Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

This protocol is significant because it provides detailed instructions on how to prepare and inject mosquito embryos for CRISPR-Cas9 genome editing. The main advantages of this micro-injection technique are that it has a high rate of mosquito survival, and allows researchers to determine the function of a gene. This protocol has been specifically tailored to inject mosquitoes that belong to the genus of Culex.

These mosquitoes transmit several pathogens, including the viruses that cause West Nile fever and St.Louis encephalitis as well as filarial nematodes, that cause canine heartworm and elephantiasis. Therefore having tools such as CRISPR-Cas9 genome editing, will allow us to better understand and control these important disease factors. Injecting freshly laid insect embryos, and getting a higher rate of survival takes lots of time, practice and patience.

Therefore we recommend injecting several embryos with a colored saline solution before injecting expensive materials to generate a mutation. Begin by back-filling the injection needles with the injection mix. Choose a needle filler or gel-loading tip that will fit easily into the back-end of the selected injection needle.

Make sure that there is a bit of space between the end of the needle filler and the injection needle. Carefully aspirate, a single small drop of the injection mix into the injection needle near the spot where it begins to taper. Then attach the injection needle to the micro injector.

Place the slide containing the embryos onto the stage of the microscope. To open the needle, use a starting injection pressure of approximately 30 Psi and adjust it as necessary to deliver an appropriate volume of injection mix. Under a dissection microscope, position the needle above the mosquito embryo, and carefully use the micromanipulator to lower the needle onto the embryo.

Once the needle is barely touching the embryo, quickly move the embryo perpendicular to the long axis of the needle. To determine if the needle was successfully opened, press the injection trigger and see whether any bubbles or injection mix escape from the needle. If not, repeat moving the mosquito embryo against the needle until it is open and the injection mix escapes when the trigger is pressed.

Position the first mosquito embryo in the center of view, ensure that it is in focus and place the needle over the first egg. Progressively increase the magnification on the microscope, carefully lowering the needle to keep it just above the first embryo. Proceed until the microscope has reached its highest magnification, and to both the embryo and the needle are in focus.

Move the embryo slightly to the left and lower the needle so that it is in the same plane of view as the embryo. Use the microscope stage to gently touch the embryo to the tip of the needle. The narrow posterior end of the mosquito embryo should deflect slightly.

Using the microscope stage, move the posterior end of the embryo onto the needle and observe the needle penetrating the chorion of the mosquito egg. The needle should just penetrate the membrane and the approximate location of the pole cells within the embryo. Placing the injection mix in this region, increases the likelihood that mutations will occur and be inherited in the germline.

Pull the injection trigger one to three times to shoot injection mix into the embryo. Deliver a small amount of injection mix, just enough that a small clearing in the embryo plasm can be detected. Use the stage to move the embryo to the right, away and off the tip of the needle.

If the injection went well, little to no fluid should leak from the embryo. Move the stage down so that the next embryo is positioned in front of the needle. And repeat the injection.

If injection mix does not appear to be coming out, or if a large amount of fluid escapes from the embryo after the needle is removed, replace the injection needle and start again. Destroy any uninjected or damaged eggs. After all embryos on the slide have been injected, wash off the Halocarbon oil by applying reverse osmosis water with a squirt bottle.

Direct the stream of water onto the top of the glass cover slip above the injected embryos and allow the water to flow down the cover slip, making sure to not direct the stream onto the embryos. Cover the slide with 150 microliters of water and place it on wet filter paper inside of a square Petri dish. Incubate the Petri dish in an environmental chamber, set to 25 to 27 degrees Celsius and 70 to 80%relative humidity with a long-day photoperiod.

This protocol was used to successfully inject embryos of the Northern house mosquito, Culex pipiens ensuring that the anterior end of the mosquito embryo extends beyond the strip of medical dressing, greatly increased larval survival and results in high-quality offspring that are capable of developing to adulthood and reproducing. Sequencing revealed, that approximately 10%of the screened mosquitoes showed a small insertion or deletion near the Cas9 cut-site of the first guide RNA, suggesting that the pole cells were successfully targeted during micro-injections. The F1 individuals successfully mated and produced viable offspring, some of which also contained the mutation.

When micro-injecting insect embryos, most people struggle with initially opening the needle and moving the embryo onto the needle. Remember that developing these skills takes time and practice. This protocol can be modified or adapted to inject the embryos of other mosquitoes or even other insects species.

Beyond using this protocol to knock-out genes with targeted CRISPR-Cas9 genome editing, the same techniques can be used to inject plasmids or transposons to either knock-in genes or generate random mutations. My lab is currently using this technique to better understand how mosquitoes measure daily and regulate their seasonal responses. However, how us researchers are using CRISPR-Cas9 genome editing to better understand the biology of several organisms.