This method can help answer key questions in the biology of the human malaria parasite, P.Falciparum, by helping to uncover protein function through conditional knockdown. The main advantage of this technique is that it allows for relatively easy generation of conditional mutants as compared to previous methods. To begin, go to Chop Chop and select Fasta Target'Under Target, paste the 200 base pairs from the three prime end of the open reading frame of a gene and 200 base pairs from the start of the gene's three prime UTR.
Under In'select P.Falciparum'and select CRISPR/Cas9'under Using'Next, click Find Target Sites'Select a gRNA sequence from the options presented, giving preference to the most efficient gRNA that is closest to the site of modification and that has the fewest off-target sites. Purchase the gRNA sequence and its reverse complement as polyacrylamide gel electrophoresis purified oligos. The gRNA sequence used to target PfH-sp70x can be found in the text protocol.
Add 40 micrograms each of pMK-U6, pUF1-Cas9, and pHA-glmS DNA into a sterile 1.5 millimeter centrifuge tube. Add one-tenth of the volume of DNA of three molar sodium acetate in water to the tube and mix it well using a vortex. Then, add 2.5 times the volume of 100 percent ethanol to the tube and mix it well using a vortex for at least 30 seconds.
Place the tube on ice or at minus 20 degrees Celsius for 30 minutes. Then, centrifuge the tube at 18, 300 times G for 30 minutes at four degrees Celsius. Following centrifugation, carefully remove the supernatant from the tube without disturbing the pellet.
Add three times the volume of 70 percent ethanol to the tube and mix it briefly using a vortex. Centrifuge the tube at 18, 300 times G for 30 minutes at four degrees Celsius. Again, carefully remove the supernatant from the tube without disturbing the pellet.
Leave the tube open and allow the pellet to air dry for 15 minutes. Store the precipitated DNA at minus 20 degrees Celsius until it is needed for transfection. To transfect RBCs, prepare 1X cytomix buffer as well as incomplete and complete RPMI as described in the text protocol.
Filter sterilize the buffer using a 0.22 micron filter. Add 380 microliters of the 1X cytomix to the DNA and vortex to dissolve. Allow the DNA to dissolve in the 1X cytomix for 10 minutes, vortexing every three minutes for 10 seconds.
In a sterile, 15 milliliter conical tube, combine 300 microliters of isolated human RBCs in the incomplete RPMI with four milliliters of 1X cytomix. Centrifuge the RBCs at 870 times G for three minutes. Then remove the supernatant from the RBC pellet.
Re-suspend the RBC pellet with the DNA-cytomix mixture and transfer it to a 0.2 centimeter electroporation cuvette. Porate the RBCs using the conditions listed in the text protocol. Following electroporation, transfer the contents from the cuvette to a 15 milliliter conical tube containing five milliliters of complete RPMI.
Centrifuge the tube at 870 times G for three minutes at 20 degrees Celsius. Then, decant the supernatant. Now, re-suspend the pellet in four milliliters of complete RPMI and transfer to one well in a six-well tissue culture plate.
Add 400 microliters of a high schizont culture to the transferred RBCs. Maintain all the cultures at 37 degrees Celsius under three percent oxygen, three percent CO2, and 94 percent nitrogen. The next day, wash the culture with four milliliters of complete RPMI.
Centrifuge the culture at 870 times G for three minutes. Aspirate the supernatant. Re-suspend the culture in four milliliters of complete RPMI.
48 hours later, wash the culture with four milliliters of complete RPMI. Then, re-suspend the culture in complete RPMI containing one micromolar DSM1 to select for the Cas9 plasmid. After this point, replace the culture medium with fresh complete RPMI plus one micromolar DSM1 every 48 hours.
Continue washing the cultures each day with complete RPMI until parasites are no longer visible by blood smear. To make a blood smear, pipette 150 microliters of culture into a 0.6 milliliter centrifuge tube. After pelleting the cells by centrifugation at 1, 700 times G for 30 seconds, aspirate off the supernatant.
Use a pipette to transfer the pelleted cells to a glass slide. Using a second glass slide held at a 45 degree angle to the first slide, smear the blood droplet. Stain the slide using a commercially available staining kit according to the manufacturer's protocol.
View the parasites using 100 times oil-immersion objective. Beginning five days post-transfection, remove two milliliters of the culture with RBCs re-suspended in the culture medium. Add back two milliliters of fresh medium and blood at two percent hematocrit.
Add fresh blood in this manner once a week until parasite reappear, as determined by thin blood smear. Perform serial dilutions of the parasite culture to achieve a final concentration of 0.5 parasites per 200 microliters. Add 200 microliters of the diluted culture to the wells of a 96-well tissue culture plate.
Maintain the cloning plate until parasites are detectable in the wells. Every 48 hours, replace the medium in the 96-well plate with fresh medium. Once a week, starting five days after beginning the cloning plate, remove 100 microliters from each well and add back 100 microliters of fresh medium plus blood.
To identify any wells containing parasites, place the 96-well plate at a 45 degree angle for approximately 20 minutes, allowing the blood to settle at an angle within the plate. Then, place the 96-well plate on a light box. Notice that the wells containing parasites contain media that is yellow in color, compared to the pink media of parasite-free wells, due to acidification of the medium by the parasites.
Using a serological pipette, move the contents of the parasite-containing wells to a 24-well tissue culture plate to allow expansion of the parasitemia. Using PCR analysis, check these clonal parasite lines for correct integration. The results of an immunofluorescence assay are shown here, demonstrating the successfully HA tagging of PfH-sp70x.
PfH-sp70x glmS parasites were fixed and stained with DAPI as a nucleus marker, as well as antibodies to HA.Membrane-associated histidine rich protein one, a marker of protein export to the host RBC. A Western Blot analysis of PfH-sp70x protein level after treatment with glucosamine is shown here. As expected, the protein level decreases during the duration of treatment.
This technique paves the way for researchers in the field of parasitology to investigate fundamental questions in the biology of the malaria parasite. It's important to remember that P.Falciparum is a blood-borne pathogen and appropriate personal protective equipment should be worn while performing this procedure.
