Paolo et al. report the first successful use of CRISPR/Cas9 in the New World Screwworm fly, Cochliomyia hominivorax (C. hominivorax), a parasite of warm blooded animals including humans, as well as a CRISPR/Cas9 protocol for the Australian sheep blowfly, Lucilia cuprina (L. cuprina).
The eradication of the New World Screwworm fly from North America is the first application of sterile insect technique (SIT) and is one of the great entomological success stories. Current production and release of sterile males at the COPEG facility, a collaboration between the Unites States Department of Agriculture (USDA) and the Panama Ministry of Agriculture and Livestock (MIDA) is used to prevent reestablishment of C. hominivorax from South America.
Lucilia cuprina, which causes flystrike, is a major pest for livestock (sheep) in Australia, and causes significant losses in the agricultural industry. In contrast to C. hominivorax, vector control strategies are limited to pesticide and preventative wool shearing or surgery on livestock.
Paulo et al. first targeted the yellow (ChY) gene in C. hominivorax. The authors hypothesized that ChY loss of function would create a non-lethal brown body phenotype, creating an easy phenotype to score.
For C. hominivorax, early syncytial embryos were injected using a high concentration of Cas9 (500ng/uL) and 2 sgRNAs targeting opposite strands. The pre-complexed RNPs were injected into embryos, resulting in 13.6 % survival and a 68.4% mutagenesis rate (9 males and 17 females) among the survivors. 2/9 founder crossings resulted in viable eggs, indicating germline tissue damage during development.
The mutated allele sequences showed a diversity of indels, with nearly a third of the sequences containing large deletions. An inbred line was maintained for over 15 generations, demonstrating that the CRISPR induced mutations were stable. Paulo et al. later used a lower concentration of Cas9 (360ng/uL), and found increased survival of injected embryos to adults (16.9% to 9.5%) but lower mutagenesis rate (55.6% to 73.7%) compared to the high Cas9 concentration (500ng/uL). Additionally, a higher frequency of germline mutations was reported with the higher Cas9 concentration. While they conclude a high concentration of Cas9 is more effective in generating mutations, high Cas9 concentrations lowered survival. Therefore, Paulo et al. suggest using an intermediate concentration of 400ng/uL for most targets unless the marker is autosomal recessive.
Paulo et al. next attempted to target the yellow gene in L. cuprina (LcY), first with injecting sgRNAs (Lc-Y-sgRNAs 1 and 2) and Cas9 mRNA. Even though the mutations were recessive, through interbreeding G0 adults, they were able to identify individuals with brown bodies. Indels associated with the cut site of sgRNA 1 but none with sgRNA 2 were found. From the generated mutants, Paulo et al. established two mutant lines, one of which, the LcY1B strain was used to compare Cas9 protein efficiency with Cas9 mRNA.
To test the mutagenic efficiency of injecting Cas9 protein in L. cuprina embryos, Paulo et al. injected Cas9 protein with the efficient sgRNA 1 from earlier, into wt-LcY1B heterozygous embryos. The heterozygotes were used to increase the frequency of visible phenotypes. They conclude that Cas9 RNP injection is an efficient method in generating mutations in L. cuprina.
The transformer gene (tra) is an attractive target gene for SIT using genetic sexing technology. Tra transcripts are sex-specifically spliced. Inhibition of tra through dsRNA or RNAi leads to transformation of females to males in many Diptera species, including L. cuprina.
Using their CRISPR protocol developed for C. hominivorax, Paulo et al. target the exon-1-intron-1 boundary region. They report 9.2% survival, phenotypically normal males, and intersexual phenotypes in 60% of the females. These intersexual phenotypes include partial to fully transformed ovipositors to male genitalia and abnormal reproductive tissues. A mutagenesis rate of 72.7% was reported from CrispRVariants analysis. They report that the observed phenotypes match RNAi data for a closely related species, confirming that CRISPR targeting of tra could be used for C. hominivorax.
The development of CRISPR protocols in the New World Screwworm fly and Australian sheep blowfly will be of significant value for researchers trying to develop novel and perhaps more cost effective vector control strategies for these two species. Additionally, gene function investigations can now be initiated for these fly species.