CRISPR-Cas9 mediated dsRNase knockout improves RNAi efficiency in the fall armyworm.

Lepidopteran insects are refractory to RNA interference (RNAi) response, especially to orally delivered double-stranded RNA (dsRNA). High nuclease activity in the midgut lumen is proposed as one of the major reasons for RNAi insensitivity. We identified three dsRNase genes highly expressed in the midgut of fall armyworm (FAW), Spodoptera frugiperda. The genomic region harboring those three dsRNase genes was deleted using the CRISPR-Cas9-mediated genome editing method. A homozygous line with deletion of three dsRNase genes was produced. dsRNA degradation by midgut lumen contents of mutant larvae was lower than in wild-type larvae. Feeding dsRNA targeting the inhibitor of apoptosis (IAP) gene increased knockdown of the target gene and mortality in mutants compared to wild-type larvae. These results suggest that dsRNases in the midgut contribute to RNAi inefficiency in FAW. Formulations that protect dsRNA from dsRNase degradation may improve RNAi efficiency in FAW and other lepidopteran insects.

Azadirachtin Inhibits Nuclear Receptor HR3 in the Prothoracic Gland to Block Larval Ecdysis in the Fall Armyworm, Spodoptera frugiperda.

Azadirachtin has been used to control agricultural pests for a long time; however, the molecular mechanism of azadirachtin on lepidopterans is still not clear. In this study, the fourth instar larvae of fall armyworm were fed with azadirachtin, and then the ecdysis was blocked in the fourth instar larval stage (L4). The prothoracic glands (PGs) of the treated larvae were dissected for RNA sequencing to determine the effect of azadirachtin on ecdysis inhibition. Interestingly, one of the PG-enriched genes, the nuclear hormone receptor 3 (HR3), was decreased after azadirachtin treatment, which plays a critical role in the 20-hydroxyecdysone action during ecdysis. To deepen the understanding of azadirachtin on ecdysis, the HR3 was knocked out by using the CRISPR/Cas9 system, while the HR3 mutants displayed embryonic lethal phenotype; thus, the stage-specific function of HR3 during larval molting was not enabled to unfold. Hence, the siRNA was injected into the 24 h L4 larvae to knock down HR3. After 96 h, the injected larvae were blocked in the old cuticle during ecdysis which is consistent with the azadirachtin-treated larvae. Taken together, we envisioned that the inhibition of ecdysis in the fall armyworm after the azadirachtin treatment is due to an interference with the expression of HR3 in PG, resulting in larval mortality. The results in this study specified the understanding of azadirachtin on insect ecdysis and the function of HR3 in lepidopteran in vivo.

CRISPR-Cas9 Genome Editing Uncovers the Mode of Action of Methoprene in the Yellow Fever Mosquito, Aedes aegypti.

Methoprene, a juvenile hormone (JH) analog, is widely used for insect control, but its mode of action is not known. To study methoprene action in the yellow fever mosquito, Aedes aegypti, the E93 (ecdysone-induced transcription factor) was knocked out using the CRISPR-Cas9 system. The E93 mutant pupae retained larval tissues similar to methoprene-treated insects. These insects completed pupal ecdysis and died as pupa. In addition, the expression of transcription factors, broad complex and Krüppel homolog 1 (Kr-h1), increased and that of programmed cell death (PCD) and autophagy genes decreased in E93 mutants. These data suggest that methoprene functions through JH receptor, methoprene-tolerant, and induces the expression of Kr-h1, which suppresses the expression of E93, resulting in a block in PCD and autophagy of larval tissues. Failure in the elimination of larval tissues and the formation of adult structures results in their death. These results answered long-standing questions on the mode of action of methoprene.

SoxC is Required for Ecdysteroid Induction of Neuropeptide Genes During Insect Eclosion.

In insects, the shedding of the old exoskeleton is accomplished through ecdysis which is typically followed by the expansion and tanning of the new cuticle. Four neuropeptides, eclosion hormone (EH), ecdysis triggering hormone (ETH), crustacean cardioactive peptide (CCAP) and bursicon (Bur) are known to control ecdysis. However, the regulation of these neuropeptide genes is still poorly understood. Here, we report that in the red flour beetle (RFB) Tribolium castaneum and the fall armyworm (FAW) Spodoptera frugiperda, knockdown or knockout of the SoxC gene caused eclosion defects. The expansion and tanning of wings were not complete. In both RFB and FAW, the knockdown or knockout of SoxC resulted in a decrease in the expression of EH gene. Electrophoretic mobility shift assays revealed that the SfSoxC protein directly binds to a motif present in the promoter of SfEH. The luciferase reporter assays in Sf9 cells confirmed these results. These data suggest that transcription factor SoxC plays a key role in ecdysteroid induction of genes coding for neuropeptides such as EH involved in the regulation of insect eclosion.

Transcript level is a key factor affecting RNAi efficiency.

Efficiency is the basis for the application of RNA interference (RNAi) technology. Actually, RNAi efficiency varies greatly among insect species, tissues and genes. Previous efforts have revealed the mechanisms for variation among insect species and tissues. Here, we investigated the reason for variable efficiency among the target genes in the same insect. First, we tested the genes sampled randomly from Tribolium castaneum, Locusta migratoria and Drosophila S2 cells for both their expression levels and sensitivity to RNAi. The results indicated that the genes with higher expression levels were more sensitive to RNAi. Statistical analysis showed that the correlation coefficients between transcript levels and knockdown efficiencies were 0.8036 (n = 90), 0.7255 (n = 18) and 0.9505 (n = 13), respectively in T. castaneum, L. migratoria and Drosophila S2 cells. Subsequently, ten genes with varied expression level in different tissues (midgut and carcass without midgut) of T. castaneum were tested. The results indicated that the higher knockdown efficiency was always obtained in the tissue where the target gene expressed higher. In addition, three genes were tested in different developmental stages, larvae and pupae of T. castaneum. The results found that when the expression level increased after insect pupation, these genes became more sensitive to RNAi. Thus, all the proofs support unanimously that transcript level is a key factor affecting RNAi sensitivity. This finding allows for a better understanding of the RNAi efficiency variation and lead to effective or efficient use of RNAi technology.

Chimeric Double-Stranded RNAs Could Act as Tailor-Made Pesticides for Controlling Storage Insects.

Double-stranded RNA (dsRNA), the unique trigger of RNA interference, could be used as potential pesticides for the management of storage insects. High species specificity greatly improves the biosafety of dsRNAs. However, there are usually more than one insect species in real circumstances. In this study, we present a new strategy that broadens the control spectrum of a formulation using single dsRNA fragments. First, effective target genes were selected for each insect pest, here including Rhyzopertha dominica and Blattella germanica. Then, a template was prepared by conjugating various fragments from each of the selected genes. With this template, a piece of chimeric dsRNA was synthesized, and, thus, regional complementary specificity for genes from different insects was harnessed. Finally, injection treatments with this chimeric dsRNA demonstrated that each gene was selectively silenced, and the insects of both species were effectively killed by continuously feeding the chimeric dsRNA. Meanwhile, the results also demonstrated that the toxicity of chimeric dsRNA for non-target organisms, including Zophobas atratus and Periplaneta americana, could be low. This is the first description of a single dsRNA fragment accurately targeting several pest species, and the method provides promise of novel tailor-made biopesticides in the future management of storage insects.

Expanding the Toolkit for Genome Editing in a Disease Vector, Aedes aegypti: Transgenic Lines Expressing Cas9 and Single Guide RNA Induce Efficient Mutagenesis.

CRISPR-Cas9 mediated genome editing methods are being used for the analysis of gene function. However, it is hard to identify gene knockout mutants for genes whose knockout does not cause distinct phenotypes. To overcome this issue in the disease vector, Aedes aegypti, a transgenic Cas9/single guide RNA (sgRNA) method, was used to knock out the eye marker gene, kynurenine 3-monooxygenase (kmo), and the juvenile hormone receptor, Methoprene-tolerant (Met). PiggyBac transformation vectors were prepared to express sgRNAs targeting kmo and Met under the control of the U6 promoter. Transgenic Ae. aegypti expressing kmo-sgRNA or Met-sgRNA under the control of the U6 promoter and enhanced green fluorescent protein (eGFP) under the control of the hr5ie1 promoter were produced. The U6-sgRNA adults were mated with AAEL010097-Cas9 adults. The progeny were screened, and the insects expressing eGFP and DsRed were selected and evaluated for mutations in target genes. About 77% and 78% of the progeny that were positive for both eGFP and DsRed in kmo-sgRNA and Met-sgRNA groups, respectively, showed mutations in their target genes.

Off-target effects of RNAi correlate with the mismatch rate between dsRNA and non-target mRNA.

RNAi is a potent technique for the knockdown of target genes. However, its potential off-target effects limit the widespread applications in both reverse genetic analysis and genetic manipulation. Previous efforts have uncovered rules underlying specificity of siRNA-based silencing, which has broad applications in humans, but the basis for specificity of dsRNAs, which are better suited for use as insecticides, is poorly understood. Here, we investigated the rules governing dsRNA specificity. Mutational analyses showed that dsRNAs with >80% sequence identity with target genes triggered RNAi efficiently. dsRNAs with ≥16 bp segments of perfectly matched sequence or >26 bp segments of almost perfectly matched sequence with one or two mismatches scarcely distributed (single mismatches inserted between ≥5 bp matching segments or mismatched couplets inserted between ≥8 bp matching segments) also able to trigger RNAi. Using these parameters to predict off-target risk, dsRNAs can be designed to optimize specificity and efficiency, paving the way to the widespread, rational application of RNAi in pest control.

Identification of a double-stranded RNA-degrading nuclease influencing both ingestion and injection RNA interference efficiency in the red flour beetle Tribolium castaneum.

RNA interference (RNAi) efficiency dramatically varies among different insects and among administration methods. Numerous studies have revealed that a poor RNAi response is usually associated with a high double-stranded RNA (dsRNA)-degrading activity. Using the red flour beetle Tribolium castaneum, we conducted genome-wide identification of genes encoding dsRNA-degrading nucleases of the DNA/RNA non-specific endonuclease superfamily. To achieve a robust RNAi response in T. castaneum, four dsRNase genes were identified in the genome that seemed to be the potential factors reducing RNAi efficacy. Analysis of biochemical properties revealed that optimal conditions for the dsRNA-degrading activity were alkaline (pH 8.0) in the absence of Mg2+ at 37 °C. The dsRNA-degrading activity was predominantly present in the gut, and via heterologous expression and RNAi experimentation, gut-specific TcdsRNase1 was confirmed as the major nuclease performing dsRNA degradation. After a knockdown of the TcdsRNase1 nuclease activity, RNAi efficiency improved from 38.6% to 58.9% and from 20.9% to 53.9% for injection and ingestion of dsRNA, respectively. Our results contribute to a comprehensive understanding of the mechanisms influencing dsRNA stability and even RNAi efficiency in T. castaneum and point to a good method for improving RNAi efficiency through downregulation of the relevant nuclease activity.

Regulation of olfactory-based sex behaviors in the silkworm by genes in the sex-determination cascade.

Insect courtship and mating depend on integration of olfactory, visual, and tactile cues. Compared to other insects, Bombyx mori, the domesticated silkworm, has relatively simple sexual behaviors as it cannot fly. Here by using CRISPR/Cas9 and electrophysiological techniques we found that courtship and mating behaviors are regulated in male silk moths by mutating genes in the sex determination cascade belonging to two conserved pathways. Loss of Bmdsx gene expression significantly reduced the peripheral perception of the major pheromone component bombykol by reducing expression of the product of the BmOR1 gene which completely blocked courtship in adult males. Interestingly, we found that mating behavior was regulated independently by another sexual differentiation gene, Bmfru. Loss of Bmfru completely blocked mating, but males displayed normal courtship behavior. Lack of Bmfru expression significantly reduced the perception of the minor pheromone component bombykal due to the down regulation of BmOR3 expression; further, functional analysis revealed that loss of the product of BmOR3 played a key role in terminating male mating behavior. Our results suggest that Bmdsx and Bmfru are at the base of the two primary pathways that regulate olfactory-based sexual behavior.

Genome editing in the fall armyworm, Spodoptera frugiperda: Multiple sgRNA/Cas9 method for identification of knockouts in one generation.

The CRISPR/Cas9 system is an efficient genome editing method that can be used in functional genomics research. The fall armyworm, Spodoptera frugiperda, is a serious agricultural pest that has spread over most of the world. However, very little information is available on functional genomics for this insect. We performed CRISPR/Cas9-mediated site-specific mutagenesis of three target genes: two marker genes [Biogenesis of lysosome-related organelles complex 1 subunit 2 (BLOS2) and tryptophan 2, 3-dioxygenase (TO)], and a developmental gene, E93 (a key ecdysone-induced transcription factor that promotes adult development). The knockouts (KO) of BLOS2, TO and E93 induced translucent mosaic integument, olive eye color, and larval-pupal intermediate phenotypes, respectively. Sequencing RNA isolated from wild-type and E93 KO insects showed that E93 promotes adult development by influencing the expression of the genes coding for transcription factor, Krüppel homolog 1, the pupal specifier, Broad-Complex, serine proteases, and heat shock proteins. Often, gene-edited insects display mosaicism in which only a fraction of the cells are edited as intended, and establishing a homozygous line is both costly and time-consuming. To overcome these limitations, a method to completely KO the target gene in S. frugiperda by injecting the Cas9 protein and multiple sgRNAs targeting one exon of the E93 gene into embryos was developed. Ten percent of the G0 larvae exhibited larval-pupal intermediates. The mutations were confirmed by T7E1 assay, and the mutation frequency was determined as >80%. Complete KO of the E93 gene was achieved in one generation using the multiple sgRNA method, demonstrating a powerful approach to improve genome editing in lepidopteran and other non-model insects.

Identification and characterization of multiple dsRNases from a lepidopteran insect, the tobacco cutworm, Spodoptera litura (Lepidoptera: Noctuidae).

RNA interference (RNAi) efficiency varies among insects. RNAi is highly efficient and systemic in coleopteran insects but quite variable and inefficient in lepidopteran insects. Degradation of double-stranded RNA (dsRNA) by double-stranded ribonucleases (dsRNases) is thought to contribute to the variability in RNAi efficiency observed among insects. One or two dsRNases involved in dsRNA digestion have been identified in a few insects. To understand the contribution of dsRNases to reduced RNAi efficiency in lepidopteran insects, we searched the transcriptome of Spodoptera litura and identified six genes coding for DNA/RNA non-specific endonucleases. Phylogenetic analysis revealed the evolutionary expansion of dsRNase genes in insects. The mRNA levels of three midgut-specific dsRNases increased during the larval stage, and the highest dsRNA-degrading activity was detected in third-instar larvae. Proteins produced via the expression of three midgut-specific dsRNases, and the widely expressed dsRNase3, in a baculovirus system showed dsRNase activity for four out of five dsRNases tested. In addition, the increase in dsRNA-degrading activity and upregulation of dsRNase1 and 2 in larvae fed on cabbage leaves suggests that the diet of S. litura can influence dsRNase expression, dsRNA stability, and thus probably RNAi efficiency. This is the first report that multiple dsRNases function together in an RNAi-recalcitrant insect. The data included in this paper suggest that multiple dsRNases coded by the S. litura genome might contribute to the lower and variable RNAi efficiency reported in this and other lepidopteran insects.

Knockout of juvenile hormone receptor, Methoprene-tolerant, induces black larval phenotype in the yellow fever mosquito, Aedes aegypti.

The yellow fever mosquito, Aedes aegypti, vectors human pathogens. Juvenile hormones (JH) control almost every aspect of an insect's life, and JH analogs are currently used to control mosquito larvae. Since RNA interference does not work efficiently during the larval stages of this insect, JH regulation of larval development and mode of action of JH analogs are not well studied. To overcome this limitation, we used a multiple single guide RNA-based CRISPR/Cas9 genome-editing method to knockout the methoprene-tolerant (Met) gene coding for a JH receptor. The Met knockout larvae exhibited a black larval phenotype during the L3 (third instar larvae) and L4 (fourth instar larvae) stages and died before pupation. However, Met knockout did not affect embryonic development or the L1 and L2 stages. Microscopy studies revealed the precocious synthesis of a dark pupal cuticle during the L3 and L4 stages. Gene expression analysis showed that Krüppel homolog 1, a key transcription factor in JH action, was down-regulated, but genes coding for proteins involved in melanization, pupal and adult cuticle synthesis, and blood meal digestion in adults were up-regulated in L4 Met mutants. These data suggest that, during the L3 and L4 stages, Met mediates JH suppression of pupal/adult genes involved in the synthesis and melanization of the cuticle and blood meal digestion. These results help to advance our knowledge of JH regulation of larval development and the mode of action of JH analogs in Ae. aegypti.

CRISPR/Cas9 mediated gene knockout reveals a more important role of PBP1 than PBP2 in the perception of female sex pheromone components in Spodoptera litura.

Three different pheromone binding proteins (PBPs) can typically be found in the sensilla lymph of noctuid moth antennae, but their relative contributions in perception of the sex pheromone is rarely verified in vivo. Previously, we demonstrated that SlitPBP3 plays a minor role in the sex pheromone detection in Spodoptera litura using the CRISPR/Cas9 system. In the present study, the roles of two other SlitPBPs (SlitPBP1 and SlitPBP2) are further verified using the same system. First, by co-injection of Cas9 mRNA/sgRNA into newly laid eggs, a high rate of target mutagenesis was induced, 51.5% for SlitPBP1 and 46.8% for SlitPBP2 as determined by restriction enzyme assay. Then, the homozygous SlitPBP1 and SlitPBP2 knockout lines were obtained by cross-breeding. Finally, using homozygous knockout male moths, we performed electrophysiological (EAG recording) and behavioral analyses. Results showed that knockout of either SlitPBP1 or SlitPBP2 in males decreased EAG response to each of the 3 sex pheromone components (Z9,E11-14:Ac, Z9,E12-14:Ac and Z9-14:Ac) by 53%, 60% and 63% (for SlitPBP1 knockout) and 40%, 43% and 46% (for SlitPBP2 knockout), respectively. These decreases in EAG responses were similar among 3 pheromone components, but were more pronounced in SlitPBP1 knockout males than in SlitPBP2 knockout males. Consistently, behavioral assays with the major component (Z9,E11-14:Ac) showed that SlitPBP1 knockout males responded in much lower percentages than SlitPBP2 knockout males in terms of orientation to the pheromone, along with reduction in close range behaviors such as hairpencil display and mating attempt. Taken together, this study provides direct functional evidence for the roles of SlitPBP1 and SlitPBP2, as well as their relative importance (SlitPBP1 > SlitPBP2) in the sex pheromone perception. This information is valuable in understanding mechanisms of sex pheromone perception and may facilitate the development of PBP-targeted pest control techniques.

A Δ9 desaturase (SlitDes11) is associated with the biosynthesis of ester sex pheromone components in Spodoptera litura.

Sex pheromone biosynthesis in moths relies on the activity of multiple enzymes, including Δ9 desaturase, which plays an important role in catalyzing desaturation at the Δ9 position of the carbon chain. However, the physiological function of moth Δ9 desaturase has not been elucidated in vivo. In this study, we used the CRISPR/Cas9 system to knockout the Δ9 desaturase gene (SlitDes11) of Spodoptera litura to analyze its role in sex pheromone biosynthesis. First, through the direct injection of SlitDes11-single guide RNA (sgRNA)/Cas9 messenger RNA into newly laid eggs, gene editing was induced in around 30% of eggs 24 h after injection and was induced in 20.8% of the resulting adult moths. Second, using a sibling-crossing strategy, insects with mutant SlitDes11 (bearing a premature stop codon) were selected, and homozygous mutants were obtained in the G5 generation. Third, pheromone gland extracts of adult female homozygous SlitDes11 mutants were analyzed using Gas chromatography (GC). The results showed that titers of all three ester sex pheromone components; Z9, E11-14:Ac, Z9,E12-14:Ac, and Z9-14:Ac; were reduced by 62.40%, 78.50%, and 72.50%, respectively. This study provides the first direct evidence for the role of SlitDes11 in sex pheromone biosynthesis in S. litura, and indicates the gene could be as potential target to disrupt sexual communication in S. litura for developing a new pollution-free insecticide.

CRISPR/Cas9-mediated PBP1 and PBP3 mutagenesis induced significant reduction in electrophysiological response to sex pheromones in male Chilo suppressalis.

Pheromone-binding proteins (PBPs) are thought to bind and transport sex pheromones onto the olfactory receptors on the dendrite membrane of olfactory neurons, and thus play a vital role in sex pheromone perception. However, the function of PBPs has rarely been demonstrated in vivo. In this study, two PBPs (PBP1 and PBP3) of Chilo suppressalis, one of the most notorious pyralid pests, were in vivo functionally characterized using insects with the PBP gene knocked out by the CRISPR/Cas9 system. First, through direct injection of PBP-single guide RNA (sgRNA)/Cas9 messenger RNA into newly laid eggs, a high rate of target-gene editing (checked with polled eggs) was induced at 24 h after injection, 21.3% for PBP1-sgRNA injected eggs and 19.5% for PBP3-sgRNA injected eggs. Second, by an in-crossing strategy, insects with mutant PBP1 or PBP3 (both with a premature stop codon) were screened, and homozygous mutants were obtained in the G3 generation. Third, the mutant insects were measured for electroantennogram (EAG) response to female sex pheromones. As a result, both PBP mutant males displayed significant reduction in EAG response, and this reduction in PBP1 mutants was higher than that in PBP3 mutants, indicating a more important role of PBP1. Finally, the relative importance of two PBPs and the possible off target effect induced by sgRNA-injection are discussed. Taken together, our study provides a deeper insight into the function of and interaction between different PBP genes in sex pheromone perception of C. suppressalis, as well as a valuable reference in methodology for gene functional study in other genes and other moth species.

Expression Profile and Functional Characterization Suggesting the Involvement of Three Chemosensory Proteins in Perception of Host Plant Volatiles in Chilo suppressalis (Lepidoptera: Pyralidae).

The high sensitivity of the olfactory system is essential for feeding and oviposition in moth insects, and some chemosensory proteins (CSPs) are thought to play roles in this system by binding and carrying hydrophobic odorants across the aqueous sensillar lymph. In this study, to identify the olfactory CSPs from a repertoire of 21 CSP members in the notorious rice pest Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), tissue expression patterns were firstly examined by quantitative real-time polymerase chain reaction (qPCR). It showed that CSP2 was antennae specific and seven more CSPs (CSP1, 3, 4, 6, 15, 16, and 17) were antennae biased in expression, suggesting their olfactory roles; while other CSPs were multiple-tissue expressed and non-antennae biased, suggesting other functions for these genes. To further determine the ligand binding specificity, three putative olfactory genes (CSP1-3) were expressed in Escherichia coli cells, and binding affinity of these three recombinant CSP proteins were measured for 35 plant volatiles by the ligand binding assays. CSP1 and CSP2 exhibited high binding affinities (Ki ≤ 10.00 µM) for four (2-tridecanone, benzaldehyde, laurinaldehyde and 2-pentadecanone) and two (2-heptanol and (+)-cedrol) host plant volatiles, respectively; the three CSPs also showed moderate binding affinity (Ki = 10.01-20.00 µM) for 16 plant volatiles. Our study suggests that the three CSPs play essential roles in the perception of host plant volatiles, providing bases for the elucidation of olfactory mechanisms in this important pyralid pest.

CRISPR/Cas9 mediated BLOS2 knockout resulting in disappearance of yellow strips and white spots on the larval integument in Spodoptera litura.

The custom-design bacterial CRISPR/Cas9 system has been recently used in some insects, indicating a powerful technique for studies on gene function and transgenic insects. However, its use in lepidopteran pests is scarce. Here, we reported a CRISPR/Cas9 system mediated mutagenesis of biogenesis of lysosome-related organelles complex1, subunit 2 (BLOS2) gene in a noctuid pest Spodoptera litura. A fragment of SlitBLOS2 was identified by analyzing a S. litura transcriptome database by local basic BLAST, and the full length cDNA was acquired by RACE strategy. To clarify the function of SlitBLOS2, CRISPR/Cas9 based target mutagenesis of SlitBLOS2 was achieved, displaying a mosaic translucent integument in 62.3-70.6% larvae of G0 generation. Further PCR-based genotype analysis demonstrated various mutations occurred at the SlitBLOS2 specific target site. A homozygote mutant individual was obtained in G1 generation, in which the yellow strips and white spots on the larval integument completely disappeared. Our study clearly demonstrates the function of SlitBLOS2 in the integument coloration, and thus provides a useful marker gene for genome editing based gene functional study and pest control strategy in S. litura as well as other lepidopteran pests.

Functional characterization of PBP1 gene in Helicoverpa armigera (Lepidoptera: Noctuidae) by using the CRISPR/Cas9 system.

Pheromone binding proteins (PBPs) are thought to play crucial roles in perception of the sex pheromones particularly in noctuid moths, but this is rarely in vivo evidenced due to lacking an effective technique. Here, we reported an in vivo functional study of PBP1 in the important lepidopteran pest Helicoverpa armigera (HarmPBP1), by using the CRISPR/Cas9 system. Efficient and heritable mutagenesis was achieved by egg injection of mixture of Cas9-mRNA and HarmPBP1-sgRNA. The TA cloning and sequencing revealed various insertion and/or deletion (indel) mutations at the target site. Among those, one mutation resulted in a premature stop codon at the target site, which led to a highly truncated protein with only 10 amino acids. The HarmPBP1 with this mutation would completely loss its function, and thus was used to select the homozygous mutant insects for functional analysis. The electroantennogram recording showed that the mutant male adults displayed severely impaired responses to all three sex pheromone components (Z11-16:Ald, Z9-16:Ald and Z9-14:Ald). Our study provides the first in vivo evidence that HarmPBP1 plays important role in perception of female sex pheromones, and also an effective methodology for using CRISPR/Cas9 system in functional genetic study in H. armigera as well as other insects.

Functional characterization of SlitPBP3 in Spodoptera litura by CRISPR/Cas9 mediated genome editing.

Functional gene analysis by using genome editing techniques is limited only in few model insects. Here, we reported an efficient and heritable gene mutagenesis analysis in an important lepidopteran pest, Spodoptera litura, using the CRISPR/Cas9 system. By using this system, we successfully obtained the homozygous S. litura strain by targeting the pheromone binding protein 3 gene (SlitPBP3), which allowed us to elucidate the role of this gene in the olfaction of the female sex pheromones. By co-injection of Cas9 mRNA and sgRNA into S. litura eggs, highly efficient chimera mutation in SlitPBP3 loci was detected both in injected eggs (39.1%) and in the resulting individual moths (87.5%). We used the mutant moths as parents to obtain the G1 offspring and the homozygous mutant strain in G2. The function of SlitPBP3 was explored by Electroantennogram (EAG) recordings with a homozygous mutant strain. The result showed that the EAG responses were significantly decreased in mutant males than in control males when treated with the major sex pheromone component (Z9,E11-14:Ac) and a minor component (Z9-14:Ac) at higher dosages. The results demonstrate that s SlitPBP3 gene plays a minor role in the perception of the female sex pheromones. Furthermore, our study provides a useful methodology with the CRISPR/Cas9 system for gene in vivo functional study, particular for lepidopteran species in which the RNAi approach is not efficient.