Specific Gene Disruption in the Major Livestock Pests Cochliomyia hominivorax and Lucilia cuprina Using CRISPR/Cas9

Autor(es):Mostrar menos -

Paulo, Daniel F. ^{[1, 2]} ; Williamson, Megan E. ^[3] ; Arp, Alex P. ^{[4, 5]} ; Li, Fang ^[3] ; Sagel, Agustin ^{[6, 7]} ; Skoda, Steven R. ^{[6, 7]} ; Sanchez-Gallego, Joel ^{[6, 7]} ; Vasquez, Mario ^{[6, 7]} ; Quintero, Gladys ^{[6, 7]} ; Perez de Leon, Adalberto A. ^{[4, 5]} ; Belikoff, Esther J. ^[3] ; Azeredo-Espin, Ana M. L. ^[1] ; Owen McMillan, W. ^[2] ; Concha, Carolina ^[2] ; Scott, Maxwell J. ^[3]

Afiliação do(s) autor(es)

^[1] Univ Estadual Campinas, Dept Genet Evolut Microbiol & Immunol, Ctr Mol Biol & Genet Engn, Campinas, SP - Brazil

^[2] Smithsonian Trop Res Inst, Lab Ecol & Evolutionary Genom, Gamboa - Panama

^[3] North Carolina State Univ, Dept Entomol & Plant Pathol, Raleigh, NC 27695 - USA

^[4] USDA ARS, Knipling Bushland US Livestock Insects Res Lab, Kerrville, TX 78029 - USA

^[5] USDA ARS, Vet Pest Genom Ctr, Kerrville, TX - USA

^[6] USDA ARS, Knipling Bushland US Livestock Insects Res Lab, Screwworm Res Site, Pacora - Panama

^[7] USDA ARS, Vet Pest Genom Ctr, Screwworm Res Site, Pacora - Panama

• Tipo de documento: Artigo Científico
• Fonte: G3-GENES, GENOMES, GENETICS; v. 9, n. 9, p. 3045-3055, SEP 2019.
• Citações Web of Science: 0

Resumo

Cochliomyia hominivorax and Lucilia cuprina are major pests of livestock. Their larvae infest warm-blooded vertebrates and feed on host's tissues, resulting in severe industry losses. 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. (AU)

• Processo FAPESP: 17/05432-7 - Desenvolvimento de uma Linhagem Mutante da Mosca da Bicheira para o Co-receptor Olfativo Utilizando o Sistema CRISPR/Cas9: Uma Investigação Funcional Acerca da Evolução da Percepção Olfativa na Família Calliphoridae.
• Beneficiário: Daniel Fernando Paulo
• Modalidade de apoio: Bolsas no Exterior - Estágio de Pesquisa - Doutorado