Yeast strains for mosquito management

Abstract

This document discloses methods for producing interfering RNA (RNA) biopesticides, such as microbial host organisms engineered to produce interfering RNA molecules that inhibit gene expression in disease-carrying mosquitoes through RNA interference. This document also discloses polynucleotides, such as expression cassettes encoding these interfering RNA molecules and facilitating integration (e.g., stable integration) into the host organism's genome. This document further discloses compositions comprising the disclosed nucleotide sequences and a host organism, and methods for using them to control mosquito populations.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 526,834, filed July 14, 2023, entitled “YEAST STRAIN FOR MOSQUITOMANAGEMENT,” the entire disclosure of which is expressly incorporated herein by reference.

[0003] sequence list reference

[0004] This application includes a sequence list XML that has been submitted electronically, and is incorporated herein by reference in its entirety. The copy of the sequence list XML was created on June 26, 2024, and is named “IU202301602WOST26.xml”, with a size of 51,544 bytes. Technical Field

[0005] This disclosure generally relates to yeast-based insecticide compositions that can be used to control, for example, reduce, disease vector populations (such as mosquitoes). This disclosure also relates to methods for preparing yeast-based insecticide compositions. Background Technology

[0006] Mosquito-borne diseases (such as malaria and dengue fever) pose a major threat to human health and negatively impact economic growth. Given the slow progress in vaccine development and distribution, mosquito control remains a primary mechanism for disease prevention. However, existing mosquito control methods are inadequate. For example, insecticide resistance is increasing, rendering some currently used options ineffective. Furthermore, there are growing concerns about the off-target effects of pesticides. For these and other reasons, existing pesticide combinations are insufficient to combat existing and future vector-borne diseases, thus necessitating the development of next-generation, environmentally safe mosquito control options.

[0007] RNA interference technology offers a more selective alternative to broad-spectrum insecticides due to its sequence-dependent mechanism of action, and has been shown to disrupt the growth (e.g., fitness) and survival of targeted insect pests such as mosquitoes. However, the delivery of interfering RNA remains challenging, and various strategies, including injection of naked double-stranded RNA (dsRNA) and oral administration such as by combining insecticidal nucleic acid molecules with artificial feed, are employed.

[0008] An emerging strategy involves exposing mosquitoes to symbiotic or attractant microorganisms (e.g., yeast) engineered to produce insecticidal nucleic acid molecules (e.g., interfering RNA). The efficient and stable integration of expression cassettes into the microbial genome influences the expression levels of desired nucleotide sequences and commercial viability. In applications using engineered microorganisms for vector control, novel methods can be used to optimize the microbial production and other properties of insecticidal nucleic acid molecules. Therefore, there is a need for improved methods for engineering microorganisms to biosynthesize insecticidal nucleic acids, such as producing microorganisms with advantageous properties, such as improved genetic stability and increased nucleic acid yield targeting mosquito genes. Furthermore, there is a need to provide engineered microbial strains suitable for large-scale fermentation and commercial applications. The aspects of the invention disclosed herein address these needs.

[0009] By incorporating via reference

[0010] Each patent, publication, and non-patent document cited in this application is incorporated herein by reference in its entirety as if it were individually incorporated and as if it were fully set forth herein. However, the purpose of such citations (whether to patents, publications, non-patent documents, or other sources of information) is generally to provide context for discussing the features of the invention. Therefore, unless specifically stated otherwise, citations should not be construed as an admission that the document or underlying information is prior art in any jurisdiction or constitutes part of common general knowledge in the art. Summary of the Invention

[0011] A first aspect of the invention includes a prototrophic mutant yeast cell having an expression cassette stably integrated into at least one location within the yeast cell genome, wherein the expression cassette contains a nucleotide sequence encoding an interfering RNA molecule capable of inhibiting gene expression in mosquitoes, and wherein the nucleotide sequence encoding the interfering RNA molecule is flanked by inverted terminal repeat sequences.

[0012] A second aspect of the invention includes an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule capable of inhibiting gene expression in mosquitoes, wherein the nucleotide sequence encoding the interfering RNA molecule is flanked by inverted terminal repeat sequences.

[0013] A third aspect of the invention includes a composition comprising yeast cells or expression cassettes of the foregoing, such as expression cassettes containing a nucleotide sequence encoding an interfering RNA molecule capable of inhibiting gene expression in mosquitoes, wherein the nucleotide sequence encoding the interfering RNA molecule is flanked by inverted terminal repeat sequences, or prototrophic mutant yeast cells having such expression cassettes stably integrated into their genome.

[0014] The fourth aspect of the invention includes a method for engineering microbial host cells to produce interfering RNA molecules, for example, using a transposase / transposon system outside of a nuclease system to stably integrate an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule into the genome of a host organism, wherein the nuclease system is used to generate deletion mutations in auxotrophic rescue genes in the host organism.

[0015] The fifth aspect of the invention includes a method for controlling mosquito populations, such as using the yeast cells, expression cassettes, and compositions described in various aspects of the invention as mosquito bio-insecticides, such as interfering RNA bio-insecticides targeting mosquitoes, for example by feeding mosquitoes the yeast cells, expression cassettes, and compositions disclosed herein, thereby disrupting the mosquitoes' adaptability and / or survival.

[0016] The first embodiment is a prototrophic mutant yeast cell having an expression cassette stably integrated into at least one location within the genome of the yeast cell, wherein the expression cassette contains a nucleotide sequence encoding an interfering RNA molecule; wherein the interfering RNA molecule is capable of inhibiting the expression of the Shaker gene in mosquitoes; and wherein the nucleotide sequence encoding the interfering RNA molecule is flanked by inverted terminal repeat sequences.

[0017] The second embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the yeast cell's genome, the expression cassette containing a nucleotide sequence encoding an interfering RNA molecule, wherein the nucleotide sequence encoding the interfering RNA molecule is flanked by a 5' inverted terminal repeat sequence and a 3' inverted terminal repeat sequence.

[0018] The third embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the yeast cell's genome, the expression cassette containing a nucleotide sequence encoding an interfering RNA molecule, wherein the nucleotide sequence encoding the interfering RNA molecule is operatively linked to a glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter, wherein the GAP promoter separates the nucleotide sequence encoding the interfering RNA molecule from a 3' inverted terminal repeat sequence.

[0019] The fourth embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the yeast cell's genome, the expression cassette containing a nucleotide sequence encoding an interfering RNA molecule, wherein the 3' flanking end of the nucleotide sequence encoding the interfering RNA molecule is a CYC1 terminator, wherein the CYC1 terminator separates the nucleotide sequence encoding the interfering RNA molecule from the 5' inverted terminal repeat sequence.

[0020] The fifth embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the genome of the yeast cell, the expression cassette comprising a GAP promoter and a CYC1 terminator, wherein the GAP promoter comprises a nucleotide sequence having at least 85% sequence identity with SEQ ID NO:11, and the CYC1 terminator comprises a nucleotide sequence having at least 85% sequence identity with SEQ ID NO:12.

[0021] The sixth embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the genome of the yeast cell, wherein the expression cassette further includes a auxotroph rescue promoter operatively linked to an auxotroph rescue gene, and wherein the auxotroph rescue promoter is flanked by a 5' inverted terminal repeat sequence.

[0022] The seventh embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the yeast cell's genome, the expression cassette containing a auxotrophic rescue promoter, wherein the auxotrophic rescue promoter is a minimal auxotrophic rescue promoter.

[0023] The eighth embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the genome of the yeast cell, the expression cassette containing a minimal auxotroph rescue promoter, wherein the minimal auxotroph rescue promoter contains a nucleotide sequence having at least 70% identity with the full length of SEQ ID NO:3.

[0024] The ninth embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the genome of the yeast cell, the expression cassette containing a minimal auxotroph rescue promoter, wherein the minimal auxotroph rescue promoter contains a nucleotide sequence that is 100% identical to the full length of SEQ ID NO:3.

[0025] The tenth embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the yeast cell's genome, the expression cassette containing a auxotroph rescue gene, wherein the auxotroph rescue gene encodes leucine.

[0026] The 11th embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the yeast cell's genome, the expression cassette containing a auxotroph rescue gene, wherein the auxotroph rescue gene is leu2.

[0027] The 12th embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the genome of the yeast cell, the expression cassette containing a auxotroph rescue promoter, wherein the auxotroph rescue promoter contains SEQ ID NO:3, and the auxotroph rescue gene is leu2.

[0028] The 13th embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the genome of the yeast cell, the expression cassette further comprising an insulator sequence.

[0029] The 14th embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the yeast cell's genome, the expression cassette containing an insulator sequence that separates the 5' end of a nucleotide sequence encoding an interfering RNA molecule from a 3' inverted terminal repeat sequence.

[0030] The 15th embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the yeast cell's genome, the expression cassette containing an insulator sequence that separates the 3' end of a nucleotide sequence encoding an interfering RNA molecule from a 5' inverted terminal repeat sequence.

[0031] The 16th embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the genome of the yeast cell, the expression cassette containing an insulator sequence, wherein the insulator sequence has 70% identity with the full length of SEQ ID NO:6.

[0032] The 17th embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the genome of the yeast cell, the expression cassette containing an insulator sequence, wherein the insulator sequence is 100% identical to the full length of SEQ ID NO:6.

[0033] The 18th embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the genome of the yeast cell, the expression cassette containing a 5' inverted terminal repeat sequence, wherein the 5' inverted terminal repeat sequence has at least about 70% identity with the full length of SEQ ID NO:5.

[0034] The 19th embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the genome of the yeast cell, the expression cassette containing a 5' inverted terminal repeat sequence, wherein the 5' inverted terminal repeat sequence is 100% identical to the full length of SEQ ID NO:5.

[0035] The 20th embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the genome of the yeast cell, the expression cassette containing a 5' inverted terminal repeat sequence, wherein the 5' inverted terminal repeat sequence consists of the full length of SEQ ID NO:5.

[0036] The 21st embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the genome of the yeast cell, the expression cassette containing a 3' inverted terminal repeat sequence, wherein the 3' inverted terminal repeat sequence has at least about 70% identity with the full length of SEQ ID NO:4.

[0037] The 22nd embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the genome of the yeast cell, the expression cassette containing a 3' inverted terminal repeat sequence, wherein the 3' inverted terminal repeat sequence is 100% identical to the full length of SEQ ID NO:4.

[0038] The 23rd embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the genome of the yeast cell, the expression cassette containing a 3' inverted terminal repeat sequence, wherein the 3' inverted terminal repeat sequence consists of the full length of SEQ ID NO:4.

[0039] The 24th implementation plan is a prototrophic yeast cell with a complementary auxotrophic mutation.

[0040] The 25th implementation is a prototrophic yeast cell with a complementary auxotrophic mutation, wherein the complementary auxotrophic mutation includes his3Δ0, leu2Δ0, trp1Δ0, ura3Δ0 or a combination thereof.

[0041] The 26th embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the genome of the yeast cell, wherein the expression cassette contains at least one, at least three, or at least five copies of a nucleotide sequence encoding an interfering RNA molecule.

[0042] The 27th implementation is a prototrophic yeast cell having an expression cassette stably integrated into the yeast cell genome, wherein the expression cassette has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 sites integrated into the yeast cell genome.

[0043] The 28th embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the genome of the yeast cell, the expression cassette containing a nucleotide sequence encoding an interfering RNA molecule, wherein the nucleotide sequence transcribed from the nucleotide sequence encoding the interfering RNA molecule contains at least 25 consecutive nucleotides, the at least 25 consecutive nucleotides being partially or completely complementary to a sequence containing at least 80%, 84%, 88%, 92%, 96%, or 100% identity with the full length of SEQ ID NO:2.

[0044] The 29th embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the genome of the yeast cell, the expression cassette containing a nucleotide sequence encoding an interfering RNA molecule, wherein the nucleotide sequence transcribed from the nucleotide sequence encoding the interfering RNA molecule contains at least 25 consecutive nucleotides that are either partially or completely complementary to the full length of SEQ ID NO:2.

[0045] The 30th embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the genome of the yeast cell, the expression cassette containing a nucleotide sequence encoding an interfering RNA molecule, wherein the nucleotide sequence encoding the interfering RNA molecule has at least 80% or at least 90% sequence similarity to the full length of SEQ ID NO:1.

[0046] The 31st embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the genome of the yeast cell, the expression cassette containing a nucleotide sequence encoding an interfering RNA molecule, wherein at least 10% or at least 20% of the sequence differences are located within the TTCAAGAGA nucleotide sequence of SEQ ID NO:1.

[0047] The 32nd embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the genome of the yeast cell, the expression cassette containing a nucleotide sequence encoding an interfering RNA molecule, wherein the nucleotide sequence encoding the interfering RNA molecule comprises the full length of SEQ ID NO:1.

[0048] The 33rd embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the genome of the yeast cell, the expression cassette containing a nucleotide sequence having at least 70%, 80% or 90% identity with SEQ ID NO:7 or its complement; or with the full length of SEQ ID NO:8 or its complement.

[0049] The 34th embodiment is a prototrophic yeast cell having an expression cassette stably integrated into the genome of the yeast cell, wherein the expression cassette consists of a nucleotide sequence having 100% identity with SEQ ID NO:7 or its complement; or with the full length of SEQ ID NO:8 or its complement.

[0050] The 35th embodiment is a prototrophic yeast cell having an expression cassette containing the full length of SEQ ID NO:7, wherein the expression cassette has a single copy of a nucleotide sequence encoding an interfering RNA molecule, wherein the nucleotide sequence encoding the interfering RNA molecule contains the full length of SEQ ID NO:1, wherein the interfering RNA molecule is capable of inhibiting the expression of the Shaker gene in mosquitoes.

[0051] The 36th embodiment is a prototrophic yeast cell having an expression cassette containing the full length of SEQ ID NO:8, wherein the expression cassette has at least three copies of a nucleotide sequence encoding an interfering RNA molecule, wherein the nucleotide sequence encoding the interfering RNA molecule contains SEQ ID NO:1, the interfering RNA molecule being able to suppress the expression of the Shaker gene in mosquitoes, and the expression cassette having a genomic integration site.

[0052] The 37th embodiment is a prototrophic yeast cell having a nucleotide sequence encoding an interfering RNA molecule, the nucleotide sequence comprising the full length of SEQ ID NO:1, wherein the nucleotide sequence encoding the interfering RNA molecule is integrated at any one or more of the following genomic sites: chromosome IV (NC_001136, position 543,705), chromosome VIII (NC_001140, position 124,029), chromosome X (NC_001142, position 181,309), chromosome XI (NC_001143, position 300,654), or chromosome XII (NC_001144, position 213,991).

[0053] The 38th implementation scheme is a prototrophic yeast cell with the genotype MATa, PiggyBac (leu2d / P TDH3 -shRNA_463-T CYC1 ), CEN / ARS (URA3 / SPBase_Sc-CO).

[0054] The 39th implementation scheme is a prototrophic yeast cell in which the nucleotide sequence encoding shRNA_463 is integrated on chromosomes IV (NC_001136, position 1,357,520), VIII (NC_001140, position 124,029), X (NC_001142, position 181,309), XI (NC_001143, position 300,654), and XII (NC_001144, position 213,991).

[0055] The 40th implementation scheme is a prototrophic yeast cell with the genotype MATa, PiggyBac (leu2d / P TDH3 -shRNA_463-T CYC1 , P TDH3 -shRNA_463-T CYC1 , P TDH3 -shRNA_463-T CYC1 ), CEN / ARS (URA3 / SPBase-Sc-CO).

[0056] The 41st implementation is a prototrophic yeast cell in which the nucleotide sequence encoding shRNA_463 is integrated at position 543,705 on chromosome IV (NC_001136).

[0057] The 42nd embodiment is a prototrophic yeast cell, wherein the nucleotide sequence encoding shRNA_463 has at least 80% or at least 90% sequence similarity to the full length of SEQ ID NO:1.

[0058] The 43rd implementation is a prototrophic yeast cell, wherein the nucleotide sequence encoding shRNA_463 contains the full length of SEQ ID NO:1.

[0059] The 44th implementation is a prototrophic yeast cell, wherein the nucleotide sequence encoding shRNA_463 consists of the full length of SEQ ID NO:1.

[0060] The 45th embodiment is a prototrophic yeast cell, wherein the yeast cell is Pichiapastoris, Saccharomyces cerevisiae, or Yarrowialipolytica.

[0061] The 46th implementation scheme is a prototrophic yeast cell, wherein the yeast cell is a Saccharomyces cerevisiae FL100 strain or a S288C strain.

[0062] The 47th implementation scheme is prototrophic yeast cells, such as interfering RNA bio-insecticides that target mosquito genes, wherein the mosquito is a species of the genera Aedes, Anopheles, or Culex.

[0063] The 48th implementation scheme is prototrophic yeast cells, such as interfering RNA bio-insecticides targeting mosquito genes, where the mosquitoes are Anopheles gambiae, Aedes aegypti, Aedes albopictus, Culex pipiens, or Culex quinquesfasciatus.

[0064] The 49th implementation is prototrophic yeast cells, in which at least 15 g / L of stem cell weight is produced after culturing in a high-cell-density fermentation medium for 72 hours.

[0065] The 50th implementation is prototrophic yeast cells, in which the yeast OD600 reaches at least 50 after being cultured in a fermentation medium for 24 hours.

[0066] The 51st implementation plan is prototrophic yeast cells, in which the yeast OD600 reaches at least 75 after culturing in a fermentation medium for 72 hours.

[0067] The 52nd implementation is prototrophic yeast cells, in which, after culturing in a fermentation medium for 72 hours relative to time = 0, the expression level of interfering RNA molecules is increased by at least 100%.

[0068] The 53rd implementation scheme is the use of prototrophic yeast cells from any of the implementation schemes as a mosquito bio-insecticide.

[0069] The 54th embodiment is an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule capable of suppressing the expression of the Shaker gene in mosquitoes, wherein the nucleotide sequence encoding the interfering RNA molecule is flanked by inverted terminal repeat sequences.

[0070] The 55th implementation is an expression cassette in which the interfering RNA molecule is an RNA construct, double-stranded RNA (dsRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), or antisense oligonucleotide.

[0071] The 56th implementation is an expression cassette in which the nucleotide sequence encoding the interfering RNA molecule is flanked by a 5' inverted terminal repeat sequence and a 3' inverted terminal repeat sequence.

[0072] The 57th embodiment is an expression cassette that further comprises a glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter, wherein the GAP promoter is operatively linked to a nucleotide sequence encoding an interfering RNA molecule; and wherein the GAP promoter separates the nucleotide sequence encoding the interfering RNA molecule from a 3' inverted terminal repeat sequence.

[0073] The 58th embodiment is an expression cassette that further includes a CYC1 terminator, wherein the CYC1 terminator is located at the 3' end flanking the nucleotide sequence encoding the interfering RNA molecule; and wherein the CYC1 terminator separates the nucleotide sequence encoding the interfering RNA molecule from the 5' inverted terminal repeat sequence.

[0074] The 59th embodiment is an expression cassette, wherein the GAP promoter contains a nucleotide sequence having at least 85% sequence identity with SEQ ID NO:11, and the CYC1 terminator contains a nucleotide sequence having at least 85% sequence identity with SEQ ID NO:12.

[0075] The 60th embodiment is an expression cassette that further includes a auxotrophic rescue promoter operatively linked to an auxotrophic rescue gene, wherein the auxotrophic rescue promoter is flanked by a 5' inverted terminal repeat sequence.

[0076] The 61st implementation is an expression box, in which the auxotrophic rescue promoter is the minimal auxotrophic rescue promoter.

[0077] The 62nd embodiment is an expression cassette in which the minimal auxotrophic rescue promoter contains a nucleotide sequence that is at least 70% identical to the full length of SEQ ID NO:3.

[0078] The 63rd embodiment is an expression cassette in which the minimal auxotrophic rescue promoter contains a nucleotide sequence that is 100% identical to the full length of SEQ ID NO:3.

[0079] The 64th embodiment is an expression cassette, wherein the auxotrophic rescue gene encodes histidine, leucine, tryptophan, or uracil.

[0080] The 65th implementation is an expression cassette, wherein the auxotroph rescue gene is leu2, ura3, his3, trp1, or a combination thereof.

[0081] The 66th implementation is an expression cassette, in which the auxotroph rescue promoter is dleu2 (SEQ ID NO:3) and the auxotroph rescue gene is leu2.

[0082] The 67th implementation is an expression box, wherein the expression box further includes an insulator sequence.

[0083] The 68th implementation is an expression cassette in which an insulator sequence separates the 5' end of the nucleotide sequence encoding the interfering RNA molecule from the 3' inverted terminal repeat sequence.

[0084] The 69th implementation is an expression cassette in which an insulator sequence separates the 3' end of the nucleotide sequence encoding the interfering RNA molecule from the 5' inverted terminal repeat sequence.

[0085] The 70th embodiment is an expression box in which the insulator sequence has 70% identity with the full length of SEQ ID NO:6.

[0086] The 71st embodiment is an expression box in which the insulator sequence is 100% identical to the full length of SEQ ID NO:6.

[0087] The 72nd embodiment is an expression cassette in which the 5' inverted terminal repeat sequence has at least about 70% identity with the full length of SEQ ID NO:5.

[0088] The 73rd embodiment is an expression box in which the 5' inverted terminal repeat sequence is 100% identical to the full length of SEQ ID NO:5.

[0089] The 74th embodiment is an expression box, wherein the 5' inverted terminal repeat sequence consists of the full length of SEQ ID NO:5.

[0090] The 75th embodiment is an expression cassette in which the 3' inverted terminal repeat sequence has at least about 70% identity with the full length of SEQ ID NO:4.

[0091] The 76th embodiment is an expression box in which the 3' reverse terminal repeat sequence is 100% identical to the full length of SEQ ID NO:4.

[0092] The 77th embodiment is an expression box, wherein the 3' inverted terminal repeat sequence consists of the full length of SEQ ID NO:4.

[0093] The 78th embodiment is an expression cassette, wherein the expression cassette contains at least one, at least three, or at least five copies of a nucleotide sequence encoding an interfering RNA molecule.

[0094] The 79th embodiment is an expression cassette, wherein the nucleotide sequence transcribed from the nucleotide sequence encoding the interfering RNA molecule comprises at least 25 consecutive nucleotides, said at least 25 consecutive nucleotides being partially or completely complementary to a sequence comprising at least 80%, 84%, 88%, 92%, 96%, or 100% identity with the full length of SEQ ID NO:2.

[0095] The 80th embodiment is an expression cassette, wherein the nucleotide sequence transcribed from the nucleotide sequence encoding the interfering RNA molecule comprises at least 25 consecutive nucleotides, said at least 25 consecutive nucleotides being partially or completely complementary to the full length of SEQ ID NO:2.

[0096] The 81st embodiment is an expression cassette in which the nucleotide sequence encoding the interfering RNA molecule has at least 80% or at least 90% sequence similarity to the full length of SEQ ID NO:1.

[0097] The 82nd embodiment is an expression cassette in which the nucleotide sequence encoding the interfering RNA molecule comprises the full length of SEQ ID NO:1.

[0098] The 83rd embodiment is an expression cassette in which the nucleotide sequence encoding the interfering RNA molecule consists of the full length of SEQ ID NO:1.

[0099] The 84th embodiment is an expression cassette, wherein the expression cassette contains a nucleotide sequence having at least 70%, 80%, or 90% identity with the full length of SEQ ID NO:7 or its complement or with the full length of SEQ ID NO:8 or its complement.

[0100] The 85th embodiment is an expression cassette, wherein the expression cassette consists of a nucleotide sequence having 100% identity with the full length of SEQ ID NO:7 or its complement or SEQ ID NO:8 or its complement.

[0101] The 86th implementation is an expression cassette, in which the expression cassette is stably integrated into the genome of the host organism via transposition.

[0102] The 87th implementation is an expression cassette, wherein the expression cassette is stably integrated into at least one or at least three sites in the host organism's genome.

[0103] The 88th implementation is an expression cassette, in which the expression cassette is integrated into one of the 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 sites in the host organism's genome.

[0104] The 89th implementation scheme is an expression cassette that is stably integrated into the genome of a host organism, wherein the host organism is a yeast cell.

[0105] The 90th implementation scheme is an expression cassette that is stably integrated into the genome of a host organism, such as Pichia pastoris, Saccharomyces cerevisiae, or Yersinia lipophila.

[0106] The 91st embodiment is an expression cassette in which the nucleotide sequence encoding the interfering RNA molecule is integrated into one or more genomic loci on any of the following chromosomes of the Saccharomyces cerevisiae FL100 strain: chromosome IV (NC_001136, position 543,705), chromosome VIII (NC_001136, position 1,357,520), chromosome VIII (NC_001140, position 124,029), chromosome X (NC_001142, position 181,309), chromosome XI (NC_001143, position 300,654), or chromosome XII (NC_001144, position 213,991).

[0107] The 92nd embodiment is a composition comprising yeast cells or expression cassettes from any of the above embodiments.

[0108] The 93rd embodiment is a composition comprising yeast cells from any of the above embodiments, wherein the yeast cells are killed by heating and / or freeze-drying.

[0109] The 94th embodiment is a composition that further comprises a sugar bait.

[0110] The 95th embodiment is a composition that further comprises an insecticide.

[0111] The 96th embodiment is a composition, wherein the composition is located inside a trap.

[0112] The 97th embodiment is a composition in which the composition is selectively insecticidal against mosquitoes.

[0113] The 98th embodiment is a selective mosquito-killing composition, wherein the mosquito is a mosquito larva or an adult mosquito.

[0114] The 99th embodiment is a composition that selectively kills mosquitoes of the genera Aedes, Anopheles, or Culex.

[0115] The 100th embodiment is a composition that selectively kills Aedes aegypti, Aedes albopictus, Anopheles gambiae, Culex pipiens salina, or Culex quinquefasciatus.

[0116] The 101st implementation is a method for stably integrating an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule into the host organism's genome using a transposase / transposon system, wherein the interfering RNA molecule suppresses gene expression in mosquitoes.

[0117] The 102nd implementation is a method for stably integrating an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule into the genome of a host organism, wherein the interfering RNA molecule is an RNA construct, double-stranded RNA (dsRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), or antisense oligonucleotide.

[0118] The 103rd implementation is a method for stably integrating an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule into the genome of a host organism, wherein a nuclease system is used to generate deletion mutations in auxotroph rescue genes in the host organism.

[0119] The 104th implementation is a method for generating deletion mutations in auxotrophic rescue genes in a host organism, wherein the auxotrophic mutations inhibit the host organism's ability to produce histidine, leucine, tryptophan, uracil, or combinations thereof.

[0120] The 105th embodiment is a method for generating deletion mutations in auxotrophic rescue genes in a host organism, wherein the auxotrophic mutations include ura3Δ0, leu2Δ0, his3Δ0, trp1Δ0, or combinations thereof.

[0121] The 106th implementation is a method for generating deletion mutations in auxotrophic rescue genes in a host organism, wherein the auxotrophic mutations are ura3Δ0, leu2Δ0, his3Δ0 and trp1Δ0.

[0122] The 106th embodiment is a method for generating deletion mutations in auxotrophic rescue genes in a host organism using a nuclease system, wherein the nuclease system comprises a dimer Clo51 endonuclease having at least about 95% sequence identity with SEQ ID NO:16.

[0123] The 107th embodiment is a method for generating deletion mutations in auxotrophic rescue genes in a host organism using a nuclease system, wherein the nuclease system comprises a dimer Clo51 endonuclease having at least about 95% sequence identity with SEQ ID NO:16.

[0124] The 108th embodiment is a method for generating deletion mutations in auxotroph rescue genes in a host organism using a nuclease system, wherein the nuclease system further comprises two guide RNAs transcribed from gRNA nucleotide sequences, the gRNA nucleotide sequences having at least 90% identity with the full length of SEQ ID NO:17 and SEQ ID NO:18; or SEQ ID NO:19 and SEQ ID NO:20; and wherein the gRNA nucleotide sequences are operatively linked to an SNR52 promoter and an SNR52 terminator, the SNR52 promoter comprising a nucleotide sequence having approximately 90% identity with SEQ ID NO:13, and the SNR52 terminator comprising a nucleotide sequence having approximately 95% identity with SEQ ID NO:14.

[0125] The 109th embodiment is a method for stably integrating an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule into the genome of a host organism using a transposase / transposon system, wherein the transposase / transposon system comprises a transposon vector and a transposase; and wherein the transposase integrates the transposon expression cassette into at least one integration site in the genome of the host organism.

[0126] The 110th implementation is a method for stably integrating an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule into the host organism's genome using a transposase / transposon system, wherein the transposase integrates the expression cassette into 2, 3, 4, 5, 6, 7, 8, 9, or 10 sites in the host organism's genome.

[0127] The 111th embodiment is a method for stably integrating an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule into the genome of a host organism using a transposase / transposon system, wherein the expression cassette further includes a auxotrophic rescue promoter and an auxotrophic rescue gene.

[0128] The 112th embodiment is a method for stably integrating an expression cassette containing a auxotrophic rescue promoter and a auxotrophic rescue gene, wherein the auxotrophic rescue promoter is flanked by a 5' inverted terminal repeat sequence and the nucleotide sequence encoding the interfering RNA molecule is flanked by a 3' inverted terminal repeat sequence.

[0129] The 113th embodiment is a method for stably integrating an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule into the genome of a host organism using a transposase / transposon system, wherein the nucleotide sequence encoding the interfering RNA molecule is operatively linked to a glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter, wherein the GAP promoter separates the nucleotide sequence encoding the interfering RNA molecule from a 3' inverted terminal repeat sequence.

[0130] The 114th embodiment is a method for stably integrating an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule into the genome of a host organism using a transposase / transposon system, wherein the 3' flanking end of the nucleotide sequence encoding the interfering RNA molecule is a CYC1 terminator, wherein the CYC1 terminator separates the nucleotide sequence encoding the interfering RNA molecule from the 5' inverted terminal repeat sequence.

[0131] The 115th embodiment is a method for stably integrating an expression cassette containing a GAP promoter and a CYC1 terminator into the genome of a host organism, wherein the GAP promoter contains a nucleotide sequence having at least 85% sequence identity with SEQ ID NO:11, and the CYC1 terminator contains a nucleotide sequence having at least 85% sequence identity with SEQ ID NO:12.

[0132] The 116th embodiment is a method for stably integrating an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule into the genome of a host organism using a transposase / transposon system, wherein the expression cassette further includes a auxotroph rescue promoter operatively linked to an auxotroph rescue gene, and wherein the auxotroph rescue promoter is flanked by a 5' inverted terminal repeat sequence.

[0133] The 117th implementation is a method for stably integrating an expression cassette into the genome of a host organism, the expression cassette comprising an auxotrophic rescue promoter operatively linked to an auxotrophic rescue gene, wherein the auxotrophic rescue promoter is a minimal auxotrophic rescue promoter.

[0134] The 118th embodiment is a method for stably integrating an expression cassette containing a minimal auxotrophic rescue promoter into the genome of a host organism, wherein the minimal auxotrophic rescue promoter contains a nucleotide sequence having at least 70% identity with the full length of SEQ ID NO:3.

[0135] The 119th embodiment is a method for stably integrating an expression cassette containing a minimal auxotrophic rescue promoter into the genome of a host organism, wherein the minimal auxotrophic rescue promoter contains a nucleotide sequence that is 100% identical to the full length of SEQ ID NO:3.

[0136] The 120th embodiment is a method for stably integrating an expression cassette into the genome of a host organism using a transposon / transposase system, wherein the expression cassette further contains an insulator sequence.

[0137] The 121st implementation is a method for stably integrating an expression cassette containing an insulator sequence into the genome of a host organism using a transposon / transposase system, wherein the insulator sequence separates the 5' end of the nucleotide sequence encoding the interfering RNA molecule from the 3' inverted terminal repeat sequence.

[0138] The 122nd embodiment is a method for stably integrating an expression cassette containing an insulator sequence into the genome of a host organism using a transposon / transposase system, wherein the insulator sequence separates the 3' end of the nucleotide sequence encoding the interfering RNA molecule from the 5' inverted terminal repeat sequence.

[0139] The 123rd embodiment is a method for stably integrating an expression cassette containing an insulator sequence into the genome of a host organism using a transposon / transposase system, wherein the insulator sequence has 70% identity with the full length of SEQ ID NO:6.

[0140] The 124th embodiment is a method for stably integrating an expression cassette containing an insulator sequence into the genome of a host organism using a transposon / transposase system, wherein the insulator sequence is 100% identical to the full length of SEQ ID NO:6.

[0141] The 125th embodiment is a method for stably integrating an expression cassette containing a 5' inverted terminal repeat sequence into the genome of a host organism using a transposon / transposase system, wherein the 5' inverted terminal repeat sequence has at least about 70% identity with the full length of SEQ ID NO:5.

[0142] The 126th embodiment is a method for stably integrating an expression cassette containing a 5' inverted terminal repeat sequence into the genome of a host organism using a transposon / transposase system, wherein the 5' inverted terminal repeat sequence is 100% identical to the full length of SEQ ID NO:5.

[0143] The 127th embodiment is a method for stably integrating an expression cassette containing a 5' inverted terminal repeat sequence into the genome of a host organism using a transposon / transposase system, wherein the 5' inverted terminal repeat sequence consists of the full length of SEQ ID NO:5.

[0144] The 128th embodiment is a method for stably integrating an expression cassette containing a 3' inverted terminal repeat sequence into the genome of a host organism using a transposon / transposase system, wherein the 3' inverted terminal repeat sequence has at least about 70% identity with the full length of SEQ ID NO:4.

[0145] The 129th embodiment is a method for stably integrating an expression cassette containing a 3' inverted terminal repeat sequence into the genome of a host organism using a transposon / transposase system, wherein the 3' inverted terminal repeat sequence is 100% identical to the full length of SEQ ID NO:4.

[0146] The 130th embodiment is a method for stably integrating an expression cassette containing a 3' inverted terminal repeat sequence into the genome of a host organism using a transposon / transposase system, wherein the 3' inverted terminal repeat sequence consists of the full length of SEQ ID NO:4.

[0147] The 131st embodiment is a method for stably integrating an expression cassette into the genome of a host organism using a transposon / transposase system, wherein the expression cassette contains at least one, at least three, or at least five copies of a nucleotide sequence encoding an interfering RNA molecule.

[0148] The 132nd implementation is a method for stably integrating an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule into the host organism's genome using a transposase / transposon system, wherein the interfering RNA molecule is capable of suppressing the expression of the Shaker gene in mosquitoes.

[0149] The 133rd embodiment is a method for stably integrating an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule into the genome of a host organism using a transposase / transposon system, wherein the nucleotide sequence transcribed from the nucleotide sequence encoding the interfering RNA molecule contains at least 25 consecutive nucleotides, said at least 25 consecutive nucleotides being partially or completely complementary to a sequence containing at least 80%, 84%, 88%, 92%, 96%, or 100% identity with the full length of SEQ ID NO:2.

[0150] The 134th embodiment is a method for stably integrating an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule into the genome of a host organism using a transposase / transposon system, wherein the nucleotide sequence transcribed from the nucleotide sequence encoding the interfering RNA molecule contains at least 25 consecutive nucleotides, said at least 25 consecutive nucleotides being partially or completely complementary to the full length of SEQ ID NO:2.

[0151] The 135th embodiment is a method for stably integrating an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule into the genome of a host organism using a transposase / transposon system, wherein the nucleotide sequence encoding the interfering RNA molecule has at least 80% or at least 90% sequence similarity to the full length of SEQ ID NO:1.

[0152] The 136th embodiment is a method for stably integrating an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule into the genome of a host organism using a transposase / transposon system, wherein the nucleotide sequence encoding the interfering RNA molecule is 100% identical to the full length of SEQ ID NO:1.

[0153] The 137th embodiment is a method for stably integrating an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule into the genome of a host organism using a transposase / transposon system, wherein the nucleotide sequence encoding the interfering RNA molecule consists of the full length of SEQ ID NO:1.

[0154] The 138th embodiment is a method for stably integrating an expression cassette into the genome of a host organism using a transposase / transposon system, wherein the expression cassette contains a nucleotide sequence having at least 70%, 80%, or 90% identity with the full length of SEQ ID NO:7 or its complement; or SEQ ID NO:8 or its complement.

[0155] The 139th embodiment is a method for stably integrating an expression cassette into the genome of a host organism using a transposase / transposon system, wherein the expression cassette consists of a nucleotide sequence having 100% identity with the full length of SEQ ID NO:7 or its complement; or SEQ ID NO:8 or its complement.

[0156] The 140th embodiment is a method for stably integrating an expression cassette into the genome of a host organism using a transposase / transposon system, wherein the transposase contains a nucleotide sequence having at least about 70% identity with SEQ ID NO:10.

[0157] The 141st embodiment is a method for stably integrating an expression cassette into the genome of a host organism using a transposase / transposon system, wherein the transposase contains a nucleotide sequence that is 100% identical to SEQ ID NO:10.

[0158] The 142nd embodiment is a method for controlling mosquito populations, comprising feeding mosquitoes with yeast cells according to any one of claims 1-52, expression cassettes according to any one of claims 54-91, or compositions according to any one of claims 92-101, thereby controlling mosquito populations.

[0159] The 143rd implementation plan is a method for controlling mosquito populations, wherein the mosquitoes are mosquito larvae or adult mosquitoes.

[0160] The 144th implementation plan is a method for controlling mosquito populations, wherein the mosquitoes are species of the genera Aedes, Anopheles, or Culex.

[0161] The 145th implementation plan is a method for controlling mosquito populations, wherein the mosquitoes are Aedes aegypti, Anopheles gambiae, or Culex quinquefasciatus. Attached Figure Description

[0162] The accompanying drawings are included to provide a further understanding of this disclosure, and are incorporated in and form part of this specification. They illustrate embodiments and, together with the description, are used to explain the principles of this disclosure.

[0163] Figure 1A A schematic diagram of Saccharomyces cerevisiae engineering, depicting precise cleavage via the dimer Cas-CLOVER and two gRNAs that target genes encoding essential amino acids required for growth.

[0164] Figure 1B A schematic diagram of Saccharomyces cerevisiae engineering, depicting the use of the Super PiggyBac transposase / transposon system to randomly integrate biopesticide cargoes with selective auxotrophic markers (PNut = nutrient promoter; N.gene = nutrient gene) into the "TTAA" site, thereby facilitating detection and positive gene integration.

[0165] Figure 2 The bar graph shows the comparative expression levels of shRNA (Sh.463) targeting the mosquito Shaker gene in the Cas-CLOVER / piggyBac synthetic yeast strain relative to the initial piggyBac integrated strain DMT9-51.1.

[0166] Figure 3 The bar graph shows a Cas-CLOVER / piggyBac synthetic yeast strain that has been down-selected and has recovered from nutritional deficiencies, expressing high levels of shRNA (Sh.463) targeting the mosquito Shaker gene, relative to the initial piggyBac integrated strain DMT4-51.1R#1.

[0167] Figure 4 Chromosome diagram showing the DMT9-56.10R#3 genome integration site.

[0168] Figure 5A , 5B Chromosome diagrams showing the DMT9-52.2R#3 genomic integration site, 5C and 5D: Figures 5A-5D This shows the stable integration site of the expression cassette encoding the insecticidal shRNA Sh.463.

[0169] Figure 6 The bar graph depicts the larval-killing activity of the Cas-CLOVER / piggyBac synthetic yeast strain expressing shRNA targeting the mosquito Shaker gene.

[0170] Figure 7 The bar graph shows the adult insecticidal activity of the Cas-CLOVER / piggyBac synthetic yeast strain expressing shRNA targeting the mosquito Shaker gene.

[0171] Figure 8 The line graph depicts the growth curve (OD600) and shRNA (Sh.463) expression of DMT9-56.10R#3 in two different high-cell-density fermentation media, HCD and DFM. The dashed line represents shRNA expression and the solid line represents optical density. Detailed Implementation

[0172] The efficacy of biopesticides that produce interfering RNA molecules has been demonstrated. However, further improvements are needed to effectively address the burden of mosquito-borne diseases. For example, improvements in methods for producing biopesticides from engineered host organisms, industrial-scale production of biopesticides, and selective delivery of biopesticides to target organisms could offer superior properties and advantages.

[0173] In one instance, an interfering RNA biocide targeted the mosquito gene Shaker, which encodes an evolutionarily conserved voltage-gated potassium channel subunit that has been shown to be essential for mosquito survival (Mysore et al., PLoS Negl Trop Dis. 2020 Jul; 14(7): e0008479). Delivery of the interfering RNA molecule Sh.463 as siRNA to adult Aedes aegypti resulted in Shaker gene silencing, leading to severe neurological and behavioral deficits and high levels of adult mortality. The siRNA was either injected or provided as an attractive toxic sugar bait (ATSB). Similarly, when Sh.463 was administered to Aedes aegypti larvae in the form of short hairpin RNA (shRNA) expressed in Saccharomyces cerevisiae (i.e., the exemplary interfering RNA biocide described herein) and formulated into dried, inactivated yeast tablets, the biocide induced neurological deficits, larval and adult mortality.

[0174] Furthermore, the selectivity of Sh.463 in various mosquito populations was confirmed. The target gene or Sh.463 lacks known target sites in humans and other non-target organisms (such as non-target arthropods). However, the target sites are conserved in different mosquito species, such as Aedes, Anopheles, and Culex, as shown in Table 1 (excerpted from Mysore et al., PLoSNegl Trop Dis. 2020 Jul; 14(7): e0008479).

[0175] Table 1. Conservation assessment of the Sh.463 target site

[0176]

[0177] Previous methods for engineering host organisms to produce interfering RNA molecules involve ligating a nucleotide sequence encoding shRNA.463 into non-integrating and integrating shuttle vectors. See, for example, Haipairai et al., Sci Rep. 2017; 7: 13223 and Mysore et al., PLoS Negl Trop Dis. 2020 Jul; 14(7): e0008479. This paper discloses an improved method for engineering interfering RNA biopesticides with exemplary advantages of efficient and stable integration into the host organism genome, enhanced expression of interfering RNA molecules, and industrial scale-up feasibility. In one aspect, this paper describes the production of interfering RNA biopesticides for controlling disease-carrying mosquitoes using a Cas-CLOVER system combined with transposon systems such as piggyBac transposons and Super piggyBac transposases. In this paper, “transposon system” is interchangeably referred to as “transposon / transposase system” or “transposase / transposon system”.

[0178] Although *Saccharomyces cerevisiae* is a natural attractant for mosquitoes, it lacks the necessary mechanisms for processing the interfering RNA molecules disclosed herein. Exemplary yeast host organisms are well-suited for gene editing via Cas-CLOVER, enabling the production of a wide variety of molecules. However, microbial genomes are typically non-redundant compared to plant and mammalian genomes. This characteristic poses a technical challenge to nuclease and transposon-mediated gene editing, as off-target cleavage or random integration into essential genes can lead to cell death, reducing the likelihood of viable clones.

[0179] Like the original CRISPR / Cas9 system, Cas-CLOVER is an RNA-guided system. While retaining the simplicity of the original Cas9 system, unlike Cas9, Cas-CLOVER uses a dimerizing nuclease called Clo051 to edit the genome. The Clo51 subunit is a obligate dimer, meaning it must dimerize to perform cleavage activity. Clo051 is associated with reduced off-target nuclease activity compared to CRISPR, such as higher fidelity, larger deletion fragments, and more efficient knock-in.

[0180] The Cas-CLOVER system was used to systematically create yeast strains with multiple gene deletions, thereby establishing a universal bioprocessing platform. These gene deletions were easily rescued using selected markers (ura3, leu2, his3, trp1). All auxotrophic marker gene knockouts were complete OFR deletions induced by homology-directed repair using donor PCR fragments (after Cas-CLOVER cleavage). The donor PCR fragments covered 200 bp upstream of the translation start site and 200 bp downstream of the stop codon. Figure 1A Combining the resulting auxotrophic engineered yeast strains with Super PiggyBac transposase / transposon technology provides efficient detection and selection of positively integrated cargoes. Figure 1B ).

[0181] In addition to being a universal bioprocessing platform for transposase integration, *Saccharomyces cerevisiae* lacks components of the RNA interference (RNAi) pathway, which enables the organism to become a cellular biofactory and delivery system for interfering with RNA molecules in order to control mosquitoes that cause hundreds of thousands of deaths globally each year. See, for example, Duman-Scheel. *Current Drug Targets*. 20(9), 942-952 and Mysore et al., *Insect Genomics: Methods and Protocols*. 1858;213-231. Humana Press.

[0182] The term "siRNA" or "small interfering RNA" refers to short double-stranded RNA molecules (e.g., approximately 19-30 base pairs in length) that function via the RNAi pathway and are less than 30 base pairs in length. Each siRNA unwinds into two single-stranded RNAs (ssRNAs), one of which is incorporated into the RNA-induced silencing complex (RISC), resulting in post-transcriptional gene silencing. siRNAs can be produced in various ways. In some cases, long dsRNAs are introduced into cells via viruses, endogenous RNA expression (i.e., microRNAs), or exogenously delivered dsRNAs. The DICER enzyme cleaves the long double-stranded RNA into siRNAs. In some preferred embodiments, the siRNA is approximately 25 bp in length. While using longer (300-400 bp) double-stranded RNA (dsRNA) molecules is one method of producing interfering RNA, the short length (21-25 bp) of tailored small interfering RNAs (siRNAs) is advantageous for designing highly specific interfering RNAs.

[0183] The terms "short hairpin RNA" and "small hairpin RNA" are both encompassed within the term "shRNA." shRNA is an artificial RNA with a secondary structure that allows a portion of the RNA strand to form a hairpin loop. shRNA can be transcribed under the control of RNA Pol-II or Pol-III promoters and folds into a structure similar to the siRNA double strand. Subsequently, shRNA is processed into siRNA by DICER.

[0184] The term "dsRNA" (double-stranded RNA) refers to a long double-stranded RNA molecule that is cleaved by the DICER enzyme into short double-stranded fragments of approximately 20-25 nucleotides of siRNA.

[0185] RNA interference (RNAi) or post-transcriptional gene silencing (PTGS) refers to the biological process by which RNA molecules interfere with or inhibit the expression of specific genes that have complementary nucleotide sequences to interfering RNA (gene-specific inhibition of gene expression). RNAi leads to post-transcriptional mRNA degradation, resulting in reduced translation and protein expression.

[0186] RNA interference (RNAi) technology employs genetic constructs encoding interfering RNA molecules, such as dsRNA and shRNA. Typically, the RNA construct contains sense and antisense sequences positioned in regions flanking the intron sequences with the correct splicing orientation to the donor and recipient splicing sites. Alternatively, spacer sequences of various lengths can be used to separate self-complementary regions of the sequences within the construct. During the processing of the gene construct transcript, the intron sequences can be spliced ​​away, allowing the sense, antisense, and splice-joining sequences to combine and form double-stranded RNA. Alternatively, when secondary structures inhibit splicing mechanisms, the intron sequences are not spliced ​​away, and the dsRNA exists in a hairpin configuration. When dsRNA is expressed in the cell, ribonucleases bind to and cleave the double-stranded RNA, initiating a cascade of events that leads to the degradation of target mRNA molecules, thereby silencing such target genes. RNA interference using shRNA is documented in Sheng et al., Front Bioeng Biotechnol. 2020 Aug 7;8:940 and generally described in Bass, Nature 411: 428-29 (2001); Elbahir et al., Nature 411: 494-98 (2001); and Fire et al., Nature 391: 806-11 (1998); and WO 01 / 75164, which also discuss methods for preparing interfering RNA.

[0187] Interfering RNA can hybridize with the full-length mRNA encoded by the target gene, or with a fragment of the target RNA or DNA (the target sequence). For example, to reduce the expression of a target gene in mosquitoes through RNA interference, an expression cassette encoding an interfering RNA molecule is stably integrated into the genome of a host organism (such as a yeast cell). This interfering RNA molecule has an mRNA sequence transcribed from the target gene, or a substantially identical sequence (including engineered sequences that do not translate proteins) or a fragment thereof.

[0188] Stable integration refers to the long-term expression of a transgene by integrating foreign DNA into the host nuclear genome. Therefore, the foreign DNA or the transgene is integrated into the genome, replicates along with the genomic DNA, and is passed on to offspring. In contrast, in transient transfection, nucleic acids are not integrated into the host cell genome. In transient systems, the foreign DNA cannot replicate independently of the host DNA and may only persist for a few days. See, for example, Fus-Kajawa, Front Bioeng Biotechnol. 2021 Jul 20;9:701031.

[0189] Although the parental strains of the RNA-interfering biocides disclosed herein (such as DMT9-52.2R#3 and DMT9-56.10R#3) have achieved stable integration (see, for example, US20220346380A1), the growth rates of these parental strains are insufficient for large-scale fermentation. The parental strains also contain galactose-inducible promoters, which increases the cost of large-scale yeast production. Compared to the parental strains, the strains disclosed herein express the disclosed shRNA at significantly higher levels, thereby reducing the yeast dosage required to kill mosquitoes. This feature promises to reduce the cost of commercial production and deployment.

[0190] The resulting genetically modified host organism can then be fed to mosquitoes to determine its ability to suppress target gene expression, resulting in reduced mosquito fitness and / or survival. While the interfering RNA sequence used for RNA interference need not be identical to the mRNA transcribed from the target sequence of the target gene, it is generally substantially identical, for example, with at least 70%, 80%, 90%, 95%, 98%, or higher identity. It is known in the art that dsRNA molecules that are not perfectly complementary to the mRNA transcribed from the target sequence (e.g., having only 95% identity with the mRNA transcribed from the target sequence) are effective in controlling pests (see, for example, Narva et al., U.S. Patent No. 9,012,722).

[0191] Target genes can be selected based on various criteria, including gene necessity, expression level, and differences from sequences of related species. Exemplary target genes include Shaker and its orthologs (such as Shaker-like genes), which encode voltage-gated potassium channel proteins. Exemplary target organisms having the target gene or its ortholog are shown in Table 2. Exemplary target genes and organisms were obtained by searching SEQ ID NO:2 in NCBI's BLAST database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0192] Table 2. Exemplary target genes and related gene IDs

[0193]

[0194] In some instances, suppressing target genes interferes with the production of essential proteins. The exemplary target gene Shaker encodes structural components of voltage-dependent potassium channels. Mutations in the conserved Drosophila Shaker gene in mosquito species such as Anopheles result in overexcitation near axonal branching points due to improper neuronal repolarization. See, for example, Salkof & Wyman, Nature. 1981;293(5829):228–30 and Lichtinghagen et al., EMBO J. 1990;9(13):4399–407. These neural defects manifest behaviorally as uncontrolled movements (Jan & Jan, J Physiol. 1997;505(Pt 2):267–82). For example, Drosophila Shaker mutants exhibit aberrant movements such as leg tremors (hence the phenotypic descriptive name Shaker (Trout & Kaplan, J Neurobiol. 1973;4(6):495–512)).

[0195] The terms “gene repression,” “gene expression downregulation,” or “suppression or suppression of gene expression” are used interchangeably to refer to a measurable or observable reduction in gene expression, or the complete elimination of gene expression detectable at the level of protein products (“gene silencing”) and / or mRNA products from the gene. In some embodiments, gene repression leads to gene silencing, referring to the ability of interfering RNA to target and degrade mRNA, resulting in translational interruption, which prevents protein expression. The ability of interfering RNA to repress or downregulate at least one gene results in suppressed or inhibited mosquito growth or maturation, or death of mosquito larvae or adults. Downregulation or repression can occur at the transcriptional or post-translational stage of target gene expression by promoting transcript turnover, cleavage, or translational disruption.

[0196] The inhibition of target gene expression can be quantified by measuring endogenous target RNA or proteins translated from target RNA, and the results of inhibition can be confirmed by examining the external characteristics of cells or organisms. Techniques for quantifying RNA and proteins are well known to those skilled in the art.

[0197] The term "gene" refers to a polynucleotide sequence containing the control and coding sequences necessary for the production of a polypeptide (protein). A polypeptide may be encoded by a full-length coding sequence or any portion of the coding sequence. A gene includes regions preceding and following the coding region (leader and trailing regions) and intervening sequences (introns) between individual coding segments (exons). The leader, trailing region, and introns contain regulatory elements (such as promoters, enhancers, etc.) essential for gene transcription and translation. A gene may be a continuous coding sequence or may contain one or more introns located between splice junctions. As used herein, a gene may include variants of a gene, including but not limited to modifications such as mutations, insertions, deletions, or substitutions of one or more nucleotides. A "target gene" is a gene that the interfering RNA of this technology targets to downregulate or repress. A "gene product" may refer to mRNA or protein expressed from a specific gene.

[0198] The terms "nucleic acid," "polynucleotide," and "oligonucleotide" refer to single-stranded or double-stranded polymers of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Monomers are generally referred to as nucleotides. Nucleic acids may include modified nucleotides that allow polymerases to read them correctly without significantly altering the expression of the polypeptide encoded by the nucleic acid.

[0199] The phrase "coding nucleic acid sequence" refers to a nucleic acid, such as DNA, that serves as a template for transcription of a specific RNA molecule (such as shRNA, dsRNA, or mRNA translated into protein). Nucleic acid sequences include full-length nucleic acid sequences as well as non-full-length sequences derived from full-length sequences. Coding sequences may include degenerate codons (relative to the natural sequence) or sequences that provide codon preference in a particular host cell.

[0200] The term "promoter" refers to a region or sequence located upstream and / or downstream of the transcription start site and involved in the recognition and binding of RNA polymerases and other proteins to initiate transcription. A "yeast promoter" is a promoter capable of initiating transcription in yeast cells. A yeast promoter can be a nucleic acid sequence initially isolated from yeast, but for the purposes of this disclosure, promoters not initially isolated from yeast are also considered "yeast promoters."

[0201] An "expression cassette" is a nucleic acid construct that, when introduced into a host cell (such as a yeast cell), leads to the transcription of RNA molecules (such as dsRNA or mRNA). An expression cassette typically includes the sequence to be expressed and sequences necessary for expression of the desired sequence, such as a promoter that is operatively linked to that sequence. Typically, the expression cassette is inserted into an expression vector to be introduced into the host cell.

[0202] The term "complementarity" or "complementarity" refers to the ability of a nucleic acid in a polynucleotide to form base pairs with another nucleic acid in a second polynucleotide. For example, the sequence AGT is complementary to the sequence TCA. Complementarity can be partial, where only some nucleic acids match according to base pairing, or complete, such as perfect complementarity or complete complementarity, where all nucleic acids match according to base pairing.

[0203] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably to refer to amino acid polymers or groups of two or more interacting or bound amino acid polymers. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acid, as well as both naturally occurring and non-naturally occurring amino acid polymers.

[0204] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that have been modified.

[0205] In the context of two or more nucleic acids or proteins in this invention, the term "identical" or percentage "identity" refers to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotides or amino acids (i.e., approximately 60% identity within a specified region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity when comparing and aligning to achieve maximum correspondence within a comparison window or specified region), as measured by using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters or by manual alignment and visual inspection. See, for example, the NCBI website: ncbi.nlm.nih.gov / BLAST / . For example, the above techniques can be used to compare the dsRNA sequence of this invention with target gene sequences in mosquitoes, taking into account the presence of uracil in the dsRNA and thymine in the DNA. Sequences having at least approximately 90% sequence identity using the above methods are referred to as "substantially identical." This definition also refers to and can be applied to complements of test sequences. This definition also includes sequences with deletions and / or additions, as well as sequences with substitutions. Optimal alignment of such sequences can be performed using any publicly available algorithm or program for determining sequence identity and alignment, such as BLAST.

[0206] In some implementations, the reduction, inhibition, or suppression of target gene expression leads to life cycle disruption, such as reduced viability, growth, development, or reproduction of target mosquitoes (including larval and adult forms). Such assessments are within the capabilities of those skilled in the art and are described, for example, in Mysore et al., PLoS Negl Trop Dis., 14(7): e0008479.

[0207] Other methods for confirming downregulated gene expression are known in the art, including but not limited to using molecular techniques to measure mRNA or protein expression, such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with microarrays, antibody binding, enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, or fluorescence activated cell analysis (FACS).

[0208] Methods for engineering yeast modification

[0209] In some respects, methods have been provided for the stable integration of multinucleotide sequences encoding interfering RNA molecules into the genome of a host organism, including the use of nuclease systems and transposase systems, wherein the interfering RNA molecules suppress gene expression in mosquitoes. Therefore, methods for engineering host organisms to express interfering RNA molecules, wherein the interfering RNA molecules are capable of suppressing gene expression in mosquitoes, are disclosed. In this document, such engineered host organisms may be referred to as “biopesticides” or “interfering RNA biopesticides.”

[0210] In some embodiments, the disclosed method includes integrating an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule into the genome of a host organism stably integrated into the host organism. In some embodiments, integrating the expression cassette into the host organism's genome includes using nuclease systems and transposon systems. In some embodiments, the interfering RNA molecule suppresses gene expression in mosquitoes.

[0211] In some embodiments, a nuclease system is used to induce mutations in genes of the host organism, such as selection marker genes. In some embodiments, the mutation is a deletion mutation. In some embodiments, the mutation is a partial or complete deletion of the open reading frame (OPF) of the selection marker gene. In some embodiments, the mutation is a complete deletion of the OPF of the selection marker gene. In some embodiments, the OPF deletion originates from homology-directed repair, such as homology-directed repair initiated by nuclease activity.

[0212] In some embodiments, the selected marker gene is a dominant marker gene, a auxotroph rescue gene, or a combination thereof. In some embodiments, the dominant marker gene is KanMX, ble, Sh ble, hph, Cat, CUP1, SFA1, dehH1, PDR3-9, AUR1-C, nat, CYH2, pat, ARO4-, OFP, SMR1, FZF1-4, DsdA, or a combination thereof, of their orthologs.

[0213]

[0214] In some embodiments, a nuclease system is used to generate auxotrophic mutants in microbial cells, such as yeast cells. In some embodiments, a nuclease system is used to generate auxotrophic mutants or multiple auxotrophic mutants. In some embodiments, a nuclease system is used to generate histidine auxotrophs, leucine auxotrophs, tryptophan auxotrophs, or uracil auxotrophs. In some embodiments, a nuclease system is used to generate multiple auxotrophic microbial cells, such as yeast cells, having any two or more of the following: histidine auxotrophs, leucine auxotrophs, tryptophan auxotrophs, and uracil auxotrophs. In some embodiments, a nuclease system is used to generate multiple auxotrophic microbial cells, such as yeast cells, having both uracil and leucine auxotrophs. In some embodiments, a nuclease system is used to generate multiple auxotrophic microbial cells (such as yeast cells) having both histidine and tryptophan auxotrophs.

[0215] In some implementations, the auxotrophic mutation originates from a complete open reading frame (CRF) deletion in an auxotrophic rescue gene. In some implementations, the CRF deletion originates from homology-directed repair, such as homology-directed repair initiated by nuclease activity. In some implementations, the nuclease is Clo51, such as Clo51 in the Cas-CLOVER system.

[0216] In some embodiments, the auxotrophic mutation includes ura3Δ0, leu2Δ0, his3Δ0, trp1Δ0, or a combination thereof. In some embodiments, the auxotrophic mutant includes the auxotrophic mutations ura3Δ0 and leu2Δ0. In some embodiments, the auxotrophic mutant includes the auxotrophic mutations ura3Δ0, leu2Δ0, his3Δ0, and trp1Δ0.

[0217] The identity of transformants can be determined by the deletion and restoration of various selection markers, such as the stable integration of polynucleotide sequences or expression cassettes disclosed herein into host organisms, such as yeast cells. For example, protrophic markers, C / N source-related markers, resistance markers, and their manipulation are described, for example, by Siewers, Methods Mol Biol. 2014;1152:3-15.

[0218] In other instances, alternative gene-editing nuclease systems can be used to produce auxotrophic yeast strains suitable for use according to the disclosed methods. In yet another instance, auxotrophic yeast strains can be provided for the transposon-mediated integration of the disclosed shRNA and expression cassette in the production of RNA-interfering biopesticides.

[0219] Auxotrophic mutants carry mutations that prevent them from synthesizing essential compounds, such as mutations in genes that promote amino acid production or nucleotide biosynthesis. Unless the culture medium is supplemented with the necessary amino acids or nucleotides, auxotrophic strains cannot grow. For such strains, transformants can be selected using plasmids carrying wild-type copies of the mutated gene, provided that the mutation reversion frequency is lower than the transformation frequency. The latter can be ensured by using irreversible knockout mutations.

[0220] Auxotrophic selection biomarkers, such as auxotrophic rescue genes, can complement auxotrophic mutations and restore the growth of auxotrophic mutants. See, for example, Ulfstedt et al., Front Plant Sci. 2017 Nov 3;8:1850. Auxotrophic rescue genes such as URA3, LEU2, or HIS3 are ubiquitous in yeast genetics, where they are used to select cells that have been successfully transformed with recombinant DNA. Auxotrophic biomarkers and their applications are further described by Yuan, PLoS ONE 2011;6(10):e25830), Solis-Escalante et al., FEMS Yeast Res. 2013 Jan; 13(1):126–139, and wiki.yeastgenome.org (“Common Auxotrophic Biomarkers”).

[0221] In some embodiments, the disclosed method includes using a nuclease system to generate auxotrophic mutants or auxotrophic mutants. In some embodiments, the nuclease system comprises at least one guide RNA (gRNA) and a nuclease such as an endonuclease. In some embodiments, the nuclease system comprises one gRNA and a nuclease. In some embodiments, the nuclease system comprises at least two gRNAs and a nuclease. In some embodiments, the nuclease system comprises two gRNAs and a nuclease. In some embodiments, the nuclease is a dimer nuclease. In some embodiments, the nuclease system comprises two guide RNAs and a dimer nuclease. In some embodiments, the nuclease system is a Cas-CLOVER.

[0222] In some embodiments, the nuclease system comprises a gRNA, such as sgRNA, transcribed from a sequence having at least about 80% or 90% identity with the full length of SEQ ID NO:17, SEQ ID NO:18, or their complements. In some embodiments, the nuclease system comprises a gRNA, such as sgRNA, transcribed from a sequence having about 75%, 80%, 85%, 90%, 95%, or 100% identity with the full length of SEQ ID NO:17, SEQ ID NO:18, or their complements. In some embodiments, the nuclease system comprises two gRNAs transcribed from the full lengths of SEQ ID NO:17 and SEQ ID NO:18.

[0223] In some embodiments, the nuclease system comprises a gRNA, such as sgRNA, transcribed from a sequence having at least about 80% or 90% identity with the full length of SEQ ID NO:19, SEQ ID NO:20, or their complements. In some embodiments, the nuclease system comprises a gRNA, such as sgRNA, transcribed from a sequence having about 75%, 80%, 85%, 90%, 95%, or 100% identity with the full length of SEQ ID NO:19, SEQ ID NO:20, or their complements. In some embodiments, the nuclease system comprises two gRNAs transcribed from the full lengths of SEQ ID NO:19 and SEQ ID NO:20.

[0224] In some embodiments, the nuclease system comprises a dimer nuclease. In some embodiments, the dimer nuclease has at least about 80%, 85%, 90%, or 95% sequence identity with the full length of SEQ ID NO:16. In some embodiments, the dimer nuclease has about 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with the full length of SEQ ID NO:16. In some embodiments, the dimer nuclease is Clo051.

[0225] Cas-CLOVER is a dimer gene editing system that exhibits higher precision and no off-target effects compared to CRISPR-Cas9. While synthetic CRISPR uses a single guide RNA (sgRNA) and a CRISPR-associated protein (Cas) to bind to and cleave the target genomic sequence, Cas-CLOVER uses two gRNAs. Cas-CLOVER uses a non-catalytically inactive Cas fusion protein, or "dead Cas" (dCas), fused with the dimer Clo051 endonuclease domain.

[0226] In the Cas-CLOVER system, two gRNAs can be designed to target a gene to produce a double-strand break, a feature similar to that found in other dimeric gene editing technologies (i.e., ZFN and TALEN). The Cas-CLOVER system requires a protospacer adjacent motif (PAM) sequence in each RNA. Both guide RNAs should be designed in a PAM-out orientation. A flexible spacer range of 15-30 nucleotides between the two guides allows the dual complex to function. The flexibility of Cas-CLOVER guide RNA design allows users to target any gene.

[0227] The dual-synthetic gRNA / Cas-CLOVER complex interacts with the left and right complementary sequences within the targeted locus, leading to dimerization of the Clo051 nuclease domain and cleavage of the targeted locus. The generation of double-strand breaks activates DNA repair mechanisms, which repair the breaks via non-homologous end joining (NHEJ) or homologous recombination (HR). Various online open-source gRNA design tools are available to those skilled in the art, including CRISPR-MIT, E-CRISP, CHOPCHOP, CRISPOR, or ZiFit.

[0228] When using Cas-CLOVER in a plasmid-based editing system, both guide RNAs should be designed with the PAM facing outwards. Specifically, when designing and creating the left guide RNA, the sequence matches the bottom strand of the selected guide sequence, read in a 5' to 3' orientation toward the PAM site. This 5' to 3' sequence should be cloned into the vector between the appropriate promoter and guide scaffold for the system. When designing and creating the right guide sequence, the sequence matches the top strand, read in a 5' to 3' orientation toward the PAM site. The 5' to 3' sequence of this guide sequence should also be cloned into the vector between the appropriate promoter and guide scaffold. Various methods exist for creating guide RNAs for cloning into vectors, including synthesizing fragments or annealing two single-stranded oligonucleotides to form a double-stranded fragment. Several other methods for cloning guide RNAs into vectors are also readily apparent to those skilled in the art. Choosing left and right guides separated by 15-30 nucleotide spacers is crucial (Demeetra, “Designing Cas-CLOVER: A Dimeric RNA Guided Targeted Nuclease for Precision Gene-Editing,” User Guide. Cas-CLOVER for Gene Editing).

[0229] An exemplary dCas9-Clo051 fusion protein (referred to in the art as the "Cas-CLOVER" protein) and the polynucleotide sequence encoding the dCas9-Clo051 fusion protein are described in detail in U.S. Patent Publication No. 2022 / 0042038, the contents of which are incorporated herein by reference in their entirety. Gene editing compositions including Cas-CLOVER and methods for gene editing using these compositions are described in detail in U.S. Patent Publications Nos. 2017 / 0107541, 2017 / 0114149, 2018 / 0187185, and US20160060610A1, the contents of which are incorporated herein by reference in their entirety.

[0230] In some embodiments, the disclosed methods include integrating an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule into the genome of a host organism via transposition. In this document, transposition refers to a transposon-mediated method for integrating a transgene (including an expression cassette carrying the transgene) into the genome of a host organism.

[0231] In some embodiments, a transposon system is used, for example, to integrate an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule into the genome of a host organism. In some embodiments, the transposon system comprises a transposon vector and a transposase. In some embodiments, the disclosed method includes integrating the expression cassette into at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 sites in the genome of a host organism. In some embodiments, the disclosed method includes integrating the expression cassette into 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 sites in the genome of a host organism.

[0232] In some embodiments, the disclosed methods include using transposon systems such as piggyBac, Super piggyBac, Sleeping Beauty, Hyperactive Sleeping Beauty, helitron transposon system, Tol2 transposon system, and TcBuster transposon system, including, for example, piggyBac-like systems or mutant transposon systems thereof, to integrate an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule into the genome of a host organism. In some embodiments, the nucleotide sequence encoding a transposase is operatively linked to a constitutive promoter. In some embodiments, the nucleotide sequence encoding a transposase is operatively linked to an ADH1 promoter or a derivative thereof (e.g., a truncated version).

[0233] In some embodiments, the disclosed methods include integrating an expression cassette containing an inverted terminal repeat sequence into the genome of a host organism via transposition, such as using a transposon system. In some embodiments, the transposon system comprises a transposon vector and a transposase. In some embodiments, the transposase is a piggyBac™ (PB) transposase, a piggyBac-like (PBL) transposase, a Super piggyBac™ (SPB) transposase polypeptide, a Sleeping Beauty transposase, a Hyperactive Sleeping Beauty (SB100X) transposase, a helitron transposase, a Tol2 transposase, a TcBuster transposase, or a mutant TcBuster transposase.

[0234] In some embodiments, the disclosed method includes integrating an expression cassette containing 5' and 3' inverted terminal repeat sequences into the genome of a host organism. In some embodiments, the inverted terminal repeat sequences are recognized by piggyBac, Super PiggyBac, or piggyBac-like transposases. In some embodiments, the inverted terminal repeat sequences are recognized by Sleeping Beauty transposases. In some embodiments, the inverted terminal repeat sequences are recognized by Helraiser transposons. In some embodiments, the inverted terminal repeat sequences are recognized by Tol2 transposases. In some embodiments, the inverted terminal repeat sequences are recognized by TcBuster transposases.

[0235] In some embodiments, the 5' inverted terminal repeat sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:5. In some embodiments, the 5' inverted terminal repeat sequence has about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:5. In some embodiments, the 5' inverted terminal repeat sequence is composed of the full length of SEQ ID NO:5.

[0236] In some embodiments, the 3' inverted terminal repeat sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:4. In some embodiments, the 3' inverted terminal repeat sequence has about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:4. In some embodiments, the 3' inverted terminal repeat sequence is composed of the full length of SEQ ID NO:4.

[0237] Inverted terminal repeats, such as transposon-specific inverted terminal repeats, can be recognized by transposases such as SuperPiggyBac transposases. PiggyBac (PB) transposons are mobile genetic elements that efficiently transpose between vectors and chromosomes via a "cut-and-paste" mechanism. During transposition, SuperPB transposases recognize transposon-specific inverted terminal repeats (ITRs) located at both ends of the transposon vector, move the contents from the original site, and efficiently integrate them into the TTAA chromosomal site. See, for example, System Biosciences, 2014, "PiggyBac Transposon System".

[0238] In some embodiments, the disclosed method includes integrating an expression cassette into the genome of a host organism, wherein the expression cassette further comprises a promoter. In some embodiments, the promoter is flanked at the 5' end of a nucleotide sequence encoding an interfering RNA molecule. In some embodiments, the promoter is operatively linked at the 5' end to a nucleotide sequence encoding an interfering RNA molecule. In some embodiments, the promoter separates the nucleotide sequence encoding the interfering RNA molecule from a 5' inverted terminal repeat sequence. In some embodiments, the promoter is a glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter or a derivative thereof.

[0239] In some embodiments, the GAP promoter comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:11. In some embodiments, the GAP promoter comprises a nucleotide sequence having 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:11. In some embodiments, the GAP promoter comprises a nucleotide sequence having 100% identity with the full length of SEQ ID NO:11. In some embodiments, the GAP promoter consists of a nucleotide sequence having 100% identity with the full length of SEQ ID NO:11.

[0240] In some embodiments, the disclosed method includes integrating an expression cassette into the genome of a host organism, wherein the expression cassette further includes a terminator such as a transcription terminator. In some embodiments, the terminator is located at the 3' end flanking the nucleotide sequence encoding the interfering RNA molecule. In some embodiments, the terminator separates the nucleotide sequence encoding the interfering RNA molecule from the 3' inverted terminal repeat sequence. In some embodiments, the terminator is a CYC1 terminator.

[0241] In some embodiments, the CYC1 terminator comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:12. In some embodiments, the CYC1 terminator comprises a nucleotide sequence having 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:12. In some embodiments, the CYC1 terminator comprises a nucleotide sequence having 100% identity with the full length of SEQ ID NO:12. In some embodiments, the CYC1 terminator consists of a nucleotide sequence having 100% identity with the full length of SEQ ID NO:12.

[0242] In some embodiments, the disclosed method includes integrating an expression cassette into the genome of a host organism, wherein the expression cassette further comprises a selection marker promoter operatively linked to a selection marker gene. In some embodiments, the selection marker is an auxotrophic marker, such as an auxotrophic rescue gene. In some embodiments, the selection marker promoter is flanked by a 5' inverted terminal repeat sequence. In some embodiments, the selection marker promoter is a minimal promoter. In some embodiments, the minimal promoter is truncated or mutated. In some embodiments, the selection marker promoter is a minimal auxotrophic rescue promoter. In this document, "auxotrophic rescue promoter" is interchangeable with "auxotrophic marker promoter."

[0243] In some embodiments, the disclosed method includes integrating an expression cassette into the genome of a host organism, wherein the expression cassette further comprises a auxotrophic rescue promoter operatively linked to an auxotrophic rescue gene. In some embodiments, the auxotrophic rescue promoter is flanked by a 5' inverted terminal repeat sequence. In some embodiments, the auxotrophic rescue promoter is a minimal auxotrophic rescue promoter. In some embodiments, the minimal auxotrophic rescue promoter is truncated or mutated.

[0244] In some embodiments, the minimal auxotroph rescue promoter is a truncated leucine promoter, and the auxotroph rescue gene is leucine. In some embodiments, the minimal promoter comprises a nucleotide sequence having at least 70%, at least 80%, or at least 90% identity with the full length of SEQ ID NO:3. In some embodiments, the minimal promoter comprises a nucleotide sequence having 70%, 73%, 77%, 80%, 83%, 87%, 90%, 93%, 97%, or 100% identity with the full length of SEQ ID NO:3. In some embodiments, the minimal promoter comprises a nucleotide sequence having 100% identity with the full length of SEQ ID NO:3. In some embodiments, the minimal promoter consists of a nucleotide sequence having 100% identity with the full length of SEQ ID NO:3.

[0245] In some embodiments, the insulator sequence separates the 5' end of the nucleotide sequence encoding the interfering RNA molecule from the 5' inverted terminal repeat sequence. In some embodiments, the insulator sequence separates the 3' end of the nucleotide sequence encoding the interfering RNA molecule from the 3' inverted terminal repeat sequence.

[0246] In some embodiments, the disclosed method includes integrating an expression cassette into the genome of a host organism, wherein the expression cassette further comprises an insulator sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:6. In some embodiments, the insulator sequence has approximately 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the full length of SEQ ID NO:6. In some embodiments, the insulator sequence consists of a nucleotide sequence having 100% identity with the full length of SEQ ID NO:6.

[0247] In some embodiments, the disclosed method includes integrating an expression cassette into the genome of a host organism, wherein the expression cassette contains at least 1, 3, 5, 7, 9, or 10 copies of a nucleotide sequence encoding an interfering RNA molecule. In some embodiments, the expression cassette contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of a nucleotide sequence encoding an interfering RNA molecule.

[0248] In some embodiments, the disclosed method includes integrating an expression cassette into the genome of a host organism, wherein the expression cassette contains a nucleotide sequence encoding an interfering RNA molecule that suppresses gene expression in *Aedes*, *Anopheles*, or *Culex* species. In some embodiments, the interfering RNA molecule suppresses gene expression in *Anopheles gambiae*, *Aedes aegypti*, *Aedes albopictus*, *Culex pipiens sharpii*, or *Culex quinquefolius*. In some embodiments, the interfering RNA molecule suppresses the expression of *Shaker* in *Aedes*, *Anopheles*, or *Culex* species, such as *Anopheles gambiae*, *Aedes aegypti*, *Aedes albopictus*, *Culex pipiens sharpii*, or *Culex quinquefolius*.

[0249] In some embodiments, the disclosed method includes integrating an expression cassette into the genome of a host organism, wherein the expression cassette contains a nucleotide sequence encoding an interfering RNA molecule targeting the Shaker gene in mosquitoes. In some embodiments, the interfering RNA molecule is capable of suppressing Shaker expression in mosquitoes. In some embodiments, the nucleotide sequence transcribed from the nucleotide sequence encoding the interfering RNA molecule is partially or completely complementary to a sequence containing at least 80%, 84%, 88%, 92%, 96%, or 100% identity with the full length of SEQ ID NO:2. In some embodiments, the interfering RNA molecule contains at least 25 consecutive nucleotides, said at least 25 consecutive nucleotides being partially or completely complementary to the full length of SEQ ID NO:2. In some embodiments, the nucleotide sequence encoding the interfering RNA molecule is integrated into the genome of a host organism via transposition, such as stable integration into the genome of a host organism.

[0250] In some embodiments, the disclosed method includes integrating an expression cassette into the genome of a host organism, wherein the expression cassette contains a nucleotide sequence encoding an interfering RNA molecule shRNA.463 (also referred to herein as “shRNA_463”) (represented by SEQ ID NO:1), the nucleotide sequence flanking an inverted terminal repeat sequence. In some embodiments, the expression cassette contains a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:1, wherein the nucleotide sequence encodes an interfering RNA molecule and is flanked by an inverted terminal repeat sequence. In some embodiments, the nucleotide sequence has 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100% identity with the full length of SEQ ID NO:1, wherein the nucleotide sequence encodes an interfering RNA molecule and is flanked by an inverted terminal repeat sequence. In some embodiments, the nucleotide sequence encoding the interfering RNA molecule consists of 100% identity with the full length of SEQ ID NO:1, wherein the nucleotide sequence encodes an interfering RNA molecule and is flanked by an inverted terminal repeat sequence. In some implementations, the inverted terminal repeat sequence is flanked at the 5' and 3' ends of the nucleotide sequence encoding the interfering RNA molecule.

[0251] In some embodiments, the disclosed method includes integrating an expression cassette into the genome of a host organism, wherein the expression cassette contains a nucleotide sequence having 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100% identity with the full length of SEQ ID NO:1, flanked at the 5' end by an inverted terminal repeat sequence having approximately 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:4. In some embodiments, the nucleotide sequence has 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100% identity with the full length of SEQ ID NO:1, flanked at the 3' end by an inverted terminal repeat sequence having approximately 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:5.

[0252] In some embodiments, the disclosed method includes integrating an expression cassette into the genome of a host organism, wherein the expression cassette contains at least 1, 3, 5, 7, 9, or 10 copies of a nucleotide sequence encoding an interfering RNA molecule, such as SEQ ID NO:1 or a sequence having at least 80% or 90% identity with SEQ ID NO:1.

[0253] In some embodiments, the disclosed method includes integrating an expression cassette into the genome of a host organism, wherein the expression cassette comprises a nucleotide sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the full length of SEQ ID NO:7 or its complement. In some embodiments, the expression cassette comprises a nucleotide sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the full length of SEQ ID NO:8 or its complement.

[0254] In some embodiments, the expression cassette comprises a nucleotide sequence having approximately 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the full length of SEQ ID NO:7 or its complement. In some embodiments, the expression cassette comprises a nucleotide sequence having approximately 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the full length of SEQ ID NO:8 or its complement.

[0255] In some embodiments, the disclosed method includes using a transposase to integrate an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule into the genome of a host organism, the transposase containing a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with the full length of SEQ ID NO:10. In some embodiments, the transposase contains a nucleotide sequence having about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity with the full length of SEQ ID NO:10.

[0256] Super piggyBac transposase is represented by SEQ ID NO:10. The activity of the piggyBac transposon system allows for the easy transfer of a target gene between two (5' and 3') inverted terminal repeat sequences in the piggyBac vector into the target genome. In some embodiments, the Super piggyBac transposase is operatively linked to a constitutive promoter. In some embodiments, the Super piggyBac transposase is operatively linked to an ADH1 promoter, such as a strong ADH1 promoter or a derivative thereof, such as a truncated version. SEQ ID NO:21 represents a full-length ADH1 promoter. In some embodiments, SEQ ID NO:10 or a nucleotide sequence having at least 70%, at least 80%, at least 90%, or at least 95% identity with the full-length SEQ ID NO:10 is operatively linked to an ADH1 promoter or a derivative thereof, such as a truncated ADH1 promoter.

[0257] In some embodiments, the disclosed method includes integrating an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule into the genome of a microbial host organism, such as a fungal cell. In some embodiments, the microbial host cell is a yeast cell. In some embodiments, the host organism is a species of *Pichia pastoris* (e.g., *Pichia pastoris*), a species of *Saccharomyces cerevisiae* (e.g., *Saccharomyces cerevisiae*), or a species of *Yersinia lipolytica* (e.g., *Yersinia lipophila*). In some embodiments, the host organism is *Saccharomyces cerevisiae*. In some embodiments, the host organism is *Saccharomyces cerevisiae* strain FL100 or *Saccharomyces cerevisiae* strain S288C.

[0258] In some embodiments, the disclosed method includes integrating an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule into the genome of *Saccharomyces cerevisiae* (e.g., strain FL100) at any one or more of the following integration sites: chromosome IV (NC_001136, position 543,705); chromosome IV (NC_001136, position 1,357,520); chromosome VIII (NC_001140, position 124,029); chromosome X (NC_001142, position 181,309); chromosome XI (NC_001143, position 300,654); and chromosome XII (NC_001144, position 213,991).

[0259] The integration of expression cassettes into the host organism's genome can be confirmed using methods known to those skilled in the art. For example, PCR and sequencing can be used to confirm the integration of expression cassettes, such as those containing nucleotide sequences encoding interfering RNA molecules.

[0260] Specifically, regarding shRNA Sh.4643, expression was confirmed by extracting total RNA from 5.6 mg of clumped yeast obtained from a culture prepared according to Hapaairai et al., Sci Rep. 2017;7(1):13223, following the manufacturer's instructions (Invitrogen, Carlsbad, CA). cDNA was prepared according to the instructions provided in the High-Capacity RNA to cDNA Kit (AppliedBiosystems, Foster City, CA), and 1 / 100 of the resulting cDNA was used as a template for PCR amplification using Clontech Labs 3P TaKaRa Taq DNA polymerase (Clontech Laboratories, MountainView, CA), as described in Mysore et al., PLoS Negl Trop Dis. 2020 Jul; 14(7):e0008479.

[0261] Genetically modified host organisms and their components

[0262] In some respects, this article provides polynucleotide sequences and genetically modified host organisms containing these sequences, designed to produce interfering RNA molecules, such as interfering RNA insecticides. These interfering RNA insecticides can interfere with gene expression in mosquitoes, such as inhibiting the expression of target genes.

[0263] Polynucleotide sequences and expression cassettes

[0264] In some embodiments, this document provides nucleotide sequences and expression cassettes containing nucleotide sequences encoding interfering RNA molecules capable of suppressing gene expression in mosquitoes. In some embodiments, the nucleotide sequence encoding the interfering RNA molecule is flanked by inverted terminal repeat sequences. In some embodiments, the nucleotide sequence encoding the interfering RNA molecule is flanked by 5' inverted terminal repeat sequences and 3' inverted terminal repeat sequences. In some embodiments, the interfering RNA molecule is an RNA construct, double-stranded RNA (dsRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), or antisense oligonucleotide.

[0265] In some embodiments, the 5' inverted terminal repeat sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:5. In some embodiments, the 5' inverted terminal repeat sequence has about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:5. In some embodiments, the 5' inverted terminal repeat sequence is composed of the full length of SEQ ID NO:5.

[0266] In some embodiments, the 3' inverted terminal repeat sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:4. In some embodiments, the 3' inverted terminal repeat sequence has about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:4. In some embodiments, the 3' inverted terminal repeat sequence is composed of the full length of SEQ ID NO:4.

[0267] In some implementations, the disclosed expression cassette contains an inverted terminal repeat sequence. The PiggyBac inverted terminal repeat element is a short inverted terminal repeat (ITR) transposable element, 2.5 kb in length, with a 13 bp ITR sequence and a 2.1 kb ORF. (See, for example, Handler et al., PNAS 1998;95(13): 7520-7525; Cary et al. Virology 1989;161, 8–17; Elick et al. Genetica 1995;97, 127–139). It is part of a subclass of ITR elements that have so far been found only in Lepidoptera and are specifically inserted into TTAA target sites. See, for example, Beames & Summers, Virology 1990;174: 354–363; Fraser et al., Virology 1995;211: 397–407; Wang & Fraser, Insect Mol Biol 1993;1: 109–116.

[0268] In some embodiments, the disclosed expression cassette also includes a promoter. In some embodiments, the promoter is flanked at the 5' end by a nucleotide sequence encoding an interfering RNA molecule. In some embodiments, the promoter is operatively linked at the 5' end to a nucleotide sequence encoding an interfering RNA molecule. In some embodiments, the promoter separates the nucleotide sequence encoding the interfering RNA molecule from a 5' inverted terminal repeat sequence. In some embodiments, the promoter is a glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter.

[0269] A promoter is a control sequence in a nucleic acid sequence that controls the initiation and rate of transcription. It can contain genetic elements that regulate proteins and molecules such as RNA polymerases and transcription factors to bind to initiate transcription of the nucleic acid sequence. The phrase "operably linked" means that expression control elements (such as promoters) are positioned and / or oriented relative to the nucleic acid to control the initiation and / or expression of the nucleic acid.

[0270] Promoters can be promoters naturally associated with nucleic acid sequences, for example, obtained by isolating a 5' non-coding sequence located upstream of a coding region. Alternatively, promoters can be recombinant or heterologous promoters, referring to promoters that are not typically associated with nucleic acid fragments in their natural environment. Such promoters can include promoters of other genes and non-naturally occurring promoters. Expression control elements can be derived from yeast of the species or strains from which RNA interference (RNAi) will be used or where the RNAi pathway will be modified. For example, if RNAi is to be used in Saccharomyces cerevisiae, it may be necessary to use Saccharomyces cerevisiae promoters to guide the expression of dsRNA. However, any expression control element capable of guiding transcription in the target cell can be used.

[0271] The promoters used can be constitutive or inducible. For example, various yeast-specific promoters can be used to regulate expression in yeast cells. Examples of inducible yeast promoters include GAL1-10, GAL1, GALL, GALS, TET, CUP1, VP16, and VP16-ER. Examples of repressible yeast promoters include Met25. Examples of constitutive yeast promoters include glyceraldehyde-3-phosphate dehydrogenase promoters (also known as GAP, GPD, and TDH3), phosphoglycerate kinase (PGK), alcohol dehydrogenase promoters (ADH), translation elongation factor-1-α promoters (TEF), cytochrome c-oxidase promoters (CYC1), and MRP7. Promoters containing steroid response elements (such as glucocorticoid response elements) that can be induced by glucocorticoids or other steroid hormones can also direct expression in yeast. Other constitutive or inducible yeast promoters can also be used, such as promoters of α-factor genes, phosphate pathway genes (such as PH05), or alcohol oxidases. In some implementations, the vector contains an expression control element called an upstream activation sequence (UAS).

[0272] Such elements can activate gene transcription from distal locations, such as up to about 1,000 to 1,200 bp from the promoter, and are considered functional equivalents of metazoan enhancers. For discussion, see, for example, Petrascheck, M, et al., Nucleic Acids Res., 33(12): 3743-3750, 2005. Expression levels achieved using inducible promoters can be modulated, for example, by controlling the amount of inducer or the length of exposure. Furthermore, mutant promoters that result in lower expression levels than wild-type promoters can be used. In some embodiments, the expression control element is derived from the species to which the element will guide expression, while in other embodiments, the expression control element is derived from a different species.

[0273] Suitable promoters for expression in yeast are well known, including, for example, the phage T7 promoter, promoters derived from GAL1 (which are galactose-induced), ADH1, TEF1 promoters, and AOX promoters (methanol-inducible promoters). Many yeast cloning vectors have been designed and are readily available. Methods for transforming Saccharomyces cerevisiae cells with exogenous DNA to produce recombinant polypeptides are also well known. Transformed cells are selected by phenotype determined by selection markers, typically drug resistance or the ability to grow in the absence of specific nutrients such as leucine. The application and use of the GAP promoter have been documented, for example, in Waterham et al., Gene. 1997 Feb 20;186(1):37-44.

[0274] Some promoters can also be used for the expression of small RNAs, such as single guide RNAs (sgRNAs) that participate in defining target sequences that define endonuclease activity. One method for preparing sgRNA involves expressing the guide RNA cloned into a plasmid vector in a host cell. In some instances, the cell uses its normal RNA polymerase to transcribe genetic information from newly introduced DNA to generate sgRNA. In some embodiments, the nucleotide sequence encoding the sgRNA is operatively linked to a promoter, such as an RNA polymerase promoter, a tRNA promoter, or a hybrid RNA polymerase-tRNA promoter. In some embodiments, the RNA polymerase promoter is an RNA pol II or RNA pol III promoter. In some embodiments, RNA pol II is an ADH promoter. In some embodiments, RNA pol III is an SNR52 promoter or a derivative thereof.

[0275] In some embodiments, the disclosed expression cassette comprises a nucleotide sequence encoding a gRNA such as sgRNA, along with its promoter and terminator. In some embodiments, the SNR52 promoter has at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:13. In some embodiments, the SNR52 promoter has about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:13. In some embodiments, the SNR52 promoter consists entirely of the full length of SEQ ID NO:13.

[0276] The SNR52 promoter is unique among Pol III promoters because it possesses a natural cleavage site that leads to the excision of the sgRNA from the primary transcript. See, for example, DiCarlo et al., Nucleic Acids Res. 2013;41: 4336-4343 and Marck et al., Nucleic Acids Res. 2006;34 (6):1816-1835. In some instances, promoters operatively linked to the nucleotide sequence encoding the sgRNA can be optimized to improve transcription efficiency and mutation frequency, for example, as described in Ng & Dean, mSphere. 2017 Mar-Apr; 2(2): e00385-16 and Schwartz et al., ACSSynth. Biol. 2016, 5, 4, 356–359.

[0277] In some embodiments, the nucleotide sequence encoding the gRNA is transcribed and terminated, such as a gRNA terminator. In some embodiments, the gRNA terminator is the SNR52 terminator. In some embodiments, the SNR52 terminator comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:14. In some embodiments, the SNR52 terminator comprises a nucleotide sequence having about 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:14. In some embodiments, the SNR52 terminator comprises a nucleotide sequence having 100% identity with the full length of SEQ ID NO:14. In some embodiments, the SNR52 terminator consists of a nucleotide sequence having 100% identity with the full length of SEQ ID NO:14.

[0278] In some embodiments, the disclosed expression cassette further includes a promoter sequence, such as the GAP promoter. In some embodiments, the GAP promoter comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:11. In some embodiments, the GAP promoter comprises a nucleotide sequence having 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:11. In some embodiments, the GAP promoter comprises a nucleotide sequence having 100% identity with the full length of SEQ ID NO:11. In some embodiments, the GAP promoter consists of a nucleotide sequence having 100% identity with the full length of SEQ ID NO:11.

[0279] In some embodiments, the disclosed expression cassette further includes a terminator, such as a transcription terminator. In some embodiments, the terminator is located at the 3' end flanking the nucleotide sequence encoding the interfering RNA molecule. In some embodiments, the terminator separates the nucleotide sequence encoding the interfering RNA molecule from the 3' inverted terminal repeat sequence. In some embodiments, the terminator is a CYC1 terminator or a derivative thereof.

[0280] In some embodiments, the disclosed expression cassette includes a CYC1 terminator comprising a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:12. In some embodiments, the disclosed expression cassette includes a CYC1 terminator comprising a nucleotide sequence having 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:12. In some embodiments, the disclosed expression cassette includes a CYC1 terminator comprising a nucleotide sequence having 100% identity with the full length of SEQ ID NO:12. In some embodiments, the disclosed expression cassette includes a CYC1 terminator composed of a nucleotide sequence having 100% identity with the full length of SEQ ID NO:12.

[0281] The CYC1 terminator is a DNA fragment with a specific structure that marks the termination of transcription. It is located on the X chromosome of Saccharomyces cerevisiae strain FL100 from 526690 bp to 526939 bp. Synthetic terminators used for heterologous gene expression in yeast (including the CYC1 terminator and its derivatives) are described in, for example, Curran et al., ACS Synthetic Biology, 2015;4(7):824-832, Mumberg et al., Gene. 1995 Apr 14;156(1):119-22, Zaret & Sherman, Cell. 1982 Mar;28(3):563-73.

[0282] In some embodiments, the disclosed expression cassette contains a nucleotide sequence encoding a gRNA such as sgRNA. In some embodiments, the nucleotide sequence terminating transcription of a sequence encoding a gRNA such as a gRNA terminator is the SNR52 terminator or a derivative thereof. In some embodiments, the disclosed expression cassette contains an SNR52 terminator containing a nucleotide sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:14. In some embodiments, the disclosed expression cassette contains an SNR52 terminator containing a nucleotide sequence having about 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:14. In some embodiments, the disclosed expression cassette contains an SNR52 terminator containing a nucleotide sequence having 100% identity with the full length of SEQ ID NO:14. In some embodiments, the disclosed expression cassette contains an SNR52 terminator consisting of a nucleotide sequence that is 100% identical to the full length of SEQ ID NO:14.

[0283] In some embodiments, the disclosed expression cassette further includes a selection marker promoter operatively linked to the selection marker gene. In some embodiments, the selection marker is a auxotrophic marker, such as an auxotrophic rescue gene. In some embodiments, the selection marker promoter is flanked by a 5' inverted terminal repeat sequence. In some embodiments, the selection marker promoter is a minimal promoter. In some embodiments, the minimal promoter is truncated or mutated. In some embodiments, the selection marker promoter is a minimal auxotrophic rescue promoter.

[0284] In some embodiments, the disclosed expression cassette further includes a auxotrophic rescue promoter operatively linked to the auxotrophic rescue gene. In some embodiments, the auxotrophic rescue promoter is flanked by a 5' inverted terminal repeat sequence. In some embodiments, the auxotrophic rescue promoter is a minimal auxotrophic rescue promoter. In some embodiments, the minimal auxotrophic rescue promoter is truncated or mutated.

[0285] In some embodiments, the minimal auxotroph rescue promoter is a truncated leucine promoter, and the auxotroph rescue gene is leucine. In some embodiments, the minimal promoter comprises a nucleotide sequence having at least 70%, at least 80%, or at least 90% identity with the full length of SEQ ID NO:3. In some embodiments, the minimal promoter comprises a nucleotide sequence having 70%, 73%, 77%, 80%, 83%, 87%, 90%, 93%, 97%, or 100% identity with the full length of SEQ ID NO:3. In some embodiments, the minimal promoter comprises a nucleotide sequence having 100% identity with the full length of SEQ ID NO:3. In some embodiments, the minimal promoter consists of a nucleotide sequence having 100% identity with the full length of SEQ ID NO:3.

[0286] In some embodiments, the disclosed expression cassette containing a minimal or truncated promoter operatively linked to a selectable marker gene increases the expression level of the interfering RNA molecule, relative to an expression cassette containing a native or full-length promoter operatively linked to the selectable marker gene. In some embodiments, the expression level of the interfering RNA molecule is increased by 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500%.

[0287] In some embodiments, the expression level of the interfering RNA molecule is increased by at least 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500% relative to the expression cassette containing the natural promoter operatively linked to the selection marker gene. In some embodiments, the expression level of the interfering RNA molecule is increased by approximately 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500% relative to the expression cassette containing the natural promoter operatively linked to the selection marker gene. In some implementations, the expression level is 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold greater than that of a natural promoter containing an expression cassette operatively linked to a selection marker gene.

[0288] Compared to natural promoters that drive auxotrophic rescue gene expression, the integration cassettes with minimal, defective, or truncated promoters described herein result in increased expression of interfering RNA molecules. Minimal promoters can be truncated or mutated compared to natural promoters. See, for example, Parker & Newstead, Protein Sci. 2014 Sep;23(9):1309-14 and Tang et al., Metabolites. 2020 Aug 6;10(8):320. Truncating endogenous promoters to remove non-essential bases is one strategy for constructing minimal promoters. However, minimally truncated promoters contain elements from endogenous promoters and are susceptible to homologous recombination. Therefore, mutagenesis, such as saturation mutagenesis, is another strategy for creating minimal promoters. See, for example, Tang et al., Metabolites. 2020 Aug 6;10(8):320.

[0289] In some embodiments, the disclosed expression cassette further includes a selection rescue gene. In some embodiments, the selection marker gene is a auxotrophic rescue gene, such as a auxotrophic marker gene for a complementary auxotrophic mutation. In some embodiments, the auxotrophic rescue gene encodes an amino acid, such as an essential amino acid. In some embodiments, the essential amino acid is histidine, leucine, tryptophan, uracil, or a combination thereof. In some embodiments, the auxotrophic rescue gene is leu2, ura3, his3, trp1, their orthologs, or a combination thereof. In some embodiments, the disclosed expression cassette includes a auxotrophic rescue promoter dleu2 operatively linked to leu2, represented by SEQ ID NO:3.

[0290] Using auxotrophic mutant host organisms helps select cells that have successfully integrated publicly available expression cassettes into the host cell genome, i.e., cell growth indicates auxotrophic complementation or protrophic restoration, such as genome integration mediated by transposons of publicly available expression cassettes. Therefore, the selection of auxotrophic rescue promoters and auxotrophic rescue genes is associated with auxotrophic mutations. Auxotrophic genes are required for growth in the absence of essential nutrients such as essential amino acids. For example, a auxotrophic URA3 gene mutation results in the inability to grow in uracil-deficient media because URA3 encodes a key enzyme in the de novo pathway of uracil biosynthesis (Lacroute, J Bacteriol 95:824–832). As described herein, growth of the ura3 mutant in uracil-deficient media can be restored if cells have been transformed with DNA containing the URA3 gene, such as by transposition. Other selection and anti-selection techniques are described, for example, Yuan, PLoSONE 2011;6(10):e25830.

[0291] In some embodiments, the disclosed expression cassette further includes an insulator sequence. In some embodiments, the insulator sequence separates the 5' end of the nucleotide sequence encoding the interfering RNA molecule from the 5' inverted terminal repeat sequence. In some embodiments, the insulator sequence separates the 3' end of the nucleotide sequence encoding the interfering RNA molecule from the 3' inverted terminal repeat sequence.

[0292] In some embodiments, the disclosed expression cassette comprises an insulator sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:6. In some embodiments, the disclosed expression cassette comprises an insulator sequence having approximately 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the full length of SEQ ID NO:6. In some embodiments, the insulator sequence consists of a nucleotide sequence having 100% identity with the full length of SEQ ID NO:6.

[0293] Insulators are genomic sequence elements that help organize the eukaryotic genome into coherent regulatory domains. Insulators can encode both enhancer blocking activity and / or chromatin barrier activity that helps delineate active and repressive chromatin domains, the enhancer blocking activity preventing interactions between enhancers located in different regulatory domains and promoters. Thus, insulators are cis-regulatory elements that block erroneous gene activation or heterochromatin diffusion imposed by remote enhancers and silencers. Therefore, insulators have been used for the stabilization of transgenes, such as those delivered by viral vectors. See, for example, Wang et al., Proc Natl Acad Sci USA. 2015 Aug 11;112(32):E4428-37. Insulator sequences have also been described in the context of transposons or transposon-mediated gene delivery. See, for example, Bire et al., PLoS One. 2013; 8(12): 1-10 and Mossine et al., PLoS One. 2013; 8(12): e85494.

[0294] In some embodiments, the disclosed expression cassette encodes an interfering RNA molecule that suppresses gene expression in mosquitoes belonging to the genera *Aedes*, *Anopheles*, or *Culex*. In some embodiments, the disclosed expression cassette contains a nucleotide sequence encoding an interfering RNA molecule that suppresses gene expression in *Anopheles gambiae*, *Aedes aegypti*, *Aedes albopictus*, *Culex pipiens sharpii*, or *Culex quinquefolius*. In some embodiments, the disclosed expression cassette contains a nucleotide sequence encoding an interfering RNA molecule that suppresses the expression of *Shaker* in *Aedes*, *Anopheles*, or *Culex* species, such as *Anopheles gambiae*, *Aedes aegypti*, *Aedes albopictus*, *Culex pipiens sharpii*, or *Culex quinquefolius*.

[0295] In some embodiments, the disclosed expression cassette contains a nucleotide sequence encoding an interfering RNA molecule, wherein the interfering RNA molecule is an RNA construct, double-stranded RNA (dsRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), or an antisense oligonucleotide. In some embodiments, the disclosed expression cassette contains inverted terminal repeat sequences flanking the nucleotide sequence encoding the interfering RNA molecule.

[0296] In some embodiments, the disclosed expression cassette contains a nucleotide sequence encoding an interfering RNA molecule targeting the Shaker gene in mosquitoes. In some embodiments, the interfering RNA molecule is capable of suppressing the expression of the Shaker gene in mosquitoes. In some embodiments, the nucleotide sequence transcribed from the nucleotide sequence encoding the interfering RNA molecule is partially or completely complementary to a sequence containing at least 80%, 84%, 88%, 92%, 96%, or 100% identity with the full length of SEQ ID NO:2. In some embodiments, the disclosed expression cassette encodes an interfering RNA molecule containing at least 25 consecutive nucleotides that are partially or completely complementary to the full length of SEQ ID NO:2. In some embodiments, the nucleotide sequence encoding the interfering RNA molecule is integrated into the genome of a host organism via transposition, such as stable integration into the genome of a host organism.

[0297] In some embodiments, the disclosed expression cassette comprises a nucleotide sequence encoding an interfering RNA molecule shRNA.463, represented by SEQ ID NO:1, wherein the nucleotide sequence is flanked by inverted terminal repeat sequences. In some embodiments, the disclosed expression cassette comprises a nucleotide sequence encoding an interfering RNA molecule that is 100% identical to the full length of SEQ ID NO:1, wherein the nucleotide sequence encodes the interfering RNA molecule and is flanked by inverted terminal repeat sequences. In some embodiments, the inverted terminal repeat sequences are located flanking the 5' and 3' ends of the nucleotide sequence encoding the interfering RNA molecule.

[0298] In some embodiments, the disclosed expression cassette comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:1, wherein the nucleotide sequence encodes an interfering RNA molecule and is flanked by inverted terminal repeat sequences. In some embodiments, the disclosed expression cassette comprises a nucleotide sequence having 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100% identity with the full length of SEQ ID NO:1, wherein the nucleotide sequence encodes an interfering RNA molecule and is flanked by inverted terminal repeat sequences. The major portion of SEQ ID NO:1 that may be modified without impairing insecticidal activity is TTCAAGAGA, which corresponds to a hairpin loop.

[0299] In some embodiments, the disclosed expression cassette comprises a nucleotide sequence having 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100% identity with the full length of SEQ ID NO:1, flanked at the 5' end by an inverted terminal repeat sequence having approximately 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:4. In some embodiments, the disclosed expression cassette comprises a nucleotide sequence having 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100% identity with the full length of SEQ ID NO:1, flanked at the 3' end by an inverted terminal repeat sequence having approximately 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the full length of SEQ ID NO:5.

[0300] In some embodiments, the disclosed expression cassette contains at least 1, 3, 5, 7, 9, or 10 copies of the nucleotide sequence encoding an interfering RNA molecule (e.g., SEQ ID NO: 1). In some embodiments, the disclosed expression cassette contains at least 1, 3, 5, 7, 9, or 10 copies of the nucleotide sequence encoding an interfering RNA molecule. In some embodiments, the disclosed expression cassette contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the nucleotide sequence encoding an interfering RNA molecule.

[0301] In some embodiments, the disclosed expression cassette comprises a nucleotide sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the full length of SEQ ID NO:7 or its complement. In some embodiments, the disclosed expression cassette comprises a nucleotide sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the full length of SEQ ID NO:8 or its complement.

[0302] In some embodiments, the disclosed expression cassette comprises a nucleotide sequence having approximately 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the full length of SEQ ID NO:7 or its complement. In some embodiments, the disclosed expression cassette comprises a nucleotide sequence having approximately 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the full length of SEQ ID NO:8 or its complement.

[0303] In some embodiments, the disclosed expression cassette comprises a nucleotide sequence that is 100% identical to the full length of SEQ ID NO:7 or its complement. In some embodiments, the disclosed expression cassette comprises a nucleotide sequence that is 100% identical to the full length of SEQ ID NO:8 or its complement.

[0304] In some embodiments, the disclosed expression cassette is stably integrated into the genome of a host organism. In some embodiments, the disclosed expression cassette is stably integrated into the genome of a microorganism, such as a fungal cell. In some embodiments, the disclosed expression cassette is stably integrated into the genome of a Pichia pastoris species, such as *Pichia pastoris*, a yeast species, such as *Saccharomyces cerevisiae*, including strains FL100 or S288C, or a *Yersinia* species, such as *Yersinia lipolytica*. In some embodiments, the disclosed expression cassette is integrated via transposition, such as stably integrated into the genome of a host organism, such as through transposon-mediated genome integration methods.

[0305] In some embodiments, a transposon system is used to integrate a publicly disclosed expression cassette into the genome of a host organism. In some embodiments, the transposon system comprises a transposon vector and a transposase. In some embodiments, the transposon system is piggyBac, Super piggyBac, Sleeping Beauty, Hyperactive Sleeping Beauty, helitron transposon system, Tol2 transposon system, TcBuster transposon system, including, for example, piggyBac-like transposon systems, or mutant transposon systems thereof.

[0306] In some embodiments, the disclosed expression cassette is integrated into the genome of a host cell via a transposase comprising a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with the full length of SEQ ID NO:10. In some embodiments, the disclosed expression cassette is integrated into the genome of a host cell via a transposase comprising a nucleotide sequence having approximately 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity with the full length of SEQ ID NO:10.

[0307] In some embodiments, the disclosed expression cassette is integrated into at least one site in the host organism's genome, such as an integration site or an integration site. In some embodiments, the disclosed expression cassette is integrated into at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 sites in the host organism's genome. In some embodiments, the disclosed expression cassette is integrated into 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 integration sites in the host cell's genome.

[0308] In some embodiments, the disclosed expression cassette is integrated into the genome of a host organism, such as *Saccharomyces cerevisiae* strain FL100, at one or more of the following integration sites: chromosome IV (NC_001136, position 543,705); chromosome IV (NC_001136, position 1,357,520); chromosome VIII (NC_001140, position 124,029); chromosome X (NC_001142, position 181,309); chromosome XI (NC_001143, position 300,654); and chromosome XII (NC_001144, position 213,991).

[0309] host organism

[0310] In some respects, this document provides host organisms containing multinucleotide sequences, such as expression cassettes, flanked by inverted terminal repeat sequences, such as host organisms containing disclosed expression cassettes, wherein the multinucleotide sequences encode interfering RNA molecules capable of suppressing gene expression in mosquitoes.

[0311] In some embodiments, the disclosed host organism is a prototrophic form, such as a prototrophic yeast cell. In some embodiments, the disclosed host organism is a prototrophic form of *Pichia pastoris*, *Saccharomyces*, or *Yersinia*. In some embodiments, the disclosed host organism is a prototrophic form of *Pichia pastoris*, *Saccharomyces cerevisiae*, or *Yersinia lipolytica*. In some embodiments, the disclosed host organism is a prototrophic *Saccharomyces cerevisiae* strain FL100. In some embodiments, the disclosed host organism is a prototrophic *Saccharomyces cerevisiae* strain S288C.

[0312] In some embodiments, the disclosed host organism comprises a auxotrophic rescue promoter flanked by 5' inverted terminal repeat sequences. In some embodiments, the disclosed host organism comprises a nucleotide sequence encoding an interfering RNA molecule flanked by 3' inverted terminal repeat sequences. In some embodiments, the expression cassette is integrated into the host cell's genome, such as being stably integrated into the host cell's genome.

[0313] In some implementations, the disclosed host organism contains a nucleotide sequence encoding an interfering RNA molecule, wherein the interfering RNA molecule is an RNA construct, double-stranded RNA (dsRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), or antisense oligonucleotide.

[0314] In some embodiments, the disclosed host organism produces interfering RNA molecules that suppress gene expression in mosquito species of the genera *Aedes*, *Anopheles*, or *Culex*. In some embodiments, the disclosed host organism produces interfering RNA molecules that suppress gene expression in *Anopheles gambiae*, *Aedes aegypti*, *Aedes albopictus*, *Culex pipiens sharpii*, or *Culex quinquefolius*. In some embodiments, the disclosed host organism produces interfering RNA molecules that suppress the expression of *Shaker* in *Aedes*, *Anopheles*, or *Culex* species, such as *Anopheles gambiae*, *Aedes aegypti*, *Aedes albopictus*, *Culex pipiens sharpii*, or *Culex quinquefolius*.

[0315] In some embodiments, the expression cassette is stably integrated into the genome of the host organism. In some embodiments, the disclosed host organism is a microorganism, such as a fungal cell, like a yeast cell. In some embodiments, the disclosed host organism is a species of *Pichia pastoris*, such as *Pichia pastoris*, a species of *Saccharomyces cerevisiae*, such as strain FL100 or S288C, or a species of *Yersinia*, such as *Yersinia lipolytica*. In some embodiments, the disclosed host organism is *Saccharomyces cerevisiae*. In some embodiments, the disclosed host organism is *Saccharomyces cerevisiae* strain FL100 or strain S288C.

[0316] The *Saccharomyces cerevisiae* strain FL100 has been described by Casaregola et al., *Yeast* 1998;14(6):551-64, and its whole genome shotgun sequence is available in GenBank accession number JRIT00000000.1. Furthermore, the *Saccharomyces cerevisiae* S288C genome has been described by Fisk et al., *Yeast*. 2006 Sep;23(12):857-65 and YeastGenome.org (GenBank Accession No.: GCF_000146045.2). Those skilled in the art can obtain general genomic details of the disclosed host organism.

[0317] In some embodiments, the disclosed host organism contains an expression cassette containing inverted terminal repeat sequences that facilitate integration of the expression cassette via a transposon system (e.g., via transposition). In some embodiments, the transposon system is piggyBac, Super piggyBac, Sleeping Beauty, Hyperactive Sleeping Beauty, helitron transposon system, Tol2 transposon system, TcBuster transposon system, including, for example, piggyBac-like transposon systems, or mutant transposon systems thereof.

[0318] In some embodiments, the disclosed host organism includes an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule, a auxotrophic rescue promoter, and an auxotrophic rescue gene, wherein the auxotrophic rescue promoter is flanked by a 5' inverted terminal repeat sequence and the nucleotide sequence encoding the interfering RNA molecule is flanked by a 3' inverted terminal repeat sequence.

[0319] In some embodiments, the genome of the disclosed host organism contains at least one genomic integration site of an expression cassette (e.g., a disclosed expression cassette). In some embodiments, the genome of the disclosed host organism contains at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 genomic integration sites of an expression cassette. In some embodiments, the genome of the disclosed host organism contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 integration sites of an expression cassette.

[0320] In some embodiments, the disclosed host organism's genome contains at least one genomic integration site encoding a nucleotide sequence of an interfering RNA molecule. In some embodiments, the disclosed host organism's genome contains at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 genomic integration sites encoding a nucleotide sequence of an interfering RNA molecule. In some embodiments, the disclosed host organism's genome contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 integration sites encoding a nucleotide sequence of an interfering RNA molecule. In some embodiments, the interfering RNA molecule suppresses the expression of the Shaker gene in mosquitoes.

[0321] In some embodiments, the disclosed host organism comprises a nucleotide sequence encoding an interfering RNA molecule targeting the Shaker gene in mosquitoes. In some embodiments, the disclosed host organism comprises a nucleotide sequence transcribed from the nucleotide sequence encoding the interfering RNA molecule, the interfering RNA molecule being partially or completely complementary to a sequence containing at least 80%, 84%, 88%, 92%, 96%, or 100% identity to the full length of SEQ ID NO:2. In some embodiments, the disclosed host organism comprises an interfering RNA molecule comprising at least 25 consecutive nucleotides, said at least 25 consecutive nucleotides being partially or completely complementary to the full length of SEQ ID NO:2. In some embodiments, the nucleotide sequence encoding the interfering RNA molecule is integrated (e.g., stably integrated) into the genome of the host organism.

[0322] In some embodiments, the disclosed host organism comprises a nucleotide sequence encoding an interfering RNA molecule shRNA.463 (represented by SEQ ID NO:1), wherein the nucleotide sequence is flanked by inverted terminal repeats. In some embodiments, the disclosed host organism comprises a nucleotide sequence having at least 80% or at least 90% identity with SEQ ID NO:1, wherein the nucleotide sequence is flanked by inverted terminal repeats. In some embodiments, the disclosed host organism comprises a nucleotide sequence having 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, or 98% identity with SEQ ID NO:1, wherein the nucleotide sequence is flanked by inverted terminal repeats. In some embodiments, the disclosed host organism comprises a nucleotide sequence encoding an interfering RNA molecule that is 100% identical to the full length of SEQ ID NO:1, wherein the nucleotide sequence is flanked by inverted terminal repeats. In some implementations, the inverted terminal repeat sequence is located on the 3' flanking side of the nucleotide sequence encoding the interfering RNA molecule.

[0323] In some embodiments, the disclosed host organism comprises a nucleotide sequence having at least 80% or at least 90% sequence similarity to the full length of SEQ ID NO:1, wherein the nucleotide sequence is flanked by inverted terminal repeats. In some embodiments, the disclosed host organism comprises a nucleotide sequence having 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, or 98% similarity to SEQ ID NO:1, wherein the nucleotide sequence is flanked by inverted terminal repeats. In some embodiments, the disclosed host organism comprises a nucleotide sequence comprising the full length of SEQ ID NO:1, wherein the nucleotide sequence is flanked by inverted terminal repeats. In some embodiments, the disclosed host organism comprises a nucleotide sequence consisting of the full length of SEQ ID NO:1, wherein the nucleotide sequence is flanked by inverted terminal repeats. In some embodiments, the inverted terminal repeats are located on the 3' end flanking of the nucleotide sequence encoding the interfering RNA molecule.

[0324] In some embodiments, the disclosed host organism comprises an expression cassette containing a nucleotide sequence having at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the full length of SEQ ID NO:7 or its complement. In some embodiments, the disclosed host organism comprises an expression cassette containing a nucleotide sequence having at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the full length of SEQ ID NO:8 or its complement.

[0325] In some implementations, the disclosed host organism is a yeast cell containing genotype

[0326] .

[0327] In some implementations, the disclosed host organism is a yeast cell containing genotype

[0328] .

[0329] In some embodiments, the disclosed host organism comprises an expression cassette containing a minimal or truncated promoter operatively linked to a selectable marker gene (e.g., leu2d). In some embodiments, the disclosed host organism containing a minimal or truncated promoter exhibits elevated levels of interfering RNA molecules compared to expression cassettes containing native or full-length promoters operatively linked to selectable marker genes. In some embodiments, the disclosed host organism contains interfering RNA molecules with at least 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500% increased expression levels of interfering RNA molecules relative to expression cassettes containing native promoters operatively linked to selectable marker genes. In some embodiments, the expression level of the interfering RNA molecule is increased by approximately 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500% relative to the expression cassette containing the natural promoter operably linked to the selectable marker gene. In some embodiments, the expression level is 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold greater relative to the expression cassette containing the natural promoter operably linked to the selectable marker gene.

[0330] Production of RNA-interfering biopesticides

[0331] In some respects, this document provides methods for culturing disclosed host organisms, such as RNA-interfering biopesticides, on an industrial scale. In some embodiments, the disclosed methods include culturing the disclosed host organism in a fermentation medium, such as a high-cell-density fermentation medium.

[0332] In some embodiments, the disclosed host organism produces a cell dry weight of at least 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, or 50 g / L after growing in a fermentation medium for about 72 hours. In some embodiments, the disclosed host organism, grown in a fermentation medium for approximately 72 hours, produces cell dry weights of approximately 0 g / L, 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, 55 g / L, 60 g / L, 65 g / L, 70 g / L, 75 g / L, 80 g / L, 85 g / L, 90 g / L, 95 g / L, or 100 g / L. In some embodiments, the fermentation medium is a high-cell-density fermentation medium.

[0333] In some embodiments, the disclosed host organism reaches an OD600 of at least about 5, 25, 50, 75, or 100 after growing in a fermentation medium for about 72 hours. In some embodiments, the disclosed host organism reaches an OD600 of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 after growing in a fermentation medium for about 72 hours. In some embodiments, the fermentation medium is a high-cell-density fermentation medium.

[0334] In some embodiments, culturing a host organism according to the methods described herein results in the production of interfering RNA molecules, such as continuous production. In some embodiments, culturing the host organism in a fermentation medium for about 72 hours results in an increase in the expression level of the interfering RNA molecules by at least about 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500% relative to time = 0 hours. In some embodiments, culturing the host organism in a fermentation medium for about 72 hours results in an increase in the expression level of the interfering RNA molecules by about 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500% relative to time = 0 hours. In some embodiments, the host organism is a yeast cell, such as *Pichia pastoris*, *Saccharomyces cerevisiae*, or *Yersinia lipolytica*. In some embodiments, the disclosed method includes culturing the host organism in a high-cell-density fermentation medium. In some embodiments, interfering RNA molecules are used as biopesticides to disrupt the adaptability and / or survival of mosquitoes, such as in mosquito population control.

[0335] In some instances, fermentation media, such as high-cell-density fermentation media, are proprietary or commercial compositions containing, for example, carbon sources such as glucose, nitrogen sources such as peptone, and amino acids necessary for yeast growth. Yeast culture methods such as high-cell-density fed-batch culture are described, for example, in Hoek et al., Biotechnol Bioeng. 2000 Jun 5;68(5):517-23 and Vogel & Todaro, Cross-Flow Filtration.

[0336] Composition

[0337] In some aspects, this document provides compositions comprising disclosed polynucleotides, expression cassettes, host organisms, or combinations thereof. In some embodiments, the disclosed compositions comprise live host organisms as disclosed herein. In some embodiments, the disclosed compositions comprise dead host organisms as disclosed herein, such as heat-inactivated and / or lyophilized interfering RNA biopesticides. In a preferred embodiment, the host organism, such as the interfering RNA biopesticide, is heat-inactivated and / or lyophilized to reduce or eliminate the ability of the host organism to grow after being released into the treatment area. In a preferred embodiment, the disclosed yeast interfering RNA biopesticide is heat-inactivated to reduce or eliminate the ability of yeast to grow after being released into the treatment area. In some embodiments, the yeast is formulated as a ready-to-use dried preparation. In some embodiments, the yeast is *Saccharomyces cerevisiae*.

[0338] In some embodiments, the disclosed composition comprises an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule, wherein the interfering RNA molecule is an RNA construct, double-stranded RNA (dsRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), or an antisense oligonucleotide. In some embodiments, the expression cassette is integrated, for example, stably integrated into the genome of a host organism, such as a yeast interfering RNA biopesticide.

[0339] In some embodiments, the disclosed composition comprises an expression cassette encoding an interfering RNA molecule that suppresses gene expression in mosquitoes of the genera *Aedes*, *Anopheles*, or *Culex*. In some embodiments, the disclosed composition comprises an expression cassette encoding an interfering RNA molecule that suppresses gene expression in *Anopheles gambiae*, *Aedes aegypti*, *Aedes albopictus*, *Culex pipiens sharpii*, or *Culex quinquefolius*. In some embodiments, the disclosed composition comprises an expression cassette encoding an interfering RNA molecule that suppresses the expression of *Shaker* in *Aedes*, *Anopheles*, or *Culex* species, such as *Anopheles gambiae*, *Aedes aegypti*, *Aedes albopictus*, *Culex pipiens sharpii*, or *Culex quinquefolius*.

[0340] In some embodiments, the disclosed composition comprises an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule that targets the Shaker gene in mosquitoes. In some embodiments, the interfering RNA molecule is capable of suppressing the expression of the Shaker gene in mosquitoes.

[0341] In some embodiments, the disclosed composition comprises a nucleotide sequence transcribed from a nucleotide sequence encoding an interfering RNA molecule, the interfering RNA molecule being partially or completely complementary to a sequence containing at least 80%, 84%, 88%, 92%, 96%, or 100% identity to the full length of SEQ ID NO:2. In some embodiments, the disclosed composition comprises an expression cassette encoding an interfering RNA molecule comprising at least 25 consecutive nucleotides, said at least 25 consecutive nucleotides being partially or completely complementary to the full length of SEQ ID NO:2. In some embodiments, the disclosed composition comprises a disclosed expression cassette integrated into the genome of a host organism. In some embodiments, the disclosed composition comprises a nucleotide sequence encoding an interfering RNA molecule that is integrated (e.g., stably integrated) into the genome of a host organism.

[0342] In some embodiments, the disclosed composition comprises an expression cassette containing a nucleotide sequence encoding the interfering RNA molecule shRNA.463 (represented by SEQ ID NO:1), wherein the nucleotide sequence is flanked by 3' inverted terminal repeats. In some embodiments, the disclosed composition comprises an expression cassette containing a nucleotide sequence having at least 80% or at least 90% identity with SEQ ID NO:1, wherein the nucleotide sequence is flanked by inverted terminal repeats. In some embodiments, the disclosed composition comprises an expression cassette containing 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, or 98% of the nucleotide sequence of SEQ ID NO:1, wherein the nucleotide sequence is flanked by inverted terminal repeats. In some embodiments, the disclosed composition comprises an expression cassette containing a nucleotide sequence having 100% identity with the full length of SEQ ID NO:1, wherein the nucleotide sequence is flanked by inverted terminal repeats. In some embodiments, the disclosed composition comprises an expression cassette containing a nucleotide sequence encoding an interfering RNA molecule that is 100% identical to the full length of SEQ ID NO:1, wherein the nucleotide sequence is flanked by inverted terminal repeat sequences. In some embodiments, the inverted terminal repeat sequences are located at the 3' end flanking of the nucleotide sequence encoding the interfering RNA molecule.

[0343] In some embodiments, the disclosed compositions comprise a nucleotide sequence encoding an interfering RNA molecule that is integrated into the genome of a host organism. In some embodiments, the disclosed compositions comprise a nucleotide sequence encoding an interfering RNA molecule that is integrated (e.g., stably integrated) into the genome of a host organism.

[0344] In some embodiments, the disclosed compositions comprise a disclosed expression cassette stably integrated into the genome of a microorganism, such as a fungal cell. In some embodiments, the disclosed compositions comprise a disclosed expression cassette stably integrated into the genome of a *Pichia* species, such as *Pichia pastoris*, a *Yeast* species, such as *Saccharomyces cerevisiae*, or a *Yeast* species, such as *Yeast lipolytica*. In some embodiments, the disclosed compositions comprise a disclosed expression cassette stably integrated into the genome of *Saccharomyces cerevisiae* strain FL100 or strain S288C.

[0345] In some embodiments, the disclosed composition comprises prototrophic yeast cells containing genotypes MATa,PiggyBac (leu2d / PTDH3-shRNA_463-TCYC1), CEN / ARS (URA3 / SPBase_Sc-CO).

[0346] In some embodiments, the disclosed composition comprises prototrophic yeast cells containing genotypes MATa,PiggyBac (leu2d / PTDH3-shRNA_463-TCYC1, PTDH3-shRNA_463-TCYC1, PTDH3-shRNA_463-TCYC1), CEN / ARS (URA3 / SPBase-Sc-CO).

[0347] In some embodiments, the disclosed composition has insecticidal properties, such as mosquitoic properties. In some embodiments, the disclosed composition, upon ingestion by a mosquito, disrupts the mosquito's adaptability and / or survival. In some embodiments, the disclosed composition kills a mosquito upon ingestion, such as a mosquito larva or adult mosquito.

[0348] In some embodiments, the disclosed composition further comprises an insecticide, such as a chemical insecticide. In some embodiments, the insecticide is DEET (N,N-diethyl-meta-toluamide deltamethrin), etofenprox, methoprene, permethrin, piperonyl butoxide, phenothrin, malathion, pyriproxyfen, or a combination thereof.

[0349] In some embodiments, the disclosed compositions comprise attractants such as microorganisms, such as bacterial or fungal cells. In some embodiments, the attractant is yeast. In some embodiments, the attractant is a species of yeast, such as *Saccharomyces cerevisiae*. In some embodiments, the disclosed compositions further comprise additional attractants, such as bait. In some embodiments, the additional attractants include sugars, octanol, plant extracts, pheromones, volatile organic compounds, carbon dioxide, lactic acid, ammonia, or combinations thereof. Mosquito attractants are described, for example, in Dormont et al., *J Chem Ecol.* 2021 May;47(4-5):351-393.

[0350] In some embodiments, the disclosed compositions further comprise a sugar bait. In some embodiments, the bait is located within a trap such as a lure trap. In some embodiments, the bait and / or trap comprises any of the nucleic acids disclosed herein, including expression cassettes, host organisms such as interfering RNA biopesticides, or compositions.

[0351] In some embodiments, the bait contains an attractant. In some embodiments, the attractant is sugar. In some embodiments, the bait is an attractant-targeted sugar bait or an attractant-toxic sugar bait (ATSB). Attractant-targeted sugar baits typically contain an attractant, including in the form of sugar, such as fruit syrup, and a toxic agent, such as a chemical pesticide. See, for example, Wongthangsiri et al., Agriculture and Natural Resources, 2018;52(4):393-398.

[0352] Lure traps are commonly used to attract and kill pests. The design and use of such traps are well known to those skilled in the art. The lure trap of the present invention can be any device that contains an interfering RNA bio-insecticide or a combination thereof, and prevents insect pests, such as mosquitoes, from escaping after contact with the trap. The trap can have various sizes, shapes, colors, and materials. The trap can be specifically designed and manufactured for use as an insect trap, or can be a container converted and adapted from other uses, such as a glass petri dish, a metal coffee can, a cardboard box, or any common plastic, metal, fiberglass, composite, or ceramic container.

[0353] How to use

[0354] In some aspects, this document provides methods for using disclosed polynucleotides, expression cassettes, host organisms such as interfering RNA bio-insecticide compositions, or combinations thereof, for example in mosquito control. In some embodiments, the disclosed methods include contacting mosquitoes with the disclosed polynucleotides, expression cassettes, host organisms, compositions, or combinations thereof, such that the contacting mosquitoes ingest the polynucleotides, expression cassettes, host organisms, compositions, or combinations thereof.

[0355] In some embodiments, the disclosed method includes contacting mosquito larvae with the disclosed polynucleotide, expression cassette, host organism, composition, or combination thereof. In some embodiments, the disclosed method includes contacting adult mosquitoes with the disclosed polynucleotide, expression cassette, host organism, composition, or combination thereof. Mosquitoes, as referred to herein, generally encompass different stages of life, such as pupae, larvae, and adults. Furthermore, contacting mosquitoes herein encompasses either ingestion or feeding of the disclosed polynucleotide, expression cassette, host organism, composition, or combination thereof to mosquitoes.

[0356] In some embodiments, the host organism is a yeast cell, such as a species of the genus *Pichia*, such as *Pichia pastoris*, a species of the genus *Saccharomyces*, such as *Saccharomyces cerevisiae*, including strain FL100 or S288C, or a species of the genus *Yersinia*, such as *Yersinia lipolytica*. In some embodiments, the host organism is *Saccharomyces cerevisiae*, such as strain FL100 or S288C.

[0357] In some embodiments, the disclosed method also includes contacting mosquitoes with an insecticide, such as a chemical insecticide. In some embodiments, the insecticide that contacts the mosquitoes is DEET (N,N-diethyl-m-toluamide), deltamethrin, methoxyprotein, permethrin, piperonyl butyl ether, permethrin, malathion, pyriproxyfen, or a combination thereof.

[0358] In some embodiments, the disclosed method further includes contacting mosquitoes with additional attractants such as bait. In some embodiments, the additional attractants include sugars, octanol, plant extracts, pheromones, volatile organic compounds, carbon dioxide, lactic acid, ammonia, or combinations thereof. Mosquito attractants are described, for example, in Dormont et al., J Chem Ecol. 2021 May;47(4-5):351-393.

[0359] In some embodiments, the bait is a sugar bait, such as an attractive targeted sugar bait or an attractive toxic sugar bait (ATSB). In some embodiments, the sugar bait contains sugar and at least one insecticide, such as a mosquito pesticide.

[0360] ATSBs (which are known in the art and commercially available) may contain sugar baits and toxic agents, such as insecticides. ATSBs are described, for example, in WO2020185583A1, WO2009150254A1, Hapairai et al., Insect Biochem MolBiol. 2020 May; 120: 103359, Mysore et al., PLoS Negl Trop Dis. 2020 Jul; 14(7): e0008479, Wongthangsiri et al., Agric.Nat. Resour. 2018;52(4):393-398, Khan et al., PLoS One. 2013 Sep 24;8(9):e77225, Fraser et al., Malar J. 2021Mar 17;20(1):15.

[0361] In some implementations, the disclosed method results in a percentage (%) mortality rate of at least about 10%, 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 100%, including any and all numerical ranges and ranges in between.

[0362] Example

[0363] Example 1: Using Cas-CLOVER and piggyBac in combination to generate yeast strains stably integrated with expression cassettes encoding the interfering RNA pesticide Sh.463

[0364] Saccharomyces cerevisiae was engineered using a combination of the Cas-CLOVER and piggyBac systems to produce Sh.463. The yeast strains incorporated multiple integrations of the Sh.463 shRNA expression cassette cargo, which involves interfering with the expression of a conserved sequence in the mosquito Shaker gene. Previously, the efficacy of the interfering RNA pesticide Sh.463, with dual larval and adult-killing effects (Mysore et al., PLoS Negl Trop Dis., 14(7): e0008479), was described, which achieved the production and delivery of Sh.463 by engineering Saccharomyces cerevisiae to express this RNA molecule. Previous studies involved the synthesis of custom DNA oligonucleotides encoding shRNA expression cassettes corresponding to the Sh.463 target sequence, followed by stable transformation of Saccharomyces cerevisiae according to a previously described method (Hapairai et al., Sci Rep. 2017;7(1):13223).

[0365] method

[0366] Yeast strains were constructed according to the method described in Brizzee et al., J. Fungi 2023, 9, 1056. Briefly, to generate auxotrophic mutants, Cas-CLOVER was expressed on a kanamycin-resistant CEN / ARS plasmid under the ScRNR2 yeast promoter. The URA3 gene was targeted using left URA3 gRNA (SEQ ID NO:17) and right URA3 gRNA (SEQ ID NO:18) expressed by the SNR52 promoter and SNR52 terminator. Both the gRNA and Cas-CLOVER were located on the same plasmid and transformed into *Saccharomyces cerevisiae* FL100 along with a homologous donor repair (HDR) fragment located 200 bp upstream of the start codon and 200 bp downstream of the stop codon in the URA3 gene.

[0367] After removing the plasmid from the strain and verifying the deletion of the intact ORF by PCR, the strain was then used to target the LEU2 gene. Cas-CLOVER was expressed under the ScRNR2 yeast promoter on the hygromycin-resistant CEN / ARS plasmid. The LEU2 gene was targeted using left LEU2 gRNA (SEQ ID NO:19) and right LEU2 gRNA (SEQ ID NO:20) expressed by the SNR52 promoter and SNR52 terminator. The guide and Cas-CLOVER were both located on the same plasmid and transformed into *Saccharomyces cerevisiae* FL100 (ura3) along with a homologous donor repair (HDR) fragment. In (0), the homologous donor repair fragment is located 200 bp upstream of the start codon and 200 bp downstream of the stop codon in the LEU2 gene. Plasmids carrying gRNA and Cas-CLOVER were removed from the strain, and the complete ORF deletion of the leu2 gene was confirmed by PCR. The resulting genotype was MATa,ura3. 0, leu2 0.

[0368] Super piggyBac transposase was cloned into a URA3 selection plasmid containing a CEN / ARS origin of replication. A basic piggyBac transposon plasmid was generated by cloning the minimal Leu2d promoter and Leu2 gene from pRS425; a plasmid map of pRS425 is available to those skilled in the art, for example, from www.snapgene.com. The nucleotide sequences encoding the minimal Leu2d promoter and Leu2 gene were inserted into the piggyBac ITR. A nucleotide sequence encoding Sh.463 shRNA (SEQ ID NO:1) was introduced by cloning a shRNA cassette into the piggyBac ITR at a multiple cloning site upstream of the LEU2 selection marker. This shRNA cassette contains nucleotide sequences encoding the GAP promoter (SEQ ID NO:11), Sh.463 shRNA (SEQ ID NO:1), and the CYC1 terminator (SEQ ID NO:12).

[0369] Following the instructions of the EZ-Yeast Transformation Kit, *Saccharomyces cerevisiae* was transformed at a transposon to transposase ratio of 3:1 (750 ng: 250 ng). After incubation at 30°C for 1 hour, the transformation solution was removed. The yeast was recovered in SD-URA medium and stirred overnight at 30°C for piggyBac selection. On the second day, the cells were cultured on CM-uracil agar plates for 2–3 days until colonies formed. Colonies were picked and placed on CM-leucine agar plates to select transformants integrating the leucine selection marker. Subsequently, the colonies from the plates were expanded into 96-well deep-well plates for amplification.

[0370] The expression of Sh.463 shRNA was verified by RNA extraction and cDNA synthesis, and the expression level of shRNA was assessed by qRT-PCR. Figure 2 Various strains expressing a range of mosquito-killing interfering RNA pesticide (IRP) levels were shown. Relative expression was compared to the control strain DMT9-51.1, which was engineered to express Sh.463 using only piggyBac integration.

[0371] Auxotrophic complementation (also known as auxotrophic complementation) was achieved by amplifying 200 bp upstream or downstream of the ura3 or leu2 gene from the parental *Saccharomyces cerevisiae* strain FL100 and transforming it into 1 μg of the nucleotide sequence encoding the minimal promoter leu2d and the leu2 gene, as described above with modifications. After incubation at 30°C for 1 hour, cells were recovered for 4 hours in YPD medium instead of selection medium, and then plated on selection plates (CM-URA or CM-LEU2). Colonies were picked and placed on seed plates with selection medium, and subsequently grown for qPCR analysis.

[0372] Example 2: ATSB assay for mosquito larval and adult mosquito killing using a Cas-CLOVER / piggyBac yeast strain exhibiting relatively high Sh.463 expression.

[0373] Yeast strains exhibiting the highest comparative levels of Sh.463 were selected for further studies in the mosquito-killing assay. These strains were engineered according to the method described in Example 1. These strains were down-selected, and their auxotrophic forms (denoted by "R") were restored. Restoration (e.g., complementation) of the auxotrophic form to transiently express the yeast strain DMT4-342 (pRS426_463, SEQ ID NO:9) showed a 10-fold reduction in Sh.463 expression levels compared to the low-expression, stably integrated piggyBac strain DMT9-51.1R by qRT-PCR. The highest-expressing strains, DMT9-52.2R #3 and DMT9-56.10R #3, were included in laboratory larval killing studies using Anopheles gambiae, Aedes aegypti, Culex quinquefolium, and Culex pipiens sharpium.

[0374] Figure 3 The Cas-CLOVER / piggyBac synthetic yeast strains, after downward selection and auxotrophic recovery, are shown, exhibiting relatively high levels of Sh.463 expression. Sh.463 shRNA expression levels were assessed by qRT-PCR, as described in Brizzee et al., J. Fungi 2023, 9, 1056, and are shown relative to the piggyBac integrative strain DMT9-51.1R#1. The initial piggyBac integrative strain used a native leucine promoter (SEQ ID NO:15) and exhibited very low expression compared to subsequent strains with minimal promoters and multiple shRNA copies. Sh.463 genotypes and auxotrophic recovery genotypes are shown in Table 3.

[0375] Table 3: Genotypes of yeast strains selected by downward selection

[0376]

[0377] Larval kill assay: The larval kill assay was performed as previously described, with 40 mg of yeast as the full dose for every 20 larvae. See, for example, Mysore et al., Insect genomics: Methods and protocols. 2019;1858:213-231. Humana Press; Hapairai et al., Scientific Reports. 2017;7:13223; Mysore et al., PLoS Negl Trop Dis., 2020;14(7): e0008479. Figure 6 The larvicidal activity of heat-inactivated Cas-CLOVER / piggyBac synthetic yeast strains expressing shRNA Sh.463 targeting the mosquito Shaker gene was demonstrated. The larvicidal activity of 50% doses of the control *Saccharomyces cerevisiae* strain DMT4-347.1R and *Saccharomyces cerevisiae* Sh.463-expressing strains DMT9-52.2R #3 and DMT9-56.10R #3 against *Anopheles gambiae*, *Aedes aegypti*, *Culex pipiens quinquefolius*, and *Culex pipiens puntata* was shown. Compared to the control treatment, the yeast treatments DMT9-52.2R #3 and DMT9-56.10R #3 induced significant larval mortality (P<0.001).

[0378] These experiments demonstrate that larvae ingesting dried, heat-inactivated DMT9-52.2R #3 and DMT9-56.10R #3 yeast strains are effective in killing mosquitoes before adulthood. Furthermore, the CLOVER / piggyBac Sh.463 yeast strain was observed to be more potent than Sh.463 produced by conventional methods as described in Mysore et al., PLoS Negl Trop Dis., 2020;14(7):e0008479. In other words, a smaller quantity of CLOVER / piggyBac Sh.463 yeast strain is needed to achieve a lethal dose compared to strains produced by the methods described by Mysore et al.

[0379] Adult-killing assay: The ATSB assay was performed as previously described to evaluate the adult-killing activity of *Saccharomyces cerevisiae* Sh.463 expression strains DMT9-52.2R #3 and DMT9-56.10R #3. See, for example, Mysore et al., *Insects* 2021;12(11):986. Here, the full dose consisted of approximately 5 μl of 0.4 μg / μl yeast in the sugar bait. Figure 7The adult-killing activity of the Cas-CLOVER / piggyBac synthetic yeast strain expressing shRNA Sh.463 was demonstrated. Adult mosquito-killing ability was demonstrated by delivering 50% dose concentrations of DMT9-52.2R #3 and DMT9-56.10R #3 yeast as ATSB to adult female Anopheles gambiae, Aedes aegypti, Aedes albopictus, Culex quinquefolius, and Culex pipiens sharpina. Both DMT9-52.2R #3 and DMT9-56.10R #3 induced significant mortality in adult female mosquitoes compared to control yeast and sugar bait (ASB) treatment (P < 0.001, ANOVA).

[0380] These experiments on adult females demonstrated the adult-killing ability of yeast strains produced using the Cas-CLOVER / piggyBac system. Similar to the larval-killing assay, a smaller dose of yeast was used to achieve a lethal dose compared to similar experiments using laboratory yeast strains in the past (unpublished data). These studies not only indicate the feasibility, but also the advantages, of using Cas-CLOVER and piggyBac-engineered Saccharomyces cerevisiae containing a stably integrated Sh.463 shRNA expression cassette for controlling Aedes, Anopheles, and Culex mosquito larvae and adults.

[0381] After completing insecticidal tests on the two optimal yeast strains, the fully recovered strains (see Table 3) were subsequently cultured by shaking on synthetic growth media and industrial fermentation media. The high growth rates achieved in these cultures indicate that the yeast performs well in industrial-scale fermentation, enabling the global deployment of these insecticides. Additional fermentation scale-up studies are described in Example 4.

[0382] Example 3: Whole genome sequencing of the Cas-CLOVER / piggyBac engineered Saccharomyces cerevisiae strain expressing mosquito-killing shRNA Sh.463

[0383] Whole-genome sequencing and external validation were performed via Oxford Nanopore Technology to determine the genomic integration sites for DMT9-52.2R #3 and DMT9-56.10R #3. For DMT9-56.10R #3, 135 sequencing reads were identified and directly mapped to the piggyBac transposon. Flanking sequences of the piggyBac ITR were searched using BLASTn (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to identify the genomic integration site. These reads identified the insecticide cargo integrated at position 543,705 on chromosome IV (NC_001136), located between the NRG1 and HEM13 genes. Figure 4 As shown.

[0384] For DMT9-52.2R #3, sequencing revealed five distinct genomic integration sites: (A) chromosome IV (NC_001136, position 1,357,520), located between the ADA2 and UTP6 genes; (B) chromosome VIII (NC_001140, position 124,029), located between the SOD2 and TDA3 genes; (C) chromosome X (NC_001142, position 181,309), located between the PBS2 and MCO6 genes; (D) chromosome XI (NC_001143, position 300,654), located within the STB6 gene; and (E) chromosome XII (NC_001144, position 213,991), located within the MLH2 gene, as shown in Figure 5.

[0385] Therefore, the insecticidal Sh.463 shRNA expression cassette stably integrates into chromosomes IV, VIII, X, XI, and XII, with two of the five genomic integration sites located intragenetically, occurring in the STB6 gene on chromosome XI and the MLH2 gene on chromosome XII, respectively. For each of these characterized yeast strains, Table 4 summarizes the integration events, Sh.463 copy number, and genomic location.

[0386] Table 4: Summary of WGS data for yeast strains DMT9-56.10R #3 and DMT9-52.2R #3

[0387]

[0388]

[0389] Example 4: Fermentation study of a Cas-CLOVER / piggyBac engineered Saccharomyces cerevisiae strain expressing mosquito-killing shRNA Sh.463

[0390] A pilot-scale fermentation study was conducted according to the method described in Brizzee et al., J. Fungi 2023, 9, 1056 to determine the economic feasibility of scaling up fermentation to an industrial scale. Confirmation of feasibility would highlight the additional advantage of this engineered yeast, which is easy to cultivate and deploy on a large scale. At fermentation scales of 5 L to 10 L, using two different high-cell-density growth fermentation media, HCD and DFM, DMT9-56.10R #3 produced cell dry weights (DCW) of 121.3 g / L and 23.5 g / L, respectively. Figure 8The 72-hour growth curves of DMT9-56.10R #3 and Sh.463 expression are shown in the presence of HCD and DFM. Relative expression was quantified by qRT-PCR and compared with expression at time point 0 (TP0). Dashed lines correspond to HCD and DFM for each culture medium condition. During the 72-hour period, the optical densities of the two fermentation media reached 173.92 and 77.81, respectively. Figure 8 (solid line). Except under high growth conditions, the expression of Sh.463 remained stable throughout the fermentation process regardless of the culture medium used. Figure 8 ,dotted line).

[0391] Fermentation is being conducted at scales of 5L to 10L because system parameters can be scaled up to large-scale fermenters, such as those exceeding 100L. Furthermore, downstream processes are currently underway to optimize the cleaning of the yeast material, similar to that used in nutrient yeast production. See, for example, Vogel & Todaro, Fermentation and Biochemical Engineering Handbook: Principles, process design and equipment. Elsevier Science. 1996;7:271-347.

[0392] Example 5: Field Test

[0393] Field assays for larvicides are currently being conducted in Trinidad and Tobago. Guiyun Yan's laboratory has described several potential field sites in Kenya (Kweka et al., PLoS One. 2012; 7(12): e52084). Several field sites exist in the area near St. Augustine, Trinidad and Tobago. The site is located on a remote stretch of Los Armadillos Road. The area around the site is completely covered by forest for several kilometers. Severson and Chadee's laboratory has conducted larval sampling experiments at these sites, where they found a large number of Aedes aegypti larvae.

[0394] Small-scale field trials: Small-scale field trials were conducted on natural mosquitoes in their natural breeding grounds. The objectives of these studies were: i) to determine the efficacy of interfering RNA larvicides in natural breeding grounds, including residual activity; ii) to identify the optimal field application dose; iii) to monitor abiotic parameters that may affect the efficacy of the larvicides; and iv) to record qualitative observations of non-target biota coexisting with mosquito larvae (WHO, 2005). Specific interfering RNA larvicides and delivery strategies deemed appropriate in simulated and semi-field trials were evaluated in the field. The habitats evaluated in these trials included natural and man-made containers not used for storing drinking water. As described in the WHO guidelines (2005), at least three replicates were randomly selected for each type of habitat for each dose of the experimental or control larvicide formulation. Samples were collected exactly before treatment, on day 2, and weekly thereafter, and the abundance of immature mosquitoes (first and second instar larvae, third and fourth instar larvae, and pupae) was monitored post-treatment until the density of fourth instar larvae in the treated habitat was comparable to that in the control container. Potential and residual activity were determined by measuring the abundance of each larval instar and pupa before and after treatment (taking into account dynamic changes occurring in the treatment and control containers). Adult emergence was monitored by sampling and counting pupal skins. Data were evaluated using ANOVA as described in the WHO (2005) guidelines.

[0395] Large-scale field trials: Validating the efficacy of larvicides deemed acceptable in small-scale field trials in natural mosquito populations in their natural breeding habitats in larger-scale field trials conducted in accordance with WHO (2005) guidelines. The objectives of these trials include: i) confirming the efficacy of larvicides at selected field application doses when applied to large plots in natural breeding habitats; ii) validating the residual activity and application intervals of the larvicides; iii) examining the ease of application and dispersal of the larvicides; iv) assessing community acceptance of this intervention; and v) detecting any unintended effects of the treatment on non-target organisms (WHO, 2005). The pre-treatment density of larvae and pupae in each larval habitat should be assessed at least twice within one week prior to treatment. The habitats assessed include those assessed in small-scale field trials, but 25 replicates are assessed for each control or experimental treatment. Samples are collected and assessed, and data are analyzed using the same general procedures described for small-scale trials. Non-target organisms coexisting with mosquito larvae are also counted and examined to determine unintended effects of the larvicide treatment. In addition, the ease of storage, handling, and application of the pesticide formulations will be assessed. Observations on the acceptance of these larvicides by residents in the area will also be recorded.

[0396] Equivalent schemes and scope

[0397] Many equivalent embodiments of the specific implementation described herein can be identified or determined by those skilled in the art through routine experiments. The scope of the invention is not intended to be limited to the foregoing, but rather as set forth in the appended claims.

[0398] In the claims, the articles "a," "an," and "the / described" may refer to one or more, unless otherwise indicated by the contrary or the context clearly states otherwise. A claim or description containing "or" between one or more members of the group is deemed satisfied if one, more than one, or all of the group members are present in, used in, or otherwise associated with a given product or method, unless otherwise stated by the contrary or the context clearly states otherwise. This invention includes embodiments in which exactly one member of the group is present in, used in, or otherwise associated with a given product or method. This invention also includes embodiments in which more than one or all of the group members are present in, used in, or otherwise associated with a given product or method.

[0399] Furthermore, it should be understood that this invention covers all variations, combinations, and arrangements in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims are incorporated into another claim. For example, any claim dependent on another claim may be modified to include one or more limitations found in any other claim dependent on the same basic claim.

[0400] When elements are presented in list form (e.g., in Markush group format), it should be understood that each subgroup of the elements is also disclosed, and any element can be removed from that group. It should be understood that, generally, when an invention or aspect of an invention is referred to as comprising specific elements, features, etc., certain embodiments or aspects of the invention consist of or are substantially composed of these elements, features, etc. For the sake of brevity, those embodiments are not explicitly described herein. It should also be noted that the term "comprising" is intended to be open-ended, allowing for the inclusion of additional elements or steps.

[0401] When a range is given, the endpoints are included. Furthermore, it should be understood that, unless otherwise stated or explicitly indicated by a person skilled in the art based on the context and understanding, values ​​expressed as ranges may take any specific value within the range or a subrange (up to one-tenth of the lower limit unit) in different embodiments of the invention, unless the context explicitly specifies otherwise.

[0402] The terms "about" or "approximately" refer to a specific value within an acceptable margin of error as determined by someone skilled in the art, which will depend in part on how the value is measured or determined, such as limitations of the measurement system. For example, according to practice in the art, "about" may mean within one standard deviation or more than one standard deviation. Alternatively, "about" may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Or, the term may mean within an order of magnitude of a value, such as within 5 times or 2 times. When a specific value is described in the application and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable margin of error for the specific value.

[0403] Furthermore, it should be understood that any particular embodiment of the invention falling within the scope of the prior art may be expressly excluded from any one or more claims. Because such embodiments are considered to be known to those skilled in the art, they may be excluded even if not expressly stated herein. Any particular embodiment of the method of the invention may be excluded from any one or more claims for any reason, whether or not related to the existence of the prior art.

[0404] sequence list

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Claims

1. A prototrophic mutant yeast cell containing an expression cassette stably integrated into at least one location within the genome of the yeast cell. The expression cassette contains a nucleotide sequence encoding an interfering RNA molecule; and the interfering RNA molecule is capable of suppressing the expression of the Shaker gene in mosquitoes; and The nucleotide sequence encoding the interfering RNA molecule is flanked by inverted terminal repeat sequences.

2. The yeast cell according to claim 1, wherein the nucleotide sequence encoding the interfering RNA molecule is flanked by a 5' inverted terminal repeat sequence and a 3' inverted terminal repeat sequence. The 5' inverted terminal repeat sequence thereon has at least about 70% identity with the full length of SEQ ID NO:5; and The 3' inverted terminal repeat sequence therein has at least about 70% identity with the full length of SEQ ID NO:

4.

3. The yeast cell of claim 1, wherein the nucleotide sequence encoding the interfering RNA molecule is operatively linked to a glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter, wherein the GAP promoter separates the nucleotide sequence encoding the interfering RNA molecule from the 5' inverted terminal repeat sequence.

4. The yeast cell according to claim 3, wherein the nucleotide sequence encoding the interfering RNA molecule is side-joined with a CYC1 terminator at the 3' end, wherein the CYC1 terminator separates the nucleotide sequence encoding the interfering RNA molecule from the 3' inverted terminal repeat sequence.

5. The yeast cell of claim 4, wherein the GAP promoter comprises a nucleotide sequence having at least 85% sequence identity with SEQ ID NO:11, and the CYC1 terminator comprises a nucleotide sequence having at least 85% sequence identity with SEQ ID NO:

12.

6. The yeast cell of claim 1, wherein the expression cassette further comprises a auxotrophic rescue promoter operatively linked to an auxotrophic rescue gene, and wherein the auxotrophic rescue promoter is side-connected to the 5' inverted terminal repeat sequence.

7. The yeast cell of claim 6, wherein the auxotrophic rescue promoter comprises a minimal auxotrophic rescue promoter, wherein the minimal auxotrophic rescue promoter comprises a nucleotide sequence having at least 70% identity with the full length of SEQ ID NO:

3.

8. The yeast cell of claim 1, wherein the auxotroph rescue gene encodes leucine.

9. The yeast cell of claim 1, wherein the expression cassette further comprises an insulator sequence that separates the 5' end of the nucleotide sequence encoding the interfering RNA molecule from the 5' inverted terminal repeat sequence; and the insulator sequence separates the 3' end of the nucleotide sequence encoding the interfering RNA molecule from the 3' inverted terminal repeat sequence, the insulator sequence having 70% identity with the full length of SEQ ID NO:

6.

10. The yeast cell of claim 1, wherein the yeast cell has a complementary auxotrophic mutation, the complementary auxotrophic mutation comprising his3Δ0, leu2Δ0, trp1Δ0, ura3Δ0 or a combination thereof.

11. The yeast cell of claim 1, wherein the expression cassette comprises at least one, at least three, or at least five copies of the nucleotide sequence encoding the interfering RNA molecule, and the expression cassette comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 sites integrated into the yeast cell genome.

12. The yeast cell of claim 1, wherein the nucleotide sequence transcribed from the nucleotide sequence encoding the interfering RNA molecule comprises at least 25 consecutive nucleotides, said at least 25 consecutive nucleotides being partially or completely complementary to a sequence comprising at least 80%, 84%, 88%, 92%, 96% or 100% identity with the full length of SEQ ID NO:

2.

13. The yeast cell of claim 1, wherein the nucleotide sequence encoding the interfering RNA molecule has at least 80% or at least 90% sequence similarity to the full length of SEQ ID NO.

1.

14. The yeast cell of claim 1, wherein the expression cassette comprises a nucleotide sequence having at least 70%, 80%, or 90% identity with the following full-length sequence: a) SEQ ID NO:7 or its complement; or b) SEQ ID NO:8 or its complement.

15. The yeast cell according to claim 1, wherein the yeast cell is Pichia pastoris, Saccharomyces cerevisiae, or Yersinia lipolytica.

16. The yeast cell according to claim 1, wherein the mosquito is a species of the genera Aedes, Anopheles, or Culex.

17. The yeast cells of claim 1, wherein 72 hours of culture in a high-cell-density fermentation medium produces at least 15 g / L of stem cell weight.

18. A composition comprising the yeast cells of claim 1, wherein the yeast cells are heat-inactivated and / or freeze-dried, the composition further comprising a sugar bait or an insecticide.

19. The composition according to claim 18, wherein the composition is selectively insecticidal against mosquitoes. The mosquitoes mentioned therein are mosquito larvae or adult mosquitoes; and The mosquitoes mentioned are species of the genera Aedes, Anopheles, or Culex.

20. A method for controlling a mosquito swarm, comprising feeding mosquitoes with the yeast cells of claim 1, thereby controlling the mosquito swarm. The mosquitoes mentioned therein are mosquito larvae or adult mosquitoes; and The mosquitoes mentioned are species of the genera Aedes, Anopheles, or Culex.

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