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As they are serious pests, considerable effort has been made to develop genomic resources and functional tools aiming to improve their management and control. Here, we report a significant addition to the pool of genome manipulation tools through the establishment of efficient CRISPR\/Cas9 protocols for the generation of directed and inheritable modifications in the genome of these flies. Site-directed mutations were introduced in the C. hominivorax and L. cuprina yellow genes (ChY and LcY) producing lightly pigmented adults. High rates of somatic mosaicism were induced when embryos were injected with Cas9 ribonucleoprotein complexes (RNPs) pre-assembled with guide RNAs (sgRNAs) at high concentrations. Adult flies carrying disrupted yellow alleles lacked normal pigmentation (brown body phenotype) and efficiently transmitted the mutated alleles to the subsequent generation, allowing the rapid creation of homozygous strains for reverse genetics of candidate loci. We next used our established CRISPR protocol to disrupt the C. hominivorax transformer gene (Chtra). Surviving females carrying mutations in the Chtra locus developed mosaic phenotypes of transformed ovipositors with characteristics of male genitalia while exhibiting abnormal reproductive tissues. The CRISPR protocol described here is a significant improvement on the existing toolkit of molecular methods in calliphorids. Our results also suggest that Cas9-based systems targeting Chtra and Lctra could be an effective means for controlling natural populations of these important pests.<\/jats:p>","DOI":"10.1534\/g3.119.400544","type":"journal-article","created":{"date-parts":[[2019,7,25]],"date-time":"2019-07-25T01:55:24Z","timestamp":1564019724000},"page":"3045-3055","source":"Crossref","is-referenced-by-count":42,"title":["Specific Gene Disruption in the Major Livestock Pests<i>Cochliomyia hominivorax<\/i>and<i>Lucilia cuprina<\/i>Using CRISPR\/Cas9"],"prefix":"10.1093","volume":"9","author":[{"given":"Daniel F","family":"Paulo","sequence":"first","affiliation":[{"name":"Centre for Molecular Biology and Genetic Engineering, Department of Genetics, Evolution, Microbiology and Immunology, University of Campinas"},{"name":"Laboratory of Ecological and Evolutionary Genomics, Smithsonian Tropical Research Institute, Gamboa, Panama"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Megan E","family":"Williamson","sequence":"additional","affiliation":[{"name":"Department of Entomology and Plant Pathology, North Carolina State University, Raleigh NC"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alex P","family":"Arp","sequence":"additional","affiliation":[{"name":"USDA-ARS, Knipling-Bushland U.S. Livestock Insects Research Laboratory and Veterinary Pest Genomics Center, Kerrville TX, and"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fang","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Entomology and Plant Pathology, North Carolina State University, Raleigh NC"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Agustin","family":"Sagel","sequence":"additional","affiliation":[{"name":"USDA-ARS, Knipling-Bushland U.S. Livestock Insects Research Laboratory and Veterinary Pest Genomics Center, Screwworm Research Site, Pacora, Panama"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steven R","family":"Skoda","sequence":"additional","affiliation":[{"name":"USDA-ARS, Knipling-Bushland U.S. Livestock Insects Research Laboratory and Veterinary Pest Genomics Center, Screwworm Research Site, Pacora, Panama"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joel","family":"Sanchez-Gallego","sequence":"additional","affiliation":[{"name":"USDA-ARS, Knipling-Bushland U.S. Livestock Insects Research Laboratory and Veterinary Pest Genomics Center, Screwworm Research Site, Pacora, Panama"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mario","family":"Vasquez","sequence":"additional","affiliation":[{"name":"USDA-ARS, Knipling-Bushland U.S. Livestock Insects Research Laboratory and Veterinary Pest Genomics Center, Screwworm Research Site, Pacora, 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Genetic Engineering, Department of Genetics, Evolution, Microbiology and Immunology, University of Campinas"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"W Owen","family":"McMillan","sequence":"additional","affiliation":[{"name":"Laboratory of Ecological and Evolutionary Genomics, Smithsonian Tropical Research Institute, Gamboa, Panama"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carolina","family":"Concha","sequence":"additional","affiliation":[{"name":"Laboratory of Ecological and Evolutionary Genomics, Smithsonian Tropical Research Institute, Gamboa, Panama"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6536-4735","authenticated-orcid":false,"given":"Maxwell J","family":"Scott","sequence":"additional","affiliation":[{"name":"Department of Entomology and Plant Pathology, North Carolina State University, Raleigh 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