Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture Research Coordination Meeting on Comparing Rearing Efficiency and Competitiveness of Sterile Male Strains Produced by Genetic, Transgenic or Symbiont-Based Technologies

The New World Screwworm fly (NWS), Cochliomyia hominivorax, is the only obligatory parasitic blowfly in the Neotropical region. Their larvae infest warm-blooded vertebrates and feed on host’s live tissues, resulting in severe livestock losses. We have developed an embryonic female lethal strain for an improved SIT control program in Panama. This strain contains a two-component system consisting of the gene promoter L. cuprina bottleneck driving expression of the tTA gene and a tetO-Lchid gene effector, both in the same insertion site in the genome. The addition of the sex-specifically spliced intron from the transformer gene within the Lchid gene ensures that only females die when insects are reared in the absence of tetracycline. This strain has been evaluated for fitness characteristics that are relevant for its performance in a mass rearing facility as well as mating characteristics relevant to their potential success in the field, showing promise for use in a future control program. CRISPR/Cas9-based genome editing has been successfully used for functional genomic studies in many organisms, and has recently emerged as a potential tool for insect pest control. Here, we report for the first time the successful establishment of an efficient CRISPR protocol for the generation of inheritable mutations in the NWS. In order to demonstrate its potential, we targeted two C. hominivorax loci: yellow, which determines the recessive unpigmented cuticle phenotype in adult flies and the olfactory coreceptor Orco, that forms a complex with all the Odorant-selective Receptors and is required for the transduction of the odor stimuli into behavioral responses. Our results show we successfully induced a high rate of somatic biallelic mutations in 46 to 68% of the surviving G0 flies. Notably, 90% of these mosaic flies transmitted their mutated alleles to the next generation (G1), resulting in mutant progenies at percentages ranging from 14 to 88%. Medium (300bp) to large (2.2Kb) genomic deletions were also induced by simultaneously microinjecting two sgRNAs targeting these genes, demonstrating that large DNA motifs or even entire exons can be removed from the NWS genome. Using diverse crossings and genotyping strategies, we found the first CRISPR-mediated homozygous mutant strains of NWS, named ChoYellow and ChoOR83b, currently being maintained at COPEG. We next used the developed protocol to target the female determining transformer locus (tra), aiming the reprograming of all progeny toward to males. Following the Cas9-RNPs microinjections, 60% of the surviving females developed partially to fully transformed ovipositors into male genitalia and abnormal reproductive tissue. Most probably, the male-like structures in these mosaic females are due to somatic mutations induced in one or both of alleles of tra in these cells. These results stand as a proof-of-concept that genome-editing strategies based on Cas9-mediated mutagenesis could be an effective mean for controlling Screwworm natural populations.