Following a decade of significant strides forward, the global malaria eradication agenda has stalled, due in part to the accelerating emergence of insecticide-resistant mosquitoes and drug-resistant malarial parasites. The World Health Organization and others have called for the development of new strategies to help defeat this devastating disease that infects over 2 million people and killing over 400,000 annually, predominantly young children in impoverished regions. Gene-drives, which can bias inheritance of desired traits, offer a novel and promising strategy either to eliminate disease causing insect vectors, or to immunize them against pathogens. Such super-Mendelian CRIPSR-based gene-drive systems encode bipartite transgenic cassettes consisting of the Cas9 endonuclease and a guide RNA (gRNA), which directs DNA cleavage at the genomic site of insertion. In reproductive cells, such targeted cutting of the homologous chromosome results in copying the drive element at the cleavage site through homology directed repair, resulting in nearly all progeny inheriting the drive element and its cargo. My group has contributed to developing the first CRIPSR-based gene drive (or active genetic) systems in flies, mosquitoes, mammals, and bacteria. We also pioneered allelic-drive systems designed to bias inheritance of a favored allelic variant at a separate genetic locus. In addition, we have developed, and extensively tested, two types of self-copying drive neutralizing systems, both of which carry gRNAs, but no source of Cas9. ERACRs delete and replace gene-drives, while e-CHACRs copy themselves while mutating and inactivating the Cas9 transgene carried on a gene-drive. Small population cage experiments in flies and mosquitoes have shown that highly efficient gene-drives rapidly spread through target populations, and that ERACRs and e-CHACRs can reliably replace (ERACRs) or halt (e-CHACRs) a gene-drive element. In this grant, we propose first to develop a flexible two-component (split-drive or CHACR) system that can be genetically converted (or hacked) into a single full-drive system. The split and full drive elements are inserted into genes essential for viability or reproduction, and also carry recoded cDNAs of the targeted genes to restore function of those loci. These recoded systems benefit greatly from a phenomenon we discovered and refer to as lethal/sterile mosaicism, which dominantly eliminates loss-of-function alleles (mistakes) in the target gene generated by imprecise DNA repair events rather than the intended copying event. Next, we will develop and test next-generation ERACR and e-CHACRs able to eliminate or halt our recoded-drives, and also test a self-limiting drive system that slowly targets Cas9 for mutagenesis. In parallel to these drive experiments, we will delve into the mechanisms and timing of the drive process using a unique set of image-based genetic elements we have developed. We anticipate that the intellectual advances an...