Project Abstract In 2020 malaria cases (241M) and deaths (627,000) reached the highest since 2012 emphasizing the urgent need for new tools for prevention, control, and elimination of this disease. Sanaria’s Plasmodium falciparum (Pf) sporozoite (SPZ) Vaccine, composed of radiation attenuated PfSPZ administered by intravenous injection, assessed in 1740 subjects aged 5 months to 61 years in 6 countries in US, Europe and Africa, is safe and protective for 18 months in Africa, while PfSPZ-CVac (chemo-attenuated) conferred 100% protection against heterologous controlled human malaria infection for at least 12 weeks. PfSPZ are produced using aseptically reared female Anopheles stephensi mosquitoes. Only females ingest blood, so in the current manufacturing process, aseptic male mosquitoes are superfluous, consuming growth medium and occupy space that could otherwise be used for production of more females at no additional cost or effort. In this project we plan to make the aseptic mosquito rearing process 2-fold more efficient (reducing costs of vaccine manufacture by 10-15%) by removing male mosquitoes from the system at the embryonic stage. This will be achieved by creating a mosquito line in which males are conditionally expressing a lethal insect-specific hybrid toxin, originally produced by spiders. The lethality will be induced on a switch, using the Tetracycline-controlled gene expression system. In our specific aims we will: 1. Establish a driver line: a transgenic A. stephensi line carrying the Tet-On transactivator (rtTA). A transgenic line will be created by inserting the rtTA, under the tight control of the vasa promoter, which is expressed in the first few hours after egg laying. The construct will be inserted using piggyBac-based germline modification. In the absence of doxycycline, rtTA should not bind to the Tetracycline responsive element (TRE) and thus the lethal gene will be inactive when expression is not wanted. 2. Establish a Y-linked docking A. stephensi line. We have identified specific Y-chromosome genomic loci which can be used to target integration of the locus of X (lox) docking sites. RFP under the 3xP3 promoter, flanked by two lox sites will be introduced to the identified Y sequences using CRISPR-Cas9 mediated homology-derived repair. 3. Establish an effector line: A. stephensi carrying Y-linked spider hybrid toxin under the control of the Tetracycline Response Element (TRE). The Y-linked docking line will be used to integrate a gene cassette containing TRE, minimal promoter and the hybrid toxin via lox sites using the cre/lox recombinase-mediated cassette exchange. In this transgenic line the lethal hybrid toxin will be expressed only in male mosquitoes when both doxycycline and rtTA are available; in the absence of doxycycline and rtTA no lethality is anticipated. 4. Generate a transgenic conditional male-lethal sexing strain of A. stephensi. Females from the driver line will be crossed with males from the effector lin...