Targeted Gene Editing in Porcine Germ Cells

Abstract

As the genetic mutations driving human disease are identified, there is an increasing need for a biomedical model that can accurately represent the disease of interest and provide a platform for potential therapeutic testing. Pigs are a better model for human disease than rodents because of their genetic and physiological similarities to humans. However, current methods to generate porcine models are both technically challenging and expensive. Germline genetic modification through gene edited spermatogonia provides an effective alternative to how these models are developed. Here we report an improved technique of gene editing in spermatogonia of pigs using CRISPR-Cas9 to generate different edits that reflect the genotypes of human diseases.

1. Introduction

Historically, the mouse is considered a foundational animal model across the majority of scientific fields, driving many pharmacological and human disease related studies. While mouse models offer advantages such as cost effectiveness and simplicity of care, they often do not fully recapitulate human physiology and in some cases do not even manifest the disease of interest. Pigs, by contrast, are genetically, physiologically, and metabolically more similar to humans than rodent models, and are quickly becoming an invaluable platform for the research of human disease (1). The use of pigs as biomedical models does, however, present a unique set of challenges. Currently, generation of gene edited pigs relies heavily on the inefficient processes of somatic cell nuclear transfer (SCNT) or zygote microinjection or electroporation of genetic editing systems such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats and CRISPR-associated-9 (CRISPR-Cas9) (2–5). The limitations of these techniques point to the need for an alternative approach, making the genetic editing and subsequent transplantation of spermatogonia a promising candidate for the generation of porcine biomedical models (6,7).

Spermatogonia is a broad term used to describe a population of male germ cells including cells at earliest stages of differentiation and the undifferentiated unipotent spermatogonial stem cell (SSC) (8). These cells can be transplanted into a germ cell depleted donor testis and restore full spermatogenesis (9,10). Their ability to recolonize and proliferate within the recipient testis niche make spermatogonia ideal targets for genetic editing. Targeted gene knock-out and the correction of disease alleles has been demonstrated in mouse and rat spermatogonia through the use of engineered nucleases and viral-mediated transgenesis, at the expense of spermatogonial viability and off-target effects, respectively (11–14). In the approach outlined below, we used CRISPR-Cas9 gene editing technology to introduce custom indels and single nucleotide polymorphisms (SNPs) into specific loci pertinent to human disease modelling in porcine spermatogonia. CRISPR-Cas9 complexes were delivered by electroporation in ribonucleoprotein (RNP) format alongside a single strand oligo donor (ssODN) repair template. We demonstrated higher viability post-transfection and increased editing efficiency when using CRISPR-Cas9 compared to TALENs in isolated spermatogonia (14,15).

2. Materials

All cell culture work and resuspension of oligos was carried out in a biological safety cabinet to prevent contamination. Analysis of editing was done on the bench top. Digestion solutions are filter sterilized prior to use. All tissue culture reagents are warmed to 37°C prior to use unless otherwise noted.

2.1. Spermatogonial Isolation and Differential Plating

1. 1x Dulbecco’s Phosphate buffered saline (DPBS) without calcium and magnesium, with 1x Penicillin-Streptomycin (100 U/ml Penicillin and 100 ug/ml Streptomycin; P/S).

2. High-glucose DMEM medium.

3. Solution A: 20 mg of Sigma Collagenase IV, 40 mg Worthington Collagenase IV in 25 ml of DMEM. 1 ml of FBS added after resuspension.

4. Solution B: 80 mg Worthington Collagenase IV, 40 mg Hyaluronidase in 35 ml of DMEM.

5. Deoxyribonuclease I (DNase I) solution at 7 mg/ml in DMEM.

6. DMEM/F12 with 5% FBS and 1x P/S.

7. Sterilized surgical tools: forceps, scissors, scoopula

8. 0.22μm filter units and syringe filters.

9. 40μm cell strainers.

10. 70μm cell strainers.

11. 0.25% Trypsin-1mM EDTA solution

12. 10cm Cell culture plates

13. Centrifuge

14. Water bath at 37°C

15. Hemocytometer or calibrated electronic cell counter

16. Cell culture incubator at 37°C and 5% CO₂

17. Beaker

18. Trypan blue viability dye

19. 50mL conical tubes

2.2. Fluorescence Activated Cell Sorting of Spermatogonia

1. Alpha Mem Advanced with 10 μg/ml holo-transferrin, 10 μg/ml insulin, 1x non-essential amino acids, 1mM Na-pyruvate, 1% FBS, 10 μM β- mercaptoethanol, 25ng/ml GFRa1 recombinant rat, 40ng/ml GDNF recombinant human, 20ng/ml EGF recombinant human, 1x P/S, 1% BSA, 1x L-glutamine, 15mM Hepes

2. Alpha Mem Advanced, 1% BSA, 10 μM β- Mercaptoethanol with 1x P/S

3. Fetal Bovine Serum with 10 μM β- mercaptoethanol

4. 40μm filter

5. 15 ml conical tubes

6. 130 μm nozzle

7. Sorter

8. Centrifuge

9. 15 ml tube rack

10. Ice bucket

11. Trypan blue viability dye

2.3. CRISPR-Cas9 and Nucleofection Reagents

1. sgRNA or crRNA for gene of interest

2. tracrRNA

3. Cas9 protein

4. Nuclease free water

5. DPBS without calcium or magnesium

6. PCR strip tubes

7. Thermocycler

8. Lonza Cell Line Nucleofector Kit V (VCA-1003)

9. Lonza Nucleofector 2b Device

10. ssODN template for gene of interest

11. Electroporation enhancer

12. 6-well tissue culture plates

13. 0.2% Gelatin in PBS

14. Spermatogonia Media: αMEM Advanced culture medium, 1% FBS, 0.1% BSA, 1X non-essential amino acids, 1mM sodium pyruvate, 15 mM HEPES, 2 mM L-glutamine, 10 μM beta-mercaptoethanol, 100 U/ml Penicillin-100 ug/ml Streptomycin, 10 ng/ml glial cell line-derived neurotrophic factor

15. Trypsin 0.25% EDTA

16. PBS without calcium and magnesium

2.4. Detection and Analysis of Editing

1. Lysis buffer: 10 mM Tris-Cl pH 8.0, 2 mM EDTA, 2.5%Tween20, 2.5% Triton-X 100, 100 mg/mL Proteinase K

2. PCR strip tubes

3. Thermocycler

4. Forward and reverse primers flanking region of interest

5. Hi-Fi Taq polymerase

6. Surveyor mutation detection kit

7. Restriction endonuclease(s)

8. Appropriate enzyme buffer

9. Nuclease free water

10. 10% TBE polyacrylamide gel.

11. 10x TBE: 500 mL of distilled water, 121.1 g Tris Base, 55.0 g Boric acid, 40.0 mL of .5M EDTA.

12. 1x TBE: diluted 10x TBE with ddH₂O.

13. 5x Ficoll Buffer: 2.5 g Ficoll-400, 1 mL 1M Tris-HCl, pH 7.4, 2 mL 0.5M EDTA, 20 mL distilled water, 25 mg orange G and xylene cyanol.

14. Electrophoresis rig

15. Staining Buffer: 130mL 1x TBE buffer, 6.5μL ethidium bromide

16. Gel imaging machine/software

17. Adobe Photoshop or similar

18. Image J

3. Methods

The workflow is schematically outlined in Figure 1. Sections 3.1 and 3.2 are described in detail in Sakib et al, 2019 (16).

Figure 1:

Schematic representation of the workflow involved in targeted gene editing of porcine spermatogonia.

3.1. Spermatogonial cell isolation

1. Dissolve the components of Solutions A, B and DNase I in 25mL, 35mL, and 10mL of DMEM respectively. Filter-sterilize each solution through a 0.22μm filter. (See Note 1)

2. Wash testes in a beaker containing DPBS with 1xP/S. Remove testes and place into a 10cm dish containing DPBS with 1xP/S.

3. Using sterile scissors and forceps, remove and discard tunica vaginalis and epididymis. Transfer testes to another 10cm dish containing DPBS with 1xP/S.

4. Weigh dish containing isolated testes and record weight.

5. Make a longitudinal cut in the tunica albuginea and using scissors and forceps, peel and scrape the parenchyma from the tunica. Remove visible mediastinum. (See Note 2). Place parenchyma into a new 10cm dish containing DMEM.

6. Weigh remaining tunica in PBS, and calculate mass of parenchyma isolated.

7. Using scissors, cut parenchyma into 1mm³ pieces, removing any remaining pieces of mediastinum that appear as white tissue. (See Note 3).

8. Once the tissue is thoroughly chopped, transfer around 7–10 g of tissue into each tube of Solution A using the scoopula. Bring total volume to 50mL with DMEM.

9. Place the Solution A tube(s) into the water bath at 37°C. Digest for 30 minutes, inverting the tube every 5 minutes (See Note 4). Add 100 ul DNase I during the digestion if strings or clumps of DNA appear.

10. After 30 minutes, centrifuge the Solution A tube(s) at 90 × g for 90 seconds.

11. Gently aspirate off the spent Solution A digestion solution and decant Solution B into the tube containing the centrifuged tissue. Bring total volume to 50mL with DMEM.

12. Place the Solution B tube(s) into the water bath at 37°C. Digest for 30 minutes, inverting the tube every 5 minutes (See Note 5).

13. After 30 minutes, centrifuge the Solution B tube(s) at 90 × g for 90 seconds.

14. Gently aspirate off the spent Solution B digestion solution.

15. Wash isolated tubules repeatedly with 10–15mL DPBS with 1xP/S. Tubules will remain in supernatant while undigested tissue settles quickly to the bottom of the tube. Collect tubules in supernatant in a new 50mL conical tube.

16. Centrifuge tubules at 90 × g for 90 seconds.

17. Resuspend tubules in 5mL of DPBS with 1xP/S. Add 500μl of DNase I and 5ml of DMEM. Add 15mL Trypsin 0.25% EDTA, and place in water bath at 37°C.

18. Check progress of digestion every 5 minutes by removing a 10μL of solution and examining under microscope. (See Notes 6 and 7).

19. Once the digestion is approaching finality, strain solution through a 70μm mesh filter into 5mL of FBS.

20. Filter once more through a 40μm mesh filter.

21. Centrifuge isolated cells at 500 × g for 5 minutes. Once finished decant and discard solution and resuspend cells in 50mL of DMEM.

22. Count viable cells using hemocytometer and trypan blue exclusion.

3.2. Differential Plating

1. Plate 25 million cells per 10cm tissue culture dish, in a final media volume of 8mL of DMEM/F12 with 5% FBS and 1xP/S.

2. Allow cells to sit in a cell culture incubator at 37°C and 5% for 1.5 hours. (Note 8).

3. Consolidate the supernatant from two plates into one new 10cm dish.

4. Allow cells to sit in a cell culture incubator at 37°C and 5% CO₂ for 1 hour.

5. Consolidate the supernatant from two plates once more into one new 10 cm dish.

6. Allow cells to sit in a cell culture incubator at 37°C and 5% CO₂ overnight.

7. The following day collect two fractions of cells: Non-adherent fraction of cells in the supernatant and the slightly-adhered fraction. Collect the former by just aspirating the supernatant into a 50mL conical tube. Collect the latter first by using a 1:20 dilution of 0.25% Trypsin-EDTA in PBS for 5 minutes at room temperature. Collect the cells that detach into a new 50mL conical containing 5mL FBS to stop the trypsin. Then followed by a 1:5 dilution of 0.25% Trypsin-EDTA in PBS for 2.5 minutes at 37°C and 2.5 minutes at room temperature. Collect the detached cells same as mentioned above.

8. Centrifuge both fractions at 500 × g for 5 minutes, resuspend in PBS and count using a hemocytometer and trypan blue.

9. Move forward with the fraction that has the highest percentage of spermatogonia, or combine the fractions if necessary, to achieve the appropriate cell number for transfection.

3.3. Preparation of CRISPR-Cas9 Reagents

For CRISPR-Cas9 experiments, either a sgRNA or a crRNA:tracrRNA complex can be used. If using sgRNA, skip steps 1 and 2. The following CRISPR design tool was used to create the guide RNAs used: http://www.rgenome.net/cas-designer/

1. To create the crRNA:tracrRNA complex, combine equimolar concentrations of crRNA for the locus of interest with tracrRNA per reaction in PCR strip tubes. In this experiment we used 200 pmol.

2. Using a thermal cycler, heat the reaction components at 95°C. Then cool the reaction to 22°C at a ramp rate of 0.1°C/sec.

3. Incubate complex or sgRNA with 175 pmol of Cas9 protein and 3.5μL of DPBS at room temperature for 15 minutes. If the complex is not being used immediately, store on ice. Total volume is 8.33μL.

3.4. Fluorescence Activated Cell Sorting of Porcine Spermatogonia

1. Prepare FACS collection tubes with 3 ml of fetal bovine serum with 10 μM β- mercaptoethanol.

2. Place rack for collection tubes on ice (Note 9).

3. Filter cells through 40μm filter.

4. Resuspend spermatogonia to a concentration of 5 × 10⁶/ ml in Alpha Mem Advanced, 1% BSA, 10 μM β- mercaptoethanol with 1x P/S in 15 ml tube.

5. Set up sorter with 2.0 ND and a 130 μm nozzle with 10 psi at lowest flow rate.

6. Place tube with enriched cells into sample collection chamber at room temperature and set up sample spin to 200 rpm.

7. Open sorter software workspace with FSC-A/SSC-A dot plot, SSC-A/SSC-H dot plot, FSC-A/FSC-H dot plot, and GFP-A PERCP-Cy5–5-A dot plot.

8. Adjust laser intensity for FSC and SSC to receive cell population cloud appearance as shown.

9. Gate ‘germ cell cloud’ according to distinct FSC-A/SS-A tightly (Figure 1, P1).

10. Remove doublets from sort from cell population P1 by gating out events falling under a linear line in an FSC-A/FSH-H dot plot and create gate P2.

11. Remove doublets from sort from cell population P2 by gating out events falling under a linear line in SSC-A/SSC-H dot plot and create gate P3.

12. Gate out dying cell from sort by plotting events on GFP-A/PERCP-CY5-5-A dot plot and gating out events that appear on a linear diagonal line in the plot, create gate P4.

13. Place 15 ml collection tubes with filled with 3 ml FBS with 10 μM β- mercaptoethanol into sample collection chamber.

14. Place ice packs into sample collection chamber.

15. Sort cells at lowest flow rate out of gate P4 in 15 ml collection tube filled with 3 ml FBS with 10 μM β- mercaptoethanol.

16. After obtaining required cell number spin down cells at 500G for 5 min immediately or store short term on the rack on ice.

17. Test purity of post sort cell population with running a sample in FACS.

18. Optional: Assess viability with trypan blue.

19. Resuspend cells at required concentration in Alpha Mem Advanced with 10 μg/ml holo-transferrin, 10 μg/ml insulin, 1x non-essential amino acids, 1mM Na-pyruvate, 1% FBS, 10 μM β- mercaptoethanol, 25ng/ml GFRa1 recombinant rat, 40ng/ml GDNF recombinant human, 20ng/ml EGF recombinant human, 1x P/S, 1%BSA, 2x L-glutamine, 15mM Hepes.

3.5. Nucleofection of Porcine Spermatogonia

Prior to nucleofection, coat 6 well tissue culture plates with 0.2% gelatin for 1–3 hours at room temperature. Aspirate off gelatin and allow plates to dry. Once dry, add spermatogonial culture medium and allow to equilibrate prior to transfection in a cell culture incubator set to 37°C and 5% CO₂. Allow nucleofection reagents to come to room temperature and set aside appropriate number of cuvettes and transfer pipettes for the reactions.

1. Centrifuge isolated spermatogonia in 15 mL tubes at 1 million cells per tube for 5 minutes at 500 × g.

2. While cells are centrifuging, mix an appropriate volume of Lonza nucleofection solution V and supplement 1. For one reaction, 90μL of solution V and 20μL of supplement 1 are used.

3. Gently aspirate off PBS wash.

4. Resuspend the cell pellets in 100μL of the transfection solution just before transfection. Gently pipette the cells up and down a few times. (See Note 10).

5. Immediately add 168 pmol of ssODN repair template (if using homology directed repair) and 0.1nmol electroporation enhancer to the RNP complex. Skip this step if you are not using a repair template.

6. Directly add the RNP complex (with ssODN if applicable) to the cells in transfection solution.

7. Gently pipette up and down and transfer solution to the Lonza nucleofection cuvettes. Gently tap the cuvette to ensure the solution has reached the bottom of the cuvette. Avoid introducing air bubbles.

8. Insert cuvette into the Amaxa nucleofector machine, and electroporate on setting X-005.

9. Remove cuvettes and collect 6 well plates that were equilibrated. Using the provided Lonza transfer pipettes, add 0.5 ml of prewarmed media to the nucleofection cuvettes, then aspirate the transfected cells gently out of the cuvette. (See Note 11).

10. Transfer cells to the appropriate wells and return the plate to the incubator and leave cells overnight.

11. The following morning, change media by collecting the media from the wells and centrifuging for 5 minutes at 500 × g. Add 1mL of fresh media back to the wells during centrifugation to ensure slightly adhered cells do not desiccate. Aspirate and resuspend pellet in 0.5mL fresh media and add back to the wells.

12. Change half of the media every other day following transfection by following step 11.

13. Five days after transfection, collect non-adherent and slightly-adhered cells with 1:5 diluted 0.25% Trypsin-EDTA in DPBS for 5 minutes at room temperature. Stop the trypsin reaction with 500μL of FBS per 1 mL of trypsin.

14. Centrifuge cells and then wash once with DPBS. Proceed to analysis of editing. If analysis is not occurring right away, store cell pellets at −80°C.

3.6. Detection of Editing

1. Lyse cell pellets using 50–100μL of lysis buffer and move lysis over to PCR strip tubes.

2. Place in thermocycler and incubate at 50°C for 60 minutes and 95°C for 15 minutes.

3. Using primers that were created to flank the gRNA target site, amplify the region of interest using Hi-Fi Taq polymerase. (See Note 12).

4. To examine the frequency of mutations caused by non-homologous end joining (NHEJ), use 10μL of the PCR product in a Surveyor mutation detection reaction.

5. To examine the frequency of mutations caused by homology directed repair (HDR), digest 10μL of PCR product with required restriction endonuclease in appropriate enzyme buffer at temperature and time dictated by the endonuclease (See Note 13).

6. Remove and place a 10% TBE polyacrylamide gel into an electrophoresis rig, and fill the rig with 1x TBE. Place the rig on ice or in a cold room.

7. Add 5x Ficoll to the reactions to a final 1X concentration to ensure they sink to the bottom of the gel wells.

8. Load 3μL of an appropriate ladder for the product size.

9. Load 6μL of a Surveyor reaction and/or 10μL of an endonuclease reaction.

10. Run the gel per manufacturer’s recommendations to ensure sufficient band separation. In these experiments 200 volts for 1 hour was used.

11. Once the time is up, remove the gel from the gel mold by cracking it with a gel removal tool and a razor blade.

12. Fully submerge gel in staining buffer in appropriate container.

13. Place on a rocker and stain for 10 minutes at room temperature.

14. Dispose of ethidium bromide solution and wash three times in DI water (See Note 14).

15. Image gel at an exposure time less than 2 seconds as not to over expose the images.

3.7. Analysis of Editing

For more information on the theory behind analysis see Guschin et al, 2010 (17).

1. Open the image of the gel in photoshop or a similar editing program.

2. Remove the colour of the image, and then invert the black and white image.

3. Change the Shadow input level to 100.

4. Save the image as a .jpg.

5. Open the editing image in Image J.

6. Using the subtract background function, set pixels to 20.

7. Select the rectangle tool and draw a box around your first lane of interest. Press the ‘1’ key. Make sure to include all bands in that lane.

8. Click and drag the rectangle to your next lane of interest, and press the ‘2’ key. Continue this until you reach your last lane. On the last lane, press the ‘3’ key.

9. Another window will appear containing the graphs of the band densities. The left peak represents the density of the wildtype band, while other peaks represent other products from either the NHEJ or HDR.

10. Select the line tool, and while holding ‘Shift’ drag a line across the graph, starting at the lower left of the wildtype peak, where the line begins to ascend.

11. Select the wand tool, and click on the area under the peak, above the recently drawn line. Do this for all peaks.

12. Another window will appear containing the area of the selection. Copy these numbers into an Excel file and label the numbers with their corresponding sample names and which band they represent (WT, edited, etc).

13. For Surveyor reactions, calculate the percent editing using the following formula:

    $$\mathrm{\%}Editing=100\times ({1-(1-(\frac{\mathrm{\Sigma }editedbands}{\mathrm{\Sigma }allbands})}^{0.5}))$$
    For endonuclease reactions, calculate the HDR rate using the following formula:

    $$\mathrm{\%}HDR=((\mathrm{\Sigma }editedbands)/(\mathrm{\Sigma }allbands))\times 100$$

4. Notes

1. It is recommended to only filter sterilize 50mL at a time as larger volume filters can get clogged by the enzymes.

2. It is easiest to hold the tunica with forceps and pull as you scrape along the tunica with scissors to collect the parenchyma.

3. It is important to remove as much as the mediastinum as possible. Because it is mainly connective tissue, it can use up much of the enzyme in solution, making the digestion of the parenchyma less efficient.

4. If floating DNA is noticed at this step, up to 500μL of DNase I can be added to the digestion solution.

5. As per note 4, DNase I can also be added at this step if required.

6. DNA will be noticed upon trypsinization. Add 1mL of DNase I along with 3mL of DMEM media without FBS. More DNase I can be added throughout this step if required.

7. At the beginning of the digestion, an aliquot of digestion solution will appear mainly as tubules. As the digestion progresses, the tubules break apart into single cells. Ideally, the digestion should be stopped when the majority of cells are in single cell format to avoid over-digestion. One can also triturate during this step to help break up tubules faster.

8. This step is effective because the somatic cells of the testis will adhere rapidly to a tissue culture dish and will remain adhered whilst the spermatogonial remain in suspension.

9. A cell population sorted for subsequent culture should be maintained at room temperature, to avoid stress caused by drastic temperature changes. Cells sorted for RNA extraction however, should be sorted at 4°C and kept on ice until further processing.

10. Cells should not remain in the nucleofection solution for longer than 15 minutes. It is recommended to only do 5–6 transfection at once to limit the time spent in this solution.

11. When aspirating the cells out of the cuvette, avoid taking the “popcorn” like bubbles/DNA that accumulate near the top of the solution in the cuvette.

12. Hi-Fi Taq polymerase is used so that there is a lower likelihood of mismatches being introduced by the polymerase, as these would be detected by the Surveyor assay and provide a false result.

13. The restriction site should be designed as part of the ssODN that was introduced upon transfection. This site should be specific to the edit.

14. Ensure to wash the gel well after ethidium bromide, as failure to do so can result in high background when imaging the gel.