WO2022221299A1

WO2022221299A1 - Insect products and methods of producing same

Info

Publication number: WO2022221299A1
Application number: PCT/US2022/024452
Authority: WO
Inventors: Aaron T. DOSSEY; Fu-Chyun CHU
Original assignee: All Things Bugs Llc
Priority date: 2021-04-12
Filing date: 2022-04-12
Publication date: 2022-10-20

Abstract

According to various aspects of this disclosure, the present disclosure relates to insect products, insect farming, genetically modified insects methods of producing the same, and uses of products and insects.

Description

INSECT PRODUCTS AND METHODS OF PRODUCING SAME CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit, under 35 U.S.C. §119(e), to United States

Provisional Patent Application Serial No. 63/173,893, filed April 12, 2021, the entire contents of which are hereby incorporated herein by this reference.

STATEMENT OF GOVERNMENT FUNDING

[0002] This invention was made with government support under contract number

140D6318C0055 awarded by The Defense Advanced Research Projects Agency. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] According to various aspects of this disclosure, the present disclosure relates to insects comprising one or more genetic modifications effecting one or more phenotypic or non-phenotypic changes relative to wild-type insects of the same species.

BACKGROUND OF THE INVENTION

[0004] Insects such as crickets and mealworms are useful as nutrient-dense, low- maintenance crops, and are an attractive avenue for genetic modification. Such modifications can increase the nutrient content of an insect, or to introduce transgenes for the production of materials not encoded by the insect genome. However, current methods of genetically modifying insects are limited in scope relative to the methods which exist for genetically modifying other organisms, such as bacterial and mammalian species. In particular, verification of genetic modification in insects requires time-consuming gene sequencing or biomolecular analysis of expression products. There remains a need for a simple, easily detectable marker of successful genetic modification in insect species. BRIEF SUMMARY OF THE INVENTION

[0005] The present disclosure provides a transgenic insect (e.g., a cricket or mealworm), and methods of producing the same. In some aspects, one or more genetic modifications are introduced into the insect using clustered regularly interspaced short palindromic repeats ("CRISPR") technology. In some aspects, genetic modification produces a white eye recessive phenotype. In some aspects, genetic modification produces a dominant green fluorescence phenotype. Transgenic crickets and mealworms bearing both genetic modifications are disclosed. In some aspects, the methods are useful for verifying successful genetic modification of insects. In some aspects, insects bearing two or more genetic modifications provide a multi-marker phenotype. In some aspects, the genetically modified insects exhibit increased production of nutrients or transgenic expression products.

[0006] In some aspects, the present disclosure provides a genetically modified insect comprising one or more genetic modifications relative to a wild-type insect of the same species. In some aspects, the present disclosure provides a genetically modified insect comprising one or more genetic modifications relative to a wild-type insect of the same species, wherein the wild-type insect of the same species is selected from a superworm, a cricket, a mealworm, a Caribbean fruit fly, a black soldier fly, a grasshopper, a locust, a katydid, a silkworm, or a caterpillar.

[0007] In some aspects, the one or more genetic modifications effect a phenotype detectable by visual inspection.

[0008] In some aspects, the genetically modified insect is an insect selected from the group consisting of: a superworm, a cricket, a mealworm, a Caribbean fruit fly, a black soldier fly, a grasshopper, a locust, a katydid, a silkworm, or a caterpillar.

[0009] In some aspects, the genetically modified insect is a cricket. In some aspects, the cricket is of the species Acheta domesticus.

[0010] In some aspects, the genetically modified insect is a mealworm. In some aspects, the mealworm is of the species Tenebrio molitor.

[0011] In some aspects, the one or more genetic modifications consist of two genetic modifications. In some aspects, the one or more genetic modifications consist of three genetic modifications. In some aspects, the one or more genetic modifications consist of four genetic modifications. In some aspects, the one or more genetic modifications consist of five genetic modifications. In some aspects, the one or more genetic modifications consist of six or more genetic modifications. In some aspects, the one or more genetic modifications consist of six to ten genetic modifications. The genetically modified insect of claim 12, wherein the one or more genetic modifications consist of six to ten genetic modifications.

[0012] In some aspects, the one or more genetic modifications are introduced by a

CRISPR/Cas9 system.

[0013] In some aspects, the one or more genetic modifications comprise an indel mutation.

[0014] In some aspects, the one or more genetic modifications comprises a gene knock out. In some aspects, the one or more genetic modifications comprise knock-out of two or more genes.

[0015] In some aspects, the gene knock-out is a knock-out of a vermilion gene. In some aspects, the gene knock-out is introduced by a CRISPR/Cas9 system using a construct encoding a polynucleotide comprising a guide RNA sequence complementary to a portion of a vermilion gene.

[0016] In some aspects, the one or more genetic modifications comprises a gene knock- in. In some aspects, the gene knock-in is introduced by a CRISPR/Cas9 system using a construct encoding a polynucleotide comprising a guide RNA sequence complementary to a portion of a vermilion gene.

[0017] In some aspects, the portion of a vermilion gene comprises exon 2 or a portion of exon 2.

[0018] In some aspects, the one or more genetic modifications alter one or more genes associated with a recessive phenotype.

[0019] In some aspects, the one or more genetic modifications alter one or more genes associated with a dominant phenotype.

[0020] In some aspects, the one or more genetic modifications comprises genomic incorporation of a transgene. In some aspects, the transgene is incorporated at the locus of a vermilion gene.

[0021] In some aspects, the transgene encodes a fluorescent protein.

[0022] In some aspects, the fluorescent protein is a green fluorescent protein. In some aspects, the green fluorescent protein is EGFP. [0023] In some aspects, the fluorescent protein is a red fluorescent protein. In some aspects, the red fluorescent protein is DsRed.

[0024] In some aspects, the phenotype detectable by visual inspection is detectable by visual inspection only after illuminating the genetically modified insect with light of one or more wavelengths corresponding to one or more excitation wavelengths characteristic of the fluorescent protein.

[0025] In some aspects, the one or more genetic modifications comprise an alteration in an eye color gene. In some aspects, the eye color gene is a vermilion gene.

[0026] In some aspects, the genetically modified insect is a cricket which expresses a fluorescent protein and has a white eye phenotype.

[0027] In some aspects, the genetically modified insect is a mealworm which expresses a fluorescent protein and has a white eye phenotype.

[0028] In some aspects, the present disclosure provides a molecular construct comprising a sequence encoding, in the 5' to 3' direction: a first guide RNA segment, a tissue-specific promoter, a phenotypic marker, and a second guide RNA segment.

[0029] In some aspects, the molecular construct further comprises a sequence encoding a transgene of interest, wherein the sequence encoding a transgene of interest is 3' of the tissue-specific promoter and 5' of the sequence encoding a phenotypic marker.

[0030] In some aspects, the sequence encoding a transgene further comprises a sequence encoding a secretion signal.

[0031] In some aspects, the phenotypic marker is a fluorescent protein.

[0032] In some aspects, the fluorescent protein is a green fluorescent protein. In some aspects, the green fluorescent protein is EGFP.

[0033] In some aspects, the fluorescent protein is a red fluorescent protein. In some aspects, the red fluorescent protein is DsRed.

[0034] In some aspects, the first guide RNA segment is complementary to a first sequence of a first gene, and the second guide RNA segment is complementary to a second sequence of a second gene.

[0035] In some aspects, the first guide RNA segment is complementary to a first sequence of a first gene, and the second guide RNA segment is complementary to a second sequence of the first gene.

[0036] In some aspects, the tissue-specific promoter is a muscle tissue-specific promoter. [0037] In some aspects, the muscle tissue-specific promoter is a muscle actin promoter.

[0038] In some aspects, the molecular construct further comprises a sequence encoding a self-cleaving peptide 5' of the sequence encoding the phenotypic marker.

[0039] In some aspects, the sequence encoding a self-cleaving peptide is 3' of the sequence encoding the transgene of interest. In some aspects, the self-cleaving peptide is a 2A self-cleaving peptide. In some aspects, the 2A self-cleaving peptide is T2A.

[0040] In some aspects, the molecular construct further comprises an SV40 sequence 3' of the sequence encoding the phenotypic marker and 5' of the sequence encoding the second guide RNA segment. The molecular construct of any one of claims 37-52, further comprising an SV40 sequence 3' of the sequence encoding the phenotypic marker and 5' of the sequence encoding the second guide RNA segment.

[0041] In some aspects, the present disclosure provides a method of producing a genetically modified insect, comprising introducing one or more genetic modifications in a wild-type insect.

[0042] In some aspects, the method comprises introducing into a wild-type insect a construct disclosed herein.

[0043] In some aspects, the method comprises introduction of two or more constructs disclosed herein, wherein the two or more constructs are not identical in sequence.

[0044] In some aspects, the method is used to produce a genetically modified insect disclosed herein.

Further GMO Insect Aspects

[0045] In some aspects, the wild-type insect is a cricket selected from the superfamily

Grylloidea. In some aspects, the cricket is Gryllodes sigillatus. In some aspects, the cricket is Gryllus bimaculatus. In some aspects, the cricket is Gryllus assimilis. In some aspects, the mealworm is of the species Zophobas morio.

[0046] In some aspects, the one or more genetic modifications comprises a gene knock out and a gene knock-in. In some aspects, the gene knock-out or gene knock-in is introduced by the use of a transposable element. In some aspects, the gene knock-out or gene knock-in is introduced using a transposase.

[0047] In some aspects, the one or more genetic modifications effect an increase in transcription of a target gene. In some aspects, the one or more genetic modifications effect a decrease in transcription of a target gene. In some aspects, the decrease in transcription is a reduction in the transcription of the target gene to a level which is not detectable by conventional reverse transcription PCR methods.

[0048] In some aspects, the one or more genetic modifications comprise a modification of a regulatory element. In some aspects, the modification of a regulatory element is a replacement of a promoter region with an exogenous sequence or an endogenous sequence.

[0049] In some aspects, the target gene is a wild-type gene. In some aspects, the target gene is a transgene. In some aspects, the transgene is selected from the group consisting of: a gene encoding an enzyme, a gene encoding a fluorescent protein, and a gene encoding a food protein.

[0050] In some aspects, the genetically modified insect produces less of a target product than a wild-type insect of the same species. In some aspects, the genetically modified insect produces more of a target product than a wild-type insect of the same species.

[0051] In some aspects, the target product is one or more of: a nutritional biomolecule, a chemical precursor to a nutritional biomolecule, an engineered biological product, a vaccine, a hormone, a flavorant, a colorant, a pigment, a fragrance, a fluorescent molecule, a peptide, a polypeptide, a polynucleotide, a lipid, a therapeutic drug product, a plastic, an insect repellant, and chitin.

[0052] In some aspects, the target product is a nutritional biomolecule. In some aspects, the nutritional biomolecule is a vitamin. In some aspects, the vitamin is vitamin A, a B vitamin, vitamin C, a D vitamin, or vitamin K.

[0053] In some aspects, the nutritional biomolecule is folic acid or folate.

[0054] In some aspects, the nutritional biomolecule is a protein.

[0055] In some aspects, the nutritional biomolecule is a fatty acid.

[0056] In some aspects, the nutritional biomolecule is an essential nutrient in humans or livestock. In some aspects, the essential nutrient is an essential amino acid.

[0057] In some aspects, the target product is an engineered biological product. In some aspects, the engineered biological product reduces immunogenicity in humans.

[0058] In some aspects, the engineered biological product is engineered chitin.

[0059] In some aspects, the engineered biological product comprises a non-natural structural feature which aids in separation of the engineered biological product from other components of the genetically modified insect. [0060] In some aspects, the target product is a vaccine. In some aspects, the vaccine is suitable for vaccination of a domesticated organism or non-domesticated organism. In some aspects, the vaccine comprises an artificial antigenic construct. In some aspects, the vaccine comprises a wild-type virus or an engineered virus, or an antigenic component thereof. In some aspects, the vaccine is for vaccination against one or more variants of: an influenza, a member of the family Coronaviridae, a henipavirus, and a coxsackievirus (e.g., hand, foot, and mouth disease virus). In some aspects, the influenza is a high pathologic avian influenza or a low pathologic avian influenza. In some aspects, the member of the family Coronaviridae is SARS-CoV-2 or a variant thereof.

[0061] In some aspects, the vaccine is for vaccination of an insect against an insect pathogen. In some aspects, the vaccine is for vaccination of a human against a human pathogen. In some aspects, the vaccine is for vaccination of a livestock species against a livestock pathogen. In some aspects, the livestock species is a bovine species, an avian species, a poultry species, a porcine species, a fish species, or an arthropod species.

[0062] In some aspects, the target product is a polypeptide. In some aspects, the polypeptide is an enzyme.

[0063] In some aspects, the polypeptide is a signaling molecule.

[0064] In some aspects, the polypeptide is a transmembrane protein. In some aspects, the transmembrane protein is a transporter. In some aspects, the transporter is an ion transport channel. In some aspects, the ion transport channel is a calcium transport channel, an iodine transport channel, or an iron transport channel.

[0065] In some aspects, the polypeptide is a therapeutic peptide. In some aspects, the polypeptide is an antimicrobial peptide. In some aspects, the polypeptide is an antiviral peptide.

[0066] In some aspects, the polypeptide is an antibody. In some aspects, the antibody is an engineered antibody.

[0067] In some aspects, the polynucleotide is a DNA molecule. In some aspects, the

DNA molecule is a plasmid.

[0068] In some aspects, the polynucleotide is an RNA molecule. In some aspects, the

RNA molecule is a tRNA, pre-mRNA, mRNA, an interfering RNA, or a ribozyme.

[0069] In some aspects, the target product is a polynucleotide. In some aspects, the polynucleotide is suitable for use as an interfering nucleic acid. In some aspects, the interfering nucleic acid is suitable for use in RNA interference. In some aspects, the interfering nucleic acid is a microRNA ("miRNA"). In some aspects, the interfering nucleic acid is a small interfering RNA ("siRNA"). In some aspects, the siRNA is transcribed under the control of one or more regulatory elements suitable for transcription of an siRNA or precursor to an siRNA. In some aspects, the one or more regulatory elements comprise a promoter suitable for transcription of an siRNA or a precursor to an siRNA. In some aspects, the interfering nucleic acid is a small hairpin RNA ("shRNA").

[0070] In some aspects, expression of the polynucleotide effects a reduction in the abundance of a target RNA. In some aspects, the interfering nucleic acid effects a reduction in the abundance of more than one (e.g., two, three, four, five) target RNA molecules having different sequences. In some aspects, the target RNA is a messenger RNA ("mRNA"). In some aspects, the target RNA is a pre-mRNA. In some aspects, expression of the polynucleotide effects a reduction in the abundance of a protein encoded by the target RNA. In some aspects, the expression of the polynucleotide effects a reduction in the activity of an enzyme or signaling molecule encoded by the target RNA. In some aspects, the expression of the polynucleotide effects a phenotypic effect on the insect. In some aspects, the target RNA is a viral RNA. In some aspects, the polynucleotide reduces a molecular or phenotypic effect of infection of the insect by a viral pathogen.

[0071] In some aspects, an interfering nucleic acid is fed to an insect disclosed herein in an amount sufficient to cause an RNA interference effect disclosed herein. In some aspects, an interfering nucleic acid is injected into an insect disclosed herein in an amount sufficient to cause an RNA interference effect disclosed herein.

[0072] In some aspects the genetically modified insect expresses an interfering nucleic acid disclosed herein. In some aspects, the interfering nucleic acid is constitutively expressed. In some aspects, the interfering nucleic acid is operably linked to an inducible promoter. In some aspects, the interfering nucleic acid is operably linked to a tissue- specific promoter.

[0073] In some aspects, the polynucleotide encodes a viral antigen. In some aspects, the viral antigen is the spike protein of SARS-CoV-2 or a variant or a portion thereof.

[0074] In some aspects, the target product is a therapeutic drug product. In some aspects, the therapeutic drug product is an antibiotic. [0075] In some aspects, the target product is administrable in a therapeutically effective amount by oral consumption of part or all of the genetically modified insect, sublingual administration of a formulation comprising the target product, rectal administration of a formulation comprising the target product, parenteral administration of a formulation comprising the target product, or inhalation of a formulation comprising the target product.

[0076] In some aspects, the one or more genetic modifications confer a difference in reproduction between the genetically modified insect and the wild-type insect of the same species. In some aspects, the difference in reproduction is an increased number of offspring.

[0077] In some aspects, the one or more genetic modifications confer a phenotypic difference between the genetically modified insect and the wild-type insect of the same species.

[0078] In some aspects, the phenotypic difference is selected from a difference in the time to progress through one or more stages of the life cycle, a difference in body weight, a difference in the ability to jump or fly, a difference in the presence of one or more anatomical structures, a difference in the color of one or more anatomical structures, a difference in the size of one or more anatomical structures, and a difference in survival rate following inoculation with a pathogen.

[0079] In some aspects, the phenotypic difference is a difference in the time to progress through one or more stages of the life cycle. In some aspects, the difference in the time to progress through one or more stages of the life cycle is a decreased amount of time to progress through one or more stages of the life cycle.

[0080] In some aspects, the phenotypic difference is a difference in body weight. In some aspects, the difference in body weight is an increased body weight.

[0081] In some aspects, the phenotypic difference is a difference in the ability to jump or fly. In some aspects, the difference in the ability to jump or fly is a reduced or eliminated ability to fly.

[0082] In some aspects, the one or more anatomical structures is an eye. In some aspects, the eye is a different color than the wild-type insect of the same species. In some aspects, the eye is white. [0083] In some aspects, the one or more anatomical structures is a wing. In some aspects, the wing is absent or modified in structure.

[0084] In some aspects, the one or more anatomical structures is an exoskeleton. In some aspects, the exoskeleton is a lighter color than the exoskeleton of a wild-type insect of the same species. In some aspects, the exoskeleton is a darker color than the exoskeleton of a wild-type insect of the same species.

[0085] In some aspects, the phenotypic difference is a difference in survival rate following inoculation with a pathogen. In some aspects, the pathogen is a viral species. In some aspects, the viral species is selected from: a species from the Family Iridoviridae , a species from the Family Parvoviridae, and a species from the Family Baculoviridae . In some aspects, the species from the family Iridoviridae is cricket iridovirus (CrIV). In some aspects, the species from the Family Parvoviridae is a species from the subfamily Densovirinae . In some aspects, the species from the subfamily Densovirinae isAcheta domesticus Densovirus. In some aspects, the viral species is cricket paralysis virus (CrPV). In some aspects, the pathogen is a bacterial species. In some aspects, the pathogen is a fungal species.

Feed Conversion Efficiency

[0086] In some aspects, the genetically modified insect is characterized by a difference relative to a wild-type insect of the same species having eaten the same amount and composition of an insect food as the genetically modified insect.

[0087] In some aspects, the difference is an increased body weight in the genetically modified insect.

[0088] In some aspects, the difference is a decreased amount of time to progress through one or more stages of the life cycle, or a different number of stages of the life cycle, for the genetically modified insect.

[0089] In some aspects, the difference is an increased rate of reproduction for the genetically modified insect.

[0090] In some aspects, the difference is decreased food consumption or water consumption by the genetically modified insect. Food Composition

[0091] In some aspects, the present disclosure provides a composition comprising a part or all of a genetically modified insect disclosed herein, wherein the composition is suitable for consumption by humans, a companion animal species, or a livestock species.

[0092] In some aspects, the composition comprises an unground portion of the insect. In some aspects, the composition comprises a ground portion of the insect.

[0093] In some aspects, the composition is substantially free of chitin.

[0094] In some aspects, the livestock species is a poultry species. In some aspects, the livestock species is a cattle species.

[0095] In some aspects, the composition is a food product. In some aspects, the food product is in the form of: a pasta, a tortilla, a protein crisp, a chip, a puffed extruded snack, duros, a pate, a butter alternative, a beverage, a shake, a cereal, or a powder. In some aspects, the cereal is an extruded cereal, a puffed cereal, a baked cereal, an O- shaped cereal, or a flake cereal.

[0096] In some aspects, the food product is defatted.

[0097] In some aspects, the food product comprises at least 1 gram of protein for every 3 grams of the food product. In some aspects, the food product comprises about 10% to about 90% protein by weight of the composition.

[0098] In some aspects, the part of the insect is an insect extract.

Method of Producing GMO Insect

[0099] In some aspects, introducing one or more genetic modifications in the wild-type insect comprises electroporation. In some aspects, the introducing one or more genetic modifications in the wild-type insect comprises lipofection. In some aspects, the introducing one or more genetic modifications in the wild-type insect comprises the use of a transposable element.

[0100] In some aspects, the step of introducing one or more genetic modifications in the wild-type insect comprises viral delivery of the polynucleotide into the first insect.

[0101] In some aspects, the step of introducing one or more genetic modifications in the wild-type insect comprises liposomal delivery of the polynucleotide into the first insect. [0102] In some aspects, the insect is at a developmental stage selected from: embryo, egg, ootheca, larva, nymph, prepupa, pupa, adult, imago, cocoon, chrystalis, maggot, caterpillar, worm, or winged stage.

Polynucleotide for use in Producing GMO Insects

[0103] In some aspects, the present disclosure provides a polynucleotide suitable for use in genetic modification of an insect.

[0104] In some aspects, the insect is a cricket or a mealworm.

[0105] In some aspects, the polynucleotide is a DNA molecule or an RNA molecule.

[0106] In some aspects, the present disclosure provides a polynucleotide vector comprising a polynucleotide disclosed herein.

[0107] In some aspects, the present disclosure provides a viral vector comprising a polynucleotide or polynucleotide vector disclosed herein.

[0108] In some aspects, the present disclosure provides a liposomal composition comprising a polynucleotide or polynucleotide vector disclosed herein.

Compositions with Carriers/Excipients

[0109] In some aspects, the present disclosure provides a composition comprising the target product produced by a genetically modified insect disclosed herein, and a pharmaceutically acceptable carrier or excipient.

Methods of Producing Products of Expression in GMO Insects

[0110] In some aspects, the present disclosure provides a method of producing the target product produced by a genetically modified insect disclosed herein, the method comprising isolating a fraction comprising the target product from the genetically modified insect. In some aspects, the method further comprises purifying the product from the fraction.

Method of Fishing

[0111] In some aspects, the present disclosure provides a fishing lure comprising a genetically modified insect, wherein the genetically modified insect emits fluorescent, luminescent, or bioluminescent light. [0112] In some aspects, the genetically modified insect is a cricket or mealworm. In some aspects, the genetically modified insect is a genetically modified insect disclosed herein.

[0113] In some aspects, the present disclosure provides a method of fishing comprising baiting a hook with a fishing lure comprising a genetically modified insect disclosed herein.

Insect Farming Apparatus

[0114] An automated insect farming apparatus comprising: a growth chamber; a nutrient delivery system; a water delivery system; a mechanism for removal of waste and/or one or more insects; a computerized system programmed to automate one or more of: nutrient delivery, water delivery, and insect removal; wherein the computerized system is operably linked to one or more of: the growth chamber, the nutrient delivery system, the water delivery system, and the mechanism for removal of waste and/or one or more insects.

[0115] In some aspects, the mechanism for removal of waste and/or one or more insects is a forced air system. In some aspects, the forced air system is an air knife system.

[0116] In some aspects, the growth chamber comprises a growth matrix. In some aspects, the growth matrix comprises a polymer. In some aspects, the growth chamber comprises a cross hatched rod matrix. In some aspects, the growth chamber comprises a vertical plate matrix.

Method of Farming

[0117] In some aspects, the present disclosure provides a method of insect farming comprising providing an insect, providing a composition of one or more nutrients to the insect, and harvesting the insect at a desired stage of development, wherein one or more steps is automated.

[0118] In some aspects, the insect is a wild-type insect or a genetically modified insect. In some aspects, the genetically modified insect is a genetically modified insect disclosed herein.

[0119] In some aspects, the insect is provided in a growth chamber. In some aspects, the insect cannot escape from the growth chamber. [0120] In some aspects, a forced air system removes the insect or a waste product from the growth chamber. In some aspects, the growth chamber is vibrated, shaken, or otherwise agitated to remove the insect or a waste product from the growth chamber.

[0121] In some aspects, one or more nutrients are provided to the insect by an automated system. In some aspects, one or more nutrients are provided to the insect by a passive system.

[0122] In some aspects, water is provided to the insect by an automated system. In some aspects, water is provided to the insect by a passive system.

[0123] In some aspects, the passive system comprises a wicking material along which water can passively travel. In some aspects, the wicking material comprises nylon or a ceramic.

[0124] In some aspects, the insect produces a metabolic waste product, and wherein a conveyor belt carries some or all of the metabolic waste product away from the insect.

Products Made from Insects

[0125] In some aspects, the present disclosure provides a composition comprising a product isolated from an insect. In some aspects, the insect is a wild-type insect or a genetically modified insect disclosed herein. In some aspects, the insect is a wild-type insect. In some aspects, the product is a target product disclosed herein. In some aspects, the product is suitable for use in a food product, a beverage product, a pet food product, a pet treat or snack, or an animal feed.

Reduced Allergencity

[0126] In some aspects, a genetically modified insect disclosed herein comprises one or more genetic alterations, wherein the genetically modified insect produces less or none of an allergen, or wherein the insect produces an allergen which elicits a reduced allergic response in a human, a domesticated animal, a livestock animal, or a wild animal.

[0127] In some aspects, the allergen is tropomyosin.

[0128] In some aspects, the allergen which elicits a reduced allergic response is a modified tropomyosin. BRIEF DESCRIPTION OF THE FIGURES

[0129] FIG. 1 A shows wild-type Acheta domesticus having a black eye phenotype.

[0130] FIG. IB shows Acheta domesticus with a knock-out "white eye" or reduced eye pigment color (or vermilion color) phenotype distinguishable from the black eye phenotype shown in FIG. 1 A.

[0131] FIG. 2 shows a knock-in EGFP construct for Acheta domesticus vermilion.

[0132] FIG. 3 A shows wild-type Acheta domesticus.

[0133] FIG. 3B shows an EGFP-expressing knock-in Acheta domesticus , having a glowing, green body under an EGFP suitable light filter, and is distinguishable from the wild-type Acheta domesticus of FIG. 3 A.

[0134] FIG. 4 shows a knock-in EGFP construct for Tenehrio motor vermilion.

[0135] FIG. 5 shows a wild-type (left) and EGFP expressing knock-in (right) Tenehrio motor , where the EGFP expressing knock-in has a glowing green body under an EGFP suitable light filter, which is distinguishable from the wild-type Tenehrio motor.

[0136] FIG. 6 shows a dual knock-in construct for both EGFP and DsRed for Tenehrio motor.

[0137] FIG. 7 shows a knock-in of GeneA and EGFP in Tenehrio motor. While GeneA expression was verified by standard molecular biology techniques (data not shown), EGFP expression was verified by visual inspection under an EGFP suitable light filter, where the left Tenehrio motor insect (left) has a glowing green body distinguishable from the wild-type Tenehrio motor insect (right).

[0138] FIG. 8 shows dual knock-in constructs for DsRed and EGFP (upper construct) or

GeneA having a secretion signal and EGFP (bottom construct).

[0139] FIG. 9 shows dual knock-in Tenehrio motor expressing EGFP and DsRed (left panel) or EGFP and GeneA having a secretion signal (right panel). Under a light filter suitable for detecting EGFP, the dual knock-in EGFP+DsRed Tenehrio motor has a green body (left panel, upper insect). Under a light filter suitable for detecting DsRed, the dual knock-in EGFP+DsRed Tenehrio motor has a red body (left panel, lower insect). Expression of GeneA having a secretion signal was verified with standard molecular biology techniques. DETAILED DESCRIPTION OF THE INVENTION

Insects of the Disclosure

[0140] Many insects are compatible with various aspects of the present disclosure. For example, whereas an aspect may describe genetic modification of Acheta domesticus , a skilled artisan will understand that such methods are applicable to other insects, including those not explicitly disclosed. Further, where genetically modification of an insect is described herein, such examples are not limiting; genetic modification can be of a wild- type insect, or an insect which has already undergone a genetic or other modification, or the progeny or other descendant of such an insect. In some aspects, the present disclosure provides for genetic manipulation of or other processing of an insect.

[0141] In some aspects, the insect is an insect of the Class Insecta.

[0142] In some aspects, the insect is an insect of the Subclass Apterygota. In some aspects, the inset is an insect of the subclass Pterygota.

[0143] In some aspects, the insect is of the Order Archaeognatha. In some aspects, the insect is of the Order Zygentoma. In some aspects, the insect is of the Order Ephemeroptera. In some aspects, the insect is of the Order Odonata. In some aspects, the insect is of the Order Orthoptera. In some aspects, the insect is of the Order Neuroptera.

In some aspects, the insect is of the Order Phasmatodea. In some aspects, the insect is of the Order Embioptera. In some aspects, the insect is of the Order Notoptera. In some aspects, the insect is of the Order Plecoptera. In some aspects, the insect is of the Order Dermaptera. In some aspects, the insect is of the Order Zoraptera. In some aspects, the insect is of the Order Mantodea. In some aspects, the insect is of the Order Blattodea. In some aspects, the insect is of the Order Psocoptera. In some aspects, the insect is of the Order Phthiraptera. In some aspects, the insect is of the Order Thysanoptera. In some aspects, the insect is of the Order Hemiptera. In some aspects, the insect is of the Order Hymenoptera. In some aspects, the insect is of the Order Strepsiptera. In some aspects, the insect is of the Order Coleoptera. In some aspects, the insect is of the Order Megaloptera. In some aspects, the insect is of the Order Raphidioptera. In some aspects, the insect is of the Order Trichoptera. In some aspects, the insect is of the Order Lepidoptera. In some aspects, the insect is of the Order Diptera. In some aspects, the insect is of the Order Siphonaptera. In some aspects, the insect is of the Order Mecoptera. In some aspects, the insect is of the Order Homoptera. In some aspects, the insect is of the Order Diplura. In some aspects, the insect is of the Order Mantophasmatodea. In some aspects, the insect is of the Order Grylloblatodea. In some aspects, the insect is of the Order Isoptera. In some aspects, the insect is of the Order Heteroptera. In some aspects, the insect is of the Order Phasmida.

[0144] In some aspects, the insect is an insect of the Family Gryllidae. In some aspects, the insect is an insect of the Family Grylloidea. In some aspects, the insect is an insect of the Family Tenebrionidae. In some aspects, the insect is an insect of the Family Tephiritidae. In some aspects, the insect is an insect of the Family Strati omyidae. In some aspects, the insect is an insect of the Family Arctiidae. In some aspects, the insect is an insect of the Family Saturniidae. In some aspects, the insect is an insect of the Family Acrididae.

[0145] In some aspects, the insect is an insect disclosed in U.S. Appl. No. 14/537,960 filed November 11, 2014, which is incorporated by reference in its entirety herein.

[0146] In some aspects, the insect is an insect disclosed in U.S. Patent No. 8,912,338, which is incorporated in its entirety herein. In some aspects, the insect is an insect of a taxonomic group (e.g., Order, Family) identified in U.S. Patent No. 8,912,338.

[0147] In some aspects, the insect is a cricket, a fly, a moth, a beetle (e.g., a mealworm), a superworm, a silkworm, a waxworm, an ant, a cicada, a scorpion, a June bug, a grasshopper, a termite, or a pill bug. In some aspects, the insect is a Banded cricket, a Jamaican field cricket (or field cricket), a differential grasshopper, a black soldier fly, a house fly, a buffalo worm, a fruit fly, a Tephritid fruit fly, a hornworm, a cabbage white fall armyworm, a corn earworm, a cabbage looper, or a butterfly. Where an informal or colloquial name is used, it will be understood by a skilled artisan which species or taxon is meant.

[0148] In some aspects, the insect is Gryllodes sigillatus , Gryllus bimaculatus , Gryllus assimilis, a species of the superfamily Grylloidea, Melanoplus differ entialis, another species of the genus Melanoplus , a species of the order Orthoptera, Zophobas morio , Hermetia illucens , Musca domestica, Alphitobius diaperinus , Drosophila sp. (e.g. I) melanogaster ), a species of the Family Tephritidae (e.g., Ceratitis capitata , Anastrepha suspensa , Anastrepha ludens , or another species in the genus Anastrepha sp.), a species of the Order Diptera, species of the Order Coleoptera, a species of the Family Tenebrionidae, a species of the Family Dermestidae, Manduca sexta, another species in the genus Manduca sp., Galleria mellonella , Achroia grisella , Pieris rapae , Bombyx mori , another species in the genus Bombyx sp ., another species in the family Bombycidae, another species in the Family Saturniidae, Spodoptera frugiperda , Helicoverpa zea , Trichoplusia ni , or another species of the Order Lepidoptera.

[0149] In some aspects, the insect is a beetle. In some aspects, the insect is a mealworm.

In some aspects, the insect is a darkling beetle. In some aspects, the insect is an insect of the family Tenebrionidae . In some aspects, the insect is Tenebrio molitor. In some aspects, the insect is Zophobas morio. In some aspects, the insect is Triboleum castinatum.

[0150] It will be understood by a skilled artisan that the nomenclature for insect species is evolving, and that the nomenclature at the time of filing controls. However, this is not intended to limit the disclosure to insect species which have thus far been discovered. An after-arising discovery of an insect, or a nomenclature change for an existing insect, does not preclude its use with one or more aspects of the present disclosure.

Definitions

[0151] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present application including the definitions will control. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents and other references mentioned herein are incorporated by reference in their entireties for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0152] Although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods and examples are illustrative only and are not intended to be limiting. Other features and advantages of the disclosure will be apparent from the detailed description and from the claims.

[0153] In order to further define this disclosure, the following terms and definitions are provided.

[0154] The singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), as well as the terms "one or more," and "at least one" can be used interchangeably herein. In certain aspects, the term "a" or "an" means "single." In other aspects, the term "a" or "an" includes "two or more" or "multiple."

[0155] The term "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 10 percent, up or down (higher or lower).

[0156] Throughout this disclosure, various aspects of this invention are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Numeric ranges recited are inclusive of the numbers defining the range and include each integer within the defined range.

[0157] Units, prefixes, and symbols are denoted in their Systeme International de Unites

(SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Where a range of values is recited, it is to be understood that each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the disclosure. Thus, ranges recited herein are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0158] Where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of the disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of a disclosure is disclosed as having a plurality of alternatives, examples of that disclosure in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of a disclosure can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0159] As used herein, the term “substantial” means more than a minimal or insignificant amount; and “substantially” means more than a minimally or insignificantly. Thus, for example, the phrase “substantially similar”, as used herein, denotes a sufficiently high degree of similarity between two numeric values or features such that one of skill in the art would consider the difference between the two values to not be of statistical significance or qualitative significance within the context of the characteristic measured by said values or features. Thus, the difference between two values that are substantially similar to each other is typically less than about 10%, and can be less than about 20%, less than about 30%, less than about 40%, or less than about 50% as a function of the reference value or comparator value.

[0160] The term "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0161] The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, formulations, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0162] The term "excipient" refers to any substance, not itself a therapeutic agent, which may be used in a composition for delivery of an active therapeutic agent to a subject or combined with an active therapeutic agent (e.g., to create a pharmaceutical composition) to improve its handling or storage properties or to permit or facilitate formation of a dose unit of the composition (e.g., formation of a hydrogel which may then be optionally incorporated into a patch). Excipients include, but are not limited to, solvents, penetration enhancers, wetting agents, antioxidants, lubricants, emollients, substances added to improve appearance or texture of the composition and substances used to form hydrogels. Any such excipients can be used in any dosage forms according to the present disclosure. The foregoing classes of excipients are not meant to be exhaustive but merely illustrative as a person of ordinary skill in the art would recognize that additional types and combinations of excipients could be used to achieve the desired goals for delivery of a drug. The excipient can be an inert substance, an inactive substance, and/or a not medicinally active substance. The excipient can serve various purposes. A person skilled in the art can select one or more excipients with respect to the particular desired properties by routine experimentation and without any undue burden. The amount of each excipient used can vary within ranges conventional in the art. Techniques and excipients which can be used to formulate dosage forms are described in Handbook of Pharmaceutical Excipients, 6th edition, Rowe et ah, Eds., American Pharmaceuticals Association and the Pharmaceutical Press, publications department of the Royal Pharmaceutical Society of Great Britain (2009); and Remington: the Science and Practice of Pharmacy, 21th edition, Gennaro, Ed., Lippincott Williams & Wilkins (2005).

[0163] The term "effective amount" or "pharmaceutically effective amount" or

"therapeutically effective amount" as used herein refers to the amount or quantity of a drug or pharmaceutically active substance which is sufficient to elicit the required or desired therapeutic response, or in other words, the amount which is sufficient to elicit an appreciable biological response when administered to a patient.

[0164] The term "treating" or "treatment" as used herein refers to the administration of a composition to a subject for therapeutic purposes.

[0165] As used herein, the term "knock-in" refers to a cell, tissue, organ, organ system, or organism, or from the same organism, comprising a polynucleotide sequence (e.g., a transgene) derived from another organism incorporated into its genome at a target locus or replacing the polynucleotide originally present at a target locus. For example, an insect which contains a bacterial gene segment integrated into its genome and replacing a corresponding target endogenous insect gene is a knock-in insect. In some aspects, polynucleotide originally present at the target locus is replaced with a gene having a wild- type or modified gene sequence. The exogenous polynucleotide can be a non-natural sequence, or a sequence from another organism, such as a gene from a bacterial, fungal, plant, livestock, or other species. In some aspects, the knock-in effects a phenotypic change in the insect. In some aspects, the knock-in is compatible with a separate knock out in the same insect.

[0166] As used herein, the term "knock-out" refers to a cell, tissue, organ, organ system, or organism, wherein a target polynucleotide sequence in its genome is removed or replaced with a polynucleotide sequence, which results in a loss or reduction in function at the locus of the target polynucleotide. As a non-limiting example, an insect which has a functional gene removed is a knock-out insect. In another non-limiting example, an insect which has a functional gene replaced with non-functional DNA is an insect. In some aspects, the knock-out effects a phenotypic change in the insect. In some aspects, the knock-out is compatible with a separate knock-in in the same insect.

[0167] As used herein, the term "CRISPR technology" refers generally to methods and compositions for genetic engineering which rely on guide polynucleotides ( e.g ., guide RNA, or "gRNA") and natural or engineered enzymes associated with activity at or near clustered randomly interspaced short palindromic repeats ("CRISPR") (e.g., wild-type or engineered Cas9, or wild-type or engineered Casl2a (formerly Cpfl)) for targeted cleavage of DNA. It is understood that CRISPR technology is a rapidly evolving field of research, and that lesser-used CRISPR enzymes or techniques, or refined methods and compositions which may later be developed, are nevertheless within the scope of this disclosure. For example, the use of a Cas9 enzyme later engineered for higher stability or endonuclease activity is encompassed within the methods of this disclosure, provided such engineering does not render the enzyme incompatible with the use in insects. In a further non-limiting example, the use of a gRNA which is itself chemically modified for increased stability (e.g, wherein the gRNA comprises a phosphorothioate backbone rather than phosphodiester backbone) is within the scope of this disclosure.

[0168] As used herein, the term "air knife" refers to an apparatus which directs air with sufficient force to displace an insect. In some aspects, the apparatus is configured to deliver a laminar sheet of air. [0169] As used herein, the term "suitable for consumption" refers to the quality of a composition of being edible and having a nutritional or therapeutic value which a skilled artisan would regard as outweighing negative features, such as toxicity of one or more components of the composition.

[0170] As used herein, the term "GFP" refers to green fluorescent protein. As a non limiting example, a sequence of GFP designated as UniProt P42212 is available online at uniprot.org/uniprot/P42212. As used herein, the term "EGFP" refers to enhanced green fluorescent protein. As a non-limiting example, a sequence of EGFP designated as UniProt C5MKY7 is available online at uniprot.org/uniprot/C5MKY7. As used herein, the term "DsRed" refers to red fluorescent protein. As a non-limiting example, a sequence of DsRed designated as UniProt Q9U6Y8 is available online at uniprot.org/uniprot/Q9U6Y8. A skilled artisan will understand that such a reporter protein (e.g., GFP, EGFP, DsRed) is useful provided the reporter function is operable (i.e., emitted light). Accordingly, it will be understood that deviations from a reported sequence are within the scope of the disclosure. In some aspects, a reporter protein (e.g., GFP, EGFP, DsRed) disclosed herein shares at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity with the sequence of a reference reporter protein. In some aspects, the reference reporter protein is UniProt P42212, UniProt Q9U6Y8, or UniProt C5MKY7.

[0171] It is understood that wherever aspects are disclosed herein with reference to a feature or features of a genetically modified insect in relative terms but without a comparator explicitly indicated, the relevant comparator is the same feature or features in a wild-type insect of the same species at the same stage of development (e.g., egg, nymph, adult, pupa), unless context dictates otherwise. As a non-limiting example, when referring to the level of transcription of a gene in a genetically modified insect, a qualitative or quantitative descriptor referring to the level of transcription of the gene (e.g, "increased transcription by 10%") is relative to the level of transcription in a wild- type insect of the same species at the same stage of development (i.e., 10% greater transcription of the gene in the genetically modified insect relative to the level of transcription of the gene in a wild-type insect of the same species at the same stage of development). [0172] It is understood that wherever aspects are described herein with the language

"comprising," otherwise analogous aspects described in terms of "consisting of' and/or "consisting essentially of are also provided.

[0173] As used herein, "3 sgRNA" refers to a composition or experiment using 3 different guide RNAs for CRISPR/Cas9 knock-in and/or knock-out.

[0174] As used herein, " 1 sgRNA" refers to a composition or experiment using only one guide RNA.

[0175] As used herein, "sgRNA" refers to single guide RNA.

[0176] In some aspects, the methods described herein employ a non-homologous end joining approach for the DNA repair portion of the CRISPR experiments for gene knock- in. It will be understood by a person of ordinary skill in the relevant art that this method can also be applicable to gene knock-out.

[0177] In some aspects, a homology directed repair approach for may be employed for knock-ins/knock-outs.

[0178] In some aspects, examples embryos (or "eggs") are injected with a cocktail of

CRISPR components. In some aspects, the cocktail of CRISPR components comprises Cas9 enzyme/protein and one or more guide RNA (sgRNA(s)). In some aspects, the one or more sgRNA(s) target an insect's genome at a desired location for knock-out. In some aspects, the cocktail further comprises an additional DNA construct for additional target knock-in.

[0179] In some aspects, insects having been injected with a construct disclosed herein are the "GO" generation. In some aspects, the GO generation are adult insects. In some aspects, GO insects are screened for the desirable phenotype and/or genotype. In some aspects, the GO insects are crossed with one another (self cross). In some aspects, the GO insects are out-crossed to wild type individuals and/or to another strain to generate the down-stream desired stains with the desired stable inheritable (or even transient) desirable genotype and/or phenotype. In some aspects, insects not bearing a desired phenotype or genotype are bred out or crossed out.

[0180] As used herein, the term "E+G0" refers to crosses of GO generation (e.g.,

CRISPR/Cas9 with construct and/or guide RNA injected embryos which have grown up to maturity) which are positive for EGFP (GFP) expression. In some aspects, insects are injected with the CRISPR cocktail comprising RNA and/or DNA of choice. In some aspects, insects injected with the CRISPR cocktail are embryos (GO generation). In some aspects, GFP expression and/or fluorescence is monitored. In some aspects, expression of a gene of interest is monitored. In some aspects, a knock-out phenotype is observed. In some aspects, insects bearing a desired genotype or phenotype (i.e., the "positives" or +'s) are mated with one another as "self-cross". In some aspects, insects bearing a desired genotype or phenotype are out-crossed with wild type or another strain.

[0181] In some aspects, the present disclosure provides a genetically modified insect comprising one or more genetic modifications relative to a wild-type insect of the same species. In some aspects, the present disclosure provides a genetically modified insect comprising one or more genetic modifications relative to a wild-type insect of the same species, wherein the wild-type insect of the same species is selected from a superworm, a cricket, a mealworm, a Caribbean fruit fly, a black soldier fly, a grasshopper, a locust, a maggot, a beetle, a pupa, a buffalo worm, a moth, a butterfly, a katydid, a silkworm, or a caterpillar. In some aspects, the wild-type insect is a superworm. In some aspects, the wild-type insect is a cricket. In some aspects, the wild-type insect is a mealworm. In some aspects, the wild-type insect is a Caribbean fruit fly. In some aspects, the wild-type insect is a black soldier fly. In some aspects, the wild-type insect is a grasshopper. In some aspects, the wild-type insect is a fly. In some aspects, the wild-type insect is a maggot. In some aspects, the wild-type insect is a beetle. In some aspects, the wild-type insect is a pupa. In some aspects, the wild-type insect is a buffalo worm. In some aspects, the wild- type insect is a moth. In some aspects, the wild-type insect is a butterfly. In some aspects, the wild-type insect is a locust. In some aspects, the wild-type insect is a katydid. In some aspects, the wild-type insect is a silkworm. In some aspects, the wild-type insect is a caterpillar.

[0182] In some aspects, the one or more genetic modifications effect a phenotype detectable by visual inspection.

[0183] In some aspects, the genetically modified insect is an insect selected from the group consisting of: a superworm, a cricket, a mealworm, a Caribbean fruit fly, a black soldier fly, a grasshopper, a locust, a katydid, a silkworm, or a caterpillar. In some aspects, the locust is Locusta migrator.

[0184] In some aspects, the genetically modified insect is a cricket. In some aspects, the cricket is of the species Acheta domesticus. [0185] In some aspects, the genetically modified insect is a mealworm. In some aspects, the mealworm is of the species Tenebrio molitor.

[0186] In some aspects, the one or more genetic modifications consist of two genetic modifications. In some aspects, the one or more genetic modifications consist of three genetic modifications. In some aspects, the one or more genetic modifications consist of four genetic modifications. In some aspects, the one or more genetic modifications consist of five genetic modifications. In some aspects, the one or more genetic modifications consist of six or more genetic modifications. In some aspects, the one or more genetic modifications consist of six to ten genetic modifications. The genetically modified insect of claim 12, wherein the one or more genetic modifications consist of six to ten genetic modifications.

[0187] In some aspects, the one or more genetic modifications are introduced by a

CRISPR/Cas9 system.

[0188] In some aspects, the one or more genetic modifications comprise an indel mutation.

[0189] In some aspects, the one or more genetic modifications comprises a gene knock out. In some aspects, the one or more genetic modifications comprise knock-out of two or more genes.

[0190] In some aspects, the gene knock-out is a knock-out of a vermilion gene. In some aspects, the vermilion gene encodes a protein which shares at least 85% sequence identity with the sequence of Uniprot Q6SIK7 (available at uniprot.org/uniprot/Q6SIK7). In some aspects, the gene knock-out is introduced by a CRISPR/Cas9 system using a construct encoding a polynucleotide comprising a guide RNA sequence complementary to a portion of a vermilion gene.

[0191] In some aspects, the gene knock-out is of a white gene. In some aspects, the vermilion gene encodes a protein which shares at least 85% sequence identity with the sequence of Uniprot PI 0090 (available at uniprot.org/uniprot/Pl 0090).

[0192] In some aspects, the one or more genetic modifications cause a detectable phenotype. In some aspects, the detectable phenotype is detectable by visual inspection.

In some aspects, the phenotype is emission of a particular wavelength. In some aspects, the particular wavelength is not detectable by visual inspection. In some aspects, the particular wavelength is a wavelength of light emitted by one or more of GFP, EGFP, and DsRed. In some aspects, the present disclosure provides a method of determining if an insect has incorporated one or more genetic modifications, the method comprising determining if the detectable phenotype is present in the insect, wherein the insect is determined to have incorporated one or more genetic modifications if the detectable phenotype is present in the insect. In some aspects, the detectable phenotype is a phenotype disclosed herein.

[0193] In some aspects, the gene knock-out is of a toxic gene, a gene that inhibits growth, a gene that slows down an insect life cycle, a color pigment gene, or an allergen gene (e.g., tropomyosin). In some aspects, an allergen gene is knocked-out and replaced with a corresponding gene which confers a reduced or eliminated allergic response. In some aspects, an allergen gene is knocked-out and a corresponding gene which confers a reduced or eliminated allergic response is knocked-in at a different locus. In some aspects, the allergen gene encodes tropomyosin. In some aspects, an engineered tropomyosin characterized by reduced immunogenicity is knocked-in. In some aspects, an engineered tropomyosin characterized by reduced immunogenicity is knocked-in and an endogenous tropomyosin gene is knocked-out.

[0194] In some aspects, the one or more genetic modifications comprises an alteration in a regulatory mechanism of a target gene. In some aspects, the regulatory mechanism is a regulatory element. In some aspects, the regulatory element in a promoter or terminator.

In some aspects, the regulatory element is a promoter. In some aspects, the regulatory element is a terminator. In some aspects, the alteration comprises removal of the regulatory mechanism. In some aspects, the alteration comprises replacement of the regulatory mechanism. In some aspects, the alteration comprises the use of CRISPR technology. In some aspects, the regulatory mechanism is a trans-acting binding element that binds a regulatory element. In some aspects, the alteration comprises a knock-out or other disruption which reduces or eliminates transcription of a gene encoding the trans acting binding element. In some aspects, the alteration in a regulatory mechanism causes a change in transcription of a gene involved in metabolism of a target product. In some aspects, the alteration in a regulatory mechanism causes a change in transcription of a gene involved in biosynthesis of a target product. In some aspects, the target product is a target product disclosed herein. In some aspects, the target product is an enzyme. In some aspects, the gene involved in biosynthesis of a target product is a gene encoding an enzyme involved in biosynthesis of the target product. In some aspects, the change in transcription is a reduction in transcription. In some aspects, the change in transcription is an elimination of transcription. In some aspects, the change in transcription is an increase in transcription. In some aspects, the increase or decrease in transcription is by about 100%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1,000%, about 10,000%, about 100,000%, or about 1,000,000%.

[0195] In some aspects, the one or more genetic modifications comprises a gene knock- in. In some aspects, the gene knock-in is introduced by a CRISPR/Cas9 system using a construct encoding a polynucleotide comprising a guide RNA sequence complementary to a portion of a gene (e.g., a vermilion gene or a white gene).

[0196] In some aspects, the portion of a vermilion gene comprises exon 2 or a portion of exon 2.

[0197] In some aspects, the one or more genetic modifications alter one or more genes associated with a recessive phenotype.

[0198] In some aspects, the one or more genetic modifications alter one or more genes associated with a dominant phenotype.

[0199] In some aspects, the one or more genetic modifications comprises genomic incorporation of a transgene. In some aspects, the transgene is incorporated at the locus of a vermilion gene.

[0200] In some aspects, the transgene encodes a fluorescent protein.

[0201] In some aspects, the fluorescent protein is a green fluorescent protein. In some aspects, the green fluorescent protein is EGFP.

[0202] In some aspects, the fluorescent protein is a red fluorescent protein. In some aspects, the red fluorescent protein is DsRed.

[0203] In some aspects, the phenotype detectable by visual inspection is detectable by visual inspection only after illuminating the genetically modified insect with light of one or more wavelengths corresponding to one or more excitation wavelengths characteristic of the fluorescent protein.

[0204] In some aspects, the one or more genetic modifications comprise an alteration in an eye color gene. In some aspects, the eye color gene is a vermilion gene. [0205] In some aspects, the genetically modified insect is a cricket which expresses a fluorescent protein and has a white eye phenotype.

[0206] In some aspects, the genetically modified insect is a mealworm which expresses a fluorescent protein and has a white eye phenotype.

[0207] In some aspects, the present disclosure provides a molecular construct comprising a sequence encoding, in the 5' to 3' direction: a first guide RNA segment, a tissue-specific promoter, a phenotypic marker, and a second guide RNA segment.

[0208] In some aspects, the molecular construct further comprises a sequence encoding a transgene of interest, wherein the sequence encoding a transgene of interest is 3' of the tissue-specific promoter and 5' of the sequence encoding a phenotypic marker.

[0209] In some aspects, the sequence encoding a transgene further comprises a sequence encoding a secretion signal.

[0210] In some aspects, the phenotypic marker is a fluorescent protein.

[0211] In some aspects, the fluorescent protein is a green fluorescent protein. In some aspects, the green fluorescent protein is EGFP.

[0212] In some aspects, the fluorescent protein is a red fluorescent protein. In some aspects, the red fluorescent protein is DsRed.

[0213] In some aspects, the first guide RNA segment is complementary to a first sequence of a first gene, and the second guide RNA segment is complementary to a second sequence of a second gene.

[0214] In some aspects, the first guide RNA segment is complementary to a first sequence of a first gene, and the second guide RNA segment is complementary to a second sequence of the first gene.

[0215] In some aspects, the tissue-specific promoter is a muscle tissue-specific promoter.

[0216] In some aspects, the muscle tissue-specific promoter is a muscle actin promoter.

[0217] In some aspects, the molecular construct comprises a sequence encoding a linker peptide. In some aspects, the sequence encoding the linker peptide is flanked by sequences encoding protein domains. In some aspects, the molecular construct comprises sequences encoding one or more linker peptides. In some aspects, the molecular construct comprises sequences encoding 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker peptides. In some aspects, the one or more linker peptides comprise a linker selected from F2A, P2A, or T2A. In some aspects, the molecular construct comprises one or more sequences encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 beta carotene biosynthesis genes, wherein a sequence encoding a linker peptide separates sequences encoding beta carotene biosynthesis genes.

[0218] In some aspects, the molecular construct comprises, in the 5’ to 3’ direction, a muscle actin promoter, a sequence encoding a first beta-carotene biosynthesis gene, a first linker sequence, a sequence encoding a second beta-carotene biosynthesis gene, a second linker sequence, a sequence encoding a third beta-carotene biosynthesis gene, a third linker sequence, a sequence encoding a fourth beta-carotene biosynthesis gene, a fourth linker sequence, sequence encoding a reporter protein, and a SV40 sequence. In some aspects, the first linker is a P2A linker. In some aspects, the P2A linker is optimized for expression in Drosophila melanogaster. In some aspects, the second linker is a T2A linker. In some aspects, the third linker is a F2A linker. In some aspects, the fourth linker is a P2A linker. In some aspects, the reporter protein is DsRed.

[0219] In some aspects, the molecular construct comprises, in the 5’ to 3’ direction, a first muscle actin promoter, a gene sharing at least 85% sequence identity with ninaB , a first SV40 sequence, a second muscle actin promoter, a retinol dehydrogenase gene, a second SV40 sequence, a PUb promoter, a sequence encoding a reporter protein, and a third SV40 sequence. In some aspects, a first linker sequence is 3’ to the first SV40 sequence and 5’ to the second muscle actin promoter. In some aspects, a second linker sequence is 3’ to the second SV40 sequence and 5’ to the PUb promoter.

[0220] In some aspects, the molecular construct comprises, in the 5’ to 3’ direction, a first muscle actin promoter, a gene sharing at least 85% sequence identity with ninaB , a sequence encoding a first linker peptide, a retinol dehydrogenase gene, a second linker peptide, a sequence encoding a reporter protein, and a first SV40 sequence.

[0221] In some aspects, the first linker peptide is P2A. In some aspects, the second linker peptide is F2A. In some aspects, the first, second, and third SV40 sequences are identical. In some aspects, the first and second muscle actin promoters are identical. In some aspects, the reporter protein is EGFP. In some aspects, a sequence encoding a linker disclosed herein can be replaced with an alternative sequence encoding a different linker. In some aspects, the different linker is a linker disclosed herein.

[0222] In some aspects, the molecular construct further comprises a sequence encoding a self-cleaving peptide 5' of the sequence encoding the phenotypic marker. [0223] In some aspects, the sequence encoding a self-cleaving peptide is 3' of the sequence encoding the transgene of interest. In some aspects, the self-cleaving peptide is a 2A self-cleaving peptide. In some aspects, the 2A self-cleaving peptide is T2A.

[0224] In some aspects, the molecular construct further comprises an SV40 sequence 3' of the sequence encoding the phenotypic marker and 5' of the sequence encoding the second guide RNA segment. The molecular construct of any one of claims 37-52, further comprising an SV40 sequence 3' of the sequence encoding the phenotypic marker and 5' of the sequence encoding the second guide RNA segment.

[0225] In some aspects, the present disclosure provides a method of producing a genetically modified insect, comprising introducing one or more genetic modifications in a wild-type insect.

[0226] In some aspects, the method comprises introducing into a wild-type insect a construct disclosed herein.

[0227] In some aspects, the method comprises introduction of two or more constructs disclosed herein, wherein the two or more constructs are not identical in sequence.

[0228] In some aspects, the method is used to produce a genetically modified insect disclosed herein.

[0229] In some aspects, the one or more genetic modifications comprise introduction of a polynucleotide which has been codon-optimized for expression. In some aspects, the polynucleotide has been codon-optimized for expression in an insect in which it is introduced. In some aspects, the polynucleotide has been codon-optimized for expression in a bacterial species. In some aspects, the polynucleotide has been codon-optimized for expression in a eukaryotic species. In some aspects, the polynucleotide has been codon- optimized for expression in an archaeal species.

Further GMO Insect Aspects

[0230] In some aspects the genetically modified insect comprises one or more genetic alterations which do not cause a phenotypic effect. In some aspects, the one or more genetic alterations is useful for a method of determining the origin of the genetically modified insect. In some aspects, the method of determining the origin of the genetically modified insect comprises determining the sequence of a locus comprising the one or more genetic alterations. In some aspects, the one or more genetic alterations comprise the addition of an identifiable barcode sequence to the genome of a wild-type or genetically modified insect.

[0231] In some aspects, the wild-type insect is a cricket selected from the superfamily

[0232] In some aspects, the genetically modified insect comprises one or more genetic alterations which establish or alter a biosynthetic pathway. In some aspects, the one or more genetic alterations comprise one or more knock-ins. In some aspects, the one or more genetic alterations comprise one or more knock-outs. In some aspects, the biosynthetic pathway is an insect biosynthetic pathway. In some aspects, the biosynthetic pathway is not an insect biosynthetic pathway. In some aspects, one or more steps of the biosynthetic pathway produces a target product within the genetically modified insect. In some aspects, the target product is a target product disclosed herein. In some aspects, one or more steps of the biosynthetic pathway produces a vitamin or precursor thereof. In some aspects, the target product is a nutritional biomolecule disclosed herein. In some aspects, the biosynthetic pathway is a lipid biosynthesis pathway. In some aspects, the biosynthetic pathway is a polynucleotide biosynthesis pathway. In some aspects, the biosynthetic pathway is an amino acid biosynthesis pathway. In some aspects, the amino acid biosynthesis pathway synthesizes an essential amino acid.

[0233] In some aspects, the biosynthetic pathway is the tricarboxylic acid (TCA) cycle pathway. Methods and examples of introducing exogenous TCA cycle pathway genes are exemplified in U.S. Patent No. 8,048,661 B2, which is incorporated by reference in its entirety herein. In some aspects, the biosynthetic pathway comprises use of one or more of an ATP-citrate lyase, a citrate lyase, a fumarate reductase, and an alpha- ketoglutarate:ferredoxin oxidoreductase.

[0234] In some aspects, the essential amino acid is an amino acid disclosed herein. In some aspects, the amino acid biosynthesis pathway synthesizes a non-proteinogenic amino acid. In some aspects, the biosynthetic pathway is an aminoacyl-tRNA biosynthesis pathway. In some aspects, the aminoacyl-tRNA biosynthesis pathway comprises an aminoacyl-tRNA synthetase which is orthogonal to the genetically modified insect. In some aspects, the aminoacyl-tRNA synthetase is an engineered aminoacyl- tRNA synthetase. In some aspects, the engineered aminoacyl-tRNA synthetase activates a non-proteinogenic amino acid. In some aspects, the aminoacyl-tRNA synthetase transfers a non-proteinogenic amino acid moiety onto a tRNA. In some aspects, a tRNA is encoded a polynucleotide which has been exogenously introduced into an insect. In some aspects, the non-proteinogenic amino acid is useful a chemical reaction in which proteinogenic amino acids cannot participate efficiently. In some aspects, the chemical reaction is click chemistry. Methods for using an orthogonal aminoacyl-tRNA/aminoacyl-tRNA synthetase system, e.g., to rapid-label cell surface proteins in living cells using click chemistry, are known in the field. See, e.g., Nikic, I. et al. (2015) Labeling proteins on live mammalian cells using click chemistry. Nat Protoc. 10(5):780-91. In some aspects, aminoacyl-tRNA biosynthesis pathway introduces an aminoacyl-tRNA which decodes a codon with an amino acid not encoded by that codon in the wild-type insect from which the genetically modified insect is derived. In some aspects, the aminoacyl-tRNA synthetase has a mutation in which alters post-transfer editing activity. In some aspects, the aminoacyl-tRNA synthetase has a mutation which alters pre-transfer aminoacyl-AMP editing activity. In some aspects, the aminoacyl-tRNA synthetase has a mutation which alters the activity of amino acid activation. In some aspects, the alteration in activity of amino acid activation is an alteration in amino acid specificity. In some aspects, the aminoacyl-tRNA synthetase has a mutation in a post-transfer aminoacyl-tRNA editing domain. Non-limiting examples and methods of orthogonal aminoacyl-tRNA/aminoacyl- tRNA synthetase systems and non-proteinogenic (or "unnatural") amino acids are disclosed in U.S. Patent No. 8,815,542 B2, which is incorporated herein in its entirety.

[0235] In some aspects, the one or more genetic modifications comprises a gene knock out and a gene knock-in. In some aspects, the gene knock-out or gene knock-in is introduced by the use of a transposable element. In some aspects, the gene knock-out or gene knock-in is introduced using a transposase.

[0236] In some aspects, the one or more genetic modifications reduce or eliminate production of an undesired product. Methods and examples of reducing or eliminating production of an undesired product (endogenous aldehyde dehydrogenase conversion of isobutyraldehyde to isobutyrate; endogenous pyruvate decarboxylase conversion of pyruvate to acetaldehyde) are exemplified in U.S. Patent No. 8,158,404 B2, which is incorporated by reference in its entirety herein. [0237] In some aspects, the one or more genetic modifications effect an increase in transcription of a target gene. In some aspects, the one or more genetic modifications effect a decrease in transcription of a target gene. In some aspects, the decrease in transcription is a reduction in the transcription of the target gene to a level which is not detectable by conventional reverse transcription PCR methods.

[0238] In some aspects, the target gene is an endogenous gene. In some aspects, the target gene is an exogenous gene. In some aspects, the target gene is introduced as a knock-in.

In some aspects, the target gene is modified relative to a wild-type sequence of the target gene. In some aspects, the target gene shares at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity with the sequence of the wild-type gene.

[0239] In some aspects, the target gene is a gene involved in the biosynthesis of a target product disclosed herein. In some aspects, the target produce is an enzyme. In some aspects, the enzyme is an enzyme disclosed herein. In some aspects, the target product is a carotenoid. In some aspects, the target product is a retinoid. In some aspects, the target product is a trans- retinal. In some aspects, target product is a 13 - v.s retinal. In some aspects, the target product is a trans retinol. In some aspects, the target product is 13 -cis retinol. In some aspects, the target product is a retinoid. In some aspects, the target product is beta carotene (b-carotene). In some aspects, the target product is a vitamin. In some aspects, the target product is Vitamin A. Certain biosynthetic pathways and corresponding genes associated with metabolism of carotenoids and vitamins (e.g., Vitamin A) are known or identifiable to a skilled artisan, and are exemplified in the literature (See, e.g., Moise, A.R. et al. (2014) Mechanistic Aspects of Carotenoid Biosynthesis. Chem Rev. 114(1): 164-193. See also Lintig, V.J. and Vogt, K. (2000). 275(16): 11915-11929.).

[0240] The nature of retinol biosynthesis will be appreciated and understood by a skilled artisan. In some aspects, retinol is synthesized from the breakdown of b-carotene. First a b-carotene 15, 15’ -monooxygenase cleaves b-carotene at the central double bond, creating an epoxide. This epoxide is then attacked by water creating two hydroxyl groups in the center of the structure. The cleavage occurs when these alcohols are reduced to the aldehydes using NADH. The resulting compound is called retinal. Retinal is then reduced to retinol by the enzyme retinol dehydrogenase. Retinol dehydrogenase is an enzyme that is dependent on NADH.

[0241] In some aspects, the target gene encodes a retinol dehydrogenase. In some aspects, the target gene is a b-carotene 15, 15’ -monooxygenase. In some aspects, the target gene encodes a phytoene synthase. In some aspects, the phytoene synthase is a daffodil (. Narcissus pseudonarcissus) phytoene synthase. In some aspects the gene encoding the phytoene synthase is PSY. In some aspects, the target gene encodes a lycopene beta- cyclase. In some aspects, the target gene encodes a carotene desaturase. In some aspects, the carotene desaturase is a Erwinia uredovora carotene desaturase. In some aspects, enzyme is Crtl, CrtB, or CrtY. In some aspects, the target gene is crtl, crtB , or crtY. In some aspects, the target gene is a beta carotene biosynthesis gene. In some aspects, the target gene encodes an enzyme which converts beta carotene or a precursor to a Vitamin A precursor. In some aspects, the gene encodes a b-carotene dioxygenase. In some aspects, the b-carotene dioxygenase shares at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or 100% sequence identity with a Drosophila melanogaster b-carotene dioxygenase (see, e.g., GENBANK™ Accession Number AI063857). In some aspects, the target gene encodes a retinoid isomerohydrolase. In some aspects, the target gene encodes a RPE65. In some aspects, the RPE65 is a human RPE65 (see, e.g., uniprot.org/uniprot/Q16518). In some aspects, the RPE65 is a Bos Taurus RPE65. In some aspects, the target gene encodes a carotenoid isomerooxygenase. In some aspects, the carotenoid isomerooxygenase is encoded by a ninaB gene (see, e.g., uniprot.org/uniprot/Q9VFS2). In some aspects, the target gene encodes In some aspects, the target gene shares at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or 100% sequence identity with a gene disclosed herein.

[0242] In some aspects, the one or more genetic modifications comprise a modification of a regulatory element. In some aspects, the modification of a regulatory element is a replacement of a promoter region with an exogenous sequence or an endogenous sequence.

[0243] In some aspects, transgene is under the control of an exogenous promoter. In some aspects, one or more transgenes are under the control of an exogenous promoter. In some aspects, the exogenous promoter is a promoter for an actin (e.g., muscle actin), polyubiquitin, or ubiquitin.

[0244] In some aspects, the target gene is a wild-type gene. In some aspects, the target gene is a transgene. In some aspects, the transgene is selected from the group consisting of: a gene encoding an enzyme, a gene encoding a light-emitting protein, a gene encoding an antigen, and a gene encoding a food protein.

[0245] In some aspects, the enzyme is a light-emitting protein. In some aspects, the light- emitting protein has the amino acid sequence of a light-emitting protein encoded by a jellyfish species, or shares at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or 100% sequence identity with the amino acid sequence of a light-emitting protein encoded by a jellyfish species.

[0246] In some aspects, the antigen is a viral, bacterial, or fungal antigen. In some aspects, the viral antigen is influenza hemagglutinin. In some aspects, the viral antigen is a coronaviral spike protein. In some aspects, the coronaviral spike protein is a SARS- CoV-2 spike protein. In some aspects, the antigen is a receptor binding protein. In some aspects, the antigen is a nucleoprotein.

[0247] In some aspects, the light-emitting protein is a green fluorescent protein ("GFP") such as EGFP, DsRed, or luciferase. In some aspects, the insect produces or is genetically modified to produce a cofactor for activity of a light-emitting protein. In some aspects, the cofactor is luciferin.

[0248] In some aspects, the food protein is ovalbumin, casein, myoglobin, hemoglobin, heme, a beta carotene enzyme, a protein involved in production of Vitamin A, gluten, or gelatin. In some aspects, the genetically modified insect produces beta carotene.

[0249] In some aspects, the food product has improved digestability relative to a food product produced from a wild-type insect of the same species. In some aspects, the food product has improved bioavailability of a component contained therein relative to a food product produced from a wild-type insect of the same species.

[0250] In some aspects, the genetically modified insect produces less of a target product than a wild-type insect of the same species. In some aspects, the genetically modified insect produces about 0.01% to about 1000%, 5% to about 900%, about 50% to about 800%, about 200% to about 700%, about 300% to about 600%, or about 400% to about 500% less of the target product than the wild-type insect of the same species. In some aspects, the genetically modified insect produces about 0.01% to about 5%, 5% to about 50%, about 50% to about 100%, about 200% to about 300%, about 300% to about 500%, or about 500% to about 1000% less of the target product than the wild-type insect of the same species. In some aspects, the genetically modified insect produces about 0.01%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 250%, about 300%, about 400%, about 500% about 600%, about 700%, about 800%, about 900%, or about 1000% less of the target product than the wild-type insect of the same species. In some aspects, the wild-type insect produces a target product which the genetically modified insect of the same species does not produce.

[0251] In some aspects, the genetically modified insect produces more of a target product than a wild-type insect of the same species. In some aspects, the genetically modified insect produces about 0.01% to about 1000%, 5% to about 900%, about 50% to about 800%, about 200% to about 700%, about 300% to about 600%, or about 400% to about 500% more of the target product than the wild-type insect of the same species. In some aspects, the genetically modified insect produces about 0.01% to about 5%, 5% to about 50%, about 50% to about 100%, about 200% to about 300%, about 300% to about 500%, or about 500% to about 1000% more of the target product than the wild-type insect of the same species. In some aspects, the genetically modified insect produces about 0.01%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 250%, about 300%, about 400%, about 500% about 600%, about 700%, about 800%, about 900%, or about 1000% more of the target product than the wild-type insect of the same species. In some aspects, the genetically modified insect produces a target product which the wild-type insect of the same species does not produce.

[0252] In some aspects, the target product is one or more of: a nutritional biomolecule, a chemical precursor to a nutritional biomolecule, an engineered biological product, a vaccine, a hormone, a flavorant, a colorant, a pigment, a fragrance, a fluorescent molecule, a peptide, a polypeptide, a polynucleotide, a lipid, a therapeutic drug product, a plastic, an insect repellant, and chitin. In some aspects, the chemical precursor is a provitamin. In some aspects, the provitamin is provitamin A.

[0253] In some aspects, the target product is a nutritional biomolecule. In some aspects, the nutritional biomolecule is a vitamin. In some aspects, the vitamin is vitamin A, a B vitamin, vitamin C, a D vitamin, or vitamin K. In some aspects, the vitamin is vitamin A.

[0254] In some aspects, the nutritional biomolecule is folic acid or folate.

[0255] In some aspects, the nutritional biomolecule is a protein. In some aspects, the protein is a peptide disclosed herein. In some aspects, the protein is a polypeptide disclosed herein. In some aspects, the protein is an enzyme disclosed herein.

[0256] In some aspects, the nutritional biomolecule is a fatty acid. In some aspects, the nutritional biomolecule is an essential fatty acid. In some aspects, the fatty acid is an omega-3 fatty acid. In some aspects, the fatty acid is an omega-6 fatty acid. In some aspects, the fatty acid is an unsaturated fatty acid. In some aspects, the fatty acid is a monounsaturated fatty acid. In some aspects, the fatty acid is a polyunsaturated fatty acid.

[0257] In some aspects, the nutritional biomolecule is an essential nutrient in humans or livestock. In some aspects, the essential nutrient is an essential amino acid. In some aspects, the essential amino acid is a human essential amino acid. In some aspects, the human essential amino acid is phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, histidine, arginine, lysine, or leucine.

[0258] In some aspects, the target product is an engineered biological product. In some aspects, the engineered biological product reduces immunogenicity in humans.

[0259] In some aspects, the engineered biological product is an engineered chitin. In some aspects, the engineered chitin reduces the risk of choking or causing abrasions when consumed. In some aspects, the engineered chitin is a softened chitin. In some aspects, the engineered chitin is a chitosan. In some aspects, the engineered chitin is suitable for use as a carrier in a drug delivery system. In some aspects, the engineered chitin is suitable for use as an antibacterial agent.

[0260] In some aspects, the engineered biological product comprises a non-natural structural feature which aids in separation of the engineered biological product from other components of the genetically modified insect. In some aspects, the engineered biological product is a protein. In some aspects, the non-natural structural feature is an affinity tag.

In some aspects, the affinity tag binds to an affinity substance with a dissociation constant sufficient to allow the engineered biological product to be separated from at least one other component of the genetically modified insect by washing a complex comprising the affinity substance bound to the affinity tag with a fluid which carries the at least one other component away from the complex. In some aspects, the affinity tag is a polyhistidine tag. In some aspects, the polyhistidine tag consists of six consecutive histidine residues.

In some aspects, the affinity tag is a FLAG tag.

[0261] In some aspects, the engineered biological product is a pigment. In some aspects, the pigment is carmine red.

[0262] In some aspects, the target product is a vaccine. In some aspects, the vaccine is suitable for vaccination of a domesticated organism or non-domesticated organism. In some aspects, the vaccine comprises an artificial antigenic construct. In some aspects, the vaccine comprises a wild-type virus or an engineered virus, or an antigenic component thereof. In some aspects, the vaccine is for vaccination against one or more variants of: an influenza, a member of the family Coronaviridae, a paramyxovirus, a henipavirus, a Nipah virus, and a coxsackievirus (e.g., hand, foot, and mouth disease virus). In some aspects, the influenza is a high pathologic avian influenza or a low pathologic avian influenza. In some aspects, the member of the family Coronaviridae is SARS-CoV-2 or a variant thereof. In some aspects the variant of SARS-CoV-2 is alpha variant, delta variant, omicron variant, or a variant thereof.

[0263] In some aspects, the vaccine is for vaccination of an insect against an insect pathogen. In some aspects, the insect pathogen is a cricket pathogen. In some aspects, the insect pathogen is a mealworm pathogen. In some aspects, the insect pathogen is a pathogen to an insect species disclosed herein. In some aspects, the vaccine is for vaccination of a human against a human pathogen. In some aspects, the vaccine is for vaccination in a domesticated animal against a domesticated animal pathogen. In some aspects, the domesticated animal is a cat or dog. In some aspects, the vaccine is for vaccination of a livestock species against a livestock pathogen. In some aspects, the livestock species is a bovine species, an avian species, a poultry species, a porcine species, a fish species, or an arthropod species. In some aspects, the bovine species is Bos taurus. In some aspects, the avian species is Callus domesticus. In some aspects, the porcine species is Sus domesticus. In some aspects, the vaccine is for vaccination against porcine epidemic diarrhea virus (PEDV). In some aspects, the vaccine is for vaccination against cricket paralysis virus (CrPV). In some aspects, the vaccine is for vaccination against an insect Iridovirus.

[0264] In some aspects, the vaccine is for vaccination of a plant against a plant pathogen.

In some aspects, the plant pathogen is Tobacco mosaic virus.

[0265] In some aspects, the present disclosure provides a method of producing a formulation comprising a vaccine. In some aspects, the vaccine is a vaccine disclosed herein. In some aspects, the formulation comprising a vaccine comprises a pharmaceutically acceptable carrier or excipient. In some aspects, the formulation comprising a vaccine is suitable for injection. In some aspects, the formulation comprising a vaccine is suitable for inhalation. In some aspects, the formulation comprising a vaccine is an aerosol. In some aspects, the formulation comprising a vaccine is in liquid form. In some aspects, the formulation comprising a vaccine is in powder form. In some aspects, the formulation comprising a vaccine is a solid dosage form. In some aspects, the formulation comprising a vaccine is a capsule or tablet. In some aspects, the formulation comprising a vaccine is suitable for vaccination of a species disclosed herein against a pathogen disclosed herein. In some aspects, the species is a human, and the pathogen is a coronavirus. In some aspects, the coronavirus is a variant of SARS-CoV-2.

[0266] In some aspects, the present disclosure provides a method of administering a formulation comprising a vaccine. In some aspects, the formulation comprising a vaccine is a formulation comprising a vaccine disclosed herein. In some aspects, the method of administering a formulation comprising a vaccine comprises administering a therapeutically effective amount of the formulation to a subject in need thereof. In some aspects, the method of administering a formulation comprising a vaccine comprises prophylactic administration of the formulation in an amount effective to reduce the risk of death or hospitalization from infection of a pathogen.

[0267] In some aspects, the target product is a hormone. In some aspects, the hormone is a growth hormone. In some aspects, the hormone is insulin. In some aspects, the hormone is an estrogen. In some aspects, the hormone is a progestogen. In some aspects, the hormone is a steroid. In some aspects, the genetically modified insect producing the hormone has softened chitin relative to a wild-type insect of the same species. In some aspects, the genetically modified insect producing the hormone has an altered life cycle progression. In some aspects, the altered life cycle progression is characterized by a longer period of time in one or more life cycle stages. In some aspects, the altered life cycle progression is characterized by a reduced period of time in one or more life cycle stages. In some aspects, the longer period of time is about 10% to about 1000%, about 20% to about 900%, about 30% to about 800%, about 40% to about 600%, about 50% to about 500%, about 60% to about 400%, about 70% to about 300%, about 80% to about 200% longer. In some aspects, the longer period of time is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000% longer. In some aspects, the reduced period of time is about 10% to about 1000%, about 20% to about 900%, about 30% to about 800%, about 40% to about 600%, about 50% to about 500%, about 60% to about 400%, about 70% to about 300%, about 80% to about 200% reduced. In some aspects, the reduced period of time is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000% reduced.

[0268] In some aspects, the target product is an enzyme. In some aspects, the polypeptide is an enzyme. In some aspects, the enzyme is an enzyme of one or more of International Union of Biochemistry and Molecular Biology (IUBMB) classification system classes EC 1, EC 2, EC 3, EC 4, EC 5, EC 6, and EC 7. In some aspects, the enzyme is an enzyme of one or more of IUBMB classification system subclasses EC 1.1, EC 1.2, EC 1.3, EC 1.4, EC 1.5, EC 1.6, EC 1.7, EC 1.8, EC 1.9, EC 1.10, EC 1.11, EC 1.12, EC 1.13, EC 1.14, EC 1.15, EC 1.16, EC 1.17, EC 1.18, EC 1.19, EC 1.20, EC 1.21, EC 1.22, EC 1.23, EC 1.97, EC 2.1, EC 2.2, EC 2.3, EC 2.4, EC 2.5, EC 2.6, EC 2.7, EC 2.8, EC 2.9, EC 2.10, EC 3.1, EC 3.2, EC 3.3, EC 3.4, EC 3.5, EC 3.6, EC 3.7, EC 3.8, EC 3.9, EC 3.10, EC 3.11, EC 3.12, EC 3.13, EC 4.1, EC 4.2, EC 4.3, EC 4.4, EC 4.5, EC 4.6, EC 4.7, EC 4.99, EC 5.1, EC 5.2, EC 5.3, EC 5.4, EC 5.5, EC 5.99, EC 6.1, EC 6.2, EC 6.3, EC 6.4, EC 6.5, EC 6.6, EC 7.1, EC 7.2, EC 7.3, EC 7.4, EC 7.5, and EC 7.6. In some aspects, the enzyme is an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, or a translocase. In some aspects, the enzyme shares at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5% , or 100% sequence identity with the amino acid sequence of a wild-type enzyme. In some aspects, the enzyme is a cellulase. In some aspects, the cellulase uses cellulose as a substrate. In some aspects, the enzyme is a lignase. In some aspects, the lignase uses lignan as a substrate. In some aspects, the enzyme is a dioxygenase. In some aspects, the enzyme is a b-carotene dioxygenase. In some aspects, the b-carotene dioxygenase is derived from an insect. In some aspects, the insect is Drosophila melanogaster. In some aspects, the enzyme is a restriction enzyme. In some aspects, the enzyme catalyzes production of a target product disclosed herein, or a precursor to the target product. In some aspects the enzyme is an insect enzyme. In some aspects, the enzyme is a human enzyme. In some aspects, the enzyme is an enzyme encoded by a domesticated animal. In some aspects, the enzyme is an enzyme encoded by a non-domesticated animal. In some aspects, the enzyme is an industrially useful enzyme. In some aspects, the industrially useful enzyme catalyzes production of a food product or additive. In some aspects, the food product or additive is a sweetener. In some aspects, the enzyme is Palatase, Lipozyme TL IM, Lipase AK Amano, Lipopan F, Cellulase, Amylase, Xylose isomerase, Resinase, Penicillin amidase, or amidase. In some aspects, the enzyme is Novozym-435, Bromelain, Noopazyme, Asparaginase, Ficin, Urokinase, b-Lactamase, or Subtilisin. In some aspects, the amylase is an a-amylase. In some aspects, the amylase is a glucoamylase. In some aspects, the enzyme is a hemicellulase.

[0269] In some aspects, the enzyme is a fusion protein. In some aspects, the fusion protein comprises an enzyme one or more additional protein domains. In some aspects the one or more additional protein domains have one or more enzymatic activities. In some aspects, the one or more additional protein domains do not have enzymatic activity. In some aspects, the one or more additional protein domains have a binding activity useful for isolation of the fusion protein. In some aspects, the enzyme is useful for conversion of an industrial waste product to one or more other chemical species. In some aspects, the enzyme substrate is a sugar or polysaccharide. In some aspects, the enzyme substrate is a sugar. In some aspects, the enzyme substrate is a polysaccharide. In some aspects, the enzyme substrate is cellulose. In some aspects, the enzyme substrate is xylose. In some aspects, the enzyme substrate is a toxin. In some aspects, the enzyme product is a flavorant. In some aspects, the enzyme substrate is a nucleic acid. In some aspects, the enzyme substrate is a peptide, polypeptide, or protein. In some aspects, the enzyme substrate is a fatty acid. In some aspects, the enzyme substrate is a small molecule. In some aspects, the polypeptide comprises a secretion signal. In some aspects, the polypeptide comprises a cleavage peptide.

[0270] In some aspects, the target product is a polypeptide. In some aspects, the polypeptide is a signaling molecule. In some aspects, the polypeptide is a cytokine.

[0271] In some aspects, the polypeptide is a transmembrane protein. In some aspects, the transmembrane protein is a transporter. In some aspects, the transporter is an ion transport channel. In some aspects, the ion transport channel is a calcium transport channel, an iodine transport channel, or an iron transport channel. In some aspects, the transmembrane protein is an engineered transmembrane protein having an amino acid sequence not found in nature. In some aspects, the transmembrane protein is a fusion protein. In some aspects, the transmembrane protein is a chimeric antigen receptor.

[0272] In some aspects, the polypeptide is a therapeutic peptide. In some aspects, the polypeptide is an antimicrobial peptide. In some aspects, the polypeptide is an antiviral peptide. In some aspects, the polypeptide is a carrier peptide which aids in delivery of a cargo material into a target cell. In some aspects, the cargo material is polypeptide, a nucleic acid, a lipid, a small molecule, or a nanoparticle. In some aspects, the therapeutic peptide is suitable for administration to a domesticated animal. In some aspects, the therapeutic peptide is suitable for administration of a livestock species. In some aspects, the therapeutic peptide is suitable for administration to a human.

[0273] In some aspects, the polypeptide is an antibody. In some aspects, the antibody is an engineered antibody. In some aspects, the antibody has an IgG structure. In some aspects, the engineered antibody is multispecific. Various non-limiting antibodies, antibody formats, and applicable methods are referred to in U.S. published application Pub. No. US 2019/0202935 Al. US 2019/0202935 A1 is incorporated by reference in its entirety herein.

[0274] In some aspects, the target product is a polynucleotide. In some aspects, the polynucleotide is a DNA molecule. In some aspects, the DNA molecule is a plasmid. In some aspects, the DNA molecule is a construct disclosed herein. In some aspects, the DNA molecule is an expression cassette. In some aspects, the expression cassette encodes more than one gene. In some aspects, the polynucleotide is a linear DNA. In some aspects, the polynucleotide is a circular DNA. [0275] In some aspects, the polynucleotide is an RNA molecule. In some aspects, the

RNA molecule is a tRNA, pre-mRNA, mRNA, an interfering RNA, or a ribozyme.

[0276] In some aspects, the polynucleotide encodes a viral antigen. In some aspects, the viral antigen is the spike protein of SARS-CoV-2 or a variant or a portion thereof. A skilled artisan will appreciate that representative sequences of the spike protein are readily available (see, e.g., NCBI Reference Sequence NC_045512.2). In some aspects, the viral antigen shares at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity with NCBI Reference Sequence NC_045512.2.

[0277] In some aspects, the target product is a lipid. In some aspects, the lipid comprises a fatty acid disclosed herein.

[0278] In some aspects, the target product is a therapeutic drug product. In some aspects, the therapeutic drug product is an antibiotic. In some aspects, the therapeutic drug product is an antibiotic or an antimicrobial. In some aspects, the antibiotic is a penicillin. In some aspects, the antibiotic or the antimicrobial is for use in humans, domesticated animals, or wild animals. In some aspects, the antibiotic is a tetracycline. In some aspects, the antibiotic is a cephalosporin. In some aspects, the antibiotic is a fluoroquinolone. In some aspects, the antibiotic is lincomycin. In some aspects, the antibiotic is a sulfonamide. In some aspects, the antibiotic is a glycopeptide antibiotic. In some aspects, the antibiotic is an aminoglycoside. In some aspects, the antibiotic is a carbapenem. In some aspects, the antibiotic is amoxicillin. In some aspects, the antibiotic is doxycycline. In some aspects, the antibiotic is cephalexin. In some aspects, the antibiotic is ciprofloxacin. In some aspects, the antibiotic is clindamycin. In some aspects, the antibiotic is metronidazole. In some aspects, the antibiotic is azithromycin. In some aspects, the antibiotic is sulfamethoxazole. In some aspects, the antibiotic is trimethoprim. In some aspects, the antibiotic is clavulanate. In some aspects, the antibiotic is levofloxacin. In some aspects, the antibiotic is vancomycin.

[0279] In some aspects, an insect extract comprises the therapeutic drug product, and the insect extract is suitable for oral, nasal, rectal, topical, or parenteral administration. In some aspects, the insect extract is in solid or powder form. In some aspects, the insect extract is in liquid form. In some aspects, topical administration comprises administration to the skin. In some aspects, administration to the skin comprises dermal or transdermal administration. In some aspects, administration to the skin comprises introduction of the insect extract through a cut, scratch, wound, or injection. In some aspects, the nasal administration comprises inhalation of an aerosol.

[0280] In some aspects, an insect extract is a processed insect extract having the therapeutic drug product substantially isolated from undesirable or inert insect components. In some aspects, the therapeutic drug product is a target product disclosed herein. In some aspects, the drug product is substantially isolated from undesirable or inert insect components wherein the ratio of the drug product to an undesirable or inert insect component is increased by 5-50%, 50-100%, 200-300%, 300-500%, or 500- 1000%. In some aspects, the drug product is substantially isolated from undesirable or inert insect components wherein the ratio of the drug product to an undesirable or inert insect component is increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 400%, 500% 600%, 700%, 800%, 900%, or 1000%.

[0281] In some aspects, the target product is a metabolite. In some aspects, the metabolite is precursor, intermediate, or product of a target product disclosed herein. In some aspects, the target product is acetoacetyl CoA thiolase, adenosine 5 '-triphosphate, capsanthin-capsorubin synthase, 4-(cytidine 5'-diphospho)-2C-methyl-D-erythritol, 4- (cytidine 5'-diphospho)-2C-methyl-D-erythritol-2-phosphate, 4-(cytidine 5'-diphospho)- 2C-methyl-D-erythritol kinase, cytidine 5 '-monophosphate, bacterial -type phytoene desaturase, carotene isomerase, dehydrosqualene synthase, b-carotene ketolase, bacterial- type lycopene b-cyclase, cytidine 5 '-triphosphate, dimethylallyl diphosphate, 1-deoxy-D- xylulose-5-phosphate, l-deoxy-D-xylulose-5-phosphate reductoisom erase, 1-deoxy-D- xylulose-5-phosphate synthase, flavin adenine dinucleotide, reduced FAD (FADH· or FADH2), flavin mononucleotide, farnesyl diphosphate, farnesyl diphosphate synthase, glyceraldehyde 3 -phosphate, geranylgeranyl diphosphate, geranylgeranyl diphosphate synthase, l-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase, 1 -hydroxy -2- methyl-2-(E)-butenyl-4-diphosphate synthase, 1 -hydroxy-2-methyl-2-(E)-butenyl-4- diphosphate, 3 -hydroxy-3 -methylglutaryl-CoA, 3 -hydroxy-3 -methylglutaryl-CoA reductase, 3 -hydroxy-3 -methylglutaryl-CoA synthase, p-hydroxyphenylpyruvate, p- hydroxyphenylpyruvate dioxygenase, nonheme diiron oxidase, IPP-DMAPP isomerase type 1, IPP-DMAPP isomerase type 2, isopentenyl diphosphate, b-carotene ketolase, plant-type lycopene cyclase, lycopene b-cyclase, lycopene e-cyclase, 2C-methyl-D- erythritol-4-phosphate cytidyltransferase, 2C-methyl-D-erythritol-2, 4-cyclodiphosphate synthase, 2C-methyl-D-erythritol-2, 4-cyclodiphosphate, 2C-methyl-D-erythritol-4- phosphate, mevalonate kinase, mevalonate, mevalonate diphosphate decarboxylase, nicotinamide adenine dinucleotide phosphate, oxidized form, nicotinamide adenine dinucleotide phosphate, neoxanthin synthase, heme-binding cytochrome P450, plant-type phytoene desaturase, phosphomevalonate kinase, phytoene synthase, squalene synthase, violaxanthin de-epoxidase, z-carotene desaturase, 15-cis^-carotene-isomerase, or zeaxanthin epoxidase.

[0282] In some aspects, the target product is a silk. In some aspects, the silk is a spider silk. In some aspects, the silk is a moth silk.

[0283] In some aspects, the target product is collagen. In some aspects, the target product is gelatin.

[0284] In some aspects, the target product is administrable in a therapeutically effective amount by oral consumption of part or all of the genetically modified insect, sublingual administration of a formulation comprising the target product, rectal administration of a formulation comprising the target product, parenteral administration of a formulation comprising the target product, or inhalation of a formulation comprising the target product. In some aspects, inhalation of the formulation comprising the target product is nasal inhalation. In some aspects, inhalation of the formulation comprising the target product is oral inhalation.

[0285] In some aspects, the one or more genetic modifications confer a difference in reproduction between the genetically modified insect and the wild-type insect of the same species. In some aspects, the difference in reproduction is an increased number of offspring. In some aspects, the difference in reproduction is an increased fecundity. In some aspects, the difference in reproduction is a production of a higher amount of eggs.

In some aspects, the difference in reproduction is an increased rate of breeding.

[0286] In some aspects, the one or more genetic modifications confer a phenotypic difference between the genetically modified insect and the wild-type insect of the same species. [0287] In some aspects, the phenotypic difference is one or more of: an increase in survival to adulthood or a harvest stage, longer survival with less food or water, less consumption of food or water, a lowered stress response, or a lowered rate of infection by an insect pathogen. In some aspects, the lowered stress response is a lowered stress response in response to overcrowding of an insect habitat with other insects.

[0288] In some aspects, the phenotypic difference is selected from a difference in the time to progress through one or more stages of the life cycle, a difference in body weight, a difference in the ability to jump or fly, a difference in the presence of one or more anatomical structures, a difference in the color of one or more anatomical structures, a difference in the size of one or more anatomical structures, and a difference in survival rate following inoculation with a pathogen.

[0289] In some aspects, the phenotypic difference is a difference in the time to progress through one or more stages of the life cycle. In some aspects, the difference in the time to progress through one or more stages of the life cycle is a decreased amount of time to progress through one or more stages of the life cycle.

[0290] In some aspects, the phenotypic difference is a difference in body weight. In some aspects, the difference in body weight is an increased body weight. In some aspects, the difference in body weight is a decreased body weight.

[0291] In some aspects, the phenotypic difference is a difference in the ability to jump or fly. In some aspects, the difference in the ability to jump or fly is a reduced or eliminated ability to jump or fly. In some aspects, the difference in the ability to jump or fly is an increased ability to jump or fly.

[0292] In some aspects, the phenotypic difference is a difference in the presence of one or more anatomical structures. In some aspects, the one or more anatomical structures is an eye. In some aspects, the eye is a different color than the wild-type insect of the same species. In some aspects, the eye is white. In some aspects, the one or more anatomical structures is a wing. In some aspects, the wing is absent or modified in structure. In some aspects, the wing is increased in size. In some aspects, the one or more anatomical structures is an exoskeleton. In some aspects, the exoskeleton is a lighter color than the exoskeleton of a wild-type insect of the same species. In some aspects, the exoskeleton is a darker color than the exoskeleton of a wild-type insect of the same species. [0293] In some aspects, the phenotypic difference is a difference in survival rate following inoculation with a pathogen. In some aspects, the pathogen is a viral species. In some aspects, the viral species is selected from: a species from the Family Iridoviridae , a species from the Family Parvoviridae, and a species from the Family Baculoviridae . In some aspects, the species from the family Iridoviridae is cricket iridovirus (CrIV). In some aspects, the species from the Family Parvoviridae is a species from the subfamily Densovirinae . In some aspects, the species from the subfamily Densovirinae isAcheta domesticus Densovirus. In some aspects, the viral species is cricket paralysis virus (CrPV).

[0294] In some aspects, the pathogen is a bacterial species. In some aspects, the bacterial species is an insect pathogen. In some aspects, the bacterial species is a human pathogen. In some aspects, the bacterial species is a domesticated animal pathogen. In some aspects, the bacterial species is a livestock pathogen. In some aspects, the bacterial species is a non-domesticated animal pathogen.

[0295] In some aspects, the pathogen is a fungal species. In some aspects, the fungal species is an insect pathogen. In some aspects, the fungal species is a human pathogen. In some aspects, the fungal species is a domesticated animal pathogen. In some aspects, the fungal species is a livestock pathogen. In some aspects, the fungal species is a non- domesticated animal pathogen.

[0296] In some aspects, the present disclosure provides a genetically modified insect, wherein the genetically modified insect emits fluorescent, luminescent, or bioluminescent light. In some aspects, the genetically modified insect is suitable for a use in entertainment or educational purposes.

Feed Conversion Efficiency

[0297] In some aspects, the genetically modified insect is characterized by a difference relative to a wild-type insect of the same species having eaten the same amount and composition of an insect food as the genetically modified insect.

[0298] In some aspects, the difference is an increased body weight in the genetically modified insect. In some aspects, the difference is a decreased body weight in the genetically modified insect.

[0299] In some aspects, the difference is a decreased amount of time to progress through one or more stages of the life cycle, or a different number of stages of the life cycle, for the genetically modified insect. In some aspects, the difference is an increased amount of time to progress through one or more stages of the life cycle, or a different number of stages of the life cycle, for the genetically modified insect.

[0300] In some aspects, the difference is an increased rate of reproduction for the genetically modified insect. In some aspects, the difference is a decreased rate of reproduction for the genetically modified insect.In some aspects, the difference is decreased food consumption or water consumption by the genetically modified insect. In some aspects, the difference is increased food consumption or water consumption by the genetically modified insect.

Food Composition

[0301] In some aspects, the present disclosure provides a composition comprising a part or all of an insect disclosed herein, wherein the composition is suitable for consumption by humans, a companion animal species, or a livestock species. In some aspects, the present disclosure provides a composition comprising a part or all of a genetically modified insect disclosed herein, wherein the composition is suitable for consumption by humans, a companion animal species, or a livestock species.

[0302] In some aspects, the composition comprises an unground portion of the insect. In some aspects, the composition comprises a ground portion of the insect.

[0303] In some aspects, the composition is substantially free of chitin. In some aspects, the weight ratio of chitin to protein in the composition is reduced relative to the weight ratio of chitin to protein in the insect from which the composition is derived. In some aspects, the weight ratio of chitin to protein in the composition is reduced relative to the weight ratio of chitin to protein in the insect from which the composition is derived, wherein the composition and the insect are dehydrated. In some aspects, the reduction in weight ratio is by 10-90%, 20-80%, 30-70%, or 40-60%. In some aspects, the reduction in weight ratio is by 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0304] In some aspects, the composition comprises modified chitin. In some aspects, the modified chitin is characterized by being softer than chitin from a wild-type insect. In some aspects, the genetically modified insect produces chitin which is characterized by being softer than chitin from a wild-type insect, wherein the genetically modified insect which has an altered life cycle progression. In some aspects, the altered life cycle progression is characterized by a longer period of time in one or more life cycle stages. In some aspects, the altered life cycle progression is characterized by a reduced period of time in one or more life cycle stages.

[0305] In some aspects, the livestock species is a poultry species. In some aspects, the livestock species is a cattle species.

[0306] In some aspects, the composition is a food product. In some aspects, the food product is in the form of: a pasta, a tortilla, a protein crisp, a chip, a puffed extruded snack, duros, a pate, a butter alternative, a beverage, a shake, a cereal, or a powder. In some aspects, the cereal is an extruded cereal, a puffed cereal, a baked cereal, an O- shaped cereal, or a flake cereal. In some aspects, the food product is in the form of a cookie. In some aspects, the food product is in the form of a powder (e.g., insect "flour"). In some aspects, the food product is fortified in one or more nutrients.

[0307] In some aspects, the food product is defatted. In some aspects, the weight ratio of fat to protein in the composition is reduced relative to the weight ratio of fat to protein in the insect from which the composition is derived. In some aspects, the weight ratio of fat to protein in the composition is reduced relative to the weight ratio of fat to protein in the insect from which the composition is derived, wherein the composition and the insect are dehydrated. In some aspects, the reduction in weight ratio is by about 10% to about 90%, about 20% to about 80%, about 30% to about 70%, or about 40% to about 60%. In some aspects, the reduction in weight ratio is by 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0308] In some aspects, the food product comprises at least 1 gram of protein for every 3 grams of the food product. In some aspects, the food product comprises about 10% to about 90% protein by weight of the composition. In some aspects, the food product comprises about 0.5% to about 99.999%, about 5% to about 95%, about 10% to about 90%, about 20% to about 80%, about 30% to about 70%, or about 40% to about 60% protein by weight of the composition. In some aspects, the food product comprises about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 99.999% protein by weight of the composition.

[0309] In some aspects, the part of the insect is an insect extract. In some aspect, the insect extract is prepared in a method of insect extract preparation. In some aspects, the method of insect extract preparation comprises combining an at least partially ground insect with a suitable solvent. In some aspects, the method of insect extract preparation comprises combining an at least partially ground insect with carbon dioxide.

Method of Producing GMO Insect

[0310] In some aspects, introducing one or more genetic modifications in the wild-type insect comprises electroporation. In some aspects, the introducing one or more genetic modifications in the wild-type insect comprises lipofection. In some aspects, the introducing one or more genetic modifications in the wild-type insect comprises the use of a transposable element. In some aspects, the use of a transposable element comprises use of a transposase.

[0311] In some aspects, the step of introducing one or more genetic modifications in the wild-type insect comprises viral delivery of the polynucleotide into the first insect.

[0312] In some aspects, the step of introducing one or more genetic modifications in the wild-type insect comprises liposomal delivery of the polynucleotide into the first insect.

[0313] In some aspects, the insect is at a developmental stage selected from: embryo, egg, ootheca, larva, nymph, prepupa, pupa, adult, imago, cocoon, chrystalis, maggot, caterpillar, worm, or winged stage.

Polynucleotide for use in Producing GMO Insects

[0314] In some aspects, the present disclosure provides a polynucleotide suitable for use in genetic modification of an insect.

[0315] In some aspects, the insect is a cricket or a mealworm.

[0316] In some aspects, the polynucleotide is a DNA molecule or an RNA molecule.

[0317] In some aspects, the present disclosure provides a polynucleotide vector comprising a polynucleotide disclosed herein. [0318] In some aspects, the present disclosure provides a viral vector comprising a polynucleotide or polynucleotide vector disclosed herein. In some aspects, the viral vector is a Baculovirus vector.

[0319] In some aspects, the present disclosure provides a liposomal composition comprising a polynucleotide or polynucleotide vector disclosed herein.

Compositions with Carriers/Excipients

[0320] In some aspects, the present disclosure provides a composition comprising the target product produced by a genetically modified insect disclosed herein, and a pharmaceutically acceptable carrier or excipient. In some aspects, the composition is in the form of a capsule, tablet (e.g., a dispersible tablet), sprinkles, troche, sachet, injectable fluid, ointment, cream, emulsion, suspension, solution, slurry, food product, or powder. In some aspects, the composition is suitable for coadministration of one or more additional therapeutic agents.

Methods of Producing Products of Expression in GMO Insects

[0321] In some aspects, the present disclosure provides a method of producing the target product produced by a genetically modified insect disclosed herein, the method comprising isolating a fraction comprising the target product from the genetically modified insect. In some aspects, the method further comprises purifying the product from the fraction.

Method of Fishing

[0322] In some aspects, the present disclosure provides a fishing lure comprising a genetically modified insect, wherein the genetically modified insect emits fluorescent, luminescent, or bioluminescent light.

[0323] In some aspects, the genetically modified insect is a cricket or mealworm. In some aspects, the genetically modified insect is a genetically modified insect disclosed herein.

[0324] In some aspects, the present disclosure provides a method of fishing comprising baiting a hook with a fishing lure comprising a genetically modified insect disclosed herein. Insect Farming Apparatus

[0325] An automated insect farming apparatus comprising: a growth chamber; a nutrient delivery system; a water delivery system; a mechanism for removal of waste and/or one or more insects; a computerized system programmed to automate one or more of: nutrient delivery, water delivery, and insect removal; wherein the computerized system is operably linked to one or more of: the growth chamber, the nutrient delivery system, the water delivery system, and the mechanism for removal of waste and/or one or more insects.

[0326] In some aspects, the automated insect farming apparatus comprises a separating mechanism that can separate objects (e.g., insects or portions thereof) from one or more of other objects (e.g., insects of different mass or shape, insects of different life cycle stages, waste, food, or water). In some aspects, the separating mechanism comprises allowing the contents of a growth chamber disclosed herein to fall into a laterally directed stream of air or low pressure system (e.g., a vacuum), wherein a first fraction of the components are directed laterally into a first chamber and a second fraction of the components fall into a second chamber.

[0327] In some aspects, the separating mechanism comprises a mechanism which combines the contents of a growth chamber disclosed herein with a liquid, wherein a first fraction of the components float and a second fraction of the components does not float.

[0328] In some aspects, the mechanism for removal of waste and/or one or more insects is a forced air system. In some aspects, the forced air system is an air knife system.

[0329] In some aspects, the mechanism for removal of waste and/or one or more insects is a mechanism which shakes, vibrates, or otherwise moves. In some aspects, the mechanism for removal of waste and/or one or more insects is suitable to separate an insect from an unwanted material. In some aspects, the unwanted material is one or more of: insect waste, feed, insect at other life stages, and other materials (e.g., growth or crawling substrates).

[0330] In some aspects, the growth chamber comprises a growth matrix. In some aspects, the growth matrix comprises a polymer. In some aspects, the growth chamber comprises a cross hatched rod matrix. In some aspects, the growth chamber comprises a vertical plate matrix. [0331] In some aspects, the automated insect farming apparatus comprises a filtering or sifting mechanism suitable for separating insects from an unwanted material. In some aspects, the filtering or sifting mechanism comprises a mesh material (e.g., a screen or sheet of material with suitably sized holes).

[0332] In some aspects, the automated insect farming apparatus comprises an automated or mechanized system that collects one or more growth chambers and places them on a conveyor, wherein the conveyor transports the one or more growth chambers to a shaker, sifter or other insect separation device or system.

[0333] In some aspects, the automated insect farming apparatus comprises an automated or mechanized system that collects one or more growth chambers and places them directly into a separation device or system.

[0334] In some aspects, the automated insect farming apparatus comprises a mechanism which returns one or more growth chambers to a rack, wheeled dolly, stack of other growth chambers, or other location for re-use.

[0335] In some aspects, the automated insect farming apparatus comprises a mechanism that transports a used growth chamber to an automated or mechanized system or device which washes, air blows, cleans, cleanses, and/or sterilizes the growth chamber.

[0336] In some aspects, the growth chamber or the used growth chamber is a tray. In some aspects, the mechanism that transports a used growth chamber returns the tray to a rack or a stack of trays. In some aspects, a growth chamber is selected from the rack or the stack of trays, and one or more insects are added to the growth chamber. In some aspects, a growth chamber is selected from the rack or the stack of trays, and the growth chamber is subjected to a processing step. In some aspects, the processing step comprises a cleaning step. In some aspects, the cleaning step comprises one or more of a washing step, a drying step, and a sanitization step. In some aspects, the drying step comprises air drying. In some aspects, the drying step comprises directing air to the growth chamber. In some aspects, the sanitization step comprises directing to the growth chamber one or more of a solvent, a detergent, a surfactant, a detergent, an alcohol, an amount of air, an amount of ozone, an amount of ultraviolet light, or an amount of heat. In some aspects, the solvent is water. In some aspects, the detergent is sodium dodecyl sulfate. In some aspects, the alcohol is ethanol. [0337] In some aspects, the automated insect farming apparatus comprises a mechanism that harvests an insect.

[0338] In some aspects, the automated insect farming apparatus comprises a mechanism that freezes an insect. In some aspects, the automated insect farming apparatus comprises a mechanism that drops an insect onto a conveyor belt or air flow system which transports the insect to the mechanism that freezes the insect. In some aspects, the insect is flash frozen. In some aspects the mechanism that freezes the insect comprises a cryogen. In some aspect, the cryogen is carbon dioxide or liquid nitrogen. In some aspects, the mechanism that freezes the insect directs cold air to the insect.

[0339] In some aspects, the automated insect farming apparatus is mounted on device comprising wheels. In some aspects, the device comprising wheels is an automobile. In some aspects, the device comprising wheels is a trailer. In some aspects, the device comprising wheels comprises an electricity generator (e.g., a battery, a gasoline-powered device, a natural gas-powered device, or a hydrogen-powered device). In some aspects, the device comprising wheels is not powered by an engine or motor. In some aspects, the device comprising wheels is of a size suitable for one person to push the device without aid.

[0340] In some aspects, the automated insect farming apparatus can be operably coupled to an insect processing apparatus. In some aspects, the insect processing apparatus is a homogenizer, tank, steam jacketed kettle, colloid mill, comitrol, spray dryer, heat dryer, or freeze dryer.

[0341] In some aspects, the automated insect farming apparatus can be operably coupled to a device which blanches, heats, cooks, treats with carbon dioxide, or otherwise euthanizes and/or processes the insect.

[0342] In some aspects, the automated insect farming apparatus comprises a vacuum which moves insects and/or waste. In some aspects, the automated insect farming apparatus a vacuum or forced air system (e.g., an air knife) separates insects from waste and/or leftover feed materials.

[0343] In some aspects, the automated insect farming apparatus comprises a sieve, which is used to separate insects from waste and/or leftover feed materials. In some aspects, the sieve can be used to separate insects by development stage and/or size. [0344] In some aspects, the automated insect farming apparatus comprises a conveyor system (e.g., belt, vibratory) which moves insects, waste materials, or leftover feed.

[0345] In some aspects, the automated insect farming apparatus comprises a mechanism which uses centrifugal force to harvest insects (e.g., remove insects and/or waste and/or leftover feed from the growth chamber at the desired time/life stage). In some aspects, centrifugal force is used to separate insects from waste and/or leftover feed.

[0346] In some aspects, the automated insect farming apparatus comprises a robot which can transport insects and/or waste and/or leftover feed. In some aspects, the robot comprises a robotic arm that can transport an insect growth chamber into a system which can transport insects and/or waste and/or leftover feed. In some aspects, the robotic arm or robot can move or shake a container containing insects, to transfer them into another device. In some aspects, a robotic arm or robot can move or shakes a container of insects and/or waste materials and/or leftover feed in order to separate those materials from one another.

[0347] In some aspects, the automated insect farming apparatus comprises a mechanism by which insect eggs are harvested/separated from feed and/or waste materials via a sieve using gravity, vibration, forced air, water or fluid flow, and/or a vacuum.

[0348] In some aspects, the automated insect farming apparatus comprises insect growth chambers which can be configured to farm insects by adding insect eggs to feed materials.

[0349] In some aspects, the growth chamber is vibrated, shaken, or otherwise agitated to remove the insect or a waste product and/or leftover feed from the growth chamber.

[0350] In some aspects, the growth chamber can be dumped, tilted, or otherwise moved to allow insects and/or waste material and/or leftover feed to move out of the chamber into a downstream device. In some aspects, the downstream device can transport insects and/or waste and/or feed. In some aspects, the downstream device separates insects from waste and/or leftover feed. In some aspects, the downstream device dries insects. In some aspects, the downstream device dries a waste product. In some aspects, the downstream device can vibrate. In some aspects, the downstream device comprises a sieve and/or screen/mesh. In some aspects, the downstream device comprises a mechanism which directs forced air. In some aspects, the downstream device comprises a mechanism which creates a low pressure environment (e.g., a vacuum). In some aspects, the downstream device comprises a downstream freezer that can freeze insects. In some aspects, the downstream device comprises a conveyor system that can transport and freeze insects. In some aspects, the downstream freezer uses a conveyor belt. In some aspects, the downstream freezer comprises a mechanism which directs forced air or creates a low pressure environment (e.g., a vacuum). In some aspects, the downstream freezer can vibrate and move insects contained therein.

[0351] In some aspects, the automated insect farming apparatus comprises a combined system that can transport insects, waste, and/or feed using a low pressure environment (e.g., a vacuum) or forced air, onto a device which separates insects from feed and waste via a sieve or vibration. In some aspects, the combined system can use forced air, a low pressure system (e.g., a vacuum), a conveyor belt, or vibration to transport insects onto a conveyor belt.

[0352] In some aspects, the combined system further deposits insects onto a conveyor belt or conveyor system which moves them into a freezer. In some aspects, the freezer is suitable for freezing one or more insects. In some aspects, the freezer is an individual quick freezing (IQF) blast/flash freezer. In some aspects, the freezer can use cryogens to accomplish freezing.

[0353] In some aspects, the automated insect farming apparatus comprises a mechanism by which water is provided to the insect through gravity or through wicking action using a porous material such as ceramics, rope, polyacrylamide, sponges or other similar material.

[0354] In some aspects, water is provided to the insect from above the insect.

[0355] In some aspects, the growth chamber is a matrix of hanging chains, ropes or other stranded materials. In some aspects, the growth chamber is a set of trays.

[0356] In some aspects, the automated insect farming apparatus comprises an air knife system which can remove insects, waste, and/or feed from the growth chamber. In some aspects, vibration is used to remove insects, waste, and/or feed from the growth chamber. In some aspects, a low pressure system (e.g., a vacuum) is used to remove insects, waste, and/or feed from the growth chamber.

[0357] In some aspects, the automated insect farming apparatus can be configured to direct air or a low pressure system (e.g., a vacuum) to separate insects by size or stage of the life cycle. [0358] In some aspects, the automated insect farming apparatus comprises a hammering mechanism, which can be used on the growth chamber to remove insects and/or waste and/or feed from the growth chamber.

[0359] In some aspects, the automated insect farming apparatus comprises a mechanism by which light is used to remove insects, waste, and/or feed from the growth chamber. In some aspects, light is used to herd insects and/or encourage their movement to the desired area of the growth chamber, into the growth chamber, out of the growth chamber and/or into or out of a downstream device.

[0360] In some aspects, the automated insect farming apparatus comprises a mechanism by which cold air is used to immobilize insects.

[0361] In some aspects, the automated insect farming apparatus comprises a mechanism by which vibrational forces can be used to immobilize insects.

[0362] In some aspects, the automated insect farming apparatus comprises a mechanism by which carbon dioxide can be used to immobilize insects. In some aspects, the automated insect farming apparatus comprises a mechanism by which carbon dioxide can be used to euthanize insects.

[0363] In some aspects, the automated insect farming apparatus comprises a mechanism by which electric shock can be used to immobilize insects. In some aspects, the automated insect farming apparatus comprises a mechanism by which electric shock can be used to euthanize insects.

[0364] In some aspects, the automated insect farming apparatus comprises a mechanism by which heat can be used to immobilize insects. In some aspects, the automated insect farming apparatus comprises a mechanism by which heat can be used to euthanize insects.

[0365] In some aspects, the automated insect farming apparatus comprises a mechanism by which heat can be used to euthanize insects and kill an unwanted organism. In some aspects, the unwanted organism is present in or on an insect. In some aspects, the unwanted organism is a pathogen. In some aspects, the unwanted organism is a virus, fungus, bacterium, nematode, or arthropod.

[0366] In some aspects, the automated insect farming apparatus comprises a mechanism by which heat can be used to remove insects, waste, and/or feed from the growth chamber. [0367] In some aspects, the automated insect farming apparatus comprises a mechanism by which forced air can be used to remove insects, waste, and/or feed from the growth chamber. In some aspects the mechanism by which forced air can be used comprises a mechanism to direct compressed air.

[0368] In some aspects, the automated insect farming apparatus comprises a mechanism by which insect eggs can be deposited into the growth chamber. In some aspects, the automated insect farming apparatus comprises a mechanism by which small nymphs can be deposited into the growth chamber. In some aspects, the automated insect farming apparatus comprises a mechanism by which small larvae can be deposited into the growth chamber.

[0369] In some aspects, the growth chamber system can be configured such that no further feed, water, or human interaction with the interior of the growth chamber is needed for an entire insect growth cycle.

[0370] In some aspects, the automated insect apparatus comprises a mechanism by which an insect has access to an insect feed. In some aspects, the insect feed comprises an agricultural or food industry byproduct. In some aspects, the insect feed comprises a plant material. In some aspects, the plant material is not a human food product. In some aspects, the insect feed comprises algae. In some aspects, the insect feed comprises a bacterial species or a component of a bacterial species. In some aspects, the insect fee comprises a yeast or other fungal species, or a component of a yeast or other fungal species.

Method of Farming

[0371] In some aspects, the present disclosure provides a method of insect farming comprising providing an insect, providing a composition of one or more nutrients to the insect, and harvesting the insect at a desired stage of development, wherein one or more steps is automated.

[0372] In some aspects, the insect is a wild-type insect or a genetically modified insect. In some aspects, the genetically modified insect is a genetically modified insect disclosed herein.

[0373] In some aspects, the insect is provided in a growth chamber. In some aspects, the insect cannot escape from the growth chamber. [0374] In some aspects, a forced air system removes the insect or a waste product from the growth chamber. In some aspects, the growth chamber is vibrated, shaken, or otherwise agitated to remove the insect or a waste product from the growth chamber.

[0375] In some aspects, one or more nutrients are provided to the insect by an automated system. In some aspects, one or more nutrients are provided to the insect by a passive system.

[0376] In some aspects, water is provided to the insect by an automated system. In some aspects, water is provided to the insect by a passive system.

[0377] In some aspects, the passive system comprises a wicking material along which water can passively travel. In some aspects, the wicking material comprises nylon or a ceramic. In some aspects, the wicking material comprises nylon. In some aspects, the wicking material comprises a ceramic.

[0378] In some aspects, the insect produces a metabolic waste product, and wherein a conveyor belt carries some or all of the metabolic waste product away from the insect.

Products Made from Insects

[0379] In some aspects, the present disclosure provides a composition comprising a product isolated from an insect. In some aspects, the insect is a wild-type insect or a genetically modified insect disclosed herein. In some aspects, the insect is a wild-type insect. In some aspects, the product is suitable for use in a food product, a beverage product, a pet food product, a pet treat or snack, or an animal feed. In some aspects, the animal feed is suitable for administration to an aquatic species, a chicken, a pig, a cow, or a sheep. In some aspects, the food product is a food ingredient. In some aspects, the food ingredient is a composition comprising a fat, an oil, a protein, or another nutrient. In some aspects, the food product is an insect powder (e.g., an insect "flour"). In some aspects, the aquatic species is a fish.

[0380] In some aspects, the product is a target product disclosed herein. In some aspects, the product is a vaccine antigen, a vaccine, a drug, an antibiotic, an antimicrobial, an enzyme, a functional peptide, a functional protein, a food additive, an antibody, a nutritional additive, a feed additive, a color pigment, a flavorant, a perfume or aroma compound, an oil, a fatty acid, a biomaterial, a spider silk, or a moth silk.

[0381] In some aspects, the product isolated from an insect can be administered at a therapeutically effective amount to a subject in need thereof, in a method of treating, preventing, or treating a sign or symptom of one or more of: a nutrient deficiency, a cancer, an infection, an autoimmune disease, and a wound.

[0382] In some aspects, the product isolated from an insect can be used to prepare a medicament for the treatment, prevention, or treatment of a sign or symptom of one or more of: a nutrient deficiency, a cancer, an infection, an autoimmune disease, and a wound. In some aspects, the product isolated from an insect can be used to prepare a medicament for the treatment, prevention, or treatment of a sign or symptom of one or more of: type I diabetes mellitus, type II diabetes mellitus, a skin condition, a sexual dysfunction, a cognitive impairment, and a digestive disease or condition. In some aspects, the sexual dysfunction is erectile dysfunction. In some aspects, the digestive disease or condition is Crohn’s disease.

[0383] In some aspects, the product isolated from an insect can be used to prepare a medicament for an improvement of athletic performance.

[0384] In some aspects, the nutrient deficiency is a vitamin deficiency. In some aspects, the vitamin deficiency is a Vitamin A deficiency. In some aspects, the subject has xerophthalmia.

Methods of Treatment

[0385] In some aspects, the present disclosure provides a method of treating or preventing a disease, condition, or sign or symptom thereof, the method comprising administering to a therapeutically amount of a composition to a subject in need thereof. In some aspects, the disease, condition, or sign or symptom thereof is a vitamin deficiency. In some aspects, the vitamin deficiency is a Vitamin A deficiency. In some aspects, the disease, condition, or sign or symptom thereof is xerophthalmia.

Reduced Allergencity

[0386] In some aspects, a genetically modified insect disclosed herein comprises one or more genetic alterations, wherein the genetically modified insect produces less or none of an allergen, or wherein the insect produces an allergen which elicits a reduced allergic response in a human, a domesticated animal, a livestock animal, or a wild animal.

[0387] In some aspects, the allergen is tropomyosin. [0388] In some aspects, the allergen which elicits a reduced allergic response is a modified tropomyosin. In some aspects, the modified tropomyosin is an engineered tropomyosin.

[0389] In some aspects, consumption of the genetically modified insect results in less of an IgE response in a human relative consumption of the same amount, by mass, of the wild-type species from which the genetically modified insect is derived.

Cell lines

[0390] In some aspects, the present disclosure provides an insect cell line, wherein the insect cell line comprises a genetic modification disclosed herein. In some aspects, the insect cell line is a cricket, black soldier fly, or mealworm cell line.

[0391] United States patent literature citations referred to herein (i.e., U.S. patents and patent applications) are incorporated by reference in their entirety. The present specification incorporates by reference in its entirety the disclosure of United States patent application number 14/537,960 ("the '960 application"), filed November 11, 2014. In some aspects, a composition disclosed herein is prepared according to one or more embodiments explicitly or implicitly disclosed in the '960 application. In some aspects, a composition disclosed herein is characterized by one or more features explicitly or implicitly disclosed in the '960 application.

[0392] It is to be understood that headers are provided solely for ease of reading, and are not intended to be limiting. Aspects disclosed under one or more headers can be applicable to or combinable with aspects disclosed under one or more other headers.

[0393] It is further noted that the claims may be drafted to exclude any optional element.

As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely", "only" and the like in connection with the recitation of claim elements, or the use of a "negative" limitation.

[0394] The following examples are illustrative and do not limit the scope of the claimed aspects. EXAMPLES

Example 1. Acheta domesticus knock-out of vermilion gene

[0395] Acheta domesticus gene knock-out was accomplished using a CRISPR/Cas9 system targeting the vermilion gene at Exon 2. 100% of the 16 GO crosses attempted with 3 sgRNA resulted in successful knock-out and a white-eye phenotype (FIG. IB). 90% of the 10 GO crosses attempted with 1 sgRNA resulted in successful knock-out and a white- eye phenotype. For comparison, wild-type Acheta domesticus has a black eye phenotype (FIG. 1A).

Example 2. Knock-in construct for Acheta domesticus

[0396] A construct for knock-out of the Acheta domesticus gene vermilion and incorporation of an EGFP gene is conceptually represented in FIG. 2.

[0397] Successful gene knock-in was observed by an increase in green fluorescence (FIG.

3B) relative to wild-type Acheta domesticus (FIG. 3 A).

Example 3. Tenebrio molitor knock-out of vermilion gene

[0398] Tenebrio molitor gene knock-out was accomplished using a CRISPR/Cas9 system targeting the vermilion gene at Exon 2. 54% of the 13 E+G0 crosses attempted with 3 sgRNA resulted in successful knock-out and a white-eye phenotype. The 13 E+G0 crosses were crosses of at least one positive GO injectee with at least one other cricket (wild type, another GO of the same injectee group, or other). Individual sgRNA attempts ranged in successful knock-out and a white-eye phenotype in from 30% to 47% of the 10 GO crosses attempted. A no-sgRNA control resulted in no knock-out.

Example 4. Knock-in construct for Tenebrio molitor

[0399] A construct for knock-out of the Tenebrio molitor gene vermilion and incorporation of an EGFP gene is conceptually represented in FIG. 4:

[0400] Successful gene knock-in was observed by an increase in green fluorescence relative to wild-type Tenebrio molitor (FIG. 5). Example 5. Dual knock-in of EGFP and GeneA in Tenebrio molitor

[0401] A dual knock-in construct for incorporation of EGFP and a gene designated

"GeneA" was prepared and is conceptually represented in FIG. 6.

[0402] Successful gene knock-in was observed by an increase in green fluorescence relative to wild-type Tenebrio molitor (FIG. 7) as well as the presence of GeneA expression products as measured by standard molecular biology techniques.

Example 6. Dual knock-in constructs for Tenebrio molitor

[0403] Dual knock-in constructs for incorporation of EGFP and either DsRed or GeneA with a secretion signal were prepared and are conceptually represented in FIG. 8.

[0404] Successful gene knock-in was observed in both cases by an increase in green fluorescence, and an increase in red fluorescence for the construct encoding DsRed (FIG. 9). Secreted EGFP resulted in higher fluorescence.

Example 7. Insect Farming - Crickets

[0405] As a non-limiting example, insects are farmed according to the following protocol, which presents a weekly routine for cricket farming. This protocol has been used with crickets farmed at Armstrong’s Cricket Farm of West Monroe, LA, using LONESTAR^® cricket feed manufactured by TFP Nutrition of Nacogdoches, TX. A skilled artisan will understand that alterations of this protocol are permitted, such as scheduling events at different times than are exemplified, or using insects other than crickets.

Monday, Wednesday, and Friday: Cricket Colony

[0406] Moisten cricket eggs in a petri dish containing a water crystal (moisture wicking polymer such polyacrylamide) and tissue paper.

[0407] After 10-15 minutes, check all egg cups for young crickets hatch and add water to the tissue paper if necessary to maintain moisture.

[0408] Transfer the young crickets from egg cups to each cage every Monday,

Wednesday, and Friday. To start a new cage, add a 1/4 egg carton, one feed dish (small petri dish with feed) and one water dish (water crystal in small petri dish).

[0409] Provide feed (or refill the small petri dish to full) and water to water dish to each cricket cage Monday, Wednesday, and Friday. [0410] Check the cage every Monday and if there are around 30-50 young crickets, stop the egg-lay colony and transfer the egg-lay colony to “back up” section.

[0411] Once the cage start showing adults, transfer the cage to “egg-lay section” and adding the egg-lay dish (150mm* 10mm petri dish fill with water crystal).

[0412] Egg-lay dish are collected from adult cages and a new egg-lay dish will replace the old one in each egg-lay cage on Monday and Friday.

Tuesday: Egg Wash

[0413] Add some water in the egg-lay dish and pour the contents of the egg-lay dish in a cup (16 oz). Add more water to wash off the eggs in the dish if there are eggs remaining and pour in the cup.

[0414] Add water in the cup to more than half cup and use a plastic pipette and stir the water.

[0415] Wait for 10-15 second and eggs will sink to the bottom. Dump away the water along with water crystals slowly to avoid loss eggs from the cup.

[0416] Repeat the wash for at least 2 more times or until the water is clear with eggs.

[0417] Transfer cleaned eggs to a piece of wet paper towel on a petri dish lid by plastic pipette.

[0418] Put the eggs in a 8 oz Deli container (e.g., model S-21216 from Uline, Pleasant

Prairie, WI) with a lid until hatch.

Monday, Wednesday, and Friday: Phenotypic Cricket Screening

[0419] For this part of the present example, crickets modified to express green fluorescent protein are screened for green fluorescence. A skilled artisan will understand that other phenotypic or even genetic markers can be used to screen successful genetic modification of a target insect. As a non-limiting example, alternatives like GFP, dsRED, and luciferase can also be used.

[0420] Transgenic cricket colonies EGFP are screened when hatched.

[0421] Put a cup (16 oz) on the top of a flat ice pack, transfer all hatched cricket from one strain into the cup and wait at least 30 second or after all crickets are knocking down.

[0422] Turn on the fluorescent light source and wait for it to warm up, turn on “shutter” and light will transfer to scope. [0423] Transfer all cricket to the black dish and place it under the fluorescent scope

(suggest use a flat ice pack as well) and use the “GFP LP” filter.

[0424] Check all crickets and only save crickets that show strong GFP expression (strong green color, can see muscle green in the abdomen) and white eye phenotype.

[0425] Put crickets exhibiting green fluorescence (indicating successful genetic modification) into their own colonies and freeze the unwanted crickets.

Example 8: Cricket Egg Microinjection to Prepare Genetically Modified Cricket via CRISPR Technology

[0426] Set up egg-lay in wild-type ("WT") cricket colony at around 10 AM for four hours to receive fresh and non-developed eggs.

[0427] Wash the eggs as described above (a regular egg wash), but do not transfer eggs.

[0428] After washing the eggs (as regular egg wash), use 70% ethanol to briefly clean the eggs for 10-15 seconds and rinse with water for at least 3 times.

[0429] To prepare the microinjection slide, cut black filter paper to 50* 10mm and lay it on a clean glass slide.

[0430] Cut another two pieces of black filter paper strips 50*2mm and lay one on the top of the other, then place in the middle of the larger piece of black filter paper.

[0431] Added some water to the filter paper until all area of the paper is wet and water is visible on the edge of the filter paper.

[0432] Use a plastic pipette to transfer some eggs (10-20 eggs) to a petri dish with some water and use a brush to transfer eggs individually to the microinjection slides.

[0433] Lay the eggs on the edge of two sides of the filter paper stripes for microinjection.

[0434] The final concentration of Knock-out or DNA knock-in microinjection solution is

100 ng/ul of DNA construct, 10 pmol/ul of sgRNA, 1 mg/ul of TrueCut Cas9 protein V2 (ThermoFisher), and 20% Phenol red buffer (Sigma Aldrich).

[0435] After mixing the solution, leave it at room temperature for 5-10 minutes as the

Cas9-sgRNA structure is formed. Then keep the solution in the ice for the microinjection.

[0436] The needle for microinjection is pulled by a P-2000 needle puller (Sutter instrument). The setting is: Heat: 335 Fil: 4 Vel: 40 Del: 240 Pul: 120.

[0437] Load 1-4 ul of microinjection solution into the back of needle with Femtotips

(Eppendorf) and make sure there is no bubble in the needle tips. [0438] Apply the needle to a FemtoJet 5247 microinjector (Eppendorf) and use the pressure 4-7 psi for holding and 9-10 psi for injection.

[0439] Inject the eggs in the two third area of the eggs from the smaller end. A red dot will be visible inside the eggs if the microinjection is successful. Add more water to the black filter paper if the eggs start getting dry during the injection process.

[0440] After injection, transfer the black filer paper from glass slides to a piece of wet paper towel and keep in a 150*10 mm petri dish.

[0441] Place the dish in a hatch chamber (Modular incubator chamber, Billups-

Rothenberg) with two wet paper towels in it for 10-12 days.

[0442] Check for cricket hatch start on Day 9. Screen and transfer hatched crickets to their colonies.

Example 9: Young Cricket Microinjection: (RNAi)

[0443] Place young (ping head) crickets in ice to knock them down.

[0444] To prepare the microinjection stage, cut black filter paper to 50* 10mm and place it on an ice pack.

[0445] Cut another 3-4 pieces of black filter paper strips 10*5mm and lay one on the top of the other, then place in the middle of the larger piece black filter paper.

[0446] Add some water to the filter paper to hold the filter paper strips in place.

[0447] Transfer one cricket at a time to the side of filter paper strips for microinjection.

[0448] Set up needle (needle and injector set up are same as embryo injection).

[0449] Inject the cricket with 2/3 of the cricket abdomen. Hold the injection until a large area of the cricket abdomen shows red color.

[0450] Transfer the injected cricket to a separate container and keep until phenotype screening.

Example 10: Insect Farming - Mealworms Mealworm Colony: (Tuesday)

[0451] As a non-limiting example, mealworms are farmed according to the following protocol, which presents a weekly routine for mealworm farming. This protocol has been used with mealworms, using wheat as food for larvae and adults, and whole wheat flour for egg-laying adults. A skilled artisan will understand that alterations of this protocol are permitted, such as scheduling events at different times than are exemplified, or using insects other than mealworms.

[0452] Place mealworm pupae sieve on a #5 sieve and place on the top of a #25 sieve on the collecting pan.

[0453] Transfer everything from one larvae box (identify if the box is ready for collecting pupae, usually most larvae are full grown size) at a time to the sieves and use the shaker to shake for 30 seconds.

[0454] Collect all pupae on the pupae sieve to pupae container (8 oz cup).

[0455] Transfer everything from #5 and #25 sieves back to the larvae box.

[0456] Discard the waste from the collecting pan and repeat the sieving process described above with another larvae box.

[0457] If the larvae are not full-grown size, check if they need more wheat bran or are housed with too much waste (and sift out waste). If the larvae are too small (smaller than #25 sieve), don’t sift.

[0458] Once all larvae boxes have been checked, place the pupae container into the pupae/adult container and use tissue paper to create a bridge to the pupae container for new emerged adults to move out.

[0459] After accumulating around 500 adults, transfer them into another container with whole wheat flour to set up adult egg-lay box.

[0460] On the top of the whole wheat flour, place two large pieces of tissue paper to cover most of the surface area on the flour, and add a dish with some water crystals (moisture wicking polymer material).

[0461] Sift out eggs from egg-lay box once a week by using the #5 sieve on top of #25 sieve. All adults will be collected by #5 sieve. Material collected by #25 sieve will contain all eggs. Flour will be collected by collecting pan.

[0462] Place flour and adults back to the egg-lay box and set it up as described above with two large pieces of tissue paper covering most of the flour surface area and a dish with water crystals added. Example 11: Microinjection to Prepare Genetically Modified Mealworm via CRISPR Technology

[0463] Sift out eggs from egg-lay box as described above in Example 8 before 10 AM then set and place the egg-lay box in incubator for 4 hours. Eggs from this sift are not be used for microinjection.

[0464] Sift out eggs from egg-lay box and use those eggs for microinjection.

[0465] Else a forceps to remove the large unwanted pieces (dead body, wheat bran, dry water crystals) from the eggs.

[0466] Transfer around 50-100 eggs into a FISHERBRAND™ Sterile Cell Strainer filter having a mesh size of 100 pm (could do more eggs if needed).

[0467] Place the filter in a dish and use 2.5% bleach (2.5 ml bleach from the bottle + 97.5 ml deoinized water) to soak and wash the eggs for 1-1.5 minutes. Make sure the bleach passes through the filter many times to rinse off the flour from eggs.

[0468] Use water to rinse away the bleach from the filter and dish at least 3 times.

[0469] Remove most of the water from this dish and use it to bring the filter with eggs to a microscope.

[0470] Place a glass cover slide in a petri dish lid and put the filter next the slide, to aid in transfer of the eggs from the filter to the slide.

[0471] Use a fine brush to transfer eggs from the filter to the edge of the glass cover slide and lay them next to each other. Try facing the small end of the eggs outside the slide.

[0472] Try to not bring the flour chunk or wheat bran with eggs to the slide.

[0473] Keep a little water under each egg and let it air dry to stick the eggs on the slide.

[0474] Don’t use any eggs showing transparency in part of the egg, or deformed eggs.

[0475] Only lay the eggs on 2 sides of the slide with the small end of the eggs facing out.

[0476] If the needle is not ready, place cover slides in a petri dish and set up the needle.

[0477] The final concentration of Knock-out or DNA knock-in microinjection solution is

100 ng/ul of DNA construct, 10 pmol/ul of sgRNA, 1 mg/ul of TureCut Cas9 protein V2 (ThermoFisher), and 20% Phenol red buffer (Sigma Aldrich).

[0478] After mixing the solution, let it set on room temperature for 5-10 minutes for

Cas9-sgRNA structure formed. Then keep the solution in the ice for the microinjection.

[0479] The needle for microinjection was pulled by P-2000 needle puller (Sutter instrument). The setting is: Heat: 350 Fil: 4 Vel: 50 Del: 255 Pul: 120. [0480] Load 1-4 ul of microinjection solution into the back of needle with Femtotips

(Eppendorf) and make sure there is no bubble in the needle tips.

[0481] Apply the needle to the FemtoJet 5247 microinjector (Eppendorf) and use the pressure 4-7 psi for holding and 9-10 psi for injection.

[0482] Inject the eggs from the smaller end. A red dot will be visible inside the eggs if the microinjection is successful.

[0483] After finish injecting all eggs from the side, turn the glass cover slide to the other side and repeat the microinjection.

[0484] After injection, transfer the glass cover slides to a 150*10 mm petri dish.

[0485] Place the dish in a hatch chamber (Modular incubator chamber, Billups-

Rothenberg) with two wet paper towels in it for 7-10 days.

[0486] Check for larvae hatch start on Day 5. And screen/transfer hatched larvae to their colonies.

Example 12: Transgenic Mealworm Phenotypic Screening: (Tuesday or Wednesday)

[0487] Using a light microscope, screen all larvae and eggs from petri dish from small pair group:

COV2 strains: Screen for EGFP (filter: GFP LP), save good expressor as +/+, not good expressor as +/-. If have good number of +/+ (20 up per week), don’t have to save +/-. R-G strains: Screen for EGFP and dsRNA (Filter: ET DSR), save good expressers for both EGFP and dsRNA

V strains: check larvae eye color, only save no eye color individuals.

[0488] Turn the fluorescent light off.

[0489] Place the larvae to their own colony cups (16 oz).

[0490] Check each small pair colony’s full size larvae cup or pupae cup for pupae and adults. Separate pupae to their pupae cup (8 oz) and adults to the small pair egg-lay cup (16 oz).

[0491] If there is no small pair egg-lay cup for the colony yet, set up one by adding 1 inch deep flour into a 16 oz cup with 2 pieces of tissue paper on the top. Add a small dish lid with 2-3 pieces of water crystals in it to the cup. Then add adults in the cup.

[0492] Sift out eggs from each small pair egg-lay cup and transfer each egg to their own petri dish. Set the egg-lay cups back and add more water crystals.

[0493] Keep the eggs until next week for screening. Example 13: Transgenic Mealworm colonies: (Monday)

[0494] Initially, check each colony and determine the colony condition to 3 types:

I: Larvae growing stage: good number of larvae in the colony and all insect still larvae and cannot find any pupae/adult yet.

II: Adult egg-lay stage: good number of insects in the colony and find at least 2 pupae/adults, set up an egg-lay cup inside the colony box.

Ill: Pay attention colony: Cannot find much insect in the colony. Set up an egg-lay cup inside the colony box once with any adult or pupae found.

[0495] Check each II and III colony. For colonies with adults, add water to the egg-lay cup and replace water crystal once a week. Sift the eggs out and transferring them into the colony once a month (first week of each month).

[0496] Check I colonies for adults/pupae first week of each month and move to II once they have pupae/adults.

[0497] Ideally every 2-3 generations, check for desired phenotypes (white eye (V strains), white eye with highly expressed EGFP (V-EGFP), COV2 (which expresses receptor binding domain of SARS-CoV-2), OVA (which expresses hen ovalbumin), white with highly expressed EGFP and dsRNA (Red-Green strains containing a fusion of ds-Red and EGFP with a T2A cleavage peptide between them)) on pupae and re-set the colonies.

Claims

WHAT IS CLAIMED IS:

1. A genetically modified insect comprising one or more genetic modifications relative to a wild-type insect of the same species, wherein the wild-type insect of the same species is selected from a superworm, a cricket, a mealworm, a Caribbean fruit fly, a black soldier fly, a grasshopper, a locust, a maggot, a beetle, a pupa, a buffalo worm, a moth, a butterfly, a katydid, a silkworm, or a caterpillar.

2. The genetically modified insect of claim 1, wherein the one or more genetic modifications effect a phenotype detectable by visual inspection.

3. The genetically modified insect of claim 1 or claim 2, wherein the genetically modified insect is a cricket or a mealworm.

4. The genetically modified insect of claim 3, wherein the genetically modified insect is a cricket.

5. The genetically modified insect of claim 3 or claim 4, wherein the cricket is of the species Acheta domesticus.

6. The genetically modified insect of claim 3, wherein the genetically modified insect is a mealworm.

7. The genetically modified insect of claim 3 or claim 6, wherein the mealworm is of the species Tenebrio molitor.

8. The genetically modified insect of any one of claims 1-7, wherein the one or more genetic modifications consist of two genetic modifications.

9. The genetically modified insect of any one of claims 1-7, wherein the one or more genetic modifications consist of three genetic modifications.

10. The genetically modified insect of any one of claims 1-7, wherein the one or more genetic modifications consist of four genetic modifications.

11. The genetically modified insect of any one of claims 1-7, wherein the one or more genetic modifications consist of five genetic modifications.

12. The genetically modified insect of any one of claims 1-7, wherein the one or more genetic modifications consist of six or more genetic modifications.

13. The genetically modified insect of claim 12, wherein the one or more genetic modifications consist of six to ten genetic modifications.

14. The genetically modified insect of any one of claims 1-13, wherein the one or more genetic modifications are introduced by a CRISPR/Cas9 system.

15. The genetically modified insect of any one of claims 1-14, wherein the one or more genetic modifications comprise an indel mutation.

16. The genetically modified insect of any one of claims 1-15, wherein the one or more genetic modifications comprises a gene knock-out.

17. The genetically modified insect of any one of claim 1-16, wherein the one or more genetic modifications comprise knock-out of two or more genes.

18. The genetically modified insect of claim 16 or claim 17, wherein the gene knock-out is a knock-out of a vermilion gene.

19. The genetically modified insect of any one of claims 16-18, wherein the gene knock-out is introduced by a CRISPR/Cas9 system using a construct encoding a polynucleotide comprising a guide RNA sequence complementary to a portion of a vermilion gene.

20. The genetically modified insect of any one of claims 1-19, wherein the one or more genetic modifications comprises a gene knock-in.

21. The genetically modified insect of claim 20, wherein the gene knock-in is introduced by a CRISPR/Cas9 system using a construct encoding a polynucleotide comprising a guide RNA sequence complementary to a portion of a vermilion gene.

22. The genetically modified insect of claim 19 or claim 21, wherein the portion of a vermilion gene comprises exon 2 or a portion of exon 2.

23. The genetically modified insect of any one of claims 1-22, wherein the one or more genetic modifications alter one or more genes associated with a recessive phenotype.

24. The genetically modified insect of any one of claims 1-23, wherein the one or more genetic modifications alter one or more genes associated with a dominant phenotype.

25. The genetically modified insect of any one of claims 1-24, wherein the one or more genetic modifications comprises genomic incorporation of a transgene.

26. The genetically modified insect of claim 25, wherein the transgene is incorporated at the locus of a vermilion gene.

27. The genetically modified insect of claim 26, wherein the transgene encodes a fluorescent protein.

28. The genetically modified insect of claim 27, wherein the fluorescent protein is a green fluorescent protein.

29. The genetically modified insect of claim 28, wherein the green fluorescent protein is EGFP.

30. The genetically modified insect of claim 28, wherein the fluorescent protein is a red fluorescent protein.

31. The genetically modified insect of claim 30, wherein the red fluorescent protein is DsRed.

32. The genetically modified insect of any one of claims 2-31, wherein the phenotype detectable by visual inspection is detectable by visual inspection only after illuminating the genetically modified insect with light of one or more wavelengths corresponding to one or more excitation wavelengths characteristic of the fluorescent protein.

33. The genetically modified insect of any one of claims 1-32, wherein the one or more genetic modifications comprise an alteration in an eye color gene.

34. The genetically modified insect of any one of claims 1-33, wherein the eye color gene is a vermilion gene.

35. The genetically modified insect of any one of claims 1-34, wherein the genetically modified insect is a cricket which expresses a fluorescent protein and has a white eye phenotype.

36. The genetically modified insect of any one of claims 1-35, wherein the genetically modified insect is a mealworm which expresses a fluorescent protein and has a white eye phenotype.

37. A molecular construct comprising a sequence encoding, in the 5' to 3' direction: a first guide RNA segment; a tissue-specific promoter; a phenotypic marker; and a second guide RNA segment.

38. The molecular construct of claim 37, further comprising a sequence encoding a transgene of interest, wherein the sequence encoding a transgene of interest is 3' of the tissue- specific promoter and 5' of the sequence encoding a phenotypic marker.

39. The molecular construct of claim 38, wherein the sequence encoding a transgene further comprises a sequence encoding a secretion signal.

40. The molecular construct of claim 37, wherein the phenotypic marker is a fluorescent protein.

41. The molecular construct of claim 38, wherein the fluorescent protein is a green fluorescent protein.

42. The genetically modified insect of claim 39, wherein the green fluorescent protein is EGFP.

43. The genetically modified insect of claim 38, wherein the fluorescent protein is a red fluorescent protein.

44. The molecular construct of claim 43, wherein the red fluorescent protein is DsRed.

45. The molecular construct of claim 37, wherein the first guide RNA segment is complementary to a first sequence of a first gene, and the second guide RNA segment is complementary to a second sequence of a second gene.

46. The molecular construct of claim 37, wherein the first guide RNA segment is complementary to a first sequence of a first gene, and the second guide RNA segment is complementary to a second sequence of the first gene.

47. The molecular construct of any one of claims 37-46, wherein the tissue-specific promoter is a muscle tissue-specific promoter.

48. The molecular construct of claim 47, wherein the muscle tissue-specific promoter is a muscle actin promoter.

49. The molecular construct of any one of claims 37-48, comprising a sequence encoding a self-cleaving peptide 5' of the sequence encoding the phenotypic marker.

50. The molecular construct of claim 49, wherein the sequence encoding a self-cleaving peptide is 3' of the sequence encoding the transgene of interest.

51. The molecular construct of claim 50, wherein the self-cleaving peptide is a 2A self cleaving peptide.

52. The molecular construct of claim 51, wherein the 2A self-cleaving peptide is T2A.

53. The molecular construct of any one of claims 37-52, further comprising an SV40 sequence 3' of the sequence encoding the phenotypic marker and 5' of the sequence encoding the second guide RNA segment.

54. A method of producing a genetically modified insect, comprising introducing one or more genetic modifications in a wild-type insect.

55. The method of claim 54, comprising introducing into a wild-type insect the construct of any one of claims 37-53.

56. The method of claim 55, comprising introduction of two or more constructs of claims 37- 53, wherein the two or more constructs are not identical in sequence.

57. The method of claim 55 or claim 56, wherein the genetically modified insect is the genetically modified insect of any one of claims 1-36.

58. The genetically modified insect of claim 3 or claim 4, wherein the wild-type insect is a cricket selected from the superfamily Grylloidea.

59. The genetically modified insect of claim 58, wherein the cricket is Gryllodes sigillatus.

60. The genetically modified insect of claim 58, wherein the cricket is Gryllus bimaculatus.

61. The genetically modified insect of claim 58, wherein the cricket is Gryllus assimilis.

62. The genetically modified insect of claim 6, wherein the mealworm is of the species

Zophobas morio.

63. The genetically modified insect of any one of claims 16-21, wherein the one or more genetic modifications comprises a gene knock-out and a gene knock-in.

64. The genetically modified insect of any one of claims 16-21, wherein the gene knock-out or gene knock-in is introduced by the use of a transposable element.

65. The genetically modified insect of any one of claims 16-21, wherein the gene knock-out or gene knock-in is introduced using a transposase.

66. The genetically modified insect of any one of claims 1-36, wherein the one or more genetic modifications effect an increase in transcription of a target gene.

67. The genetically modified insect of any one of claims 1-36, wherein the one or more genetic modifications effect a decrease in transcription of a target gene.

68. The genetically modified insect of claim 67, wherein the decrease in transcription is a reduction in the transcription of the target gene to a level which is not detectable by conventional reverse transcription PCR methods.

69. The genetically modified insect of any one of claims 1-36, wherein the one or more genetic modifications comprise a modification of a regulatory element.

70. The genetically modified insect of claim 69, wherein the modification of a regulatory element is a replacement of a promoter region with an exogenous sequence or an endogenous sequence.

71. The genetically modified insect of any one of claims 1-36, wherein the target gene is a wild-type gene.

72. The genetically modified insect of any one of claims 1-36, wherein the target gene is a transgene.

73. The genetically modified insect of claim 72, wherein the transgene is selected from the group consisting of: a gene encoding an enzyme, a gene encoding a fluorescent protein, and a gene encoding a food protein.

74. The genetically modified insect of any one of claims 1-22, wherein the genetically modified insect produces less of a target product than a wild-type insect of the same species.

75. The genetically modified insect of any one of claims 1-24, wherein the genetically modified insect produces more of a target product than a wild-type insect of the same species.

76. The genetically modified insect of claim 74 or claim 76, wherein the target product is one or more of: a nutritional biomolecule, a chemical precursor to a nutritional biomolecule, an engineered biological product, a vaccine, a hormone, a flavorant, a colorant, a pigment, a fragrance, a fluorescent molecule, a peptide, a polypeptide, a polynucleotide, a lipid, a therapeutic drug product, a plastic, an insect repellant, and chitin.

77. The genetically modified insect of claim 76, wherein the nutritional biomolecule is a vitamin.

78. The genetically modified insect of claim 77, wherein the vitamin is vitamin A, a B vitamin, vitamin C, a D vitamin, or vitamin K.

79. The genetically modified insect of claim 76, wherein the nutritional biomolecule is folic acid or folate.

80. The genetically modified insect of claim 76, wherein the nutritional biomolecule is a protein.

81. The genetically modified insect of claim 76, wherein the nutritional biomolecule is a fatty acid.

82. The genetically modified insect of claim 76, wherein the nutritional biomolecule is an essential nutrient in humans or livestock.

83. The genetically modified insect of claim 82, wherein the essential nutrient is an essential amino acid.

84. The genetically modified insect of claim 76, wherein the engineered biological product reduces immunogenicity in humans.

85. The genetically modified insect of claim 76, wherein the engineered biological product is engineered chitin.

86. The genetically modified insect of claim 76, wherein the engineered biological product comprises a non-natural structural feature which aids in separation of the engineered biological product from other components of the genetically modified insect.

87. The genetically modified insect of claim 76, wherein the vaccine is suitable for vaccination of a domesticated organism or non-domesticated organism.

88. The genetically modified insect of claim 76, wherein the vaccine comprises an artificial antigenic construct.

89. The genetically modified insect of claim 76, wherein the vaccine comprises a wild-type virus or an engineered virus, or an antigenic component thereof.

90. The genetically modified insect of claim 76, wherein the vaccine is for vaccination against one or more variants of: an influenza, a member of the family Coronaviridae, a henipavirus, and a coxsackievirus (e.g., hand, foot, and mouth disease virus).

91. The genetically modified insect of claim 90, wherein the influenza is a high pathologic avian influenza or a low pathologic avian influenza.

92. The genetically modified insect of claim 90, wherein the member of the family Coronaviridae is SARS-CoV-2 or a variant thereof.

93. The genetically modified insect of claim 76, wherein the vaccine is for vaccination of an insect against an insect pathogen.

94. The genetically modified insect of claim 76, wherein the vaccine is for vaccination of a human against a human pathogen.

95. The genetically modified insect of claim 76, wherein the vaccine is for vaccination of a livestock species against a livestock pathogen.

96. The genetically modified insect of claim 95, wherein the livestock species is a bovine species, an avian species, a poultry species, a porcine species, a fish species, or an arthropod species.

97. The genetically modified insect of claim 76, wherein the polypeptide is an enzyme.

98. The genetically modified insect of claim 76, wherein the polypeptide is a signaling molecule.

99. The genetically modified insect of claim 76, wherein the polypeptide is a transmembrane protein.

100. The genetically modified insect of claim 99, wherein the transmembrane protein is a transporter.

101. The genetically modified insect of claim 100, wherein the transporter is an ion transport channel.

102. The genetically modified insect of claim 101, wherein the ion transport channel is a calcium transport channel, an iodine transport channel, or an iron transport channel.

103. The genetically modified insect of claim 76, wherein the polypeptide is a therapeutic peptide.

104. The genetically modified insect of claim 76, wherein the polypeptide is an antimicrobial peptide.

105. The genetically modified insect of claim 76, wherein the polypeptide is an antiviral peptide.

106. The genetically modified insect of claim 76, wherein the polypeptide is an antibody.

107. The genetically modified insect of claim 106, wherein the antibody is an engineered antibody.

108. The genetically modified insect of claim 26, wherein the polynucleotide is a DNA molecule.

109. The genetically modified insect of claim 108, wherein the DNA molecule is a plasmid.

110. The genetically modified insect of claim 76, wherein the polynucleotide is an RNA molecule.

111. The genetically modified insect of claim 110, wherein the RNA molecule is a tRNA, pre- mRNA, mRNA, an interfering RNA, or a ribozyme.

112. The genetically modified insect of claim 110, wherein the polynucleotide encodes a viral antigen.

113. The genetically modified insect of claim 112, wherein the viral antigen is the spike protein of SARS-CoV-2 or a variant or a portion thereof.

114. The genetically modified insect of claim 76, wherein the therapeutic drug product is an antibiotic.

115. The genetically modified insect of claim 76, wherein the target product is administrable in a therapeutically effective amount by: oral consumption of part or all of the genetically modified insect, sublingual administration of a formulation comprising the target product, rectal administration of a formulation comprising the target product, parenteral administration of a formulation comprising the target product, or inhalation of a formulation comprising the target product.

116. The genetically modified insect of any one of claims 1-36, wherein the one or more genetic modifications confer a difference in reproduction between the genetically modified insect and the wild-type insect of the same species.

117. The genetically modified insect of claim 116, wherein the difference in reproduction is an increased number of offspring.

118. The genetically modified insect of any one of claims 1-36, wherein the one or more genetic modifications confer a phenotypic difference between the genetically modified insect and the wild-type insect of the same species.

119. The genetically modified insect of claim 118, wherein the phenotypic difference is selected from: a difference in the time to progress through one or more stages of the life cycle; a difference in body weight; a difference in the ability to jump or fly; a difference in the presence of one or more anatomical structures; a difference in the color of one or more anatomical structures; a difference in the size of one or more anatomical structures; and a difference in survival rate following inoculation with a pathogen.

120. The genetically modified insect of claim 119, wherein the difference in the time to progress through one or more stages of the life cycle is a decreased amount of time to progress through one or more stages of the life cycle.

121. The genetically modified insect of claim 119, wherein the difference in body weight is an increased body weight.

122. The genetically modified insect of claim 119, wherein the difference in the ability to jump or fly is a reduced or eliminated ability to fly.

123 The genetically modified insect of claim 119, wherein the one or more anatomical structures is an eye.

124. The genetically modified insect of claim 123, wherein the eye is a different color than the wild-type insect of the same species.

125. The genetically modified insect of claim 124, wherein the eye is white.

126. The genetically modified insect of claim 119, wherein the one or more anatomical structures is a wing.

127. The genetically modified insect of claim 126, wherein the wing is absent or modified in structure.

128. The genetically modified insect of claim 119, wherein the one or more anatomical structures is an exoskeleton.

129. The genetically modified insect of claim 128, wherein the exoskeleton is a lighter color than the exoskeleton of a wild-type insect of the same species.

130. The genetically modified insect of claim 128, wherein the exoskeleton is a darker color than the exoskeleton of a wild-type insect of the same species.

131. The genetically modified insect of claim 119, wherein the pathogen is a viral species.

132. The genetically modified insect of claim 131, wherein the viral species is selected from: a species from the Family Iridoviridae , a species from the Family Parvoviridae, and a species from the Family Baculoviridae .

133. The genetically modified insect of claim 132, wherein the species from the family Iridoviridae is cricket iridovirus (CrIV).

134. The genetically modified insect of claim 132, wherein the species from the Family Parvoviridae is a species from the subfamily Densovirinae .

135. The genetically modified insect of claim 134, wherein the species from the subfamily Densovirinae is Acheta domesticus Densovirus.

136. The genetically modified insect of claim 131, wherein the viral species is cricket paralysis virus (CrPV).

137. The genetically modified insect of claim 119, wherein the pathogen is a bacterial species.

138. The genetically modified insect of claim 119, wherein the pathogen is a fungal species.

139. The genetically modified insect of any one of claims 1-36, wherein the genetically modified insect is characterized by a difference relative to a wild-type insect of the same species having eaten the same amount and composition of an insect food as the genetically modified insect.

140. The genetically modified insect of claim 139, wherein the difference is an increased body weight in the genetically modified insect.

141. The genetically modified insect of claim 139, wherein the difference is a decreased amount of time to progress through one or more stages of the life cycle, or a different number of stages of the life cycle, for the genetically modified insect.

142. The genetically modified insect of claim 139, wherein the difference is an increased rate of reproduction for the genetically modified insect.

143. The genetically modified insect of claim 139, wherein the difference is decreased food consumption or water consumption by the genetically modified insect.

144. A composition comprising a part or all of the genetically modified insect of any one of claims 1-36, wherein the composition is suitable for consumption by humans, a companion animal species, or a livestock species.

145. The composition of claim 144, wherein the composition comprises an unground portion of the insect.

146. The composition of claim 144 or claim 145, wherein the composition comprises a ground portion of the insect.

147. The composition of any one of claims 144-146, wherein the composition is substantially free of chitin.

148. The composition of any one of claims 144-147, wherein the livestock species is a poultry species.

149. The composition of any one of claims 144-148, wherein the livestock species is a cattle species.

150. The composition of any one of claims 144-149, wherein the composition is a food product.

151. The composition of claim 150, wherein the food product is in the form of: a pasta, a tortilla, a protein crisp, a chip, a puffed extruded snack, duros, a pate, a butter alternative, a beverage, a shake, a cereal, or a powder.

152. The composition of claim 151, wherein the cereal is an extruded cereal, a puffed cereal, a baked cereal, an O-shaped cereal, or a flake cereal.

153. The composition of any one of claims 150-152, wherein the food product is defatted.

154. The composition of any one of claims 150-153, wherein the food product comprises at least 1 gram of protein for every 3 grams of the food product.

155. The composition of any one of claims 150-154, wherein the food product comprises about 10% to about 90% protein by weight of the composition.

156. The composition of any one of any one of 144-155, wherein the part of the insect is an insect extract.

157. The method of any one of claims 54-57, wherein introducing one or more genetic modifications in the wild-type insect comprises electroporation.

158. The method of any one of claims 54-57, wherein the introducing one or more genetic modifications in the wild-type insect comprises lipofection.

159. The method of any one of claims 54-57, wherein the introducing one or more genetic modifications in the wild-type insect comprises the use of a transposable element.

160. The method of any one of claims 54-57, wherein the step of introducing one or more genetic modifications in the wild-type insect comprises viral delivery of the polynucleotide into the first insect.

161. The method of any one of claims 54-57, wherein the step of introducing one or more genetic modifications in the wild-type insect comprises liposomal delivery of the polynucleotide into the first insect.

162. The method of any one of claims 54-57, wherein the insect is at a developmental stage selected from: embryo, egg, ootheca, larva, nymph, prepupa, pupa, adult, imago, cocoon, chrystalis, maggot, caterpillar, worm, or winged stage.

163. A polynucleotide suitable for use in genetic modification of an insect.

164. The polynucleotide of claim 163, wherein the insect is a cricket or a mealworm.

165. The polynucleotide of claim 122, wherein the polynucleotide is a DNA molecule or an RNA molecule.

166. A polynucleotide vector comprising the polynucleotide of any one of claims 163-165.

167. A viral vector comprising the polynucleotide of any one of claims 163-165 or the polynucleotide vector of claim 166.

168. A liposomal composition comprising the polynucleotide of any one of claims 163-165 or the polynucleotide vector of claim 166.

169. A composition comprising: the target product produced by the genetically modified insect of any one of claims 74-138, and a pharmaceutically acceptable carrier or excipient.

Methods of Producing Products of Expression in GMO Insects

170. A method of producing the target product produced by the genetically modified insect of any one of claims 74-138, the method comprising isolating a fraction comprising the target product from the genetically modified insect.

171. The method of claim 170, further comprising purifying the product from the fraction.

172. A fishing lure comprising: a genetically modified insect; wherein the genetically modified insect emits fluorescent, luminescent, or bioluminescent light.

173. The fishing lure of claim 172, wherein the genetically modified insect is a cricket or mealworm.

174. The fishing lure of claim 172 or claim 173, wherein the genetically modified insect is the genetically modified insect of any one of claims 1-36.

175. A method of fi shing compri sing : baiting a hook with the fishing lure of any one of claims 172-174.

176. An automated insect farming apparatus comprising: a growth chamber; a nutrient delivery system; a water delivery system; a mechanism for removal of waste and/or one or more insects; a computerized system programmed to automate one or more of: nutrient delivery; water delivery; and insect removal; wherein the computerized system is operably linked to one or more of: the growth chamber; the nutrient delivery system; the water delivery system; and the mechanism for removal of waste and/or one or more insects.

177. The automated insect farming apparatus of claim 176, wherein the mechanism for removal of waste and/or one or more insects is a forced air system.

178. The automated insect farming apparatus of claim 177, wherein the forced air system is an air knife system.

179. The automated insect farming apparatus of any one of claims 176-178, wherein the growth chamber comprises a growth matrix.

180 The automated insect farming apparatus of claim 179, wherein the growth matrix comprises a polymer.

181. The automated insect farming apparatus of any one of claims 176-180, wherein the growth chamber comprises a cross hatched rod matrix.

182. The automated insect farming apparatus of any one of claims 176-180, wherein the growth chamber comprises a vertical plate matrix.

183. A method of insect farming comprising: providing an insect; providing a composition of one or more nutrients to the insect; and harvesting the insect at a desired stage of development; wherein one or more steps is automated.

184. The method of claim 183, wherein the insect is a wild-type insect or a genetically modified insect.

185. The method of claim 183, wherein the genetically modified insect is the genetically modified insect of any one of claims 1-36.

186. The method of any one of claims 183-185, wherein the insect is provided in a growth chamber.

187. The method of claim 186, wherein the insect cannot escape from the growth chamber.

188. The method of claim 186 or claim 187, wherein a forced air system removes the insect or a waste product from the growth chamber.

189. The method of any one of claims 186-188, wherein the growth chamber is vibrated, shaken, or otherwise agitated to remove the insect or a waste product from the growth chamber.

190. The method of any one of claims 183-189, wherein one or more nutrients are provided to the insect by an automated system.

191. The method of any one of claims 183-190, wherein water is provided to the insect by an automated system.

192. The method of any one of claims 183-191, wherein one or more nutrients are provided to the insect by a passive system.

193. The method of any one of claims 183-192, wherein water is provided to the insect by a passive system.

194. The method of claim 193, wherein the passive system comprises a wicking material along which water can passively travel.

195. The method of claim 194, wherein the wicking material comprises nylon or a ceramic.

196. The method of any one of claims 183-195, wherein the insect produces a metabolic waste product, and wherein a conveyor belt carries some or all of the metabolic waste product away from the insect.

197. A composition comprising a product isolated from an insect.

198. The composition of claim 197, wherein the insect is a wild-type insect or the genetically modified insect of any one of claims 1-36.

199. The composition of claim 197, wherein the insect is a wild-type insect.

200. The composition of any one of claims 197-199, wherein the product is suitable for use in a food product, a beverage product, a pet food product, a pet treat or snack, or an animal feed.

201. The genetically modified insect of any one of claims 1-36, comprising one or more genetic alterations, wherein the insect produces less or none of an allergen, or wherein the insect produces an allergen which elicits a reduced allergic response in a wild-type insect, a genetically modified insect, a human, a domesticated animal, a livestock animal, or a wild animal.

202. The genetically modified insect of claim 201, wherein the allergen is tropomyosin.

203. The genetically modified insect of claim 201, wherein the allergen which elicits a reduced allergic response is a modified tropomyosin.