Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

It is difficult to use the host infection stage of T.cruzi in experiments because amastigote is an obligate intracellular parasite. Our culturing technique allows the amastigote to temporarily replicate outside of the host cell, so we can perform experiments directly on this clinically relevant stage of the parasite. Demonstrating the procedure will be Yukie Akutsu, a technician from our institute.

Maintain a host parasite co-culture according to the manuscript directions. When the Cas9-expressing parasite is ready for differentiation, collect the supernatant into a conical tube, and check for sample quality under a microscope. If there is a significant number of extracellular amastigotes, perform a swim-out procedure to isolate the trypomastigotes.

Spin down the mixture of trypomastigotes and amastigotes for fifteen minutes as 2, 100 times G.Then discard most of the supernatant, leaving 0.5 to 1 milliliter of medium in the tube. Loosely cap the tube, and incubate the pellet at 37 degrees Celsius for one to two hours, which will allow the active trypomastigotes to swim out of the pellet. After the incubation, transfer the supernatant with the trypomastigotes to a 1.5 milliliter microcentrifuge tube.

Spin it down, and resuspend the pellet with 5 milliliters of DMEM. Transfer the parasite to a T-25 culture flask, and incubate the flask at 37 degrees Celsius under 5%carbon dioxide in a humidified incubator. Around 95%of the parasites will differentiate into amastigotes after 24 hours.

Centrifuge the culture of extracellular amastigotes for 15 minutes at 2, 100 times G, and discard the supernatant. Resuspend the pellet with electroporation buffer containing provided supplement solution to a final cell density of one times 10 to the 8th cells per milliliter. Aliquot 100 microliters of the resuspended parasites into 1.5 liter microcentrifuge tubes.

Then add five to 10 micrograms of Guide RNA, and gently mix by pipetting. Transfer the mixture to a two millimeter gap electroporation cuvette, and apply a pulse with the electroporation device. Then, transfer the cuvette contents into a T-25 flask, containing five milliliters of prewarmed LIT medium, leave the cap loose, and incubate the flask at 37 degrees Celsius under 5%carbon dioxide.

Monitor the cell growth either by continuation of axenic culturing or as intracellular amastigotes after host cell infection. If monitoring the cell growth as axenic amastigotes, gently shake the flask to resuspend the extracellular amastigotes into the solution, and mix 20 microliters of the culture with one microliter of propidium iodide solution in a microcentrifuge tube. It is important not to leave the culture flask outside of the incubator for longer than necessary because the temperature is one of the factors that enables axenic proliferation of amastigote.

Apply the sample onto a hemocytometer, and observe it under a fluorescence microscope. Then count the number of viable amastigotes that are not stained by propidium iodide. To monitor the growth of knockout cells as intracellular amastigotes, seed host 3T3 cells in a 12-well plate with DMEM.

One day after electroporation collect the knockout amastigotes by centrifugation, discard the supernatant, and resuspend the parasites with two milliliters of DMEM. Remove the medium from the host cell culture, and add the resuspended amastigotes. Incubate the plate at 37 degrees Celsius under 5%carbon dioxide for two days, which will allow the amastigotes to establish an infection.

After the two days, wash away the extracellular amastigotes outside of host cells with DMEM, and add fresh DMEM. Continue the incubation for an additional two days, and then visualize the nuclei of the host cells and the intracellular amastigotes. It has been demonstrated that T.Cruzi amastigotes transfected with Guide RNA against the essential gene TcCGM1 show a significant growth defect compared with a control group.

When monitoring growth as intracellular amastigotes, the fraction of host nuclei associated with T.Cruzi nuclei is significantly lower in the TcCGM1 knockout group compared to the control. On the contrary, transfection of Guide RNA against one of paraflagellar rod proteins TcPAR1 does not significantly affect cell growth after four days of axenic culturing or inhibit the growth of intracellular amastigotes. However, five days post-infection, the number of trypomastigotes that emerge out of the host cell is significantly lower in TcPAR1 knockout co-culture compared to the control co-culture.

Furthermore, the differentiated trypomastigotes inside the host cell in the control group appeared more active than the ones in the knockout group. This method can also be used to study the effects of exogenous genes on amastigote stage instead of transfecting Guide RNA into Cas9-expressing amastigote, you can transfect the plasmid into a wild-type amastigote to express an exogenous gene.