Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles (“Nanoblades”)

1. sgRNA Design and cloning

NOTE: Guidelines for the design of sgRNAs can be obtained from multiple sources such as https://blog.addgene.org/how-to-design-your-grna-for-crispr-genome-editing or from Hanna and Doench¹⁰.

1. Once the 20 nucleotide sgRNA sequences have been designed, order the following single-stranded DNA oligonucleotides:
   1. Forward: 5' caccgNNNNNNNNNNNNNNNNNNNN 3' (N corresponds to the targeted locus without the protospacer-adjacent motif (PAM) sequence)
   2. Reverse: 5' aaacNNNNNNNNNNNNNNNNNNNNc 3' (N correspond to the reverse-complement of the targeted locus without the PAM sequence)
      NOTE: No special modifications are required when ordering the oligonucleotides (no requirement for 5' phosphate).
2. Hybridize the two DNA oligonucleotides in a 0.2 mL polymerase chain reaction (PCR) tube by mixing 5 µL of annealing buffer (500 mM NaCl; 100 mM Tris-HCl; 100 mM MgCl2; 10 mM DTT; pH 7.9 at 25 °C), 1 µL of each DNA oligonucleotide (100 µM stock solution in water), and 42 µL of water.
3. On a PCR block, incubate samples at 95 °C for 15 s and then decrease the temperature to 20 °C with a ramp of 0.5 °C/s. Keep at room temperature or store at -20 °C.
   NOTE: The protocol can be paused here.
4. Digest 10 µg of the BLADE or SUPERBLADE sgRNA expression plasmids with 10 units of BsmBI-v2 restriction enzyme for 3 h at 55 °C in a total reaction volume of 50 µL.
   NOTE: The digested vector should release a DNA insert of ~1.9 kb and a second DNA fragment of ~3.3 kb.
5. Load the restriction reaction on a 1% agarose gel stained with 5 µg/mL of ethidium bromide (or a safer alternative DNA gel stain).
   NOTE: Wear appropriate protection gear when manipulating ethidium bromide, which is suspected of causing genetic defects.
   1. On an ultraviolet (UV) table set at a wavelength of 312 nm (to avoid damaging the DNA), cut the 3.3 kb DNA fragment from the gel, and place it in a 1.5 mL microcentrifuge tube.
      NOTE: Wear appropriate protection gear (gloves and UV protection goggles) when manipulating ethidium bromide and working on the UV table.
   2. Extract DNA from the sliced gel containing the 3.3 kb DNA fragment using a dedicated DNA gel extraction kit (see the Table of Materials). Quantify the amount of purified DNA using a spectrophotometer.
      ​NOTE: The protocol can be paused here.
6. Ligate the hybridized forward and reverse DNA oligonucleotides from step 1.2 to the BsmB1-digested, gel-purified BLADES or SUPERBLADE vector from step 1.5.2. For this, add 2 µL of T4 DNA ligase buffer, 50 ng of the gel-purified vector (from step 1.5.2), 1 µL of the hybridized DNA oligonucleotides (from step 1.2), water to make up the volume to 19 µL, and 1 µL of T4 DNA ligase. Incubate the reaction at 25 °C for 10 min.
   1. Transform the ligation product into competent bacteria (see the Table of Materials) as described in¹¹. Plate the transformed bacteria on an ampicillin Luria Bertani agar plate and incubate overnight at 37 °C.
   2. Select several isolated colonies on the agar plate to perform DNA minipreparation¹¹ (see the Table of Materials), and perform Sanger sequencing using a U6 forward primer (5' GACTATCATATGCTTACCGT 3') to check for correct ligation of the sgRNA variable sequence.
      ​NOTE: Other sgRNA expression plasmids can be used if they do not code for the Cas9 protein, which could interfere with Nanoblade production.

2. Plasmid preparation

1. Perform maxipreparation (see the Table of Materials) of all required plasmids, and prepare 10 µg aliquots at 1 µg/mL to store at -20 °C. Avoid repeated freeze/thawing cycles of the plasmids; use aliquots twice before discarding them.

3. Nanoblade preparation

1. On Day 1, seed between 3.5 and 4 × 10⁶ HEK293T cells (see the Table of Materials) in 10 mL of Dulbecco's modified Eagle medium (DMEM) containing high glucose, sodium pyruvate, L-glutamine, 10% fetal bovine serum (FBS), and penicillin/streptomycin in a 10 cm cell culture dish. Move the 10 cm plate gently backward and forward, then from right to left (repeat this sequence 5x) to distribute cells homogeneously over the culture dish. Incubate cells at 37 °C in a cell incubator with 5% CO₂.
   NOTE: All procedures related to the handling of cultured cells and Nanoblades should be performed under a cell culture laminar flow hood to avoid their contamination.
2. Day 2: Plasmid transfection
   1. Cells should be 70-80% confluent 24 h after plating (Figure 1A). Replace the medium with 10 mL of fresh DMEM containing high glucose, sodium pyruvate, L-glutamine, 10% FBS (penicillin and streptomycin can be omitted although it is not mandatory) before transfection.
      NOTE: At this step, it is important that the cells are not confluent. Otherwise, transfection efficiency as well as particle production could be reduced.
   2. For each 10 cm plate, prepare the following quantities of plasmids in a 1.5 mL tube: 0.3 µg pCMV-VSV-G, 0.7 µg pBaEVRless, 2.7 µg MLV Gag/Pol, 1.7 µg BIC-Gag-Cas9, 4.4 µg of BLADES or SUPERBLADES plasmid encoding the cloned sgRNA (or 2.2 µg each if using two sgRNAs).
   3. Add 500 µL of transfection buffer (see the Table of Materials), vortex for 10 s, and then centrifuge for 1 s. Add 20 µL of the transfection reagent (see the Table of Materials), vortex the tube for 1 s, and then centrifuge for 1 s.
   4. Incubate for 10 min at room temperature, and add the entire solution dropwise to the cells in DMEM medium using a P1000 pipettor. Move the 10 cm plate gently backward and forward, then from right to left (repeat this sequence 5x) to uniformly distribute the transfection reagent over the cells. Incubate cells at 37 °C for at least 40 h in a cell incubator with 5% CO₂.
      NOTE: If desired, medium can be changed 4 h after transfection.
3. On Day 3, check the morphology of the transfected cells under the microscope.
   NOTE: Producer cells will begin to fuse. This is a normal occurrence due to the expression of fusogenic viral envelopes (Figure 1B,C).
4. Day 4: Harvesting Nanoblades
   NOTE: At least 40 h after transfection, the cells would have fused together because of expression of the fusogenic viral envelopes, and sometimes, the cells are completely detached from the plate support (Figure 1D).
   1. Collect 9 mL of the culture medium supernatant using a 10 mL pipette.
      NOTE: Nanoblades are VLPs capable of delivering the Cas9 protein and its associated sgRNA into primary cells and in vivo. Although they are not considered genetically modified organisms as they are devoid of genetic material, they can induce genetic changes. Therefore, they must be manipulated with caution to avoid any contact with users (especially if they are programmed to target tumor suppressor genes). Users are advised to follow their local safety guidelines for the manipulation of retroviral vectors and work in a BSL-2 level laboratory when preparing VLPs and performing transduction experiments. Nanoblades can be inactivated with 70% ethanol or 0.5% of sodium hypochlorite. It is also advisable to treat all plastic waste (pipette tips, tissue culture plates, centrifugation tubes) with 0.5% sodium hypochlorite for at least 10 min to inactivate the Nanoblades.
   2. Centrifuge the collected supernatant at 500 × g for 5 min to remove cellular debris and recover the supernatant without disturbing the cell pellet.
      ​NOTE: If Nanoblades are meant to be used on primary cells, filter the supernatant using a 0.45 µm or 0.8 µm filter. Be aware that this step drastically reduces the Nanoblade titer as a significant fraction will be blocked in the filter membrane.
   3. Pellet the Nanoblades overnight (12-16 h) in a swinging bucket rotor at 4,300 × g or at 209,490 × g in an ultracentrifuge for 75 min at 4 °C (see the Table of Materials).
      ​NOTE: If target cells can grow in DMEM, it is possible to incubate them directly with the supernatant obtained after step 3.4.2 without concentrating the Nanoblades.
5. Day 5: Resuspension and storage of Nanoblades
   1. After centrifugation, slowly aspirate the medium and resuspend the white pellet with 100 µL of cold 1x phosphate-buffered saline (PBS). Cover the tube with parafilm, and incubate for 1 h at 4 °C with gentle agitation before resuspending the pellet by pipetting up and down.
      NOTE: A white viscous material may appear upon resuspension; this is normal and does not significantly affect the efficiency of transduction.
   2. Store the Nanoblades at 4 °C if planning on using them within four weeks. Otherwise, snap-freeze the Nanoblades in liquid nitrogen and store them at -80 °C.
      ​NOTE: Wear protection goggles and cryogenic gloves when manipulating liquid nitrogen. Snap-freezing and storage at -80 °C leads to a significant decrease in Nanoblade efficiency. Moreover, thawed Nanoblades should not be frozen again. The protocol can be paused here.

4. Concentration of Nanoblades on a sucrose-cushion

NOTE: As an alternative to overnight centrifugation or ultracentrifugation (step 3.4.3), the Nanoblades can be concentrated on a sucrose cushion. This yields a purer fraction of Nanoblades, although the total amount recovered will be lower.

1. Prepare a 10% sucrose solution (weight to volume) in 1x PBS, and filter it through a 0.2 µm syringe filter (see the Table of Materials).
2. Begin the process of concentrating the Nanoblades on the sucrose cushion.
   1. Place 9 mL of VLP-containing sample (from step 3.4.3) into an ultracentrifuge tube (see the Table of Materials). Using a 3 mL syringe and cannula, slowly layer 2.5 mL of the 10% sucrose under the sample, trying not to mix the VLP-containing sample and the sucrose solution.
   2. Alternatively, place 2.5 mL of 10% sucrose into an ultracentrifuge tube (see the Table of Materials). Tilt the tube and slowly add the 9 mL of VLP-containing sample (from step 3.4.3) with a low-speed pipettor. During this operation, progressively raise the tube to a vertical position.
3. Centrifuge the samples at 209,490 × g in an ultracentrifuge for 90 min at 4 °C.
   NOTE: This technique can be adapted for low-speed centrifugation (4,300 × g) overnight as described in ¹².
4. After centrifugation, remove the supernatant carefully and place the tube upside down on tissue paper to remove any remaining liquid. After 1 min, add 100 µL of 1x PBS and place the tube at 4 °C with a parafilm cover in a tube holder on an agitation table for 1 h (see the Table of Materials) before resuspending the pellet by pipetting up and down.
   ​NOTE: The protocol can be paused here.

5. Monitoring Cas9 loading within Nanoblades by dot-blot

1. Prepare the dilution buffer by adding 1 volume of lysis buffer containing a non-ionic surfactant (see the Table of Materials) in 4 volumes of 1x PBS. Dilute 2 µL of concentrated Nanoblades in 50 µL of dilution buffer, vortex briefly, and transfer 25 µL of this mixture into a new tube containing 25 µL of dilution buffer. Repeat this operation to have 4 tubes of Nanoblade dilutions (2-fold dilution steps).
2. For the standard controls, dilute 2 µL of recombinant Cas9 nuclease (see the Table of Materials) into 50 µL of dilution buffer, vortex briefly, and proceed to make eight serial dilutions (2-fold dilution for each step).
3. Carefully spot 2.5 µL of each VLP dilution and 2.5 µL of each standard onto a nitrocellulose membrane with a multichannel pipet (a larger volume may result in overlapping spots).
   NOTE: A methanol-treated polyvinyldifluoride membrane may also be used.
4. Once the particles are absorbed onto the membrane, block the membrane with 1x Tris-buffered saline containing a non-ionic surfactant (TBS-T) supplemented with non-fat dry-milk (5% w/v) for 45 min at room temperature.
   NOTE: The protocol can be paused here, and the membrane stored at 4 °C in 1x TBS-T.
5. Discard the 1x TBST supplemented with non-fat dry-milk, and incubate the membrane overnight at 4 °C with the Cas9-horseradish peroxidase antibody (1/1000 dilution in 1x TBST, 5% milk). Wash the membrane 3x with TBS-T, and visualize the signal using an enhanced chemiluminescent substrate kit.
6. Quantify the dot intensity for the Nanoblades and recombinant Cas9 standard dilutions using the proprietary software provided with the gel imaging station or imageJ¹³. Define a linear curve linking dot intensity to the Cas9 concentration. Using the function of the obtained curve, extrapolate the Cas9 content in each preparation.
   NOTE: The amount of recombinant Cas9 protein control can saturate the reading for the most concentrated samples of the standard dilution set (Figure 2). It is therefore advised, when defining the linear curve, to remove the reading from the undiluted samples (and sometimes that of the first dilution steps) if they are not in the linear range with respect to the known concentration of Cas9 that was spotted. Similarly, when extrapolating the amount of Cas9 within the Nanoblade samples, only use the readings that are within the linear range of the standard curve.

6. Transduction of target cells with Nanoblades (procedure for transduction in a 12-well plate)

1. In a 12-well plate, seed 100,000-200,000 cells (either primary or immortalized adherent cells) per well in 1 mL of the appropriate cell culture medium. Allow the cells to adhere to the plate surface before transduction.
2. In a 1.5 mL microcentrifuge tube, add 5-20 µL of concentrated Nanoblades (from step 3.5.1 or 4.4) to 500 µL of cell culture medium, and mix by pipetting up and down with a P1000 pipettor. Remove the medium from cells, and replace it with the 500 µL of this Nanoblade mixture.
   NOTE: Transduction must be optimized for each cell type. It is important to use the smallest possible volume of medium (while avoiding drying of the target cells) so that the Nanoblades remain highly concentrated. Adherent cells must be transduced directly while attached to the plate (do not transduce in suspension as this will significantly decrease transduction efficiency). Some cells tolerate prolonged exposure to Nanoblades (24-48 h) while others are very sensitive and may form small syncytia. In this case, Nanoblades must be incubated with cells only for 4-6 h before replacing the medium. Spinoculation¹⁴ can also improve transduction for cells grown in suspension. Adjuvants such as cationic polymers (see the Table of Materials) can also improve transduction efficiency in some cell types.
3. After 4-6 h of cell incubation in a low volume of medium containing Nanoblades, increase the volume of medium to the normal amount (1 mL if working with a 12-well plate), or replace it with fresh medium if the cells are sensitive to VLPs.
   NOTE: Cell medium containing Nanoblades must be inactivated with 0.5% sodium hypochlorite for 10 min before discarding it. Use gloves and protective goggles when manipulating sodium hypochlorite. If Nanoblades induce cell death, adapt the amount and total time of exposure to reduce cell mortality.

7. Measuring CRISPR efficiency at the targeted locus by T7 endonuclease assay

1. Design PCR primers to amplify a 400-700 base-pair (bp) region encompassing the CRISPR-cleavage site.
   NOTE: The cleavage site should be distant from the amplicon edge by at least 200 bp and should be slightly shifted from the center of the amplicon so that upon T7 endonuclease cleavage, 2 fragments of different sizes will be released.
2. Extract genomic DNA from cells treated with Nanoblades targeting the gene of interest and from control cells treated with Nanoblades programmed with a control sgRNA (see the Table of Materials).
   ​NOTE: The protocol can be paused here.
3. Using 150 ng of genomic DNA as a template, program a PCR reaction of 30 µL volume (final volume) by following the manufacturer's protocol. Check that the PCR amplification yields a single amplicon of the expected size by running a 2% agarose gel stained with 5 µg/mL of ethidium bromide (or a safer alternative DNA gel stain).
   ​NOTE: Wear appropriate protection gear when manipulating ethidium bromide, which is suspected of causing genetic defects. The protocol can be paused here.
4. Heteroduplex generation and digestion
   1. In a 0.2 mL PCR tube, add 5 µL of the enzyme buffer (provided with the T7 endonuclease I), 20 µL of water, and 24 µL of the PCR product from step 7.3. Allow heteroduplex formation by heating the samples to 94 °C over 3 min and then by decreasing the temperature (2 °C per min) to reach 40 °C.
   2. Add 0.5 µL of T7-endonuclease I at room temperature to each heteroduplex tube, including the control. Incubate at 37 °C for 15 min. Load the resulting reaction in a 2.5% (weight/volume) agarose gel stained by ethidium bromide. After migration, image the gel on a UV transilluminator.
      NOTE: Wear appropriate protection gear when manipulating ethidium bromide, which is suspected of causing genetic defects. Use UV-protection goggles when using the UV transilluminator.
   3. Measure cleavage efficiencies by analyzing the image resulting from the digestion reaction to quantify the intensity of each band with appropriate software (see the Table of Materials).

8. Measuring CRISPR efficiency at the targeted locus by Sanger sequencing and TIDE analysis

NOTE: As an alternative to the T7 endonuclease assay, CRISPR efficiency can be monitored by analysis and deconvolution of Sanger sequencing traces based on the TIDE protocol¹⁵.

1. Perform Sanger sequencing of PCR amplicons from step 7.3 (include a control condition corresponding to untreated cells) using either the forward or reverse PCR primer.
2. Analyze the Sanger sequencing traces of the control condition (untreated cells) and Nanoblade-treated samples using the TIDE server (https://tide.nki.nl) and following their analysis guidelines.

9. Nanoblade complex-formation with ssODN donors for homology-directed repair (procedure for transduction in a 12-well plate)

NOTE: Guidelines for the design of ssODN for efficient homology-directed repair mediated editing have been described previously¹⁶.

1. In a 12-well plate, seed 100,000-200,000 cells per well in 1 mL of the appropriate cell culture medium. Allow the cells to adhere to the plate surface before transduction.
2. Prepare 100 µL of a solution of the cationic polymer (see the Table of Materials) at 8 µg/mL in 1x PBS.
   1. Mix 19 µL of the cationic polymer solution with 100 pmol of the ssODN template. Add 20 µL of concentrated Nanoblades (from step 3.5.1 or 4.4), and incubate for 15 min on ice.
   2. Remove the complexed Nanoblades/ssODN from ice, and add 500 µL of cell culture medium (at 37 °C). Remove the medium from target cells (from step 9.1), and add the 500 µL of medium containing the complexed Nanoblades/ssODN. Allow the cells to proliferate for 48 h before genotyping.
3. Extract genomic DNA from a fraction of the cell population using a dedicated extraction kit (see the Table of Materials).
   1. Design PCR primers to amplify a 400-700 bp region encompassing the knock-in site.
      ​NOTE: PCR primers should not overlap with the homology arms of the ssODN to avoid false-positive results resulting from the PCR amplification of any residual ssODN still present within target cells.
   2. Using 150 ng of genomic DNAs from control cells (untreated) or Nanoblade-treated-cells as a template, program a 30 µL PCR reaction following the manufacturer's protocol.
      ​NOTE: ssODN traces may be present in the cell medium several days after transduction with the complex. This ssODN may serve as a partial template for PCR assays attempting to screen for the correct integration. Hence, it is advisable to passage the cells at least twice after transduction, to avoid eventual false-positive assays.
   3. Load 5 µL of the control and Nanoblade-treated PCR reactions in a 1% (weight/volume) agarose gel stained by ethidium bromide. After migration, image the gel on a UV-transilluminator.
      NOTE: If homology recombination is successful and corresponds to the insertion of more than 1 bp of genetic material, there should be a difference in the molecular weight of the PCR amplicons between the control and the Nanoblade-treated sample. As the efficiency of HDR does not reach 100%, two bands should be visible in the Nanoblade-treated sample (one of similar size to the control PCR amplicon corresponding to the unedited allele and one of higher molecular weight corresponding to the knock-in allele, see Figure 3B middle panel).
4. Perform Sanger sequencing of the control and Nanoblade-treated PCR amplicons.
5. Quantify knock-in efficiency using the TIDER protocol¹⁷.

10. Nanoblade delivery in vivo

1. Deliver up to 25 µL of concentrated Nanoblades from step 3.5.1 through retro-orbital injection or up to 100 µL through tail vein injection, as described in¹⁸, if working with mice.
   NOTE: All procedures involving animal experimentation (including Nanoblade injections for genome editing purposes) require an approved protocol from a local ethics committee.
2. For the generation of transgenic mice, use a micro-injector to deliver from 1 pL to 10 pL of concentrated Nanoblades from step 4.4 into the perivitelline space of mouse oocytes as described previously¹⁸.
   NOTE: For perivitelline injection, it is essential to purify and concentrate Nanoblades on a sucrose cushion to avoid clogging of the micro-injector.