Specific gene disruption in the major livestock pests Cochliomyia hominivorax and Lucilia cuprina using CRISPR/Cas9

Authors: PAULO, DANIEL - Universidade De Campinas (UNICAMP); WILLIAMSON, MEGAN - North Carolina State University; Arp, Alex; LI, FANG - North Carolina State University; SAGEL, AGUSTIN - US Department Of State; Skoda, Steven; SANCHEZ-GALLEGO, JOEL - US Department Of State; Vasquez, Homero; QUINTERO, GLADYS - US Department Of State; Perez De Leon, Adalberto - Beto; BELIKOFF, ESTHER - North Carolina State University; AZEREDO-ESPIN, ANA - Universidade De Campinas (UNICAMP); MCMILLAN, W - Smithsonian Tropical Research; CONCHA, CAROLINA - Smithsonian Tropical Research; SCOTT, MAXWELL - North Carolina State University

Submitted to: Genes, Genomes, Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/23/2019
Publication Date: 7/24/2019
Citation: Paulo, D.F., Williamson, M., Arp, A.P., Li, F., Sagel, A., Skoda, S.R., Sanchez-Gallego, J., Vasquez, H., Quintero, G., Perez De Leon, A.A., Belikoff, E.J., Azeredo-Espin, A.M., Mcmillan, W.O., Concha, C., Scott, M.J. 2019. Specific gene disruption in the major livestock pests Cochliomyia hominivorax and Lucilia cuprina using CRISPR/Cas9. Genes, Genomes, Genetics. Volume 9, Issue 7. https://doi.org/10.1534/g3.119.400544.
DOI: https://doi.org/10.1534/g3.119.400544

Interpretive Summary: The New World screwworm and the Australian sheep blowfly are major pests of livestock where they remain established. Due to their economic impact, considerable effort has been made to develop genomic resources, including functional genomic tools, to enhance area-wide pest management. Here, we add a new approach with the adaptation of an efficient tool, technically known as CRISPR/Cas9, to edit the genome of screwworms and blowflies. A high proportion of adults that developed from embryos injected with components of the genome editing tool carried mutations at the targeted yellow or transformer genes. This approach expands our ability to study the role of specific genes in the unique biology and behavior of screwworms and blowflies. Genome editing presents the opportunity to advance the sterile insect technique through biotechnology-enhanced strategies to keep regions of the Americas free of screwworm where this high-consequence livestock pest was eradicated.

Technical Abstract: 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 in the ChY and LcY 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 gene 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 gene drive systems targeting Chtra and Lctra could be an effective means for controlling natural populations of these important pests.