As a model insect, D. melanogaster is at the forefront of genetic analysis, and the application of CRISPR/Cas9 technology in Drosophila has promoted the use of genome editing technology in other insects (Bassett and Liu, 2014). In 2013, Gratz et al. first used CRISPR/Cas9 technology to introduce mutations into the Drosophila genome, generating a 4.6 kb deletion in the yellow locus by utilizing two target sgRNAs and a single-stranded oligonucleotide donor (ssODN) template (Gratz et al., 2013a). Furthermore, they discussed the potential applications of CRISPR/Cas9-mediated genome editing technology and described the benefits of generating designer flies on demand (Gratz et al., 2013b). In the same year, researchers described a method of increasing the frequency of HR using a reintegration vector (Baena-Lopez et al., 2013). Yu et al. compared the efficiencies of TALEN- and CRISPR/Cas9-mediated HDR mechanisms, constructed an easy-to-screen platform, and developed three HDR approaches for precise mutagenesis (Yu et al., 2014). Researchers have reported the construction of two transgene vectors expressing the Cas9 protein and an sgRNA and the subsequent crossing of flies carrying these transgenes to obtain an active sgRNA-Cas9 complex in the germline as well as the injection of an sgRNA-encoding plasmid into transgenic-Cas9 flies, which allowed the knockout or knock-in of different target genes (Kondo and Ueda, 2013; Gratz et al., 2014; Port et al., 2014; Ren et al., 2014a; Sebo et al., 2014; Xue et al., 2014a,b). Three methods have been used to induce HDR in flies; several researchers injected donor template and sgRNA plasmids into transgenic Cas9 embryos (Gratz et al., 2014; Ren et al., 2014b; Zhang X. et al., 2014), while Port et al. injected a donor template plasmid into transgenic embryos containing Cas9 and an sgRNA (Port et al., 2014), and Gokcezade et al. injected donor template, Cas9 and sgRNA plasmids into non-transgenic individuals (Gokcezade et al., 2014). Subsequently, the Port group compared these three methods of facilitating HDR using the same gRNA and donor plasmid and found that the use of transgenic individuals produced a higher knock-in frequency than non-transgenic individuals (Port et al., 2015).

Scientists are constantly looking for new fly research methods, which has undoubtedly accelerated the development of this model organism as well as other Drosophila species. Several studies have reported the use of genome editing techniques to introduce mutations in Drosophila cell lines (Böttcher et al., 2014; Gao et al., 2015; Lin et al., 2015; Mabashi-Asazuma et al., 2015; Kunzelmann et al., 2016), and a GFP-expressing cell line and comprehensive workflow were established for analyzing functional Drosophila genes. Thus, research at the cellular level will facilitate the optimization of the gene editing system and the establishment of a simple and convenient platform (Kunzelmann et al., 2016). In 2015, Gantz et al. developed a mutagenic chain reaction (MCR) method that could produce autocatalytic mutations and used this method to convert heterozygous mutations to homozygous mutations (Gantz and Bier, 2015). The CRISPR/Cas9 system was used to introduce a site-specific mutation (G275E) into the nicotinic acetylcholine receptor (nAChR) Dα6 subunit, and although the spinosad insecticide resistance level of flies with the G275E mutation (66-fold higher than non-mutated flies) was lower than that of individuals with a Dα6-null mutation (311-fold higher than non-mutated flies), it sufficiently demonstrated that the G275E mutation is directly related to spinosad resistance (Zimmer et al., 2016). Another study reported the development of a method for scarless allele replacement in Drosophila, which was shown to be suitable for single or multiple allelic substitutions in regions of interest (Lamb et al., 2016).

Some studies focused on specific genes and the defective phenotypes observed when these genes were knocked out using CRISPR/Cas9 technology. Bassett et al. targeted the yellow gene using the CRISPR/Cas9 system and found that the system was highly efficient and concentration dependent, as the efficiency of gene editing increased with increasing concentrations of the sgRNA; however, the adult survival rate was reduced when higher sgRNA concentrations were used (Bassett et al., 2013). Yu et al. also targeted the yellow gene and demonstrated greatly increased efficiency (Yu et al., 2013). Based on the use of CRISPR/Cas9 technology in D. melanogaster, researchers introduced site-specific mutations into the white (w) and Sex lethal (Sxl) genes of Drosophila suzukii. The mutant phenotype of white eyes was generated at a low efficiency, which may have occurred because the flies were injected with plasmid DNA encoding Cas9 and the sgRNA other than mRNA, in contrast to mRNA, the plasmid DNA needs to experience a transcription process in vivo. In addition, it may also has been due to the specificity of the white gene and Drosophila species. Mutation of the Sxl gene resulted in abnormal genitalia and reproductive tissues in female individuals (Li and Scott, 2016). Another study revealed the critical role of succinyl-CoA synthetase/ligase (SCS), which is associated with metabolites in Drosophila. The authors generated a mutation in the SCS alpha subunit (Scsα) using the CRISPR/Cas9 system and observed that Scsα-deficient individuals exhibited developmental delays, impaired locomotor activity and increased mortality under starvation conditions (Quan et al., 2017). Thus, as stated above, Scsα is essential for proper energy metabolism in Drosophila. Asaoka et al. utilized the CRISPR/Cas9 system to establish linear ubiquitin E3 ligase (LUBEL)-deficient flies, which exhibited reduced survival and defective climbing in response to heat (Asaoka et al., 2016). A sex-specific gene has also been studied in Drosophila using CRISPR/Cas9. To ensure that the transcriptional activity of the male X chromosome is equal to that of the two female X chromosomes, the male-specific lethal (MSL) complex is recruited to regions of the X chromosome; this process is dependent on the chromatin-linked adapter for MSL proteins (CLAMP) zinc finger protein. Urban et al. used the CRISPR/Cas9 system to generate mutations in the clamp gene in flies and found that clamp-null males and females died at different developmental stages and that the expression of a transcription factor was altered in a sex-specific manner (Urban et al., 2016). Also in Drosophila, the troponin C (TpnC) gene was shown to be associated with muscle formation using CRISPR/Cas9 technology (Chechenova et al., 2017). Typically, mutations in the coding region of a gene are studied; however, a recent paper reported that CRISPR/Cas9 technology could be used to mutant transcription factor binding sites in enhancer regions of the Alk locus in Drosophila, thereby mediating the expression of the Alk gene (Mendoza-Garcia et al., 2017).

Mosquitoes can spread many diseases that pose a serious threat to human health, such as Zika, malaria, dengue, chikungunya, and filariasis (Gabrieli et al., 2014; Reegan et al., 2017). For the past several decades, synthetic insecticides have been used to control vector mosquitoes. Not only do these insecticides affect the ecological environment (Bayen, 2012), they also induce resistance in vector mosquitoes (Tikar et al., 2009). As a result of their blood-triggered reproductive strategy, female mosquitoes must feed on blood before mating with male individuals, and they then lay their eggs in water. A female mosquito can lay eggs 3–4 times in her life, producing 150–250 individuals each time. Understanding this breeding mechanism can help us to control the mosquito population. Aedes aegypti is a vector of many human-borne arboviruses, such as yellow fever, dengue, and chikungunya (Weaver and Barrett, 2004). Targeting mosquito genes of interest in vivo using a genome editing tool has become popular, and ZFNs and TALENs have been successfully used in mosquitoes (Aryan et al., 2013; Smidler et al., 2013). In 2015, scientists first used CRISPR/Cas9 technology to modify the genome of A. aegypti (Dong et al., 2015), targeting the ECFP gene in transgenic A. aegypti mosquitoes harboring DsRed and ECFP marker genes and obtaining individuals that expressed DsRed but not ECFP. Kistler et al. injected optimal sgRNA-Cas9 mixtures into A. aegypti embryos and demonstrated that CRISPR/Cas9 could induce different mutations using diverse repair mechanisms (Kistler et al., 2015). Basu et al. established a platform for the rapid screening of candidate sgRNAs using a transient embryo assay, which enhanced the efficiency of gene editing and improved HR-mediated repair rates (Basu et al., 2015).

Non-coding microRNAs can regulate a series of physiological processes, including ecdysis, metamorphosis, embryogenesis, and host-pathogen interactions (Lucas et al., 2013, 2015b), and are regarded as a new frontier in mosquito biology. miRNA-1174 is responsible for sugar and blood uptake in A. aegypti (Liu S. et al., 2014). microRNA-8 is expressed in the fat body and can regulate the A. aegypti reproductive process (Lucas et al., 2015a). The functional verification of a microRNA is usually performed using an RNA interference technique. A recent study targeted microRNA-309 using the CRISPR/Cas9 system, resulting in a severe loss of ovarian function, growth retardation, substantially decreased follicle numbers, and a reduced egg hatching rate in mutant A. aegypti individuals. Additionally, the authors identified that the homeobox 4 (SIX4) gene was a direct target of miR-309 (Zhang et al., 2016). These phenotypic defects were consistent with the effects caused by antagomir silencing (a novel class of chemically engineered oligonucleotides) which can silence the expression of endogenous gene, but the results mediated by CRISPR/Cas9 were more efficient and clear. Because only female mosquitoes feed on blood and transmit pathogens (Papathanos et al., 2009), the conversion of female mosquitoes into harmless male individuals has shown promise as a new vector management strategy. Male determination in A. aegypti is controlled by the M factor, which is a dominant male-determining factor located on the Y chromosome in the M locus. Hall et al. targeted an M factor functional gene, Nix, in A. aegypti using the CRISPR/Cas9 system, which led to the feminization of Nix⁻ males (Hall et al., 2015). In Anopheles gambiae, which is the major vector of malaria, researchers utilized CRISPR/Cas9 technology to target three genes related to a female-sterility phenotype (Hammond et al., 2016). In Anopheles stephensi, Gantz et al. established an efficient autonomous CRISPR/Cas9-mediated gene drive system originating from the MCR method and demonstrated that the progeny of males and females derived from transgenic males exhibited a high frequency of germ-line gene conversion, likely as a result of HDR (Gantz et al., 2015). The CRISPR/Cas9 method has also been used to reveal the relationship between detoxifying enzymes and insecticide resistance in Culex quinquefasciatus (Itokawa et al., 2016); when the cytochrome P450 gene CYP9M10 was targeted in a resistant strain of C. quinquefasciatus, the individuals carrying no functional CYP9M10 copy exhibited an ~110-fold reduction in permethrin resistance, demonstrating that CYP9M10 is a crucial factor associated with pyrethroid resistance. Overall, these studies show that the CRISPR/Cas9-mediated genome editing system can be used to effectively modify the mosquito genome and potentially to control the population of mosquitoes and disease transmission. The biosafety risks of this technology related to the release of CRISPR/Cas9-edited insects into the environment should be considered when selecting the most suitable, environmentally friendly method of implementing the gene driven system (Esvelt et al., 2014; Oye et al., 2014; Alphey, 2016; Champer et al., 2016; Taning et al., 2017).

The lepidopteran insect B. mori is an important, economically valuable species as well as a model organism in scientific research. It is one of the earliest insect models in which a genomic editing tool was used to verify a gene function in vivo. Researchers compared three different genomic engineering techniques (ZFNs, TALENs, and CRISPR/Cas9) (Daimon et al., 2014) and found that the most convenient and effective method was the CRISPR/Cas9 system. The BmBLOS2 gene was targeted to verify the application of CRISPR/Cas9 in B. mori; when this gene is mutated, the larval integument is changed from opaque to translucent (Wang et al., 2013; Liu Y. et al., 2014). Generally, mutation strategies cause small indels in a coding sequence; however, Liu et al. have reported the use of a method that can induce 3 kb fragment variations in non-coding sequences using a CRISPR system with two sgRNAs in the B. mori cell line BmNs, they designed two 20-bp sgRNAs adjacent to the PAM site with the typical sequence NGG (where N represents any base), the 3 kb fragment locates between both sgRNAs. They also designed six sgRNAs to target six different genes and cotransfected these six sgRNAs and Cas9 into BmNs cell line. Ultimately, the targeted mutations were detected for all target loci, indicating that CRISPR/Cas system can induce multiple genes mutations simultaneously. Therefore, the CRISPR/Cas9 system enables the production of large-fragment or multiple-gene mutations simultaneously in B. mori (Liu Y. et al., 2014; Ma et al., 2014). In another study, the authors took advantage of a notable mutant phenotype of the Bm-ok gene, which allowed homozygous mutants to be easily screened and the mutation efficiency of CRISPR/Cas9 technology to be calculated (Wei et al., 2014).

The Wnt1 signaling pathway is important for embryonic development in insects. According to previous reports, the Wnt1 gene is responsible for the formation of spot patterns (Yamaguchi et al., 2013) and normal abdominal segments in B. mori (Yamaguchi et al., 2011). The CRISPR/Cas9 system provides an ideal strategy for studying the Wnt1 signaling pathway. Zhang et al. obtained BmWnt1 gene knockout individuals and observed that the mutants exhibited defects in body segmentation and pigmentation in a dose-dependent manner and that the Hox homologous genes were down-regulated in BmWnt1-deficient individuals (Zhang et al., 2015). Studies have shown that the Fem piRNA is the primary female determining factor in B. mori. Xu et al. used a transgenic-based CRISPR/Cas9 system to elucidate the sex determination mechanism of male individuals. They generated a series of mutations in the BmSxl, Bmtra2, Bmlmp, BmlmpM, BmPSI, and BmMasc genes and studied individuals with each mutation, ultimately determining that the BmPSI gene is a critical auxiliary factor in determining the male sex in B. mori (Xu et al., 2017). Liu et al. indicated that deletion of BmOrco gene by utilizing CRISPR/Cas9 technology severely disrupts the olfactory system, furthermore, the homozygous mutant was unable to respond to sex pheromones (Liu Q. et al., 2017). Many reports have shown that the CRISPR/Cas9 system can be used to integrate donor DNA into the B. mori genome, but this process depends on several factors. A recent study indicated that the lack of NHEJ-related factors, such as BmKu70, BmKu80, BmLigIV, BmXLF, and BmXRCC4, increased HR efficiency mediated by CRISPR/Cas9 in B. mori (Ma et al., 2014; Zhu et al., 2015).

Several studies have successfully used CRISPR/Cas9 technology in gene therapy by disrupting a viral gene, such as HIV-1 or hepatitis B genes (Ebina et al., 2013; Seeger and Sohn, 2014; Liao et al., 2015; Pellagatti et al., 2015). However, there is currently no method that can completely eliminating a viral genome in insects. B. mori is the only host of BmNPV, which was exploited by scientists to construct a virus-induced CRISPR/Cas9 system, with the Cas9 nuclease being activated upon infection with the virus (Dong et al., 2016). This new system inhibited viral proliferation within a short time, and, importantly, the optimized system provided new ideas regarding the establishment of a virus-free transgenic line in B. mori. In addition, researchers targeted the BmNPV ie-1 and me53 genes in another study and successfully introduced mutations into these two genes; this method not only contributes to modern sericulture but also opens up options for future anti-viral therapies (Chen et al., 2017). Increasing numbers of functional genes have been identified by researchers using the CRISPR/Cas9 system in B. mori. Juvenile hormone (JH) and 20-hydroxyecdysone (20E) are responsible for insect growth and development. A loss-of-function analysis of B. mori JH esterase (BmJHE) was performed using a CRISPR/Cas9 transgenic system, and the mutant individuals exhibited an extended life span; this information could be used to extend the larval stage of B. mori to increase silk production (Zhang Z. et al., 2017). Furthermore, in another study, it was demonstrated that the E75 isoforms of the 20E primary response gene mediated the regulation of steroidogenesis and developmental timing in B. mori. Of the three isoforms of E75, E75A/C is a positive transcriptional activator and induces the biosynthesis of ecdysteroid. Interestingly, scientists demonstrated that E75B had an opposite effect using CRISPR/Cas9 technology, as the synthesis of ecdysteroid was increased in B. mori E75B mutants (Li K. et al., 2016).

S. litura is an omnivorous and destructive pest that is found throughout the world. Abdominal-A (abd-A) is an important gene for development in insects, and, using RNA interference in B. mori, scientists demonstrated that Bmabd-A is primarily responsible for normal development of the third to sixth body segments (Pan et al., 2009). Another study showed that as a transcription factor, Bmabd-A interacts with BmPOUM2 during larva-to-pupa metamorphosis (Deng et al., 2012). Based on the axial patterning of the Drosophila cardiac tube, the Ubx gene is expressed in the aorta, and the homeotic genes abd-A and abd-B are expressed in the heart, indicating the genetic and functional diversity of this organ (Ponzielli et al., 2002). Recently, in S. litura, scientists targeted the Slabd-A gene using the CRISPR/Cas9 system and obtained Slabd-A-deficient individuals, which exhibited abnormal body segmentation and anomalous pigmentation (Bi et al., 2016). The same gene, abd-A, was also knocked out in P. xylostella using CRISPR/Cas9; injecting eggs with 500 ng/μl Cas9 mRNA resulted in a higher mutation rate (91%) for the Pxabd-A gene in G0 than injecting 300 ng/μl Cas9 mRNA (Huang et al., 2016). Phenotype analysis indicated that the Pxabd-A gene plays an essential role in promoting segmentation and gonad development in P. xylostella (Huang et al., 2016). The function of this gene is analogous to that in other Lepidoptera insects. Because the mutation mechanisms of the Pxabd-A gene are relatively complex, it is difficult to analyze their effects, and there are many issues to be resolved, such as the lower mutation rate in G2 and other roles of the Pxabd-A gene in insects.

Chitin is a natural amino polysaccharide involved in the formation of the extracellular matrix. The synthesis of chitin is primarily confined to epithelial cells under the cuticle and within the gut giving rise to peritrophic matrices of arthropods, some other invertebrates (nematode egg shells and some protozoan cyst walls) and fungal septa (septa, spores, and cell walls). Chitin synthesis is a target of many insecticides, fungicides, and acaricides (Merzendorfer, 2013). Many arthropod pests pose a serious threat to human health and life, including A. gambiae, the main vector of malaria (Hammond et al., 2016), A. aegypti is a potent vector of dengue, yellow fever, and chikungunya viruses (Kistler et al., 2015). The diamondback moth, P. xylostella, is a global lepidopterous pest of brassicaceous vegetables and was the first pest to evolve field resistance to Bt insecticides (Guo et al., 2015); it later developed basic resistance to almost all chemical insecticides. There are many chitin synthesis inhibitors. Benzoylureas (BPUs), buprofezin, and etoxazole are commonly used and share a set of molecular modes of action (MoAs) by directly interacting with chitin synthase. Initially, the acaricide etoxazole was used to inhibit chitin biosynthesis in the two-spotted spider mite, Tetranychus urticae. A single non-synonymous SNP was found in the chitin synthase 1 gene (CHS1) at position 1017, where an isoleucine (I) was replaced with a phenylalanine (F), and scientists demonstrated that this single amino acid replacement is related to etoxazole resistance (Van Leeuwen et al., 2012). At the same position as the spider mite I1017F mutation, Douris et al. identified that a mutation (I1042M) in the CHS1 gene of BPU-resistant P. xylostella. Similarly, this is an amino acid substitution of isoleucine (I) to methionine (M), and the authors introduced both substitution mutations (I1056M/F) of the CHS1 gene into Drosophila using the CRISPR/Cas9 system combined with the HDR pathway. These mutations corresponded to I1042M and I1017F in P. xylostella and T. urticae, respectively. The Drosophila lines that were homozygous for these mutations (I1056M/F) were highly resistant to BPUs, etoxazole and buprofezin (Douris et al., 2016); therefore, the CRISPR/Cas9 system provided a method of verifying MoA-resistance mechanisms in vivo and identified mechanisms that were shared across species.

The olfactory system of insects is very sensitive and depends on odor binding proteins to identify a variety of odorant substances. Pheromone binding proteins are the most important type of odor binding protein (Vogt and Riddiford, 1981). To illustrate the function of the pheromone binding protein gene in S. litura, researchers first mutated the pheromone binding protein 3 gene (SlitPBP3) using CRISPR/Cas9 genome editing technology and obtained SlitPBP3 mutant individuals. Compared with wild-type males, the mutant males exhibited a significantly decreased response to sex pheromone components (Zhu et al., 2016). CRISPR/Cas9 genome editing tools were also used to target the olfactory receptor co-receptor (Orco) gene in Spodoptera littoralis, and the knockout individuals failed to respond to plant odors and sex pheromones (Koutroumpa et al., 2016). Compared with RNA interference techniques that are not suitable for Lepidoptera insects, the CRISPR/Cas9 system provides a more in-depth and stable method for studying functional genes.

Bacillus thuringiensis (Bt) insecticides and Bt transgenic crops are widely used for pest control (Bravo et al., 2011). However, the evolution of resistance to Bt toxins has threatened the long-term development of pest management. A cadherin-like receptor was identified as a receptor of the Bt Cry1A toxin in several Lepidoptera insects (Wu, 2014). The cadherin-related receptor interacts with the Cry1Ab protoxin, which facilitates proteolytic cleavage and forms a pre-pore oligomeric structure in Manduca sexta (Gómez et al., 2002). Genetic linkage, cell toxicity and RNA interference experiments have demonstrated that cadherin is involved in Cry1Ac resistance in several Lepidoptera insects. Wang et al. first targeted exon 9 of the cadherin gene by injecting a mixture of Cas9 mRNA and an sgRNA into Helicoverpa armigera eggs. Using this CRISPR/Cas9 genome manipulation system, they obtained HaCad gene mutant individuals, which exhibited 549-fold higher resistance to Cry1Ac compared with a control strain (Wang J. et al., 2016). This result provided direct functional evidence that HaCad is a key receptor for Cry1Ac and is related to Cry1Ac resistance. CRISPR/Cas9 was also used in H. armigera to mutate four pigment genes, white, brown, scarlet, and ok; these mutations caused various physiological phenotypes in H. armigera (Khan et al., 2017). Recently, Chang et al. have demonstrated that the antagonist-mediated optimization of mating time ensures maximum fecundity using CRISPR/Cas9 system in H. armigera, which provides a new strategy to destroy pest mating (Chang et al., 2017).

The pine caterpillar moth, Dendrolimus punctatus, is a devastating forest pest in China and Southeast Asia, inducing severe defoliation and reducing resin production. Researchers first induced mutations in the Wnt-1 gene using the CRISPR/Cas9 system to precisely and efficiently modify gene expression in D. punctatus. Previous reports indicated that the Wnt-1 gene is associated with segmentation and development. In this paper, multiple mutant phenotypes were observed, such as abnormal posterior segments, defective legs, and head deformation (Liu H. et al., 2017). Therefore, the genome editing technology can be used to manipulate the genome of D. punctatus and, importantly, may provide a management strategy for a major defoliator.

The eyespot wings of nymphalid butterflies are colorful and appealing. Initially, the eyespot was located on the ventral hindwing and subsequently moved to dorsal wing surfaces. This apparent evolutionary mechanism is closely related to the function of the eyespot, which plays an important role in predation, courtship and sexual dimorphism (Monteiro, 2015). The eyespot pattern is regulated by a series of homologous genes (such as Spalt, North, Dll, and en) that form a single origin of expression and regulatory network (Reed and Serfas, 2004; Oliver et al., 2012). CRISPR/Cas9 is useful for studying the eyespot color patterns of the nymphalid butterflies Junonia coenia and Vanessa cardui. Spalt and Distal-less (Dll) are transcription factor genes that have been shown to play essential roles in promoting and inhibiting the formation of eyespot development, respectively (Zhang and Reed, 2016). Perry and colleagues verified the three-way stochastic choices that expand butterfly color vision using the CRISPR/Cas9 system (Perry et al., 2016). Recently, CRISPR/Cas9 was used to modify the Papilio machaon genome. The authors targeted four Abd-B genes and injected sgRNA/Cas9 mixtures into freshly collected P. machaon eggs; the mutants exhibited four pairs of extra prolegs on segments A7-A9, which were not observed in wild-type individuals (Li X. Y. et al., 2016). Marker et al. also defined the critical function of the clk gene in controlling migration behavior using TALENs and CRISPR/Cas9 genome editing tools in Danaus plexippus. They reported that injecting fewer than 100 eggs is sufficient to recover mutant progeny and to generate monarch knockout lines in ~3 months (Markert et al., 2016). In 2016, scientists integrated comparative genomics and CRISPR/Cas9 technology in two highly heterozygous and closely related butterflies, Papilio xuthus and P. machaon. They demonstrated interesting evolution patterns in butterflies and knocked out three genes that produce obvious phenotypes, Abd-B, ebony, and fz, using CRISPR/Cas9 technology (Li et al., 2015). This research provided a valuable genomic and genetic technology for studying butterflies and other insects. Soon after, Zhang et al. utilized comparative RNA-Seq technology to identify eight candidate genes associated with melanin pigmentation in four butterfly species and then knocked out these candidate genes to verify their function using the CRISPR/Cas9 system (Zhang L. et al., 2017). Thus, the combination of comparative genomics and CRISPR/Cas9 technology will become a powerful tool for discovering novel genes and revealing the behavioral mechanisms of non-model organisms.

Locusts are important agricultural pests, and in the early days of locust outbreaks, governments issued policies to eradicate locusts. The traditional eradication tactics rely on synthetic insecticides, which increase the financial and environmental costs. Scientists also consider locusts to be important insects at the molecular level. One study showed that developmental synchrony is the foundation of gregarious behavior, migration and copulation. A miRNA (miR-276) gene was found to facilitate the synchrony of egg development and to directly determine the density of locusts (He et al., 2016). The olfactory system plays an important role in insects. Chemical pheromone signals are first received by peripheral tissues and processed in antennal nerve tissues, finally, olfactory and other sensory organs integrate the signals in the brain to direct insect behaviors, such as foraging, feeding, mating, and spawning (Leal, 2013). To further study the functional genes of Locusta migratoria in vivo, researchers used the CRISPR/Cas9 technique to modify the locust genome using an sgRNA targeting the odorant receptor co-receptor (Orco) gene. By microinjecting Cas9-mRNA and Orco-sgRNA into locust eggs, the authors achieved highly efficient mutation rates in the Orco gene, and they also successfully established Orco homozygous and heterozygous mutant lines (Li Y. et al., 2016). This is the first time the CRISPR/Cas9 system was used in locusts for genome editing, and the results not only suggested new ideas for locust control but also provided guidelines for using the technology in other insects from the orthopteran clade such as crickets.

Learning and memory are important functions of the human brain. Arthropods have a complex “brain” (Strausfeld, 2012) that enables them to adapt to different external environments. The central mechanisms of learning and memory are hotspot issues in insect neurobiology. Many studies have shown that the insect "brain" possesses associative learning abilities. In mammals, dopamine neurons are thought to play an important role in mediating both appetitive and aversive reinforcement. In insects, octopamine has been shown to participate in appetitive reinforcement, while dopamine has been shown to mediate aversive reinforcement. Honeybees have a strong olfactory learning and memory ability. In 2007, scientists demonstrated that octopamine and dopamine promoted appetitive and aversive reinforcement, respectively, in olfactory-based associative learning (Vergoz et al., 2007). Contrary to this result in honeybees, scientists proposed that dopamine in mushroom bodies mediates not only appetitive reinforcement but also aversive reinforcement in Drosophila (Krashes et al., 2009; Liu et al., 2012). To resolve these contradictions, Awata et al. knocked out the type 1 dopamine receptor gene (Dop1) using CRISPR/Cas9 technology in crickets (Gryllus bimaculatus). Dop1 gene knockout crickets were defective in aversive learning with sodium chloride punishment but not in appetitive learning with water or sucrose reward (Awata et al., 2015). This study illustrated that CRISPR/Cas9 system is valuable in studies of associative learning and can be used to knock out related genes in arthropods.

The red flour beetle, Tribolium castaneum (Coleoptera), is a major pest of stored agricultural products. Beetles feed on grains, such as maize, wheat, and rice, lowering the nutritional value of food. Additionally, their secretions contain the carcinogen benzoquinone. The advent of a T. castaneum genomic database (Tribolium Genome Sequencing Consortium, 2008; Kim et al., 2010) has prompted researchers to look for genetic-based methods of eradication instead of the traditional fumigation; these methods could improve pest management and reduce the damage to the environment. Some approaches have been reported, including transgenesis (Pavlopoulos et al., 2004; Berghammer et al., 2009), RNA interference technology (Gilles and Averof, 2014), heat shock-mediated mis-expression of genes (Schinko et al., 2012), the GAL4/UAS system (Schinko et al., 2010), and the targeting of compensation mechanisms that are activated after disruption (Perkin et al., 2017). Subsequently, a simpler and faster genome editing tool, the CRISPR/Cas9 system, was first used in T. castaneum (Gilles et al., 2015). In this paper, Gilles et al. demonstrated that mutations in the E-cadherin gene caused severe defects in dorsal closure; moreover, they also achieved efficient homology-directed knock-in via the CRISPR-mediated system. In a sense, the use of this genome editing system in different species lays the foundation for establishing a transgenic method based on the CRISPR/Cas9 system, which could shorten the developmental gap between model organisms and non-model creatures.

Hymenoptera is the third largest clade of insects after Coleoptera and Lepidoptera and includes bees, ants, wasps, sawflies (Davis et al., 2010). The vast majority of hymenopterans are beneficial pollinators or predatory natural enemies of pests. The parasitoid wasp, Nasonia vitripennis, is not only an important natural enemy of pests but is also an ideal experimental insect. Li et al. used the CRISPR/Cas9 genome editing system to target the eye pigmentation gene cinnabar in N. vitripennis. They injected sgRNA/Cas9 mixtures into collected eggs, and effective and heritable mutants were obtained (Li et al., 2017). These results demonstrated that the gene manipulation system can be utilized to study haplo-diploid sex determination, axis pattern formation and other biological behaviors in N. vitripennis.

Tetranychus urticae is a polyphagous mite that can carry and transmit virus and can travel by wind and water and on insects, birds, humans, animals, all kinds of farm tools and flower seedlings. Mitochondrial respiratory complex I plays a key role in the synthesis and transfer of ATP in T. urticae, and NADH: ubiquinone oxidoreductase is crucial for oxidative energy conversion (Wirth et al., 2016). Mitochondrial electron transport inhibitors (METIs) including quinazolines, pyrimidinamines, pyrazoles, and pyridazinones are widely used to block the quinone binding receptor of complex I. Because of their frequent utilization, resistance to METI-Is evolves quickly (Stumpf and Nauen, 2001; Van Pottelberge et al., 2009). Bajda et al. identified an H92R amino substitution in the PSST homolog of complex I that is associated with METI-I resistance in a resistant T. urticae strain (Bajda et al., 2017). Subsequently, introduced the H92R mutation into the Drosophila PSST homolog using the CRISPR/Cas9 genome editing tool, but failed to obtain homozygous mutant fly individuals, indicating that the H92A amino substitution is lethal in Drosophila but not in T. urticae and that the mechanism of lethality requires further study (Bajda et al., 2017).

Palaemon carinicauda (falsely called Exopalaemon carinicauda previously) is an economically important shrimp that belongs to the order Decapoda of crustaceans. Previously, there were no studies regarding gene-specific manipulation in this species. Recently, scientists knocked out the EcChi4 gene in shrimp using the CRISPR/Cas9 gene editing system and revealed the function of this gene in vivo. This is the first time that the microinjection method has been used in P. carinicauda, and the mutations were shown to be transmitted to subsequent generations. The use of CRISPR/Cas9 technology in shrimp provides a foundation for knockout or knock-in studies of genes of interest related to their development, growth, metabolism, and reproduction (Gui et al., 2016). This gene editing system will certainly become a robust tool for manipulating the genomes of decapods.

Homeotic genes (Hox genes) can regulate the specialized biological phenotypes of many arthropod species (Hughes and Kaufman, 2002). Therefore, scientists are committed to studying how Hox proteins lead to morphological diversity (Pearson et al., 2005). It has been noted that Hox genes play a role in generating limb diversity (Jung et al., 2014), but the precise functions of these genes have rarely been studied in crustaceans. Martin et al. combined CRISPR/Cas9 and RNAi technology to analyze six Hox genes in Parhyale hawaiensis (Crustacean, Amphipoda). They targeted the six Hox genes Dfd, Antp, Abd-A, Abd-B, Scr, and Ubx, and injected Cas9/sgRNA mixtures into one-cell embryos of P. hawaiensis. The results revealed the function of these six genes in different appendage regions of P. hawaiensis, and they also indicated the different phenotypic effects caused by deficiencies in different Hox genes (Martin et al., 2016).

Daphnia magna (Crustacea, Cladocera) is a member of the zooplankton found in fresh water. Because they are easy to culture are parthenogenetic clones and have a short generation time, they are attractive for scientific research (Hebert, 1978). Interestingly, D. magna is able to detect ecotoxicity and environment status through its feeding behaviors (Zhu et al., 2008). A cDNA library was constructed, and the expressed sequence tags of D. magna were determined (Watanabe et al., 2005). Scientists utilized the published genetic information to analyze the eye phenotype gene eyeless by injecting Cas9/sgRNA mixtures into eggs and obtained mutants with defects in the eyeless gene accompanied with a deformed eye phenotype (Nakanishi et al., 2014). This is the first example of genetic modification in D. magna; therefore, the experimental results may be of great value to future studies in this species.