Targeting Sex Determination to Suppress Mosquito Populations

Reviewer #1 (Public Review):

Precision guided sterile insect technology (pgSIT) is a means of mosquito vector control that aims to simultaneously kill females while generating sterile males for field release. These sterile males are expected to mate with 'wild' females resulting in very few eggs being laid or low hatching rates. Repeated releases are expected to result in the suppression of the mosquito population. This method avoids cumbersome sex-sorting while generating the sterile males. Importantly, until release, the two genetic elements that bring about female lethality and male sterility - the Cas9 and the gRNA carrying mosquitoes - are maintained as separate lines. They are crossed only prior to release, and therefore, this approach is considered to be more safe than gene drives.

The authors had made a version of this pgSIT in their 2021 paper where they targeted *β-Tubulin 85D*, which is only expressed in the male testes and its loss-of-function results in male sterility. In that pgSIT, they did not have female lethality, but generated flightless females by simultaneously targeted *myosin heavy chain,* which is expressed only in the female wings. Here the authors argue, that the survival of females is not ideal, and so modify their 2021 approach to achieve female lethality/sterility.

To do this, they target two genes - the female specific isoform of Dsx and intersex. They use multiple gRNAs against these genes and validate their ability to cause female lethality/sterility. Having verified that these do indeed affect female fertility, they combine gRNAs against Dsx and ix to generate female lethality/sterility and use *β-Tubulin 85D* to generate male sterility (previously validated). When these gRNA mosquitoes are crossed to Cas9 and the progeny crossed to WT (the set-up for pgSIT), they find that very few eggs are laid, larval death is high, and what emerges are males or intersex progeny that are sterile.

As this is the requirement for pgSIT, the authors then test if it is able to induce population suppression. To do this, they conduct cage trials and find that only when they use 20:1 or 40:1 ratio of pgSIT:WT cages, does the population crash in 4-5 generations. They model this pgSIT's ability to suppress a population in the wild. Unfortunately, I was not able to assess what parameters from their pgSIT were used in the model and therefore the predicted efficacy of their pgSIT, (though the range of 0-.1 is not great, given that the assessment is between 0-0.15).

Finally, they also develop a SENSR with a rapid fluorescence read-out for detecting the transgene in the field. They show that this sensor is specific and sensitive, detecting low copy numbers of the transgene. This would be important for monitoring any release.

Overall, the data are clear and well presented. The manuscript is well written (albeit likely dense for the uninitiated!). I had concerns about the efficacy of generating the pgSIT animals - the overall number of eggs hatched from the gRNA (X) Cas9 cross appears to be low, therefore, very large numbers of parental animals would have to be reared and crossed to obtain enough sterile males for the SIT. In addition to this, I was concerned about the intersex progeny that can blood-feed. These could potentially contribute to the population and it would be useful to see the data that suggest that these numbers are low and the animals will not be competent in the field.

Reviewer #2 (Public Review):

This is a thorough and convincing body of work that represents an incremental but significant improvement on iterations of this method of CRISPR-based Sterile Insect Technique ('pgSIT'). In this version, compared to previous, the authors target more genes than previously, in order to induce both female inviability (targeting the genes intersex and doublesex, compared to fem-myo previously) and male sterility (targeting a beta-tubulin, as previously in the release generation.
The characterization of the lines is extensive and this data will be useful to the field. However, what is lacking is some context as to how this formulation compares to the previous iteration. Mention is made of the possible advantage of removing most females, compared to just making them flightless (as previously) but there is no direct comparison, either experimental, or theoretical i.e. imputing the life history traits into a model. For me this is a weakness, yet easily addressed. In a similar vein, much is made in alluding to the 'safety concerns of gene drive' and how this is a more palatable half-way house, just because it has CRISPR component within it; it is not. It would be much more sensible, and more informative, to compare this pgSIT technology to RIDL (release of insects carrying a dominant lethal), which is essentially a transgene-based version of the Sterile Insect Technique, as is the work presented here.

The authors achieve impressive results and show that these strains, under a scenario of high levels of release ratios compared to WT, could achieve significant local suppression of mosquito populations. The sensitivity analysis that examines the effect of changing different biological or release parameters is well performed and very informative.

The authors are honest in acknowledging that there are still challenges in bringing this to field release, namely in developing sexing strains and optimizing release strategies - a question I have here is how to actually release eggs, and could variability in the efficiency of this aspect be modelled in the sensitivity analysis? It seems to me like this could be a challenge and inherently very variable.

Reviewer #3 (Public Review):

Summary and Strengths:

The manuscript by Li et al. presents an elegant application of sterile insect technology (pgSIT) utilizing a CRISPR-Cas9 system to suppress mosquito vector populations. The pgSIT technique outlined in this paper employs a binary system where Cas9 and gRNA are conjoined in experimental crosses to yield sterile male mosquitoes. Employing a multiplexed strategy, the authors combine multiple gRNA to concurrently target various genes within a single locus. This approach successfully showcases the disruption of three distinct genes at different genomic positions, resulting in the creation of highly effective sterile mosquitoes for population control. The pioneering work of the Akbari lab has been instrumental in developing this technology, previously demonstrating its efficacy in Drosophila and Aedes aegypti.

By targeting the female-specific splice isoform (exon-5) of doublesex in conjunction with intersex and β-tubulin, the researchers induce female lethality, leading to a predominance of sterile male mosquitoes. This innovation is particularly noteworthy as the deployment of sterile mosquitoes on a large scale typically requires substantial investment in sex sorting. However, this study circumvents this challenge through genetic manipulation.

Weaknesses:

One notable concern arising from this manuscript pertains to the absence of data regarding the potential off-target effects of the gRNA. Given the utilization of multiple gRNA, the risk of unintended mutations in non-target areas of the genome increases. With around 1% of males still capable of producing fertile offspring, understanding the frequency of unintended genome targeting becomes crucial. Such mutations could potentially become fixed within the natural population.
The experiments are well-conceived, featuring suitable controls and repeated trials to yield statistically significant data. However, a primary issue with the manuscript lies in its data presentation. The authors' graphical representations are intricate and demand considerable attention to discern the nuances, especially due to the striking similarity between the symbols representing different genotypes. As it stands, the manuscript primarily caters to experts within the field, thereby warranting improvements in data visualization for broader comprehension.