High-Efficiency Gene Disruption in Primary Bone Marrow-Derived Macrophages Using Electroporated Cas9-sgRNA Complexes

Our laboratory studies, the interaction between mycobacterium tuberculosis and the host immune system with a focus on the initial interaction between the bacterium and the macrophage. There's a variety of gene disruption techniques used in macrophage, including RNAi technologies and CRISPR technologies. One of the major challenges is the low efficiency of gene disruption in macrophages.

This protocol enables the rapid and high efficiency genetic manipulation of primary macrophages without the need for cloning and plasmid construction. These methods should be broadly applicable in immunology, enabling researchers to disrupt genes in primary macrophages for a wide array of studies. To begin, set the parameters of the pipette station placed in the laminar flow hood to 1, 900 volts, 20 milliseconds in one pulse.

Carefully remove a transfection tube from the package to keep it sterile and fill it with 3 milliliters of E buffer. To prepare the cells for electroporation remove the growth medium from the cells and wash them using PBS to remove the serum and divalent cations that promote adhesion. Then, add a mixture of PBS and 1 millimolar EDTA to the cells and incubate at room temperature for about five minutes until the cells begin to detach.

Pipette up and down gently to detach the cells and transfer the cells to a low protein binding 15 milliliter tube. Pellet the cells at 500 G for five minutes. After centrifugation quickly remove most of the supernatant, leaving about 1 milliliter of the supernatant in the tube.

Resuspend the cells in the remaining supernatant and transfer it to a low protein binding 1.5 milliliter microfuge tube. Remove a small aliquot of cells to count and pellet the remaining cells by centrifuging at 500G at room temperature for five minutes. Count the cells during centrifugation.

Take 1x sgRNA and 20 millimolar Cas9 solution, and warm the tubes to room temperature. After centrifugation, remove all the supernatant from the cell pellet and resuspend the cells in PBS with calcium chloride and magnesium chloride at a concentration of 40 million cells per milliliter. For the sgRNA Cas9 ribonucleoprotein complex assembly, add 2.5 microliters of sgRNA to 1 microliter of the diluted Cas9 in sterilized PCR tubes, and dispense the sgRNA slowly with mixing for about 15 seconds to prevent precipitation.

Incubate for five minutes at room temperature to allow the ribonucleoprotein complex formation. To deliver the ribonucleoprotein complex by electroporation prepare the electroporation tip by depressing the plunger to extend the stem. Then, insert the stem into the tip.

Resuspend the cells by, by pipetting up and down. Load the electroporation tip with the cells and withdraw the full 10 microliters volume capacity of the tip. Starting with the negative control sgRNA transfer 10 microliters of cells in the electroporation tip to the sample tube containing 3.5 microliters of ribonucleoprotein.

Pipette up and down three times to mix well and draw 10 microliters of the mixture back into the tip. Place the tip into the electroporation station, lowering it into the buffer and push Start on the touchscreen display. After completion, the display will indicate a successful pulse.

Remove the pipette from the device and place the cells in a dry well of the uncoated 12 well plate. Rinse the tip by pipetting up and down twice in 15 milliliters of PBS with calcium chloride and magnesium chloride. Set aside the pipette with the tip still attached.

Immediately add 1 milliliter of the antibiotic-free medium aliquoted earlier to the cells, and gently shake the plate to mix. The representative Sanger sequencing chromatogram of the Rosa 26 locus from the bone marrow derived macrophages electroporated with a controlled non-targeting sgRNA Cas9 ribonucleoprotein Rosa 26 specific sgRNA and Src and Cblb genes in the edited macrophages are shown here. The analysis of the targeted genes by PCR and Sanger sequencing shows multiple nucleotides at each position downstream of the Cas9 cleavage site.

These results validate the attainment of a high editing efficiency for multiple targeted genes.