Precision-Guided Sterile Insect Technique

Ying Yan, Ph.D. is a postdoctoral researcher at Justus-Liebig-University Gießen, Germany where he has been working on building male-only strains of economically important agricultural pests, which will facilitate the area-wide Genetic Pest Management programs (GPM). He also working on developing novel GPM strategies involving different genome-editing methods.
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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).

Sterile Insect Technique – Theory

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.

The Drosophila somatic sex-determination hierarchy. The ratio of X chromosomes to sets of autosomes determines the on/off state of the Sex-lethal (Sxl) gene. In females, where the X:A ratio is 1, active SXL protein is made and its production is maintained via autoregulation. The presence of SXL causes splicing of the transformer (tra) pre-mRNA such that active TRA protein is made. When TRA is present with the protein product of the transformer-2 (tra-2) gene, the pre-mRNA of the doublesex (dsx) gene is spliced into its female-specific form, which encodes the DSX F protein. Similarly, the pre-mRNAs from the 5′-most promoter of the fruitless (fru) gene are spliced in a female-specific manner, and do not produce any detectable protein (three other promoters of fru produce transcripts that do not differ between the sexes). DSX F interacts with the products of the hermaphrodite (her) and intersex (ix) genes to activate female terminal differentiation and to repress male terminal differentiation. In males, where the X:A ratio is 0.5, no active SXL is made, so the tra pre-mRNA is spliced into its default, male-specific form, which does not produce active TRA protein. Although it is present in males, TRA-2 cannot act without active TRA, so the dsx and fru pre-mRNAs are spliced into default, male-specific forms. The male-specific DSX M protein activates male terminal differentiation and represses female terminal differentiation, interacting to some extent with HER. Although ix is expressed in males, like tra-2 it has no detectable function. The male-specific FRU M protein activates male courtship behavior. Arrows indicate positive regulation, bars indicate negative regulation and gray shading of gene names indicates that active proteins are not produced in the given sex. 

Sex determination in Drosophila shoing the role of Sxl in female development. Image credit:

This report provides several points of significance:

  1. 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.
  2. The pgSIT males do carry the transgenes, but are sterile. So the transgenes do not persist in the environment once the release stops.
  3. 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.
  4. 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.
  5. 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)

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