RNP assembly

Protocol for Cas9 RNP-mediated gene editing in human primary dendritic cells

This protocol is designed for editing of human primary monocyte-derived dendritic cells by electroporation (aka nucleofection) of CRISPR/Cas9 ribonucleoprotein (RNP) complexes, i.e. Cas9 complexed with a target-complementary guide RNA. With active guide RNAs, we have observed editing efficiencies >80%, as assessed by deep sequencing of edited loci, with recovery of ~50% of cells (although cell recovery varies from donor to donor).

To generate dendritic cells, we isolate monocytes from fresh peripheral blood mononuclear cells (PBMCs) by negative magnetic selection, followed by differentiation with GM-CSF and IL-4 for 7 days. At this point, the cells should display dendritic cell morphology and express several markers for monocyte-derived dendritic cells including HLA-DR, CD11b, CD11c, CD80, CD83, CD86, and CD205. Once dendritic cells are obtained, the editing procedure consists of preparation of the dendritic cells for the procedure, assembly of the Cas9 RNPs, mixing cells with RNPs, and nucleofection using the Lonza 4D Nucleofector (required: X unit; optional: 96-well shuttle). The general editing protocol may also work for other dendritic cell subtypes/monocyte-derived dendritic cells obtained through other protocols, but we have not tested any of these cell types.

We have obtained the best results using sgRNAs from Synthego, and the remainder of the protocol is tailored to these reagents. We observe efficient editing with high viability in Nucleofector solution P3 (Lonza) with pulse codes DJ-108, DJ-107, DJ-110, CP-107, as well as multiple other pulse codes as well as in Nucleofector solution P1 (Lonza) with pulse code CB-128. All pulse codes are for the 4D Nucleofector; unfortunately we cannot map these to specific pulse parameters that are easily transferable to other electroporation devices. Our current recommendation is to use pulse code DJ-108 in solution P3. We also recommend including appropriate pulse/no RNP and no pulse/no RNP controls. (Note that electroporation conditions generally are not transferable to Cas9/guide RNA reagents from other companies.)

Reagents

• Dendritic cell differentiation medium:
  • RPMI 1640 supplemented with 10% FBS, 1% Pen/Strep/Gln (optional), 50 ng/mL GM-CSF, 20 ng/mL IL-4. Add cytokines the day the media will be used.
  • We source GM-CSF and IL-4 from Gemini Bio
  • All our work has been done with this differentiation medium – other DC differentiation procedures exist, but we have not tested if the obtained DCs can be nucleofected using the same conditions.
• to isolate monocytes, we have been using kits from StemCell, in particular either the Monocyte Enrichment kit (https://www.stemcell.com/easysep-human-monocyte-enrichment-kit.html ) or the Monocyte Isolation kit (https://www.stemcell.com/easysep-human-monocyte-isolation-kit.html ) with corresponding magnetic stand.
• centrifuge tubes 
• Corning CellStripper dissociation reagent
• fluorescently labeled anti-CD14 antibody and antibodies against dendritic cell markers of interest to assess differentiation and homogeneity (and flow cytometry tubes)
• Lonza P3 Primary Cell Nucleofector kit and nucleofection cuvettes
• purified Cas9
• guide RNAs against genes of interest
• fresh PBMCs (for example from AllCells)
• TE buffer
• PBS

Equipment

• General tissue culture equipment
• Lonza 4D Nucleofector with a X-Unit (96-well shuttle recommended for large-scale experiments)
• Cell counter (we use the Countess automated hemocytometer)

Procedure
 

1) Isolate monocytes from PBMCs and differentiate into dendritic cells

1. Count PBMCs, for example using the Countess automated hemocytometer. Calculate total cell number.
   - Optional: save two aliquots of ~0.5 x 10⁶ cells for staining with anti-CD14 antibody.
2. Isolate CD14+ monocytes from fresh PBMCs following StemCell Monocyte kit instructions. (We usually perform the optional additional separation step for purity.)
3. Count monocytes. Calculate total cell number.
   - Optional: save two aliquots of ~0.5 x 10⁶ cells for staining with anti-CD14 antibody.
4. Plan to seed ~30-39 x 10⁶ monocytes per T150 flask (scale accordingly if using flasks of different size).
5. Spin isolated monocytes for 10 min @ 200 x g in centrifuge tube. Aspirate supernatant.
6. Resuspend pelleted monocytes to ~1-1.3 x 10⁶ cells per mL in dendritic cell differentiation medium. Seed in appropriate number of T150 flasks (30-35 mL each).
   - Optional: stain pre-separation and post-separation cells with fluorescently labeled anti-CD14 antibody and assess monocyte recovery and population purity by flow cytometry.
7. Allow for cells to differentiate over the course of 7 days. Replace medium every 2-3 days. To replace the medium:
   - Carefully tilt the flask with cells and aspirate ~75-90% of the medium and transfer into a centrifuge tube. 
   - Immediately and carefully add fresh differentiation medium to flask by letting it run down the base of the flask.
   - Spin aspirated medium for 10 min @ 200 x g. Aspirate supernatant and resuspend in 1-2 mL of fresh differentiation medium. Add back to flask dropwise.
8. After 7 days, assess differentiation status, for example by brightfield microscopy and/or antibody staining.
    

2) Nucleofection of dendritic cells (perform all steps in a tissue culture hood unless otherwise indicated)

The number of cells to nucleofect depends on the downstream assays. In general, it seems that increasing cell number can help mitigate some of the nucleofection toxicity. We have nucleofected 0.4 x 10⁶ cells per well in 16-well cuvettes or 96-well plates. After nucleofection, we transfer cells from each cuvette well into 2 wells of a 96-well plate.

1. Program the Nucleofector instrument with the desired pulse codes (e.g. P3, DJ-108).
2. Prepare media and target plates for cells after nucleofection
   - prepare fresh dendritic cell differentiation medium with cytokines and warm to 37 °C.
   - prepare target plates. E.g. for 96-well plates, fill 2 wells per electroporation with 50 uL differentiation medium. Pre-warm these plates to 37 °C
3. Prepare nucleofection solutions
   - prepare nucleofection solutions by adding nucleofection supplement to nucleofection solution, following instructions from Lonza
   The timing of the next steps can be tricky. Preparing the cells takes ~60-90 min, but once the cells are dissociated, try to proceed immediately to nucleofection. The RNP generation step requires a ~10-20 min incubation step. For best results, prepare the sgRNAs ahead of time and store at –30 or –80 °C. Then time the RNP assembly and cell preparation such that the 10-20 min RNP assembly incubation ends when the cells are ready.
4. Prepare RNPs
   - dissolve Synthego sgRNAs to 100 µM in TE (in an RNase-free environment, not in TC hood)
   - dilute to 25 µM working stock in RNase-free water (not in TC hood)
   - mix sgRNA and purified Synthego Cas9 directly in nucleofector solution (in TC hood) at a molar ratio of 2.5:1, incubate at RT for 10-20 min. (note: this molar ratio has worked well in our hands, but further excess of sgRNA could increase editing efficiency in some cases.)
   - per nucleofection, prepare 20 uL of Cas9 RNP. To nucleofect 0.4 x 106 cells, we use 20 pmol Cas9 complexed with 50 pmol sgRNA and filled to 20 uL with nucleofector solution (P1 or P3)
   - Example spreadsheet:

      e. Prepare cells
         - Swirl or rock flask with dendritic cells gently. Aspirate supernatants from flask and transfer to 50 mL centrifuge tube.
         - Add 3 mL CellStripper solution to flask (for T150 flask, scale by surface area for other flasks), incubate 15 min @ 37 °C, 5% CO₂. 
         - Tap flask 2-3 times with a flat hand. Observe under microscope to check dissociation. Aspirate dissociated cells into separate centrifuge tube. If some cells are still attached, repeat dissociation with 3 mL CellStripper for 5-10 min.
        - Spin detached cells for 10 min @ 90 x g.
        - Aspirate supernatant, combine with flask supernatant (for example by resuspending pellet directly in non-attached cell suspension)
        - Count cells, for example using the Countess automated hemocytometer

       f. Perform nucleofections

         - Calculate number of cells required for nucleofections. (e.g. 0.4 x 10⁶ cells per well in 16-well cuvette strips or 96-well plates)

         - Handle separate batches of cells if using different nucleofector solutions.

         - Transfer media containing appropriate number of cells (with 5% excess) into a 50 mL centrifuge tube. (NOTE: smaller tubes can be used, but do not use a table-top centrifuge for Eppendorf tubes for the centrifiguation step as it will not hold the 90 x g speed consistently)

         - Spin 10 min @ 90 x g. Aspirate supernatant. Resuspend cells in 2-5 mL PBS (~200-500 uL per reaction).

         - Spin 10 min @ 90 x g.

         - Aspirate supernatant, removing as much PBS as possible.

         - Gently resuspend cells in nucleofection solution, 5 uL nucleofector solution per reaction (plus 5% excess)

         - Transfer 5 uL cells into nucleofection cuvette 

         - Add pre-formed Cas9 RNP or nucleofector solution (no RNP control) to each corresponding well of the cuvette, 20 uL per well. Mix by gently pipetting twice. Be careful to not introduce air bubbles.

         - Immediately nucleofect using pre-programmed Lonza 4D nucleofector or 96-well shuttle (pulse codes listed above).

      g. Post-nucleofection treatment

         - Immediately after nucleofection (up to 5 min), add 75 uL pre-warmed DC differentiation medium (with cytokines) to each well of the nucleofection cuvette by letting the media run down the side of the cuvette (be careful to ensure that the media actually touches the cell suspension instead of having an air bubble between the two layers). Do not mix.

         - Incubate at 37 °C for at least 1 hour.

         - Mix cells and transfer into prepared target plates, ~50 uL each into two wells of a 96-well plate (~100 uL final volume, ~100k cells after accounting for cell death)

       h. Maintain cells by periodically replenishing medium every 2-3 days. Genotyping can be performed 3-5 days after editing. Timing of other assays may vary.

         - To harvest for genotyping:
             o Pipet cells up and down in 96-well plate, transfer to V-bottom 96-well plate.
             o Add 25 uL CellStripper to all wells, incubate 15 min @ 37 °C, 5% CO₂
             o Confirm dissociation under the microscope. Pipet dissociated cells up and down, then transfer to V-bottom 96-well plate with supernatants. 
             o Repeat dissociation step, if necessary.
             o spin 10 min @ 300 x g to pellet cells.
             o Aspirate supernatants and resuspend pellets in QuickExtract. Proceed with genomic DNA extraction and library preparation. For accurate genotyping, isolate genomic DNA corresponding to at least 5,000 genomic copies.

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