Advances in CRISPR/Cas9-Based Gene Editing Technology in Mosquitoes

Abstract

As global warming increases, mosquito activity areas are expanding, alongside changes in the natural environment and the misuse of insecticides, thus further increasing mosquito resistance and exposing the world to an elevated risk of mosquito-borne diseases. With advancements in gene editing technology, researchers are continually attempting to modify various mosquito genes and validate their functional roles with CRISPR-Cas9 technology. Here, we review recent applications of CRISPR-Cas9-based gene editing technology in mosquito-borne disease prevention and control, as well as mosquito population suppression, phenotyping, and resistance. We further discuss the development of new techniques, such as ReMOT Control and DIPA-CRISPR, which differ from conventional embryo microinjection and should be expanded in future insect gene editing research.

Main article text

INTRODUCTION

With the development of gene sequencing technology, the exploration of genome sequence function has recently become a new research direction [1,2]. The development of convenient and accurate gene editing tools has facilitated the investigation of gene function. To date, three types of gene editing tools have been developed: zinc-finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology, and clustered regulatory interspaced short palindromic repeats (CRISPR) technology. The first gene editing tool used by researchers was ZFN, a chimeric fusion structure consisting of a zinc finger protein and the shearing domain of the Fok-I nucleic acid endonuclease. The zinc finger protein structural domain recognizes the target site and relies on the action of Fok-I to cut DNA double strands and form double-strand breaks. TALEN is derived from ZFN and likewise uses Fok-I nucleic acid endonuclease to cleave sequences. However, owing to the difficulty, complexity, and cost of design, neither gene editing method has been widely used in laboratory settings [3,4]. CRISPR-Cas, an immune system widely found in bacteria and most archaea, confers resistance to viral or exogenous sequences [5]. This technology quickly became the state of the art in the field of gene editing, because of its advantages of efficiency, convenience, speed, and affordability [6].

Currently, the CRISPR-Cas9 system most widely used in scientific research is that of Streptococcus pneumonia [7]. This system consists of three main parts: CRISPR-derived RNA (cr-RNA) [8], trans-activating cr-RNA (tracr-RNA) [9,10], and nucleic acid endonuclease 9, or the Cas9 protein [11]. The complete CRISPR-Cas9 system can achieve targeted cleavage of genes. After further exploration, researchers successfully chimerized cr-RNA and tracr-RNA, thus forming a complex called single-guide RNA (sg-RNA), which undergoes complementary base pairing with target DNA sequences. The use of sgRNA not only preserves the DNA cleavage function of Cas9 endonuclease but also simplifies the composition of the CRISPR system [6].

Double-strand breaks induce either nonhomologous end joining (NHEJ) or homologous directed repair (HDR) in vivo [12]. The NHEJ repair mechanism is the most frequent type of break repair at double-strand breaks. Because of its error-prone nature, random insertions or deletions of bases at the DNA break usually lead to DNA changes [13], which result in frameshift mutations in the original protein-coding strand, thereby disrupting the original protein composition. In contrast, HDR can be induced in vivo when a repair template is introduced. HDR consists primarily of the newly introduced target gene and homologous sequences at both ends of the cut site with the host template. HDR can be used to knock in a target fragment at the CRISPR cleavage site to achieve to expression of a specific gene [14], but it is much less efficient than NHEJ, and screening for an appropriate sgRNA and selecting the length of the homologous sequences are essential [15,16] (Fig 1).

FIGURE 1 |

CRISPR-Cas9 system cleavage and repair pathway. cr-RNA+tracr-RNA or sgRNA binds the Cas9 protein and forms the RNA-Cas9 RNP, which specifically recognizes the PAM in the gene sequence. Cleavage of double stranded DNA upstream of the PAM by the RuvC and HNH nuclease domains of the Cas9 protein forms double strand breaks. These double-strand breaks induce either NHEJ or HDR. NHEJ repair occurs through two main routes: random deletion or random insertion. HDR occurs through the introduction of repair templates containing homologous arms and a target sequence, and results in directed repair. Current mosquito gene editing strategies are based on embryo injection editing and ovary-targeted editing. This figure was created with BioRender.com.

In recent years, CRISPR-Cas9 technology has been applied to a variety of research fields, and has been widely used for gene editing in plants, including Oryza sativa [17,18], Triticum aestivum [19,20], and Arabidopsis thaliana [21]; in microorganisms, such as bacteria [22–24], fungi [25–27], and archaea [28]; and in animals, such as mosquitoes [29], zebrafish [30], mice [31], rabbits [32], and many others. This technology has even been used in human gene therapy [33].

CRISPR/CAS9 TECHNOLOGY IN MOSQUITOES

Mosquitoes may carry dengue fever, Zika, yellow fever, and chikungunya fever, and other flaviviruses of the arbovirus group [34], which cause widespread epidemics and morbidity, primarily in the tropics. With the intensification of global warming, epidemic trends have begun to emerge in some non-tropical countries [35], thus severely endangering human lives [36]. Suppressing mosquito populations or replacing wild populations with mosquitoes with diminished ability to act as vectors for viral pathogens would dramatically decrease the risk of insect-borne disease transmission. Since 1998, researchers have sought to genetically manipulate mosquitoes with ZFN and TALEN [37–39]. These research efforts have greatly accelerated with the maturation of CRISPR-Cas9 gene editing technology. Cas9 protein-nucleic acid complexes enable mosquito genes to be broken for gene editing. This process can result in random repair (i.e., NHEJ) or precision repair through the introduction of plasmids containing homologous sequences during the editing process (i.e., HDR); the latter technique is commonly used in gene-drive technology. Gene editing strategies have been developed for various mosquito species such as Aedes aegypti [40], Anopheles stephensi [41], Anopheles gambiae [29], and Culex burnetii [42]. Current directions in mosquito gene editing focus primarily on infectious disease prevention, population suppression, phenotype screening, and decreasing mosquito resistance to insecticides (Fig 2).

FIGURE 2 |

Application of CRISPR-Cas9 technology in mosquitoes and verification of gene functions. Illustrations of recent mosquito gene editing experiments with CRISPR-Cas9 technology. Currently, the main research directions for mosquito gene editing are as follows: ① infectious disease control, including anti-viral (AeRel1, Obp10, Obp22, GCTL-3, and CFAV-EVEs genes), anti-bacterial (OUT7B and Obp10 genes), and anti-plasmodium (CTL-4, Scorpine, SGS-1, LRIM-1, mosGILT, and FREP1 genes) targets; ② population suppression, including reproductive damage (CRVP379, Dsx, CYC, β-tubulin, Nix, GCTL-3, Fruitless, and miR-277/309 genes), pupation rate (5-HTR7A gene), and eclosion rate (E93, Myo-fem, AcAmt, Stretchin, and Act-4 genes) targets; ③ phenotypic screening, including olfaction (KMO, CYC, AcAmt, Gr22/23/24, and IR8a genes), appearance (Spn-F, White, Kh, DsRed, Op1/2, KMO, aaNAT1, Met, Ae-Forked, and 5-HTR2B genes), and activity (Stretchin, AeAct, PPK301, Myo-fem, and Spn-F genes) targets; and ④ insecticide resistance (V402L, L1014F, nAChR α6, mJHBP, NPYLR7, and CYP9M10 genes) targets. This figure was created with BioRender.com.

Prevention and control of mosquito-borne infectious diseases

The difficulty in developing vaccines for mosquito-borne pathogens and the increasing resistance of mosquitoes to insecticides have led to continued failure to effectively prevent the spread of mosquito-borne infectious diseases; therefore, a novel, safe, convenient, and efficient method to control mosquitoes is urgently needed [43–45]. With the advent of gene editing technology, researchers aim to regulate mosquito genes to prevent the reproduction and spread of infectious pathogens (arboviruses/parasites/bacteria). When a mosquito bites a pathogen-infected host, the pathogens enter the mosquito and reproduce, then further spread after the mosquito bites another healthy host [46,47]. Similarly to their mechanism of invasion in the human body, pathogens require receptor-mediated access to mosquito cells and the ability to escape the mosquito’s natural immune response to proliferate. On the basis of these characteristics, researchers have identified a series of key regulatory factors associated with pathogen replication and receptor-mediated access, through the analysis of the mosquito transcriptome and pathogenesis [48–51]. These findings can be applied to regulate immune effects in mosquitos through gene editing techniques. Currently, arboviruses and malaria remain the most critical mosquito-borne human health concerns. Major advances in this area are briefly reviewed below.

In the field of malaria research, Dong et al. have found that knocking out the fibrinogen-associated protein FREP1 gene in Anopheles gambiae inhibits Plasmodium falciparum and Plasmodium berghei infection. In addition, deletion of this gene significantly decreases blood-sucking, reproduction, and hatching efficiency; delays pupation; and shortens the mosquito lifespan, probably because the mosquito FREP1 gene functions similarly to fibrinogen proteins in other organisms, by directly affecting various developmental processes [52]. In another similar study, knockout of the immune response gene leucine-rich protein LRIM1 in Anopheles stephensi has been found to inhibit Plasmodium sporogony development via the melanization and phagocytosis of ookinetes. Plasmodium infection rates in mosquitoes subsequently decrease, together with the diversity and abundance of mosquito midgut microbiota. Additionally, deletion of the LRIM1 gene affects the reproductive capacity of mosquitoes, through a significant decrease in egg laying and hatching rates in females [53]. In 2022, the Simões research team reported that knockout of the CTL4 gene in Anopheles gambiae significantly increases Plasmodium falciparum suppression in mosquitoes, to a much greater extent than observed in gene-edited Anopheles mosquitoes with FREP1 host factor deletion and overexpression of the REL2 transcription factor [51,52]. Importantly, mosquitoes lacking CTL4 have elevated susceptibility to bacterial infection. Bacterial challenge assays have suggested that CTL4, in a melanization-independent manner, antagonizes systemic bacterial infections in mosquitoes [54]. Moreover, mutating the Anopheles gambiae lysosomal thiol reductase mosGILT34 [55] or inserting a known anti-microbial peptide, scorpion scorpine [56], has been shown to affect mosquito resistance to Plasmodium, thus enabling control of the spread of malaria epidemics. In addition, knockdown of the Aedes aegypti salivary gland surface protein SGS1 gene effectively attenuates the ability to carry Plasmodium gallinaceum, an avian malaria parasite. This discovery has provided new ideas for the prevention and treatment of human malaria diseases [57].

Dong’s research team has inhibited the transmission of arboviruses by disrupting the mosquito Obp10 and Obp22 genes, which encode odor-binding proteins that attenuate the secretion of virus particles into saliva; their exact mechanism of action is not understood and may work in conjunction with specific ligands [58]. The AaRel1 transcription factor mediates the antiviral effects of the mosquito Toll immune pathway. Overexpression of this gene in Aedes aegypti through CRISPR technology has been found to significantly suppress dengue virus serotype 2 titer in mosquitoes. Theoretically, this inhibition should also be effective against Zika virus [59]. Li’s team has found that mutation of GCTL-3 in Aedes aegypti also enhances suppression of both dengue II and Zika viruses [60]. A possible reason for this finding may be that GCTL-3 gene mutation causes a lack of Serratia marcescens, a bacterium that promotes viral transmission, in the mosquito midgut [61]. With advances in sequencing technology, host genomes have been discovered to frequently contain fragments of viral genomes known as endogenous viral elements (EVEs). EVEs integrated into mosquito genomes are believed to be closely associated with the corresponding viral infections. Suzuki’s team has used CRISPR-Cas9 technology to knock out the EVE gene of the cell fusion virus CFAV in Aedes aegypti, thus enhancing replication of CFAV in the ovaries, and has experimentally demonstrated that the interaction of RNAs from EVEs and viruses through the piRNA pathway inhibits viral replication, thereby suppressing viral transmission by mosquitoes [62]. Editing the non-retroviral EVE locus may provide new ideas for future prevention and control of mosquito-borne infectious diseases.

Mosquito population suppression

To control the risks inherent in artificially regulating mosquito populations, researchers have focused on altering mosquito motility, disrupting their sex-determining genes or reproductive functions, and then releasing the genetically edited mosquitoes into the wild to interfere with reproduction, through a method known as the sterile insect technique (SIT). The advent of gene drivers has altered the traditional Mendelian laws of inheritance by biasing the heritability of insects containing specific genes toward supra-Mendelian heritability (>50%), and has enabled the modification of populations, but their development has been slow, because of difficulties in application [63]. The emergence of CRISPR-Cas9, combined with gene-drive technology, has greatly decreased difficulties in population manipulation and opened new possibilities for mosquito population control. Consequently, researchers are continually searching for genes that regulate the physiological activity or sex determination of mosquitoes, to suppress their numbers or modify entire populations.

Numerous research teams are currently targeting male mosquitoes. Some have shown that knocking out the β2-tubulin (B2t) gene in Aedes aegypti significantly inhibits the fertility of male mosquitoes, most of which fail to produce progeny after mating with females [64], whereas knocking out the B2t gene in Anopheles gambiae causes X chromosome fragmentation in males and significantly increases the proportion of male mosquitoes in the population [65]. The presence of the fruitless gene in insects is a major driver of male courtship and mating behavior. The Basrur team has used CRISPR-Cas9 to knock out the fruitless gene in Aedes aegypti, thus preventing males from mating and reproducing. Interestingly, male mosquitoes lacking the fruitless gene are strongly attracted to human hosts and exhibit altered male feeding behavior; these findings warrant more intensive study [66]. Hall et al. have knocked out the male determinant Nix gene in Aedes aegypti, and subsequently observed incomplete male genital development and thus decreased reproductive capacity [67].

Studies are increasingly being conducted on female rather than male loci. Knocking out the tweedledee and tweedledum genes in Aedes aegypti effectively increases the activity and preservation time of eggs; females robustly retain eggs in their ovaries until freshwater is found, thus facilitating the search for areas suitable for egg-laying [68]. Female reproductive function is affected by mutations in the cysteine-rich toxin CRVP379 gene [69], β-Tubulin 85D gene [70], and core clock CYC gene [71] in Aedes aegypti; the kynurense hydroxylase kh gene in Anopheles stephensi [72]; and the dsx gene in Anopheles gambiae [73,74]. Mutations in miR-277 and miR-309 both affect ovarian development in Aedes aegypti, thus leading to population suppression [75,76], whereas mutations in the 5-hydroxytryptamine receptor 5-HTR7A gene [77] and myo-fem gene cause restricted motility in females, thereby affecting their normal reproductive function and decreasing their fertility [70].

Mosquito phenotypic screening

Genes determining the phenotypic characteristics of mosquitoes have been an additional research focus. These genes broadly regulate alterations in olfaction, motility, and appearance. Alterations in olfaction directly contribute to mosquitoes’ ability to discriminate between human hosts. CRISPR-based studies have shown that mutations in the odor-binding protein co-receptor Orco alter mosquito olfaction and attenuate mosquito localization to human hosts [78,79]. Disruption of the core clock gene CYC in Aedes aegypti eliminates the characteristic circadian patterns of locomotor activity and significantly decreases the response to human host odor and mating success [71], whereas the taste receptor gene Gr23/24 [80] and the pro-ionotropic receptor Ir8a gene [81] significantly alter olfactory ability in mosquitoes. Moreover, knocking out the ammonium transporter protein AcAmt gene, which is involved in multiple systems of mosquito olfaction, reproduction, and ammonia metabolism, severely affects motility [82]. Similarly, knockout of the muscle protein Stretchin gene [83], actin AeAct-4, and myo-fem gene all affect mosquitoes’ flight ability [84,85], thus further influencing physiological functions. Mutations in the White [86,87], Kh [86,88], Cardinal [86], Yellow [86,88], Ebony [86], DsRed [89], Kmo [90,91], Met [91,92], and ECFP genes [93] result in altered eye characteristics and pigmentation. Mutations in spn-F [94] affect the formation of bristle and falcate scales. Moreover, mutations in the aromatic amine-N-acetyltransferase aaNAT1 [95] result in an altered appearance, wherein the outer surface of the skeleton of mutant mosquitoes is rough, and the cuticle is dark in color. Nix [96] knockout males show feminization. The forked gene Ae-Forked [97] is required for bristle elongation. Finally, knockout of the 5-hydroxytryptamine receptor 5-HTR2B gene [98] leads to decreased body size, postponed development, shortened lifespan, retarded ovarian growth, and markedly diminished lipid accumulation. Together, these insights have demonstrated how CRISPR-Cas9 technology has enabled researchers to explore previously unknown functions of mosquito genes.

Mosquito insecticide resistance-related gene studies

Mutations at sites on the pyrethroid voltage-gated sodium channel VGSC can alter insecticide resistance. A study using CRISPR-Cas9 technology has demonstrated that mutations at both the V402L and L1014F sites in Anopheles gambiae can increase mosquito resistance to pyrethroids. Additionally, the L1014F mutation increases mosquito resistance to permethrin by 9.9-fold and to dichlorodiphenyltrichloroethane (DDT) by 24-fold [99,100]. In contrast, deletion of the cytochrome P450 gene CYP9M10 in Aedes aegypti results in a more than 100-fold decrease in mosquito resistance to pyrethroid [42]. The Lan research team has used CRISPR-Cas9 technology to mutate the nAChR α6 subunit of the nicotinic acetylcholine receptor in Anopheles gambiae, thus resulting in a 320-fold increase in mosquito resistance to spinosad [101]. Moreover, mutations in the mJHBP gene, encoding a juvenile-protective hormone-binding protein in Aedes aegypti, enhance mosquito susceptibility to bacteria, and significantly increase sepsis and mortality in mosquitoes attacked by bacteria [102].

Researchers are increasingly using CRISPR-Cas9 technology to target mosquito genes with gene editing to elucidate their functions. Table 1 summarizes the literature on mosquito gene editing based on CRISPR-Cas9, including the species of mosquito edited, the countries and research sponsors, the edited genes and their functions, and the survival rates of eggs after editing and the gene editing frequency. Notably, researchers use differing methods for calculating gene editing frequencies. These methods can be divided into two calculation modes: No. (mosquitoes with successful gene editing)/No. (injected mosquito eggs) and No. (mosquitoes with successful gene editing)/No. (mosquito eggs surviving after injection). The chosen calculation method leads to differences in the results of gene editing frequencies.

TABLE 1 |

CRISPR/Cas9 in mosquito research.

Year	Species	Country and sponsor	Gene(s)	Function in mosquitoes	Survival and GEF a	Reference
Mosquito-borne Infectious Disease Control
2023	Aedes aegypti	United States of America University of California San Diego	AaRel I	Suppression of DENV2 titer	Unknown	[59]
2023	Aedes aegypti	China Chinese Academy of Sciences	OTU7B	Enhanced resistance to fungal infection	Unknown	[103]
2022	Anopheles gambiae	United States of America Johns Hopkins University	CTL-4	Suppression of Plasmodium falciparum	Unknown	[54]
2021	Anopheles gambiae	United Kingdom Imperial College London	Scorpine	Suppression of Plasmodium falciparum	7.0% (62/889)/1.2% (11/889) b	[56]
2021	Aedes aegypti	United States of America Texas A&M University	SGS1	Decreased Plasmodium sporozoite invasion of salivary glands	19% (87/445)/1.1% (5/445) b	[57]
2021	Anopheles stephensi	United States of America Sanaria Inc. Rockville	LRIM I	Suppression of Plasmodium sporozoite development	3.5% (19/549)/0.1% (1/549) b	[53]
2021	Aedes aegypti	United States of America Johns Hopkins University	Obp10/22	Suppression of DENV2 and ZIKV transmission	53.4% (103/193)/50% (3/6) c	[58]
2020	Anopheles gambiae	United States of America Yale University School of Medicine	mosGILT	Decreased oocyst numbers after Plasmodium infection	15% (87/580)/4.5% (26/580) b	[55]
2020	Aedes aegypti	China National Tsing Hua University	GCTL-3	Decreased DENV-2 infection rate	26% (210/795)/1.1% (2/210) c	[60]
2020	Aedes aegypti	France Institut Pasteur	CFAV-EVEs	Enhanced CFAV replication in ovaries	Unknown	[62]
2018	Anopheles gambiae	United States of America Johns Hopkins University	FREP I	Suppression of Plasmodium infection	10%/3.5% (28/800) b	[52]
Mosquito Population Suppression
2023	Aedes aegypti	United States of America Rockefeller University	Tweedledum	Enhanced egg retention	Unknown	[68]
2022	Aedes aegypti	China Hainan University	5-HTR7A	Abnormal pupa development	7.7% (14/180)/60% (6/10) c	[77]
2022	Aedes aegypti	United States of America Johns Hopkins Bloomberg School	CRVP379	Decreased reproductive capacity in females	33.87% (83/245)/5.7% (14/245) b	[69]
2022	Aedes aegypti	United States of America University of Kentucky	E93	Completed pupal ecdysis, pupal death	25% (150/600)/unknown	[104]
2022	Aedes aegypti	Pakistan Government College University Faisalabad	DsxF1/F2	Decreased fecundity rate and hatching rate	51.11% (230/450), 54.00% (243/450)/36.59%, 29.36%	[105]
2022	Aedes aegypti	United States of America Texas A&M University	CYC	Decreased egg hatching rate and adult survival rate	Unknown	[71]
2021	Aedes aegypti	United States of America University of California	Beta-tubulin	Suppression of fertility	Unknown 2.6% (18/700)/0.4% (3/700) b	[70,64]
2021	Aedes aegypti	United States of America University of California	Myo-fem	Decreased flight capacity	Unknown	[70]
2021	Anopheles gambiae	United States of America Vanderbilt University	AcAmt	Decreased insemination rate Increased mortality during eclosion	Unknown	[82]
2021	Aedes aegypti	United States of America Texas A&M University	Stretchin	Decreased flight capacity	7.1% (57/800)/0.25% (2/800) b	[83]
2020	Aedes albopictus	China Southern Medical University	Nix	Feminization of males	28.7% (286/996)/3.6% (36/996) b	[96]
2020	Anopheles stephensi	United States of America University of California	Kh	Decreased survival rate Impaired reproductive ability	51.4% (259/504)/unknown	[72]
2020	Aedes aegypti	China National Tsing Hua University	GCTL-3	Decreased reproductive ability	Unknown	[60]
2020	Anopheles gambiae	United Kingdom Imperial College London	Dsx	Production of unisex populations	Unknown	[73]
2020	Aedes aegypti Culex quinquefasciatus	United Kingdom The Pirbright Institute	Act-4	Interference with flight muscle function	0.7% (6/915)/ 17% (1/6) c	[84]
2020	Aedes aegypti	United States of America The Rockefeller University	Fruitless	Failure of males to mate	Unknown	[66]
2018	Anopheles gambiae	United Kingdom Imperial College London	Dsx	Intersex phenotype and complete sterility	Unknown	[74]
2017	Aedes aegypti	United States of America University of California	miR-277	Suppression of ovarian development	41%/unknown	[75]
2016	Anopheles gambiae	United Kingdom Imperial College London	AGAP007280 AGAP011377 AGAP005958	Recessive female sterility phenotype	10.3% (56/540)/7.1% (4/56) c 4.2% (21/500)/4.8% (1/21) c 12.3% (49/400)/2.0% (1/49) c	[29]
2016	Anopheles gambiae	United Kingdom Imperial College London	Beta-tubulin	Extreme male bias among progeny	Unknown	[65]
2016	Aedes aegypti	United States of America University of California	miR-309	Impaired ovarian development	51.5% (103/200)/64.1% (66/103) c	[76]
2015	Aedes aegypti	United States of America Virginia Polytechnic Institute and State University	Nix	Virilization of females	Unknown	[67]
Mosquito Phenotypic Screening
2022	Anopheles sinensis	China Chongqing Normal University	Orco	Impaired mosquito sensitivity to human hosts	Unknown	[78]
2022	Aedes aegypti	Israel Ben-Gurion University of the Negev	Spn-F	Tapered epithelial cellular extension structures	Unknown	[94]
2022	Aedes aegypti	United States of America Texas A&M University	CYC	Diminished response to host odor	Unknown	[71]
2021	Culex quinquefasciatus	United States of America University of California San Diego	White /Kh /Cardinal /Yellow /Ebony	Appearance change	10.01% (34/337)/unknown 11.5% (46/400)/unknown 11.0% (32/290)/unknown 11.4% (25/220)/unknown 31.3% (90/288)/unknown	[86]
2021	Culex quinquefasciatus	United Kingdom The Pirbright Institute	DsRed	White eye phenotype	14.8% (57/384)/0%	[89]
2021	Aedes aegypti	United States of America University of California	Op1/Op2	Abolished vision-guided target attraction	Unknown	[106]
2021	Culex pipiens pallens	China Nanjing Medical University	Kmo	White and mosaic eye phenotypes	Unknown (ReMOT)	[90]
2021	Aedes aegypti	China Hainan University	aaNAT1	Cuticular pigmentation formation	5.6% (17/302)/unknown	[95]
2021	Anopheles gambiae	United States of America Vanderbilt University	AcAmt	Peripheral neuron sensitivity to ammonia	Unknown	[82]
2021	Aedes aegypti	United States of America Texas A&M University	Stretchin	Abnormal open wing posture	Unknown	[83]
2021	Aedes aegypti	United States of America University of Kentucky	Kmo/Met	Mosaic eye phenotypes	Unknown/ 0.6% (3/500) b	[91]
2020	Aedes aegypti	United States of America Texas A&M University	AeAct-4/Myo-fem	Flightlessness in females	20.4% (83/407)/unknown	[85]
2020	Aedes aegypti Aedes albopictus	China Southern Medical University	Nix	Feminization and deformities in males	34.9% (213/609)/14.1% (30/213) c 34.7% (279/805)/24.0% (67/279) c 24.7% (246/996)/14.6% (36/246) c (Data from multiple experiments)	[96]
2020	Aedes aegypti	Israel Ben-Gurion University of the Negev	Ae-Forked	Required for bristle elongation	Unknown	[97]
2020	Anopheles coluzzii	United States of America Vanderbilt University	Gr22/23/24	Required for CO2 sensitivity	Unknown	[80]
2020	Aedes aegypti Culex quinquefasciatus	United Kingdom The Pirbright Institute	Act-4	Interference with flight muscle function	0.7% (6/915)/unknown 4.0% (67/1802)/unknown 9.0% (68/736)/unknown (Data from multiple experiments)	[84]
2020	Anopheles coluzzii	United States of America Vanderbilt University	Orco	Decreased attraction to human odors	Unknown	[79]
2019	Aedes aegypti	United States of America The Rockefeller University	Ppk301	Altered spawning capacity	Unknown	[107]
2019	Aedes aegypti	United States of America University of Kentucky	Kmo-A Kmo-B Kmo-C Kmo-D	Black larval phenotype	48.2% (281/583)/0% (0/255) c 27.4% (131/478)/8.2 (8/97) c 33.3% (72/216)/6% (4/65) c 22.1% (62/280)/0% (0/62) c	[92]
2019	Aedes albopictus	China Southern Medical University	Kh Yellow	Eye and body pigmentation defects	72.91% (148/203)/51.35% (76/138) c 87.03% (161/185)/32.92% (53/161) c	[88]
2019	Aedes aegypti	United States of America Florida International University	Ir8a	Decreased attraction to human odors	Unknown	[81]
2018	Anopheles gambiae	United States of America University of California	White	White and mosaic eye phenotypes	37% (69/185)/46% (32/69) c 32% (81/251)/59% (48/81) c 21% (47/219)/87% (41/47) c 19% (33/177)/88% (29/33) c 11% (24/228)/92% (22/24) c	[87]
2018	Aedes aegypti	United States of America University of California	5-HTR2B	Decreased body size	40%/unknown	[98]
2015	Aedes aegypti	United States of America University of Missouri	ECFP	Changes in eye characteristics	11.7% (73/626)/5.5% (4/73) c	[93]
Mosquito Resistance
2022	Anopheles gambiae	United Kingdom Liverpool School of Tropical Medicine	V402L	Decreased mortality after exposure to pyrethroids and DDT	Unknown	[99]
2021	Anopheles gambiae	United Kingdom Liverpool School of Tropical Medicine	L1014F	Decreased mortality after exposure to pyrethroids and DDT	Unknown	[100]
2021	Anopheles gambiae	China Hainan University	nAChR alpha6	Decreased mortality after exposure to spinosad	Unknown	[101]
2020	Aedes aegypti	United States of America NIH/NIAID Laboratory of Malaria and Vector Research	mJHBP	Decreased antimicrobial resistance	16.7% (20/120)/unknown	[102]
2016	Culex quinquefasciatus	Japan National Institute of Infectious Diseases	CYP9M10	Decreased mortality after exposure to pyrethroids	Unknown/3% (4/128) c	[42]

^aSurvival and GEF indicates egg survival rate/gene editing frequencies.

^bNo. (mosquitoes with successful gene editing)/No. (injected mosquito eggs).

^cNo. (mosquitoes with successful gene editing)/No. (mosquito eggs survived after injection).

DEVELOPMENT OF CRISPR-CAS9 DERIVED TECHNOLOGIES

New technologies in mosquitoes

Currently, CRISPR-Cas9-based mosquito editing techniques rely on the microinjection [15] of sgRNA and Cas9 protein or Cas9 mRNA into individual embryos within mosquito eggs [108–110]. This process is technically difficult and costly; requires rigorous experimental equipment and experimenter handling; and has extremely low injection success rates, thus causing irreparable damage to mosquito eggs [111]. To overcome these difficulties, Lule-Chávez has used an innovative particle inflow gun technique to transfer Cas9 protein and sgRNA into Aedes aegypti and Anopheles gambiae eggs via particle bombardment. This process not only increases transformation efficiency and survival rates, but also is faster, simpler, and less expensive than micro-injection [112].

The Chaverra-Rodriguez research team has pioneered the development of a technique called receptor-mediated ovary transduction of cargo (ReMOT Control), which bypasses the step of mosquito egg embryo injection (Fig 3). For introducing targeted heritable mutations, high concentrations of Cas9 sgRNA complexes are directly injected into the hemolymph in female Aedes aegypti 24 hours after blood feeding [113]. Notably, the Cas9 protein used in ReMOT Control is not a commercial protein but a Cas9 fusion protein with a Drosophila melanogaster yolk protein tag (DmYP), which has been shown to effectively target the ovaries of some insects. Compared with ordinary commercial Cas9 proteins, this fusion Cas9 protein has stronger targeting properties and therefore shows better gene editing performance. This technique has been used to edit the Kmo, Kh, ECFP, and DsRed genes of Anopheles stephensi. Given the differences in developmental characteristics between Anopheles stephensi and Aedes aegypti, injection 48 hours after blood feeding has been shown to efficiently induce mutations in Anopheles stephensi progeny. In addition, the use of saponin substantially increases the efficiency of gene knockout, achieving results similar to those with use of chloroquine; therefore, the agents used to mediate intranuclear endosomal escape may influence the effects of ReMOT [114]. Li’s team has used the ReMOT Control technique to knock out the Kmo gene in Culex paleus and have found that 24 h after blood feeding is a preferable injection time for this species [90].

FIGURE 3 |

Difference between ReMOT Control technology and traditional embryo Micro-injection. A Traditional CRISPR-Cas9-based gene editing in mosquitoes relies on micro-injection of sgRNA+Cas9 RNP into freshly laid eggs to achieve gene editing in mosquitoes. B ReMOT Control bypasses the step of mosquito egg injection and directly injects sgRNA+fusion Cas9 RNP into the hemolymph of female mosquitoes after blood feeding; the RNP circulates to the developing ovaries, thus achieving gene editing in offspring. This figure was created with BioRender.com.

New technologies in other vectors

Ticks carry and transmit a variety of viruses and microorganisms [115]. Researchers have attempted to genetically edit tick eggs with CRISPR-Cas9; however, the development of gene editing in ticks has been hindered by their hard chorionic membrane, high intra-egg pressure, and the female tick’s gene’s organ, which encases the eggs in a waxy substance [116,117]. The advent of ReMOT Control technology has substantially overcome these problems, thereby enabling successful editing of the genes of some tick progeny through direct injection of Cas9 protein complexes into female ticks. All injected ticks have been reported to survive. To date, researchers have successfully edited the genes of Nasonia vitripennis [118], the red flour beetle Tribolium castaneum [119], and the Silverleaf Whitefly [120] with ReMOT Control technology. Moreover, this technique has been effectively used in the treatment of the notable disease vector Rhodnius prolixus. The development of mosquito gene editing has been greatly advanced by the emergence of ReMOT Control technology, which overcomes many of the drawbacks of microscopic embryo injection and enables widespread use of the CRISPR system. However, current research is limited and requires continuous screening and optimization of injection conditions for different species to achieve higher editing efficiency.

Recently, Shirai’s research team has pioneered the development of direct parental CRISPR (DIPA-CRISPR), a new technology based on ReMOT Control. The primary difference between this method and ReMOT Control is that the DIPA technique uses commercial Cas9 proteins rather than fusion proteins with DmYP tags. Shirai’s team has discovered that co-injection of nontagged Cas9 protein with the sgRNA protein complex into female Blattella germanica effectively delivers the protein to developing oocytes and alters the progeny’s genes. This method may potentially be used to eradicate cockroaches at the genetic level [121]. The team has also successfully used this protocol to edit the cardinal gene of Tribolium castaneum and change its eye color, thus demonstrating its broad applicability to certain insects [120]. Given the lack of a requirement to express tagged fusion Cas9 proteins, the researchers believe that DIPA-CRISPR technology can be used in laboratory settings to expand the scope of gene editing in insects more easily and effectively. This technique could theoretically also be applied to mosquitoes, thereby further simplifying the experimental process and allowing more laboratories perform gene editing.

DISCUSSION

As global warming increases, the spread of mosquito-borne viruses is gradually increasing each year, and the task of preventing and controlling mosquitoes and mosquito-borne infectious diseases is critical. Mosquito-borne flaviviruses have similar sequences and structures, and extensive antibody cross-reactivity has slowed vaccine development. For example, no effective preventive measures are available for mosquito-borne dengue fever, which causes many infections and deaths every year. The emergence of gene editing has provided new hope for mosquito control and prevention.

The advent of CRISPR gene editing technology has intensified the modification of mosquito genes in recent years. Researchers have discovered a series of genes associated with mosquito phenotypes, resistance, and viral interaction. Several public databases in China and other countries contain transcriptomic data from mosquito-related experiments, with an aim to gather experimental data to discover more genetic loci associated with mosquito physiology and virus transmission, thus advancing the study of mosquito gene function. Gene editing for mosquitoes is focused primarily on physiological functions and vector efficacy. However, with the aid of genomic data on mosquitoes or other vector organisms, future research may encompass a more expansive range of topics, including a deeper exploration of gene function and its potential applications in the prevention and control of vector-borne diseases. Moreover, researchers have optimized experimental methods. Hsing-Han Li has summarized the various experimental methods used for mosquito-based CRISPR gene editing and evaluated their advantages and disadvantages, thus providing a valuable reference for researchers [122]. New technology for gene editing continues to emerge, to address the shortcomings of traditional embryo injections, simplify the difficulties experimental manipulation, and enable more laboratories to participate in modifying insects’ genes. In the future, the optimization and innovation of new technologies such as ReMOT Control and DIPA-CRISPR may be used to screen target genes applicable to mosquito oocytes, whereas precise yolk formation time may be considered for different mosquito species to achieve higher gene editing efficiency. Notably, these traditional and novel techniques have been successfully used for gene editing in other disease vectors, thus facilitating the investigation of gene function and regulation.

Although irradiated mosquito SIT technology has been implemented in many countries, its high cost and the mating difficulties between irradiated male mosquitoes and wild female mosquitoes have prevented this technology from achieving satisfactory results in mosquito control. With the advent of CRISPR technology, scientists have continued to attempt to create gene-drive mosquitoes, to achieve replacement of wild-type mosquitos with genetically modified mosquitoes by altering the sex genes and sterilizing offspring, or by altering the ability to transmit pathogens to decrease the spread of disease [123]. As with other CRISPR-Cas9 gene editing, the potential for off-target effects of genes must be considered. Mosquito gene knockout also necessitates the use of whole-genome sequencing and off-target site prediction software to mitigate the risk of off-target effects. Additionally, off-target site prediction based on AI modeling and bioinformatics is currently available to enhance the stability of gene editing. However, gene-drive methods, such as insect irradiation SIT and even Wolbachia, require the release of large numbers of mosquitoes to alter wild populations. Whether gene-drive mosquitoes might affect other populations or the environment, and whether they are safe, remains unknown. However, these gene-drive mosquitoes have been found to work satisfactorily in small-scale laboratory replacement [124], and several countries have established mosquito factories to release Wolbachia-carrying mosquitoes in the wild, which is indeed effective in decreasing mosquito populations. The release of mosquitoes in the wild that are unable to transmit the virus, thereby replacing wild populations, has been shown to limit viral transmission. Nonetheless, whether these viruses might infect other new vectors that might subsequently infect humans is an important consideration. Additionally, the absence of the virus in mosquitoes might possibly result in the evolution of new phenotypes and potentially lead to attenuation of gene-drive suppression. Whether the decrease in mosquito numbers achieved with gene-drives might increase the number of remaining vector organisms, thereby elevating the risk of other disease transmission, additionally remains unclear. How to regulate these engineered mosquitoes, and what policies and regulations should be put in place, will require further consideration [125–127]. Discussions are ongoing as to whether releasing gene-drive mosquitoes into the wild on a large scale is ethical. Finally, the release of gene-drive mosquitoes might raise fears among the public [128] (such as whether their release might change the diet of traditional mosquitoes, transmit new pathogens, or alter human genetics through bites). The release of gene-drive mosquitoes to replace wild populations, and further prevent and control the spread of arboviruses, will require implementation of robust regulatory frameworks and policies to oversee the process and assess its efficacy, to maintain a balance between the application and effects of this strategy. A positive balance in which the potential benefits of releasing gene-drive mosquitoes outweigh the potential harms might indicate that the latter are not significant, that the likelihood of their occurrence is tolerable, and that reliable mitigation strategies could address the potential harms. How to best address these questions warrants further in-depth study by researchers and government regulators.

ACKNOWLEDGEMENTS

This research received no external funding.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

REFERENCES

1. Chen XG, Jiang X, Gu J, Xu M, Wu Y, Deng Y, et al.. Genome sequence of the Asian Tiger mosquito, Aedes albopictus, reveals insights into its biology, genetics, and evolution. Proc Natl Acad Sci U S A. 2015. Vol. 112(44):E5907–E5915

2. Neafsey DE, Waterhouse RM, Abai MR, Aganezov SS, Alekseyev MA, Allen JE, et al.. Mosquito genomics. Highly evolvable malaria vectors: the genomes of 16 Anopheles mosquitoes. Science. 2015. Vol. 347(6217):1258522

3. Kim YG, Shi Y, Berg JM, Chandrasegaran S. Site-specific cleavage of DNA-RNA hybrids by zinc finger/FokI cleavage domain fusions. Gene. 1997. Vol. 203(1):43–49

4. Moscou MJ, Bogdanove AJ. A simple cipher governs DNA recognition by TAL effectors. Science. 2009. Vol. 326(5959):1501

5. Jiang F, Doudna JA. CRISPR-Cas9 structures and mechanisms. Annu Rev Biophys. 2017. Vol. 46:505–529

6. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012. Vol. 337(6096):816–821

7. Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A. 2012. Vol. 109(39):E2579–E2586

8. van der Oost J, Westra ER, Jackson RN, Wiedenheft B. Unravelling the structural and mechanistic basis of CRISPR-Cas systems. Nat Rev Microbiol. 2014. Vol. 12(7):479–492

9. Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada Z, et al.. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 2011. Vol. 471(7340):602–607

10. Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014. Vol. 346(6213):1258096

11. Heler R, Samai P, Modell JW, Weiner C, Goldberg GW, Bikard D, et al.. Cas9 specifies functional viral targets during CRISPR-Cas adaptation. Nature. 2015. Vol. 519(7542):199–202

12. Bae S, Kweon J, Kim HS, Kim JS. Microhomology-based choice of Cas9 nuclease target sites. Nat Methods. 2014. Vol. 11(7):705–706

13. Gantz VM, Bier E. The mutagenic chain reaction: a method for converting heterozygous to homozygous mutations. Science. 2015. Vol. 348(6233):442–444

14. Roth TL, Li PJ, Blaeschke F, Nies JF, Apathy R, Mowery C, Yu R, et al.. Pooled knockin targeting for genome engineering of cellular immunotherapies. Cell. 2020. Vol. 181(3):728–744.e21

15. Li M, Li T, Liu N, Raban RR, Wang X, Akbari OS. Methods for the generation of heritable germline mutations in the disease vector Culex quinquefasciatus using clustered regularly interspaced short palindrome repeats-associated protein 9. Insect Mol Biol. 2020. Vol. 29(2):214–220

16. Li M, Bui M, Yang T, Bowman CS, White BJ, Akbari OS. Germline Cas9 expression yields highly efficient genome engineering in a major worldwide disease vector, Aedes aegypti . Proc Natl Acad Sci U S A. 2017. Vol. 114(49):E10540–E10549

17. Lowder LG, Zhang D, Baltes NJ, Paul JW 3rd, Tang X, Zheng X, et al.. A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol. 2015. Vol. 169(2):971–985

18. Mikami M, Toki S, Endo M. Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice. Plant Mol Biol. 2015. Vol. 88(6):561–572

19. Zhang Y, Liang Z, Zong Y, Wang Y, Liu J, Chen K, et al.. Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nat Commun. 2016. Vol. 7:12617

20. Shan Q, Wang Y, Li J, Gao C. Genome editing in rice and wheat using the CRISPR/Cas system. Nat Protoc. 2014. Vol. 9(10):2395–2410

21. Feng Z, Mao Y, Xu N, Zhang B, Wei P, Yang DL, et al.. Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis . Proc Natl Acad Sci U S A. 2014. Vol. 111(12):4632–4637

22. Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol. 2013. Vol. 31(3):233–239

23. Zhang K, Duan X, Wu J. Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system. Sci Rep. 2016. Vol. 6:27943

24. Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol. 2015. Vol. 81(7):2506–2514

25. Shao Y, Lu N, Wu Z, Cai C, Wang S, Zhang LL, et al.. Creating a functional single-chromosome yeast. Nature. 2018. Vol. 560(7718):331–335

26. Liu R, Chen L, Jiang Y, Zhou Z, Zou G. Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system. Cell Discov. 2015. Vol. 1:15007

27. Liu Q, Gao R, Li J, Lin L, Zhao J, Sun W, et al.. Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering. Biotechnol Biofuels. 2017. Vol. 10:1

28. Nayak DD, Metcalf WW. Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans . Proc Natl Acad Sci U S A. 2017. Vol. 114(11):2976–2981

29. Hammond A, Galizi R, Kyrou K, Simoni A, Siniscalchi C, Katsanos D, et al.. A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae . Nat Biotechnol. 2016. Vol. 34(1):78–83

30. Auer TO, Duroure K, De Cian A, Concordet JP, Del Bene F. Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. Genome Res. 2014. Vol. 24(1):142–153

31. Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, et al.. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat Methods. 2014. Vol. 11(4):399–402

32. Song Y, Yuan L, Wang Y, Chen M, Deng J, Lv Q, et al.. Efficient dual sgRNA-directed large gene deletion in rabbit with CRISPR/Cas9 system. Cell Mol Life Sci. 2016. Vol. 73(15):2959–2968

33. Lu Y, Xue J, Deng T, Zhou X, Yu K, Deng L, et al.. Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer. Nat Med. 2020. Vol. 26(5):732–740

34. Gaunt MW, Sall AA, Lamballerie X, Falconar AKI, Dzhivanian TI, Gould EA. Phylogenetic relationships of flaviviruses correlate with their epidemiology, disease association and biogeography. J Gen Virol. 2001. Vol. 82(Pt 8):1867–1876

35. Gascon J, Bern C, Pinazo MJ. Chagas disease in Spain, the United States and other non-endemic countries. Acta Trop. 2010. Vol. 115(1-2):22–27

36. Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, et al.. The global distribution and burden of dengue. Nature. 2013. Vol. 496(7446):504–507

37. Jasinskiene N, Coates CJ, Benedict MQ, Cornel AJ, Rafferty CS, James AA, et al.. Stable transformation of the yellow fever mosquito, Aedes aegypti, with the Hermes element from the housefly. Proc Natl Acad Sci U S A. 1998. Vol. 95(7):3743–3747

38. Allen ML, O’Brochta DA, Atkinson PW, Levesque CS. Stable, germ-line transformation of Culex quinquefasciatus (Diptera: Culicidae). J Med Entomol. 2001. Vol. 38(5):701–710

39. Smidler AL, Terenzi O, Soichot J, Levashina EA, Marois E. Targeted mutagenesis in the malaria mosquito using TALE nucleases. PLoS One. 2013. Vol. 8(8):e74511

40. Kistler KE, Vosshall LB, Matthews BJ. Genome engineering with CRISPR-Cas9 in the mosquito Aedes aegypti . Cell Rep. 2015. Vol. 11(1):51–60

41. Gantz VM, Jasinskiene N, Tatarenkova O, Fazekas A, Macias VM, Bier E, et al.. Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi . Proc Natl Acad Sci U S A. 2015. Vol. 112(49):E6736–E6743

42. Itokawa K, Komagata O, Kasai S, Ogawa K, Tomita T. Testing the causality between CYP9M10 and pyrethroid resistance using the TALEN and CRISPR/Cas9 technologies. Sci Rep. 2016. Vol. 6:24652

43. Pryce J, Richardson M, Lengeler C. Insecticide-treated nets for preventing malaria. Cochrane Database Syst Rev. 2018. Vol. 11(11):Cd000363

44. Ranson H, Lissenden N. Insecticide resistance in African Anopheles mosquitoes: a worsening situation that needs urgent action to maintain malaria control. Trends Parasitol. 2016. Vol. 32(3):187–196

45. Moyes CL, Vontas J, Martins AJ, Ng LC, Koou SY, Dusfour I, et al.. Contemporary status of insecticide resistance in the major Aedes vectors of arboviruses infecting humans. PLoS Negl Trop Dis. 2017. Vol. 11(7):e0005625

46. Franz AW, Kantor AM, Passarelli AL, Clem RJ. Tissue barriers to arbovirus infection in mosquitoes. Viruses. 2015. Vol. 7(7):3741–3767

47. McMeniman CJ, Corfas RA, Matthews BJ, Ritchie SA, Vosshall LB. Multimodal integration of carbon dioxide and other sensory cues drives mosquito attraction to humans. Cell. 2014. Vol. 156(5):1060–1071

48. Volohonsky G, Hopp AK, Saenger M, Soichot J, Scholze H, Boch J, et al.. Transgenic expression of the anti-parasitic factor TEP1 in the malaria mosquito anopheles gambiae. PLoS Pathog. 2017. Vol. 13(1):e1006113

49. Moreira LA, Ito J, Ghosh A, Devenport M, Zieler H, Abraham EG, et al.. Bee venom phospholipase inhibits malaria parasite development in transgenic mosquitoes. J Biol Chem. 2002. Vol. 277(43):40839–40843

50. Isaacs AT, Jasinskiene N, Tretiakov M, Thiery I, Zettor A, Bourgouin C, et al.. Transgenic Anopheles stephensi coexpressing single-chain antibodies resist Plasmodium falciparum development. Proc Natl Acad Sci U S A. 2012. Vol. 109(28):E1922–E1930

51. Dong Y, Das S, Cirimotich C, Souza-Neto JA, McLean KJ, Dimopoulos G. Engineered Anopheles immunity to Plasmodium infection. PLoS Pathog. 2011. Vol. 7(12):e1002458

52. Dong Y, Simões ML, Marois E, Dimopoulos G. CRISPR/Cas9-mediated gene knockout of Anopheles gambiae FREP1 suppresses malaria parasite infection. PLoS Pathog. 2018. Vol. 14(3):e1006898

53. Inbar E, Eappen AG, Alford RT, Reid W, Harrell RA, Hosseini M, et al.. Knockout of Anopheles stephensi immune gene LRIM1 by CRISPR-Cas9 reveals its unexpected role in reproduction and vector competence. PLoS Pathog. 2021. Vol. 17(11):e1009770

54. Simões ML, Dong Y, Mlambo G, Dimopoulos G. C-type lectin 4 regulates broad-spectrum melanization-based refractoriness to malaria parasites. PLoS Biol. 2022. Vol. 20(1):e3001515

55. Yang J, Schleicher TR, Dong Y, Park HB, Lan J, Cresswell P, et al.. Disruption of mosGILT in Anopheles gambiae impairs ovarian development and Plasmodium infection. J Exp Med. 2020. Vol. 217(1):e20190682

56. Hoermann A, Tapanelli S, Capriotti P, Del Corsano G, Masters EK, Habtewold T, et al.. Converting endogenous genes of the malaria mosquito into simple non-autonomous gene drives for population replacement. Elife. 2021. Vol. 10:e58791

57. Kojin BB, Martin-Martin I, Araújo HRC, Bonilla B, Molina-Cruz A, Calvo E, et al.. Aedes aegypti SGS1 is critical for Plasmodium gallinaceum infection of both the mosquito midgut and salivary glands. Malar J. 2021. Vol. 20(1):11

58. Dong S, Ye Z, Tikhe CV, Tu ZJ, Zwiebel LJ, Dimopoulos G. Pleiotropic odorant-binding proteins promote aedes aegypti reproduction and flavivirus transmission. mBio. 2021. Vol. 12(5):e0253121

59. Bui M, Dalla Benetta E, Dong Y, Zhao Y, Yang T, Li M, et al.. CRISPR mediated transactivation in the human disease vector Aedes aegypti . PLoS Pathog. 2023. Vol. 19(1):e1010842

60. Li HH, Cai Y, Li JC, Su MP, Liu WL, Cheng L, et al.. C-type lectins link immunological and reproductive processes in Aedes aegypti . iScience. 2020. Vol. 23(9):101486

61. Wu P, Sun P, Nie K, Zhu Y, Shi M, Xiao C, et al.. A gut commensal bacterium promotes mosquito permissiveness to arboviruses. Cell Host Microbe. 2019. Vol. 25(1):101–112.e5

62. Suzuki Y, Baidaliuk A, Miesen P, Frangeul L, Crist AB, Merkling SH, et al.. Non-retroviral endogenous viral element limits cognate virus replication in Aedes aegypti ovaries. Curr Biol. 2020. Vol. 30(18):3495–3506.e6

63. Sinkins SP, Gould F. Gene drive systems for insect disease vectors. Nat Rev Genet. 2006. Vol. 7(6):427–435

64. Chen J, Luo J, Wang Y, Gurav AS, Li M, Akbari OS, et al.. Suppression of female fertility in Aedes aegypti with a CRISPR-targeted male-sterile mutation. Proc Natl Acad Sci U S A. 2021. Vol. 118(22):e2105075118

65. Galizi R, Hammond A, Kyrou K, Taxiarchi C, Bernardini F, O’Loughlin SM, et al.. A CRISPR-Cas9 sex-ratio distortion system for genetic control. Sci Rep. 2016. Vol. 6:31139

66. Basrur NS, De Obaldia ME, Morita T, Herre M, von Heynitz RK, Tsitohay YN, et al.. Fruitless mutant male mosquitoes gain attraction to human odor. Elife. 2020. Vol. 9:e63982

67. Hall AB, Basu S, Jiang X, Qi Y, Timoshevskiy VA, Biedler JK, et al.. SEX DETERMINATION. A male-determining factor in the mosquito Aedes aegypti . Science. 2015. Vol. 348(6240):1268–1270

68. Venkataraman K, Shai N, Lakhiani P, Zylka S, Zhao J, Herre M, et al.. Two novel, tightly linked, and rapidly evolving genes underlie Aedes aegypti mosquito reproductive resilience during drought. Elife. 2023. Vol. 12:e80489

69. Tikhe CV, Cardoso-Jaime V, Dong S, Rutkowski N, Dimopoulos G. Trypsin-like inhibitor domain (TIL)-harboring protein is essential for Aedes aegypti reproduction. Int J Mol Sci. 2022. Vol. 23(14):7736

70. Li M, Yang T, Bui M, Gamez S, Wise T, Kandul NP, et al.. Suppressing mosquito populations with precision guided sterile males. Nat Commun. 2021. Vol. 12(1):5374

71. Shetty V, Meyers JI, Zhang Y, Merlin C, Slotman MA. Impact of disabled circadian clock on yellow fever mosquito Aedes aegypti fitness and behaviors. Sci Rep. 2022. Vol. 12(1):6899

72. Adolfi A, Gantz VM, Jasinskiene N, Lee HF, Hwang K, Terradas G, et al.. Efficient population modification gene-drive rescue system in the malaria mosquito Anopheles stephensi . Nat Commun. 2020. Vol. 11(1):5553

73. Simoni A, Hammond AM, Beaghton AK, Galizi R, Taxiarchi C, Kyrou K, et al.. A male-biased sex-distorter gene drive for the human malaria vector Anopheles gambiae . Nat Biotechnol. 2020. Vol. 38(9):1054–1060

74. Kyrou K, Hammond AM, Galizi R, Kranjc N, Burt A, Beaghton AK, et al.. A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes. Nat Biotechnol. 2018. Vol. 36(11):1062–1066

75. Ling L, Kokoza VA, Zhang C, Aksoy E, Raikhel AS. MicroRNA-277 targets insulin-like peptides 7 and 8 to control lipid metabolism and reproduction in Aedes aegypti mosquitoes. Proc Natl Acad Sci U S A. 2017. Vol. 114(38):E8017–E8024

76. Zhang Y, Zhao B, Roy S, Saha TT, Kokoza VA, Li M, et al.. microRNA-309 targets the Homeobox gene SIX4 and controls ovarian development in the mosquito Aedes aegypti . Proc Natl Acad Sci U S A. 2016. Vol. 113(33):E4828–E4836

77. Li M, Zhang L, Wu Y, Li Y, Chen X, Chen J, et al.. Deletion of the serotonin receptor 7 gene changed the development and behavior of the mosquito, Aedes aegypti . Insects. 2022. Vol. 13(8):671

78. Wang Y, He X, Qiao L, Yu Z, Chen B, He Z. CRISPR/Cas9 mediates efficient site-specific mutagenesis of the odorant receptor co-receptor (Orco) in the malaria vector Anopheles sinensis . Pest Manag Sci. 2022. Vol. 78(8):3294–3304

79. Sun H, Liu F, Ye Z, Baker A, Zwiebel LJ. Mutagenesis of the orco odorant receptor co-receptor impairs olfactory function in the malaria vector Anopheles coluzzii . Insect Biochem Mol Biol. 2020. Vol. 127:103497

80. Liu F, Ye Z, Baker A, Sun H, Zwiebel LJ. Gene editing reveals obligate and modulatory components of the CO₂ receptor complex in the malaria vector mosquito, Anopheles coluzzii . Insect Biochem Mol Biol. 2020. Vol. 127:103470

81. Raji JI, Melo N, Castillo JS, Gonzalez S, Saldana V, Stensmyr MC, et al.. Aedes aegypti mosquitoes detect acidic volatiles found in human odor using the IR8a pathway. Curr Biol. 2019. Vol. 29(8):1253–1262.e7

82. Ye Z, Liu F, Ferguson ST, Baker A, Pitts RJ, Zwiebel LJ. Ammonium transporter AcAmt mutagenesis uncovers reproductive and physiological defects without impacting olfactory responses to ammonia in the malaria vector mosquito Anopheles coluzzii . Insect Biochem Mol Biol. 2021. Vol. 134:103578

83. Chae K, Valentin C, Dawson C, Jakes E, Myles KM, Adelman ZN. A knockout screen of genes expressed specifically in Ae. aegypti pupae reveals a critical role for stretchin in mosquito flight. Insect Biochem Mol Biol. 2021. Vol. 132:103565

84. Navarro-Payá D, Flis I, Anderson MAE, Hawes P, Li M, Akbari OS, et al.. Targeting female flight for genetic control of mosquitoes. PLoS Negl Trop Dis. 2020. Vol. 14(12):e0008876

85. O’Leary S, Adelman ZN. CRISPR/Cas9 knockout of female-biased genes AeAct-4 or myo-fem in Ae. aegypti results in a flightless phenotype in female, but not male mosquitoes. PLoS Negl Trop Dis. 2020. Vol. 14(12):e0008971

86. Feng X, Kambic L, Nishimoto JHK, Reed FA, Denton JA, Sutton JT, et al.. Evaluation of gene knockouts by CRISPR as potential targets for the genetic engineering of the mosquito Culex quinquefasciatus . Crispr J. 2021. Vol. 4(4):595–608

87. Li M, Akbari OS, White BJ. Highly efficient site-specific mutagenesis in malaria mosquitoes using CRISPR. G3 (Bethesda). 2018. Vol. 8(2):653–658

88. Liu T, Yang WQ, Xie YG, Liu PW, Xie LH, Lin F, et al.. Construction of an efficient genomic editing system with CRISPR/Cas9 in the vector mosquito Aedes albopictus . Insect Sci. 2019. Vol. 26(6):1045–1054

89. Purusothaman DK, Shackleford L, Anderson MAE, Harvey-Samuel T, Alphey L. CRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say. Sci Rep. 2021. Vol. 11(1):14964

90. Li X, Xu Y, Zhang H, Yin H, Zhou D, Sun Y, et al.. ReMOT control delivery of CRISPR-Cas9 ribonucleoprotein complex to induce germline mutagenesis in the disease vector mosquitoes Culex pipiens pallens (Diptera: Culicidae). J Med Entomol. 2021. Vol. 58(3):1202–1209

91. Zhu GH, Albishi NM, Chen X, Brown RL, Palli SR. Expanding the toolkit for genome editing in a disease vector, Aedes aegypti: transgenic lines expressing Cas9 and single guide RNA induce efficient mutagenesis. Crispr J. 2021. Vol. 4(6):846–853

92. Zhu GH, Jiao Y, Chereddy S, Noh MY, Palli SR. Knockout of juvenile hormone receptor, Methoprene-tolerant, induces black larval phenotype in the yellow fever mosquito, Aedes aegypti . Proc Natl Acad Sci U S A. 2019. Vol. 116(43):21501–21507

93. Dong S, Lin J, Held NL, Clem RJ, Passarelli AL, Franz AW. Heritable CRISPR/Cas9-mediated genome editing in the yellow fever mosquito, Aedes aegypti . PLoS One. 2015. Vol. 10(3):e0122353

94. Djokic S, Bakhrat A, Li M, Akbari OS, Abdu U. Scale-type-specific requirement for the mosquito Aedes aegypti Spindle-F homologue by regulating microtubule organization. Insect Mol Biol. 2022. Vol. 31(2):216–224

95. Zhang L, Li MZ, Chen ZH, Tang Y, Liao CH, Han Q. Arylalkalamine N-acetyltransferase-1 functions on cuticle pigmentation in the yellow fever mosquito, Aedes aegypti . Insect Sci. 2021. Vol. 28(6):1591–1600

96. Liu P, Jin B, Li X, Zhao Y, Gu J, Biedler JK, et al.. Nix is a male-determining factor in the Asian tiger mosquito Aedes albopictus . Insect Biochem Mol Biol. 2020. Vol. 118:103311

97. Djokic S, Bakhrat A, Tsurim I, Urakova N, Rasgon JL, Abdu U. Actin bundles play a different role in shaping scales compared to bristles in the mosquito Aedes aegypti . Sci Rep. 2020. Vol. 10(1):14885

98. Ling L, Raikhel AS. Serotonin signaling regulates insulin-like peptides for growth, reproduction, and metabolism in the disease vector Aedes aegypti . Proc Natl Acad Sci U S A. 2018. Vol. 115(42):E9822–E9831

99. Williams J, Cowlishaw R, Sanou A, Ranson H, Grigoraki L. In vivo functional validation of the V402L voltage gated sodium channel mutation in the malaria vector An. gambiae . Pest Manag Sci. 2022. Vol. 78(3):1155–1163

100. Grigoraki L, Cowlishaw R, Nolan T, Donnelly M, Lycett G, Ranson H. CRISPR/Cas9 modified An. gambiae carrying kdr mutation L1014F functionally validate its contribution in insecticide resistance and combined effect with metabolic enzymes. PLoS Genet. 2021. Vol. 17(7):e1009556

101. Lan J, Wang Z, Chen Z, Zhang L, Zhao J, Guan Q, et al.. Identification of the Aedes aegypti nAChR gene family and molecular target of spinosad. Pest Manag Sci. 2021. Vol. 77(4):1633–1641

102. Kim IH, Castillo JC, Aryan A, Martin-Martin I, Nouzova M, Noriega FG, et al.. A mosquito juvenile hormone binding protein (mJHBP) regulates the activation of innate immune defenses and hemocyte development. PLoS Pathog. 2020. Vol. 16(1):e1008288

103. Wang Y, Chang M, Wang M, Ji Y, Sun X, Raikhel AS, et al.. OTU7B modulates the mosquito immune response to Beauveria bassiana infection via deubiquitination of the toll adaptor TRAF4. Microbiol Spectr. 2023. Vol. 11(1):e0312322

104. Zhu GH, Gaddelapati SC, Jiao Y, Koo J, Palli SR. CRISPR-Cas9 genome editing uncovers the mode of action of methoprene in the yellow fever mosquito, Aedes aegypti . Crispr J. 2022. Vol. 5(6):813–824

105. Ranian K, Kashif Zahoor M, Zulhussnain M, Ahmad A. CRISPR/Cas9 mediated sex-ratio distortion by sex specific gene editing in Aedes aegypti . Saudi J Biol Sci. 2022. Vol. 29(4):3015–3022

106. Zhan Y, Alonso San Alberto D, Rusch C, Riffell JA, Montell C. Elimination of vision-guided target attraction in Aedes aegypti using CRISPR. Curr Biol. 2021. Vol. 31(18):4180–4187.e6

107. Matthews BJ, Younger MA, Vosshall LB. The ion channel ppk301 controls freshwater egg-laying in the mosquito Aedes aegypti . Elife. 2019. Vol. 8:e43963

108. Carballar-Lejarazú R, Tushar T, Pham TB, James AA. Microinjection method for Anopheles gambiae embryos. J Vis Exp. 2021. Vol. 173:e62591

109. Meuti ME, Harrell R. Preparing and injecting embryos of culex mosquitoes to generate null mutations using CRISPR/Cas9. J Vis Exp. 2020. Vol. 163:e61651

110. Criscione F, O’Brochta DA, Reid W. Genetic technologies for disease vectors. Curr Opin Insect Sci. 2015. Vol. 10:90–97

111. Adelman ZN, Jasinskiene N, James AA. Development and applications of transgenesis in the yellow fever mosquito, Aedes aegypti . Mol Biochem Parasitol. 2002. Vol. 121(1):1–10

112. Lule-Chávez AN, Carballar-Lejarazú R, Cabrera-Ponce JL, Lanz-Mendoza H, Ibarra JE. Genetic transformation of mosquitoes by microparticle bombardment. Insect Mol Biol. 2021. Vol. 30(1):30–41

113. Chaverra-Rodriguez D, Macias VM, Hughes GL, Pujhari S, Suzuki Y, Peterson DR, et al.. Targeted delivery of CRISPR-Cas9 ribonucleoprotein into arthropod ovaries for heritable germline gene editing. Nat Commun. 2018. Vol. 9(1):3008

114. Macias VM, McKeand S, Chaverra-Rodriguez D, Hughes GL, Fazekas A, Pujhari S, et al.. Cas9-mediated gene-editing in the malaria mosquito Anopheles stephensi by ReMOT control. G3 (Bethesda). 2020. Vol. 10(4):1353–1360

115. Hinckley AF, Connally NP, Meek JI, Johnson BJ, Kemperman MM, Feldman KA, et al.. Lyme disease testing by large commercial laboratories in the United States. Clin Infect Dis. 2014. Vol. 59(5):676–681

116. Santos VT, Ribeiro L, Fraga A, de Barros CM, Campos E, Moraes J, et al.. The embryogenesis of the tick Rhipicephalus (Boophilus) microplus: the establishment of a new chelicerate model system. Genesis. 2013. Vol. 51(12):803–818

117. Booth TF. Wax lipid secretion and ultrastructural development in the egg-waxing (Gene’s) organ in ixodid ticks. Tissue Cell. 1989. Vol. 21(1):113–122

118. Chaverra-Rodriguez D, Dalla Benetta E, Heu CC, Rasgon JL, Ferree PM, Akbari OS. Germline mutagenesis of Nasonia vitripennis through ovarian delivery of CRISPR-Cas9 ribonucleoprotein. Insect Mol Biol. 2020. Vol. 29(6):569–577

119. Shirai Y, Daimon T. Mutations in cardinal are responsible for the red-1 and peach eye color mutants of the red flour beetle Tribolium castaneum. Biochem Biophys Res Commun. 2020. Vol. 529(2):372–378

120. Heu CC, McCullough FM, Luan J, Rasgon JL. CRISPR-Cas9-based genome editing in the silverleaf whitefly (Bemisia tabaci). Crispr J. 2020. Vol. 3(2):89–96

121. Shirai Y, Piulachs MD, Belles X, Daimon T. DIPA-CRISPR is a simple and accessible method for insect gene editing. Cell Rep Methods. 2022. Vol. 2(5):100215

122. Li HH, Li JC, Su MP, Liu KL, Chen CH. Generating mutant Aedes aegypti mosquitoes using the CRISPR/Cas9 system. STAR Protoc. 2021. Vol. 2(2):100432

123. Willcox RR. Genetics of vectors and insecticide resistance: report of a WHO Scientific Group. World Health Organ Tech Rep Ser. 1964. Vol. 268:1–40

124. Hammond A, Pollegioni P, Persampieri T, North A, Minuz R, Trusso A, et al.. Gene-drive suppression of mosquito populations in large cages as a bridge between lab and field. Nat Commun. 2021. Vol. 12(1):4589

125. Committee on Gene Drive Research in Non-Human Organisms: Recommendations for Responsible Conduct, Board on Life Sciences, Division on Earth and Life Studies, National Academies of Sciences, Engineering, and Medicine. Gene Drives on the Horizon: Advancing Science, Navigating Uncertainty, and Aligning Research with Public Values. Washington, DC: National Academies Press. 2016

126. Teem JL, Ambali A, Glover B, Ouedraogo J, Makinde D, Roberts A. Problem formulation for gene drive mosquitoes designed to reduce malaria transmission in Africa: results from four regional consultations 2016-2018. Malar J. 2019. Vol. 18(1):347

127. Ahuja V. Regulation of emerging gene technologies in India. BMC Proc. 2018. Vol. 12 Suppl 8:14

128. Gurwitz D. Gene drives raise dual-use concerns. Science. 2014. Vol. 345(6200):1010