Insect sex sorting method

Abstract

Provided herein are methods of sex sorting a plurality of insects based on sex-specific gene expression, the methods comprising (a) generating an exogenous nucleic acid molecule; (b) delivering the exogenous nucleic acid molecule into an insect body of the plurality of insects, wherein the exogenous nucleic acid molecule comprises a promoter region, a gender-specific splicing module, a reporter gene, and a transcription terminator; (c) detecting gender specific gene expression of the reporter gene; and (d) sorting the insects of the plurality of insects based on the detection of the gender-specific gene expression in step (c), thereby sex-sorting the insects based on the gender-specific gene expression.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 418,783, filed on October 24, 2022. The disclosure of the prior application is considered part of the disclosure of the present application and is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically as an XML file named "15670-0368WO1.XML". The size of the XML file, created on October 23, 2023, is 77,956 bytes. The material in the XML file is hereby incorporated by reference in its entirety. Background Art

[0005] Sex separation is the rate-limiting step in insect release methods for biological control. For many insect species (e.g., Aedes aegypti, Anopheles spp., Culex spp., Drosophila melanogaster, Anopheles gambiae, Cx. quinquefasciatus), it is not considered possible to separate males and females early in development. Systems that allow high-throughput sex sorting are needed because this would enable the use of sterile insects to be expanded to many species, thereby limiting the need for harmful pesticides. Summary of the Invention

[0006] Provided herein are methods for sex sorting a plurality of insects based on sex-specific gene expression, the method comprising (a) generating an exogenous nucleic acid molecule; (b) delivering the exogenous nucleic acid molecule into an insect from the plurality of insects, wherein the exogenous nucleic acid molecule comprises a promoter region, a sex-specific splicing module, a reporter gene, and a transcription terminator; (c) detecting sex-specific gene expression of the reporter gene; and (d) sorting the insects from the plurality of insects based on the detection of the sex-specific gene expression in step (c), thereby sex sorting the insects based on the sex-specific gene expression.

[0007] In some embodiments, the exogenous nucleic acid molecule further comprises a piggyBac inverted terminal repeat sequence located at each end of the effector region. In some embodiments, the promoter region comprises an Hr5IE1 promoter or an OpIE-1 promoter.

[0008] In some embodiments, step (b) comprises integrating the exogenous nucleic acid molecule into the genome of the insect. In some embodiments, step (d) comprises sorting the insect at the larval stage.

[0009] In some embodiments, the gender-specific splicing module comprises an endogenous gender-specific exon sequence and a truncated gender-specific intron sequence. In some embodiments, the gender-specific splicing module is a male-specific splicing module. In some embodiments, the gender-specific splicing module is a female-specific splicing module. In some embodiments, the insect is Aedes aegypti, Drosophila melanogaster, Drosophila suzukii, Ceratitis capitata, or Anastrepha ludens. In some embodiments, the gender-specific splicing module is derived from AaeDsx, traF of C. capitata, traF of D. melanogaster, or traF of D. suzukii. In some embodiments, the gender-specific splicing module is derived from AaeDsx, and wherein the male-specific splicing module comprises Exon 4, Exon 6, or any combination thereof. In some embodiments, the gender-specific splicing module is derived from AaeDsx, and wherein the female-specific splicing module comprises Exon 4, Exon 5b, Exon 6, or any combination thereof. In some embodiments, Exon 5b is an engineered Exon 5b, wherein one or more stop codons are excluded from the Exon 5b.

[0010] In some embodiments, the reporter gene comprises a DsRed gene, an EGFP gene, or any combination thereof. In some embodiments, the insect is sorted as male based on expression of the EGFP gene. In some embodiments, the insect is sorted as female based on expression of the DsRed gene. In some embodiments, the transcriptional terminator comprises a SV40 poly(A) signal.

[0011] Also provided herein are methods of identifying the gender of an insect based on gender-specific gene expression, the method comprising (a) generating an exogenous nucleic acid; (b) delivering the exogenous nucleic acid molecule into an insect, wherein the exogenous nucleic acid molecule comprises a promoter region, a gender-specific splicing module, a reporter gene, and a transcriptional terminator; and (c) identifying gender-specific gene expression of the reporter gene, whereby the gender of the insect is identified based on the gender-specific gene expression.

[0012] In some embodiments, the exogenous nucleic acid molecule further comprises piggyBac inverted terminal repeat sequences at each end of the effector region. In some embodiments, the promoter region comprises a Hr5 IE1 promoter or an OpIE-1 promoter.

[0013] In some embodiments, step (b) comprises integrating the exogenous nucleic acid molecule into the genome of the insect. In some embodiments, step (d) comprises sorting the insects at the larval stage.

[0014] In some embodiments, the gender-specific splicing module comprises an endogenous gender-specific exon sequence and a truncated gender-specific intron sequence. In some embodiments, the gender-specific splicing module is a male-specific splicing module. In some embodiments, the gender-specific splicing module is a female-specific splicing module. In some embodiments, the insect is Aedes aegypti, Drosophila melanogaster, Drosophila suzukii, Ceratitis capitata, or Anastrepha ludens. In some embodiments, the gender-specific splicing module is derived from AaeDsx, traF of C. capitata, traF of D. melanogaster, or traF of D. suzukii. In some embodiments, the gender-specific splicing module is derived from AaeDsx, and wherein the male-specific splicing module comprises Exon 4, Exon 6, or any combination thereof. In some embodiments, the gender-specific splicing module is derived from AaeDsx, and wherein the female-specific splicing module comprises Exon 4, Exon 5b, Exon 6, or any combination thereof. In some embodiments, Exon 5b is an engineered Exon 5b, wherein one or more stop codons are excluded from the Exon 5b.

[0015] In some embodiments, the reporter gene comprises a DsRed gene, an EGFP gene, or any combination thereof. In some embodiments, the insects are sorted as male based on expression of the EGFP gene. In some embodiments, the insects are sorted as female based on expression of the DsRed gene. In some embodiments, the transcriptional terminator comprises a SV40 poly(A) signal. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1A A SEPARATOR construct using a gender-specific splicing module of AaDsx in Aedes aegypti is shown. The gender-specific splicing module of AaDsx was used to construct a SEPARATOR in A. aegypti. Expression of the SEPARATOR was driven by the constitutive baculovirus promoter Hr5Ie1. The male-specific splicing product has an EGFP coding sequence in-frame, while inclusion of a stop codon in Exon 5b prevents in-frame expression of the female DsRed. SV40 pA was used as a polyadenylation signal.

[0017] Figure 1BAn exemplary schematic diagram of generating homozygotes by crossing GFP-positive males with GFP-negative females is shown. GFP-positive larvae are sorted and the sex of each larva is determined at the pupal stage. GFP-positive males are then crossed with GFP-negative females to generate homozygotes.

[0018] Figure 1C Shows the use of Figure 1B Using the strategy in [15 generations], 100% of GFP-positive mosquito larvae were exclusively male in 15 generations. Mosquitoes were sorted based on GFP signal in the larval stage, and the sex ratio was examined using microscopy based on morphological differences in the shape of the genital lobes in the pupal stage, which are unique to each sex.

[0019] Figure 1D Photographs of embryos, larvae, pupae, and adults of wild-type (Liverpool) and SEPARATOR mosquitoes collected and photographed using a fluorescent stereomicroscope (Leica M165FC) are shown. Eggs 24-48 hours after laying were treated with a 30% NaOCl solution (final concentration approximately 3.6% available chlorine) for 15-30 minutes to remove the chorion and visualize the embryos. The eggs were hatched in deionized water in a vacuum chamber, and the resulting hatched larvae were then collected as L1 larvae.

[0020] Figure 1E The developmental stages of mosquitoes collected and photographed using a fluorescence stereo microscope (Leica M165FC) are shown. The image consists of two panels: the upper panel shows a bright field image, while the lower panel shows a GFP / mCH channel image.

[0021] Figure 2A Shows the use of complex object parameter analyzers and sorters Schematic diagram of a large-scale sex sorting process. The eggs of transgenic mosquitoes that have been engineered using the SEPARATOR system are incubated in a vacuum chamber filled with deionized water to hatch. 24 hours after hatching, the eggs are Larvae expressing GFP are screened using an instrument. GFP-positive larvae are then carefully sorted and raised in a controlled environment until they reach adulthood. Once mature, their sex is verified using various methods.

[0022] Figure 2B Shown are exemplary sorting results determined by the opacity and size of the larvae, followed by the intensity of their GFP expression. Eggs of transgenic mosquitoes genetically engineered to carry the SEPARATOR system were incubated in a vacuum chamber using deionized water. After 24 hours of incubation, the hatched larvae were separated by To ensure accurate sorting, larvae were selected based on both their opacity and size, and then sorted according to the intensity of their GFP expression.

[0023] Figures 3A-3C A comparison of the transcriptomes across the larval and pupal stages of mosquitoes is shown. Using the GFP signal, the SEPARATOR mosquito was used to individually isolate male and female mosquitoes at the L1 larval stage. After isolation, total RNA extraction and RNAseq analysis were performed. Analysis of the early pupal (EP), mid-pupa (MP), and late pupal (LP) stages was performed using data from a previous study. Sex identification at the pupal stage relied on sex-specific morphological differences. Sex-enriched genes were identified using DESeq2 and then GO enrichment analysis was performed. Shared genes between the L1 larval stage and all other comparisons were determined and a graph was created as shown in the UpSet plot ( Figure 3A ) and male mosquitoes ( Figure 3B ) and female mosquitoes ( Figure 3C ) to represent these shared genes.

[0024] Figures 4A-4B The SEPARATOR is shown to be compatible with sterile insect techniques including radiation-based sterile insect techniques (SIT, Figure 4A ) and the Wolbachia-based Incompatible Insect Technology (IIT, Figure 4B ). The process of generating radiation-induced sterile male mosquitoes begins with the culture of mosquitoes. Larger female pupae are screened out using a sieve. However, it should be noted that after the first sex sorting, some smaller female pupae may still be present. After the mosquitoes are irradiated, the newly emerged adults are screened out using an image recognition AI that is trained to distinguish the morphology of female from male adults during the second sex sorting. The SEPARATOR method utilizes a male-specific reporter gene (GFP) to positively select for male L1 larvae. This can be done using This is achieved by an instrument capable of high-throughput selection at a rate of up to 10 larvae per second. By removing female larvae early in the development process, the SEPARATOR supports more efficient production of males for SIT applications. In addition, the SEPARATOR allows the transport and release of irradiated sex-sorted pupae. This means that adult male mosquitoes can emerge directly into the environment without incurring additional fitness costs due to handling and transportation ( Figure 4A The Wolbachia-based Incompatible Insect Technology (IIT) uses a two-step sex sorting method to sort the currently used Wolbachia-infected male mosquitoes. The process of generating Wolbachia-infected SEPARATOR mosquitoes is relatively simple as it involves crossing Wolbachia-infected female mosquitoes with SEPARATOR male mosquitoes. Instrumental positive selection of resulting Wolbachia infected male L1 larvae expressing the male-specific reporter gene (GFP) Figure 4B ).

[0025] Figure 5A The relative position of the primer target sites, i.e. the 3’ end of the Hr5Ie1 promoter sequence and the 5’ end of the EGFP coding sequence, i.e. the SEPARATOR construct, is shown.

[0026] Figure 5B The PCR products visualized by gel electrophoresis and the splice junctions subsequently verified by sequencing are shown. The resulting splicing pattern is depicted in the right panel.

[0027] Figure 5C The relative levels of the non-sex-specifically regulated exons (exon 4 and exon 6) and the female-specific exons (exon 5a, exon 5b) of SEPARATOR determined by RNA sequencing (RNAseq) analysis are shown.

[0028] Figure 6 The gender-specific RNA splicing pattern of SEPARATOR verified by RNA sequencing analysis is shown. The splicing pattern of SEPARATOR was verified by RNAseq analysis on both GFP positive and GFP negative mosquitoes, with three biological replicates for each condition. The RNAseq reads were aligned to the different genotypes and the position of the exons is shown at the bottom.

[0029] Figure 7 The coverage distribution of the three chromosomes (Chr1, Chr2 and Chr3) and the SEPARATOR transgene (1174D) in SEPARATOR mosquitoes is shown. The center line represents the median, while the first and third quartiles define the boundaries of the box. The upper and lower thin lines extend from the box to the observed highest and lowest values, but not more than 1.5 times the interquartile range (IQR) from the box. Based on the sequencing depth, the coverage of chromosomes 1, 2 and 3 was 6.31, 6.30 and 6.08, respectively, while the coverage of the SEPARATOR transgene was 16.14. This indicates that the SEPARATOR transgene (1174D) is present in three copies from the coverage analysis.

[0030] Figure 8 The data processing, including the size and optical density standard (Ext / Tof), fluorescence (GFP / RFP) of the larvae, DBSCAN clustering and the final determination of GFP positive larvae is shown. Data processing, including the size and optical density standard (Ext / Tof), fluorescence (GFP / RFP) of the larvae, DBSCAN clustering and the final determination of GFP positive larvae.

[0031] Figures 9A-9BTranscriptional profiles and expression analysis, including PCA analysis, of L1-stage GFP-positive and GFP-negative larvae in SEPARATOR mosquitoes are shown. Figure 9A ) and hierarchical clustering of six samples for RNA sequencing ( Figure 9B ).

[0032] Figures 9C-9E MA diagram showing different expression patterns between GFP-positive and GFP-negative larvae at the L1 stage in SEPARATOR mosquitoes ( Figure 9C ), and upregulated genes ( Figure 9D ) and down-regulated genes ( Figure 9E ) of the enriched Gene Ontology (GO) terms.

[0033] Figure 10A Shown is a comparison of transcriptomes of L1-stage GFP-positive (male, L1M) and GFP-negative (female, L1F) larvae from SEPARATOR mosquitoes.

[0034] Figure 10B Shown is a transcriptome comparison between larvae from SEPARATOR mosquitoes and adult mosquitoes (pupae and cadavers) from the Matthews RNA-seq dataset.

[0035] Figure 11 Figure 2 shows the identification of male-enriched genes from different developmental stages in a transcriptome comparative analysis. L1 larvae from the SEPARATOR mosquito were included in the transcriptome comparative analysis. In addition, L3 and L4 larvae, as well as early pupae (EP), mid-pupae (MP), late pupae (LP), and adult mosquito carcasses (adult mosquitoes) from the Matthews RNA-seq dataset, were also included.

[0036] Figure 12 Figure 2 shows the identification of female-enriched genes from different developmental stages in a transcriptome comparative analysis. L1 larvae from the SEPARATOR mosquito were included in the transcriptome comparative analysis. In addition, L3 and L4 larvae, as well as early pupae (EP), mid-pupae (MP), late pupae (LP), and adult mosquito carcasses (adult mosquitoes) from the Matthews RNA-seq dataset, were also included.

[0037] Figure 13 Shown are the results of a gene ontology (GO) analysis of sex-enriched genes at different developmental stages. For the transcriptome comparison analysis, L1 larvae from the SEPARATOR mosquito were included. In addition, L3 and L4 larvae, as well as early pupae (EP), mid-pupae (MP), late pupae (LP), and adult mosquito carcasses (adult mosquitoes) from the Matthews RNA-seq dataset, were also included.

[0038] Figures 14A-14B A set of unique genes associated with the larval stage is shown to be identified and isolated using gene expression analysis and clustering methods. Using mfuzz clustering analysis with integrated developmental stage data, specific genes associated with the LI or L2-L4 stages are identified. Notably, cluster 17 is composed primarily of genes expressed in LI ( Figure 14A ), and cluster 1 exhibits gene expression primarily in the L2-L4 stages ( Figure 14B ).

[0039] Figures 15A-15B A schematic of the vector plasmid encoding the sex-sorting gene system including the male splice gene expression system ( Figure 15A ) and the dual marker gene expression system ( Figure 15B ) is shown.

[0040] Figures 16A-16D An exemplary sex-sorting machine for Drosophila is shown. Figure 16A Sex-specific alternative splicing of the transformer (tra) and resulting proteins in D. melanogaster, D. suzukii, D. melanogaster, and D. suzukii are shown. Figure 16B Splicing of the female-specific transformer (TraF) intron is shown to result in the production of a functional dsRed protein in females but not males. Figure 16C An exemplary schematic of the sex-sorting machine constructs engineered and tested in the study is shown. The TraF intron from D. melanogaster, D. suzukii, D. melanogaster, and D. suzukii is inserted into the coding sequence of dsRed or eGFP after the ATG translation start codon. Figure 16D Fluorescent expression of females and males carrying the respective constructs is shown.

[0041] Figures 17A-17C Expression of the Opie2-TraF-dsRed or Hr5ie1-TraF-eGFP transgenes in D. melanogaster at different developmental stages ( Figure 17A LI-L3 larval stages, Figure 17B pupal stages, Figure 17C adult stages) can be observed under white light, RFP, and GFP filters is shown.

[0042] Figure 18 Female selection efficiency of all six sex-sorting machines in different life stages of D. melanogaster is shown, which gives female-specific fluorescence, and the number of scored flies is indicated for each bar.

[0043] Figures 19A-19B Adaptation cost of all eight sex-sorting machines is shown to be accessed by two parameters: Figure 19A ) egg hatch rate, and ( Figure 19B ) Adult survival. In the CctraF-dsRed line, a fitness cost was observed in the adult survival parameter (*p<0.05, ***p<0.001, Student's t-test with equal variance).

[0044] Figure 20A Shown is a comparison of female-specific intron splice donor and acceptor sites in transformants from Drosophila melanogaster, Drosophila suzukii, Ceratitis capitata, and Anastrepha mexicana. Figure 20B Sequence alignments of the 5' beginning (upper panel) and 3' end (lower panel) of the intron are shown.

[0045] Figure 21 Shown is a protein alignment of transformant proteins in Drosophila melanogaster, Drosophila suzukii, Ceratitis capitata, and Anastrepha mexicana.

[0046] Figures 22A-22C Shows the splicing pattern of the traF intron in Drosophila melanogaster. The gel electrophoresis image shows ( Figure 22A )dsRed-traF genomic DNA PCR ( Figure 22B ) dsRed-traF cDNA. ML: molecular ladder. Figure 22C The sequencing results of cDNA from each band are shown.

[0047] Figures 23A-23B Shown is the SEPARATOR system for sex sorting of Mediterranean fruit flies. Figure 23A Schematic diagram of the sex element (Separator) cassettes 795H1 and 795K1, produced by alternative RNA-splicing of a transgenic observable reporter. These cassettes contain two functional elements: the components Hr5IE1-eGFP-SV40 and Opie2-DsRed-p10, with the DsRed coding sequence separated by a transformant (tra) intron. The tra introns in 795H1 and 795K1 are derived from the Mediterranean fruit fly (Ceratitis fruit fly) and Anastrepha mexicana (Anaesthesia mexicana), respectively. Figure 23BShown in the SEPARATOR strain, males only produce eGFP, while females express both eGFP and DsRed. Under strong light and green fluorescent protein (GFP) and red fluorescent protein (RFP) filters, representative images of the larval stage, pupal stage and adult stage of wild-type (WT) and homozygous female and male individuals are compared with each other. All images are taken for the H-002 strain carrying the 795H1 construct. Images of all life stages are taken under strong light and green fluorescent protein (GFP) and red fluorescent protein (RFP) filters.

[0048] Figures 24A-24C Characterization of transgenic SEPARATOR lines is shown. Figure 24A Shown are stacked graphs showing the sex distribution of the fluorescent phenotype of four homozygous transgenic lines at passages 9 and 10, where more than 3,500 adult flies were screened. Only the expected DsRed+ / GFP+ females and DsRed- / GFP+ males were observed, while no DsRed+ / GFP+ males or DsRed- / GFP+ females were observed. Figure 24B Shown are intra-strain egg laying rates for all four lines compared to wild type by egg laying rate within 5 hours. Figure 24C Shown are egg hatch rates within the strain cross for all four strains as compared to wild type by hatch rate of eggs laid within 5 hours. Figures 24A-24C It is shown that the H-001 and H-002 lines have the 795H1 cassette carrying the endogenous C. melitensis transformant (tra) intron, while K-001 and K-002 carry the 795K1 cassette with the Anastrepha mexicana tra intron. Figure 24A ) Chi-squared test showed that there was no statistically significant sex ratio distortion in any of the four lines. ( Figures 24B-24C ) Dunn test Statistical significance was established as follows: p<0.05=* and the significance of wild type-transgenic lines is shown on the graph. ( Figures 24B-24C ) Bars represent mean values, while dots represent raw values ​​of replicates. SEPARATOR stands for sex element generated by alternative RNA splicing of a transgenic observable reporter gene.

[0049] Figure 25 Integration maps for 4 unique lines of the 795H1 and 795K1 constructs are shown. Lines H-001 and H-002 contain the 795H1 cassette, while lines K-001 and K-002 contain the 795K1 cassette.

[0050] Figure 26Images of 795H1 and 795K1 homozygous females are shown, where the DsRed signal of the homozygous female carrying the Anastrepha mexicana transformant (tra) intron containing the 795K1 cassette is weaker (left) than the signal of the homozygous female carrying the Mediterranean fruit fly tra intron containing the 795H1 cassette (center). These images were taken simultaneously with the image of the wild-type female (right).

[0051] Figure 27 Images of transgenic and wild-type eggs are shown, where the egg images demonstrate wild-type eggs and homozygous SEPARATOR eggs, both expressing GFP and varying degrees of DsRed.

[0052] Figures 28A-28D shows that the corresponding Figure 28A ) Mediterranean fruit fly and ( Figure 28B ) the transformant (tra) intron of Anastrepha mexicana, showing ( Figure 28A ) carries 795H1 and ( Figure 28B ) Diagram of the expected sex-specific DsRed splicing pattern in flies carrying 795K1. The forward and reverse primers used in PCR amplifications, specific for the exogenous elements of the two constructs, were located in ( Figure 28A )and( Figure 28B ) are shown as F and R. Figures 28C-28D Annotated electrophoresis gel images of female and male genomic DNA (gDNA) and female and male cDNA amplifications. A DNA ladder was run in the leftmost lane, and a negative control was run in the rightmost lane. DETAILED DESCRIPTION

[0053] Provided herein are methods for sex sorting a plurality of animals (e.g., insects) based on sex-specific gene expression, the methods comprising (a) generating an exogenous nucleic acid molecule; (b) delivering the exogenous nucleic acid molecule into an insect from the plurality of insects, wherein the exogenous nucleic acid molecule comprises a promoter region, a sex-specific splicing module, a reporter gene, and a transcription terminator; (c) detecting sex-specific gene expression of the reporter gene; and (d) sorting the insects from the plurality of insects based on detecting the sex-specific gene expression in step (c).

[0054] Also provided herein is a method for identifying the sex of an insect based on sex-specific gene expression, the method comprising (a) generating an exogenous nucleic acid molecule; (b) delivering the exogenous nucleic acid molecule into the insect, wherein the exogenous nucleic acid molecule comprises a promoter region, a sex-specific splicing module, a reporter gene, and a transcription terminator; and (c) identifying sex-specific gene expression of the reporter gene.

[0055] It should be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" refer to one or more (i.e., at least one) of the grammatical objects of the article unless the context clearly dictates otherwise. For example, "a cell" encompasses one or more cells.

[0056] As used herein, the terms "about" and "approximately" when used to modify a quantity specified as a value or range indicate the stated value and reasonable deviations from the value known to those skilled in the art, e.g., ±20%, ±10%, or ±5%, within the intended meaning of the stated value.

[0057] As used herein, "delivery", "gene delivery", "gene transfer", "transduction" can refer to the introduction of exogenous polynucleotides into a host cell, regardless of the method used for the introduction. Such methods include a variety of well-known techniques, such as vector-mediated gene transfer (e.g., viral infection / transfection, or various other protein-based or lipid-based gene delivery complexes) and technologies that facilitate the delivery of "naked" polynucleotides (e.g., electroporation, "gene gun" delivery, and various other techniques for introducing polynucleotides). The introduced polynucleotides can be maintained in the host cell stably or transiently. Stable maintenance generally requires that the introduced polynucleotides contain an origin of replication compatible with the host cell or are integrated into a replicon of the host cell, such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.

[0058] In some embodiments, the polynucleotide can be inserted into the host cell by a gene delivery molecule. Examples of gene delivery molecules can include, but are not limited to, liposomes, micellar biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as baculoviruses, adenoviruses and retroviruses, phages, cosmids, plasmids, fungal vectors and other recombinant vectors commonly used in the art, which have been described for expression in various eukaryotic and prokaryotic hosts and can be used for gene therapy as well as simple protein expression.

[0059] As used herein, “engineered” or “genetically engineered” when referring to an organism (e.g., an insect) refers to an organism that comprises a nucleic acid sequence (e.g., DNA, RNA, or mRNA) that is not present in, or is present at different levels in, other similar organisms that are not engineered under similar conditions (exogenous nucleic acid), or is an organism that comprises a polypeptide expressed from the nucleic acid. In some embodiments, a genetically engineered organism is altered from its native state by the introduction of an exogenous nucleic acid, or is a progeny of such an altered organism. In some embodiments, a genetically engineered organism comprises an exogenous nucleic acid (e.g., DNA, RNA, or mRNA).

[0060] As used herein, the term “endogenous” refers to any substance and process originating from inside a life system such as an organism, tissue, or cell.

[0061] As used herein, the term “endogenous” refers to any material introduced from outside or originating outside a cell, tissue, or organism that is not produced by or derived from the same cell, tissue, or organism into which it was introduced.

[0062] As used herein, the term “expression” refers to the process of transcription of a polynucleotide into mRNA and / or the subsequent translation of the transcribed mRNA into a peptide, polypeptide, or protein. In some embodiments, if the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell. Expression levels of a gene can be determined by measuring the amount of mRNA or protein in a cell or tissue sample; further, expression levels of multiple genes can be determined to establish an expression profile of a particular sample.

[0063] As used herein, the terms “nucleic acid” and “nucleotide” are intended to be consistent with their use in the art and include naturally occurring species or functional analogs thereof. Naturally occurring nucleic acids typically have a phosphodiester bond-containing backbone. Analog structures can have alternative backbone linkages including any of a variety of backbone linkages known in the art. Naturally occurring nucleic acids typically have deoxyribose (e.g., present in deoxyribonucleic acid (DNA)) or ribose (e.g., present in ribonucleic acid (RNA)).

[0064] Nucleic acid can contain any nucleotide in the multiple analogs with these sugar moieties known in the art.Nucleic acid can include natural or non-natural nucleotides.In this respect, natural deoxyribonucleic acid can have one or more bases selected from the group consisting of: adenine (A), thymine (T), cytosine (C) or guanine (G), and ribonucleic acid can have one or more bases selected from the group consisting of: uracil (U), adenine (A), cytosine (C) or guanine (G).The useful non-natural bases that can be included in nucleic acid or nucleotide are known in the art.

[0065] The term "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or a combination thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides with similar binding properties to the reference nucleic acid. Unless otherwise indicated, a specific nucleic acid sequence also implicitly encompasses complementary sequences as well as explicitly stated sequences. In some embodiments of any of the isolated nucleic acids described herein, the isolated nucleic acid is DNA. In some embodiments of any of the isolated nucleic acids described herein, the isolated nucleic acid is RNA.

[0066] As used herein, the term "plurality" can refer to a state of having multiple (e.g., more than one) different types of things (e.g., cells, genomic sequences, subjects, systems, or proteins). In some embodiments, the plurality of genomic sequences can be more than one genomic sequence, wherein each genomic sequence is different from the other.

[0067] Methods for sex sorting and identification of insect sex

[0068] Provided herein are methods for sex sorting insects based on sex-specific gene expression, the methods comprising (a) generating an exogenous nucleic acid molecule; (b) delivering the exogenous nucleic acid molecule into an insect from a plurality of insects, wherein the exogenous nucleic acid molecule comprises a promoter region, a sex-specific splicing module, a reporter gene, and a transcription terminator; (c) detecting sex-specific gene expression of the reporter gene; and (d) sorting the insects from the plurality of insects based on the detection of the sex-specific gene expression in step (c), thereby sex sorting the insects based on the sex-specific gene expression.

[0069] Also provided herein are methods of identifying the sex of an insect based on sex-specific gene expression, the methods comprising (a) generating an exogenous nucleic acid molecule; (b) delivering the exogenous nucleic acid molecule into an insect, wherein the exogenous nucleic acid molecule comprises a promoter region, a sex-specific splicing module, a reporter gene, and a transcriptional terminator; and (c) identifying sex-specific gene expression of the reporter gene, whereby the sex of the insect is identified based on the sex-specific gene expression.

[0070] As used herein, an “insect” can refer to any member of the largest class of the arthropod phylum, itself the largest animal phylum. Insects have a segmented body, legs on joints, and an external skeleton (e.g., exoskeleton). In some embodiments, an insect can include a bed bug, a housefly, a clothes moth, a Japanese beetle, an aphid, a mosquito, a flea, a horse fly, a yellowjacket, a butterfly, or a moth. In some embodiments, an insect can be a mosquito from the genus Aedes, Culex, Anopheles, or Ochlerotatus. In some embodiments, a mosquito can include Aedes aegypti, Aedes albopictus, Ochlerotatus triseriatus (Aedes triseriatus), Anopheles stephensi, Anopheles albimanus, Anopheles gambiae, Anopheles quadrimaculatus, Anopheles freeborni, Culex species, or Culiseta melanura. In some embodiments, an insect can include a fruit fly selected from the group consisting of: Medfly (Mediterranean fruit fly), Mexfly (Mexican fruit fly), Oriental fruit fly (Bactrocera dorsalis), Olive fruitfly (Bactrocera oleae), Melon fly (Bactrocera cucurbitae), Natal fruit fly (Ceratitis rosa), Cherry fruit fly (Rhagoletis cerasi), Queensland fruitfly (Bactrocera tyroni), Peach fruit fly (Bactrocera zonata), Caribbean fruit fly (Anastrepha suspensa), Oriental fruit fly (Bactrocera dorsalis), West Indian fruit fly (Anastrepha obliqua), New World screwworm (Cochliomyia hominivorax), Old World screw worm (Chrysomya bezziana), and Mediterranean fruit fly (Ceratitis capitata).hominivorax), Old World screwworm (Chrysomya bezziana), Australian sheep blowfly / greenbottle fly (Lucilia cuprina), pink bollworm (Pectinophora gossypiella), European gypsy moth (Lymantria dispar), Navel orange worm (Amyelois transitella), Peach twig borer (Anarsia lineatella), rice stem borer (Tryporyza incertulas), noctuid moth, Heliothinae, Japanese beetle (Papilla japonica), White-fringed beetle (Graphognatus spp.), Boll weevil (Anthonomous grandis), Colorado potato beetle (Leptinotarsa ​​decern lineata), vine mealybug (Planococcus ficus), Asian citrus psyllid (Diaphorina citri), Spotted wing drosophila (Drosophila suzukii), Bluegreen sharpshooter (Graphocephala atropunctata), Glassywinged sharpshooter (Flomalodisca vitripennis), Light brown applemoth (Epiphyas postvittana)), Bagradabug) (Bagrada hilaris), Brown marmorated stink bug (Halyomorpha halys), Asian Gypsy Moth selected from the group of Lymantria dispar asiatica, Lymantria dispar japonica, Lymantria albescens, Lymantria umbrosa, and Lymantria postalba, Asian longhorned beetle (Anoplophora glabripennis), Coconut Rhinoceros Beetle (Oryctes rhinoceros), Emerald Ash Borer (Agrilus planipennis), European Grapevine Moth (e.g., Lobesia botrana), European Gypsy Moth (Lymantria dispar), False Codling Moth (Thaumatotibia leucotreta), Fire Ant selected from Solenopsis invicta Buren and S. richteri Forel, Old World Bollworm (Flelicoverpa armigera), Spotted Lanternfly (Lycorma delicatula), Africanized honeybee (Apis mellifera scutellata), Fruit and shoot borer (Leucinodes orbonalis), Corn Rootworm (Diabrotica spp.), Western corn rootworm (Diabrotica virgifera), Whitefly (Bemisia tabaci), Flouse Fly (Musca domestica), Fruit Fly (Drosophila melanogaster), Mediterranean Fruit Fly (Ceratitis capitata), and Yellow Fever Mosquito (Aedes aegypti).Domestica), Green Bottle Fly (Luciola copperensis), Silk Moth (Bombyx mori), Red Scale (Aonidiella aurantia), Dog Heartworm (Dirofilaria immitis), Southern Pine Beetle (Dendroctonus frontalis), Avocado Thrips (Thysanoptera spp.), Botfly (Botfly) selected from the genera Oestridae and Dermatobia hominis), Florse Fly (Tabanus sulcifrons), Florn Fly (Flaematobia irritans), Liriomyia fasciatus (Cochliomyia spp. ... macellaria (C. macellaria), screwworm flies of C. hominivorax, C. aldrichi or C. minima, tsetse flies (Glossina spp.), warble flies selected from Flypoderma bovis or Hypoderma lineatum, spotted lanternfly (Cyprinus spp.), Khapra beetle (Trogoderma granarium), honeybee mite (Varroa destructor), termites, hemlock woolly adelgid (Adelges tsugae), walnut twig beetle (Pityophthorus truncatus), juglandis), European wood wasp (Sirex noctilio), Pink-spotted bollworm (Pectinophora scutigera), Two-spotted spidermite (Tertanychus urticae), Diamondback moth (Diamondbackmoth (plutella xylostella), taro caterpillar (spodoptera litura), red flour beetle (tribolium castaneum), green peach aphid (Myzus persicae), cotton aphid (aphis gossypii), brown planthopper (nilaparvata lugens), beet armyworm (spodotetrae xigua), western flower thrips (frankliniella occidentalis), codling moth (cydia pomonella), cowpea weevil (callosobruchus maculatus), pea aphid (Pea spp. In some embodiments, the insect is an Aedes aegypti. In some embodiments, the insect is a Drosophila melanogaster. In some embodiments, the insect is a Drosophila suzukii. In some embodiments, the insect is a Mediterranean fruit fly. In some embodiments, the insect is an Anastrepha mexicana. See, e.g., Davydova et al., doi.org / 10.1101 / 2023.09.29.560088, 2023; Liu et al., bioRxiv. 2023 Aug 14:2023.08.11.553026; and Weng et al., bioRxiv. 2023 Jul 12:2023.06.16.545348, which are incorporated herein by reference in their entireties.

[0071] In certain embodiments, any method in the methods described herein can utilize sex-specific expression by the sex-specific alternative splicing (SSAS) of a reporter gene. In certain embodiments, any method in the methods described herein can utilize male-specific expression by the sex-specific alternative splicing (SSAS) of a reporter gene. In certain embodiments, any method in the methods described herein can utilize female-specific expression by the sex-specific alternative splicing (SSAS) of a reporter gene. In certain embodiments, insects are carried out sex sorting and / or the sex of identifying insects can be identified as males. In certain embodiments, insects are carried out sex sorting and / or the sex of identifying insects can be identified as females.

[0072] The term "RNA splicing" refers to the process in molecular biology in which newly prepared precursor messenger RNA (pre-mRNA) transcripts are converted into mature messenger RNA (mRNA). It works by removing all introns (non-coding regions of RNA) and splicing the exons (coding regions) back together. For nuclear-encoded genes, splicing occurs in the cell nucleus during or shortly after transcription. For those eukaryotic genes that contain introns, splicing is generally required to produce mRNA molecules that can be translated into proteins. For many eukaryotic introns, splicing occurs in a series of reactions catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins (snRNPs). The term "alternative splicing" refers to a cellular process in which exons from the same gene are joined together in different combinations, resulting in different but related mRNA transcripts. The resulting mRNA can be translated to produce different proteins with different structures and functions, but derived from the same parent gene.

[0073] In some embodiments, the sex-specific expression of the reporter gene comprises sex-specific alternative splicing of the reporter gene, wherein the reporter gene is a harmless fluorescent marker. In some embodiments, the reporter gene comprises a DsRed gene, an EGFP gene, or any combination thereof.

[0074] In certain embodiments, any of the methods described herein can sex sort insects during early larval development. In certain embodiments, any of the methods described herein can sex sort insects as mature embryos. In certain embodiments, any of the methods described herein can be used for high-throughput sorting. In certain embodiments, high-throughput sorting comprises sorting insects at a speed of at most 740 larvae / minute (e.g., at most 300 larvae / minute, at most 400 larvae / minute, at most 500 larvae / minute, at most 600 larvae / minute, or at most 700 larvae / minute).

[0075] In some embodiments, any of the methods described herein can be used to sex sort insects without relying on sex chromosome linkage. In some embodiments, any of the methods described herein comprises a sex-specific gene expression system that is genetically stable and not susceptible to breakage by meiotic recombination or chromosomal rearrangements. In some embodiments, any of the methods described herein is portable to other species because it utilizes transposable elements, promoters, and markers that are portable across species.

[0076] In some embodiments, any of the methods described herein can be referred to as a sex element generated by alternative RNA splicing of a transgenic observable reporter gene ("SEPARATOR").

[0077] In certain embodiments, any of the methods described herein can be used for high-throughput sex selection of Aedes mosquitoes or other insects. In certain embodiments, any of the methods described herein can be a positive selection system. In certain embodiments, any of the methods described herein can positively select male mosquito L1 larvae. In certain embodiments, any of the methods described herein can positively select male L1 larvae expressing dominant male-specific reporter genes (e.g., EGFP). In certain embodiments, any of the methods described herein does not rely on the morphological differences between female mosquitoes and male mosquitoes during the pupal stage and the adult stage to distinguish two sexes. In certain embodiments, any of the methods described herein does not need to distinguish the size difference between female pupae and male pupae.

[0078] In certain embodiments, any one of the methods described herein can realize large-scale automated process. In certain embodiments, any one of the methods described herein does not require a large amount of manpower and time. In certain embodiments, any one of the methods described herein can be transplantable across multiple species.

[0079] In some embodiments, any of the methods described herein comprise gender sorting the plurality of insects based on gender-specific gene expression. As used herein, the term “gender sorting” refers to sorting and separating a plurality of insects (e.g., A. aegypti, D. melanogaster, D. suzukii, C. capitata, or A. obliqua) into two groups (e.g., males and females). In some embodiments, an insect of the plurality of insects can be identified as a male insect. In some embodiments, an insect of the plurality of insects can be identified as a female insect. In some embodiments, once an insect is identified as a male insect, it can be removed from the plurality of insects. In some embodiments, once an insect is identified as a female insect, it can be removed from the plurality of insects. In some embodiments, gender sorting can comprise sorting insects at the larval stage by fluorescence and separating the larvae into two groups (e.g., EGFP positive and EGFP negative). In some embodiments, gender sorting can comprise automated gender sorting, wherein bulk larvae (e.g., thousands of larvae) are sorted using fluorescence-based flow cytometry, and wherein male larvae expressing a reporter gene (e.g., EGFP) can form a distinct cluster and are visibly separated from female larvae that do not express the reporter gene. In some embodiments, gender sorting can comprise sorting insects at the larval stage by fluorescence by performing various methods known in the art. For example, such methods can include, but are not limited to, fluorescence-activated cell sorting (FACS), flow cytometry, automated fluorescence microscopy, microfluidic technology in conjunction with a fluorescence-based assay, or a fluorescence microplate reader. See, e.g., Marois et al., Malar J. 2012 Aug 28; 11:302; Midkiff et al., Molecules. 2019 Dec; 24(23):4292, which are incorporated by reference herein in their entirety.

[0080] In some embodiments, gender sorting can further comprise sorting the plurality of insects based on a distinct size difference between female insects and male insects. In some embodiments, female insects can be larger in size compared to male insects. In some embodiments, gender sorting can comprise sorting the plurality of insects to be size sorted through a sieve. In some embodiments, gender sorting can further comprise utilizing visual cues of morphological sexual dimorphism to identify and separate an insect from the plurality of insects.

[0081] In some embodiments, any of the methods described herein comprises detecting and / or identifying sex-specific gene expression. In some embodiments, detecting and / or identifying sex-specific gene expression can include using RNA sequencing to identify genes that exhibit sex-specific expression patterns. In some embodiments, detecting and / or identifying sex-specific gene expression can further include performing a comprehensive analysis of differential gene expression (DGE), wherein it can be found that specific genes are differentially expressed according to the sex and / or developmental stage of the insect. In some embodiments, sex-specific genes can exhibit male-enriched expression patterns in the early L1 larval stage of the insect. In some embodiments, sex-specific genes can exhibit female-enriched expression patterns in the early L1 larval stage of the insect. In some embodiments, RNA sequencing can be performed in the L1 larval stage of the insect. In some embodiments, RNA sequencing can be performed in the L3 larval stage of the insect. In some embodiments, RNA sequencing can be performed in the L4 larval stage of the insect. In some embodiments, RNA sequencing can be performed in the pupal stage of the insect. In some embodiments, RNA sequencing can be performed in the adult stage of the insect.

[0082] In some embodiments, any of the methods described herein can detect fluorescent reporter genes (e.g., EGFP and DsRed) by fluorescence detectors in any type of flow cytometer or sorter. In some embodiments, any of the methods described herein comprises a sorter that collects selected insects. In some embodiments, any of the methods described herein comprises the use of a sophisticated object parameter analyzer and sorter that allows for scalable, high-throughput sex selection of insects.

[0083] Exogenous nucleic acid molecules

[0084] Provided herein are exogenous nucleic acid molecules comprising a promoter region, a sex-specific splicing module, a reporter gene, and a transcription terminator.

[0085] In some embodiments of any of the exogenous nucleic acid molecules described herein, the exogenous nucleic acid molecule includes a promoter region (e.g., any exemplary promoter in the exemplary promoters described herein). As used herein, the term "promoter" can refer to a DNA sequence recognized by an enzyme / protein in a mammalian cell, which is necessary for initiating transcription of the connected coding sequence. A promoter generally refers to a nucleotide sequence to which, for example, RNA polymerase and / or any associated factors bind, and transcription begins at the sequence. A promoter can be a constitutive, inducible, or tissue-specific promoter (e.g., a brain-specific promoter). A promoter can be an exogenous promoter operably connected to an isolated nucleic acid. A promoter can also be a genomic sequence, wherein the promoter is close to the transcription start site and at least partially controls the expression of the associated gene product. A promoter within the genome can be located proximal (e.g., within 2000 nucleotides) or distal (e.g., greater than 2000 nucleotides) of the transcription start site. Non-limiting exemplary promoters include CMV, CBA, CAG, Cbh, EF-1α, PGK, UBC, GUSB, UCOE, hAAT, TBG, desmin, MCK, C5-12, NSE, synaptophysin, PDGF, MecP2, CaMKII, mGluR2, NFL, NFH, nβ2, PPE, ENK, EAAT2, GFAP, MBP and U6 promoters. See, for example, U.S. Patent No. 11,512,327, which is incorporated by reference in its entirety. In some embodiments, the promoter region may include the Hr5IE1 promoter. In some embodiments, the promoter region may include the OpIE-1 promoter.

[0086] In some embodiments of any of the exogenous nucleic acid molecules described herein, the exogenous nucleic acid molecule comprises a gender-specific splicing module. In some embodiments, the gender-specific splicing module can comprise an endogenous gender-specific exon sequence and a truncated intron sequence. In some embodiments, the gender-specific splicing module is a male-specific splicing module. In some embodiments, the male-specific splicing module comprises Exon 6, Exon 6, or any combination thereof. In some embodiments, the gender-specific splicing module is a female-specific splicing module. In some embodiments, the female-specific splicing module comprises Exon 4, Exon 5b, Exon 6, or any combination thereof. In some embodiments, the female-specific splicing module comprises an engineered Exon 5b, wherein the engineered Exon 5B is engineered to exclude one or more stop codons from Exon 5b. In some embodiments, the gender-specific splicing module is derived from the Aedes aegypti doublesex gene (AaeDsx). In some embodiments, the gender-specific splicing module is derived from the Mediterranean fruit fly transformer (traF). In some embodiments, the gender-specific splicing module is derived from the Drosophila melanogaster traF. In some embodiments, the gender-specific splicing module is derived from the Drosophila suzukii traF.

[0087] In some embodiments of any of the exogenous nucleic acid molecules described herein, the exogenous nucleic acid molecule comprises a reporter gene. As used herein, the term “reporter gene,” often simply referred to as “reporter,” refers to a gene that a researcher ligates to a regulatory sequence of another gene of interest in a bacterium, cell culture, animal, or plant. Such genes are called reporter genes because their expression confers a property on the organism in which they are expressed that is easily recognized and measured, or because they are selectable markers. Reporter genes are often used as an indication that a certain gene has been taken up or expressed by a cell or population of organisms. Commonly used reporter genes that induce visually identifiable properties often involve fluorescent and luminescent proteins. Examples of reporter genes can include, but are not limited to, genes encoding fluorescent proteins (e.g., GFP, dsRed, YFP, RFP, mCherry, and EGFP), luciferases (e.g., firefly luciferase, Renilla luciferase), beta-galactosidase (e.g., LacZ, ), HaloTag, and GUS (beta-glucuronidase). (Lippincott-Schwartz and Patterson, Sci. 300:87-91; Contag and Bachmann, Annu. Rev. Biomed. Eng. 4:235-260; Salehi et al., Hum. Gene Ther. 20(1):21-30; Giepmans et al., Sci. 312(5771):217-224; Marathe and McEwen, Gene. 154(1):105-107). In some embodiments, the reporter gene comprises a DsRed gene, an EGFP gene, or any combination thereof.

[0088] In some embodiments, the exogenous nucleic acid molecule comprises a coding sequence for one or more reporter genes. In some embodiments, the exogenous nucleic acid molecule comprises a coding sequence for the one or more reporter genes such that the reporter gene can be expressed in a gender-specific manner. In some embodiments, the exogenous nucleic acid molecule comprises coding sequences for EGFP and DsRed, wherein EGFP and DsRed are expressed in a gender-specific manner. In some embodiments, the exogenous nucleic acid molecule comprises a DsRed coding sequence in frame with female-specific splicing modules (exon 4, engineered exon 5b, and exon 6), thereby controlling female-specific DsRed expression. In some embodiments, the exogenous nucleic acid molecule comprises an EGFP coding sequence in frame with male-specific products (exon 4 and exon 6), thereby controlling male-specific EGFP expression.

[0089] In some embodiments, any of the methods described herein identifies the sex of an insect based on the expression of a sex-specific gene. In some embodiments, insects can be sorted as male based on the expression of a male-specific gene. In some embodiments, insects can be sorted as male based on the expression of an EGFP gene. In some embodiments, insects can be sorted as female based on the expression of a female-specific gene. In some embodiments, insects can be sorted as female based on the expression of a DsRed gene.

[0090] In some embodiments, the exogenous nucleic acid molecule comprises a transcription terminator. As used herein, a "transcription terminator" is a nucleic acid sequence that marks the end of a gene or operator in genomic DNA during transcription. This sequence mediates transcription termination by providing a signal in the newly synthesized transcript RNA that triggers the process of releasing the transcript RNA from the transcription complex. In some embodiments, the exogenous nucleic acid molecule comprises a transcription terminator, wherein the transcription terminator comprises an SV40 poly (A) signal.

[0091] In some embodiments, any of the methods described herein includes a delivery step comprising integrating the exogenous nucleic acid molecule into the genome of the insect. In some embodiments, the exogenous nucleic acid molecule is integrated into the genome of the insect via a vector.

[0092] Gene delivery vectors

[0093] In some embodiments of any of the methods described herein, the exogenous nucleic acid molecule is introduced into the genome of the insect via a vector. For example, the vector can be an expression vector, wherein the expression vector comprises a promoter sequence operably linked to a sequence encoding a molecule (e.g., a nucleic acid molecule). Non-limiting examples of vectors include plasmids, transposons (e.g., DNA transposons, RNA transposons, or retrotransposons and class III transposons), cosmids, and viral-derived vectors (e.g., any adenovirus-derived vector (AV), cytomegalovirus-derived vector (CMV), simian virus-derived vector (SV40), adeno-associated virus-derived vector (AAV), lentiviral vectors, and retroviral vectors), as well as any Vectors. For example, the vector can include sufficient cis-acting elements for expression, wherein other elements for expression can be supplied by the host mammalian cells or in an in vitro expression system. Skilled practitioners can select appropriate vectors and mammalian cells to introduce any of the spatial analysis reagents described herein.

[0094] In certain embodiments, retroviral vectors and adeno-associated virus vectors can be used as recombinant gene delivery systems for the transfer of exogenous nucleic acid molecules. These vectors provide efficient delivery of genes into cells, and stably integrate the transferred nucleic acid into the chromosomal DNA of the host cell. The protocol for producing recombinant retroviruses and infecting cells in vitro with such viruses can be found in Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, (1989), Sections 9.10-9.14, and other standard laboratory manuals. Suitable retroviral examples include pLJ, pZIP, pWE, and pEM known to those skilled in the art. Examples of suitable packaging virus strains for the preparation of ecotropic and dual-tropic retroviral systems include ΨCrip, ΨCre, Ψ2, and ΨAm. Retroviruses have been used to introduce various genes into many different cell types in vitro, including epithelial cells (see, e.g., Eglitis et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Proceedings of the National Academy of Sciences of the United States of America 85:3014-3018; Armentano et al. (1990) Proceedings of the National Academy of Sciences of the United States of America 87:6141-6145; Huber et al. (1991) Proceedings of the National Academy of Sciences of the United States of America 88:8039-8043; Ferry et al. (1991) Proceedings of the National Academy of Sciences of the United States of America 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application No. WO 89 / 07136; PCT Application No. WO 89 / 02468; PCT Application No. WO 89 / 05345; and PCT Application No. WO 92 / 07573).

[0095] In some embodiments, another viral gene delivery system used in the methods of the present invention can utilize an adenovirus-derived vector. The genome of the adenovirus can be manipulated so that it encodes and expresses the gene product of interest, but is inactivated in terms of its ability to replicate in the normal lytic viral life cycle. See, for example, Berkner et al., BioTechniques 6:616 (1988); Rosenfeld et al., Science 252:431-434 (1991); and Rosenfeld et al., Cell 68:143-155 (1992). Suitable adenovirus vectors derived from adenovirus strain Ad 5 type d1324 or other adenovirus strains (e.g., Ad2, Ad3, or Ad7, etc.) are known to those skilled in the art. Recombinant adenovirus may be advantageous in some cases because it cannot infect non-dividing cells and can be used to infect a variety of cell types, including epithelial cells (Rosenfeld et al., (1992) ibid). In addition, the viral particles are relatively stable and easy to purify and concentrate, and as described above, can be modified to affect the infectivity spectrum. Additionally, the introduced adenoviral DNA (and the foreign DNA contained therein) is not integrated into the genome of the host cell, but remains in a free state, thereby avoiding potential problems that may occur due to in situ insertional mutagenesis, where the introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). In addition, relative to other gene delivery vectors, the adenoviral genome has a larger carrying capacity for foreign DNA (up to 8 kilobases) (Berkner et al., supra; Haj-Ahmand and Graham, J. Virol. 57:267 (1986).

[0096] In some embodiments, helper-dependent (HDAd) vectors can also be generated in which all adenoviral sequences are deleted except for the DNA replication origin at each end of the viral DNA and the packaging signal at the 5' end of the genome downstream of the left packaging signal. HDAd vectors are constructed and propagated in the presence of a replication-competent helper adenovirus that provides the early and late proteins required for replication.

[0097] In some embodiments, another viral vector system for delivering nucleic acids is an adeno-associated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as adenovirus or herpes virus, as a helper virus for efficient replication and production of the life cycle. It is also one of the few viruses that can integrate its DNA into non-dividing cells and exhibits a high frequency of stable integration (see, e.g., Flotte et al., Am. J. Respir. Cell. Mol. Biol. 7:349-356 (1992); Samulski et al., J. Virol. 63:3822-3828 (1989); and McLaughlin et al., J. Virol. 62:1963-1973 (1989). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. The space for foreign DNA is limited to about 4.5 kb. AAV vectors, such as those described in Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985), can be used to introduce DNA into cells. AAV vectors have been used to introduce a variety of nucleic acids into different cell types (see, e.g., Hermonat et al., Proc. Natl. Acad. Sci. USA 81 :6466-6470 (1984); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1985); Wondisford et al., Mol. Endocrinol. 2:32-39 (1988); Tratschin et al., J. Virol. 51 :611-619 (1984); and Flotte et al., J. Biol. Chem. 268:3781-3790 (1993). The identification of Staphylococcus aureus (SaCas9) and other smaller Cas9 enzymes that can be packaged into highly stable and efficient adeno-associated virus (AAV) vectors, are easily produced, FDA-approved, and tested in multiple clinical trials, opens new avenues for therapeutic gene editing.

[0098] In some embodiments, a vector (e.g., a gene delivery vector) may include any of the exogenous nucleic acid molecules described herein. In some embodiments, the gene delivery vector is a transposon, wherein the transposon includes a DNA transposon, an RNA transposon, or a retrotransposon and a class III transposon. Examples of DNA transposons include Tc1 / mariner, piggyBac, hAT, and Helitron. DNA transposons are generally described in the following literature: for example, Wicker et al., Nat. Rev. Genet. 8 (12): 973-982; Feschotte et al., Annu. Rev. Genet. 41 (1): 331-368; and Munoz-Lopez et al., Curr. Genomics 11 (2): 115-128. RNA transposons, also known as retrotransposons, are mainly divided into long terminal repeats (LTRs) and non-long terminal repeats (non-LTRs). RNA transposons are generally described in the literature, for example, by Finnegan. Curr. Biol. 22(11): R432-R437; Dombroski et al., Mol. Cell. Biol. 14(7): 4485-92; and Sanchez et al., Nat. Commun. 8(1): 1283. Examples of class III transposons include the foldback (FB) element of Drosophila melanogaster, the TU element of Strongylocentrotus purpuratus, and the miniature inverted repeat transposable element. Class III transposons are generally described in the literature, for example, by Boutanaev and Osbourn, PNAS. 115(28): E6650-E6658; Kaminker et al., Geno. Biol. 3(12): research0084. In some embodiments, the gene delivery vector is a piggyBac transposon.

[0099] As used herein, the term "piggyBac" refers to the PiggyBac (PB) transposon, which is a mobile genetic element with high transposition between a vector and a chromosome by a "cut and paste" mechanism. During transposition, the PB transposase recognizes the transposon-specific inverted terminal repeats (ITRs) located at both ends of the transposon vector and moves the contents from the original site and integrates them into the TTAA chromosomal site. The activity of the PiggyBac transposon system enables the gene of interest between the two ITRs in the PB vector to be easily moved into the target genome. In some embodiments of any of the methods described herein, the exogenous nucleic acid molecule may further include a piggyBac inverted terminal repeat located at each end of the effector region.

[0100] As used herein, "effector region" or "effector domain" may refer to a protein interaction region / domain that can play a role in transcriptional regulation through its ability to: (i) interact with the basal transcription machinery and general co-activators, (ii) interact with other transcription factors to allow cooperative binding, and (iii) directly or indirectly recruit histone and chromatin modifying enzymes.

[0101] In some embodiments of any of the methods described herein, the exogenous nucleic acid molecule is introduced into the genome of the insect via a vector, wherein the vector comprises SEQ ID NO: 1. In some embodiments of any of the methods described herein, the exogenous nucleic acid molecule is introduced into the genome of the insect via a vector, wherein the vector comprises SEQ ID NO: 2.

[0102] SEQ ID NO: 1 - Plasmid sequence of vector 1174D

[0103]

[0104] SEQ ID NO: 2 - Plasmid sequence of vector 1174I

[0105]

[0106] Examples

[0107] The present disclosure is further described in the following examples, which do not limit the scope of the present disclosure.

[0108] Aedes aegypti

[0109] Molecular cloning and transgenics

[0110] To create the endogenous AaeDsx splicing module construct, fragments of endogenous exons and introns from genomic DNA of Aedes aegypti were amplified using PCR. Plasmid 1122I, which previously contained mCherry and EGFP, was then linearized using the restriction enzyme PacI. The linearized 1122I plasmid and fragments of endogenous exons and introns were used in the Gibson enzyme assembly method to construct the 1174CX plasmid. To open the reading frame for the female-specific product and allow in-frame expression of the mCherry coding sequence, the endogenous exon 5b was replaced by an engineered exon 5b with the stop codon removed. The sequence of the engineered exon 5b was generated using The gene fragment was synthesized by the service. The endogenous exon 5b was removed using the restriction enzymes PmlI and SnaBI, and then the fragment containing the engineered exon 5b was integrated into the cut 1174CX plasmid using Gibson assembly to generate the 1174C plasmid.

[0111] To generate a plasmid for sex-specific expression of DsRed and EGFP, fragments of the engineered exon 5b and DsRed coding sequences from the 1174C plasmid and the previous 874Y plasmid were amplified. Then, overlapping PCR was used to fuse the two fragments together. Finally, ω PCR was used to replace the mCherry coding sequence from the 1174C plasmid to the sequence of the 3xP3 promoter with the DsRed coding sequence to obtain the 1174D plasmid. During each cloning step, a single colony was selected and cultured in LB medium containing ampicillin. Then, the plasmid was extracted (using the Zymo Research Zyppy plasmid miniprep kit) and subsequently subjected to Sanger sequencing. The final plasmid was prepared to the maximum extent (using the Zymo Research ZymoPURE II plasmid large-scale preparation kit) and fully sequenced by Primordium. All primers are listed in Table 1. The complete annotated plasmid sequence and plasmid DNA are available at Addgene (ID: 200012).

[0112] Transgenic lines are produced by microinjecting preblastoderm stage embryos with a mixture of piggyBac plasmids and transposase helper plasmids. Four days after microinjection, G0 embryos hatched, and surviving pupae were separated and sexed. Pupae were placed in separate male and female cages at a ratio of 5:1, with wild-type male pupae in female cages and wild-type female pupae in male cages. After allowing several days of development and mating, blood meals were provided and eggs were collected, aged, and hatched. Fluorescent marker-positive larvae were isolated using a fluorescent stereomicroscope. In order to isolate individual insertion events, male transformants with fluorescent markers were hybridized with female transformants without fluorescent markers, and individual strains were established. Individual genetic sex strains (1174D) were maintained as a mixture of homozygotes and heterozygotes, and wild-type individuals were regularly eliminated. Genetic sex strains (1174D) were homozygous by mating of a single pair of siblings for approximately ten generations, with individuals with the brightest marker expression selected for each generation.

[0113] Mosquito rearing and maintenance

[0114] Aedes aegypti is obtained from the Liverpool strain, which has been previously used to generate a reference genome. These mosquitoes are raised in an incubator at 30°C, 20-40% humidity, and the light / dark cycle in the cage is 12 hours (Bugdorm, 24.5x24.5x24.5cm). Adult mice are arbitrarily administered a sucrose solution of 10% (m / V), and female mice are fed a blood meal of anesthetized mice for approximately 15 minutes. Egg-laying matrix is ​​provided about 3 days after the blood meal. Eggs are collected, aged for approximately 4 days to allow embryonic development, and then hatched in deionized water in a vacuum chamber. Approximately 400 larvae are raised in a plastic container (Sterilite, 34.6x21x12.4cm, the U.S.) equipped with approximately 3 liters of deionized water, and fed fish food (TetraMin Tropical Flakes, Tetra Werke, Melle, Germany). For genetic crosses, pupae were separated and sexed under a microscope based on sex-specific morphological differences in the shape of the genital lobes (located below the paddle at the end of the pupal abdominal segment) to ensure the virginity of the female pupae before they were released into cages for eclosion. These general rearing procedures were followed unless otherwise stated. To increase the number of homozygotes in the 1174D transgenic line, both high-intensity GFP pupae and female GFP-negative pupae were transferred to cages and allowed to mate after eclosion. Female mosquitoes were fed a blood meal, and five adult female mosquitoes were transferred to oviducts for colonization and egg collection. The eggs from each colony were hatched and reared. Colonies with a higher proportion of female EGFP-negative and male EGFP-positive were selected for colonization of the next generation. After several rounds of colonization, colonies with 100% males in the strong EGFP-positive group and 100% females in the GFP-negative group were selected and propagated for expansion.

[0115]

[0116] Fluorescence sorting, sexing, and imaging

[0117] To determine The exact number of larvae in a cluster is determined based on the optical density and size measurements of the individuals, "log(EXT)" and "log(TOF)". The raw data was filtered to remove outliers such as egg debris and dust. The data was then further refined by filtering based on the individual fluorescence measurements of "log(first fluorescence)" and "log(second fluorescence)." Finally, the fluorescence measurements were automatically clustered and denoised using Density-Based Spatial Clustering of Applications with Noise (DBSCAN). Sorting was performed primarily as described for Anopheles larvae. The Aedes eggs adhered to their oviposition paper were briefly rinsed to remove dust and debris, immersed in deionized water in a small container, and catalytically incubated for 30-60 minutes under partial vacuum (25% of atmospheric pressure) in a vacuum chamber. They were then incubated overnight at 28°C to maximize larval hatching. The following day, the resulting unfed newborn larvae were transferred to a large particle flow cytometer equipped with a multi-line argon laser (488, 514 nm) and a diode laser (670 nm). The larvae were analyzed and sorted using the Biosort 5281 software using a 488 nm filter and the following acquisition parameters: green PMT 500, red PMT 600, delay 8; width 6, pure mode with superdrop. The flow rate was maintained between 20 and 70 objects per second by adjusting the concentration of larvae in the sample. Larvae identified as male (GFP positive) were distributed in petri dishes. Under these conditions, the sorting rate ranged from 4000 to 7400 larvae in 10 minutes (system initialization + 6 minutes and system cleaning and shutdown 6 minutes), and the total number of sorted larvae was limited by the number of available larvae. Recording during sorting Sorted larvae were counted using the software. For quality control of sorted larvae, the instrument's reservoirs and fluids were carefully rinsed, and the sorted larvae were placed back into the machine for analysis. In some experiments, subjects falling outside the GFP-positive gate were collected in "enrichment" mode to remove GFP-negative contaminants from the pool of GFP-positive larvae and verified by microscopy. Mosquitoes were examined, scored, and imaged using a Leica M165FC fluorescence stereomicroscope equipped with a Leica DMC2900 camera. For higher-resolution images, a Leica DM4B upright microscope equipped with a VIEW4K camera was used. To distinguish male from female mosquito pupae, sex-specific morphological differences in the shape of the genital lobes, located below the paddle at the terminal end of the pupal abdominal segments, were observed microscopically. This approach ensured that both male and female pupae were included in the selection process.

[0118] Determination of genomic integration sites

[0119] To confirm the transgene insertion site, Oxford Nanopore genomic DNA sequencing was performed. Genomic DNA was extracted from five adult male and five adult female SEPARATORs using the Blood and Cell Culture DNA Midi Kit (Qiagen, Catalog No. / ID: 13343) according to the manufacturer's protocol. Sequencing libraries were prepared using the Oxford Nanopore SQK-LSK110 Genomic Library Kit and sequenced on a single MinION flow cell (R9.4.1) for 72 hours. Base calling was performed using ONT Guppy base calling software version 6.4.6, using the dna_r9.4.1_450bps_sup model, generating 3.03 million reads above a quality threshold of Q≧10, with an N50 of 7941 bp and a total yield of 11.08 Gb. To identify the transgene insertion site, nanopore reads were mapped to a plasmid carrying SEPARATOR (1174D, Addgene as plasmid number 200012) using minimap2 and further aligned to the AaegL5.0 genome (GCF_002204515.2). Subsequently, the average coverage depth of the three autosomes and the transgene was calculated using samtools, and the results were visualized in R. The coverage depths of chr1, chr2, and chr3 were determined to be 6.31, 6.30, and 6.08, respectively. Interestingly, the coverage depth of the SEPARATOR transgene was significantly higher, reaching 16.14. Based on the coverage analysis, the SEPARATOR transgene appears to exist in three copies ( Figure 7 ). By checking the read alignment using the Integrated Genomics Viewer (IGV), the exact insertion site was determined. It is noteworthy that three copies of the SEPARATOR construct sequence were identified. The three integration sites are NC_035109.1:92046983, NC_035108.1:444508475, and NC_035107.1:299022928. This finding is consistent with the results of the coverage depth analysis, further supporting the existence of three insertion sites. The second integration site on NC_035108.1 overlaps with the AAEL005024 gene, which is currently classified as an uncharacterized protein. However, the other two integration sites do not overlap with any known genes. Nanopore sequencing data have been deposited in NCBI SRA (PRJNA985064).

[0120] RNA sequencing (RNA-Seq) analysis

[0121] To quantify target gene reduction and expression from transgenes, as well as to assess overall expression patterns, Illumina RNA sequencing was performed. Total RNA was extracted in biological triplicate from 50 GFP-positive male mosquitoes and 50 GFP-negative female mosquitoes at the L1 larval stage using the miRNeasy Tissue / Cell Advanced Mini Kit (Qiagen, catalog number / ID: 217604) according to the manufacturer's protocol (6 samples in total). Genomic DNA was removed using the gDNA Eliminator column provided with the kit. RNA integrity was assessed using the RNA 6000 Pico Kit for Bioanalyzer (Agilent Technologies, catalog number / ID: 5067-1513), and mRNA was isolated from approximately 1 μg of total RNA using the NEBNext Poly(A) mRNA Magnetic Separation Module (NEB, catalog number / ID: E7490). RNA-seq libraries were constructed using the NEBNext Ultra II RNA Library Preparation Kit (NEB, catalog number / ID: E7770) for Illumina according to the manufacturer's protocol. In brief, by incubation at 94 DEG C for 15 minutes in the first strand buffer, mRNA is fragmented to an average size of 200nt. Then random primers and ProtoScript II reverse transcriptase are used to synthesize cDNA, followed by second strand synthesis using NEB second strand synthase mixture. The resulting DNA fragments are end-repaired, dA-tailed and connected to NEBNext hairpin adapters (NEB, catalog number / ID: E7335). After connection, the adapters are converted to "Y" shape by treatment with USER enzyme, and Agencourt AMPure XP beads (Beckman Coulter (Beckman Coulter) number A63880) are used to size-select the DNA fragments to generate fragments of size between 250-350bp. The DNA connected to the adapters is PCR amplified, and then AMPure XP beads are cleaned up. Libraries were quantified using the Qubit dsDNA HS kit (ThermoFisher Scientific, catalog number / ID: Q32854) and size distribution was confirmed using the High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, catalog number / ID: 5067-4626). Libraries were sequenced in paired-end mode on an Illumina NextSeq2000 with a read length of 50 nt per library and a sequencing depth of 25 million reads. Base calling and FASTQ generation were performed using DRAGEN 3.8.4. Reads were mapped to the AaegL5.0 genome (GCF_002204515.2) supplemented with the SEPARATOR transgene sequence using STAR.On average, approximately 97.4% of reads were mapped. RNA-Seq data were analyzed using an integrated web application called iDEP75. TPM values ​​were calculated from the counts generated by feature counts and combined. Hierarchical clustering of the data showed that for each genotype, all replicates clustered together, as expected. Figures 9A-9B ). DESeq2 was then used to perform differential expression analysis on males (GFP positive) and females (GFP negative) at the L1 larval stage ( Figure 9C For each DESeq2 comparison, gene ontology enrichment was performed on the differentially expressed genes. Figures 9D-9E Illumina RNA sequencing data have been deposited in NCBI-SRA (PRJNA985064). For comparative transcriptome analysis, 47 files representing six developmental stages (L3 larvae, L4 larvae, early pupae, mid-pupae, late pupae, and adult carcasses) were retrieved from SRA. These files were then aligned to the AaegL5 genome (GCF_002204515.2) using STAR.

[0122] Example 1 - Generation of a Sex-Sorted Gene Expression System

[0123] To generate a sex-specific gene expression system in Aedes aegypti, a transformation vector was constructed in which piggyBac inverted terminal repeats flanked the effector region. The effector region allows genomic integration via the piggyBac inverted terminal repeats. An effector region containing a promoter region, sex-specific splicing elements, a reporter gene, and a transcriptional terminator was constructed for sex-specific gene expression. The Aedes aegypti double-sex gene (Aadsx) was selected as a candidate gene for constructing the sex-specific splicing element. A synthetic start codon with a Kozak sequence was followed by the constitutive baculovirus promoter Hr5IE1 to initialize the reading frame. A single nucleotide was inserted at the beginning of exon 4, and the stop codon in exon 5b was eliminated to open the reading frame. Because the endogenous introns 4 and 6 are too large for plasmid construction, truncated intron sequences were used to construct the Aadsx splicing module. Overlapping open reading frames were designed to independently express DsRed and EGFP via ribosomal frameshifting, which is regulated by sex-specific RNA splicing. The SV40 poly(A) signal is a transcription termination signal. Figure 15A The results showed that all mosquitoes expressing EGFP were male. However, no mosquitoes expressing DsRed were observed (Table 2).

[0124]

[0125]

[0126] Based on the results of male splicing products, a dual-marker expression system was further designed for sex sorting. The constitutive baculovirus promoter OpIE-2 was used to express the DsRed marker in both sexes. The constitutive baculovirus promoter Hr5IE1 and the sex-specific splicing elements from Aadsx were used to regulate male-specific EGFP expression. Truncated introns (intron 4 and intron 6) and endogenous female-specific exons (exon 5a and exon 5b) were incorporated into the splicing module to maintain sex-specific RNA splicing. A schematic diagram of the vector plasmid encoding the dual-marker expression system is shown in Figure 2. Figure 15B The results showed that all DsRed-positive mosquitoes were female, while mosquitoes with both markers (DsRed and EGFP) were male. The results are shown in Table 3.

[0127]

[0128] Example 2 - Engineering SEPARATOR

[0129] To generate SEPARATOR, a sex-specific alternatively spliced ​​intron from the Aedes aegypti doublesex gene (AaeDsx) was utilized. Figure 1A and Figure 5A Dsx is a highly conserved transcription factor involved in sex determination in insects. In Aedes aegypti, the male-specific AaeDsx intron is approximately 26.5 kb, which is a bit too large for the task. Therefore, the intron was truncated by retaining the splicing factor binding sites, including the Tra / Tra-2 and RNA-binding protein 1 (RBP1) binding sites, to preserve the sex specificity of the intron. This resulted in a smaller AaeDsx intron, 4.5 kb ( Figure 1A and Figure 5A ). The reading frame was initiated by adding a start codon with a Kozak sequence and expression was performed using the constitutive Hr5IE1 AcMNPV baculovirus promoter previously shown to be functional in many species. To enable reading, nine stop codons located in endogenous exon 5b were excluded. The coding sequences for EGFP and DsRed were strategically designed to overlap, allowing them to be expressed in a sex-specific manner. The DsRed coding sequence was designed to have the female-specific product (exon 4, engineered exon 5b, and exon 6) in frame to control female-specific DsRed expression. Additionally, a male-specific splice product involving exon 4 and exon 6 was designed to have the EGFP coding sequence in frame ( Figures 5A-5C ).

[0130] The SEPARATOR construct was then introduced into the mosquito genome to generate genetically sexed strains via the piggyBac transposon. The intended plan was to ensure that all GFP-expressing mosquitoes were male, while those expressing DsRed were female. Interestingly, the results after microinjection revealed that all 55 EGFP-expressing larvae were male at the G0 pupal stage (Table 4). However, no DsRed-expressing larvae were observed in G0. All pupae from G0 were sexed, and the resulting adult mosquitoes were crossed with wild-type mosquitoes, and stable transgenic lines were selected by fluorescent markers (G1). Similar results were observed after G1, with 100% of EGFP-positive larvae being male.

[0131]

[0132] To verify the sex-specific splicing pattern of the AaeDsx splicing module, a combined analysis was performed using reverse transcription polymerase chain reaction (RT-PCR) and RNA sequencing. Fifty EGFP-positive L1 larvae and fifty EGFP-negative L1 larvae were collected for further analysis. For RT-PCR, primers designed to target the 3' end of the Hr5IE1 promoter and the 5' end of the EGFP sequence were used ( Figure 5A ). Subsequently, Sanger sequencing was performed to analyze the obtained PCR products. The results showed that the truncated intron 4, engineered exon 5b, intron 6 and exon 6 sequences showed sex-specificity in RNA splicing ( Figure 5B The results of Sanger sequencing and RNA sequencing (RNA-seq) analysis were comparable and consistent with the expected splicing pattern ( Figure 5B and Figure 6 ). Sex-specific RNA splicing patterns showed that the female spliced ​​product contained the DsRed coding sequence in frame, while the male spliced ​​product underwent a (-1) frameshift, resulting in the DsRed coding sequence being out of frame and containing the EGFP coding sequence in frame. Interestingly, both RT-PCR and RNA-seq analyses revealed that the major product observed in females contained exon 4, exon 5b, and exon 6 ( Figures 5B-5C and Table 5). Notably, these exons were found to have DsRed coding sequences in frame ( Figure 5B and Figure 6 Therefore, it can be inferred that the expression level of female-specific transcripts and the splicing pattern of female-specific products do not pose any obstacles to DsRed expression. However, no DsRed signal was observed in transgenic larvae.

[0133] The transgene integration site was determined ( Figure 7) and generated homozygous lines. To this end, larvae were sorted by fluorescence to separate them into EGFP-positive and EGFP-negative groups, and the sex ratios of these two groups were examined at the pupal / adult stage. EGFP-positive males were crossed with EGFP-negative females to enrich for homozygous ( Figure 1B ). A total of 3635 EGFP+ larvae were manually screened over 15 generations and the resulting sex was confirmed at the pupal and adult stages. Notably, 100% of the GFP+ larvae were male ( Figure 1C and Table 4). In summary, the results indicate that the SEPARATOR technique is an efficient and effective method for separating male (EGFP+) and female (EGFP-) mosquitoes, which has important implications for the widespread adaptation of SIT for mosquito control.

[0134]

[0135] Example 3 - Measuring the effectiveness of sex sorting at scale

[0136] To evaluate the applicability of SEPARATOR throughout the mosquito life cycle, we further explored the timing of EGFP expression. We found that strong EGFP signals were expressed from late embryonic stages to the adult stage of mosquitoes ( Figures 1D-1E In addition, when compared to wild-type controls (Liverpool), the EGFP intensity of SEPARATOR mosquitoes was strong enough to distinguish EGFP-positive (males) from EGFP-negative (females) under pooled conditions from early stages of the mosquito life cycle ( Figure 1D Based on these results, it can be concluded that SEPARATOR is a powerful and robust mosquito sex sorting system.

[0137] To evaluate the suitability of SEPARATOR for automated sex sorting, we used Several thousand batches of first-instar larvae were sorted by fluorescence-based flow cytometry. This produced fluorescence images in which EGFP-expressing male larvae formed distinct clusters, clearly separated from GFP-negative female larvae ( Figures 2A-2B and Figure 8 ). At flow rates ranging from 20 to 70 objects per second, EGFP-positive larvae were selected and sorted in pure mode. Even at such high flow rates, 70-80% of EGFP-positive larvae were recovered, resulting in a sorting speed of 740 larvae / min (Table 6). A total of 108,570 larvae were sorted from G7 to G9 generations. After single sorting, 0.1-0.45% EGFP-negative (female) larval contamination was observed in male populations (Table 6). Although slower flow rates can result in more complete male recovery and minimize or eliminate female contamination, high sorting speed represents a trade-off between recovery rate and production speed required for large-scale production. To address the problem of female contamination, additional measures were taken. By quality control sorting, the contamination rate of EGFP-negative larvae (females) was successfully reduced to 0.01-0.03% (Table 6). This was achieved by using A second round of sorting was performed on the sorted larvae (a total of 78,861 individuals at G9). This second sorting phase was specifically targeted at objects deviating from the EGFP-positive gate, using an "enrichment mode" to enhance precision. These experiments were performed using the old SELECT instrument model from 2005. With a more modern instrument with upgraded laminar flow and electronic controls, further reduction of these reported female contamination rates is expected. In summary, this method efficiently and effectively sorts large numbers of larvae, providing valuable insights into the performance of the sex-sorting system.

[0138]

[0139] Example 4 - Identification of Sex-Enriched Genes

[0140] Previously, determining the sex of mosquitoes during the larval stage was challenging, requiring reliance on sex-specific morphological features of pupae and adults for accurate identification. Therefore, early transcriptomic analyses focused on studying aspects of the pupal and adult stages related to sex. While some limited studies successfully distinguished the sex of L3 and L4 larval stages by assessing the expression of the male-determining factor Nix, this approach required individual PCR testing of larvae to detect Nix and determine the sex of individual mosquitoes, followed by RNA-seq analysis.

[0141] To gain valuable insights into the molecular mechanisms controlling sex determination and differentiation during early developmental stages of A. aegypti, SEPARATOR was employed to separate male and female L1 larvae. Subsequently, RNA sequencing was performed to identify genes exhibiting sex-specific expression patterns. Using the data collected, a comprehensive analysis of differential gene expression (DGE) was performed. The results of the study revealed that, at the early L1 larval stage, 1082 genes exhibited male-enriched expression patterns, while 634 genes exhibited female-enriched expression patterns Figure 9C ​). Subsequently, enrichment analysis was performed on the sex-enriched genes. Among the male-enriched genes, two distinct clusters emerged based on gene ontology (GO) terms. The first cluster was associated with cilia and microtubule formation (including terms such as cilia, axonemal, microtubule-based process, and cell protrusion), while the second cluster was associated with cuticle formation Figure 9D ). In addition, among the female-enriched genes, several distinct clusters emerged based on GO terms including immune response, metabolic process, and cuticle formation Figure 9E

[0142] Following the RNA-seq analysis of L1 larvae, publicly available transcriptome data of the L1 larval stage was compared with data from L3, L4, pupal, and adult stages of mosquitoes. To determine the overlap between the SEPARATOR set and the other six comparison sets, UpSet plots were generated to represent the shared sex-enriched genes across all developmental stages Figure 11 and Figure 12 ). In the current analysis, a large number of genes were found that were not detected in any of the comparisons performed in previous studies. This finding suggests that some of these genes can represent early-expressed genes that were later turned off in subsequent stages, making them difficult to identify in previous studies.

[0143] To further explore the sex-enriched genes during various developmental stages of mosquitoes, GO enrichment analysis was performed on the sex-enriched gene lists of different developmental stages. However, at the L3 and L4 larval stages, a limited number of genes were observed to show sex-enrichment patterns. In addition, upon analyzing Matthews’ RNA-seq dataset, it was found that the expression level of Nix, a well-known male-determining factor between L3 males and L3 females, did not show significant difference. This difference can be attributed to the lower sequencing depth of Matthews’ RNA-seq data (approximately 7 million reads per sample) compared to the current L1 stage dataset (approximately 25 million reads per sample) (Figure 10). Furthermore, to ensure consistency with samples from other stages, the dataset from adult mosquitoes that had been dissected and separated to obtain independent samples was excluded. Therefore, GO enrichment analysis was performed on the sex-enriched gene lists of L1 larvae and pupal stages of mosquitoes. The published results showed that genes involved in cilia and microtubule formation were consistently enriched in males throughout the developmental stages of mosquitoes from L1 larvae to late pupae. Notably, during the early to mid-pupal stages, GO terms related to cytoskeletal organization were identified. In addition, during the late pupal stage of mosquito development, GO terms related to sperm cell development and sperm DNA condensation were identified (Figure 3 and Figure 13 ​These results indicate that sperm development in mosquitoes is a continuous process from the early developmental stage (L1 larvae) to the late developmental stage (late pupae). For female-enriched genes, a significant emphasis was observed on GO terms related to DNA replication and DNA repair specifically in female pupae (Figures 3 and Figure 13 ).

[0144] Previously, determining the sex of larvae was a challenge. Therefore, previous transcriptome analyses relied on separating the sexes after the pupal stage, which resulted in a loss of sex-specific samples during early developmental stages. Here, published sex-specific RNA-seq results were compared with a previously collected dataset of RNA-seq data from different developmental stages. To assess the expression of sex-specific genes during early development (before the pupal stage), genes identified by mfuzz cluster analysis were first analyzed, with a particular focus on those designated as L1 or L2-L4 specific. Cluster 17 primarily included genes expressed in L1, while cluster 1 encompassed genes expressed in L2-L4 ( Figures 14A-14B ). Using a membership cutoff of 0.75, cluster 17 was found to contain 268 genes, and cluster 1 was found to contain 383 genes. Of these, 73 (27%) and 134 (35%) were determined to be sex-specifically expressed. To extend the present study beyond the mfuzz cluster, genes that showed no expression (TPM values ​​below 1) in carcasses, testes, ovaries, or pupae, but showed TPM values ​​above 1 or 10 in first-instar larvae were examined. These genes are considered to be early-expressed genes that were not detected in later stages. 210 and 93 such genes were identified in the corresponding datasets, respectively. Of these, 76 (36%) and 46 (49%) were identified as sex-specifically expressed.

[0145] Drosophila melanogaster

[0146] Molecular cloning

[0147] All gene constructs were generated using the Gibson enzyme assembly method. Construct 795G was created using an existing plasmid containing piggyBac, attB-docking sites, and the Opie2 promoter that regulates dsRed. The plasmid was subsequently linearized with XhoI and NotI enzymes. The Hr5Ie1 promoter and eGFP were cloned into the linearized plasmid to prepare 795G for use as a control plasmid. To generate female-specific dsRed (795H-K), plasmid 795G was linearized with AvrII and BamHI to allow intron insertion into dsRed. Alternatively, to generate female-specific eGFP (795L-O), 795G was linearized with MluI and BsrGI to allow intron insertion into eGFP. The traF intron was amplified from the genomic DNA of Drosophila melanogaster, Drosophila suzukii, Ceratitis melanogaster, or Anastrepha mexicana using the primers listed in Table 7.

[0148] Reverse transcription PCR (RT-PCR) of female-specific spliced ​​transcripts

[0149] To obtain spliced ​​transcripts of the four traF introns, female- and male-specific dsRed mRNAs were screened. Total RNA from ten primary females or males of w-, 795G, H, I, J, and K was extracted using the miRNeasy Tissue / Cell Advanced Kit (Qiagen). TURBO TM DNA-free (Invitrogen) was DNase treated and then the cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific TM cDNA synthesis was performed using primers 795.s2F and 795.s2R (Table 7). Genomic DNA (gDNA) was amplified using primers 795.s3F and 795.s1R, and cDNA was amplified using primers 795.s3F and 795.s1R. gDNA samples were run on a 1% TAE agarose gel, and cDNA samples were run on a 2% TAE agarose gel.

[0150] Table 7: Primer sequences

[0151]

[0152]

[0153] Rearing and fly transgenesis

[0154] Transgenic flies were maintained under standard conditions at 25°C with a 12-hour light / dark cycle and fed an Old Bloomington Molasses Recipe. Embryo injections were performed in the laboratory according to standard injection protocols. Plasmids diluted to 300–350 ng / μL in water were inserted into chromosome 2 at position P{CaryP}attP40 (Bloomington accession number 25709). Recovered transgenic lines were balanced on chromosome 2 using the single-chromosome balancer line w1118; CyO / sna[Sco]. Multiple independent lines were generated for each plasmid and tested for sex-specific fluorescence. Homozygous transgenic lines containing two copies of the transgene were used to assess sex sorting efficiency. Sex-sorting lines harboring CctraF introns 795H and 795L have been deposited at the Bloomington Drosophila Stock Center (BDSC accession number pending).

[0155] Genetics and sex selection

[0156] To evaluate the efficiency of fluorescent sex selection, ten virgin female flies were crossed with ten male flies in a fly vial. Every 12 hours, the parental flies were transferred to a fresh vial, and the number of embryos produced was scored. After hatching, the larvae or pupae were scored and transferred to different vials based on their fluorescent markers. After the adult progeny eclosed, their sex and fluorescent markers were recorded. Flies were scored using a Leica M165FC fluorescence stereomicroscope. Images were captured with a View4K camera. Each genetic cross was established five times using different parental flies.

[0157] Adaptability estimation

[0158] The adaptability of the sex-sorted strains was assessed based on two parameters: egg hatchability (from embryo to larva) and adult survival (from larva to adult). To assess egg hatchability, flies were allowed to lay eggs in fly vials for 24 hours, and the number of eggs laid in each fly vial was recorded. After 24 hours of egg laying, the number of larvae was recorded. To assess adult survival, the number of female and male flies that successfully emerged was recorded.

[0159] Statistical analysis

[0160] Statistical analysis was performed in Prism 9 from GraphPad Software, LLC. Three to five biological replicates were used to generate statistical means for comparison.

[0161] Example 5 - Development of female-specific expression of fluorescent proteins

[0162] To engineer female-specific expression of a reporter gene for positive selection in females, sex-specific alternative splicing of a conserved sex-determining gene was developed. In female Drosophila melanogaster, the translator (tra) intron between exons 1 and 2 is spliced ​​out, resulting in a functional tra protein. In males, alternative tra splicing generates a premature stop codon, thereby terminating the tra protein. This female-specific alternative splicing mechanism occurs not only in Drosophila but also in Bactrocera and Anastrepha, indicating that it is highly conserved in Diptera ( Figure 16A 、 Figures 20A-20B 、 Figure 21 Therefore, traF from Drosophila melanogaster, Drosophila suzukii, Ceratitis melanogaster, or Anastrepha was inserted into the fluorescent protein coding sequence to test female-specific fluorescent protein expression ( Figure 16B ).

[0163] Two sets of dual fluorescent marker constructs were generated, encoding fluorescent markers for both sexes and a female-specific fluorescent marker ( Figure 16C ). The constructs were cloned into plasmids containing attP recombination sites and piggyBac (PB) transposable elements. Group 1 constructs had eGFP fluorescence expressed under the ubiquitous promoter Hr5Ie1 (Hr5Ie1-eGFP) as a selectable marker for transgenics. To promote constitutive expression of female-specific fluorescent proteins, another ubiquitous promoter Opie2 was used to express dsRed, and traF was inserted immediately downstream of the ATG translation start codon of dsRed (Opie2-ATG-traF-dsRed). The constructs in Group 2 had the opposite marker configuration, where traF was inserted downstream of the ATG translation start codon of eGFP under the promoter Hr5Ie1 (Hr5Ie1-ATG-traF-eGFP) for female-specific fluorescence expression, and Opie2-dsRed was used as a selectable marker for transgenics. A total of nine constructs were created: one control construct (795G) and eight experimental constructs, with four constructs in each group ( Figure 16D ).

[0164] Example 6 - DmtraF, DstraF and CctraF produce female-specific fluorescence

[0165] Because the transgene integration site may affect gene expression, all nine constructs were integrated into the same site (BDSC No. 25709) via phiC31attP integration on chromosome 2. Nine homozygous transgenic lines were established. Six constructs containing DmtraF, DstraF, and CctraF produced female-specific fluorescence (795H, I, J, L, M, and N, Figures 17A-17CThese results indicate that inserting traF into the coding sequence of a fluorescent protein can produce female-specific fluorescence. However, for the construct carrying AltraF (795K and O), both females and males exhibited the expected female-specific fluorescence ( Figures 17A-17C This result suggests that AltraF is spliced ​​out in both females and males, rather than being spliced ​​out in a female-specific manner.

[0166] To verify the alternative splice variant, adult flies were collected for RT-PCR analysis to obtain fluorescent protein transcripts. Primers were designed to anneal to the 5'UTR region at the 3' end of the Opie2 promoter and the 3' end of the dsRed sequence (Table 7). Multiple bands were obtained from RT-PCR samples of CctraF males and both DmtraF and DstraF males ( Figures 22A-22B Sequencing of these bands showed that CctraF, DmtraF, and DstraF produced functional dsRed expression only in females, whereas AltraF had dsRed expression in both males and females ( Figure 22C Molecular results obtained from RT-PCR analysis were consistent with observations in flies, confirming that CctraF, DmtraF, and DstraF exhibit female-specific splicing in Drosophila melanogaster. This result demonstrates the feasibility of this fluorescent sex sorting method, as these female-specific splicing events allow for positive selection of either sex.

[0167] Example 7 - Confirmation of sex-specific fluorescence at multiple developmental stages

[0168] Next, the fluorescence intensity and sex specificity were assessed across multiple life stages. Six constructs exhibiting female-specific fluorescence expression (795H, I, J, L, M, and N) were evaluated. In two CctraF transgenic lines, female-specific fluorescence was observed as early as the first instar larvae (L1) life stage: 795H and 795L ( Figure 17A 、 Figure 18 ). Female-specific fluorescence was also observed in the third instar larvae (L3) of DmtraF 795I and CctraF 795L, and in the pupal stage of DmtraF 795M and DstraF 795N. Although the introns of DmtraF 795I and DmtraF 795M, and CctraF 795L and DstraF795N lines were identical, female-specific fluorescence was detected earlier in the lines carrying the female-specific dsRed marker. This result may be due to the deeper tissue penetrance and lower autofluorescence of red fluorescent protein (RFP). Notably, the female-specific fluorescence intensity differed between introns. CctraF showed the highest brightness, followed by DmtraF and DstraF ( Figure 18 This was unexpected because CctraF is a foreign / non-native intron in D. melanogaster and may hinder successful intron recognition and splicing efficiency.

[0169] Example 8 - Evaluating the Adaptability of Sex Sorted Lines

[0170] Strain fitness is critical for scalability. Fluorescent proteins have documented fitness costs in genetically engineered organisms, but including traF in their coding sequence was expected to have minimal impact on the fitness of sex-sorted strains. Therefore, egg hatching and larval to adult survival of all eight homozygous sex-sorted strains were compared to a control strain (795G) containing a fluorescent reporter gene lacking the traF intron. The results showed that flies carrying the Opie2-ATG-traF-dsRed construct had significantly higher hatching rates compared to the 795G control ( Figures 19A-19B This effect may be attributed to the fitness cost associated with dsRed functioning as a tetramer. After the addition of the traF intron, the expression level of dsRed was reduced. Therefore, the fitness cost was reduced, which in turn led to an improvement in hatching rate. Lower larval to adult survival was observed only in the CctraF-dsRed 795H line (p < 0.05, Student's t-test with equal variance, Figures 19A-19B These results suggest that the traF intron does not impose a significant fitness cost on the strain, making it suitable for potential large-scale insect population control programs.

[0171] Mediterranean fruit fly

[0172] Plasmid design and construction

[0173] Both 795H1 and 795K1 were constructed by Gibson assembly into an existing piggyBac plasmid containing Opie2 expressing DsRed and Hr5Ie1 expressing eGFP. The plasmid was linearized using AvrII and BamHI restriction enzymes. The tra intron of the Mediterranean fruit fly or Anastrepha mexicana was PCR amplified from the corresponding genomic DNA preparations and then inserted immediately into the DsRed coding sequence downstream of the ATG start codon (Table 8). The complete sequence maps and plasmids are deposited at Addgene.org (795H1 is numbered 205482 and 795K1 is numbered 205485).

[0174] Table 8: Primer Summary

[0175]

[0176] Mediterranean fruit fly rearing

[0177] The wild-type Benakeion strain of Mediterranean fruit fly used in this article was obtained from the Saccone laboratory (University of Naples "Federico II"). Adult food consists of a mixture of yeast and glucose in equal proportions, and larvae are maintained on a carrot-based diet. Flies are always maintained at 26°C and 65% relative humidity under a 12:12 hour light:dark cycle.

[0178] Mediterranean fruit fly germline transformation

[0179] Plasmids were microinjected into wild-type Benakeion strain embryos. Donor 795H1 and 795K1 piggyBac plasmids (500 ng / ml) were microinjected together with a helper plasmid (300 ng / ml) encoding the ihyPBase transposase. Hatched G0 larvae were manually transferred to a larval diet, and the surviving adults were hybridized with the original wild-type flies in a peer-to-peer manner. Fluorescence microscopy was used to identify marker-positive G1 offspring in the pupal development stage, and the selected adults were hybridized separately with wild-type flies. Homozygous transgenic lines were maintained by hybridization of 10 male and 20 female siblings after establishment.

[0180] inverse PCR

[0181] In selected marker-positive G1 individuals, unique piggyBac construct integration was analyzed by inverse PCR. Briefly, genomic DNA (gDNA) was first extracted using a modified protocol. The inverse PCR protocol was adapted from that using Sau3AI (New England Biolabs). ) and HinP1I(New England Initial gDNA digestion with 1% β-catenin and 1% β-catenin restriction enzymes was performed using established protocols. PiggyBac-specific primers were used for sequential PCR amplifications. Sanger sequencing was performed using services from Genewiz Inc., and the results were analyzed using the latest Mediterranean fruit fly genome assembly (GenBank GCA_905071925.1).

[0182] DsRed splicing confirmation

[0183] Adult males and females from transgenic lines were collected separately in TRIzol reagent (Ambion). RNA was extracted and cDNA synthesis was performed using the Maxima H Minus First-Strand cDNA Synthesis Kit with dsDNase (ThermoFisher). Simultaneously, gDNA was extracted as described above. Phusion High-Fidelity PCR Master Mix with HF Buffer (New England Biolabs) was used using primers designed in Geneious Prime 2023.1.2. ) or RedTaq DNA polymerase 2X master mix (VWR Life Science) were used for PCR (Table 8). Amplicons were visualized using 1% agarose gel.

[0184] Sex sorting assay

[0185] For all homozygous strains, two consecutive generations G9 and G10 were screened to confirm system efficiency. Parental hybridization was set up between 10 male and 20 female homozygous individuals, and eggs were collected twice at intervals of 3 days for each hybridization. Progeny was raised under normal conditions until adulthood. In adulthood, all flies were assessed by 3 phenotypic parameters. These include phenotypic sex characteristics (male or female), and by screening the fluorescent marker phenotype determined by using GFP (GFP+ or GFP-) and RFP (DsRed+ or DsRed-) filters respectively. All imaging and fluorescence screening used MVX-ZB10 macro zoom fluorescence microscope system (Olympus Corporation (Olympus)).

[0186] Adaptability assay

[0187] To assess the fitness cost of two copies of the sex-sorting gene cassette, the number of eggs laid and the egg hatching rate of flies carrying the homozygous cassette and wild-type flies were evaluated. Genetic crosses between 15 males and 25 females from each strain (H-001, H-002, K-001, K-002) were established in biological triplicate. Simultaneously, crosses between 15 males and 25 female wild-type Benakeion strain adults were also established in triplicate. After 5 days, eggs laid within a 5-hour window were placed on black filter paper on larval diet and counted using ImageJ. Hatch rate was also determined by counting the remaining unhatched eggs 4 days after the first egg retrieval.

[0188] Example 9 - Establishment and Characterization of Fluorescent SEPARATOR Lines

[0189] To engineer the SEPARATOR gene cassette for the Mediterranean fruit fly, the transformant (tra) intron of the Mediterranean fruit fly or Anastrepha mexicana was used to generate the 795H1 or 795K1 constructs, respectively. Importantly, both constructs included the DsRed coding sequence separated by the tra intron and expressed under the Opie2 promoter and the dominant Hr5-IE1-eGFP marker ( Figure 23A Germline transformation was used to induce piggyBac-dependent integration of the 795H1 and 795K1 constructs (Table 9). Two independent lines of each construct (H-001 and H-002 for 795H1; K-001 and K-002 for 795K1) were successfully characterized by inverse PCR ( Figure 25 and Table 10). Thereafter, all lines were homozygous by sib-crossing at G2. In routine screening for eight consecutive generations (from G3 to G10), no DsRed+ / GFP+ males or DsRed- / GFP+ females were found in any of the four homozygous lines. This indicates that DsRed splicing is sex-specific and that the tra intron selection was therefore successful in both the Mediterranean fruit fly and the Mexican fruit fly. For verification, the lines were amplified at G9 and G10 and the entire population was characterized ( Figure 24A As expected, no DsRed+ / GFP+ males or DsRed- / GFP+ females were detected. Only two phenotypes of transgenic individuals were found: DsRed+ / GFP+ females (48.8%) and DsRed- / GFP+ males (51.2%) in four lines (Table 11). A chi-square test of the observed and expected sex ratios showed no statistical significance in any of the four lines. Overall, in G9 and G10, 100% of transgenic flies carrying the Mediterranean fruit fly (795H1) and the Mexican fruit fly (795K1) tra intron expressed the expected fluorescent phenotype, and a total of 3,787 flies were counted.

[0190] Table 9: Summary of injections of 795H1 and 795K1 constructs

[0191]

[0192] Table 10: Determination of genomic integration by inverse PCR

[0193]

[0194] Table 11: All adult flies of the G9 and G10 generations were phenotyped by sex and screened for GFP and DsRed fluorescent markers.

[0195]

[0196]

[0197] Example 10 - Adaptation of the Fluorescent SEPARATOR Line

[0198] To assess whether the SEPARATOR cassette is associated with significant fitness costs, egg laying rates and egg hatch rates were assessed for the four homozygous lines (Table 12). Sibling crosses of homozygous individuals of each line were performed along with wild type controls. Although no statistically significant differences between wild type and transgenic lines were determined by the Kruskal-Wallis test and Dunn's multiple comparison test, egg laying rates between wild type and transgenic lines were variable Figure 24B ). Notably, however, the H-002 line had a significantly reduced egg production. At the same time, the egg hatch rate of the line carrying 795H1 with the endogenous tra intron was similar to that of the wild type Figure 24C ). The egg hatch rate of the 795K1 line containing the exogenous intron was reduced compared to non-transgenic flies. Specifically, the egg hatch rate of K-001 was significantly reduced (p = 0.019), while the egg hatch rate of K-002 was not significantly reduced (p = 0.072).

[0199] Table 12: Egg laying and hatching data

[0200]

[0201] Example 11 - Fluorescence Pattern of SEPARATOR Strain

[0202] It was observed that the GFP fluorescence signal of the 4 homozygous lines was significantly similar. However, the DsRed signal of females carrying the endogenous Mediterranean fruit fly tra intron (H-001 and H-002) was visually more intense compared to females with the Mexican fruit fly tra intron (K-001 and K-002), independent of the GFP fluorescence intensity of the homozygous individuals Figure 26 ). The fluorescence marker expression pattern throughout the life cycle of the Mediterranean fruit fly was further investigated Figure 23B ). A clear distinction between males and females was observed immediately upon hatching as larvae and became more and more apparent throughout the later stages of the life cycle. Specifically, the signal was easily recognizable in the first, second and third instar larvae, pupae and adult development stages. In the late egg stage, GFP fluorescence was very clear in all individuals, while DsRed fluorescence was more difficult to distinguish Figure 27 ). This is because all eggs had DsRed signal of different intensities, stronger than that of their wild type counterparts.

[0203] Additionally, reverse transcription PCR (RT-PCR) was performed to amplify Opie2-DsRed for molecular verification of the sex-specific nature of DsRed splicing. cDNA and gDNA synthesized from total RNA were used to verify this phenomenon from adult males and females carrying 795H1 or 795K1 ( Figures 28A-28D ). Similar gDNA-derived fragments of size were observed in both sexes, and different cDNA-derived fragments of size were observed in males and females. The female-specific cDNA-derived fragments of both the Mediterranean fruit fly (795H1) and the Mexican fruit fly (795K1) tra introns were expected to be amplified as a single shortest fragment. One of the two male-specific transcripts was detected in the 795H1 male, while multiple bands were observed in the 795K1 male. For males carrying 795K1, distinguishing these is more challenging because owing to the presence of 3 male-specific exons, there are 5 alternative male-specific tra transcripts in the fruit fly. It is inferred that the DsRed in these two boxes is similar to the splicing mode of the tra gene, and thus DsRed translation is interrupted by the male-specific stop codon. It is also concluded that, although the sequence similarity is incomplete, the Mexican fruit fly tra intron is functionally suitable for replacing its endogenous counterpart in the Mediterranean fruit fly.

Claims

1. A method for sex sorting multiple insects based on sex-specific gene expression, the method comprising: (a) generating an exogenous nucleic acid molecule; (b) delivering the exogenous nucleic acid molecule into an insect among the plurality of insects, wherein the exogenous nucleic acid molecule comprises a promoter region, a sex-specific splicing module, a reporter gene, and a transcription terminator; (c) detecting sex-specific gene expression of the reporter gene; and (d) sorting the insects from the plurality of insects based on the detection of the sex-specific gene expression in step (c), The insects are thereby sex sorted based on the sex-specific gene expression.

2. The method of claim 1, wherein the exogenous nucleic acid molecule further comprises a piggyBac inverted terminal repeat sequence located at each end of the effector region. 3 . The method according to claim 1 , wherein the promoter region comprises Hr5IE1 promoter or OpIE-1 promoter.

4. The method according to any one of claims 1 to 3, wherein step (b) comprises integrating the exogenous nucleic acid molecule into the genome of the insect.

5. The method according to any one of claims 1 to 4, wherein step (d) comprises sorting the insects at the larval stage. 6 . The method according to any one of claims 1 to 5 , wherein the sex-specific splicing module comprises an endogenous sex-specific exon sequence and a truncated sex-specific intron sequence.

7. The method of claim 6, wherein the sex-specific splicing module is a male-specific splicing module.

8. The method of claim 6, wherein the sex-specific splicing module is a female-specific splicing module.

9. The method of any one of claims 1 to 8, wherein the insect comprises Aedes aegypti, Drosophila melanogaster, Drosophila suzukii, Ceratitis capitata, or Anastrepha ludens.

10. The method of claim 9, wherein the sex-specific splicing module is derived from the Aedes aegypti doublesex gene (AaeDsx), the Mediterranean fruit fly transformant (traF), the Drosophila melanogaster traF, or the Drosophila suzukii traF.

11. The method of claim 10, wherein the sex-specific splicing module is derived from AaeDsx, and wherein the male-specific splicing module comprises exon 4, exon 6, or any combination thereof.

12. The method of claim 10, wherein the sex-specific splicing module is derived from AaeDsx, and wherein the female-specific splicing module comprises exon 4, exon 5b, exon 6, or any combination thereof.

13. The method of claim 12, wherein exon 5b is an engineered exon 5b, wherein one or more stop codons are excluded from the exon 5b. 14 . The method according to claim 1 , wherein the reporter gene comprises a DsRed gene, an EGFP gene, or any combination thereof.

15. The method of claim 14, wherein the insects are sorted as males based on the expression of the EGFP gene.

16. The method of claim 14, wherein the insects are sorted as female based on expression of the DsRed gene.

17. The method of any one of claims 1 to 16, wherein the transcription terminator comprises an SV40 poly(A) signal.

18. A method for identifying the sex of an insect based on sex-specific gene expression, the method comprising: (a) generating exogenous nucleic acid; (b) delivering the exogenous nucleic acid molecule into an insect, wherein the exogenous nucleic acid molecule comprises a promoter region, a sex-specific splicing module, a reporter gene, and a transcription terminator; and (c) identifying sex-specific gene expression of said reporter gene, The sex of the insect is thereby identified based on the sex-specific gene expression.

19. The method of claim 18, wherein the exogenous nucleic acid molecule further comprises a piggyBac inverted terminal repeat sequence located at each end of the effector region.

20. The method according to claim 18 or 19, wherein the promoter region comprises Hr5IE1 promoter or OpIE-1 promoter.

21. The method according to any one of claims 18 to 20, wherein step (b) comprises integrating the exogenous nucleic acid molecule into the genome of the insect.

22. The method according to any one of claims 18 to 21, wherein step (d) comprises sorting the insects at the larval stage.

23. The method of any one of claims 18 to 22, wherein the sex-specific splicing module comprises an endogenous sex-specific exon sequence and a truncated sex-specific intron sequence.

24. The method of claim 23, wherein the sex-specific splicing module is a male-specific splicing module.

25. The method of claim 23, wherein the sex-specific splicing module is a female-specific splicing module.

26. The method of any one of claims 18 to 25, wherein the insect is Aedes aegypti, Drosophila melanogaster, Drosophila suzukii, Ceratitis capitata, or Anastrepha mexicana.

27. The method of claim 26, wherein the sex-specific splicing module is derived from the Aedes aegypti doublesex gene (AaeDsx), the Mediterranean fruit fly transformant (traF), the Drosophila melanogaster traF, or the Drosophila suzukii traF.

28. The method of claim 27, wherein the sex-specific splicing module is derived from AaeDsx, and wherein the male-specific splicing module comprises exon 4, exon 6, or any combination thereof.

29. The method of claim 27, wherein the sex-specific splicing module is derived from AaeDsx, and wherein the female-specific splicing module comprises exon 4, exon 5b, exon 6, or any combination thereof.

30. The method of claim 29, wherein exon 5b is an engineered exon 5b, wherein one or more stop codons are excluded from the exon 5b.

31. The method according to any one of claims 18 to 30, wherein the reporter gene comprises a DsRed gene, an EGFP gene, or any combination thereof.

32. The method of claim 31 , wherein the insects are sorted as males based on expression of the EGFP gene.

33. The method of claim 31 , wherein the insects are sorted as female based on expression of the DsRed gene.

34. The method of any one of claims 18 to 33, wherein the transcription terminator comprises an SV40 poly(A) signal.

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