Abstract An ideal tool for eliminating Plasmodium falciparum (Pf), the causative agent of 99% of all malaria deaths, would be a highly effective vaccine that prevents blood stage infection and thereby prevents both disease and transmission. Sanaria has developed two effective vaccines that are phase 2 clinical trials; Pf sporozoite (SPZ) vaccine that prevents Pf blood stage infection in >80% of recipients and PfSPZ Challenge (infectious PfSPZ) used in PfSPZ-CVac, a vaccination approach utilizing PfSPZ Challenge administered in the presence of an anti-malarial drug such as chloroquine which prevents blood stage infection in 100% of the volunteers. Both these vaccines are on an aggressive timeline to commercialization. PfSPZ used in these products are extracted from aseptically reared mosquitoes. Increasing the number of PfSPZ/mosquito will reduce the cost of goods. This project will develop a line of immune deficient Anopheles stephensi mosquitoes highly susceptible to PfSPZ infections. Sanaria and the Univ. of Maryland Institute for Bioscience and Biotechnology Research used transgenic technologies to introduce and express genes that resulted in the down-regulation of REL2 immune pathway in A. stephensi, including Caspar (negative regulator of Rel2), Rel2 (transcription factor) and LRIM1 (effector). Reduction of LRIM1 expression using transgenic RNA interference resulted in increased Pf infections of Pf oocysts and PfSPZ (3.6-fold increase) in non-aseptic A. stephensi. These results are encouraging because transgenic RNA interference results in hypomorphic phenotypes. Null alleles are expected to have stronger phenotypes with even more intense infections. We will use CRISPR/Cas9 technologies to create in A. stephensi null alleles through mutagenesis of LRIM1 and three additional immune genes (TEP1, LL3 and JNK) that are implicated in limiting Pf infections in Anopheles mosquitoes. These immune deficient A. stephensi lines, will provide a stable and much more efficient PfSPZ-production platform for Sanaria's manufacturing process. We will: 1) Generate mosquito lines null for LRIM1, TEP1, LL3 and JNK using CRISPR/Cas9 driven gene specific mutagenesis; 2) Test the mutant lines and select at least one line that produce ?2- fold higher PfSPZ infection intensities compared to wild type, adapt this line to aseptic PfSPZ manufacturing process and test the potency of PfSPZ manufactured in this selected line; 3) test the prevalence and intensity of Pf infection in A. stephensi mutant lines when infected with other strains of Pf; 5) Submit a Biologics Master File to the FDA describing the strain and its incorporation into Sanaria's manufacturing process.