Genetics
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Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis
Chapters
Summary November 17th, 2020
This protocol details the steps of CRISPR/Cas9 targeted mutagenesis in sand flies: embryo collection, injection, insect rearing, and identification as well as selection of mutations of interest.
Transcript
Targeted mutagenesis has been adapted to sandflies only recently and is a crucial technique for understanding the role and function of genes of interest in these vector insects. Embryo microinjection is a very important step in insect genome editing, but it needs to be adapted to species that you are working with, in our case, sandflies. Compared to the other insect species, sand fly embryos are very small.
Take a long time to develop and are particularly sensitive to the level of humidity. Five days before the injection, blood feed female sandflies as for routine colony maintenance. One day post feeding, capture the blood-fed females in plaster pods with a side port in groups of 100 to 150 and feed the flies with a 30%sucrose solution.
On day five post-blood feeding, place a white piece of moist filter paper into the egg-laying chamber. And use a mouth aspirator to collect and transfer about 10 females from the plaster pod to the egg-laying chamber. After 30 to 60 minutes, remove the Petri dish and filter paper from the egging chamber.
Taking care of that the filter paper remains moist and continuing to collect new groups of females and embryos throughout this period. When all of the embryos have been collected, use a stereo microscope and a very fine paint brush to carefully transfer about 50 embryos onto individual pieces of moist black filter paper, place onto microscope slides, topped with a single cover slip per slide. Add enough water to each filter paper to keep the embryos moist without floating or being sucked under the cover slips and line the embryos against the cover slips.
Two to three hours after the start of the egg collection, place a slide under a dissecting microscope and use the paintbrush to carefully add water to the back end of the filter paper. Back load 0.5 to one microliters of load injection mix into a micro injection needle and mount the needle into a pneumatic injection controller, set to 30 pounds per square inch of pressure under the microscope. Depress the trigger to expel any air from the needle tip.
When the air has been released, slowly increase the hold pressure. When the injection material starts to flow, decrease the hold pressure until it is just below the point of the injection mix flowing from the needle and insert the needle into the side of the first embryo. Using the cover slip behind the embryo as a back stop, deliver a small amount of injection mix into the embryo before gently removing the needle.
Immediately after removing the needle, press the injector to remove any back-filled material from the needle tip and proceed to the next embryo. When all of the embryos have been injected, count the number of injected embryos to keep a running tally and add water to the filter paper so that the cover slip floats. Then use a probe to hold the filter paper in place while pulling the cover slip away from the embryos.
And he was filter paper to blot away the excess water from the slide. Transfer the filter paper onto a stack of moist filter paper in a new Petri dish and manually transfer each embryo onto moistened, small sized plaster pods without crowding. Then cover the pods with a screen and store the pods in a 26 degree incubator at a 70%humidity.
As with most successful microinjection protocols, a good microinjection needle suited to the embryos being injected is important. Good sharp needles can easily penetrate the embryo without allowing the material to escape post-injection. The pulled needles should not have a taper that is too long.
Otherwise, the lumen of the needle becomes very narrow for a major portion of the taper, making it difficult to get the injection pressure high enough to force the material through the needle. Post-injection reared embryos that are healthy should be retained, while damaged fungus contaminated, desiccated or deformed embryos should be discarded. Potential mutant alleles can be identified in post-injection reared individuals by PCR analysis of the region surrounding the expected cutting sites.
After mutant line establishment using the injected embryos, genotyping PCR allows the identification of homozygous mutant sibling crosses. It's essential to have a good needle to be gentle and manipulating and injecting the embryos. And to make sure that the embryos are hydrated throughout the entire procedure.
Genome editing in some flies is just getting started. This technique is really a first step that should be followed by more complex genome modification strategies.
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