The scope of our research involves establishing genetically-modified mosquito lines using the CRISPR-Cas9 technology. The use of CRISPR-Cas9 technology for mosquito population suppression, PgSIT, as well as for the establishment of the binary expression systems for spatial temporal control of gene expression.
We have obtained both knockout and knockin lines using the CRISPR-Cas9 technology. Our knockin lines include insertion of the QF transactivator that allows spatial temporal control of downstream genes, such as fluorescent markers, GFP, for tissue-specific labeling and reporters of neuronal activity, G-CaMP6.
The establishment of novel mosquito mutant lines will allow a deeper understanding of gene function and nerve, physiological, and behavioral implications. This may lead to the development of novel ways to prevent mosquito-borne disease transmission. Our lab has developed the technology for mosquito population suppression that relies upon the CRISPR-Cas9 technology to knockout genes related to male fertility and female viability. Also, we have established multiple mosquito lines for the study of mosquito sensory resistant, specifically olfaction and vision.
[Interviewer] To prepare the injection construct for knockout mutants, dilute the Cas9 protein to the desired concentration using the Cas9 Dilution Buffer. Then dilute the aliquot of in vitro synthesized guide RNA or gRNA with ultrapure water. Premix the diluted Cas9 protein with each gRNA to form a ribonucleoprotein complex. Then combine the multiple pre-mixed ribonucleoprotein complex solutions. For homology-directed repair-mediated gene cassette insertions, dilute and mix the Cas9 protein and gRNAs. Dilute the donor plasmid with ultrapure water and combine all the constructs. Next, use a quartz filament to prepare the microinjection needles. Using the following program. pull the needles with a laser micropipette puller. After setting up a manual aspirator, moisten white circle filter papers and place them either on the inside wall or on top of moist cotton inside the collector. Place five to 10 female mosquitoes that were blood-fed five to 10 days ago into the collector. Then place the collector in the dark for 45 minutes. Afterward, remove the mosquitoes from the collector. After incubation, remove the filter papers to harvest the embryos. Select pre-blastoderm stage embryos that are light gray from the harvesting paper. Transfer selected embryos with a wet brush to double-sided sticky tape placed on top of a cover slip. Align the embryos in parallel, ensuring they are side by side with all posterior ends facing the front while their surrounds remain wet. During alignment, add Halocarbon Oil 700 onto the embryos to prevent desiccation. On the microinjector, set up a compensation pressure of 300 hectopascals and an injection pressure of 500 hectopascals. Using a micro-loader, load three microliters of the injection construct into a needle. Place the cover slip with aligned embryos on a microscope slide and position it under the microscope for injection. Next, secure a needle into the needle holder with a micro-manipulator at a 10 degree angle towards the posterior end of the embryos. Open the needle gently by lightly touching its tip to the edge of the cover slip. Then inject the embryo with the plasmid construct. After injecting Aedes agyptie mosquitoes with injection construct, use lint-free disposable wipes to remove the oil surrounding the embryos. Add deionized water to rinse the embryos. Transfer the rinsed embryos onto a wet filter paper and place the filter paper on a wet tissue inside a Karat nine-ounce cup. Then place wet cotton at the bottom of the cup to maintain moisture. After keeping the embryos moist for three to four days, transfer the filter paper with embryos to approximately three liters of deionized water in a Sterilite six-quart pan for hatching. Once the G zero larvae hatch, add fish food mixed with water to the pan. Screen the G zero larvae for the fluorescent marker at the third to fourth instar larval stage. Separate the larvae based on their fluorescent status. Maintain fluorescent-positive and fluorescent-negative larvae in separate pans. Separate the injected mosquitoes by sex when they pupate and identify males by their smaller size, more prominent and pointed genital lobe, and broader paddles. Identify females by their larger size, less pronounced genital lobe, and narrower paddles. After pooling fluorescent-positive or fluorescent negative mosquitoes of each sex, outcross each pool with mosquitoes of the opposite sex from the wild type Liverpool A. agyptie strain at a ratio of three to five wild type individuals for every fluorescent screened individual, allowing them to mate for four days. After three to four days, screen the G1 larvae for the fluorescent marker at the third to fourth instar larval stages.
