Right after the 2019 New Year, Kandul et al (2019) described a novel CRISPR-based technology which they termed as “precision guided sterile insect technique (pgSIT)”. The idea of traditional SIT was planted in 1930s, which uses radiation to induce sterility in insect pests prior to release. This technology has been used for decades to combat insect pests, and a notable example is the eradication program of the New World screwworms Cochliomyia hominivorax in North and Central America.
Considerable efforts have been made to improve SIT or develop alternative strategies using different approaches, including incompatible insect technique (IIT), the release of insects carrying a dominant lethal (RIDL) or female-specific RIDL (fsRIDL), sex-ratio distortion (SRD), gene drive and so on. As a follow up, pgSIT uses the powerful tool of CRISPR to achieve simultaneous sexing and sterilization thus produce only sterile adult males. This idea was successfully demonstrated in Drosophila melanogaster.
Specifically, there are two major components for the pgSIT that developed by Kandul et al (2019). One is the Cas9-expressing line, which use either nanos, vasa or Ubiquitin 63E promoter (first two are germline-specific and the last one is ubiquitous) to drive Cas9 expression.
The other component is the single-guide (sg) or double-guide (dg) RNA line, which individually or simultaneously target sex determination genes such as sex lethal (Sxl), transformer (tra) or doublesex (dsxF), as well as genes active during spermatogenesis such as βTubulin 85D (βTub), fuzzy onions (fzo), protamine A (ProtA) or spermatocyte arrest (sa).
Notably, the crosses between homozygous dgRNAβTub,Sxl strain with each Cas9 strain resulted in 100% female lethality and 100% male sterility due to simultaneous disruption of sxl and βTub, respectively. Similar results were obtained from the crosses between each Cas9 strain and dgRNAβTub,Tra and dgRNAβTub,DsxF strain, except the females were not dead but rather transformed into sterile intersexes.
pgSIT showed complete penetrance since maternal deposition of Cas9/gRNA complexes into embryos is sufficient to achieve mutations. A βTub-promoter-GFP report line was also generated in this study to visualize the testes and spermatids. This provided a clear examination for the sterility of both male and intersex that produced from different crosses.
To further evaluate the potential of pgSIT in field condition, the release scenario of IIT, RIDL, fsRIDL and pgSIT that targeting Aedes aegypti, were simulated using the MGDrivE simulation framework. The results suggested pgSIT has greater potential to eliminate local Ae. aegypti populations than other methods.
This report provides several points of significance:
- Fitness of pgSIT males is not compromised, which is a critical factor for field application. According to the report this also indicated that the off-target numbers from CRISPR system are likely low, if there are any.
- The pgSIT males do carry the transgenes, but are sterile. So the transgenes do not persist in the environment once the release stops.
- The eggs, but not adults, are to be released for pgSIT. This not only eliminate the work of manually sexing and sterilizing males, but also simplify the distribution scheme for insects which diapause during the egg stage, such as Ae. aegypti and Ae. albopictus. pgSIT should also be quite effective for insect with density-dependent effect, since the hatched larvae would compete for resources with wild insects.
- Accumulation of resistance is unlikely for pgSIT since homozygous Cas9 and gRNA strains are raised separately, and then mated to produce sterile males. Although this would require a sexing system, which is a general issue for many SIT programs.
- Due to the flexibility and simplicity of CRISPR system, pgSIT holds great promise to be transferred to other insect agricultural pests and disease vectors.
It would be exciting to see in the future that pgSIT could be imported to other species for practical use. It maybe also worthy to systematically evaluate if pgSIT is compatible with other strategies such as gene drive.
Nikolay P. Kandul, Junru Liu, Hector M. Sanchez C., Sean L. Wu, John M. Marshall & Omar S. Akbari (2019). Transforming insect population control with precision guided sterile males with demonstration in flies. Nature Communicationsvolume 10, Article number: 84 (2019)