Malaria is one of the deadliest diseases on the planet, killing approximately 600,000 people annually, and is transmitted by the bite of an anopheline mosquito. Anophelines, and the diseases they transmit, have changed the course of history and the fate of nations, and their successful control promises to end the transmission of malaria. With the advent of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technologies, the study and control of these deadly pests have been revolutionized. As the release of genetically modified anophelines is being considered, here we outline the advances in CRISPR/Cas9 technologies and how they have revolutionized the study of anopheline basic biology and the development of innovative vector control strategies. We outline the major findings of CRISPR-based basic biological research into traits relevant for vector control including, but not limited to, olfaction, chemosensation, neurobiology, and reproduction. Further, we summarize the advancements in CRISPR-based innovative vector control strategies, such as the precision-guided sterile insect technique (pgSIT), inherited female elimination by genetically encoded nucleases to interrupt alleles (IFEGENIA), X-shredder, Y-linked editors, and gene drives. All in all, this review summarizes the basic biological and vector control research undertaken using CRISPR since its advent approximately a decade ago.

Gene drives are selfish genetic elements which promise to be powerful tools in the fight against vector-borne diseases such as malaria. We previously proposed population replacement gene drives designed to better withstand the evolution of resistance by homing through haplolethal loci. Because most mutations in the wild-type allele that would otherwise confer resistance are lethal, only successful drive homing permits the cell to survive. Here we outline the development and characterization of two ΦC31-Recombination mediated cassette exchange (RMCE) gene drive docking lines with these features in Anopheles gambiae, a first step towards construction of robust gene drives in this important malaria vector. We outline adaption of the technique HACK (Homology Assisted CRISPR knockin) to knock-in two docking site sequences into a paired haplolethal-haplosufficient (Ribosome-Proteasome) locus, and confirm that these docking lines permit insertion of drive-relevant transgenes. We report the first anopheline proteasome knockouts, and identify ribosome mutants that reveal a major hurdle that such designs must overcome to develop robust drives in the future. Although we do not achieve drive, this work provides a new tool for constructing future evolution-robust drive systems and reveals critical challenges that must be overcome for future development of gene drives designed to target haplolethal loci in anophelines and, potentially, other metazoans.

Mosquito control has successfully reduced the burden of malaria globally, but current vector control technologies cannot achieve malaria elimination. Precision guided sterile insect technique (pgSIT) is one of the most promising interventions being developed for malaria elimination. Mass release of genetically sterile males can act as a chemical-free species-specific insecticide. Before translating pgSIT from the bench to the field, however, it is essential to understand the potential costs and capabilities of this technology in a malaria-endemic region to determine if further investment into research and development of this technology is worthwhile. Therefore, we estimated the health outcomes and costs of a pgSIT program working jointly with current interventions to control the Anopheles gambiae malaria vector in the Upper River region of The Gambia. The pgSIT intervention in this region is predicted to prevent approximately 230 deaths and about 48,000 sick days per year. We have provided a range of costs that include risks associated with research and development, the facility, mass rearing efficiency, and distribution. This intervention should save disability-adjusted life years (DALY) at 11-94 USD per year and will prevent cases at 10-86 USD per infection. These estimates predict that pgSIT will cost 0.36-3.03 USD per person in the treated region annually. The cost per DALY shows life-saving at a cost comparable to current interventions in the Upper River region.

Controlling the principal African malaria vector, the mosquito Anopheles gambiae , is considered essential to curtail malaria transmission. However, existing vector control technologies rely on insecticides, which are becoming increasingly ineffective. Sterile insect technique (SIT) is a powerful suppression approach that has successfully eradicated a number of insect pests, yet the A. gambiae toolkit lacks the requisite technologies for its implementation. SIT relies on iterative mass releases of nonbiting, nondriving, sterile males which seek out and mate with monandrous wild females. Once mated, females are permanently sterilized due to mating-induced refractoriness, which results in population suppression of the subsequent generation. However, sterilization by traditional methods renders males unfit, making the creation of precise genetic sterilization methods imperative. Here, we introduce a vector control technology termed precision-guided sterile insect technique (pgSIT), in A. gambiae for inducible, programmed male sterilization and female elimination for wide-scale use in SIT campaigns. Using a binary CRISPR strategy, we cross separate engineered Cas9 and gRNA strains to disrupt male-fertility and female-essential genes, yielding >99.5% male sterility and >99.9% female lethality in hybrid progeny. We demonstrate that these genetically sterilized males have good longevity, are able to induce sustained population suppression in cage trials, and are predicted to eliminate wild A. gambiae populations using mathematical models, making them ideal candidates for release. This work provides a valuable addition to the malaria genetic biocontrol toolkit, enabling scalable SIT-like confinable, species-specific, and safe suppression in the species.

Rapid spread of insecticide resistance among anopheline mosquitoes threatens malaria elimination efforts, necessitating development of alternative vector control technologies. Sterile Insect Technique (SIT) has been successfully implemented in multiple insect pests to suppress field populations by the release of large numbers of sterile males, yet it has proven difficult to adapt to Anopheles vectors. Here we outline adaptation of a CRISPR-based genetic sterilization system to selectively ablate male sperm cells in the malaria mosquito Anopheles gambiae. We achieve robust mosaic biallelic mutagenesis of zero population growth (zpg, a gene essential for differentiation of germ cells) in F1 individuals after intercrossing a germline-expressing Cas9 transgenic line to a line expressing zpg-targeting gRNAs. Approximately 95% of mutagenized males display complete genetic sterilization, and cause similarly high levels of infertility in their female mates. Using a fluorescence reporter that allows detection of the germline leads to a 100% accurate selection of spermless males, improving the system. These males cause a striking reduction in mosquito population size when released at field-like frequencies in competition cages against wild type males. These findings demonstrate that such a genetic system could be adopted for SIT against important malaria vectors.

Precision-guided sterile insect technique (pgSIT) is an extremely promising vector control intervention that can reduce and potentially eliminate the unacceptable malaria burden, particularly in sub-Saharan Africa. The deployment of pgSIT shows the greatest promise and does not have a near peer competitor in the fight to eradicate malaria. Here we explore the cost effectiveness of using this approach in Africa using mathematical modeling and economical analysis. Overall, we find that pgSIT represents a cost-effective and promising approach to A. gambiae control in The Gambia, with the potential to deliver significant economic and social benefits.Summary Precision-guided sterile insect technique (pgSIT) is an extremely promising vector control intervention that can reduce and potentially eliminate the unacceptable malaria burden, particularly in sub-Saharan Africa. pgSIT is a safe, innovative, and highly targeted approach to mosquito control that combines the principles of the sterile insect technique (SIT) with advanced state-of-the-art technologies of genetic engineering ([Akbari et al. 2023][1]; M. [Li et al. 2021][2]; [Kandul, Liu, and Akbari 2021][3]; [Kandul et al. 2019][4], [2022][5]). The technique involves inundative releases of genetically sterile male mosquitoes into the environment to mate with their wild counterparts, sterilizing them in the process. After multiple releases, this method can suppress, and even eradicate pest populations without the use of chemical pesticides or other less specific agents. The use of pgSIT has the potential to be a lasting, safe, cost-effective, sustainable, and environmentally friendly method to suppress a target species, acting as a chemical-free, species-specific insecticide.Before this work is considered for field application, however, we need to have a robust, data-driven modeling framework that will accurately predict the outcome of pgSIT release scenarios for malaria control, and we need an assessment of the costs and health benefits of implementing this technology in a region in Africa. This cost assessment evaluates the economic feasibility of both capacity building and establishing the infrastructure for a pgSIT facility in The Gambia to control the deadly malaria mosquito vector Anopheles gambiae . We focused on the Upper River region (URR) for three key reasons: (1) this area is known to have the highest per capita malaria rates in The Gambia (2) this region has more comprehensive historical and current data on malaria incidence and prevalence, malaria-associated healthcare and prevention costs, and human demographic data and (3) its location could feasibly demonstrate that a single pgSIT production facility can support phased suppression on a regional scale and then upon local extinction be repurposed to support vector suppression on a country or continent-wide scale.The pgSIT treatment of the URR (∼2069 km2) is predicted to prevent approximately 230 deaths and about 48,000 sick days per yea r. This estimate is based on a model for localized extinction of A. gambiae , reaching full epidemiological impact by the third year. There are multiple ways to calculate the value of this intervention monetarily, from the value of statistical life (VSL) to quality adjusted life years (QALY), which ranges from 367 million to 880 million USD saved in the first ten years of the facility being active. Other metrics such as willingness-to-pay (WTP), estimates the willingness of locals to contribute to malaria prevention financially, and estimates based on gross domestic product ( GDP) growth predict this model to save either 53 million or 551 million USD , respectively, in the first ten years of intervention. This model assumes localized extinction of A. gambiae by the second year of intervention with repeated releases to maintain extinction despite seasonal mosquito migration from beyond the treatment area. Localized extinction, however, is expected to have a year-to-year suppression effect making it easier to suppress mosquito populations with reduced sterile male releases in subsequent years. It is, therefore, likely an underestimate of the costs and benefits of pgSIT sterile male releases. In later years, the release of sterile males from this facility could be redirected to new areas to expand the suppression region. Additionally, this facility would have a significant off-season where the facility is not producing sterile male A. gambiae . This off-season could, therefore, produce sterile males to suppress A. gambiae populations in other regions with seasonal malaria during the off-season in The Gambiae. It could even be used to mass produce pgSIT sterile males for other mosquito species, such as the dengue vector, Aedes aegypti , which have eggs that can be stored for many months and consequently can be stockpiled to aid in the suppression of dengue outbreaks, which are also common in the area. Initially, however, the off-season can be used to build local capacity for genetically engineered (GE) mosquitos, which would consist of training staff, optimizing procedures, and troubleshooting any issues that arose during the higher production phases.The costs for this approach will vary as there are some unknowns and variability in the expected efficiencies of the facility, equipment, and procedures. In particular, mosquito survival rates and fecundity may vary more widely at scale and selection of the rearing equipment and protocols, and the mosquito sorting technology will dictate production levels and procedures. This variability is factored into the cost, and, therefore, a range of technologies and costs are considered. The expected start-up costs range from 6 to 11.5 million USD , which includes all necessary development, field trials, equipment, facility construction, staffing, and other establishment costs. The major upfront costs vary by facility size and capital equipment, much of which is dependent on mosquito rearing efficiencies. The annual estimated operational costs range from approximately 315,000 USD, depending on equipment selection and the size of the facility. Annually, this intervention costs less than 1 USD per person to suppress mosquitoes in the URR and prevent malaria transmission, which is about 6% of a malaria prevention WTP based on current interventions or 0.3-11% when using the VSL or QALY metrics, with most variation from the VSL calculation method. This intervention costs 15 to 124 USD (2022) to save one life-year and prevents malaria infections at 13 to 113 USD per case prevented, making this method competitive with many current interventions. WTP demonstrates that this facility could be made locally sustainable long term by local funding.Overall, the estimates of the capital and operational costs associated with the production of pgSIT sterile males, and the construction and management of the production facility indicate the cost savings associated with the annual decrease in morbidity and mortality (value of life) resulting from the use of pgSIT are significantly higher than the implementation costs. These estimates suggest that pgSIT represents a cost-effective and promising approach to A. gambiae control in The Gambia, with the potential to deliver significant economic and social benefits.### Competing Interest StatementThis work was supported by funding from an Open Philanthropy award (309937-0001). The views, opinions, and/or findings expressed are those of the authors and should not be interpreted as representing the official views or policies of the U.S. government. Figures were created using www.BioRender.com.* ABS : acrylonitrile butadiene styrene ACT : artemisinin-based combination treatment AI : artificial intelligence β 2-tubulin : beta-2 tubulin Cas9 : CRISPR associated protein 9 CHIRPS : Climate Hazards Group InfraRed Precipitation with Station COPAS Complex Object Parametric Analyzer and Sorter CRISPR : clustered regularly interspaced short palindromic repeats DNA : deoxyribonucleic acid dsx : doublesex GBP : British pound GDP : gross domestic product GE : genetically engineered GMD : Gambian dalasi HDR : homology directed repair fle femaleless : a female essential gene gRNA : guide RNA IAEA : International Atomic Energy Agency Ifegenia : inherited female elimination by genetically encoded nucleases to interrupt alleles ICL : Imperial College London IPTp : intermittent preventive treatment IRR : internal rate of return IRS : indoor residual spraying L1 : first instar larvae LLIN : long last insecticidal nets LSHTM : London School of Hygiene and Tropical Medicine MGDrivE Mosquito Gene Drive Explorer MRC : Medical Research Council NPV : net present value pgSIT : precision guided sterile insect technique pgSIT 2.0 : pgSIT incorporating SEPARATOR technology to support fluorescent sex specific sorting of the pgSIT lines PPP : purchasing power parity RIDL : Release of Insects carrying a Dominant Lethal RNA : ribonucleic acid ROI : return on investment SEPARATOR : Sexing Element Produced by Alternative RNA-splicing of A Transgenic Observable Reporter SIT : sterile insect technique SMC : seasonal malaria chemoprevention QALY : quality adjusted life years DALY : disability adjusted life years URR : Upper River region USD : United States dollar USTD : United States Transportation Department VSL : value of statistical life VTOL : vertical take-off and landing WHO : World Health Organization WTP : willingness to pay zpg : zero population growth [1]: #ref-3 [2]: #ref-60 [3]: #ref-52 [4]: #ref-54 [5]: #ref-53

Malaria is among the world’s deadliest diseases, predominantly affecting Sub-Saharan Africa and killing over half a million people annually. Controlling the principal vector, the mosquito Anopheles gambiae , as well as other anophelines, is among the most effective methods to control disease spread. Here, we develop a genetic population suppression system termed Ifegenia (inherited female elimination by genetically encoded nucleases to interrupt alleles) in this deadly vector. In this bicomponent CRISPR-based approach, we disrupt a female-essential gene, femaleless ( fle ), demonstrating complete genetic sexing via heritable daughter gynecide. Moreover, we demonstrate that Ifegenia males remain reproductively viable and can load both fle mutations and CRISPR machinery to induce fle mutations in subsequent generations, resulting in sustained population suppression. Through modeling, we demonstrate that iterative releases of nonbiting Ifegenia males can act as an effective, confinable, controllable, and safe population suppression and elimination system.

ABSTRACT Rapid spread of insecticide resistance among anopheline mosquitoes threatens malaria elimination efforts, necessitating development of alternative vector control technologies. Sterile Insect Technique (SIT) has been successfully implemented in multiple insect pests to suppress field populations by the release of large numbers of sterile males, yet it has proven difficult to adapt to Anopheles vectors. Here we outline adaptation of a CRISPR-based genetic sterilization system to selectively ablate male sperm cells in the malaria mosquito Anopheles gambiae . We achieve robust mosaic biallelic mutagenesis of z ero population growth ( zpg , a gene essential for differentiation of germ cells) in F1 individuals after intercrossing a germline-expressing Cas9 transgenic line to a line expressing zpg -targeting gRNAs. Approximately 95% of mutagenized males display complete genetic sterilization, and cause similarly high levels of infertility in their female mates. Using a fluorescence reporter that allows detection of the germline leads to a 100% accurate selection of spermless males, improving the system. These males cause a striking reduction in mosquito population size when released at field-like frequencies in competition cages against wild type males. These findings demonstrate that such a genetic system could be adopted for SIT against important malaria vectors.

The development of an extensive toolkit for potential point-of-care diagnostics that is expeditiously adaptable to new emerging pathogens is of critical public health importance. Recently, a number of novel CRISPR-based diagnostics have been developed to detect SARS-CoV-2. Herein, we outline the development of an alternative CRISPR nucleic acid diagnostic utilizing a Cas13d ribonuclease derived from Ruminococcus flavefaciens XPD3002 (CasRx) to detect SARS-CoV-2, an approach we term SENSR (sensitive enzymatic nucleic acid sequence reporter) that can detect attomolar concentrations of SARS-CoV-2. We demonstrate 100% sensitivity in patient-derived samples by lateral flow and fluorescence readout with a detection limit of 45 copy/μL. This technology expands the available nucleic acid diagnostic toolkit, which can be adapted to combat future pandemics.

Mosquitoes transmit pathogens that kill >700,000 people annually. These insects use body heat to locate and feed on warm-blooded hosts, but the molecular basis of such behavior is unknown. Here, we identify ionotropic receptor IR21a, a receptor conserved throughout insects, as a key mediator of heat seeking in the malaria vector Anopheles gambiae Although Ir21a mediates heat avoidance in Drosophila, we find it drives heat seeking and heat-stimulated blood feeding in Anopheles At a cellular level, Ir21a is essential for the detection of cooling, suggesting that during evolution mosquito heat seeking relied on cooling-mediated repulsion. Our data indicate that the evolution of blood feeding in Anopheles involves repurposing an ancestral thermoreceptor from non-blood-feeding Diptera.

Since its first emergence from China in late 2019, the SARS-CoV-2 virus has spread globally despite unprecedented containment efforts, resulting in a catastrophic worldwide pandemic. Successful identification and isolation of infected individuals can drastically curtail virus spread and limit outbreaks. However, during the early stages of global transmission, point-of-care diagnostics were largely unavailable and continue to remain difficult to procure, greatly inhibiting public health efforts to mitigate spread. Furthermore, the most prevalent testing kits rely on reagent- and time-intensive protocols to detect viral RNA, preventing rapid and cost-effective diagnosis. Therefore the development of an extensive toolkit for point-of-care diagnostics that is expeditiously adaptable to new emerging pathogens is of critical public health importance. Recently, a number of novel CRISPR-based diagnostics have been developed to detect COVID-19. Herein, we outline the development of a CRISPR-based nucleic acid molecular diagnostic utilizing a Cas13d ribonuclease derived from Ruminococcus flavefaciens (CasRx) to detect SARS-CoV-2, an approach we term SENSR (Sensitive Enzymatic Nucleic-acid Sequence Reporter). We demonstrate SENSR robustly detects SARS-CoV-2 sequences in both synthetic and patient-derived samples by lateral flow and fluorescence, thus expanding the available point-of-care diagnostics to combat current and future pandemics.

Transmission of malaria parasites occurs when a female Anopheles mosquito feeds on an infected host to acquire nutrients for egg development. How parasites are affected by oogenetic processes, principally orchestrated by the steroid hormone 20-hydroxyecdysone (20E), remains largely unknown. Here we show that Plasmodium falciparum development is intimately but not competitively linked to processes shaping Anopheles gambiae reproduction. We unveil a 20E-mediated positive correlation between egg and oocyst numbers; impairing oogenesis by multiple 20E manipulations decreases parasite intensities. These manipulations, however, accelerate Plasmodium growth rates, allowing sporozoites to become infectious sooner. Parasites exploit mosquito lipids for faster growth, but they do so without further affecting egg development. These results suggest that P. falciparum has adopted a non-competitive evolutionary strategy of resource exploitation to optimize transmission while minimizing fitness costs to its mosquito vector. Our findings have profound implications for currently proposed control strategies aimed at suppressing mosquito populations.

If they are able to spread in wild populations, CRISPR-based gene-drive elements would provide new ways to address ecological problems by altering the traits of wild organisms, but the potential for uncontrolled spread tremendously complicates ethical development and use. Here, we detail a self-exhausting form of CRISPR-based drive system comprising genetic elements arranged in a daisy chain such that each drives the next. “Daisy-drive” systems can locally duplicate any effect achievable by using an equivalent self-propagating drive system, but their capacity to spread is limited by the successive loss of nondriving elements from one end of the chain. Releasing daisy-drive organisms constituting a small fraction of the local wild population can drive a useful genetic element nearly to local fixation for a wide range of fitness parameters without self-propagating spread. We additionally report numerous highly active guide RNA sequences sharing minimal homology that may enable evolutionarily stable daisy drive as well as self-propagating CRISPR-based gene drive. Especially when combined with threshold dependence, daisy drives could simplify decision-making and promote ethical use by enabling local communities to decide whether, when, and how to alter local ecosystems.

When scientists alter the genome of an organism, we typically reduce its ability to reproduce in the wild. This limitation has prevented researchers from rendering wild insects unable to spread disease, programing pests to ignore our crops, using genetics to precisely remove environmentally damaging invasive species, and much more. Gene drive occurs when a vertically transmitted genetic element reliably spreads through a population over generations despite providing no reproductive advantage to each host organism. Until recently, scientific efforts to take advantage of this natural phenomenon achieved only limited success. The advent of CRISPR genome editing has dramatically accelerated efforts to harness gene drive. Small groups of scientists may now be capable of unilaterally altering entire wild populations, and through them, the shared environment. Determining whether, when, and how to develop gene drive interventions responsibly will be a defining challenge of our time. Here we describe capabilities, safeguards, applications, and opportunities relevant to gene drive technologies.

BACKGROUND:Malaria parasites, transmitted by the bite of an anopheline mosquito, pose an immense public health burden on many tropical and subtropical regions. The most important malaria vectors in sub-Saharan Africa are mosquitoes of the Anopheles gambiae complex including An. gambiae (sensu stricto). Given the increasing rates of insecticide resistance in these mosquitoes, alternative control strategies based on the release of genetically modified males are being evaluated to stop transmission by these disease vectors. These strategies rely on the mating competitiveness of release males, however currently there is no method to determine male mating success without sacrificing the female. Interestingly, unlike other insects, during mating An. gambiae males transfer their male accessory glands (MAGs) seminal secretions as a coagulated mating plug which is deposited in the female atrium.RESULTS:Here we exploit this male reproductive feature and validate the use of a MAG-specific promoter to fluorescently label the mating plug and visualize the occurrence of insemination in vivo. We used the promoter region of the major mating plug protein, Plugin, to control the expression of a Plugin-tdTomato (PluTo) fusion protein, hypothesizing that this fusion protein could be incorporated into the plug for sexual transfer to the female. Anopheles gambiae PluTo transgenic males showed strong red fluorescence specifically in the MAGs and with a pattern closely matching endogenous Plugin expression. Moreover, the fusion protein was integrated into the mating plug and transferred to the female atrium during mating where it could be visualized microscopically in vivo without sacrificing the female. PluTo males were equally as competitive at mating as wild type males, and females mated to these males did not show any reduction in reproductive fitness.CONCLUSION:The validation of the first MAG-specific promoter in transgenic An. gambiae facilitates the live detection of successful insemination hours after copulation has occurred. This provides a valuable tool for the assessment of male mating competitiveness not only in laboratory experiments but also in semi-field and field studies aimed at testing the feasibility of releasing genetically modified mosquitoes for disease control.