Gene Editing of Primary Rhesus Macaque B Cells

All animal procedures and experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. A summary of the following protocols is presented in Figure 1. Male and female rhesus macaques (Macaca mulatta) of Indian genetic origin aged 2-8 years old were housed and cared for in accordance with the guidelines of the Committee on the Care and Use of Laboratory Animals in a biosafety level 2 facility.

CAUTION: All experiments were performed in compliance with the universal precautions for bloodborne pathogens, with sterile/aseptic techniques and proper biosafety level 2 equipment in laminar flow hoods.

1. rAAV6 production

1. Prepare the reagents for rAAV6 production.
   1. Design and clone the homology-directed repair template in between the inverted terminal repeats (ITRs) of AAV2 in the vector pAAV using standard techniques. Ensure the homology arms are of at least ~250 bp on either side, but as little as 60 bp may suffice, although longer homology arms are preferred if the construct design allows for it. If target sequences of any of the used sgRNAs are present in the HDRT, remove them using silent mutations, which are most effective in the protospacer adjacent motif or seed region of the target site.
      NOTE: Gene synthesis combined with Gibson assembly for efficient cloning³² can be performed. Prepare a Maxiprep of a correct clone for transfection. For sgRNA design, CHOPCHOP³³ is recommended, and a list of more tools can be found at https://zlab.bio/guide-design-resources. The maximum packaging capacity for AAV including ITRs is ~4.7 kb. AAV6 is the most commonly used serotype for editing hematopoietic cells, particularly B cells⁹. Other serotypes of AAV for the gene editing of rhesus macaque B cells have not been tested, but AAV2⁸ and AAV-DJ^10,11 have been used in mouse studies.
   2. Prepare 293AAV culture medium and production medium according to Table 1 and Table 2. Sterile-filter through a 0.2 µm polyethersulfone (PES) membrane filter unit. Store at 4 °C.
   3. Prepare 1x polyethylenimine (PEI) solution (1 mg/mL, 100 mL).
   4. In a 250 mL glass beaker, heat ~ 70 mL of H₂O in a microwave for ~30 s, and then add 100 mg of PEI. Add a magnetic stirrer, and stir until the PEI is mostly dissolved.
   5. Adjust the pH to 7 with 1 M HCl, then top up to 100 mL with H₂O, wait 10 min, check the pH again, and adjust if necessary.
   6. Sterile-filter the PEI solution through a 0.2 µm PES membrane filter unit, aliquot, and store −20 °C. After thawing, the solution can be stored at 4 °C for up to 2 months.
   7. Prepare 5x polyethylene glycol (PEG)/NaCl solution.
   8. Weigh out 400 g of PEG 8,000 and 24 g of NaCl.
   9. Add a magnetic stirrer to a 2 L glass beaker, add the weighed out PEG 8,000 and NaCl, and rinse with ~ 550 mL of deionized water.
   10. Stir with heating, and bring to a boil or 80-90 °C until fully dissolved.
   11. Adjust the pH to ~7.4 with 1 M NaOH, then adjust the volume to 1 L using a measuring cylinder, and transfer it into a 2 L glass bottle with the magnetic stirrer.
   12. Autoclave the bottle, magnetic stirrer, and solution in a water bath for 30 min at 121 °C.
   13. After autoclaving, cool the solution in a cold room while stirring using the magnetic stirrer to prevent separation into different phases. Aliquot if necessary, and store at 4 °C.
   14. Prepare the formulation buffer.
   15. Mix 500 mL of DPBS with 50 µL of 10% Pluronic F-68. Sterile-filter through a 0.2 µm PES membrane filter unit, and store at room temperature (RT).
2. Cell culture, transfection, and transduction for rAAV6 production
   1. Thaw, culture, and freeze 293AAV cells as described by the manufacturer using the above 293AAV culture medium and Trypsin-EDTA for splitting. It is recommended to freeze some early passages and use the cells for AAV production before they reach passage 40.
   2. For rAAV6 production, seed four 15 cm cell culture dishes with 5 x 10⁶ cells in 30 mL each. The cells are ready for transfection usually 1-2 days after seeding when they reach 80%-90% confluence.
   3. Thaw a Maxiprep of pAAV plasmid containing the HDRT to be packaged into AAV6. Resuspend 85.6 µg of the pAAV plasmid in 3 mL of pure DMEM medium.
   4. Dissolve 342 µL of 1 mg/mL PEI solution in 3 mL of pure DMEM medium. Incubate both solutions for 10 min at RT.
   5. Mix both 3 mL tubes into one tube of ~6.4 mL of transfection mix, and incubate for 20 min at RT.
   6. Meanwhile, thaw the tetracycline-enabled, self-silencing helper vector RepCap6 from the −80 °C freezer in a 37 °C water bath. To transduce the 293AAV cells, add the helper vector at a multiplicity of infection (MOI) of 25 using the median tissue culture infectious dose (TCID₅₀) and assuming 1.15 x 10⁷ cells/dish; typically, 2-10 µL is used per 15 cm dish. Rock and swirl the dishes gently to distribute.
   7. After the incubation of the transfection mix, add 1.6 mL of it dropwise across each of the four 15 cm dishes. Incubate at 37 °C and 5% CO₂ overnight.
      NOTE: Alternatively, if the rAAV6 vectors of interest are already available, these vectors can be used to provide the viral genome to be packaged, which negates the need for any plasmids with this system and yields comparable rAAV6 titers. For this approach, the 293AAV cells are co-transduced with the desired rAAV6 at an MOI of 50 (based on rAAV6 genome copies [GC]/mL) along with the helper vector.
   8. The next day, carefully aspirate and discard the culture medium, and replace with 30 mL of prewarmed production medium. Incubate for another 96 h before harvesting. No further medium change is recommended to maximize the yields.
3. Harvest and purification of the recombinant AAV6 from the medium
   1. Without dislodging cells from the dish, collect all the cell supernatant into a filter unit with a 0.2 µm PES membrane at least 50% bigger than the volume of medium to be filtered. Then, filter the supernatant.
      NOTE: If higher yields of rAAV6 are desired, the cells can be harvested and the rAAV extracted from the cell pellet using commercial kits or established protocols^34,35. Since AAV6 is mostly secreted into the medium³⁶, only supernatant was used, reducing labor, cost, and time.
   2. Add 5x PEG/NaCl solution to the filtered supernatant at 25% of the collected volume; this is typically 30 mL if four 15 cm dishes of 30 mL are used.
   3. Mix well by inverting, and then incubate overnight at 4 °C to precipitate the viral particles.
      NOTE: AAV particles are stable for up to 2 days in this solution.
   4. Precool a swing bucket centrifuge with 250 mL of tube inserts to 4 °C. Prepare a 4 mL centrifugal filter unit with a 100 kDa cutoff and a 0.22 µm hydrophilic PES syringe filter by pretreating each membrane with 2 mL of 10% Pluronic F-68 for at least 1 h at RT.
   5. Transfer the AAV-PEG/NaCl mixture into a 250 mL tube, centrifuge at 2,500 x g for 1 h at 4 °C, and then carefully remove the entire supernatant by aspiration.
   6. Resuspend the beige to white viral pellet by vortexing in 4 mL of 1 M HEPES until fully resuspended. If necessary, let it stand for 5 min, and vortex again. Resuspend using a 5 mL serological pipette, and transfer the total volume into a 15 mL tube.
   7. In a fume hood, add an equal volume of chloroform to the virus suspension-typically 4 mL.
   8. Vigorously vortex for 2 min, and then centrifuge at 1,000 x g for 5 min at RT.
   9. Collect the top layer (AAV-containing supernatant) in a new 50 mL tube, and discard the bottom layer (chloroform).
      CAUTION: Chloroform-containing solutions are hazardous waste. Follow institutional guidelines for its disposal.
   10. Place the AAV-containing supernatant under a fume hood, and let the remaining chloroform evaporate for 30 min.
   11. Meanwhile, wash the pretreated centrifugal filter unit and syringe filter.
   12. Add 1.5 mL of formulation buffer to the pretreated centrifugal filter unit. Centrifuge at 3,500 x g for 10 min at 15 °C in a swinging bucket rotor. Repeat this step with 4 mL of formulation buffer to wash the membrane.
   13. Rinse the syringe filter twice with 5 mL of formulation buffer using a 5 mL syringe.
   14. Load the ~4 mL of AAV-containing supernatant from the chloroform extraction into a 5 mL syringe, attach the washed syringe filter, and filter directly into the centrifugal filter unit.
   15. Centrifuge at 3,500 x g for 25 min at 15 °C, and then confirm that the AAV solution in the filter is around between 50-100 µL. If the volume of the solution is >100 µL, continue to centrifuge.
   16. After removing the filtrate, add 4 mL of formulation buffer inside the cup of the centrifugal filter unit, and mix the solution uniformly by pipetting. Centrifuge at 3,500 x g for 25 min at 15 °C, and then confirm that the AAV solution in the filter is between 50-100 µL. If the volume of the solution is >100 µL, continue to centrifuge. Repeat this step for another wash.
   17. After the final centrifugation, confirm the volume of the solution is 50-70 µL; if not, continue to centrifuge. Transfer the preparation to a 1.5 mL tube. Aliquot if desired, and store at −80 °C.
4. Recombinant AAV6 titer determination by qPCR
   NOTE: The qPCR primers anneal in the ITR region and should, thus, be suitable for all constructs cloned into pAAV.
   1. Thaw an aliquot of the rAAV6 to be titered and an aliquot of the AAV6 reference material. The AAV6 reference material should be close to 4 x 10¹¹ GC/mL; otherwise, adjust the dilutions accordingly.
   2. Perform a DNase I digest to remove any remaining free plasmid DNA in the rAAV6 preparation by combining 2.0 µL of the sample or AAV6 reference material with 15.6 µL of nuclease-free H₂O, 2.0 µL of 10x DNase I buffer, and 0.4 µL of DNase I.
   3. Gently mix and incubate for 30 min at 37 °C, and then transfer to ice. This is dilution 1 (see Table 3).
   4. Prepare fivefold serial dilutions of all the samples and the AAV6 reference material as in Table 3 below with water.
   5. Prepare a SYBR Green qPCR master mix. Per well, mix 4.7 µL of nuclease-free water with 10 µL of SYBR Green master mix, 0.15 µL of ITR primer forward at 100 µM, and 0.15 µL of ITR primer reverse at 100 µM.
      NOTE: Each sample is measured in duplicate, with 16 wells for the reference standard, 8 wells per sample, and 2 wells for a no-template control. Prepare 10% more master mix to account for pipetting error.
   6. In an optical 96-well or 384-well reaction plate, load 15 µL/well of the SYBR Green qPCR master mix.
   7. Next, load 5 µL of samples and AAV6 reference material or nuclease-free water for the no-template control. For the AAV6 reference standard, load dilution 2 to dilution 9. For samples, load dilution 5 to dilution 8. Measure each dilution in duplicate. Avoid bubbles.
   8. Seal the loaded plate with optical transparent film, centrifuge at 800 x g for 1 min at RT, and load the plate into the qPCR instrument with the appropriate 96-well or 384-well setup.
   9. Set up and run the qPCR instrument using SYBR detection with the following cycling conditions: 98 °C for 3 min, then 40 cycles of 98 °C for 15 s and 58 °C for 30 s, followed by a melting curve.
   10. Analyze the data with the instrument's software using the AAV6 reference material's concentration in genome copies per millimeter (GC/mL) as the standard curve (see Table 3). Calculate the sample's final concentration by multiplying by the dilution factor.
   11. Ensure the standard curve R² is close to 1.0, the PCR efficiency is 90%-110%, the baseline has been removed, the melting curve shows a single peak, the C_t values change in accordance with the dilutions, and the duplicates are within 0.5 C_t; otherwise, exclude outliers. Expect yields as in Figure 2.

2. Preparing B cell media and stimuli

1. Prepare thawing medium: Combine RPMI-1640 with 20% FCS. Sterile-filter through a 0.2 µm PES membrane filter unit. Store at 4 °C.
2. Prepare B cell culture medium: Combine the reagents in Table 4, and then sterile-filter through a 0.2 µm PES membrane filter unit. Store at 4 °C.
3. Resuspend each of the B cell stimulants in Table 5 at stock concentrations in B cell culture medium, except CpG ODN which should be resuspended in nuclease-free water. Store at −80 °C.
4. If performing non-B cell negative depletion (optional step 4), prepare DPBS (no calcium, no magnesium) with 2% FCS (DPBS 2% FCS). Sterile-filter through a 0.2 µm PES membrane filter unit. Store at 4 °C.

3. Preparation and culture of rhesus macaque B cells

NOTE: Cryopreserved rhesus macaque PBMCs or splenocytes are used to set up the cell culture^30,31.

1. Prewarm thawing medium and B cell culture media in a 37 °C water bath. Thaw the B cell stimulants from Table 5 on ice.
2. Prepare an appropriately sized tube containing prewarmed thawing medium. This should ideally be more than 10-fold the volume of the thawed cells.
3. Thaw one to two cryovials of PBMCs or splenocytes at a time in a 37 °C water bath, and decant into the prepared tube with prewarmed medium. Rinse the cryotubes to collect all the cells.
4. Centrifuge the cells at 200 x g for 10 min at RT.
   NOTE: These centrifugation settings reduce the platelet contamination while preserving the PBMC yields. Higher speeds such as 350 x g for 5 min may be used.
5. Resuspend the cells in 10 mL thawing medium for washing.
6. Repeat step 3.4 and step 3.5 for a total of three centrifugations to remove the freezing medium. After the last centrifugation, resuspend the cells at an estimated ~5 x 10⁶ cells/mL in B cell culture medium.
   NOTE: The above protocol cultures whole PBMC or splenocyte preparations with contamination by other cells. If purer B cell cultures are required, albeit at significantly reduced total B cell yields, continue with step 4. No differences have been observed in editing efficiencies between the two methods.
7. Dilute an aliquot of 10 µL of cells as necessary with B cell culture medium for counting. Count using a hemocytometer and trypan blue staining, combining equal volumes of resuspended cells and trypan blue 0.4% solution.
8. Adjust the cell concentration to 3 x 10⁶ cells/ mL with B cell culture medium according to the cell count. Then, add the B cell stimulants to their final concentrations according to Table 5, and mix.
9. Transfer the cells to an appropriate cell culture dish. Overall, 0.6 x 10⁶-0.7 x 10⁶ cells/cm² is recommended. Incubate the cells at 37 °C with 5% CO₂ for 48 h ± 2 h.

4. Optional negative depletion of non-B cells

NOTE: Yield and purity depend on the input percentage of B cells among the PBMCs, which can differ drastically among individual rhesus macaques²⁷. Expect 80%-95% purity, 60% efficiency, and 1 x 10⁶-1.5 x 10⁶ cells from 1 x 10⁷ PBMCs.

1. After the last wash (step 3.6), resuspend the cells at 1 x 10⁸ cells/mL in DPBS 2% FCS and human Fc-block diluted 1:200. The cell counts are based on the number of thawed cells.
2. Incubate for 15 min on ice to block the Fc receptors, and then add the biotinylated antibodies in Table 6. Incubate for another 20 min on ice.
3. Top up the tube with DPBS 2% FCS, and spin at 200 x g for 10 min at 4 °C.
4. Resuspend the cells in DPBS 2% FCS at 80% of the volume from step 4.1 (i.e., 80 µL per 1 x 10⁷ cells).
5. Add magnetic streptavidin beads to the cell suspension at 20% of the volume from step 4.1 (i.e., 20 µL of beads per 1 x 10⁷ cells).
6. Incubate the cells for 15 min on ice, and agitate occasionally.
7. Meanwhile, per 1 x 10⁸ cells, prepare a magnetic separator with a large magnetic depletion column and a pre-separation filter. Rinse the pre-separation filter and column with 2 mL of DPBS 2% FCS by gravity flow, and discard the flow-through. Install a 15 mL collection tube.
   NOTE: The use of other columns such as positive selection columns or other magnetic bead purification systems may drastically reduce the purity.
8. After incubation, top up the cells to 0.5 mL with DPBS 2% FCS if the volume is <0.5 mL. If the volume is ≥0.5 mL, simply proceed.
9. Load the cell suspension into the pre-separation filter on the prepared column, and collect the flow-through into the 15 mL tube.
10. Elute the unbound enriched B cells twice by adding 1 mL of DPBS 2% FCS into the pre-separation filter. Collect the unbound cells into the same tube by gravity flow.
    NOTE: Additional elution may marginally increase the yield. The purity and efficiency may be evaluated by the flow cytometry of the input cells, enriched cells, and cells retained on the column. To obtain the cells retained on the column, remove the column from the magnet, and flush with 3 mL of DPBS 2% FCS using the provided plunger. If desired, evaluate the purity by flow cytometry as in Figure 3 using the reagents in Table 7.
11. Centrifuge the enriched B cells at 200 x g for 10 min at 4 °C.
12. Resuspend the cells at an estimated ~5 x 10⁶ cells/ mL in B cell culture medium, and continue at step 3.7.

5. Primary rhesus macaque B cell gene editing

1. After activating the rhesus macaque B cells for 48 h ± 2 h, prepare the reagents for electroporation and transduction.
   1. Prewarm DMSO, nuclease-free duplex buffer, buffer T, and buffer E (10 µL electroporation kit) or E2 (100 µL electroporation kit) from the electroporation kit to RT.
   2. Thaw the rAAV6 HDRT and B cell stimulants from Table 5 on ice.
2. Resuspend the CRISPR-Cas9 sgRNAs at 100 µM in duplex buffer. Reconstitute for 10 min at RT, and mix by vortexing and flicking. Keep the reconstituted sgRNAs on ice until use. Store at −80 °C.
   NOTE: CRISPR-Cas9 sgRNAs can be designed with various online tools (see 1.1.1) and can vary drastically in their cutting efficiency. Empirical testing of the cutting efficiency is recommended using assays such as TIDE³⁷ or ICE³⁸.
3. Per 10 µL electroporation, prepare 550 µL of B cell culture medium with all the stimulants from Table 5, and add 1% DMSO. Scale the volumes 10-fold for 100 µL electroporations. Optionally, 10% of this medium can be prepared without antibiotic-antimycotic, which slightly increases the cell viability after transfection.
4. Per 10 µL electroporation, prepare a well of a 48-well cell culture plate with 50 µL of the B cell culture medium with stimulants and without antibiotic-antimycotic, if using it. For 100 µL electroporations, pipette 500 µL into the wells of a 6-well plate.
5. Add rAAV6 HDRT to the medium in the wells, up to 20% of the volume in the well. Aim for MOIs ranging from 1 x 10⁵-1 x 10⁶ based on the number of cells per transfection (10 µL electroporation: 5 x 10⁵ cells; 100 µL electroporation: 5 x 10⁶ cells) and the GC in the rAAV6 preparation. High rAAV6 stock concentrations of 5 x 10¹³ GC/mL to 5 x 10¹⁴ GC/mL are recommended to achieve high MOIs with low volumes.
   NOTE: Lower MOIs may lead to reduced editing efficiency, and MOIs of 5 x 10⁵ are generally close to the maximum editing efficiencies we have seen. An influence of varying MOIs on B cell viability has not been observed. It is recommended to include controls without rAAV6 HDRT, without RNP transfection, and without both.
6. Prewarm the prepared dishes and the remaining medium by transferring them into an incubator at 37 °C with 5% CO₂.
7. Per 10 µL electroporation, prepare 1.15 µL of ribonucleoprotein (RNP): Mix 0.4 µL of 61 µM Cas9 with 0.75 µL of 100 µM sgRNA in duplex buffer. Prepare extra (30% more for a single electroporation is recommended) due to pipetting error and to avoid bubbles when loading the electroporation tips. Scale 10-fold for 100 µL tips.
8. Incubate the RNP for at least 15 min at RT before mixing with the cells. After incubation, multiple RNPs can be combined if more than one locus is to be targeted simultaneously. No significant differences in efficiency have been observed with up to three loci at the same time.
9. Meanwhile, prepare the cells for electroporation. Keep the cells at RT at all times to avoid temperature shocks. Harvest the cells after 48 h ± 2 h of culture into an appropriate vessel. Rinse the dishes with DPBS to collect the maximum number of cells.
10. Centrifuge the cells at 200 x g for 10 min at RT. Discard the supernatant, and resuspend the cells in DPBS at ~2 x 10⁶ cells/mL.
11. Combine 10 µL of trypan blue 0.4% solution with 10 µL of the cell suspension, and count using a hemocytometer.
    NOTE: At this point, due to loss during harvesting and washing, expect about 60% of the cells that were put in culture 48 h ± 2h earlier.
12. Meanwhile, centrifuge the cells at 200 x g for 10 min at RT. Discard the supernatant, making sure to minimize any remaining DPBS. Resuspend the cells in prewarmed (RT) buffer T at 5.55 x 10⁷ cells/mL based on the above cell count.
13. Set up the transfection system by turning the machine on and setting it to 1,350 V, 15 ms, and 1 pulse. Place the pipette station inside the laminar flow hood
14. For each set of 10 electroporations, prepare a transfection tube with 3 mL of buffer E (for 10 µL transfections) or E2 (for 100 µL transfections). Insert the tube into the pipette station.
15. Per 10 µL electroporation, combine 1.15 µL of RNP with 9 µL of cells. Make sure to have a sufficient volume (+ 30%) to avoid aspirating air into the electroporation tip. Incubate at RT for 1-2 min before electroporation.
16. Aspirate 10 µL or 100 µL of RNP and cell mixture into the appropriately sized electroporation tip on an electroporation pipette, insert the loaded pipette into the pipette station, and start the electroporation. Make sure the tips are completely free of air bubbles to prevent arcing. Watch during electroporation to verify that arcing does not occur.
17. Immediately eject the electroporated cells into the prepared, prewarmed, small volume of medium with or without rAAV6 inside the 48-well (10 µL transfections) or 6-well plate (100 µL transfections). Repeat steps 5.15-5.17 with the remaining samples. Add control samples without transfection to the culture wells.
18. Incubate the cells at 37 °C with 5% CO₂ for 4 h ± 2 h, and then add the prepared, prewarmed B cell culture medium containing stimulants, DMSO, and antibiotic/antimycotic: 450 µL for 10 µL transfections or 4.5 mL for 100 µL transfections.
19. Continue the incubation at 37 °C with 5% CO₂ for 12-24 h. Then, change the medium to B cell culture medium containing stimulants and antibiotic/antimycotic without DMSO if extended culturing is desired. Analysis of the genomic DNA can be done after 24 h. Digital droplet PCR using a primer outside the homology arm and a primer inside the insert can be used to quantify the editing efficiency³⁹. Perform PCRs to amplify the insertion site and Sanger sequencing to verify correct editing.
20. For analysis of the protein levels, culture the cells for 40-48 h after electroporation to allow protein expression changes, and perform an analysis by flow cytometry using the reagents in Table 7.