the plate in a 37°C incubator, transfer desired clones to 15 mL Falcon tubes in 5 mL of medium, spin the cells down at 350 × g, aspirate the supernatant, wash the cells once with 1 mL of PBS, take up the cells in 100 µL of ESC medium, and transfer to an appropriate well size in sufficient medium (usually 48- or 24-well format).


  9. qPCR screening for correctly targeted ESC clones

    It is recommended to establish specific and sensitive qPCR protocols at the very beginning of the project as part of homology template design because it is vital that correctly manipulated ESC clones can be identified by well-established screening strategies. It may be valuable to get a DNA template synthesized that corresponds to the genomic locus after correct insertion of the homology template, which can serve as a positive control for specific and sensitive amplification of the mutated locus. Screening conditions can be even better simulated if genomic DNA from wildtype ESCs (prepared as described in steps 1 to 3 below) is spiked in when establishing PCR conditions. Test serial dilutions of the positive control templates down to a copy number of 1. Detectable amplification of 10 copies of the positive control template is usually sensitive enough to be able to later detect positive ESC clones by screening genomic DNA of ESC clones. To prevent false-positive signals during screening, be careful to avoid contamination of the work place and of equipment with “positive control” DNA fragments or positive screening amplicons; positive control DNA or amplicons should never be handled where later steps of the protocol take place (ESC culture, transfection, clone picking, and screening PCR).

    1. To prepare genomic DNA of ESC clones from Plate #1 (from Procedure H step 6) for qPCR screening, remove medium from the 96-well plate, wash cells once with PBS, and then add 200 µL of nuclease-free water to each well. In a 96-well PCR cycler, incubate the plate at 95°C for 15 min.

    2. Add Proteinase K to each well to a final concentration of 200 µg/mL and incubate at 55°C for 90 min for protein digestion. Heat plate to 95°C for 15 min to inactivate Proteinase K. Samples are now ready for screening.

    3. Design PCR screening primers that are specific for the amplification of correctly targeted sequences for the identification of successfully manipulated ESC clones. In case of site directed mutagenesis, one PCR primer should be mutation-specific by covering the mutation site (according to the design of the homology template as described in Procedure B), and the other primer should be located outside the homology template sequence. See, for example, Figure S1B in O'Neill et al. (2021). Potential positive clones will amplify at lower cycle threshold (Ct) values during qPCR. In the case of inserting multiple mutations within one ESC clone, qPCR protocols and mutation specific primers will need to be designed for each mutation site, and each qPCR screening will need to be performed separately.

    4. Perform qPCR according to your established protocol(s). Most likely, the majority of genomic ESC DNA samples will generate background signals at high Ct values (>35), depending on the specificity of the PCR protocol. However, a subset of samples will give rise to robust amplification at much lower Ct values (Figure 6). Those clones are candidates with genomic modifications which need to be further analyzed by sequencing (O’Neill et al., 2021, Figure S1C, D).



      Figure 6. ESC screening by qPCR.

      Clones are screened using a mutation-specific primer which only amplifies the correctly mutated target sequence. Negative clones (black curves) are excluded due to their high threshold cycle (Ct) value. Potential positive clones (grey/red curves) have lower Ct values. Prospective positive clones are verified by sequencing (potentially in combination with TOPO cloning) to find a clone with all mutations correctly inserted (red curve).


    5. For sequencing, the genomic region of interest is amplified with primers flanking the targeted site. The purified PCR product can then directly be used for sequencing. Sequencing data are often difficult to interpret since both alleles of the targeted gene can be altered differently, yielding two overlapping chromatograms that cannot be unequivocally assigned to one or the other allele. This can even be the case if online tools are used, such as CRISPID (Dehairs et al., 2016). In this case, a reliable technique to elucidate the sequence status of the distinct alleles is TOPO cloning of the above-mentioned PCR product. TOPO cloning results in E. coli clones that carry only one or the other allelic fragment, which can then be sequenced separately.

    6. ESC clones with confirmed genomic modification(s) can be recovered by thawing from plate #2 (see Procedure H, step 6, and step 7) and should be characterized with respect to healthy growth and good ESC morphology. Selected ESC clones are expanded and frozen in several aliquots and are now ready for embryo injection for creation of chimeric mice and subsequent germline transmission.

Recipes

  1. 10× Annealing buffer

    10 mM Tris

    1 mM EDTA

    50 mM NaCl

    pH 7.5

  2. MEF medium

    500 mL of DMEM

    50 mL of FBS (regular)

    500 µL of 50 mM β-Mercaptoethanol

    5 mL of NEAA

    5 mL of Pen/Strep

  3. ESC medium

    500 mL of KnockoutTM DMEM

    90 mL of FBS (ESC culture tested)

    1.2 mL of 50 mM β-Mercaptoethanol

    6 mL of NEAA

    6 mL of Glutamax

    6 mL of Pen/Strep

    90 µL of LIF/ESGRO (1,500 U/mL)

  4. 2× MEF Freezing medium

    20% DMSO

    30% FBS (regular)

    50% DMEM (with 1% Pen/Strep)

  5. 2× ESC Freezing medium

    20% DMSO

    30% FBS (ESC culture tested)

    50% DMEM (with 1% Pen/Strep)

  6. 10× MMC stock

    Dissolve MMC powder in MEF medium for a stock concentration of 100 µg/mL, sterile filter, and store at 4°C

  7. 10× DNA loading buffer

    34% Glycerol

    0.5% SDS

    10 mM EDTA

    2.5 mg/mL Bromophenol blue

    2.5 mg/mL Xylene cyanol

Acknowledgments

This protocol was first used in a publication in Science Immunology (O'Neill et al., 2021). This work was supported by Deutsche Forschungsgemeinschaft (ID 210592381 – SFB 1054 A04, ID 360372040 – SFB 1335 P07) and Deutsche Krebshilfe (No. 70112622). D.K. is a scientific advisor of Monopteros Therapeutics Inc., Boston.

Ethics

Pregnant mice used for the generation of Mouse Embryonic Fibroblasts from 12.5 dpc embryos were housed and handled in accordance with the guidelines of the Federation of European Laboratory Animal Science Association.

Competing interests

The authors declare no competing interests.

References

  1. Cohen, J. (2016). Any idiot can do it. Genome editor CRISPR could put mutant mice in everyone’s reach. Science. https://www.science.org/content/article/any-idiot-can-do-it-genome-editor-crispr-could-put-mutant-mice-everyones-reach.
  2. Cui, Y., Xu, J., Cheng, M., Liao, X. and Peng, S. (2018). Review of CRISPR/Cas9 sgRNA Design Tools. Interdiscip Sci 10(2): 455-465.
  3. Dehairs, J., Talebi, A., Cherifi, Y. and Swinnen, J. V. (2016). CRISP-ID: decoding CRISPR mediated indels by Sanger sequencing. Sci Rep 6: 28973.
  4. Doench, J. G., Fusi, N., Sullender, M., Hegde, M., Vaimberg, E. W., Donovan, K. F., Smith, I., Tothova, Z., Wilen, C., Orchard, R., et al. (2016). Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34(2): 184-191.
  5. González Castro, N., Bjelic, J., Malhotra, G., Huang, C. and Alsaffar, S. H. (2021). Comparison of the Feasibility, Efficiency, and Safety of Genome Editing Technologies. Int J Mol Sci 22(19): 10355.
  6. Hall, B., Cho, A., Limaye, A., Cho, K., Khillan, J. and Kulkarni, A. B. (2018). Genome Editing in Mice Using CRISPR/Cas9 Technology. Curr Protoc Cell Biol 81(1): e57.
  7. Muñoz-Santos, D., Montoliu, L. and Fernandez, A. (2020). Generation of Genetically Modified Mice Using CRISPR/Cas9. Methods Mol Biol 2110: 129-138.
  8. O'Neill, T. J., Seeholzer, T., Gewies, A., Gehring, T., Giesert, F., Hamp, I., Grass, C., Schmidt, H., Kriegsmann, K., Tofaute, M. J., et al. (2021). TRAF6 prevents fatal inflammation by homeostatic suppression of MALT1 protease. Sci Immunol 6(65): eabh2095.
  9. Oji, A., Noda, T., Fujihara, Y., Miyata, H., Kim, Y. J., Muto, M., Nozawa, K., Matsumura, T., Isotani, A. and Ikawa, M. (2016). CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice. Sci Rep 6: 31666.
  10. Paquet, D., Kwart, D., Chen, A., Sproul, A., Jacob, S., Teo, S., Olsen, K. M., Gregg, A., Noggle, S. and Tessier-Lavigne, M. (2016). Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533(7601): 125-129.
  11. Qin, W., Kutny, P. M., Maser, R. S., Dion, S. L., Lamont, J. D., Zhang, Y., Perry, G. A. and Wang, H. (2016). Generating Mouse Models Using CRISPR-Cas9-Mediated Genome Editing. Curr Protoc Mouse Biol 6(1): 39-66.
  12. Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A. and Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system.Nature Protocols 8(11): 2281-2308.
  13. Sanson, K. R., Hanna, R. E., Hegde, M., Donovan, K. F., Strand, C., Sullender, M. E., Vaimberg, E. W., Goodale, A., Root, D. E., Piccioni, F., et al. (2018). Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nat Commun 9(1): 5416.

简介

[摘要]虽然 CRISPR - Cas9 基因组编辑可以直接在单细胞小鼠受精卵中进行,但对于更复杂的修饰(例如插入两个loxP位点、顺式多个突变或更长 DNA 的精确插入或缺失)的靶向效率序列通常仍然很低(Cohen,2016) 。因此,在小鼠胚胎干细胞 (ESCs) 中靶向和验证正确的基因组修饰并随后注射到早期小鼠胚胎中可能仍然是可取的,允许在将完全表征和基因定义的 ESC 克隆转移到体外进行大规模筛选。种系。该程序可以减少动物数量,具有成本效益并符合动物福利法规的 3R 原则。在这里,我们证明在转染同源模板和 PX458 CRISPR - Cas9 质粒后,可以用流式细胞仪对 EGFP 阳性 ESC 进行分选,以富集表达 CRISPR - Cas9 的细胞。细胞分选避免了抗生素选择,因此允许更温和的培养条件和更快的 ESC 克隆生长,然后通过 qPCR 筛选正确的基因组修饰。与在琼脂糖凝胶上分析 PCR 样品相比,qPCR 筛选更方便且耗时更少。阳性 ESC 克隆通过 PCR 分析和测序验证,可用于注射到早期小鼠胚胎中,以产生具有生殖系传递的嵌合小鼠。因此,我们在这里描述了一个简单而直接的 CRISPR - Cas9 定向基因靶向 ESC 的协议。

图形概要:




[背景] 由于 CRISPR-Cas9 技术的简单、速度和低成本,它的出现引起了人们对基因编辑的兴趣激增。 CRISPR-Cas9 依赖于 RNA-DNA 结合,大大简化了以前的基因编辑方法,如锌指核酸酶 (ZFN) 和转录激活因子样效应核酸酶 (TALEN),后者依赖于感兴趣位点的蛋白质-DNA 结合因此对设计来说更麻烦,而且不是普遍有用的 (冈萨雷斯卡斯特罗等人,2021) 。由于其在体内的功效,CRISPR-Cas9 已迅速成为开发基因工程小鼠突变体的标准方法。由单向导 RNA (sgRNA)、同源模板和 Cas9 蛋白组成的 CRISPR-Cas9 机器可以被注入受精卵以产生嵌合后代(Qin et al. , 2016; Hall et al. , 2018; Muñoz-Santos et al., 2018)等人,2020) 。然而,根据所需基因修饰的性质,正确诱变的功效可能会有很大差异,伴随着小鼠数量的增加和创建新小鼠品系时的相关负担。
利用 CRISPR-Cas9 技术创建突变小鼠系的另一种策略是增强小鼠胚胎干细胞 (ESC) 中的经典基因靶向 (Oji等人,2016 年) 。 CRISPR - Cas9 介导的 DNA 双链断裂诱导同源定向修复 (HDR),以便更有效地将复杂或多种遗传改变引入种系。可以将正确突变的 ES Cs注射到早期小鼠胚胎中以产生嵌合小鼠(秦和王,2019) 。因此,胚胎干细胞中的离体基因编辑可以减少用于生成特定基因修饰系的小鼠数量,旨在根据 3R 原则(替换、减少和改进)改进工作流程。
在这里,我们报告了一种优化的工作流程,用于在小鼠 ESC 中诱导靶向突变,以便随后在小鼠中进行种系传播。我们建立了这一方案,以一步在小鼠Malt1基因座的外显子 7 和外显子 17 的两个位置引入特定点突变(O'Neill等人,2021) 。这两个错义突变在 MALT1A 蛋白的两个 TRAF6 结合基序内的氨基酸位置 325 和 814 处产生谷氨酸到丙氨酸交换,并特异性地消除与泛素连接酶 TRAF6 的相互作用。这种简单而直接的协议通常对几乎任何类型的小鼠种系靶向诱变都有用。简而言之,使用基于脂质的转染系统用 CRISPR-Cas9 机器转染鼠 ESC。成功的lipofected细胞通过细胞分选通过 EGFP 标记进行富集,并使用特定于目标突变位点的引物通过 qPCR 筛选。与利用电穿孔和随后通过抗生素抗性选择的经典方案相比,脂质转染和细胞分选的使用更方便、更快速地产生突变 ESC 克隆。由此产生的 ESC 克隆在注射到 C57BL/6 小鼠胚胎中后具有潜在的全能性并且能够进行种系传递,从而产生用于生成所需小鼠系的嵌合小鼠。该协议为寻求开发具有复杂、困难或多个突变位点的小鼠模型的研究人员提供了一个框架,包括删除大段 DNA、为条件模型插入外显子侧翼loxP位点以及在特定位点插入报告基因。该工作流程允许对 ESC 克隆进行高通量筛选,并全面减少小鼠数量和相关负担。

关键字:CRISPR-Cas9, 诱变, 鼠胚胎干细胞, mESC, 基因靶标, 基因工程小鼠突变体, GEMMs



材料和试剂


细胞系和寡核苷酸
1.R1/E (129S1/X1) 鼠胚胎干细胞 (ESC)
2.鼠胚胎饲养 (MEF) 细胞(由野生型小鼠制备)
3.Top10 化学感受态大肠杆菌(内部生产(转化效率:5 × 10 6 )或市售细胞[例如,XL10-Gold Ultracompetent 细胞(Agilent,目录号:200317)]
4.同源模板 - Ultramer DNA Oligos,2 纳摩尔规模 (IDT)
5.单向导 (sg) RNA 寡核苷酸 - 正向和反向 (Eurofins, 100 µM)
6.qPCR 引物(Eurofins,100 µM)
7.PX458 载体(pSpCas9(BB)-2A-GFP)( Addgene ,目录号:48138)


材料
1.10 cm 培养皿(Corning,目录号:353003)
2.移液器吸头 - 各种
3.96孔平底板(Corning,目录号:3997)
4.96孔圆底板(Greiner,目录号:6500101)
5.3 mL FACS 管(Falcon,目录号:352063)
6.6孔板( ThermoFisher Scientific,目录号:140685)
7.100 µm细胞过滤器( Neolab ,目录号:GF-0061)
8.Lipofectamine 3000转染试剂盒(Invitrogen,目录号:L3000-001)
9.BbsI限制酶(NEB,目录号:R0539L,10 单位/µL)
10.T4 DNA连接酶( Thermo Fisher Scientific,目录号:EL0011 )
11.NEB缓冲区2.1(10 × )(NEB,目录号:B7202S)
12.无菌的无核酸酶水(VWR,目录号:PD092)
13.NucleoSpin Gel and PCR Clean-up kit( Macherey- Nagel,目录号:7400609.50)
14.SOC 培养基(Life Technologies,目录号:15544034)
15.氨苄青霉素(Gibco,目录号:11593027)
16.LB琼脂(Carl Roth,目录号:X9963)
17.LB(Luria/Miller)培养基(Carl Roth,目录号:X968.3)
18.Miniprep试剂盒(DNA分离试剂盒)( Macherey- Nagel,目录号:740499.250)
19.丝裂霉素C(MMC)(Sigma-Aldrich,目录号: M4287-5x2mg)
20.DPBS( ThermoFisher Scientific,目录号:14190144)
21.OptiMEM培养基( ThermoFisher Scientific, 目录号:31985062)
22.蛋白酶K( ThermoFisher Scientific,目录号:26160)
23.胰蛋白酶-EDTA 0.05%( ThermoFisher Scientific,目录号:25300-054)
24.DMSO(Carl Roth,目录号:A994.1)
25.DMEM 培养基(Gibco,目录号: 41966-129)
26.Knockout TM DMEM 培养基(Life Technologies,目录号: 10829018)
27.Glutamax(Invitrogen,目录号: 35050061)
28.NEAA (Gibco,目录号: 11140-035)
29.笔/链球菌(Gibco,目录号: 15140-122)
30.胎牛血清(FBS) (Gibco,目录号: 10270-106) - MEF 细胞培养的定期收费
31.ESC FBS(经过 ESC 培养测试以保持全能性)
32.β-巯基乙醇( Thermo Fisher,目录号: 31350010)
33.LIF/ESGRO (Millipore,目录号: ESG1107)
34.EDTA(Carl Roth,目录号:8043.1)
35.氯化钠(NaCl)(Carl Roth,目录号:3957.2)
36.Tris(Carl Roth,目录号:AE15.2)
37.1 × SYBR TM Safe DNA Gel Stain( ThermoFisher Scientific,目录号:S33102)
38.溴化乙锭溶液0.07%( PanReac AppliChem,目录号:1239-45-8)
39.甘油(Carl Roth,目录号:4043.3)
40.十二烷基硫酸钠(SDS)(Carl Roth,目录号:2326.2)
41.溴酚蓝(Sigma-Aldrich,115-39-9)
42.二甲苯氰(Sigma-Aldrich,2650-17-1)
43.10 ×退火缓冲液(见配方)
44.MEF 培养基(见配方)
45.ESC 培养基(见配方)
46.2 × MEF 冷冻培养基(见配方)
47.2 × ESC 冷冻培养基(见配方)
48.10 × MMC 库存(见食谱)
49.10 × DNA 上样缓冲液(参见配方)


设备


1.显微镜(Carl Zeiss TM , Axiovert 40 CFL,配备Zeiss TM A-Plan 2.5X/0.06 物镜和Zeiss TM E-PI 10x/20 目镜)
2.荧光显微镜(Life Technologies,EVOS FL)
3.Vortex(Merck,Vortex Genie 2,目录号: Z258423-1EA)
4.凝胶成像系统/紫外透照仪 (INTAS)
5.Nanodrop TM 2000 ( ThermoFisher Scientific, ND-2000)
6.微量移液器(Eppendorf,可变容量)
7.多通道微量移液器(Eppendorf,可变容量)
8.台式冷却离心机(Eppendorf,5417R)
9.无菌工作台( Thermofisher Scientific、 HERASafe KS/Telstar、Telstar Bio II A)
10.LightCycler ®实时荧光定量 PCR 仪(Roche, LightCycler 480 II)
11.高压灭菌器(各种)
12.细胞分选仪( MoFlo 、 Cytomation 、Summit 4.3 软件)
13.电泳室(Carl Roth,Midi)
14.电泳电源(Sigma-Aldrich,Consort EV243)


软件


1.MoFlo ®计算机软件(Summit 4.3 软件,Beckman Coulter); FlowJo (BD)
2.LightCycler ® 480 软件 ( https://lifescience.roche.com/en_de/products/lightcycler14301-480-software-version-15.html )
3.CRISPR-Cas9 引导设计软件,例如BROAD Institute 的CRISPick (还有许多其他选项可供选择,参见Cui等人, 2018 年)


程序


该协议的总体目标是通过同时引入一个或多个突变甚至可能在同一基因内来操纵小鼠胚胎干细胞 (ESC) 的生殖系。为此,用一种或多种编码 sgRNA 的 CRISPR 质粒转染 ESC,在诱变位点引入双链 DNA 断裂。包含所需突变的同源模板与指南共转染以诱导同源定向修复 (HDR)。之后,生长、分离和筛选源自单细胞的 ESC 克隆,以便对基因组序列进行正确的遗传操作。


A.选择 CRISPR 单向导 RNA (sgRNA) 序列(CRISPR 向导)
使用 CRISPR-Cas9 sgRNA 设计程序在目标基因位点附近找到最佳 spCas9 ( S. pyogenes Cas9) 切割位点和相应的 20 个核苷酸引导序列,例如,来自 Broad 研究所的CRISPick基于Doench及其同事开发的算法(Doench等人,2016 年;桑森等人,2018 年) 。重要的是,Cas9 切割位点(位于化脓性链球菌PAM 序列 5'-NGG上游 3 至 4 个碱基对)尽可能靠近突变位点,因为突变效率和切割到-突变距离已被描述(Paquet et al. , 2016) 。为了提高 PX458 质粒的 U6 RNA 聚合酶 III 启动子对引导 RNA 的表达,如果20-nt 引导序列尚未以“ G” (Ran等人,2013 年) 。对于克隆,必须将引导寡核苷酸(具有BbsI 5'-CACC 粘性末端)和相应的反向互补引导寡核苷酸(具有BbsI 5'-AAAC 粘性末端)退火以获得可以克隆到的双链寡核苷酸PX458载体(pSpCas9 主干)的 BbsI 位点(图 1) 。请注意,引导序列不包括 5'-NGG PAM 序列本身。有关详细信息,请参阅Ran等人。 (2013 年) 。




图1 。指导 RNA 寡核苷酸设计示例取自O'Neill等人。 (2021) ,图 S1,Malt1 外显子 17 靶向。
( Doench et al. , 2016 )鉴定了一个 20 bp 的指导序列 ADDIN EN.CITE ,随后用于设计正向寡核苷酸。一个“额外的 G”(蓝色)被添加到 20-bp 引导序列的 5' 末端,然后是BbsI 5'-CACC 粘性末端(绿色)。反向寡核苷酸是 20 bp 引导序列的互补序列加上与“额外 G(蓝色)”互补的“C”,并具有 5'-AAAC 粘性末端(绿色)。退火的寡核苷酸具有粘性末端突出端(绿色),用于连接到PX458 CRISPR 质粒的 BbsI 位点。


B.ssODN同源模板设计
对于点突变的产生或小插入或缺失的引入,可以使用大约 200 bp 的单链寡脱氧核糖核苷酸( ssODN ) 作为 HDR 的同源模板。这些可以从专门从事高精度 DNA 合成的公司订购。所需的更改应放置在ssODN的中心。如果可能,将 Cas9 引导至目标位点的 PAM 序列应在同源模板序列内进行静默突变,以防止同源模板或成功修饰的目标基因座被 Cas9 定向切割。或者,或者除了 PAM 突变之外,还可以在 20-nt 引导序列中引入沉默突变,这同时有助于突变等位基因特异性 PCR 引物的设计(没有特定数量的沉默突变可以被普遍推荐;在我们手中,四种沉默突变被证明是合适的,如图2 所示)。此外,可以通过静默诱变在同源模板内生成人工限制位点,为筛选 ESC 克隆提供了额外的选择。图2显示了一个示例 HDR 。我们想提一下,对于另一个项目,我们通过使用总长度约为 1,400 bp 的线性双链 DNA 同源模板和位于诱变位点两侧的 700 bp 同源臂实现了成功的靶向(这可能对更大规模的遗传操作有用例如删除或插入更长的 DNA 片段)。




图2 。取自O'Neill等人的同源模板设计示例。 (2021 年) 。
Malt1 外显子 17 用“反义指南”序列靶向,如图 1 所示。所需的靶向突变以橙色显示。 “CCC”三联体密码子(代表“GGG”PAM 序列,因为这里使用了反义向导)(灰色)不能被静默突变。相反,插入了沉默突变(蓝色)以防止 sgRNA 与同源模板结合。为了比较,上面显示了同源区的野生型序列,灰色的“GGG”PAM 和框表示的 20-nt 指导序列。野生型序列中的近似预期切割位点用双线表示。


C.将 CRISPR 引导寡核苷酸克隆到 PX458 质粒中
该协议使用质粒P X458,其中包含一个用于瞬时共表达 SpCas9 和 EGFP 的盒式磁带和一个可将定制设计的 CRISPR 引导寡核苷酸连接到其中以生成在 U6 控制下表达的单个引导序列的盒式磁带RNA 聚合酶 III 启动子( Ran等人,2013 年) 。当转染到 ESC 中时, P X458 引导结构会导致 sgRNA、Cas9 和 EGFP 的表达。 EGFP 阳性 ESC 可以通过 FACS 进行富集,用于在饲养细胞上进一步培养和单个ESC克隆的生长。


1.退火正向和反向 sgRNA 寡核苷酸。通过混合设置退火反应:
感觉引导寡核苷酸 (100 µM)2 µL
反义引导寡核苷酸 (100 µM)2 µL
退火缓冲液 (10 × )2 µL
水(无核酸酶)14 µL


在加热块中将混合物加热至 95°C 5 分钟。关闭加热块,让混合物冷却至室温(约 3 小时)。在无核酸酶水中稀释退火寡核苷酸 1:400。
2.PX458向量以进行引导插入。设置以下限制性消化反应:
PX4582微克
NEB 2.1 缓冲区 (10 × )2 µL
BbsI (10 单位/µL)1.5µL
水(无核酸酶)总共加到 20 µL


在加热块上在 37°C 下孵育 1 小时。将限制反应与 2 µL 10 × DNA 上样缓冲液混合,并在 1% 琼脂糖凝胶(含有 0.5 µg/mL 溴化乙锭或 1 × SYBR TM Safe DNA Gel Stain)上以 120 V 分离 1 小时。切出线性化的 PX458 载体片段 (9,270 bp),用凝胶纯化试剂盒纯化,并在Nanodrop TM上测量浓度。
3.将退火的寡核苷酸(来自步骤 1;1:400 稀释 = 25 nM )连接到 PX458 载体(来自步骤 2)中。设置以下连接组合:
PX458(线性化)100 纳克
退火引导寡核苷酸 (25 nM )2 µL
T4 连接酶 (5 单位/µL)2 µL
T4 连接酶缓冲液 (5 × )2 µL
水(无核酸酶)总共加到 10 µL


在室温下孵育连接混合物 15 分钟。
4.为了转化为大肠杆菌,将 2 μL 的结扎 sgRNA 指南(从步骤 3)添加到 25 μL 化学感受态大肠杆菌。混合物在冰上孵育 5 分钟,42°C 热休克 40 秒,然后置于冰上 5 分钟。将大肠杆菌与 150 µL 不含抗生素的 SOC 培养基混合,并在 37°C 下孵育 1 小时。将总细胞体积涂抹在含有氨苄青霉素的 LB 板上,并在 37°C 下孵育过夜。选择菌落并接种到 5 mL 含有氨苄青霉素的 LB 培养基中,并在 37°C 和 180 rpm 下生长过夜。使用 DNA 分离试剂盒分离质粒 DNA,并通过Nanodrop TM量化 DNA 。在对正确的引导插入进行测序验证后,PX458 CRISPR 引导质粒可用于转染到鼠 ESC 中,以共表达 sgRNA 和 Cas9,并以 EGFP 作为报告基因,通过流式细胞仪细胞分选成功转染和富集。


D.小鼠胚胎成纤维细胞 (MEF) 的制备和培养
ESC 需要在一层小鼠胚胎成纤维细胞 (MEF) 上培养,MEF 作为饲养细胞。 MEFs 由 12.5 dpc (性交后天数)野生型小鼠胚胎产生,可以传代四次,然后通过丝裂霉素 C (MMC) 处理抑制生长,这样它们就可以作为 ESC 培养的饲养细胞。
1.收获 MEF
在第 12.5 天从野生型小鼠胚胎中制备小鼠 MEF 细胞。简而言之,在含有 PBS 的细胞培养皿中,将每个胚胎与卵黄囊分开,取出并丢弃头部和肝脏。随后,分别处理每个胚胎:将剩余的组织通过 100 µm 筛网筛入 MEF 培养基中,并将一个胚胎的细胞转移到 6 厘米的细胞培养皿中。让 MEF 细胞在一夜之间生长。第二天,洗去未贴壁的死细胞,让细胞进一步生长直至汇合(大约 2 天)。
2.传代 MEF
通过用 PBS 洗涤细胞、加入 2 mL 胰蛋白酶并在 37°C 下孵育 5 分钟,从 6 cm 培养皿表面分离粘附的 MEF 细胞。通过添加 MEF 培养基停止胰蛋白酶化。将收获的 MEF 从 6 厘米的盘子转移到一个 15 厘米的盘子上,让其在培养箱中生长。汇合后,从 15 厘米板(使用 8 毫升胰蛋白酶)到四个 15 厘米的盘子进行第二次通道。 MEF 细胞现在可以在液氮中冷冻(参见步骤 3)或用丝裂霉素 C (MMC) 处理以用于 ESC 培养(参见步骤 4)。
3.冷冻和解冻 MEF
从一个汇合的 15 厘米培养皿(约 2 × 10 7 个细胞)中收获 MEF。将收获的细胞放入 2 mL 的 MEF 培养基中并放在冰上。加入 2 mL 冰冷的 2 ×冷冻培养基 (1:1) 并将 1 mL 等分试样(包含约 5 × 10 6 个细胞)转移到冷冻小瓶中,将其放入聚苯乙烯泡沫塑料盒中,在 -80 °C 冰箱。 24 小时后,将冷冻细胞转移到液氮中进行长期储存。解冻时,将冷冻小瓶漂浮在 37°C 水浴中,然后将解冻的细胞悬液转移到 10 mL 的 PBS 中。 350离心机 × g 5 分钟,将细胞沉淀放入 10 mL MEF 培养基中,将细胞分布到 4 个 10 cm 培养皿中,并在 37°C/5% CO 2下孵育。要么让细胞生长 2 天以进一步传代,要么在第二天使用 MMC 治疗(参见步骤 4)。
4.用丝裂霉素 (MMC) 治疗 MEF
用作 MMC 处理的饲养细胞层的 MEF 细胞密度为每 10 cm 培养皿2.5 × 10 6 个细胞。用 MMC(MEF 培养基中 10 µg/mL)处理 MEF 细胞,并在 37°C 下孵育 2 至 3 小时以抑制进一步增殖。去除含有 MMC 的培养基,用 PBS 彻底清洗细胞 3 次,然后加入新鲜的 MEF 培养基。经 MCC 处理的 MEF 现在可用作 ESC 生长的基础层(并且应在 MMC 处理后的 2 天内使用)。


E.ESC 培养(R1/E 细胞)
胚胎干细胞必须在允许维持全能性的条件下培养。为此,ESCs 在一层饲养细胞上培养,并且必须在含有 FBS 的 ESC 培养基中保持适当的细胞密度,该培养基已被批准用于 ESC 培养(可以从商业来源购买或从专门从事 ESC 培养的实验室获得)。鼠 R1/E ESC 总是在 MMC 处理的 MEF 的汇合(但不太密集)饲养层上生长(大约 40,000 个饲养细胞/cm 2 )。
1.在 ESC 培养基中的饲养细胞上每 cm 2播种大约 30,000 个 R1/E ESC。每天更换培养基,并且 ESC 通常在培养 2 天后以适当的细胞密度和形态分裂:当 ESC 形成许多具有锋利边缘的不同菌落时,达到这种条件,这些菌落共同覆盖了培养皿表面的大约三分之一不相互接触(图 3)。对于分裂,用 PBS 洗涤 ESC 并用胰蛋白酶处理 5 分钟,通过添加 ESC 培养基使胰蛋白酶失活。通过用电动移液器强力移液获得单细胞悬液:将细胞悬液吸入 10 mL 移液管中,将移液器的尖端压在细胞培养皿的底部,然后将细胞悬液从移液管中挤出。重复此操作 10 次。在新鲜的饲养细胞上计数细胞并接种 30,000 个细胞/cm 2 。根据一些经验,可能不必总是计数 ESC,并且在显微镜下目视检查细胞后,可以估计可接受的分裂因子,对于 ESC 在最佳生长条件和细胞密度下通常为 1:6(a 1:6 的分割因子意味着从细胞培养板中收获细胞,并将 1/6 的细胞转移到相同格式的新细胞培养板中)。




图3 。 R1/E ESC 的形态。
R1/E 细胞形成不同的菌落并覆盖大约三分之一的板表面。


2.胚胎干细胞可以在液氮中冷冻:为此,胚胎干细胞如上所述被胰蛋白酶消化,细胞被计数,以350 ×g的速度旋转,在胚胎干细胞培养基中调整为5 × 10 6 个细胞/mL,并在冰上短暂预冷。加入等体积的冰冷 2 × ESC 冷冻培养基,混合后,将 1 mL 细胞悬浮液分配到冷冻管中,在聚苯乙烯泡沫塑料盒内于 -80°C 下冷冻,然后转移到液氮中。稍后解冻时,将冷冻小瓶置于 37°C 水浴中,当内容物为液体时,将细胞转移到 15 mL 管中的 10 mL PBS 中,以 350 × g旋转,取出细胞沉淀在 ESC 培养基中。将来自每个冷冻管的细胞(2.5 × 10 6 个细胞)接种到 10 cm 培养皿中的饲养细胞上。


F.胚胎干细胞的转染
报告的协议通常使用电穿孔转染带有质粒和同源模板的 ESC。电穿孔需要大量的 DNA (20 µg) 以及许多细胞(通常在 10 cm 培养皿中生长大约 5 × 10 6 个细胞),并且由于电穿孔条件恶劣,许多细胞会死亡。脂质转染被证明更温和,允许使用更少的细胞数量(200,000 个细胞在 6 孔板中生长)和更少量的 DNA(2 µg 或更少)。根据流式细胞仪中 EGFP 阳性细胞的百分比测量,这导致大约 5% 的转染效率,而在荧光显微镜下只能看到一小部分具有高 EGFP 表达的转染细胞(图 4)。根据制造商的方案,通过 Lipofectamine 3000 试剂盒使用 lipofection 以 6 孔格式进行 ESC 转染。
1.在转染前大约 16 小时,在 2 mL 的 ESC 培养基(在饲养细胞上)中的 6 孔板的每孔中播种 200,000 个 ESC。
2.在转染当天,在管 A 和管 B 中制备混合物:
A 管:125 µL OptiMEM培养基 + 5 µL Lipofectamine 3000 试剂。
管 B:125 µL OptiMEM培养基 + 6 µL P3000 试剂 + 6 µg DNA(PX458 质粒和同源模板,均匀分布至最大总 DNA 量 6 µg)。
3.将管 A 和 B 的内容物混合在一起,在室温下孵育 15 分钟。
4.将整个转染量转移到 6 孔中的 ESC(步骤 1)。
5.将 ESC 与 DNA-脂质体复合物一起孵育 6 小时。
6.孵育后,用新鲜的 ESC 培养基更换培养基,让细胞在培养箱中生长 24 小时,然后通过 FACS 对 EGFP 阳性细胞进行分类。


G.基于 FACS 的 ESC 细胞分选和单克隆 ESC 克隆生长
成功转染的 ESC 表达由 PX458 质粒编码的 EGFP,因此可以通过 FACS 富集到 95% 以上(图 4B)。只需将 2000 个分类的 EGFP 阳性 ESC 接种到 10 厘米培养皿的饲养层上,即可生长多达 100 个 ESC 克隆。与使用带有抗生素抗性盒的 DNA 构建体进行选择的经典方案相比,FACS 分选的 ESC 不需要用抗生素处理,因此 ESC 板不包含培养中的数百万垂死细胞,这必须小心冲走。单克隆 ESC 克隆通过每隔一天更换的 ESC 培养基生长,并在 7 天内准备好采摘,这比使用电穿孔和抗生素选择的经典方案快大约 2 天。
1.用 PBS清洗 6 孔板(来自程序 F,步骤 6)中的 ESC 1 × 。
2.加入 500 μL 的胰蛋白酶,孵育 5 分钟,加入 500 μL 的 ESC 培养基,上下大力吸管,生成含有 ESC 和饲养细胞的单细胞悬浮液。
3.将细胞悬液转移到新的 6 孔板中。在 1 小时内,大多数饲养细胞但不是 ESC 将重新粘附。轻轻取出 ESC 并转移到 3 mL 管中进行细胞分选。
4.使用配备 100 μm 喷嘴的细胞分选机,将 EGFP 阳性 ESC 分类到 ESC 培养基中。将 2000 个分选的细胞放在一个 10 厘米的盘子上,该盘子预先铺有 3 × 10 6 MMC 处理的 MEF 细胞,并允许细胞生长约 7 天,每隔一天更换一次培养基(图 5A)。预计每 10 cm 培养皿最多可生长 100 个 ESC 菌落。因此,建议每个条件播种 10 个 10 厘米的培养皿,以获得足够数量的菌落进行筛选(我们建议筛选大约 500 个菌落)。




图4 。用 PX458 脂转染 24 小时后的 ESC。
(A) 在荧光显微镜下看到的具有高 EGFP 表达的细胞。 (B) 与未转染的细胞 (灰色曲线)相比, 流式细胞术可以灵敏地检测具有低至中等 EGFP 表达 (黑色曲线) 的 ESC 。细胞分选导致表达 EGFP 的细胞富集到 95% 以上(绿色曲线)。


H.选择 ESC 克隆
当不同的 3 维菌落在 10 厘米板上生长并在显微镜下可以看到时,可以在细胞分选后大约 7 天挑选单克隆 ESC 克隆(图 5A)。
1.在采摘菌落之前,在 100 μL 的 ESC 培养基(每孔约 10,000 个细胞)中播种 2 个带有 MMC 处理的 MEF 细胞的平底 96 孔板。将盘子放在培养箱中,直到需要。
2.用 10 mL 的 PBS 清洗含有 ESC 菌落的 10 厘米盘子,并在盘子中加入 10 mL 的新鲜 PBS。
3.将显微镜放在无菌工作台下,以可视化单个 ESC 菌落(图 5B)。确保有足够的空间使用 100 μL 移液器从侧面接近板。用移液器吸头轻轻地将每个菌落从表面移开并将其拉起,从而将每个菌落与饲养层分离 在 50 µL 的体积中。应小心提取整个 ESC 菌落,以避免 ESC 分布到板上,从而避免其他单克隆菌落的交叉污染。




图5 。细胞采摘工作流程和设置。
(A) 来自细胞分选的 EGFP 阳性细胞以每 10 厘米培养皿 2000 个细胞的密度镀在一层 MMC 处理的 MEF 细胞上。在 7 天的生长期后,可以从每个 10 cm 培养皿中挑取 50 – 100 个单克隆 ESC 克隆到重复的 96 孔板中,分别用于冷冻或 qPCR 筛选。 (B) 在无菌细胞培养罩下进行克隆采摘的显微镜设置。


4.将挑选的菌落转移到圆底 96 孔板中。挑选 96 个克隆来填充板和筛选所需的尽可能多的板(我们通常选择五个 96 孔板)。尽快进行第 5 步(建议协同工作:一个人采摘,另一个人处理采摘的克隆)。
5.收集 96 个菌落后,在每个圆底 96 孔中加入 30 µL 胰蛋白酶,其中含有挑选的 ESC 克隆,并在 37°C 下孵育板 10 分钟。在每口井中加入 100 μL 的 ESC 培养基以停止胰蛋白酶化。
6.使用多通道微量移液器,在每个孔中用力上下移液,将 ESC 克隆聚集体分离成单个细胞,并将每个孔的内容物分成两个含有 MEF 细胞的平底板(在步骤 1 中准备)。将两个板孵育 2 天。板 #1 用于筛选,板 #2 被冷冻,以便以后回收通过 qPCR 筛选和序列分析鉴定的阳性克隆。
7.在 96 孔板中冷冻 ESC 之前,检查每个 96 孔以判断细胞密度并记录解冻时应接种的细胞培养板格式(这可以通过 1 :6 拆分因子,如程序 E 步骤 1) 中所述。对于在 96 孔板中冷冻 ESC,在板 #2(从步骤 6 开始)中用 PBS洗涤细胞 1倍,通过每孔添加 30 µL胰蛋白酶对细胞进行胰蛋白酶消化,在培养箱中孵育 5 分钟,与 70 µL ESC 混合培养基停止胰蛋白酶化反应,用移液管用力重悬细胞。将板在冰上短暂预冷,然后加入 100 µL 冰冷的 2 × ESC 冷冻培养基,在 -80°C 的聚苯乙烯泡沫塑料盒内混合和冷冻板,然后转移到液氮中。对于 96 孔板中的细胞解冻,将板在 37°C 培养箱中解冻,将所需克隆转移到 5 mL 培养基中的 15 mL Falcon 管中,以 350 × g离心细胞,吸出上清液,洗涤细胞一次用 1 mL 的 PBS,将细胞吸收到 100 μL 的 ESC 培养基中,然后转移到足够的培养基(通常是 48 或 24 孔格式)中适当的孔大小。


I.qPCR 筛选正确靶向的 ESC 克隆
建议在项目一开始就建立特定和敏感的 qPCR 协议,作为同源模板设计的一部分,因为通过完善的筛选策略可以识别正确操作的 ESC 克隆至关重要。在正确插入同源模板后,合成与基因组位点相对应的 DNA 模板可能是有价值的,它可以作为突变位点特异性和灵敏扩增的阳性对照。如果在建立 PCR 条件时加入来自野生型 ESC 的基因组 DNA(如以下步骤 1 至 3 所述制备),则可以更好地模拟筛选条件。测试将阳性对照模板连续稀释至拷贝数 1。可检测到的 10 个阳性对照模板的扩增通常足够灵敏,以后能够通过筛选 ESC 克隆的基因组 DNA 来检测阳性 ESC 克隆。为防止筛选过程中出现假阳性信号,请注意避免“阳性对照”DNA 片段或阳性筛选扩增子污染工作场所和设备;阳性对照 DNA 或扩增子绝不应在协议的后续步骤(ESC 培养、转染、克隆挑选和筛选 PCR)进行处理。
1.要从板 #1(来自程序 H 步骤 6)制备 ESC 克隆的基因组 DNA 用于 qPCR 筛选,请从 96 孔板中取出培养基,用 PBS 洗涤细胞一次,然后向每个孔中加入 200 µL无核酸酶水好。在 96 孔 PCR 循环仪中,将板在 95°C 下孵育 15 分钟。
2.将蛋白酶 K 添加到每个孔中,最终浓度为 200 µg/mL,并在 55°C 下孵育 90 分钟以进行蛋白质消化。将板加热至 95°C 15 分钟以灭活蛋白酶 K。样品现已准备好进行筛选。
3.设计 PCR 筛选引物,专门用于扩增正确靶向的序列,以识别成功操作的 ESC 克隆。在定点诱变的情况下,一个 PCR 引物应该是突变特异性的,覆盖突变位点(根据程序 B 中描述的同源模板设计),另一个引物应该位于同源模板序列之外。例如,参见O'Neill等人的图 S1B。 (2021 年) 。在 qPCR 期间,潜在的阳性克隆将以较低的循环阈值 (Ct) 值扩增。如果在一个 ESC 克隆中插入多个突变,则需要为每个突变位点设计 qPCR 协议和突变特异性引物,并且每个 qPCR 筛选都需要单独进行。
4.根据您建立的协议执行 qPCR。大多数基因组 ESC DNA 样本很可能会产生高 Ct 值 (>35) 的背景信号,具体取决于 PCR 协议的特异性。然而,样本子集会在低得多的 Ct 值下产生稳健的扩增(图 6)。这些克隆是具有基因组修饰的候选者,需要通过测序进一步分析(O'Neill等人,2021,图 S1C,D)。




图6 。通过 qPCR 筛选 ESC。 
使用仅扩增正确突变的靶序列的突变特异性引物筛选克隆。阴性克隆(黑色曲线)由于其高阈值循环 (Ct) 值而被排除在外。潜在的阳性克隆(灰色/红色曲线)具有较低的 Ct 值。通过测序(可能与 TOPO 克隆结合)验证预期阳性克隆,以找到正确插入所有突变的克隆(红色曲线)。


5.对于测序,感兴趣的基因组区域用目标位点侧翼的引物进行扩增。纯化后的 PCR 产物可直接用于测序。测序数据通常很难解释,因为目标基因的两个等位基因可以不同地改变,产生两个重叠的色谱图,不能明确地分配给一个或另一个等位基因。如果使用在线工具,例如 CRISPID (Dehairs et al. , 2016),甚至会出现这种情况。在这种情况下,阐明不同等位基因序列状态的可靠技术是上述 PCR 产物的 TOPO 克隆。 TOPO 克隆产生仅携带一个或另一个等位基因片段的大肠杆菌克隆,然后可以单独对其进行测序。
6.具有确认基因组修饰的 ESC 克隆可以通过从板 #2 中解冻来恢复(参见程序 H,步骤 6 和步骤 7),并且应以健康生长和良好ESC形态为特征。选定的 ESC 克隆在几个等分试样中进行扩展和冷冻,现在可以用于胚胎注射,以创建嵌合小鼠和随后的种系传递。


食谱


1.10 ×退火缓冲液
10 毫米三
1 毫米乙二胺四乙酸
50 毫米氯化钠
酸碱度 7.5
2.MEF 培养基
500 毫升 DMEM
50 mL FBS(常规)
500 µL 50 mM β-巯基乙醇
5 毫升的 NEAA
5 毫升笔/链球菌 
3.ESC培养基
500 毫升的Knockout TM 记忆体
90 mL FBS(经 ESC 培养测试)
1.2 mL 50 mM β-巯基乙醇
6 毫升的 NEAA
6 毫升谷氨酰胺
6 毫升笔/链球菌
90 µL LIF/ESGRO ( 1,500 U/mL)
4.2 × MEF 冷冻培养基
20% 二甲基亚砜
30% FBS(常规)
50% DMEM(含 1% 笔/链球菌)
5.2 × ESC 冷冻培养基
20% 二甲基亚砜
30% FBS(ESC 培养测试)
50% DMEM(含 1% 笔/链球菌)
6.10× MMC 库存
将 MMC 粉末溶解在 MEF 培养基中,储备浓度为 100 µg/mL,无菌过滤,并储存在 4°C
7.10× DNA 上样缓冲液
34% 甘油
0.5% SDS
10 毫米乙二胺四乙酸
2.5 毫克/毫升溴酚蓝
2.5 毫克/毫升二甲苯氰


致谢


该协议首次用于《科学免疫学》的出版物中 (奥尼尔等人,2021 年) 。这项工作得到了 Deutsche Forschungsgemeinschaft (ID 210592381 – SFB 1054 A04, ID 360372040 – SFB 1335 P07) 和 Deutsche Krebshilfe (No. 70112622) 的支持。 DK 是波士顿 Monopteros Therapeutics Inc. 的科学顾问。


伦理


用于从 12.5 dpc胚胎中产生小鼠胚胎成纤维细胞的怀孕小鼠按照欧洲实验动物科学协会联合会的指导方针进行饲养和处理。


参考


1.科恩,J. (2016)。任何白痴都可以做到。基因组编辑器 CRISPR 可以让突变小鼠触手可及。科学。 https://www.science.org/content/article/any-idiot-can-do-it-genome-editor-crispr-could-put-mutant-mice-everyones-reach 。
2.Cui, Y.、Xu, J.、Cheng, M.、Liao, X. 和 Peng, S. (2018)。审查 CRISPR/Cas9 sgRNA 设计工具。 跨学科科学10(2):455-465。
3.Dehairs, J.、 Talebi , A.、 Cherifi , Y. 和Swinnen , JV (2016)。 CRISP-ID:通过 Sanger 测序解码 CRISPR 介导的插入缺失。 科学代表6:28973。
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5.González Castro, N., Bjelic , J., Malhotra, G., Huang, C. 和Alsaffar , SH (2021)。比较基因组编辑技术的可行性、效率和安全性。 国际分子科学杂志 22(19):10355。
6.Hall, B., Cho, A., Limaye, A., Cho, K., Khillan , J. 和 Kulkarni, AB (2018)。使用 CRISPR/Cas9 技术对小鼠进行基因组编辑。 电流 原始细胞生物学81(1):e57。
7.Muñoz -Santos, D.、Montoliu, L. 和 Fernandez, A. (2020)。使用 CRISPR/Cas9 生成转基因小鼠。方法 Mol Biol 2110:129-138。
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9.Oji, A., Noda, T., Fujihara , Y., Miyata, H., Kim, YJ, Muto, M., Nozawa, K., Matsumura, T., Isotani , A. 和Ikawa , M. (2016 )。 CRISPR/Cas9 介导的 ES 细胞基因组编辑及其在小鼠嵌合分析中的应用。 科学代表6:31666。
10.Paquet, D., Kwart , D., Chen, A., Sproul, A., Jacob, S., Teo, S., Olsen, KM, Gregg, A., Noggle , S. 和 Tessier-Lavigne, M. (2016 年)。使用 CRISPR/Cas9 有效引入特定纯合和杂合突变。 自然533(7601):125-129。
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Copyright: © 2022 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. O'Neill, T. J., Krappmann, D. and Gewies, A. (2022). Optimized CRISPR-Cas9-based Strategy for Complex Gene Targeting in Murine Embryonic Stem Cells for Germline Transmission. Bio-protocol 12(10): e4423. DOI: 10.21769/BioProtoc.4423.
  2. O'Neill, T. J., Seeholzer, T., Gewies, A., Gehring, T., Giesert, F., Hamp, I., Grass, C., Schmidt, H., Kriegsmann, K., Tofaute, M. J., et al. (2021). TRAF6 prevents fatal inflammation by homeostatic suppression of MALT1 protease. Sci Immunol 6(65): eabh2095.
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