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Published in final edited form as: Curr Opin Insect Sci. 2024 Jul 15;65:101235. doi: 10.1016/j.cois.2024.101235

Gene editing in agricultural, health, and veterinary pest arthropods: recent advances

Madhusudan Manjunatha 1, Michael Pham 2, Monika Gulia-Nuss 2, Andrew Nuss 1,2,*
PMCID: PMC11528696  NIHMSID: NIHMS2016304  PMID: 39019112

Abstract

Pest arthropods cause significant crop damage or are vectors of pathogens for both plants and animals. The current standard of pest management prevents against crop losses and protects human and animal health, but shortcomings exist, such as insecticide resistance and environmental damage to nontarget organisms. New management methods are therefore needed. The development of new tools, such as site-specific gene editing, has accelerated the study of gene function and phenotype in non-model arthropod species and may enable the development of new strategies for pathogen and arthropod control. Here, the most recent developments in gene editing in arthropod pests are briefly reviewed. Additionally, technological advances that could be applicable to new species or enhance the success rates of gene editing in species with already established protocols are highlighted.

Keywords: Gene editing, pest, vector, arthropod, CRISPR/Cas9

Introduction

Arthropods are the most diverse animal phylum, making up over 80% of all known animal species. A small fraction of these have drastic impacts on public health and food production. Currently 40% of world crop production is lost to arthropod agricultural pests, either directly or through the pathogens they vector [1,2]. Additionally arthropod disease vectors (e.g., mosquitoes, sandflies, and ticks), are responsible for 17% of all infectious diseases and result in over 700,000 deaths globally per year [3]. These problems are predicted to increase, with climactic changes resulting in elevated arthropod population growth rates and range expansion. Together, arthropod pests represent significant global health and agricultural burdens despite current control methods.

Numerous methods are currently employed to manage pest arthropod populations, most notably the use of chemical pesticides. However, chemical controls have drawbacks, including the development of pesticide resistance and environmental impacts on beneficial and other non-target species. The development of gene-editing tools, notably CRISPR/Cas9 and related techniques, has advanced the idea of more targeted approaches for pest population management, particularly those enabling gene drive and sterile insect strategies [4,5]. However, applying these tools to non-model organisms requires extensive research investments, such as establishing reliable lab rearing protocols and troubleshooting idiosyncratic life history details, as well as the development of functional tools necessary for genetic modification, including marker phenotypes, tissue- or developmental-dependent promoters, and embryo injection protocols, among others. In this mini-review, we highlight cutting-edge gene editing developments within the last two years in pest arthropods of medical, veterinary, and agricultural importance (Table 1). This brief snapshot provides insight into current arthropod genome-editing developments and what the future might hold.

Table 1:

CRISPR/Cas9 developments in Arthropods of Medical, Veterinary, and Agricultural Importance. Studies beyond the scope of this review (not in the past 2 years) are labelled with just the year in parentheses.

Species (common name) Order CRISPR Knockout (KO) Genes Targeted Methodology CRISPR Knock-in Comments
C. hominivorax (New World Screwworm) Diptera Yellow, transformer (KO in 2019) Embryo injection No U6 promoters tested in L. cuprina16
L. cuprina (Australian sheep blowfly)16 Yellow (KO in 2019) Embryo injection (0.68–1.17%*) No C. hominivorax U6 promoters functional in L. cuprina16
Z. cucurbitae (melon fly) White 32 Embryo injection (66.7%*) No Percent of visible mutants out of surviving adults at G0. Unified protocol for tephritid species
B. dorsalis (oriental fruit fly) Sex peptide receptor 33 Embryo injection (75%*) No Percent of sterile females to total females at G0
L. hesperus (western tarnished plant bug) Hemiptera White, cinnabar, cardinal19 Embryo injection (86%*) No
β-tubulin 20 Embryo injection No
R. prolixus (Kissing bug)18 Rp-scarlet, Rp-white, and Rp-yellow ReMOT Control DIPA-CRISPR No ReMOT Control (with chloroquine) worked whereas DIPA-CRISPR did not work in this species
D. citri (Asian citrus psyllid)22 White, kynurenine hydroxylase Embryo injection (1.4%*) ReMOT Control (3.1%*) BAPC (0%*) DIPA-CRISPR (8.8%*) No Methods used for the 1st time in this species and in situ microinjection on host plant
N. lugens (brown plant hopper)24 NlugGr23a (putative fecundity-related Gr) Embryo injection No Caused sterility in mutant males but did not affect mating behavior or morphology
S. furcifera (white-backed planthopper) Tryptophan 2,3-dioxygenase 14 Thorax injection of the 3rd instar nymphs (19.76%*) No Nanoparticle-based CRISPR-Cas13d for disruption of mRNA expression
Tryptophan 2,3-dioxygenase 23 DIPA-CRISPR (56.7%*) No Highest gene editing efficiency was achieved 2 days posteclosion
H. vitripennis (glassy-winged sharpshooter)21 White, cinnabar Embryo injection (58.9–80%*) No Methods used for the 1st time in this species and in situ microinjection on host plant
O. furnacalis (Asian corn borer)29 Lepidoptera Ovarian serine protease Embryo injection (28.42%*) No
P. interpunctella (Indianmeal moth) White 26 Embryo injection (12–32%*) No
White 25 Embryo injection (38%*) Yes A single-stranded oligo DNA nucleotide (ssODN) was used to rescue the one bp white spontaneous deletion
P. xylostella (diamondbac k moth) Yellow 31 Embryo injection (40%*) Yes 1st single CRISPR/Cas 9-based gene drive construct for this species and only non-mosquito/cell line example dsRNA knockdown of Ku70 to improve knock-in efficiency
Ovary-serine protease 30 Embryo injection No
S. frugiperda (fall armyworm)28 Sfyellow-y, sfebony, sflaccase2, sfscarlet, and sfok Embryo injection No
T. absoluta (tomato leaf miner)27 Cinnabar (Zygote) embryo injection (31.9%*) No 1st CRISPR/Cas 9-based genome editing in this species
T. castaneum (red flour beetle) Coleoptera Vermilion 34 Embryo injection Yes Cas9 transgenic line having constructs pB-hs-Cas9-hs and pBhs-Cas9-GFP-nanos
Cardinal 10 DIPA-CRISPR (53.2%*) ReMOT Control (0.26%*) Yes Knock-in of ssODN short homology arms (96 bp) introducing a restriction site and mutating the protospacer adjacent motif
H. hebetor (ectoparasitic wasp)7 Hymenoptera White Embryo injection No Combination of two sgRNA yielded higher mutation rate (84.62%*)
F. occidentalis (western flower thrips)9 Thysanoptera White, white-like BAPC + Saponin (SYNCAS) (24.07–32.05%*) Yes ssODN of 170 bp for white gene was used for knock-in
T. urticae (red spider mite or two-spotted spider mite) Trombidiformes Phytoene desaturase (KO in 2020) DIPA-CRISPR (0.485%*) No
Phytoene desaturase CYP384A1, Antennapedia 9 BAPC + Saponin (SYNCAS) (5–6%*) Yes ssODN of 192 bp for chitin synthase 1 gene was used for knock-in
I. scapularis (blacklegged tick or deer tick)17 Ixodida Proboscipedi a, Chitinase Embryo injection (0.9%*) ReMOT Control (1.7–4.2%*) No First genetic transformation in any tick species
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*

Transformation efficiency data as reported in the cited paper. Note that calculation of efficiency varies widely by reporting study, ranging from transformation efficiency of all injected individuals to transformation efficiency of only the survivors of injections, to screening of progeny from maternal uptake transformation techniques.

New technological improvements in gene editing in arthropods

Many techniques reported in this review are applications of existing insect gene-editing techniques applied to new species, yet several recent technological innovations have been applied for the first time in pest arthropods. For instance, a new embryo-screening method used shed pupal cases of Bactrocera dorsalis (oriental fruit fly) [6] or Habrobracon hebetor (an ectoparasitic wasp) [7] to screen for edited genes. This method may be useful in other species where a marker phenotype is not readily available, and prevents damage to edited individuals, in addition to reducing costs of maintaining non-edited animals.

Embryo injection is an acute challenge for several arthropods, as certain life history traits or egg morphology make embryo injections highly infeasible. In an innovation for CRISPR-mediated transgenesis not involving direct embryo injection, a method similar to Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) was employed using cell-penetrating peptide (CPP) tags. This was used to deliver a fluorescent protein to several tissues and cell types (including bacteriocytes) in Acyrthosiphon pisum, the pea aphid [8]. Unlike ReMOT Control, this technique allows delivery of a Cas9-sgRNA ribonucleoprotein complex (RNP) to embryos in species that are non-vitellogenic. Another method used Branched Amphiphilic Peptide Capsules (BAPC) together with saponin in a synergistic interaction to deliver the RNP in the two-spotted spider mite, Tetranychus urticae (Fig. 1). This method, termed SYNCAS, was also used in the same study to successfully edit and achieve knock-in in the Western flower thrip, Frankliniella occidentalis [9]**. Yet another technique involved injecting high concentrations of RNP into the hemocoel of adult females before oviposition without using any type of carrier molecule and was used to edit the red flour beetle, Tribolium casteneum, and the German cockroach, Blattella germanica [10]**. This was termed direct parental (DIPA-) CRISPR by the authors, although this strategy has been used previously by other groups [11,12]. However, a novel development in the newer work was to achieve CRISPR knock-in events when co-injecting the RNP together with single stranded oligo DNA nucleotides (ssODNs) with homology arms [10]. However, DIPA-CRISPR may not work in every arthropod system [13]. Together, these advancements may facilitate gene-editing in species where embryo injection is difficult.

Figure 1:

Figure 1:

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Highlight of recent, novel gene editing techniques in arthropods. Maternal transformations encompasses techniques in which gravid females are injected with a RNP for uptake into the oocytes and editing of the developing embryo, often with the help of a yolk peptide to facilitate oocyte uptake. SYNCAS is represented here in the two spotted spider mite, Tetranychus urticae. RNA-editing in arthropods has recently been achieved using Cas13. The example here depicts the action of REAPER, a system designed in Aedes aegypti for activation in response to Chikungunya virus in a blood meal, which then activates Cas13d that degrades viral specifically, and mosquito vector RNA nonspecifically, which results in mosquito mortality.

In an innovative departure from using CRISPR/Cas9, the CRISPR-Cas13 system (specifically Cas13d) was recently used for RNA editing in in the white-backed planthopper (WBPH), Sogatella furcifera, a migratory rice pest in Asia [14]* and injections were used to disrupt SfTO transcripts. Notably, Cas13d demonstrated a quicker phenotypic onset and decreased levels of SfTO transcript compared to RNA interference (RNAi). This suggests the CRISPR-Cas13d system has potential advantages over the current widespread usage of conventional RNAi techniques in terms of specificity and immediate outcomes. In addition, CRISPR-Cas13 was recently used to target chikungunya virus in Ae. aegypti, through a strategy known as REAPER (vRNA Expression Activates Poisonous Effector Ribonuclease) (Fig. 1). In this strategy, transgenic Ae. aegypti, upon taking up viral RNA in a blood meal, expressed CRISPR-13 targeted to specific arboviruses. This significantly diminished viral replication and even generated sufficient off-target collateral RNA cleavage to kill the majority of infected mosquitoes, preventing viral transmission to new hosts [15]**.

Gene editing in arthropods of medical and veterinary importance

Genome editing has been successfully demonstrated in several medically and veterinary important arthropod species, most notably mosquitoes. However, work has recently expanded to additional medically and veterinary important arthropod species. Of interest is New World Screwworm fly, Cochliomyia hominvorax, a myiasis causing parasite of warm-blooded animals. The eradication of this pest in North America last century was the first application of sterile insect technique (SIT) and is one of the greatest non-chemical pest management success stories. Although earlier methods relied on the release of irradiated sterile males, recent efforts are geared towards the development of genetically-controlled conditional lethal female strains to simplify rearing protocols and reduce costs. Recently, several U6 promoters were screened for use in C. hominovorax using the related pest, Lucillia cuprina, the Australian sheep blowfly [16]. The identification of optimal U6 promoters could be used in developing CRISPR/Cas9-based genetic lines such as sexing strains.

Gene editing in Ixodes scapularis ticks was reported for the first time, via two different methods: embryo injection and ReMOT Control [17]*. Among non-insect arthropods, I. scapularis is the most significant vector of public health concern in the United States due to its ability to transmit multiple pathogens, yet gene editing in this species was previously stymied by challenging egg morphology. Successful embryo injection required several technical advancements, including chemical treatments to soften the chorion and reduce the intraovular pressure as well as wax removal through draining of the wax gland of the mother tick. ReMOT Control involved injection of gravid females with a yolk protein or vitellogenin tagged Cas9 protein that directed the Cas9 into the ovaries. Edits to the homeobox gene, Proboscipedia, and a chitinase gene were demonstrated. This work represents a large step forward in a vector that was previously intractable to genome editing.

Another gene editing first was reported for the kissing bug, Rhodnius prolixus, an important vector of Trypanosoma cruzi, the causative agent of Chagas disease [18]. This was achieved using ReMOT Control. P2C (derived from Drosophila melanogaster Yolk Protein 1) and BtKV (derived from a vitellogenin binding sequence in Bemisia tabaci) were used for delivering RNPs to oocytes, with BtKV being slightly more effective in inducing edits. Potential phenotypic markers were targeted, with knockout of Rp-scarlet (Rp-sca) and Rp-white (Rp-w) resulting in red and white eyes, respectively. The Rp-yellow (Rp-y) gene was also edited, resulting in a yellow cuticle phenotype. DIPA-CRISPR was also attempted, but did not yield any visible phenotypes. This study opens the door for gene editing in triatomine bugs and potentially new Chagas disease control strategies.

Gene editing in arthropods of agricultural importance

The promise of gene-editing technologies for management of agriculturally relevant arthropods is keenly recognized. Recently, a massive increase in research has emerged for a variety of agricultural pests, focused mainly on various stages of genome editing tool development including identification of phenotype markers, promoters, fertility disruption, and facilitation of CRISPR-Cas9 delivery to germline cells. A brief sample of new developments in genetically edited agricultural pests is included below and in Table 1.

As noted above, SYNCAS was recently used for gene editing in the highly polyphagous and widespread two-spotted spider mite, T. urticae. This approach improved knockout efficiency over previous methods to edit this mite. Two genes were targeted for knockout as a proof of concept, CYP384A1 and Antennapedia (Antp), which incurred “lemon” phenotypes (a yellow body color due to the absence of ketocarotenoids) and severe leg deformities, respectively. Further, knock-ins were also achieved by SYNCAS coupled with enhancer V2 (an HDR stimulating agent) to induce an I1017F mutation to the chitin synthase 1 gene (CHS1) which conferred etoxazole resistance [9]. These advancements facilitate gene editing in an arthropod which is challenging to manipulate due to its small size.

In Hemiptera, advancements have recently been reported for Lygus hesperus, the western tarnished plant bug, an agricultural pest in Mexico and the western United States. RNAi screening of the eye pigments cardinal (LhCd), cinnabar (LhCn), and white (LhW) demonstrated that LhW knockdown was fatal to developing embryos in L. hesperus, but knockdowns of LhCd and LhCn produced viable insects with bright red eye phenotypes [19]. Subsequent CRISPR/Cas9 editing created heritable germline knockouts of LhCd (Card) and LhCn (Cinn), with a stable red eye phenotype, particularly in the Cinn strain. Further, RNAi screening and subsequent CRISPR/Cas9 knockout of β-tubulin2 in L. hesperus resulted in males with non-viable sperm that could mate but were effectively sterile, an important step in developing genetically tractable sterile-male strategies in this pest species [20].

Similar to the work in L. hesperus, advancements in phenotype markers and successful CRISPR/Cas9 gene editing were recently made for Homalodisca vitripennis, the glassy-winged sharpshooter (GWSS), a leafhopper vector in several crops, particularly grapes. A conserved FAD-binding domain region of cinnabar (cn) and the region of the ABC-transporter-like domain (conserved AAA domain) of white (w) were targeted for editing and resulted in white eye phenotypes. This was aided by in situ embryo microinjections of embryos on host plant tissue (in contrast to removing and positioning the eggs), to reduce disruptions to the eggs and enhance survival [21].

Huanglongbing (HLB), a devastating citrus disease caused by Candidatus Liberibacter asiaticus, is primarily spread by the insect vector Diaphorina citri (Asian citrus psyllid). Knockout of two eye pigmentation genes, white (w) and kynurenine hydroxylase (kh), in this insect each produced observable phenotypic variations in eye color and may serve as phenotypic markers of gene editing. Notable innovations in the embryo injection protocol include techniques to prevent needle blockage and, similar to the GWSS work noted above, injecting eggs attached to plant tissue, both of which improved embryo survival and facilitated the first successful use of CRISPR-Cas9 gene editing in D. citri. In addition, the ReMOT Control technique was used to inject adult females and successfully edit offspring, resulting in noticeable eye mutants with somatic mosaicism [22].

The eye pigment gene tryptophan 2,3-dioxygenase (SfTO) was targeted in S. furcifera [23]. Mutation of this gene resulted in a white eye phenotype. Instead of embryo injections, this group used DIPA-CRISPR [10], as described above, and injected RNPs into the hemocoel of adult females before oviposition to edit offspring.

Advancements have also been made to curb reproduction in the brown planthopper (BHP), Nilaparvata lugens, a major rice pest. CRISPR-Cas9 facilitated knockout of NlugGr23a, a putative fecundity-related gustatory receptor, which resulted in sterility in homozygous males [24]. As with strategies to disrupt reproduction in other insects, this target may have utility in sterile insect approaches.

In Lepidoptera, phenotype markers were explored in Plodia interpunctella, the Indianmeal moth, a stored products pest, by mutating the white gene using CRISPR/Cas9. Knockout of white resulted in white-eye mutants, and subsequent rescue with homology directed repair (HDR) restored the brown-pigmented eye phenotype [25]. Subsequent work demonstrated that, in contrast to some other moth species, the white eye phenotype did not cause embryonic lethality [26]. Similarly, the Tacinnabar gene was mutated in Tuta absoluta, the tomato leafminer, using CRISPR/Cas9. This may serve as a phenotypic marker of editing, with knockouts displaying red eye coloration in adults, and is a first for genome editing in this species [27]. Also, several studies in recent years have reported editing in Spodoptera frugiperda, the fall armyworm, and an additional knockout screen of pigment related genes (Sfebony, Sfyellow-y, Sflaccase2, and Sfscarlet) resulted in mutants with distinct eye and cuticle phenotypes [28].

CRISPR/Cas9-mediated mutagenesis is also being used to manipulate reproduction in lepidopterans. In Ostrinia furnacalis, a pest of maize in Asia and beyond, knockout of ovary serine protease (OfOsp) induced female-specific sterility [29]. Knockout of ovary serine protease was also performed in Plutella xylostella, the diamondback moth, a pest of crucifers, particularly cabbage, and this also induced female sterility with shorter ovarioles, reduced egg development and fragile eggs [30]. These may be of use in genetic pest control.

Although gene knockout through CRISPR/Cas9-induced mutagenesis is becoming more frequent, gene knock-ins are more complex. However, recent progress has been made using CRISPR/Cas9 to develop knock-in gene drive constructs in P. xylostella [31]*. This particular approach targeted the Pxyellow gene, previously demonstrated as a phenotypic marker in this species. A plasmid construct was then inserted via HDR. This construct contained a Cas9 gene (under the Pxnanos promoter) and an EGFP marker gene (HR5IE1 promoter). In addition, dsRNA of Pxku70 was co-injected to suppress non-homologous end-joining (NHEJ) to increase the probability of HDR.

Among Diptera, development of marker genes to easily screen for phenotypes was recently explored in the melon fly (Zeugodacus cucurbitae) and oriental fruit fly (Bactrocera dorsalis) [32]. As with many other insects noted above, the white eye (we) gene was targeted, and heritable knockout lines of we were established. In addition, knockout of sex peptide receptor (Bdspr) disrupted fertility in female B. dorsalis, making it a potential target for SIT [33].

In Coleoptera, a Cas9-expressing line of the model beetle Tribolium castaneum (red flour beetle) was recently developed to simplify CRISPR/Cas9-editing [34]*. Lines generated from these experiments were effective at generating germline mutants after injections of sgRNAs, not containing Cas9, suggesting they will be a useful tool for further manipulation of the T. castaneum genome. As noted above, knock-in by DIPA-CRISPR was also achieved in this species in a separate study, although knock-in efficiency was low at 0.36% overall [10].

Parasitoids are an important biocontrol component for agricultural pests and genetic manipulation of these beneficials could confer useful traits, such as insecticide resistance. However, embryo injection is particularly challenging for many parasitoids because eggs are laid directly on or inside of hosts. Despite this challenge, successful CRISPR/Cas9-mediated mutagenesis was recently reported in the hymenopteran H. hebetor, an ectoparasitic wasp that parasitizes several caterpillars including P. interpunctella [7].

Conclusions

Overall, pest arthropod genome editing, a long-desired dream in the entomological community, has become a reality for the study and genetic manipulation of arthropods, largely aided by the development of CRISPR-Cas9. This has ushered in a new era, enabling the investigation of gene function and the development of new, targeted methods for controlling pest populations and vector-borne diseases that have advantages over the current standard of pest management. Although many obstacles remain, the future is bright for the use of genome modification in developing new management tools to prevent disease transmission and crop damage by pest arthropods.

Highlights.

  • Recent genome editing technological developments pioneered specifically in pest arthropods.

  • Highlights of genome editing milestones in vector arthropods within the last two years.

  • New genome editing updates in agricultural pests.

Funding:

This publication was made possible by grants from the National Science Foundation (NSF) under Grant No. 2236698 to ABN and National Institutes of Health-National Institute of Allergy and Infectious Diseases (NIH-NIAID) grants R21AI176352 and R01AI172943 to MG-N, ABN, and MP.

Footnotes

Conflicts of Interest:

The authors declare no conflict of interest.

This work has not been previously published and has not been submitted to another journal for publication. No artificial intelligence tools have been used in the generation of this manuscript. All authors have provided their approval for this submission.

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