We have developed a simple and inexpensive protocol to load Cas9/single-guide RNA (sgRNA) ribonucleoprotein complexes within virus-like particles. These particles, called "Nanoblades", allow efficient delivery of the Cas9/sgRNA complex in immortalized and primary cells as well as in vivo.
Nanoblades allowed rapid, efficient, and dose-dependent delivery of the Cas9 and guide RNA complex, both in immortalized and in primary cells, and in the absence of anti-transgene. The main advantage of this method is that nanoblades are easy and relatively inexpensive to prepare. They can deliver the Cas9 and guide RNA complex in a dose-dependent and transient manner, thus limiting off-target effects, while allowing for efficient genome editing.
The preparation protocol for nanoblades is very simple. However, the quality of the producer cells, as well as their origin and their seeding before transfection, are essential in order to obtain high yields of virus-like particles. Demonstrating the procedure will be Laura Guiguettaz, an engineer from my laboratory.
On day one, seed 3.5 to 4 million HEK 293T cells in 10 milliliters of modified DMEM and penicillin streptomycin in a 10-centimeter cell culture dish. After 24 hours, when the cells reach 70%confluence, replace medium with fresh DMEM and use a P-1000 pipette to add transfection reagent solution in a drop-wise manner. Start harvesting nanoblades on day four by collecting the culture medium supernatant with a 10-milliliter pipette.
It is normal that the color of the culture medium turns to yellow at this stage. Centrifuge the collected supernatant at 500 G for five minutes to removes cellular debris, and recover nine milliliters of the supernatant without disturbing the cell pellet. Pellet the nanoblades in an ultracentrifuge at 209, 490 G for 75 minutes at four degrees Celsius.
After centrifugation, slowly aspirate the medium and resuspend the white pellet with 100 microliters of cold PBS. Cover the tube with parafilm and incubate for one hour at four degrees Celsius with gentle agitation. Then, resuspend the pellet again by pipetting up and down.
Prepare a 10%sucrose solution in PBS and filter it through a 0.2-micrometer syringe filter. Place 2.5 milliliters of 10%sucrose into an ultracentrifuge tube. Tilt the tube and slowly add the nine milliliters of VLP-containing sample with a low-speed pipette while progressively raising the tube to a vertical position.
Place tubes in the rotor buckets, then centrifuge the samples at 209, 490 G for 90 minutes at four degrees Celsius. After centrifugation, removed the supernatant carefully and placed the tube upside down on tissue paper to remove any remaining liquid. For didactic purposes and better visualization of the viral pellet, nanoblades loaded with the fluorescent protein mCherry can be prepared.
After one minute, add 100 microliters of PBS. Place the tube with a parafilm cover in a tube holder on an agitation table for one hour at four degrees Celsius, and resuspend the pellet by pipetting up and down. Prepare the dilution buffer by adding one volume of lysis buffer containing a non-ionic surfactant in four volumes of PBS.
Dilute two microliters of concentrated nanoblades in 50 microliters of dilution buffer and vortex briefly. Transfer 25 microliters of the mixture into a new tube containing 25 microliters of dilution buffer and repeat the procedure to have six tubes of nanoblade dilutions. Make the standard control by adding two microliters of recombinant Cas9 nuclease into 50 microliters of dilution buffer, vortex it briefly, and make eight serial dilutions.
Spot 2.5 microliters of each VLP dilution, and 2.5 microliters of each standard onto a nitrocellulose membrane carefully with a multichannel pipette. Once the particles are absorbed, block the membrane with TBST supplemented with 5%nonfat dry milk for 45 minutes at room temperature. Discard the TBST and incubate the membrane overnight at four degrees Celsius with the Cas9 horseradish peroxidase antibody.
Wash the membrane three times with TBST and visualize the signal using an enhanced chemiluminescent substrate kit. For transduction of target cells with nanoblades, replace the cell culture medium with 500 microliters of the medium containing purified nanoblades. After four to six hours of cell incubation, replace the medium to the normal amount of fresh medium.
In the protocol, cells were seeded for homogenous distribution with 70 to 80%confluence on the day of transfection, forming syncytia leading to larger cells with multiple nuclei after 24 hours. 40 hours after transfection, most cells formed syncytia and started detaching from the plate. The amount of Cas9 loaded within nanoblades was quantified using a dot blot on a nitrocellulose membrane, which displayed enhanced chemiluminescence compared to the reference recombinant Cas9.
A calibration curve was plotted for the quantified chemiluminescence signal for recombinant Cas9 dilutions against the known amount of Cas9. Then, Cas9 protein concentration in each nanoblade preparation was mapped, revealing different concentrations of Cas9 from batch to batch. The T7 endonuclease assay demonstrated that the efficiency of nanoblades can differ from batch to batch.
For example, one batch was observed to have 20%overall editing efficiency, while the other batch displayed 60%efficiency. Flag-DDX3 proteins were immuno-precipitated using anti-flag agarose beads, followed by Western blot analysis of the recovered proteins using an anti-flag antibody. Site-directed insertion of the flag tag in the DDX3 locus was also assayed by PCR using either primers flanking the insertion site, or forward and reverse primers specific to the DDX3 locus downstream of the flag insertion site.
It is important to place the cells at a correct confluence, and to obtain an even distribution of the cells. Upon nanoblade centrifugation, it is also important to resuspend the pellet and to obtain an homogeneous sample of concentrated viral particles. Nanoblades have enabled scientists to study the mechanism of double-stranded DNA break repair by delivering the Cas9 and guide RNA complex to specific genomic loci in a rapid and coordinated manner.
They have also made it possible to inactivate long noncoding RNAs in primary cells of the innate immune system in order to study their role.
