The genetic control method, which is environmentally friendly and species-specific, has effectively reduced or eliminated pests in many areas. One essential requirement to control a species is the identification of its genetic and molecular elements. Such elements, however, are rarely available in Zeugodacus cucurbitae, a very destructive insect pest worldwide.

In this study, we knocked out the white pupae (wp) gene in Z. cucurbitae and generated a wp(-) strain, which has a white pupae phenotype. The white puparium color was successfully restored to brown by inserting the wp gene rescue allele into the genome of the wp(-) strain using piggyBac transgenic technology. The potential wp promoter was then truncated to drive the expression of the wp gene and the puparium color was rescued even by the 605 bp sequence upstream of its transcription initiation site. Further fertility tests showed that knocking out or rescuing the wp gene had no effect on the reproduction of adult flies. In addition, we identified six U6 promoters and tested their promoter activities in the embryos of Z. cucurbitae. The ZcU6-2 and ZcU6-1 promoters exhibited significantly higher activity than the others and are suitable for use in CRISPR technology-based genetic control methods.

Our work first shows the success of applying piggyBac transgenic technology in Z. cucurbitae. Our results demonstrate a highly efficient transgenic screening marker by puparium color and the promoter activity of multiple ZcU6 promoters, facilitating the construction of transgenic strains that are used for genetic control of tephritid species. © 2025 Society of Chemical Industry.

Immature anophelines inhabit aquatic environments with diverse physicochemical properties and microorganisms. In insectary settings, ammonia accumulation in larval rearing trays can lead to high larval mortality. Bacterial communities in these trays may influence ammonia levels through nitrification and denitrification. While symbiotic bacteria are known to be crucial for nutrition, digestion, reproduction, and immune responses in anophelines, the microbial communities specifically associated with Anopheles coluzzii larvae have not been characterised.

Building on a study examining ammonia-capturing zeolite and water changes for rearing Anopheles coluzzii, this research characterised the bacterial communities using 16S rRNA gene sequencing to identify species linked to larval survival and phenotypic quality. Functional filters were applied to identify bacteria related to ammonia nitrification and their impact on larval development. qPCR was used to validate the sequencing data for the 10 most significant bacteria. Water changes significantly reduced bacterial diversity and abundance, improving adult mosquito development and quality. In contrast, untreated trays showed a higher abundance of potentially harmful bacteria, adversely affecting development. Applying zeolite increased nitrifying bacteria presence, benefiting mosquito growth while lowering toxic bacteria levels-trends confirmed by qPCR.

This study offers insights into the bacterial communities in mosquito larval-rearing water, highlighting species that could enhance ammonia nitrification and overall rearing success.

Gene drives are genetic elements with positively biased transmission and may be useful tools to suppress mammalian pests that threaten biodiversity worldwide. While gene drives are progressing in mice, less is known about their potential for invasive rat control. A recent report has provided the first data on germline gene conversion in rats, demonstrating that modest homing rates (up to 67%) can be achieved in females. Here, we apply these empirically derived values to investigate the potential of various gene drive strategies to suppress an island population of 200,000 rats, using our stochastic, spatially explicit, individual-based modelling framework. Standard homing drives embedded in haplosufficient fertility or viability genes failed to eradicate, but achieved permanent population suppression. In contrast, a two-target design with a homing rescue (HR) drive embedded in a haplolethal gene that also targets an independent fertility or viability gene demonstrated considerable suppression potential. Remarkably, an HR drive targeting a haplosufficient female fertility gene showed robust eradication even at the relatively low homing rates previously demonstrated in rats. Interestingly, homing rate had a relatively low influence on eradication probability while cutting efficiency at the haplolethal gene was critical. Further, as long as the latter was similar to the cutting and subsequent knockout of the unlinked female fertility gene, then eradication could be achieved across a range of homing rates. Together, these results suggest that modest homing rates, such as have been demonstrated in rats and other species, can potentially be leveraged for population suppression, offering new opportunities for gene drive development.

Gene drive technology presents a promising approach to controlling malaria vector populations. Suppression drives are intended to disrupt essential mosquito genes whereas modification drives aim to reduce the individual vectorial capacity of mosquitoes. Here we present a highly efficient homing gene drive in the African malaria vector Anopheles gambiae that targets the microRNA gene mir-184 and combines suppression with modification. Homozygous gene drive (miR-184D) individuals incur significant fitness costs, including high mortality following a blood meal, that curtail their propensity for malaria transmission. We attribute this to a role of miR-184 in regulating solute transport in the mosquito gut. However, females remain fully fertile, and pure-breeding miR-184D populations suitable for large-scale releases can be reared under laboratory conditions. Cage invasion experiments show that miR-184D can spread to fixation thereby reducing population fitness, while being able to propagate a separate antimalarial effector gene at the same time. Modelling indicates that the miR-184D drive integrates aspects of population suppression and population replacement strategies into a candidate strain that should be evaluated further as a tool for malaria eradication.

Anopheles stephensi, a major Asian malaria vector, is invading Africa and has been implicated in recent outbreaks of urban malaria. Control of this species is key to eliminating malaria in Africa. Genetic control strategies, and CRISPR/Cas9-based gene drives are emerging as promising species-specific, environmentally friendly, scalable, affordable methods for pest control. To implement these strategies, a key parameter to optimize for high efficiency is the spatiotemporal control of Cas9 and the gRNA. Here, we assessed the ability of four RNA Pol III promoters to bias the inheritance of a gene drive element inserted into the cd gene of An. stephensi. We determined the homing efficiency and examined eye phenotype as a proxy for non-homologous end joining (NHEJ) events in somatic tissue. We found all four promoters to be active, with mean inheritance rates up to 99.8%. We found a strong effect of the Cas9-bearing grandparent (grandparent genotype), likely due to maternally deposited Cas9.

The increasing burden of Aedes aegypti-borne diseases, particularly dengue, is a growing global concern, further exacerbated by climate change. Current control strategies have proven insufficient, necessitating novel approaches. Synthetic homing endonuclease gene (sHEG) drives represent one of the few emerging technologies with the potential to offer a cost-effective and equitable solution to this escalating public health challenge. However, despite multiple attempts, the homing efficiencies of Ae. aegypti sHEG systems lag behind those achieved in Anopheles mosquitoes. We discuss key insights from efforts to develop sHEGs in Ae. aegypti and highlight critical factors that may unlock further advances in this species.

Sterile insect technique (SIT) has become a key component of efficient pest control. Fruit fly pests from the Drosophilidae and Tephritidae families pose a substantial and overwhelmingly increasing threat to the agricultural industry, aggravated by climate change and globalization among other contributors. In this review, we discuss the advances in genetic engineering aimed to improve the SIT-mediated fruit fly pest control. This includes SIT enhancement strategies such as novel genetic sexing strain and female lethality approaches. Self-pervasive X-shredding and X-poisoning sex distorters, alongside gene drive varieties are also reviewed. The self-limiting precision-guided SIT, which aims to tackle female removal and male fertility via CRISPR/Cas9, is additionally introduced. By using examples of existing genetic tools in the fruit fly pests of interest, as well as model species, we illustrate that the population control intensity may be modulated depending on strategy selection.

Global health security threats in the post-coronavirus disease 2019 era include dense urban populations, increased human-animal proximity, migration driven by political or economic instability, climate change, humanitarian crises, antimicrobial resistance (AMR), and the misuse of biological research-including the accidental or intentional release of high-risk pathogens. The foundational preparation for these threats is to establish a robust, resilient public health system based on universal health coverage. The World Health Organization's International Health Regulations must continue to promote global solidarity by maintaining core capacities such as surveillance, national laboratories, and epidemiological investigations of emerging infectious diseases, with timely reporting and information sharing within the global health security community. A One Health approach is essential for addressing AMR. Infection prevention and control must be enhanced to reduce healthcare-associated infections in medical facilities. Additionally, regulations concerning biosafety and biosecurity should address dual-use research of concern as well as the accidental or intentional release of highrisk pathogens from laboratories. Global health security is a collective responsibility because these threats know no borders and require coordinated action.

The β-tubulin gene is essential for reproductive development, especially for male fertility, in different insects including Bombyx mori and Drosophila melanogaster. Targeting reproductive genes such as β-tubulin offers a promising approach to pest control that is more sustainable than chemical pesticides. However, there is limited research on the functional role of β-tubulin in Plutella xylostella, a highly damaging pest of vegetable crops. In the present study, we first identified and cloned the β-tubulin gene in P. xylostella (Pxβtubulin-1). Pxβtubulin-1 protein contains two conserved domains of Tubulin and Tubulin-C, and β-tubulin were conserved in the Lepidoptera. Spatiotemporal expression analysis revealed that Pxβtubulin-1 was highly expressed in male pupae, adult males, and testes, suggesting its testis-specific function. Using CRISPR/Cas9 technology, we generated two homozygous Pxβtubulin-1 mutant strains of P. xylostella. Mutant strains exhibited significantly lower egg production and hatching rates compared with the wild type. Dissection and measurement of reproductive organs revealed that the testes and bursa copulatrix in mutant strains were significantly reduced in size compared with the wild type. In conclusion, Pxβtubulin-1 is vital for male fertility as it influences the development of reproductive organs and can be a potential target for the control of P. xylostella.

The Plasmodium infection cycle in mosquitoes relies on numerous host factors in the vector midgut, which can be targeted with therapeutics. The mosquito prefoldin complex is needed to fold proteins and macromolecular complexes properly. Here we show that the conserved Anopheles mosquito prefoldin (PFDN)-chaperonin system is a potent transmission-blocking target for multiple Plasmodium species. Silencing any prefoldin subunit or its CCT/TRiC partner via RNA interference reduces Plasmodium falciparum oocyst loads in the mosquito midgut, as does co-feeding mosquitoes with PFDN6-specific antibody and gametocytes. Inhibition of the PFDN-CCT/TRiC chaperonin complex results in the loss of epithelial and extracellular matrix integrity, which triggers microorganism-mediated anti-Plasmodium immune priming and compromises the parasite's laminin-based immune evasion. Mouse malaria transmission-blocking vaccine and antibody co-feeding assays support its potential as a multispecies transmission-blocking target for P. falciparum and Plasmodium vivax. Further study is needed to determine the potential of this system as a transmission-blocking vaccine target.

Despite the application of control measures, mosquito-borne diseases continue to pose a serious threat to human health. In this context, exploiting Wolbachia, a common symbiotic bacterium in insects, may offer effective solutions to suppress vectors or reduce their competence in transmitting several arboviruses. Many Wolbachia strains can induce conditional egg sterility, known as cytoplasmic incompatibility (CI), when infected males mate with females that do not harbor the same Wolbachia infection. Infected males can be mass-reared and then released to compete with wild males, reducing the likelihood of wild females encountering a fertile mate. Furthermore, certain Wolbachia strains can reduce the competence of mosquitoes to transmit several RNA viruses. Through CI, Wolbachia-infected individuals can spread within the population, leading to an increased frequency of mosquitoes with a reduced ability to transmit pathogens. Using artificial methods, Wolbachia can be horizontally transferred between species, allowing the establishment of various laboratory lines of mosquito vector species that, without any additional treatment, can produce sterilizing males or females with reduced vector competence, which can be used subsequently to replace wild populations. This manuscript reviews the current knowledge in this field, describing the different approaches and evaluating their efficacy, safety, and sustainability. Successes, challenges, and future perspectives are discussed in the context of the current spread of several arboviral diseases, the rise of insecticide resistance in mosquito populations, and the impact of climate change. In this context, we explore the necessity of coordinating efforts among all stakeholders to maximize disease control. We discuss how the involvement of diverse expertise-ranging from new biotechnologies to mechanistic modeling of eco-epidemiological interactions between hosts, vectors, Wolbachia, and pathogens-becomes increasingly crucial. This coordination is especially important in light of the added complexity introduced by Wolbachia and the ongoing challenges posed by global change.

Genetic control strategies are promising solutions for control of pest populations and invasive species. Methods utilizing repeated releases of males such as sterile insect technique (SIT), release of insects carrying a dominant lethal (RIDL), self-limiting gene drives, and gene disruptors are highly controllable methods, ensuring biosafety. Although models of these strategies have been built, detailed comparisons are lacking, particularly for some of the newer strategies. Here, we conducted a thorough comparative assessment of self-limiting genetic control strategies by individual-based simulation models. Specifically, we find that repeated releases greatly enhance suppression power of weak and self-limiting gene drives, enabling population elimination with even low efficiency and high fitness costs. Moreover, dominant female sterility further strengthens self-limiting systems that can either use gene drive or disruptors that target genes without a mechanism to bias their own inheritance. Some of these strategies are highly persistent, resulting in relatively low release ratios even when released males suffer high fitness costs. To quantitatively evaluate different strategies independent from ecological impact, we proposed constant-population genetic load, which achieves over 95% accuracy in predicting simulation outcomes for most strategies, though it is not as precise in a few frequency-dependent systems. Our results suggest that many new self-limiting strategies are safe, flexible, and more cost-effective than traditional SIT and RIDL, and thus have great potential for population suppression of insects and other pests.

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.

Neglected tropical diseases (NTDs) include more than a dozen of diseases which despite their fatality receive less attention from the research community worldwide. High cost diagnosis of these diseases and lack of trained community which can accurately interpret them is the major drawback in the healthcare system. Nowadays, in the genetic engineering era more emphasis is given to the modern gene editing tools such as Transcription Activator-Like Effector Nucleases (TALENS), Zinc Finger Nucleases (ZFNs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) due to their unique tailoring molecular machinery. This review article details the applicability of CRISPR/Cas9 as a modern gene editing tool in case of NTD parasites such as trypanosomatids with an aim to target their virulent genes. It has been observed through a number of studies that knocking in/out virulent genes of these parasites have led to a significant decrease in infectivity, growth rates along with morphological defects. The article also mentions various advanced CRISPR/Cas based diagnostics such as Specific High-Sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) and SHERLOCK4HAT which can detect parasite concentration as low as 2 attomolar/L (aM: 10- 18) and 1 parasite/µL respectively. This review also enlists various regulatory and biosafety issues, for example ecological imbalance which can arise as a consequence of CRISPR/Cas based gene drives employed to target parasitic vectors. Despite its wide applications, CRISPR/Cas is associated with several limitations like off-target effects and ecological imbalance to name a few.

Anopheles stephensi is a competent malaria vector mainly present in southern Asia and the Arabian Peninsula. Since 2012, it has invaded several countries of eastern Africa, creating an emerging risk of urban transmission. Urgent efforts are required to develop novel and more efficient strategies for targeted vector control. CRISPR/Cas9-based homing gene drives have been proposed as attractive alternative strategies. Gene drives have the potential to spread a desired trait through a population at higher rates than via normal Mendelian inheritance, even in the presence of a fitness cost. Several target genes have been suggested and tested in different mosquito vector species such as Anopheles gambiae and Aedes aegypti. Several promising suppression drives have been developed in An. gambiae that target the sex determination gene doublesex (dsx).

In this study, a geographically confineable gene drive system targeting dsx was developed (dsxgRNA). Here, a transgenic line which expresses Cas9 under the control of the endogenous zpg promoter was generated. Separately a transgenic line which expresses a gRNA targeting the female specific exon of dsx was inserted into that same target site. The reproductive fitness of males and females heterozygous and homozygous for this element was determined. A series of experimental crosses was performed to combine the two elements and assess the homing rate of the dsx element in a split drive system.

The drive was able to home in a super-Mendelian rate comparable to those obtained by an autonomous drive in this species. Although inheritance rates as high as 99.8% were observed, potentially providing very potent gene drive, dominant effects on male and female fertility were observed, which would be sufficient to hinder spread of such a drive. Molecular analysis indicated that the gRNA expressing insertion disrupted normal splicing of dsx.

These results should be considered when proposing the viability of dsx as a target gene for a population suppression gene drives in Anopheles stephensi. Although high homing rates were observed, the fitness defects found in both males and females carrying the transgene would likely prohibit this drive from functioning in the field.

Arthropod melanization is a crucial defense mechanism mediated by a complex cascade of CLIP domain serine proteases (CLIPs). In this study, it was confirmed that microRNA-11903a (miR-11903a) targets Aedes-CLIPB9 (AeCLIPB9) by bioinformatics prediction and dual-luciferase reporter assays. Following intrathoracic injection of miR-11903a agomir and antagomir, Real-time quantitative polymerase chain reaction confirmed that AeCLIPB9 is negatively regulated by miR-11903a. Spatiotemporal expression analysis revealed that miR-11903a is most abundant in 4th instar larvae, followed by pupae and adults, and highly expressed in the wings, head, and midgut of female adults. Following pathogen infection, AeCLIPB9 and miR-11903a exhibited opposite expression trends, indicating their potential roles in mosquito innate immunity. To further investigate the relationship between AeCLIPB9 and miR-11903a, double-strand CLIPB9 was synthesized and RNA interference was performed. Seven-d survival assays revealed that both AeCLIPB9 and miR-11903a were crucial immune factors in fighting pathogens. Finally, longevity assays demonstrated that miR-11903a influenced mosquito lifespan.

At present, the application of CRISPR/Cas9 technology for genetic manipulation in insects is predominantly concentrated on Diptera model species, including Drosophila and mosquitoes. In contrast, non-model insects such as the brown planthoppers (BPH, Nilaparvata lugens), a major insect pest of rice, have received less attention in genetic manipulation due to insufficient tools. Here, the analysis of spatiotemporal expression patterns revealed that β2-tubulin in BPH (NlB2t) was predominantly concentrated in male adults and male testis, exhibiting high expression levels. Knockdown of NlB2t expression by using RNAi resulted in the obstruction of male testis development. Mating between the RNAi-treated males and wild-type females led to a notable reduction in the number of eggs laid and the hatching rate of those eggs by 58.2% and 50.6%, respectively. The longevity of RNAi males significantly increased, and females that had previously mated with RNAi males exhibited a diminished inclination for re-mating with wild-type males. The dual-luciferase reporter assay demonstrated robust promoter activity in the upstream 943 bp of NlB2t, capable of driving Cas9 protein expression in vivo and effectively inducing target gene knockout. These findings elucidated that NlB2t may be a key gene in BPH male testis development and reproduction, as a promising target for sterilization. Its upstream promoter serves as a germline promoter, significantly facilitating the development of genetic control tools based on CRISPR/Cas9 technology in BPH.

Insecticides play a crucial role as the primary means of controlling agricultural pests, preventing significant damage to crops. However, the misuse of these insecticides has led to the development of resistance in insect pests against major classes of these chemicals. The emergence of resistance poses a serious threat, especially when alternative options for crop protection are limited for farmers. Addressing this challenge and developing new, effective, and sustainable pest management approaches is not merely essential but also critically important. In the absence of alternative solutions, understanding the root causes behind the development of resistance in insects becomes a critical necessity. Without this understanding, the formulation of effective approaches to combat resistance remains elusive. With insecticides playing a vital role in global food security and public health, understanding and mitigating resistance are paramount. Given the growing concern over insect resistance to insecticides, this review addresses a crucial research gap by thoroughly examining the causes, mechanisms, and potential solutions. The review examines factors driving resistance, such as evolutionary pressure and excessive pesticide use, and provides a detailed analysis of mechanisms, including detoxifying enzyme overproduction and target site mutations. Providing an analysis of potential solutions, it discusses integrated pest management, strategic insecticide rotation, and the use of new pest control technologies and biological agents. Emphasizing the urgency of a multifaceted approach, the review provides a concise roadmap for sustainable pest management, guiding future research and applications.

Gene drives are alleles that can bias the inheritance of specific traits in target populations for the purpose of modification or suppression. Here, we construct a homing suppression drive in the major urban malaria vector Anopheles stephensi targeting the female-specific exon of doublesex, incorporating two gRNAs and a nanos-Cas9 to reduce functional resistance and improve female heterozygote fitness. Our results show that the drive was recessive sterile in both females and males, with various intersex phenotypes in drive homozygotes. Both male and female drive heterozygotes show only moderate drive conversion, indicating that the nanos promoter has lower activity in A. stephensi than in Anopheles gambiae. By amplicon sequencing, we detect a very low level of resistance allele formation. Combination of the homing suppression drive and a vasa-Cas9 line boosts the drive conversion rate of the homing drive to 100%, suggesting the use of similar systems for population suppression in a continuous release strategy with a lower release rate than SIT or fsRIDL techniques. This study contributes valuable insights to the development of more efficient and environmentally friendly pest control tools aimed at disrupting disease transmission.

The convoluted relationships between plants, viruses, and arthropod vectors housing bacterial endosymbionts are pivotal in the spread of harmful plant viral diseases. Endosymbionts play key roles in: manipulating host responses, influencing insect resistance to pesticides, shaping insect evolution, and bolstering virus acquisition, retention, and transmission. This interplay presents an innovative approach for developing sustainable strategies to manage plant diseases. Recent progress in targeting specific endosymbionts through genetic modifications, biotechnological advancements, and RNA interference shows potential for curbing viral spread and disease progression. Additionally, employing synthetic biology techniques like CRISPR/Cas9 to engineer endosymbionts and disrupt crucial interactions necessary for viral transmission in arthropod vectors holds promise for effective control measures. In this review, these obligate and facultative bacterial cruxes have been discussed to elaborate on their mechanistic involvement in the regulation and/or inhibition of tripartite pathways of interactions. Furthermore, we provide an in-depth understanding of endosymbionts' synergistic and antagonistic effects on: insect biology, plant immunity, and virus acquisition and transmission. Finally, we point out open questions for future research and provide research directions concerning the deployment of genetically engineered symbionts to affect plant-virus-vector interactions for sustainable disease management. By addressing existing knowledge gaps and charting future research paths, a deeper comprehension of the role of endosymbionts in plant-virus-vector interactions can pave the way for innovative and successful disease management strategies. The exploration of antiviral therapies, paratransgenesis, and pathogen-blocking tactics using engineered endosymbionts introduces pioneering solutions for lessening the impact of plant viral diseases and green pest management.

Malaria remains a significant public health challenge, particularly in low- and middle-income countries, despite ongoing efforts to eradicate the disease. Recent advancements, including the rollout of malaria vaccines, such as RTS,S/AS01 and R21/Matrix-M™, offer new avenues for prevention. However, the rise of resistance to anti-malarial medications necessitates innovative strategies. This review explores the potential integration of CRISPR/Cas9 gene drive technology with malaria vaccination efforts to enhance vector control and reduce transmission. By employing gene drive mechanisms for population suppression and replacement of malaria-transmitting Anopheles mosquitoes, combined with the immunogenic properties of vaccines, a synergistic approach can be established. This paper discussed the need for integrated strategies to address the biological complexities of malaria and socio-economic factors influencing its prevalence. Challenges such as regulatory hurdles, community acceptance, ecological impacts, and sustainable funding are examined, alongside strategies for implementation within existing malaria control programmes. This integrated approach could significantly contribute to achieving the World Health Organization's targets for malaria reduction by 2030, ultimately enhancing public health outcomes and supporting broader socio-economic development.

Vitellogenin (Vg), a yolk protein precursor, plays an important role in the oocyte development of insects and is an important target of genetic pest management. Vg is synthesized in the fat body, transported through haemolymph and accumulates in developing oocytes. In this regard, the eggplant shoot and fruit borer, Leucinodes orbonalis (Lepidoptera: Crambidae) is the major pest in South and South East Asia and a serious concern for farmers. Therefore, in the present study, we have cloned and characterized Vg from L. orbonalis (LoVg) for further applications. The cloned Vg consisted of 5,370 base pairs encoding 1,790 amino acid residues long protein. Further, sequence alignment revealed that LoVg has three conserved domains: a Vitellogenin N domain (LPD-N), a domain of unknown function protein families (DUF1943), and a von Willebrand factor type D domain (VWD). Using phylogenetic analysis, it was found that LoVg evolved alongside homologous proteins from different insects. The real-time expression levels of LoVg were significantly greater in female adults followed by the pupal stage. This suggests that Vg production and absorption in L. orbonalis occurs in the later pupal stage. Our studies showed that editing LoVg using CRISPR/Cas9 did not affect the total number of eggs laid but affected egg hatchability. These studies help us to design newer approaches in insect pest management through genetic suppression for sustainable pest management.

Genetic modification for the control of mosquitoes is frequently touted as a solution for a variety of vector-borne diseases. There has been some success using non-insecticidal methods like sterile or incompatible insect techniques to control arbovirus diseases. However, control by genetic modifications to reduce mosquito populations or create mosquitoes that are refractory to infection with pathogens are less developed. The advent of CRISPR-Cas9-mediated gene drives may advance this mechanism of control. In this review, use and progress of gene drives for vector control, particularly for malaria, is discussed. A brief history of population suppression and replacement gene drives in mosquitoes, rapid advancement of the field over the last decade and how genetic modification fits into the current scope of vector control are described. Mechanisms of alternative vector control by genetic modification to modulate mosquitoes' immune responses and anti-parasite effector molecules as part of a combinational strategy to combat malaria are considered. Finally, the limitations and ethics of using gene drives for mosquito control are discussed.

Synthetic CRISPR-Cas9 gene drive has been developed to control harmful species. However, resistance to Cas9 gene drive can be acquired easily when DNA repair mechanisms patch up the genetic insults introduced by Cas9 and incorporate mutations to the sgRNA target. Although many strategies to reduce the occurrence of resistance have been developed so far, they are difficult to implement and not always effective.

Here, Cas12f1, a recently developed CRISPR-Cas system with minimal potential for causing mutations within target sequences, has been explored as a potential platform for yielding low-resistance in gene drives. We construct Cas9 and Cas12f1 gene drives in a fast-replicating DNA virus, HSV1. Cas9 and Cas12f1 gene drives are able to spread among the HSV1 population with specificity towards their target sites, and their transmission among HSV1 viruses is not significantly affected by the reduced fitness incurred by the viral carriers. Cas12f1 gene drives spread similarly as Cas9 gene drives at high introduction frequency but transmit more slowly than Cas9 gene drives at low introduction frequency. However, Cas12f1 gene drives outperform Cas9 gene drives because they reach higher penetration and induce lower resistance than Cas9 gene drives in all cases.

Due to lower resistance and higher penetration, Cas12f1 gene drives could potentially supplant Cas9 gene drives for population control.

Pheromone-binding proteins (PBPs) are mainly responsible for binding and transporting hydrophobic pheromone molecules across the aqueous sensilla lymph to the receptor proteins. The preference of each PBP is believed to be different for each pheromone component within a single species. Significantly higher expression level of PBP1 and PBP2 in the male antennae of Spodoptera frugiperda suggesting that SfruPBP1 and SfruPBP2 might play important roles in pheromone perception. However, the preference of these two PBP to the three main pheromone components Z9-14: Ac, Z7-12: Ac and Z11-16: Ac have not been determined. In this study, a fluorescence competitive binding assay revealed that the binding intensities of SfruPBP1 and SfruPBP2 to Z9-14: Ac or Z7-12: Ac was comparable. We then used the CRISPR/Cas9 system to individually or simultaneously knock out PBP1 and PBP2 in S.frugiperda. The result of courtship behavior indicated that SfruPBP1 and SfruPBP2 were indispensable and played equal roles in perceiving the pheromones Z9-14: Ac and Z7-12: Ac for orientation, wing vibration, and hair-pencil display. Compared with Z9-14:Ac and Z7-12: Ac, Z11-16: Ac showed higher or medium binding intensities with SfruPBP1 and SfruPBP2 but played a minor role in inducing the wing vibration behavior. The results of this study are valuable for elucidating the mechanisms involved in sex pheromone perception and may facilitate the development of PBP-targeted pest control techniques.

Aedes mosquitoes are important virus vectors. We provide a toolkit for CRISPR-Cas9-editing of difficult-to-knockdown gene previously shown to be refractory to siRNA silencing in mosquito cells, which is pivotal in understanding vector biology, vector competence, host-pathogen interactions and in gene annotations. Starting from database searches of Ae. albopictus and the C6/36 cell line whole genome shotgun sequences for the prohibitin 2 (PHB2) gene, primers were designed to confirm the gene sequence in our laboratory-passaged C6/36 cell line for the correct design and cloning of CRISPR RNA into an insect plasmid vector to create a single guide RNA for the PHB2 gene target. After transfection of this plasmid vector into the C6/36 cells, cell clones selected by puromycin and/or limiting dilution were analyzed for insertions and deletions (INDELs) using PCR, sequencing and computational sequence decomposition. From this, we have identified mono-allelic and bi-allelic knockout cell clones. Using a mono-allelic knockout cell clone as an example, we characterized its INDELs by molecular cloning and computational analysis. Importantly, mono-allelic knockout was sufficient to reduce >80 % of PHB2 expression, which led to phenotypic switching and the propensity to form foci but was insufficient to affect growth rate or to inhibit Zika virus infection.•We provide a toolkit for CRISPR-Cas9-editing of the virus vector, Aedes albopictus C6/36 cell line•We validate this using a difficult-to-knockdown gene prohibitin 2•This toolkit is pivotal in understanding vector biology, vector competence, host-pathogen interactions and in gene annotations.

Novel technologies are needed to combat anopheline vectors of malaria parasites as the reductions in worldwide disease incidence has stalled in recent years. Gene drive-based approaches utilizing Cas9/guide RNA (gRNA) systems are being developed to suppress anopheline populations or modify them by increasing their refractoriness to the parasites. These systems rely on the successful cleavage of a chromosomal DNA target site followed by homology-directed repair (HDR) in germline cells to bias inheritance of the drive system. An optimal drive system should be highly efficient for HDR-mediated gene conversion with minimal error rates. A gene-drive system, AgNosCd-1, with these attributes has been developed in the Anopheles gambiae G3 strain and serves as a framework for further development of population modification strains. To validate AgNosCd-1 as a versatile platform, it must perform well in a variety of genetic backgrounds.

We introduced or introgressed AgNosCd-1 into different genetic backgrounds, three in geographically-diverse Anopheles gambiae strains, and one each in an An. coluzzii and An. arabiensis strain. The overall drive inheritance, determined by presence of a dominant marker gene in the F2 hybrids, far exceeded Mendelian inheritance ratios in all genetic backgrounds that produced viable progeny. Haldane's rule was confirmed for AgNosCd-1 introgression into the An. arabiensis Dongola strain and sterility of the F1 hybrid males prevented production of F2 hybrid offspring. Back-crosses of F1 hybrid females were not performed to keep the experimental design consistent across all the genetic backgrounds and to avoid maternally-generated mutant alleles that might confound the drive dynamics. DNA sequencing of the target site in F1 and F2 mosquitoes with exceptional phenotypes revealed drive system-generated mutations resulting from non-homologous end joining events (NHEJ), which formed at rates similar to AgNosCd-1 in the G3 genetic background and were generated via the same maternal-effect mechanism.

These findings support the conclusion that the AgNosCd-1 drive system is robust and has high drive inheritance and gene conversion efficiency accompanied by low NHEJ mutation rates in diverse An. gambiae s.l. laboratory strains.

Forests worldwide are under increasing pressure from climate change and emerging diseases, threatening their vital ecological and economic roles. Traditional breeding approaches, while valuable, are inherently slow and limited by the long generation times and existing genetic variation of trees. CRISPR technologies offer a transformative solution, enabling precise and efficient genome editing to accelerate the development of climate-resilient and productive forests. This review provides a comprehensive overview of CRISPR applications in forestry, exploring its potential for enhancing disease resistance, improving abiotic stress tolerance, modifying wood properties, and accelerating growth. We discuss the mechanisms and applications of various CRISPR systems, including base editing, prime editing, and multiplexing strategies. Additionally, we highlight recent advances in overcoming key challenges such as reagent delivery and plant regeneration, which are crucial for successful implementation of CRISPR in trees. We also delve into the potential and ethical considerations of using CRISPR gene drive for population-level genetic alterations, as well as the importance of genetic containment strategies for mitigating risks. This review emphasizes the need for continued research, technological advancements, extensive long-term field trials, public engagement, and responsible innovation to fully harness the power of CRISPR for shaping a sustainable future for forests.

Gene mutation correction was challenging until the discovery of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas). CRISPR is a new era for genome modification, and this technology has bypassed the limitations of previous methods such as zinc-finger nuclease and transcription activator-like effector nuclease. Currently, this method is becoming the method of choice for gene-editing purposes, especially therapeutic gene editing in diseases such as cardiovascular, neurological, renal, genetic, optical, and stem cell, as well as blood disorders and muscular degeneration. However, finding the optimum delivery system capable of carrying this large complex persists as the main challenge of this technology. Therefore, it would be ideal if the delivery vehicle could direct the introduction of editing functions to specific cells in a multicellular organism. Exosomes are membrane-bound vesicles with high biocompatibility and low immunogenicity; they offer the best and most reliable way to fill the CRISPR/Cas9 system delivery gap. This review presents the current evidence on the molecular mechanisms and challenges of CRISPR/Cas9-mediated genome modification. Also, the role of CRISPR/Cas9 in the development of treatment and diagnosis of numerous disorders, from malignancies to viral infections, has been discussed. Lastly, the focus is on new advances in exosome-delivery technologies that may play a role in CRISPR/Cas9 delivery for future clinical settings.

Gene drive technology has the potential to address major biological challenges. Well-studied homing suppression drives have been shown to be highly efficient in Anopheles mosquitoes, but for other organisms, lower rates of drive conversion prevent elimination of the target population. To tackle this issue, we propose a gene drive design that has two targets: a drive homing site where drive conversion takes place, and a distant site where cleavage induces population suppression. We model this design and find that the two-target system allows suppression to occur over a much wider range of drive conversion efficiency. Specifically, the cutting efficiency now determines the suppressive power of the drive, rather than the conversion efficiency as in standard suppression drives. We construct a two-target drive in Drosophila melanogaster and show that both components of the gene drive function successfully. However, cleavage in the embryo from maternal deposition as well as fitness costs in female drive heterozygotes both remain significant challenges for both two-target and standard suppression drives. Overall, our improved gene drive design has the potential to ease problems associated with homing suppression gene drives for many species where drive conversion is less efficient.

A considerable fraction of population in the world suffers from rare diseases. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its related Cas proteins offer a modern form of curative gene therapy for treating the rare diseases. Hereditary transthyretin amyloidosis, hereditary angioedema, duchenne muscular dystrophy and Rett syndrome are a few examples of such rare diseases. CRISPR/Cas9, for example, has been used in the treatment of β-thalassemia and sickle cell disease (Frangoul et al., 2021; Pavani et al., 2021) [1,2]. Neurological diseases such as Huntington's have also been focused in some studies involving CRISPR/Cas (Yang et al., 2017; Yan et al., 2023) [3,4]. Delivery of these biologicals via vector and non vector mediated methods depends on the type of target cells, characteristics of expression, time duration of expression, size of foreign genetic material etc. For instance, retroviruses find their applicability in case of ex vivo delivery in somatic cells due to their ability to integrate in the host genome. These have been successfully used in gene therapy involving X-SCID patients although, incidence of inappropriate activation has been reported. On the other hand, ex vivo gene therapy for β-thalassemia involved use of BB305 lentiviral vector for high level expression of CRISPR biological in HSCs. The efficacy and safety of these biologicals will decide their future application as efficient genome editing tools as they go forward in further stages of human clinical trials. This review focuses on CRISPR/Cas based therapies which are at various stages of clinical trials for treatment of rare diseases and the constraints and ethical issues associated with them.

Gene drives have great potential for suppression of pest populations and removal of exotic invasive species. CRISPR homing suppression drive is a powerful but unconfined drive, posing risks of uncontrolled spread. Thus, developing methods for confining a gene drive is of great significance. Tethered drive combines a confined system such as Toxin-Antidote Recessive Embryo drive with a strong drive such as a homing suppression drive. It can prevent the homing drive from spreading beyond the confined drive and can be constructed readily, giving it good prospects for future development. However, we have found that care must be taken when deploying tethered drive systems in some scenarios. Simulations of tethered drive in a panmictic population model reveal that successful deployment requires a proper release ratio between the two components, tailored to prevent the suppression drive from eliminating the confined system before it has the chance to spread. Spatial models where the population moves over a one-dimensional landscape display a more serious phenomenon of drive wave interference between the two tethered drive components. If the faster suppression drive wave catches up to the confined drive wave, success is still possible, but it is dependent on drive performance and ecological parameters. Two-dimensional simulations further restrict the parameter range for drive success. Thus, careful consideration must be given to drive performance and ecological conditions, as well as specific release proposals for potential application of tethered drive systems.

Rhipicephalus (Boophilus) microplus is globally one of the most economically important ectoparasites of cattle costing the agriculture industry billions of dollars annually. Resistance to chemical control measures has prompted the development of novel methods of control. Recent advancements in genetic control measures for human and other animal vectors have utilized sex determination research to manipulate sex ratios, which have shown promising results in mosquitoes namely Aedes aegypti and Anopheles stephensi. Here, we use R. (B.) microplus as a model to provide foundational research to allow similar avenues of investigation in ticks using R. (B.) microplus as a model. Karyotypes for R. (B.) microplus show an XX:XO sex determining system with the largest chromosome being the sex chromosome. Using flow cytometric methods, the size of the sex chromosome was estimated at 526.91 Mb. All measures to identify the sex chromosome within the cattle tick genome assembly associated sex chromosomal characteristics to two chromosomes. This discrepancy between the assembly and karyotypes of the tick led to generating a new genome assembly with a single adult male specimen. The two chromosomes in question aligned with a single scaffold within the new genome that had a length of 513.29 Mb and was the first time the sex chromosome was identified in an Ixodid genome assembly.

Anopheles mosquitoes are the sole vector of human malaria, the most burdensome vector-borne disease worldwide. Strategies aimed at reducing mosquito populations and limiting their ability to transmit disease show the most promise for disease control. Therefore, gaining an improved understanding of mosquito biology, and specifically that of the immune response, can aid efforts to develop new approaches that limit malaria transmission. Here, we use a genome-wide CRISPR screening approach for the first time in mosquito cells to identify essential genes in Anopheles and identify genes for which knockout confers resistance to clodronate liposomes, which have been widely used in mammals and arthropods to ablate immune cells. In the essential gene screen, we identified a set of 1280 Anopheles genes that are highly enriched for genes involved in fundamental cell processes. For the clodronate liposome screen, we identified several candidate resistance factors and confirm their roles in the uptake and processing of clodronate liposomes through in vivo validation in Anopheles gambiae, providing new mechanistic detail of phagolysosome formation and clodronate liposome function. In summary, we demonstrate the application of a genome-wide CRISPR knockout platform in a major malaria vector and the identification of genes that are important for fitness and immune-related processes.

CRISPR base editors can introduce point mutations into DNA precisely, and cytosine base editors (CBEs) catalyze C to T transitions. While CBEs have been thoroughly explored in cell culture and organisms such as mice, little is known about DNA base editing in insects. In this study, we evaluated germline editing rates of three different CBEs expressed under actin (ubiquitous) or nanos (germline) promoters utilizing Drosophila melanogaster. The original Rattus norvegicus-derived cytosine deaminase APOBEC1 (rAPO-1) displayed high base editing rates (~99%) with undetectable indel formation. Additionally, we show that base editors can be used for generating male sterility and female lethality. Overall, this study highlights the importance of promoter choice and sex-specific transmission for efficient base editing in flies while providing new insights for future genetic biocontrol designs in insects.

Population replacement refers to the process by which a wild-type population of insect pests is replaced by a population possessing modified traits or abilities. Effective population replacement necessitates a gene drive system capable of spreading desired genes within natural populations, operating under principles akin to super-Mendelian inheritance. Consequently, releasing a small number of genetically edited insects could potentially achieve population control objectives. Currently, several gene drive approaches are under exploration, including the newly adapted CRISPR-Cas genome editing system. Multiple studies are investigating methods to engineer pests that are incapable of causing crop damage or transmitting vector-borne diseases, with several notable successful examples documented. This review summarizes the recent advancements of the CRISPR-Cas system in the realm of population replacement and provides insights into research methodologies, testing protocols, and implementation strategies for gene drive techniques. The review also discusses emerging trends and prospects for establishing genetic tools in pest management.

Protozoan parasitic diseases pose a substantial global health burden. Understanding the pathogenesis of these diseases is crucial for developing intervention strategies in the form of vaccine and drugs. Manipulating the parasite's genome is essential for gaining insights into its fundamental biology. Traditional genomic manipulation methods rely on stochastic homologous recombination events, which necessitates months of maintaining the cultured parasites under drug pressure to generate desired transgenics. The introduction of mega-nucleases (MNs), zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs) greatly reduced the time required for obtaining a desired modification. However, there is a complexity associated with the design of these nucleases. CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas (CRISPR associated proteins) is the latest gene editing tool that provides an efficient and convenient method for precise genomic manipulations in protozoan parasites. In this chapter, we have elaborated various strategies that have been adopted for the use of CRISPR-Cas9 system in Plasmodium, Leishmania and Trypanosoma. We have also discussed various applications of CRISPR-Cas9 pertaining to understanding of the parasite biology, development of drug resistance mechanism, gene drive and diagnosis of the infection.

Population-scale genome modification can alter the composition or fate of wild populations. Synthetic gene drives provide one set of tools, but their use is complicated by scientific, regulatory, and social issues associated with transgene persistence and flow. Here we propose an alternative approach. An Allele Sail consists of a genome editor (the Wind) that introduces DNA sequence edits, and is inherited in a Mendelian fashion. Meanwhile, the edits (the Sail) experience an arithmetic, Super-Mendelian increase in frequency. We model this system and identify contexts in which a single, low frequency release of an editor brings edits to a very high frequency. We also identify conditions in which manipulation of sex determination can bring about population suppression. In regulatory frameworks that distinguish between transgenics (GMO) and their edited non-transgenic progeny (non-GMO) Allele Sails may prove useful since the spread and persistence of the GM component can be limited.

CRISPR-Cas9 homing gene drives are designed to induce a targeted double-stranded DNA break at a wild type allele ('recipient'), which, when repaired by the host cell, is converted to the drive allele from the homologous ('donor') chromosome. Germline localisation of this process leads to super-Mendelian inheritance of the drive and the rapid spread of linked traits, offering a novel strategy for population control through the deliberate release of drive individuals. During the homology-based DNA repair, additional segments of the recipient chromosome may convert to match the donor, potentially impacting carrier fitness and strategy success. Using Anopheles gambiae strains with variations around the drive target site, here we assess the extent and nature of chromosomal conversion. We show both homing and meiotic drive contribute as mechanisms of inheritance bias. Additionally, over 80% of homing events resolve within 50 bp of the chromosomal break, enabling rapid gene drive transfer into locally-adapted genetic backgrounds.

Due to limitations in conventional disease vector control strategies including the rise of insecticide resistance in natural populations of mosquitoes, genetic control strategies using CRISPR gene drive systems have been under serious consideration. The identification of CRISPR target sites in mosquito populations is a key aspect for developing efficient genetic vector control strategies. While genome-wide Cas9 target sites have been explored in mosquitoes, a precise evaluation of target sites focused on coding sequence (CDS) is lacking. Additionally, target site polymorphisms have not been characterized for other nucleases such as Cas12a, which require a different DNA recognition site (PAM) and would expand the accessibility of mosquito genomes for genetic engineering. We undertook a comprehensive analysis of potential target sites for both Cas9 and Cas12a nucleases within the genomes of natural populations of Anopheles gambiae and Aedes aegypti from multiple continents. We demonstrate that using two nucleases increases the number of targets per gene. Also, we identified differences in nucleotide diversity between North American and African Aedes populations, impacting the abundance of good target sites with a minimal degree of polymorphisms that can affect the binding of gRNA. Lastly, we screened for gRNAs targeting sex-determination genes that could be widely applicable for developing field genetic control strategies. Overall, this work highlights the utility of employing both Cas9 and Cas12a nucleases and underscores the importance of designing universal genetic strategies adaptable to diverse mosquito populations.

Vector-borne diseases (VBDs) impose devastating effects on human health and a heavy financial burden. Malaria, Lyme disease, and dengue fever are just a few examples of VBDs that cause severe illnesses. The current strategies to control VBDs consist mainly of environmental modification and chemical use, and to a small extent, genetic approaches. The genetic approaches, including transgenesis/genome modification and gene-drive technologies, provide the basis for developing new tools for VBD prevention by suppressing vector populations or reducing their capacity to transmit pathogens. The regulatory elements such as promoters are required for a robust sex-, tissue-, and stage-specific transgene expression. As discussed in this review, information on the regulatory elements is available for mosquito vectors but is scant for other vectors.

CRISPR/Cas9 manipulations are possible in many insects and ever expanding. Nonetheless, success in one species and techniques developed for it are not necessarily applicable to other species. As such, the development and expansion of CRISPR-based (clustered regularly interspaced short palindromic repeats) genome-editing tools and methodologies are dependent upon direct experimentation. One useful technique is Cas9-dependent homologous recombination, which is a critical tool for studying gene function but also for developing pest related applications like gene drive. Here, we report our attempts to induce Cas9 homology directed repair (HDR) and subsequent gene drive in Tribolium castaneum (Herbst; Insecta: Coleoptera: Tenebrionidae). Utilizing constructs containing 1 or 2 target gRNAs in combination with Cas9 under 2 different promoters and corresponding homology arms, we found a high incidence of CRISPR/Cas9 induced mutations but no evidence of homologous recombination. Even though the generated constructs provide new resources for CRISPR/Cas9 modification of the Tribolium genome, our results suggest that additional modifications and increased sample sizes will be necessary to increase the potential and detection for HDR of the Tribolium genome.

Elimination of malaria has become a United Nations member states target: Target 3.3 of the sustainable development goal no. 3 (SDG3). Despite the measures taken, the attainment of this goal is jeopardized by an alarming trend of increasing malaria case incidence. Globally, there were an estimated 241 million malaria cases in 2020 in 85 malaria-endemic countries, increasing from 227 million in 2019. Malaria case incidence was 59, which means effectively no changes in the numbers occurred, compared with the baseline 2015. Jennifer Doudna-co-inventor of CRISPR/Cas9 technology-claims that CRISPR holds the potential to lessen or even eradicate problems lying in the centre of SDGs. On the same note, CRISPR/Cas9-mediated mosquito-targeting gene drives (MGD) are perceived as a potential means to turn this trend back and put momentum into the malaria elimination effort. This paper assessed two of the critical elements of the World Health Organization Genetically modified mosquitoes (WHO GMM) Critical Pathway framework: the community and stakeholders' engagement (inability to employ widely used frameworks, segmentation of the public, 'bystander' status, and guidelines operationalization) and the regulatory landscape (lex generali, 'goldilocks dilemma', and mode of regulation) concerning mosquito-oriented gene drives (MGD) advances. Based on the assessment findings, the author believes that CRISPR/Cas-9-mediated MGD will not contribute to the attainment of SDG3 (Target 3.3), despite the undisputable technology's potential. This research pertains to the state of knowledge, legal frameworks, and legislature, as of November 2022.