CRISPR to go – a guide to gene editing Tephritid flies at remote facilities

Leigh Boardman, Ph.D. is a postdoctoral research at the University of Florida, Gainesville, Florida, USA and is interested in integrative and comparative biology, genotype-phenotype interactions and the molecular mechanisms underlying organismal tolerance to environmental stressors,
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Sim et al. (2018) recently published a paper in Insect Molecular Biology describing CRISPR/Cas 9 methods for gene editing in three genera of Tephritid fruit flies: Anastrepha, Bactrocera, and Ceratitis. CRISPR/Cas9 allows for targeted gene editing through homology driven repair or non-homologous end join (NHEJ) repair.

While CRISPR/Cas9 has been successfully demonstrated in Bactocera tyroni (Choo et al., 2018) and Ceratitis capitata (Meccariello et al., 2017), this paper signifies the first example of CRISPR/Cas 9 in an Anastrepha fruit fly, and the first in B. dorsalis. The paper by Sim et al. (2018) also provides detailed methodology for using CRISPR/Cas 9 to create heritable germline mutations in Tephritid fruit flies in quarantine facilities both with and without molecular biology capabilities.

Anastrepha ludens.
image credit:  Jack Dykinga, USDA Agricultural Research Service – This image is Image Number 1322089 at Insect Images, a source for entomological images operated by The Bugwood Network at the University of Georgia and the USDA Forest Service

Tephritidae fruit flies are amongst the most destructive pests, causing both immediate damage to commodities, and downstream quarantine and trade restrictions. With a wide host distribution, and repeated interceptions, population suppression and control of these flies is an ongoing battle in many regions of the world.

To date, genetic means of suppressing the reproduction, or reducing fitness of pest populations, have relied on the Sterile Insect Technique in which a non-lethal dose of radiation is used to sterilize insects. These sterile insects are then released into the target area to mate with wild insects, thereby suppressing the population size.

In SIT, and other manners of genetic manipulation, the availability of a genetic sexing strain (GSS) enables easy separation of males and females and facilitates the release of male-only strains. This has been shown to increase the efficacy of many SIT programs. These GSS currently exist for A. ludens, B. dorsalis, C. capitata, Zeugodacus (Bactrocera) cucurbitae, and effective CRISPR/Cas9 methods could extend GSS to include other Tephritid species, which could be used for SIT programs.

 

Bactrocera dorsalis compound eyes. a) wild type, b) white-eye mutant.
image credit: Handler and McCombs (2000) Insect Molecular Biology (2000) 9(6), 605 – 612

Sim et al. (2018) used sgRNAs to target the white gene in A. ludens, B. dorsalis, and C. capitata. Successful white-eye mutants arose in each of the species, and at least one mutant line was developed for each species. The mutant white-eye phenotypes arose from point mutations, frameshift mutation, and premature stop codons which caused deletions in the white gene. The adult survival rates were 2.1% (A. ludens), 1.5-2.3% (B. dorsalis), and 1.4-2.2% (C. capitata), with mutation rates varying from 9.1% (A. ludens) to 0-3.6% (B. dorsalis), and 7.7-28% (C. capitata).

This research was completed at both the USDA-ARS Daniel K. Inouye Pacific Basin Agricultural Research Center (Hawaii), and San Miguel Petapa Fruit Fly Rearing and Quarantine facility (Guatemala). These facilities have very different resources available, and the successful implementation of CRISPR/Cas9 at a resource-limited institution represents progress towards the broader use of this technique.

Non-Homologous End Joining, NHEJ

With a suitcase full of equipment that included injection apparatus and portable molecular biology apparatus; and refrigeration, compressed air, and support staff provided by the San Miguel Petapa facility, the authors were able to successfully demonstrate CRISPR/Cas9 in A. ludens. In contrast to B. dorsalis and C. capitata, injection times for A. ludens were significantly longer after egg laying (up to 240 min vs. 90 min); and, due to the elongated shape of the eggs and response to dechorionation, injections were targeted at the middle of the embryo (instead of the posterior end). This shows that even amongst closely related genera, species optimization of the technique is critical.

The authors noted that because their methods used CRISPR/Cas9 with NHEJ repair, these flies may not be subject to regulations on transgenic organisms. This, together with the demonstrated use of CRISPR/Cas9 in remote facilities, has the potential to broaden the impacts of this technology, and improve current pest control solutions.

Sim, S.B. et al (2018). “The ABCs of CRISPR in Tephritidae: Developing methods for inducing heritable mutations in the genera Anastrepha, Bactrocera, and Ceratitis”. In: Insect Molecular Biology. doi: 10.1111/imb.12550

Choo, A. et al. (2018). “CRISPR/Cas9-mediated mutagenesis of the white gene in the tephritid pest Bactrocera tryoni”. In: Journal of Applied Entomology 142.1-2, pp. 52–58. rssN: 0931-2048.

Meccariello, A. et al. (2017). “Highly efficient DNA-free gene disruption in the agricultural pest Ceratitis capitata by CRISPR-Cas9 ribonucleoprotein complexes”. In: Scientific Reports 7. rssN: 2045-2322.

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