The genome editing revolution creates new opportunities to improve pest management strategies, yet biosafety concerns over off-target effects and the use of foreign DNA present roadblocks to the development of modified organisms destined for field use. In a recent addition to the pre-print server BioRxiv, Meccariello et al. demonstrate the feasibility of using a DNA-free approach to apply CRISPR/Cas9 to a common agricultural pest, the Mediterranean fruit fly (medfly), Ceratitis capitata. This approach makes use of Cas9 ribonucleoprotein complexes pre-loaded with gene-specific sgRNAs (RNPs), which circumvents the need to inject plasmid constructs, and has been shown to greatly reduce off-target mutations (Kim et al., 2014).
The “medfly” is a highly invasive and devastating pest due to its tolerance for many environments and wide range of host plants. It has a particularly high impact on citrus growers in the Mediterranean and coffee growers in South America. While it is not an established pest in the continental United States, this is only because of extensive eradication campaigns performed upon detection of an infestation. Sterile Insect Technique (SIT) is widely used to control medfly populations. However, efforts to make SIT more effective and economical for medfly control have relied upon transgenic lines, which are subject to regulations governing genetically modified organisms. Thus, a gene editing approach that does not rely on transgenes, such as that developed by Meccariello et al.., is critical for making improvements to SIT, while alleviating biosafety concerns.
Encouraged by the successful production of germline mutants in Drosophila melanogaster using RNPs (Lee et al., 2014), Meccariello et al.. further optimized this approach by using a single-guide RNA rather than separate transactivating and CRISPR RNAs (Jinek et al., 2012). Additionally, they modified their injection buffer to improve Cas9 solubility, using lessons learned by the zebrafish field (Burger et al., 2016). They initially targeted the eye pigmentation gene, white, with three different sgRNAs. While success varied by sgRNA, in one instance 50% of injected flies that survived to adulthood exhibited mosaic eye color, a phenotype that requires biallelic targeting. Additionally, 6 out of 9 males crossed to white mutant females sired white mutant offspring (2 of these 9 flies were sterile). Rate of transmission varied from 1-100%, demonstrating potential for high Cas9 activity in the germline. Additionally, crossing G0 flies amongst themselves lead to successful recovery of mutant flies (2-8%), which is important when known mutant lines are not available for crossing, as was the case for white.
While white was selected for its easily scored and non-lethal phenotype, this phenotype is not evident until the fly reaches adulthood. The authors report very few of their injected flies survive to adulthood, and demonstrated that Cas9 injected on its own had high toxicity. This left some open questions about the overall activity level of the RNPs, which are expected to be active very soon after injection, but short-lived. To address this, Meccariello et al. also targeted the pair-rule gene, paired, which is required for the formation of every other segment in not only Drosophila embryos, but essentially all insects studied to date. Therefore, the authors could confidently predict that mutations to paired would impact medfly embryonic development and result in easy to score cuticle phenotypes (Drosophila pair-rule mutants are missing every other segment and die prior to hatching). 54-65% of injected medfly embryos failed to hatch, and 90% of those exhibited mosaic cuticle defects reminiscent of the pair-rule phenotype, including reduced overall size and segment number. This prevalence of partial paired mutant phenotypes indicates a high rate of early-induced biallelic mutations.
This proof-of-principle study, in which multiple genes were targeted successfully and germline transmission of mutant alleles was achieved, is an important first-step towards developing functional genomics approaches for the analysis of the traits that allow the medfly to be so prolifically invasive. Additional optimization to reduce Cas9 toxicity will greatly improve feasibility. The biggest remaining challenge will be to implement homology-directed recombination, a necessary step for producing custom SIT lines, while keeping the process plasmid-free.
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Meccariello, A., Monti, S., Romanelli, A., Colonna, R., Primo, P., Inghilterra, M.G., Corsano, G.D., Ramaglia, A., Iazzetti, G., Chiarore, A., Patti, F., Heinze, S.D., Salvemini, M., Lindsay, H., Chiavacci, E., Burger, A., Robinson, M., Mosimann, C., Bopp, D., Saccone, G., 2017. Highly efficient DNA-free gene disruption in the agricultural pest Ceratitis capitata by CRISPR-Cas9 RNPs. bioRxiv 127506. doi:10.1101/127506